基于反向遗传学构建的RSV反基因组质粒及其应用

摘要

本发明公开基于反向遗传学构建的RSV反基因组质粒，所述RSV反基因组质粒为如序列表SEQ ID No.1所示的质粒pBRB‑RSV‑rLong/A2cp、如序列表SEQ ID No.2所示的质粒pBRB‑RSV‑rLong/A2cpts或如序列表SEQ ID No.3所示的质粒pBRB‑RSV‑rLong/A2cptsΔSH。本发明构建的重组质粒经拯救获得的重组RSV病毒具有明显的减毒效果，可用于制备RSV减毒活疫苗。

说明书

技术领域

本发明涉及分子生物学和生物医学领域。更具体地，涉及一种基于反向遗传学构建的RSV反基因组重组质粒及其构建方法和应用。

背景技术

人呼吸道合胞病毒(Human Respiratory Syncytial Viru，RSV)是世界范围内引起婴幼儿和两岁以下儿童下呼吸道感染的最重要的病毒性病原，同时也在老年人和免疫缺陷人群引起严重感染。目前，利巴韦林和帕利珠单抗是仅有的用于RSV治疗和预防的药物，然而利巴韦林由于喷雾给药途径、药效不佳及存在致畸作用，阻碍了其在临床应用，帕利珠单抗主要用于2岁以下高危儿童且且价格昂贵，也未得到广泛使用。因而，开发新的RSV疫苗成为亟待解决的问题。

由于FI-RSV的失败，自20世纪60年代以来，获得在体内具有毒力减弱(attenuation,att)表型的RSV减毒活疫苗一直备受关注。现在认为，RSV减毒活疫苗的主要优势在于：减毒活疫苗能经滴鼻免疫诱导机体产生系统和局部免疫,因此可同时预防上、下呼吸道感染；减毒活疫苗能引起与自然感染相同的免疫应答，在嗣后的自然感染中不会产生疾病增强作用；在母传抗体存在的情况下，经鼻免疫的减毒活疫苗仍然具有在新生儿体内进行有效复制的能力。

RSV减毒活疫苗的研究主要经历了两个阶段，一是在20世纪近30年的时间里，主要采用传统的生物学方法获得RSV减毒活疫苗,包括宿主范围(host-range,hr)突变、冷传代(cold passage,cp)突变及化学诱变后的生物学筛选等，这些方法费时、费力，而且具有较大的偶然性；二是近10年来主要采用基因工程方法或反向遗传学(reverse genetics)获得重组RSV疫苗病毒，该方法首先利用分子生物学方法获得RSV反基因组感染性DNA克隆，在插入各种已知的突变或缺失病毒的非必需基因后，经拯救获得重组RSV疫苗病毒或RSV减毒活疫苗株。

反向遗传学不仅加深了人们对传统生物学方法获得的各种表型(att,ca,ts和hr)减毒活疫苗的遗传学机制的认识，同时大大加快了新型减毒活疫苗的研制步伐，突破了以传统生物学方法获得高度稳定的减毒株时存在的障碍，产生了第二代RSV减毒活疫苗或重组RSV，使RSV减毒活疫苗的研制工作发展到了一个崭新的阶段。

因此，通过反向遗传学技术进行构建和拯救RSV减毒疫苗病毒株是研发新的RSV疫苗的核心。

发明内容

本发明的第一个目的在于提供基于反向遗传学构建的RSV反基因组pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒。

本发明的第二个目的在于提供一种上述质粒的构建方法。

本发明的第三个目的在于提供上述质粒在拯救重组RSV的应用。

为达到上述目的，本发明采用下述技术方案：

本发明一种基于反向遗传学构建的RSV反基因组质粒，所述质粒为如序列表SEQID No.1所示的质粒pBRB-RSV-rLong/A2cp、如序列表SEQ ID No.2所示的质粒pBRB-RSV-rLong/A2cpts或如序列表SEQ ID No.3所示的质粒pBRB-RSV-rLong/A2cptsΔSH。

进一步，所述质粒pBRB-RSV-rLong/A2cp在pBRB载体中装入重组序列获得的。

其中，所述的pBRB载体的序列如SEQ ID No.4所示；

所述重组序列的全长为15405nt：1-6nt为MluI克隆位点，7-26nt为T7启动子,27-67nt为锤头状核酶序列，68-15181nt为修饰后的RSV反基因组DNA序列，15182-15270nt为丁型肝炎核酶序列，15271-15399nt为T7终止子,15400-15405nt为NruI克隆位点；

所述修饰后的RSV反基因组DNA序列为在RSV Long株反基因组DNA序列的基础上删除了SH基因的非编码区112个nt(RSVLong株反基因组4497-4608nt)并在SH基因编码区末端引入4处同义突变：SH186C>T、SH189C>T、SH192A>T、SH194,195AG>GA，以增加RSVLong反基因组DNA结构的稳定性；在如下位置引入A2株上的五个冷传代(cp)突变：N 799G>A,aa267Val>Ile；F653,654AA>CG,aa218Glu>Ala；F1568,1569CA>TT,aa523Thr>Ile；L 956,957GT>AC,aa319Cys>Tyr；5068,5070C>T,T>C,aa1690His>Tyr。

进一步，所述质粒pBRB-RSV-rLong/A2cpts是在质粒pBRB-RSV-rLong/A2cp的基础上在如下位置再引入两个温度敏感型(ts)突变：M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu。

进一步，所述质粒pBRB-RSV-rLong/A2cptsΔSH是在质粒pBRB-RSV-rLong/A2cpts的基础上删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。

进一步，上述对修饰后的的RSV反基因组DNA序列是对RSV Long株反基因组DNA序列(GenBank:AY911262.1)进行遗传学改造或修饰，即在保留了RSV Long株反基因组前导序列(Leader region/genomic promotor)、转录起始信号(Gene start signal，GS)、转录终止信号(Gene end signal，GE)和尾随序列(Trailer region/antigenomic promoter)等顺式作用元件及10个基因序列的基础上，在相应的位点引入五个cp突变(N 799G>A,aa267Val>Ile；F653,654AA>CG,aa218Glu>Ala；F1568,1569CA>TT,aa523Thr>Ile；L 956,957GT>AC,aa319Cys>Tyr；L 5068,5070C>T,T>C,aa1690His>Tyr)或两个ts突变(M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu)，删除SH基因的非编码区112个核苷酸(RSVLong株反基因组4497-4608nt)或SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)，并分别在反基因组两端添加了T7启动子终止子、锤头状核酶(hammerhead ribozyme，HHr)和丁型肝炎核酶(HDV ribozyme,HDVr)，获得了基于反向遗传学技术构建的RSV反基因组重组质粒。

本发明进一步提供了所述反基因组pBRB-RSV-rLong/A2cp质粒的构建方法，优选的，基于反向遗传学构建的RSV反基因组pBRB-RSV-rLong/A2cp质粒的构建方法包括以下步骤：

1)改造pBR322载体，将ClaI-NheI酶切位点之间序列替换为ClaI*-MluI-XbaI-HindIII-SacI-AsuII-NheI限制性内切酶多克隆位点，改造后载体命名为pBRB，序列如SEQID No.4所示；

2)按SEQ ID No.1所示序列，分段化学合成RSVLong株反基因组及附属表达原件，即分别合成DNA片段MluI-AsuII(A)、AsuII-NheI(B)、NheI-NruI(C)；所述A段含有T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt，删除了SH基因的非编码区112个核苷酸(RSVLong株反基因组4497-4608nt)及A2cp突变N 799G>A,aa267Val>Ile；所述B段含有RSVLong株反基因组5179-11006nt及A2cp突变F653,654AA>CG,aa218Glu>Ala、F1568,1569CA>TT,aa523Thr>Ile、L 956,957GT>AC,aa319Cys>Tyr；所述C段含有RSVLong株反基因组11001-15226n、A2cp突变L5068,5070C>T,T>C,aa1690His>Tyr及丁型肝炎核酶序列、T7终止子序列；

3)按C、B、A顺序，依次将各片段按常规分子克隆方法连接到pBRB载体上，获得质粒pBRB-RSV-rLong/A2cp，序列如SEQ ID No.1所示。

所述质粒pBRB-RSV-rLong/A2cpts的构建方法与pBRB-RSV-rLong/A2cp大体相同，其区别在于：步骤2)合成B段基因除含有RSVLong株反基因组5179-11006nt及3个A2cp突变外，还含有两个ts突变(M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu)。

所述质粒pBRB-RSV-rLong/A2cptsΔSH的构建方法与质粒pBRB-RSV-rLong/A2cpts大体相同，其区别在于：步骤2)中合成的A片段除含有T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt及A2cp突变N 799G>A,aa267Val>Ile外，还删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。

在本发明中，所述步骤2)各个基因片段的合成方法包括但不限于全基因合成方法或RT-PCR扩增法，优选为全基因合成方法；

所述的基因片段的连接载体不限于以上pBRB载体，也可以连接在其他的载体上。

所述重组质粒中各个基因片段的连接顺序不限于C-B-A顺序，其他的连接顺序也在本发明的保护范围之内。

采用相似或相同的制备原理，RSV反基因组也可以连接至其他的表达载体中或修改的表达载体中，表达载体的修改方式不限于本申请例举的插入多克隆位点(MCS)方法。

本发明还提供了上述RSV反基因组质粒pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH经拯救获得的重组RSV，拯救的方法，包括以下步骤：

1)分别转染上述pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒和四种辅助蛋白质粒(pCITE-N,P,L,M2-1)至宿主细胞(BHK T7/9)培养；

2)盲传至宿主细胞(Vero)继续培养至病毒空斑形成。

本发明进一步提供了所述重组RSV在制备抗RSV药物中的应用。

优选的，所述应用为所述重组RSV在制备RSV疫苗上的应用。

本发明利用实时定量PCR检测连续传代后重组病毒的基因组拷贝数、免疫酶法检测空斑形成单位(PFU/mL)来确定重组病毒的复制能力和病毒滴度，利用突变位点测序分析来检测连续传代后重组病毒的稳定性。

本发明的有益效果如下：

1.本发明构建的重组质粒以RSV Long株为母本，引入RSV A2株冷传代(cp)突变和温度敏感型(ts)突变，为后续RSV基因组改造或修饰奠定了基础。

2.本发明构建的重组质粒经拯救获得的重组RSV病毒具有明显的减毒效果，可用于制备RSV减毒活疫苗。

附图说明

下面结合附图对本发明的具体实施方式作进一步详细的说明。

图1示出载体pBRB的酶切鉴定电泳图，其中M代表分子量标记物。

图2示出RSV基因片段pBRB-RSV-A；pBRB-RSV-B；pBRB-RSV-C质粒的酶切鉴定电泳图，其中M代表分子量标记物，A：pBRB-RSV-A，B：pBRB-RSV-B，C：pBRB-RSV-C；。

图3示出重组pBRB-RSV-rLong/A2cp质粒的酶切鉴定电泳图，其中M代表分子量标记物；1：NruⅠ、NheⅠ；2：AsuⅡ、NheⅠ；3：AsuⅡ、SacⅠ；4：SacⅠ、MluⅠ；5：EcoRⅤ；6：SpeⅠ；7：AflⅡ。

图4示出质粒共转染BHK-T7/9细胞后于倒置显微镜下(NIKON ECLIPSE TE 2000-S×100)观察到的细胞融合。

图5示出盲传Vero细胞后于倒置显微镜下(NIKON ECLIPSE TE 2000-S×100)观察到的细胞融合。

图6示出免疫酶法检测重组RSV病毒连续传代后病毒滴度即空斑形成单位(PFU/mL)。

图7示出实时定量PCR检测重组病毒连续传代后病毒N基因的拷贝数。

图8示出第10、20、30代RSV-rLong/A2cp重组病毒突变位点的局部测序结果。

图9示出第10、20、30代RSV-rLong/A2cpts重组病毒突变位点的局部测序结果。

图10示出第10、20、30代RSV-rLong/A2cptsΔSH重组病毒突变位点的局部测序结果。

具体实施方式

为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。

实施例1：pBRB-RSV-rLong/A2cp质粒的构建

1)将载体pBR322改造为pBRB，即化学合成两条单链DNA；5’-CGACGCGTTACGTCTAGAGGTCAAGCTTGAGCGAGCTCGGCGTTCGAAGGCGG-3’、5’-CTAGCCGCCTTCGAACGCCGAGCTCGCTCAAGCTTGACCTCTAGACGTAACGCGT-3’，将其混合退火后连入到pBR322的ClaI/NheI位点间，替换原有片段，获得新的多克隆位点(MCS)ClaI*-MluI-XbaI-HindIII-SacI-AsuII-NheI，将改造后的载体pBRB用进行SacⅠ酶切鉴定，琼脂糖凝胶电泳可见4.2kb的目的条带(图1)，序列如SEQ ID No.4所示。

2)RSV-rLong/A2cp反基因组的基因片段合成

根据已知RSVLong株反基因组及表达附属元件序列，将其分成A(MluI-AsuII)、B(AsuII-NheI)、C(NheI-NruI)3段，分别化学合成，其中A段包括T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt、A2cp突变：N 799G>A,aa267Val>Ile及删除了SH基因的非编码区的112nt(RSVLong株反基因组4497-4608nt)并在SH基因编码区末端引入4处同义突变，即SH186C>T、SH189C>T、SH192A>T、SH194,195AG>GA；B段包括RSVLong株反基因组5179-11006nt及A2cp突变：F653,654AA>CG,aa218Glu>Ala、F1568,1569CA>TT,aa523Thr>Ile、L956,957GT>AC,aa319Cys>Tyr；C段包括RSVLong株反基因组11001-15226n及L 5068,5070C>T,T>C,aa1690His>Tyr A2cp突变、丁型肝炎核酶序列、T7终止子序列。合成后的片段分别插入pBRB载体中，命名为pBRB-RSV-A(1-5135nt)、pBRB-RSV-B(5135-10956nt)、pBRB-RSV-C(10956-15405nt)质粒，将获得的质粒分别用MluI、AsuII(图2A)；AsuII、NheI(图2B)；NruI、NheI(图2C)进行酶切鉴定，分别于5143bp、5828bp、4456bp处得到目的条带(图2)，即基因片段。

3)pBRB-RSV-rLong/A2cp质粒构建

利用AsuⅡ和NheⅠ内切酶双酶切pBRB-RSV-C质粒和pBRB-RSV-B质粒，分别回收C质粒的7.9kb载体片段和B质粒的5.8kb插入片段，用T4DNA连接酶连接，转化感受态大肠杆菌DH5ɑ，经筛选后可获得pBRB-RSV-CB质粒；利用MluⅠ和AsuⅡ内切酶双酶切pBRB-RSV-CB和pBRB-RSV-A质粒，分别回收前者的13.7kb片段和后者的5.1kb片段，用T4DNA连接酶连接，转化感受态大肠杆菌DH5ɑ，经筛选后可获得pBRB-RSV-CBA质粒，即pBRB-RSV-rLong/A2cp质粒。将最终获得的重组质粒分别用NruI/NheI(1)、AsuII/NheI(2)、AsuII/SacI(3)、SacI/MluI(4)、EcoRV(5)、SpeI(6)、AflII(7)限制性内切酶进行酶切鉴定，分别在相应位置得到目的条带(图3)。经全基因测序后发现pBRB-RSV-rLong/A2cp质粒序列与预期相符，如SEQID No.1所示。

实施例2：pBRB-RSV-rLong/A2cpts质粒的构建

1)载体pBR322改造与实施例1相同，改造后的载体pBRB序列如SEQ ID No.4所示。

2)RSV-rLong/A2cpts反基因组的基因片段合成

RSV-rLong/A2cpts基因序列及分段合成与实施例1基本相同，只是B段基因在实施例1基础上增加了两个ts突变(M2 9T>C；L2491,2492CA>TT,aa831Gln>Leu)。合成后基因片段克隆及酶切鉴定同实施例1。

3)pBRB-RSV-rLong/A2cpts质粒构建

质粒构建过程同实施例1，获得的重组质粒pBRB-RSV-rLong/A2cpts经酶切及测序鉴定后与预期相符，质粒全基因序列如SEQ ID No.2所示。

实施例3：pBRB-RSV-rLong/A2cptsΔSH质粒的构建

1)载体pBR322改造与实施例1相同，改造后的载体pBRB序列如SEQ ID No.4所示。

2)RSV-rLong/A2cptsΔSH反基因组的基因片段合成

RSV-rLong/A2cptsΔSH基因序列及分段合成与实施例2基本相同，只是A段基因在实施例2基础上还删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。合成后基因片段克隆及酶切鉴定同实施例2。

3)pBRB-RSV-rLong/A2cptsΔSH质粒构建

质粒构建过程同实施例2，获得的重组质粒pBRB-RSV-rLong/A2cptsΔSH经酶切及测序鉴定后与预期相符，质粒全基因序列如SEQ ID No.3所示。

实施例4重组病毒的拯救

将BHK T7/9细胞接种在6孔板，24h后分别转染所述pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒和四种辅助蛋白质粒(pCITE-N,P,L,M2-1)至宿主细胞(BHK T7/9)于33℃下培养，其中实验组1转染pBRB-RSV-rLong/A2cp和pCITE-N,P,L,M2-1，实验组2转染pBRB-RSV-rLong/A2cpts和pCITE-N,P,L,M2-1，实验组3转染pBRB-RSV-rLong/A2cptsΔSH和pCITE-N,P,L,M2-1，mock组不转染质粒。转染96h后盲传至Vero细胞继续放置于33℃下培养，实验组在转染(图4)和盲传(图5)时出现细胞融合，初步判断成功拯救出重组病毒。

实施例5重组病毒的鉴定

利用免疫酶法和实时定量PCR(引物如序列表SEQ ID No.5H和SEQ ID No.6所示，表1)检测连续传代后重组病毒的滴度(图6，空斑形成单位)和复制能力(图7，N基因的拷贝数)，结果都显示拯救出的重组病毒与野生型RSV病毒相比复制能力明显降低(*P<0.05；**P<0.01)，表明重组病毒在体外明显减毒。设计引物RT-PCR检测第10、20、30代重组病毒突变位点，所用引物如表1所示，，测序结果显示pBRB-RSV-rLongA2cp(图8)、pBRB-RSV-rLong/A2cpts(图9)、pBRB-RSV-rLong/A2cptsΔSH(图10)突变位点没有变化，说明拯救出的重组病毒在体外连续传代的过程中是稳定的。

表1检测重组病毒突变位点的引物

显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

SEQUENCE LISTING

<110> 北京交通大学

<120> 基于反向遗传学构建的RSV反基因组质粒及其应用

<130> JLC17I0280E

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 18815

<212> DNA

<213> 质粒pBRB-RSV-rLong/A2cp

<400> 1

acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60

aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120

aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180

ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240

aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300

atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360

tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420

ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480

gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540

atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600

aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660

atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720

accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780

actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840

gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900

attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960

cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020

gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080

agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140

gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200

acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260

tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320

gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380

cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440

accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500

acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560

ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620

ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680

ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740

cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800

ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860

tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920

gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980

cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040

gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100

ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160

cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220

ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280

gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340

aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400

aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460

gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520

aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580

acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640

cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700

cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760

agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820

aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880

gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940

atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000

agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060

aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120

tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180

accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240

gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300

acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360

tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420

cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480

ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540

gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600

gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660

atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720

ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780

atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840

aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900

gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960

gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020

aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080

aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140

tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200

caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260

ataagttaat aaaaatatac acatggggca aataatcatc ggaggaaatc caactaatca 4320

caatatctgt taacatagac aagtcaacac accagacaga atcaaccaat ggaaaataca 4380

tccataacaa tagaattctc aagcaaattc tggccttact ttacactaat acacatgatc 4440

acaacaataa tctctttgct aatcataatc tccatcatga ctgcaatact aaacaaactt 4500

tgtgaatata acgtattcca taacaaaacc tttgagttac caagagctcg agttaatact 4560

tgataaagta gttaattaaa aatagtcata acaatgaact aggatatcaa gactaacaat 4620

aacgttgggg caaatgcaaa catgtccaaa aacaaggacc aacgcaccgc taagacacta 4680

gaaaagacct gggacactct caatcattta ttattcatat catcgggctt atataagtta 4740

aatcttaaat ctatagcaca aatcacatta tccattctgg caatgataat ctcaacttca 4800

cttataatta cagccatcat attcatagcc tcggcaaacc acaaagtcac actaacaact 4860

gcaatcatac aagatgcaac aagccagatc aagaacacaa ccccaacata cctcactcag 4920

gaccctcagc ttggaatcag cttctccaat ctgtctgaaa ttacatcaca aaccaccacc 4980

atactagctt caacaacacc aggagtgaag tcaaacctgc aacccacaac agtcaagact 5040

aaaaacacaa caacaaccca aacacaaccc agcaagccca ctacaaaaca acgccaaaac 5100

aaaccaccaa acaaacccaa taatgatttt cacttcgaag tgtttaactt tgtaccctgc 5160

agcatatgca gcaacaatcc aacctgctgg gctatctgca aaagaatacc aaacaaaaaa 5220

ccaggaaaga aaaccaccac caagcctaca aaaaaaccaa ccttcaagac aaccaaaaaa 5280

gatctcaaac ctcaaaccac taaaccaaag gaagtaccca ccaccaagcc cacagaagag 5340

ccaaccatca acaccaccaa aacaaacatc acaactacac tgctcaccaa caacaccaca 5400

ggaaatccaa aactcacaag tcaaatggaa accttccact caacctcctc cgaaggcaat 5460

ctaagccctt ctcaagtctc cacaacatcc gagcacccat cacaaccctc atctccaccc 5520

aacacaacac gccagtagtt attaaaaaac atattatcac aaaaggcctt gaccaactca 5580

aacagaatca aaataaactc tggggcaaat aacaatggag ttgccaatcc tcaaagcaaa 5640

tgcaattacc acaatcctcg ctgcagtcac attttgcttt gcttctagtc aaaacatcac 5700

tgaagaattt tatcaatcaa catgcagtgc agttagcaaa ggctatctta gtgctctaag 5760

aactggttgg tatactagtg ttataactat agaattaagt aatatcaagg aaaataagtg 5820

taatggaaca gatgctaagg taaaattgat aaaccaagaa ttagataaat ataaaaatgc 5880

tgtaacagaa ttgcagttgc tcatgcaaag cacaacagca gcaaacaatc gagccagaag 5940

agaactacca aggtttatga attatacact caacaatacc aaaaaaacca atgtaacatt 6000

aagcaagaaa aggaaaagaa gatttcttgg ttttttgtta ggtgttggat ctgcaatcgc 6060

cagtggcatt gctgtatcta aggtcctgca cttagaagga gaagtgaaca agatcaaaag 6120

tgctctacta tccacaaaca aggccgtagt cagcttatca aatggagtta gtgtcttaac 6180

cagcaaagtg ttagacctca aaaactatat agataaacaa ttgttaccta ttgtgaataa 6240

gcaaagctgc agaatatcaa atatagcgac tgtgatagag ttccaacaaa agaacaacag 6300

actactagag attaccaggg aatttagtgt taatgcaggt gtaactacac ctgtaagcac 6360

ttacatgtta actaatagtg aattattgtc attaatcaat gatatgccta taacaaatga 6420

tcagaaaaag ttaatgtcca acaatgttca aatagttaga cagcaaagtt actctatcat 6480

gtccataata aaagaggaag tcttagcata tgtagtacaa ttaccactat atggtgtgat 6540

agatacacct tgttggaaat tacacacatc ccctctatgt acaaccaaca caaaagaagg 6600

gtcaaacatc tgtttaacaa gaactgacag aggatggtac tgtgacaatg caggatcagt 6660

atctttcttc ccacaagctg aaacatgtaa agttcaatcg aatcgagtat tttgtgacac 6720

aatgaacagt ttaacattac caagtgaagt aaatctctgc aatgttgaca tattcaatcc 6780

caaatatgat tgtaaaatta tgacttcaaa aacagatgta agcagctccg ttatcacatc 6840

tctaggagcc attgtgtcat gctatggcaa aactaaatgt acagcatcca ataaaaatcg 6900

tggaatcata aagacatttt ctaacgggtg tgattatgta tcaaataaag gggtggacac 6960

tgtgtctgta ggtaacacat tatattatgt aaataagcaa gaaggcaaaa gtctctatgt 7020

aaaaggtgaa ccaataataa atttctatga cccattagta ttcccctctg atgaatttga 7080

tgcatcaata tctcaagtca atgagaagat taaccagagt ttagcattta ttcgtaaatc 7140

cgatgaatta ttacatcatg taaatgctgg taaatcaacc attaatatca tgataactac 7200

tataattata gtgattatag taatattgtt atcattaatt gctgttggac tgctcctata 7260

ctgtaaggcc agaagcacac cagtcacact aagcaaggat caactgagtg gtataaataa 7320

tattgcattt agtaactgaa taaaaatagc acctaatcat gttcttacaa tggtttacta 7380

tctgctcata gacaacccat ctatcattgg attttcttaa aatctgaact tcatcgaaac 7440

tcttatctat aaaccatctc acttacacta tttaagtaga ttcctagttt atagttatat 7500

aaaacacaat tgaataccag attaacttac tatctgtaaa aatgagaact ggggcaaata 7560

tgtcacgaag gaatccttgc aaatttgaaa ttcgaggtca ttgcttgaat ggtaagagat 7620

gtcattttag tcataattat tttgaatggc caccccatgc actgctcgta agacaaaact 7680

ttatgttaaa cagaatactt aagtctatgg ataaaagtat agatacctta tcagaaataa 7740

gtggagctgc agagttggac agaacagaag agtatgctct tggtgtagtt ggagtgctag 7800

agagttatat aggatcaata aataatataa ctaaacaatc agcatgtgtt gccatgagca 7860

aactcctcac tgaactcaat agtgatgata tcaaaaaact gagagacaat gaagagctaa 7920

attcacccaa gataagagtg tacaatactg tcatatcata tattgaaagc aacaggaaaa 7980

acaataaaca aactatccat ctgttaaaaa gattgccagc agacgtattg aagaaaacca 8040

tcaaaaacac attggatatc cacaagagca taaccatcaa caacccaaaa gaattaactg 8100

ttagtgatac aaatgaccat gccaaaaata atgatactac ctgacaaata tccttgtagt 8160

ataacttcca tactaataac aagtagatgt agagtcacta tgtataatcg aaagaacaca 8220

ctatatttca atcaaaacaa cccaaataac catatgtact caccgaatca aacattcaat 8280

gaaatccatt ggacctcaca agacttgatt gacacaattc aaaattttct acagcatcta 8340

ggtgttattg aggatatata tacaatatat atattagtgt cataacactc aatcctaata 8400

ctgaccatat cgttgaatta ttaattcaaa taattcaagc tgtgggacaa aatggatccc 8460

attattaatg gaaattctgc taatgtttat ctaaccgata gttatttaaa aggtgttatc 8520

tctttctcag agtgtaatgc tttaggaagt tacatattca atggtcctta tctcaaaaat 8580

gattatacca acttaattag tagacaaaat ccattaatag aacacatgaa tctaaagaaa 8640

ctaaatataa cacagtcctt aatatctaag tatcataaag gtgaaataaa attagaagag 8700

cctacttatt ttcagtcatt acttatgaca tacaagagta tgacctcgtt ggaacagatt 8760

gctaccacta atttacttaa aaagataata agaagagcta tagaaataag tgatgtcaaa 8820

gtctatgcta tattgaataa actagggctt aaagaaaagg acaagattaa atccaacaat 8880

ggacaggatg aagacaactc agttattacg accataatca aagatgatat actttcagct 8940

gttaaggata atcaatctca tcttaaagca gacaaaaatc actctacaaa acaaaaagac 9000

acaatcaaaa caacactctt gaagaaatta atgtgttcaa tgcagcatcc tccatcatgg 9060

ttaatacatt ggtttaattt atacacaaaa ttaaacaaca tattaacaca gtatcgttca 9120

aatgaggtta aaaaccatgg gtttatattg atagataatc aaactcttag tggatttcaa 9180

tttattttga atcaatatgg ttgtatagtt tatcataagg aactcaaaag aattactgtg 9240

acaacctata atcaattctt gacatggaaa gatattagcc ttagtagatt aaatgtttgt 9300

ttaattacat ggattagtaa ctgcttgaac acattaaata aaagcttagg cttaagatgc 9360

ggattcaata atgttatctt gacacaacta ttcctttatg gtgattacat actaaagcta 9420

tttcacaatg aggggttcta cataataaaa gaggtagagg gatttattat gtctctaatt 9480

ttaaatataa cagaagaaga tcaattcaga aaacgatttt ataatagtat gctcaacaac 9540

atcacagatg ctgctaataa agctcagaaa aatctgctat caagagtatg tcatacatta 9600

ttagataaga cagtatccga taatataata aatggcagat ggataattct attaagtaag 9660

ttccttaaat taattaagct tgcaggtgac aataacctta acaatctgag tgaactatat 9720

tttttgttca gaatatttgg acacccaatg gtagatgaaa gacaagccat ggatgctgtt 9780

aaagttaatt gcaatgagac caaattttac ttgttaagca gtttgagtat gttaagaggt 9840

gcctttatat atagaattat aaaagggttt gtaaataatt acaacagatg gcctacttta 9900

agaaatgcta ttgttttacc cttaagatgg ttaacttact ataaactaaa cacttatcct 9960

tctttgttgg aacttacaga aagagatttg attgtgttat caggactacg tttctatcgt 10020

gagtttcggt tgcctaaaaa agtggatctt gaaatgatta taaatgataa agctatatca 10080

ccccctaaaa atttgatatg gactagtttc cctagaaatt atatgccgtc acacatacaa 10140

aactatatag aacatgaaaa attaaaattt tccgagagtg ataaatcaag aagagtatta 10200

gagtattatt taagagataa caaattcaat gaatgtgatt tatacaactg tgtagttaat 10260

caaagttatc tcaacaaccc taatcatgtg gtatcattga caggcaaaga aagagaactc 10320

agtgtaggta gaatgtttgc aatgcaaccg ggaatgttca gacaggttca aatattggca 10380

gagaaaatga tagctgaaaa cattttacaa ttctttcctg aaagtcttac aagatatggt 10440

gatctagaac tacaaaaaat attagaattg aaagcaggaa taagtaacaa atcaaatcgc 10500

tacaatgata attacaacaa ttacattagt aagtgctcta tcatcacaga tctcagcaaa 10560

ttcaatcaag catttcgata tgaaacgtca tgtatttgta gtgatgtgct ggatgaactg 10620

catggtgtac aatctctatt ttcctggtta catttaacta ttcctcatgt cacaataata 10680

tgcacatata ggcatgcacc cccctatata agagatcata ttgtagatct taacaatgta 10740

gatgaacaaa gtggattata tagatatcac atgggtggta ttgaagggtg gtgtcaaaaa 10800

ctatggacca tagaagctat atcactattg gatctaatat ctctcaaagg gaaattctca 10860

attactgctt taattaatgg tgacaatcaa tcaatagata taagcaaacc agtcagactc 10920

atggaaggtc aaactcatgc tcaagcagat tatttgctag cattaaatag ccttaaatta 10980

ctgtataaag agtatgcagg cataggtcac aaattaaaag gaactgagac ttatatatca 11040

cgagatatgc aatttatgag taaaacaatt caacataacg gtgtatatta ccctgctagt 11100

ataaagaaag tcctaagagt gggaccgtgg ataaacacta tacttgatga tttcaaagtg 11160

agtctagaat ctataggtag tttgacacaa gaattagaat atagaggtga aagtctatta 11220

tgcagtttaa tatttagaaa tgtatggtta tataatcaaa ttgctctaca attaaaaaat 11280

catgcgttat gtaacaataa attatatttg gacatattaa aggttctgaa acacttaaaa 11340

acctttttta atcttgataa tattgataca gcattaacat tgtatatgaa tttacccatg 11400

ttatttggtg gtggtgatcc caacttgtta tatcgaagtt tctatagaag aactcctgat 11460

ttcctcacag aggctatagt tcactctgtg ttcatactta gttattatac aaaccatgac 11520

ttaaaagata aacttcaaga tttgtcagat gatagattga ataagttctt aacatgcata 11580

atcacgtttg acaaaaaccc taatgctgaa ttcgtaacat tgatgagaga tcctcaagct 11640

ttagggtctg agagacaagc taaaattact agtgaaatca atagactggc agttacagag 11700

gttttgagta cagctccaaa caaaatattc tccaaaagtg cacaacatta taccactaca 11760

gagatagatc taaatgatat tatgcaaaat atagaaccta catatcctca cgggctaaga 11820

gttgtttatg aaagtttacc cttttataaa gcagagaaaa tagtaaatct tatatcaggt 11880

acaaaatcta taactaacat actggaaaag acttctgcca tagacttaac agatattgat 11940

agagccactg agatgatgag gaaaaacata actttgctta taaggatact tccattggat 12000

tgtaacagag ataaaagaga aatattgagt atggaaaacc taagtattac tgaattaagc 12060

aaatatgtta gggaaagatc ttggtcttta tccaatatag ttggtgttac atcacccagt 12120

atcatgtata caatggacat caaatataca acaagcacta tagctagtgg cataattata 12180

gagaaatata atgttaacag tttaacacgt ggtgagagag gaccaactaa accatgggtt 12240

ggttcatcta cacaagagaa aaaaacaatg ccagtttata atagacaagt tttaaccaaa 12300

aaacaaagag atcaaataga tctattagca aaattggatt gggtgtatgc atctatagat 12360

aacaaggatg aattcatgga agaactcagc ataggaaccc ttgggttaac atatgaaaag 12420

gccaaaaaat tatttccaca atatttaagt gtcaactatt tgcatcgcct tacagtcagt 12480

agtagaccat gtgaattccc tgcatcaata ccagcttata gaacaacaaa ttatcacttt 12540

gacactagcc ctattaatcg catattaaca gaaaagtatg gtgatgaaga tattgacata 12600

gtattccaaa actgtataag ctttggcctt agcttaatgt cagtagtaga acaatttact 12660

aatgtatgtc ctaacagaat tattctcata cctaagctta atgagataca tttgatgaaa 12720

cctcccatat tcacaggtga tgttgatatt cacaagttaa aacaagtgat acaaaaacag 12780

catatgtttt taccagacaa aataagtttg actcaatatg tggaattatt cttaagtaac 12840

aaaacactca aatctggatc tcatgttaat tctaatttaa tattggcaca taaaatatct 12900

gactattttc ataatactta cattttaagt actaatttag ctggacattg gattctaatt 12960

atacaactta tgaaagattc taaaggtatt tttgaaaaag attggggaga gggatatata 13020

actgatcata tgtttattaa tttgaaagtt ttcttcaatg cttataagac ctatctcttg 13080

tgttttcata aaggttatgg caaagcaaaa ctggagtgtg atatgaacac ttcagatctt 13140

ctatgtgtat tggaattaat agacagtagt tattggaagt ctatgtctaa ggtattttta 13200

gaacaaaaag ttatcaaata cattcttagc caagatgcaa gtttacatag agtaaaagga 13260

tgtcatagct tcaaattatg gtttcttaaa cgtcttaatg tagcagaatt tacagtttgc 13320

ccttgggttg ttaacataga ttatcatcca acacatatga aagcaatatt aacttatata 13380

gatcttgtta gaatgggatt gataaatata gatagaatac acattaaaaa taaacacaaa 13440

ttcaatgatg aattttatac ttctaatctc ttttacatta attataactt ctcagataat 13500

actcatctat taactaaata cataaggatt gctaattcag aattagaaaa taattacaac 13560

aaattatatc atcctacacc agaaacccta gagaatatac tagccaatcc gattaaaagt 13620

aatgacaaaa agacactgaa cgactattgt ataggtaaaa atgttgactc aataatgtta 13680

ccattgttat ctaataagaa gcttgttaaa tcgtctgcaa tgattagaac caattacagc 13740

aaacaagacc tgtacaatct attccctacg gttgtgatcg atagaattat agatcattca 13800

ggtaatacag ccaaatccaa ccaactttac actactactt cccatcaaat atctttagtg 13860

cacaatagca catcacttta ttgcatgctt ccttggcatc atattaatag attcaatttt 13920

gtatttagtt ctacaggttg taaaattagt atagagtata ttttaaaaga ccttaaaatt 13980

aaagatccta attgtatagc attcataggt gaaggagcag ggaatttatt attgcgtaca 14040

gtggtggaac ttcatcctga cataagatat atttacagaa gtctgaaaga ttgcaatgat 14100

catagtttac ctattgagtt tttaaggcta tacaatggac atatcaacat tgattatggt 14160

gaaaatttga ccattcctgc tacagatgca accaacaaca ttcattggtc ttatttacat 14220

ataaagtttg ctgaacctat cagtcttttt gtatgtgatg ccgaattgcc tgtaacagtc 14280

aactggagta aaattataat agaatggagc aagcatgtaa gaaaatgcaa gtactgttcc 14340

tcagttaata aatgtacgtt aatagtaaaa tatcatgctc aagatgatat tgatttcaaa 14400

ttagacaata taactatatt aaaaacttat gtatgcttag gcagtaagtt aaagggatcg 14460

gaggtttact tagtccttac aataggtcct gcaaatatat ttccagtatt taatgtagta 14520

caaaatgcta aattgatact atcaagaacc aaaaatttca tcatgcctaa gaaagctgat 14580

aaagagtcta ttgatgcaaa tattaaaagt ttgataccct ttctttgtta ccctataaca 14640

aaaaaaggaa ttaatactgc attgtcaaaa ctaaagagtg ttgttagtgg agatatacta 14700

tcatattcta tagctggacg gaatgaagtt ttcagcaata aacttataaa tcataagcat 14760

atgaacatct taaagtggtt caatcatgtt ttaaatttca gatcaacaga actaaactat 14820

aaccatttat atatggtaga atctacatat ccttacctaa gtgaattgtt aaacagcttg 14880

acaactaatg aacttaaaaa actgattaaa atcacaggta gtctgttata caactttcat 14940

aatgaataat gaataaagat cttataataa aaattcctat agctatacac tagcactgta 15000

ttcaattata gttattaaaa aattaaaaat catataattt tttataaaaa taacttttag 15060

tgaactaatc ctaaagttat cattttgatc taggaggaat aaatttaaat cccaatctaa 15120

ttggtttata tgtgtattaa ctaaactacg agatattagt ttttgacact ttttttctcg 15180

tgggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccga aggaggacgc 15240

acgtccactc ggatggctaa gggagagcca ctgctaacaa agcccgaaag gaagctgagt 15300

tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 15360

tgaggggttt tttgctgaaa ggaggaacta tatccggatt cgcgacgcga ggctggatgg 15420

ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca 15480

tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc 15540

ttaccagcct aacttcgatc actggaccgc tgatcgtcac ggcgatttat gccgcctcgg 15600

cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc 15660

ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca 15720

cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 15780

gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg 15840

cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct 15900

gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc 15960

gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac 16020

atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 16080

caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac 16140

atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat 16200

ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt 16260

aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa 16320

tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg 16380

ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga 16440

acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct 16500

cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 16560

agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 16620

tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 16680

cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 16740

ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 16800

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 16860

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 16920

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 16980

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 17040

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 17100

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 17160

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 17220

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 17280

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 17340

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 17400

aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 17460

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 17520

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 17580

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 17640

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 17700

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 17760

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 17820

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 17880

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 17940

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 18000

ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 18060

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 18120

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 18180

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 18240

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 18300

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 18360

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 18420

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 18480

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 18540

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 18600

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 18660

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 18720

gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 18780

cttcaagaat tctcatgttt gacagcttat catcg 18815

<210> 2

<211> 18815

<212> DNA

<213> 质粒pBRB-RSV-rLong/A2cpts

<400> 2

acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60

aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120

aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180

ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240

aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300

atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360

tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420

ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480

gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540

atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600

aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660

atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720

accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780

actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840

gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900

attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960

cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020

gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080

agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140

gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200

acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260

tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320

gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380

cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440

accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500

acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560

ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620

ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680

ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740

cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800

ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860

tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920

gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980

cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040

gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100

ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160

cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220

ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280

gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340

aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400

aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460

gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520

aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580

acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640

cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700

cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760

agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820

aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880

gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940

atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000

agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060

aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120

tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180

accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240

gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300

acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360

tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420

cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480

ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540

gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600

gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660

atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720

ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780

atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840

aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900

gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960

gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020

aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080

aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140

tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200

caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260

ataagttaat aaaaatatac acatggggca aataatcatc ggaggaaatc caactaatca 4320

caatatctgt taacatagac aagtcaacac accagacaga atcaaccaat ggaaaataca 4380

tccataacaa tagaattctc aagcaaattc tggccttact ttacactaat acacatgatc 4440

acaacaataa tctctttgct aatcataatc tccatcatga ctgcaatact aaacaaactt 4500

tgtgaatata acgtattcca taacaaaacc tttgagttac caagagctcg agttaatact 4560

tgataaagta gttaattaaa aatagtcata acaatgaact aggatatcaa gactaacaat 4620

aacgttgggg caaatgcaaa catgtccaaa aacaaggacc aacgcaccgc taagacacta 4680

gaaaagacct gggacactct caatcattta ttattcatat catcgggctt atataagtta 4740

aatcttaaat ctatagcaca aatcacatta tccattctgg caatgataat ctcaacttca 4800

cttataatta cagccatcat attcatagcc tcggcaaacc acaaagtcac actaacaact 4860

gcaatcatac aagatgcaac aagccagatc aagaacacaa ccccaacata cctcactcag 4920

gaccctcagc ttggaatcag cttctccaat ctgtctgaaa ttacatcaca aaccaccacc 4980

atactagctt caacaacacc aggagtgaag tcaaacctgc aacccacaac agtcaagact 5040

aaaaacacaa caacaaccca aacacaaccc agcaagccca ctacaaaaca acgccaaaac 5100

aaaccaccaa acaaacccaa taatgatttt cacttcgaag tgtttaactt tgtaccctgc 5160

agcatatgca gcaacaatcc aacctgctgg gctatctgca aaagaatacc aaacaaaaaa 5220

ccaggaaaga aaaccaccac caagcctaca aaaaaaccaa ccttcaagac aaccaaaaaa 5280

gatctcaaac ctcaaaccac taaaccaaag gaagtaccca ccaccaagcc cacagaagag 5340

ccaaccatca acaccaccaa aacaaacatc acaactacac tgctcaccaa caacaccaca 5400

ggaaatccaa aactcacaag tcaaatggaa accttccact caacctcctc cgaaggcaat 5460

ctaagccctt ctcaagtctc cacaacatcc gagcacccat cacaaccctc atctccaccc 5520

aacacaacac gccagtagtt attaaaaaac atattatcac aaaaggcctt gaccaactca 5580

aacagaatca aaataaactc tggggcaaat aacaatggag ttgccaatcc tcaaagcaaa 5640

tgcaattacc acaatcctcg ctgcagtcac attttgcttt gcttctagtc aaaacatcac 5700

tgaagaattt tatcaatcaa catgcagtgc agttagcaaa ggctatctta gtgctctaag 5760

aactggttgg tatactagtg ttataactat agaattaagt aatatcaagg aaaataagtg 5820

taatggaaca gatgctaagg taaaattgat aaaccaagaa ttagataaat ataaaaatgc 5880

tgtaacagaa ttgcagttgc tcatgcaaag cacaacagca gcaaacaatc gagccagaag 5940

agaactacca aggtttatga attatacact caacaatacc aaaaaaacca atgtaacatt 6000

aagcaagaaa aggaaaagaa gatttcttgg ttttttgtta ggtgttggat ctgcaatcgc 6060

cagtggcatt gctgtatcta aggtcctgca cttagaagga gaagtgaaca agatcaaaag 6120

tgctctacta tccacaaaca aggccgtagt cagcttatca aatggagtta gtgtcttaac 6180

cagcaaagtg ttagacctca aaaactatat agataaacaa ttgttaccta ttgtgaataa 6240

gcaaagctgc agaatatcaa atatagcgac tgtgatagag ttccaacaaa agaacaacag 6300

actactagag attaccaggg aatttagtgt taatgcaggt gtaactacac ctgtaagcac 6360

ttacatgtta actaatagtg aattattgtc attaatcaat gatatgccta taacaaatga 6420

tcagaaaaag ttaatgtcca acaatgttca aatagttaga cagcaaagtt actctatcat 6480

gtccataata aaagaggaag tcttagcata tgtagtacaa ttaccactat atggtgtgat 6540

agatacacct tgttggaaat tacacacatc ccctctatgt acaaccaaca caaaagaagg 6600

gtcaaacatc tgtttaacaa gaactgacag aggatggtac tgtgacaatg caggatcagt 6660

atctttcttc ccacaagctg aaacatgtaa agttcaatcg aatcgagtat tttgtgacac 6720

aatgaacagt ttaacattac caagtgaagt aaatctctgc aatgttgaca tattcaatcc 6780

caaatatgat tgtaaaatta tgacttcaaa aacagatgta agcagctccg ttatcacatc 6840

tctaggagcc attgtgtcat gctatggcaa aactaaatgt acagcatcca ataaaaatcg 6900

tggaatcata aagacatttt ctaacgggtg tgattatgta tcaaataaag gggtggacac 6960

tgtgtctgta ggtaacacat tatattatgt aaataagcaa gaaggcaaaa gtctctatgt 7020

aaaaggtgaa ccaataataa atttctatga cccattagta ttcccctctg atgaatttga 7080

tgcatcaata tctcaagtca atgagaagat taaccagagt ttagcattta ttcgtaaatc 7140

cgatgaatta ttacatcatg taaatgctgg taaatcaacc attaatatca tgataactac 7200

tataattata gtgattatag taatattgtt atcattaatt gctgttggac tgctcctata 7260

ctgtaaggcc agaagcacac cagtcacact aagcaaggat caactgagtg gtataaataa 7320

tattgcattt agtaactgaa taaaaatagc acctaatcat gttcttacaa tggtttacta 7380

tctgctcata gacaacccat ctatcattgg attttcttaa aatctgaact tcatcgaaac 7440

tcttatctat aaaccatctc acttacacta tttaagtaga ttcctagttt atagttatat 7500

aaaacacaat tgaataccag attaacttac tatctgtaaa aatgagaact ggggcaaaca 7560

tgtcacgaag gaatccttgc aaatttgaaa ttcgaggtca ttgcttgaat ggtaagagat 7620

gtcattttag tcataattat tttgaatggc caccccatgc actgctcgta agacaaaact 7680

ttatgttaaa cagaatactt aagtctatgg ataaaagtat agatacctta tcagaaataa 7740

gtggagctgc agagttggac agaacagaag agtatgctct tggtgtagtt ggagtgctag 7800

agagttatat aggatcaata aataatataa ctaaacaatc agcatgtgtt gccatgagca 7860

aactcctcac tgaactcaat agtgatgata tcaaaaaact gagagacaat gaagagctaa 7920

attcacccaa gataagagtg tacaatactg tcatatcata tattgaaagc aacaggaaaa 7980

acaataaaca aactatccat ctgttaaaaa gattgccagc agacgtattg aagaaaacca 8040

tcaaaaacac attggatatc cacaagagca taaccatcaa caacccaaaa gaattaactg 8100

ttagtgatac aaatgaccat gccaaaaata atgatactac ctgacaaata tccttgtagt 8160

ataacttcca tactaataac aagtagatgt agagtcacta tgtataatcg aaagaacaca 8220

ctatatttca atcaaaacaa cccaaataac catatgtact caccgaatca aacattcaat 8280

gaaatccatt ggacctcaca agacttgatt gacacaattc aaaattttct acagcatcta 8340

ggtgttattg aggatatata tacaatatat atattagtgt cataacactc aatcctaata 8400

ctgaccatat cgttgaatta ttaattcaaa taattcaagc tgtgggacaa aatggatccc 8460

attattaatg gaaattctgc taatgtttat ctaaccgata gttatttaaa aggtgttatc 8520

tctttctcag agtgtaatgc tttaggaagt tacatattca atggtcctta tctcaaaaat 8580

gattatacca acttaattag tagacaaaat ccattaatag aacacatgaa tctaaagaaa 8640

ctaaatataa cacagtcctt aatatctaag tatcataaag gtgaaataaa attagaagag 8700

cctacttatt ttcagtcatt acttatgaca tacaagagta tgacctcgtt ggaacagatt 8760

gctaccacta atttacttaa aaagataata agaagagcta tagaaataag tgatgtcaaa 8820

gtctatgcta tattgaataa actagggctt aaagaaaagg acaagattaa atccaacaat 8880

ggacaggatg aagacaactc agttattacg accataatca aagatgatat actttcagct 8940

gttaaggata atcaatctca tcttaaagca gacaaaaatc actctacaaa acaaaaagac 9000

acaatcaaaa caacactctt gaagaaatta atgtgttcaa tgcagcatcc tccatcatgg 9060

ttaatacatt ggtttaattt atacacaaaa ttaaacaaca tattaacaca gtatcgttca 9120

aatgaggtta aaaaccatgg gtttatattg atagataatc aaactcttag tggatttcaa 9180

tttattttga atcaatatgg ttgtatagtt tatcataagg aactcaaaag aattactgtg 9240

acaacctata atcaattctt gacatggaaa gatattagcc ttagtagatt aaatgtttgt 9300

ttaattacat ggattagtaa ctgcttgaac acattaaata aaagcttagg cttaagatgc 9360

ggattcaata atgttatctt gacacaacta ttcctttatg gtgattacat actaaagcta 9420

tttcacaatg aggggttcta cataataaaa gaggtagagg gatttattat gtctctaatt 9480

ttaaatataa cagaagaaga tcaattcaga aaacgatttt ataatagtat gctcaacaac 9540

atcacagatg ctgctaataa agctcagaaa aatctgctat caagagtatg tcatacatta 9600

ttagataaga cagtatccga taatataata aatggcagat ggataattct attaagtaag 9660

ttccttaaat taattaagct tgcaggtgac aataacctta acaatctgag tgaactatat 9720

tttttgttca gaatatttgg acacccaatg gtagatgaaa gacaagccat ggatgctgtt 9780

aaagttaatt gcaatgagac caaattttac ttgttaagca gtttgagtat gttaagaggt 9840

gcctttatat atagaattat aaaagggttt gtaaataatt acaacagatg gcctacttta 9900

agaaatgcta ttgttttacc cttaagatgg ttaacttact ataaactaaa cacttatcct 9960

tctttgttgg aacttacaga aagagatttg attgtgttat caggactacg tttctatcgt 10020

gagtttcggt tgcctaaaaa agtggatctt gaaatgatta taaatgataa agctatatca 10080

ccccctaaaa atttgatatg gactagtttc cctagaaatt atatgccgtc acacatacaa 10140

aactatatag aacatgaaaa attaaaattt tccgagagtg ataaatcaag aagagtatta 10200

gagtattatt taagagataa caaattcaat gaatgtgatt tatacaactg tgtagttaat 10260

caaagttatc tcaacaaccc taatcatgtg gtatcattga caggcaaaga aagagaactc 10320

agtgtaggta gaatgtttgc aatgcaaccg ggaatgttca gacaggttca aatattggca 10380

gagaaaatga tagctgaaaa cattttacaa ttctttcctg aaagtcttac aagatatggt 10440

gatctagaac tacaaaaaat attagaattg aaagcaggaa taagtaacaa atcaaatcgc 10500

tacaatgata attacaacaa ttacattagt aagtgctcta tcatcacaga tctcagcaaa 10560

ttcaatcaag catttcgata tgaaacgtca tgtatttgta gtgatgtgct ggatgaactg 10620

catggtgtac aatctctatt ttcctggtta catttaacta ttcctcatgt cacaataata 10680

tgcacatata ggcatgcacc cccctatata agagatcata ttgtagatct taacaatgta 10740

gatgaacaaa gtggattata tagatatcac atgggtggta ttgaagggtg gtgtcaaaaa 10800

ctatggacca tagaagctat atcactattg gatctaatat ctctcaaagg gaaattctca 10860

attactgctt taattaatgg tgacaatcaa tcaatagata taagcaaacc agtcagactc 10920

atggaaggtc aaactcatgc tttagcagat tatttgctag cattaaatag ccttaaatta 10980

ctgtataaag agtatgcagg cataggtcac aaattaaaag gaactgagac ttatatatca 11040

cgagatatgc aatttatgag taaaacaatt caacataacg gtgtatatta ccctgctagt 11100

ataaagaaag tcctaagagt gggaccgtgg ataaacacta tacttgatga tttcaaagtg 11160

agtctagaat ctataggtag tttgacacaa gaattagaat atagaggtga aagtctatta 11220

tgcagtttaa tatttagaaa tgtatggtta tataatcaaa ttgctctaca attaaaaaat 11280

catgcgttat gtaacaataa attatatttg gacatattaa aggttctgaa acacttaaaa 11340

acctttttta atcttgataa tattgataca gcattaacat tgtatatgaa tttacccatg 11400

ttatttggtg gtggtgatcc caacttgtta tatcgaagtt tctatagaag aactcctgat 11460

ttcctcacag aggctatagt tcactctgtg ttcatactta gttattatac aaaccatgac 11520

ttaaaagata aacttcaaga tttgtcagat gatagattga ataagttctt aacatgcata 11580

atcacgtttg acaaaaaccc taatgctgaa ttcgtaacat tgatgagaga tcctcaagct 11640

ttagggtctg agagacaagc taaaattact agtgaaatca atagactggc agttacagag 11700

gttttgagta cagctccaaa caaaatattc tccaaaagtg cacaacatta taccactaca 11760

gagatagatc taaatgatat tatgcaaaat atagaaccta catatcctca cgggctaaga 11820

gttgtttatg aaagtttacc cttttataaa gcagagaaaa tagtaaatct tatatcaggt 11880

acaaaatcta taactaacat actggaaaag acttctgcca tagacttaac agatattgat 11940

agagccactg agatgatgag gaaaaacata actttgctta taaggatact tccattggat 12000

tgtaacagag ataaaagaga aatattgagt atggaaaacc taagtattac tgaattaagc 12060

aaatatgtta gggaaagatc ttggtcttta tccaatatag ttggtgttac atcacccagt 12120

atcatgtata caatggacat caaatataca acaagcacta tagctagtgg cataattata 12180

gagaaatata atgttaacag tttaacacgt ggtgagagag gaccaactaa accatgggtt 12240

ggttcatcta cacaagagaa aaaaacaatg ccagtttata atagacaagt tttaaccaaa 12300

aaacaaagag atcaaataga tctattagca aaattggatt gggtgtatgc atctatagat 12360

aacaaggatg aattcatgga agaactcagc ataggaaccc ttgggttaac atatgaaaag 12420

gccaaaaaat tatttccaca atatttaagt gtcaactatt tgcatcgcct tacagtcagt 12480

agtagaccat gtgaattccc tgcatcaata ccagcttata gaacaacaaa ttatcacttt 12540

gacactagcc ctattaatcg catattaaca gaaaagtatg gtgatgaaga tattgacata 12600

gtattccaaa actgtataag ctttggcctt agcttaatgt cagtagtaga acaatttact 12660

aatgtatgtc ctaacagaat tattctcata cctaagctta atgagataca tttgatgaaa 12720

cctcccatat tcacaggtga tgttgatatt cacaagttaa aacaagtgat acaaaaacag 12780

catatgtttt taccagacaa aataagtttg actcaatatg tggaattatt cttaagtaac 12840

aaaacactca aatctggatc tcatgttaat tctaatttaa tattggcaca taaaatatct 12900

gactattttc ataatactta cattttaagt actaatttag ctggacattg gattctaatt 12960

atacaactta tgaaagattc taaaggtatt tttgaaaaag attggggaga gggatatata 13020

actgatcata tgtttattaa tttgaaagtt ttcttcaatg cttataagac ctatctcttg 13080

tgttttcata aaggttatgg caaagcaaaa ctggagtgtg atatgaacac ttcagatctt 13140

ctatgtgtat tggaattaat agacagtagt tattggaagt ctatgtctaa ggtattttta 13200

gaacaaaaag ttatcaaata cattcttagc caagatgcaa gtttacatag agtaaaagga 13260

tgtcatagct tcaaattatg gtttcttaaa cgtcttaatg tagcagaatt tacagtttgc 13320

ccttgggttg ttaacataga ttatcatcca acacatatga aagcaatatt aacttatata 13380

gatcttgtta gaatgggatt gataaatata gatagaatac acattaaaaa taaacacaaa 13440

ttcaatgatg aattttatac ttctaatctc ttttacatta attataactt ctcagataat 13500

actcatctat taactaaata cataaggatt gctaattcag aattagaaaa taattacaac 13560

aaattatatc atcctacacc agaaacccta gagaatatac tagccaatcc gattaaaagt 13620

aatgacaaaa agacactgaa cgactattgt ataggtaaaa atgttgactc aataatgtta 13680

ccattgttat ctaataagaa gcttgttaaa tcgtctgcaa tgattagaac caattacagc 13740

aaacaagacc tgtacaatct attccctacg gttgtgatcg atagaattat agatcattca 13800

ggtaatacag ccaaatccaa ccaactttac actactactt cccatcaaat atctttagtg 13860

cacaatagca catcacttta ttgcatgctt ccttggcatc atattaatag attcaatttt 13920

gtatttagtt ctacaggttg taaaattagt atagagtata ttttaaaaga ccttaaaatt 13980

aaagatccta attgtatagc attcataggt gaaggagcag ggaatttatt attgcgtaca 14040

gtggtggaac ttcatcctga cataagatat atttacagaa gtctgaaaga ttgcaatgat 14100

catagtttac ctattgagtt tttaaggcta tacaatggac atatcaacat tgattatggt 14160

gaaaatttga ccattcctgc tacagatgca accaacaaca ttcattggtc ttatttacat 14220

ataaagtttg ctgaacctat cagtcttttt gtatgtgatg ccgaattgcc tgtaacagtc 14280

aactggagta aaattataat agaatggagc aagcatgtaa gaaaatgcaa gtactgttcc 14340

tcagttaata aatgtacgtt aatagtaaaa tatcatgctc aagatgatat tgatttcaaa 14400

ttagacaata taactatatt aaaaacttat gtatgcttag gcagtaagtt aaagggatcg 14460

gaggtttact tagtccttac aataggtcct gcaaatatat ttccagtatt taatgtagta 14520

caaaatgcta aattgatact atcaagaacc aaaaatttca tcatgcctaa gaaagctgat 14580

aaagagtcta ttgatgcaaa tattaaaagt ttgataccct ttctttgtta ccctataaca 14640

aaaaaaggaa ttaatactgc attgtcaaaa ctaaagagtg ttgttagtgg agatatacta 14700

tcatattcta tagctggacg gaatgaagtt ttcagcaata aacttataaa tcataagcat 14760

atgaacatct taaagtggtt caatcatgtt ttaaatttca gatcaacaga actaaactat 14820

aaccatttat atatggtaga atctacatat ccttacctaa gtgaattgtt aaacagcttg 14880

acaactaatg aacttaaaaa actgattaaa atcacaggta gtctgttata caactttcat 14940

aatgaataat gaataaagat cttataataa aaattcctat agctatacac tagcactgta 15000

ttcaattata gttattaaaa aattaaaaat catataattt tttataaaaa taacttttag 15060

tgaactaatc ctaaagttat cattttgatc taggaggaat aaatttaaat cccaatctaa 15120

ttggtttata tgtgtattaa ctaaactacg agatattagt ttttgacact ttttttctcg 15180

tgggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccga aggaggacgc 15240

acgtccactc ggatggctaa gggagagcca ctgctaacaa agcccgaaag gaagctgagt 15300

tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 15360

tgaggggttt tttgctgaaa ggaggaacta tatccggatt cgcgacgcga ggctggatgg 15420

ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca 15480

tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc 15540

ttaccagcct aacttcgatc actggaccgc tgatcgtcac ggcgatttat gccgcctcgg 15600

cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc 15660

ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca 15720

cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 15780

gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg 15840

cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct 15900

gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc 15960

gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac 16020

atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 16080

caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac 16140

atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat 16200

ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt 16260

aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa 16320

tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg 16380

ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga 16440

acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct 16500

cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 16560

agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 16620

tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 16680

cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 16740

ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 16800

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 16860

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 16920

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 16980

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 17040

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 17100

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 17160

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 17220

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 17280

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 17340

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 17400

aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 17460

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 17520

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 17580

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 17640

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 17700

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 17760

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 17820

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 17880

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 17940

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 18000

ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 18060

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 18120

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 18180

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 18240

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 18300

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 18360

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 18420

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 18480

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 18540

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 18600

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 18660

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 18720

gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 18780

cttcaagaat tctcatgttt gacagcttat catcg 18815

<210> 3

<211> 18485

<212> DNA

<213> 质粒pBRB-RSV-rLong/A2cpts∆SH

<400> 3

acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60

aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120

aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180

ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240

aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300

atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360

tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420

ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480

gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540

atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600

aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660

atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720

accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780

actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840

gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900

attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960

cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020

gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080

agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140

gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200

acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260

tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320

gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380

cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440

accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500

acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560

ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620

ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680

ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740

cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800

ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860

tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920

gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980

cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040

gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100

ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160

cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220

ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280

gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340

aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400

aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460

gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520

aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580

acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640

cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700

cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760

agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820

aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880

gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940

atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000

agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060

aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120

tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180

accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240

gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300

acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360

tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420

cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480

ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540

gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600

gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660

atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720

ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780

atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840

aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900

gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960

gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020

aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080

aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140

tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200

caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260

ataagttaat aaaaatatac acatgagctc aacgttgggg caaatgcaaa catgtccaaa 4320

aacaaggacc aacgcaccgc taagacacta gaaaagacct gggacactct caatcattta 4380

ttattcatat catcgggctt atataagtta aatcttaaat ctatagcaca aatcacatta 4440

tccattctgg caatgataat ctcaacttca cttataatta cagccatcat attcatagcc 4500

tcggcaaacc acaaagtcac actaacaact gcaatcatac aagatgcaac aagccagatc 4560

aagaacacaa ccccaacata cctcactcag gaccctcagc ttggaatcag cttctccaat 4620

ctgtctgaaa ttacatcaca aaccaccacc atactagctt caacaacacc aggagtgaag 4680

tcaaacctgc aacccacaac agtcaagact aaaaacacaa caacaaccca aacacaaccc 4740

agcaagccca ctacaaaaca acgccaaaac aaaccaccaa acaaacccaa taatgatttt 4800

cacttcgaag tgtttaactt tgtaccctgc agcatatgca gcaacaatcc aacctgctgg 4860

gctatctgca aaagaatacc aaacaaaaaa ccaggaaaga aaaccaccac caagcctaca 4920

aaaaaaccaa ccttcaagac aaccaaaaaa gatctcaaac ctcaaaccac taaaccaaag 4980

gaagtaccca ccaccaagcc cacagaagag ccaaccatca acaccaccaa aacaaacatc 5040

acaactacac tgctcaccaa caacaccaca ggaaatccaa aactcacaag tcaaatggaa 5100

accttccact caacctcctc cgaaggcaat ctaagccctt ctcaagtctc cacaacatcc 5160

gagcacccat cacaaccctc atctccaccc aacacaacac gccagtagtt attaaaaaac 5220

atattatcac aaaaggcctt gaccaactca aacagaatca aaataaactc tggggcaaat 5280

aacaatggag ttgccaatcc tcaaagcaaa tgcaattacc acaatcctcg ctgcagtcac 5340

attttgcttt gcttctagtc aaaacatcac tgaagaattt tatcaatcaa catgcagtgc 5400

agttagcaaa ggctatctta gtgctctaag aactggttgg tatactagtg ttataactat 5460

agaattaagt aatatcaagg aaaataagtg taatggaaca gatgctaagg taaaattgat 5520

aaaccaagaa ttagataaat ataaaaatgc tgtaacagaa ttgcagttgc tcatgcaaag 5580

cacaacagca gcaaacaatc gagccagaag agaactacca aggtttatga attatacact 5640

caacaatacc aaaaaaacca atgtaacatt aagcaagaaa aggaaaagaa gatttcttgg 5700

ttttttgtta ggtgttggat ctgcaatcgc cagtggcatt gctgtatcta aggtcctgca 5760

cttagaagga gaagtgaaca agatcaaaag tgctctacta tccacaaaca aggccgtagt 5820

cagcttatca aatggagtta gtgtcttaac cagcaaagtg ttagacctca aaaactatat 5880

agataaacaa ttgttaccta ttgtgaataa gcaaagctgc agaatatcaa atatagcgac 5940

tgtgatagag ttccaacaaa agaacaacag actactagag attaccaggg aatttagtgt 6000

taatgcaggt gtaactacac ctgtaagcac ttacatgtta actaatagtg aattattgtc 6060

attaatcaat gatatgccta taacaaatga tcagaaaaag ttaatgtcca acaatgttca 6120

aatagttaga cagcaaagtt actctatcat gtccataata aaagaggaag tcttagcata 6180

tgtagtacaa ttaccactat atggtgtgat agatacacct tgttggaaat tacacacatc 6240

ccctctatgt acaaccaaca caaaagaagg gtcaaacatc tgtttaacaa gaactgacag 6300

aggatggtac tgtgacaatg caggatcagt atctttcttc ccacaagctg aaacatgtaa 6360

agttcaatcg aatcgagtat tttgtgacac aatgaacagt ttaacattac caagtgaagt 6420

aaatctctgc aatgttgaca tattcaatcc caaatatgat tgtaaaatta tgacttcaaa 6480

aacagatgta agcagctccg ttatcacatc tctaggagcc attgtgtcat gctatggcaa 6540

aactaaatgt acagcatcca ataaaaatcg tggaatcata aagacatttt ctaacgggtg 6600

tgattatgta tcaaataaag gggtggacac tgtgtctgta ggtaacacat tatattatgt 6660

aaataagcaa gaaggcaaaa gtctctatgt aaaaggtgaa ccaataataa atttctatga 6720

cccattagta ttcccctctg atgaatttga tgcatcaata tctcaagtca atgagaagat 6780

taaccagagt ttagcattta ttcgtaaatc cgatgaatta ttacatcatg taaatgctgg 6840

taaatcaacc attaatatca tgataactac tataattata gtgattatag taatattgtt 6900

atcattaatt gctgttggac tgctcctata ctgtaaggcc agaagcacac cagtcacact 6960

aagcaaggat caactgagtg gtataaataa tattgcattt agtaactgaa taaaaatagc 7020

acctaatcat gttcttacaa tggtttacta tctgctcata gacaacccat ctatcattgg 7080

attttcttaa aatctgaact tcatcgaaac tcttatctat aaaccatctc acttacacta 7140

tttaagtaga ttcctagttt atagttatat aaaacacaat tgaataccag attaacttac 7200

tatctgtaaa aatgagaact ggggcaaaca tgtcacgaag gaatccttgc aaatttgaaa 7260

ttcgaggtca ttgcttgaat ggtaagagat gtcattttag tcataattat tttgaatggc 7320

caccccatgc actgctcgta agacaaaact ttatgttaaa cagaatactt aagtctatgg 7380

ataaaagtat agatacctta tcagaaataa gtggagctgc agagttggac agaacagaag 7440

agtatgctct tggtgtagtt ggagtgctag agagttatat aggatcaata aataatataa 7500

ctaaacaatc agcatgtgtt gccatgagca aactcctcac tgaactcaat agtgatgata 7560

tcaaaaaact gagagacaat gaagagctaa attcacccaa gataagagtg tacaatactg 7620

tcatatcata tattgaaagc aacaggaaaa acaataaaca aactatccat ctgttaaaaa 7680

gattgccagc agacgtattg aagaaaacca tcaaaaacac attggatatc cacaagagca 7740

taaccatcaa caacccaaaa gaattaactg ttagtgatac aaatgaccat gccaaaaata 7800

atgatactac ctgacaaata tccttgtagt ataacttcca tactaataac aagtagatgt 7860

agagtcacta tgtataatcg aaagaacaca ctatatttca atcaaaacaa cccaaataac 7920

catatgtact caccgaatca aacattcaat gaaatccatt ggacctcaca agacttgatt 7980

gacacaattc aaaattttct acagcatcta ggtgttattg aggatatata tacaatatat 8040

atattagtgt cataacactc aatcctaata ctgaccatat cgttgaatta ttaattcaaa 8100

taattcaagc tgtgggacaa aatggatccc attattaatg gaaattctgc taatgtttat 8160

ctaaccgata gttatttaaa aggtgttatc tctttctcag agtgtaatgc tttaggaagt 8220

tacatattca atggtcctta tctcaaaaat gattatacca acttaattag tagacaaaat 8280

ccattaatag aacacatgaa tctaaagaaa ctaaatataa cacagtcctt aatatctaag 8340

tatcataaag gtgaaataaa attagaagag cctacttatt ttcagtcatt acttatgaca 8400

tacaagagta tgacctcgtt ggaacagatt gctaccacta atttacttaa aaagataata 8460

agaagagcta tagaaataag tgatgtcaaa gtctatgcta tattgaataa actagggctt 8520

aaagaaaagg acaagattaa atccaacaat ggacaggatg aagacaactc agttattacg 8580

accataatca aagatgatat actttcagct gttaaggata atcaatctca tcttaaagca 8640

gacaaaaatc actctacaaa acaaaaagac acaatcaaaa caacactctt gaagaaatta 8700

atgtgttcaa tgcagcatcc tccatcatgg ttaatacatt ggtttaattt atacacaaaa 8760

ttaaacaaca tattaacaca gtatcgttca aatgaggtta aaaaccatgg gtttatattg 8820

atagataatc aaactcttag tggatttcaa tttattttga atcaatatgg ttgtatagtt 8880

tatcataagg aactcaaaag aattactgtg acaacctata atcaattctt gacatggaaa 8940

gatattagcc ttagtagatt aaatgtttgt ttaattacat ggattagtaa ctgcttgaac 9000

acattaaata aaagcttagg cttaagatgc ggattcaata atgttatctt gacacaacta 9060

ttcctttatg gtgattacat actaaagcta tttcacaatg aggggttcta cataataaaa 9120

gaggtagagg gatttattat gtctctaatt ttaaatataa cagaagaaga tcaattcaga 9180

aaacgatttt ataatagtat gctcaacaac atcacagatg ctgctaataa agctcagaaa 9240

aatctgctat caagagtatg tcatacatta ttagataaga cagtatccga taatataata 9300

aatggcagat ggataattct attaagtaag ttccttaaat taattaagct tgcaggtgac 9360

aataacctta acaatctgag tgaactatat tttttgttca gaatatttgg acacccaatg 9420

gtagatgaaa gacaagccat ggatgctgtt aaagttaatt gcaatgagac caaattttac 9480

ttgttaagca gtttgagtat gttaagaggt gcctttatat atagaattat aaaagggttt 9540

gtaaataatt acaacagatg gcctacttta agaaatgcta ttgttttacc cttaagatgg 9600

ttaacttact ataaactaaa cacttatcct tctttgttgg aacttacaga aagagatttg 9660

attgtgttat caggactacg tttctatcgt gagtttcggt tgcctaaaaa agtggatctt 9720

gaaatgatta taaatgataa agctatatca ccccctaaaa atttgatatg gactagtttc 9780

cctagaaatt atatgccgtc acacatacaa aactatatag aacatgaaaa attaaaattt 9840

tccgagagtg ataaatcaag aagagtatta gagtattatt taagagataa caaattcaat 9900

gaatgtgatt tatacaactg tgtagttaat caaagttatc tcaacaaccc taatcatgtg 9960

gtatcattga caggcaaaga aagagaactc agtgtaggta gaatgtttgc aatgcaaccg 10020

ggaatgttca gacaggttca aatattggca gagaaaatga tagctgaaaa cattttacaa 10080

ttctttcctg aaagtcttac aagatatggt gatctagaac tacaaaaaat attagaattg 10140

aaagcaggaa taagtaacaa atcaaatcgc tacaatgata attacaacaa ttacattagt 10200

aagtgctcta tcatcacaga tctcagcaaa ttcaatcaag catttcgata tgaaacgtca 10260

tgtatttgta gtgatgtgct ggatgaactg catggtgtac aatctctatt ttcctggtta 10320

catttaacta ttcctcatgt cacaataata tgcacatata ggcatgcacc cccctatata 10380

agagatcata ttgtagatct taacaatgta gatgaacaaa gtggattata tagatatcac 10440

atgggtggta ttgaagggtg gtgtcaaaaa ctatggacca tagaagctat atcactattg 10500

gatctaatat ctctcaaagg gaaattctca attactgctt taattaatgg tgacaatcaa 10560

tcaatagata taagcaaacc agtcagactc atggaaggtc aaactcatgc tttagcagat 10620

tatttgctag cattaaatag ccttaaatta ctgtataaag agtatgcagg cataggtcac 10680

aaattaaaag gaactgagac ttatatatca cgagatatgc aatttatgag taaaacaatt 10740

caacataacg gtgtatatta ccctgctagt ataaagaaag tcctaagagt gggaccgtgg 10800

ataaacacta tacttgatga tttcaaagtg agtctagaat ctataggtag tttgacacaa 10860

gaattagaat atagaggtga aagtctatta tgcagtttaa tatttagaaa tgtatggtta 10920

tataatcaaa ttgctctaca attaaaaaat catgcgttat gtaacaataa attatatttg 10980

gacatattaa aggttctgaa acacttaaaa acctttttta atcttgataa tattgataca 11040

gcattaacat tgtatatgaa tttacccatg ttatttggtg gtggtgatcc caacttgtta 11100

tatcgaagtt tctatagaag aactcctgat ttcctcacag aggctatagt tcactctgtg 11160

ttcatactta gttattatac aaaccatgac ttaaaagata aacttcaaga tttgtcagat 11220

gatagattga ataagttctt aacatgcata atcacgtttg acaaaaaccc taatgctgaa 11280

ttcgtaacat tgatgagaga tcctcaagct ttagggtctg agagacaagc taaaattact 11340

agtgaaatca atagactggc agttacagag gttttgagta cagctccaaa caaaatattc 11400

tccaaaagtg cacaacatta taccactaca gagatagatc taaatgatat tatgcaaaat 11460

atagaaccta catatcctca cgggctaaga gttgtttatg aaagtttacc cttttataaa 11520

gcagagaaaa tagtaaatct tatatcaggt acaaaatcta taactaacat actggaaaag 11580

acttctgcca tagacttaac agatattgat agagccactg agatgatgag gaaaaacata 11640

actttgctta taaggatact tccattggat tgtaacagag ataaaagaga aatattgagt 11700

atggaaaacc taagtattac tgaattaagc aaatatgtta gggaaagatc ttggtcttta 11760

tccaatatag ttggtgttac atcacccagt atcatgtata caatggacat caaatataca 11820

acaagcacta tagctagtgg cataattata gagaaatata atgttaacag tttaacacgt 11880

ggtgagagag gaccaactaa accatgggtt ggttcatcta cacaagagaa aaaaacaatg 11940

ccagtttata atagacaagt tttaaccaaa aaacaaagag atcaaataga tctattagca 12000

aaattggatt gggtgtatgc atctatagat aacaaggatg aattcatgga agaactcagc 12060

ataggaaccc ttgggttaac atatgaaaag gccaaaaaat tatttccaca atatttaagt 12120

gtcaactatt tgcatcgcct tacagtcagt agtagaccat gtgaattccc tgcatcaata 12180

ccagcttata gaacaacaaa ttatcacttt gacactagcc ctattaatcg catattaaca 12240

gaaaagtatg gtgatgaaga tattgacata gtattccaaa actgtataag ctttggcctt 12300

agcttaatgt cagtagtaga acaatttact aatgtatgtc ctaacagaat tattctcata 12360

cctaagctta atgagataca tttgatgaaa cctcccatat tcacaggtga tgttgatatt 12420

cacaagttaa aacaagtgat acaaaaacag catatgtttt taccagacaa aataagtttg 12480

actcaatatg tggaattatt cttaagtaac aaaacactca aatctggatc tcatgttaat 12540

tctaatttaa tattggcaca taaaatatct gactattttc ataatactta cattttaagt 12600

actaatttag ctggacattg gattctaatt atacaactta tgaaagattc taaaggtatt 12660

tttgaaaaag attggggaga gggatatata actgatcata tgtttattaa tttgaaagtt 12720

ttcttcaatg cttataagac ctatctcttg tgttttcata aaggttatgg caaagcaaaa 12780

ctggagtgtg atatgaacac ttcagatctt ctatgtgtat tggaattaat agacagtagt 12840

tattggaagt ctatgtctaa ggtattttta gaacaaaaag ttatcaaata cattcttagc 12900

caagatgcaa gtttacatag agtaaaagga tgtcatagct tcaaattatg gtttcttaaa 12960

cgtcttaatg tagcagaatt tacagtttgc ccttgggttg ttaacataga ttatcatcca 13020

acacatatga aagcaatatt aacttatata gatcttgtta gaatgggatt gataaatata 13080

gatagaatac acattaaaaa taaacacaaa ttcaatgatg aattttatac ttctaatctc 13140

ttttacatta attataactt ctcagataat actcatctat taactaaata cataaggatt 13200

gctaattcag aattagaaaa taattacaac aaattatatc atcctacacc agaaacccta 13260

gagaatatac tagccaatcc gattaaaagt aatgacaaaa agacactgaa cgactattgt 13320

ataggtaaaa atgttgactc aataatgtta ccattgttat ctaataagaa gcttgttaaa 13380

tcgtctgcaa tgattagaac caattacagc aaacaagacc tgtacaatct attccctacg 13440

gttgtgatcg atagaattat agatcattca ggtaatacag ccaaatccaa ccaactttac 13500

actactactt cccatcaaat atctttagtg cacaatagca catcacttta ttgcatgctt 13560

ccttggcatc atattaatag attcaatttt gtatttagtt ctacaggttg taaaattagt 13620

atagagtata ttttaaaaga ccttaaaatt aaagatccta attgtatagc attcataggt 13680

gaaggagcag ggaatttatt attgcgtaca gtggtggaac ttcatcctga cataagatat 13740

atttacagaa gtctgaaaga ttgcaatgat catagtttac ctattgagtt tttaaggcta 13800

tacaatggac atatcaacat tgattatggt gaaaatttga ccattcctgc tacagatgca 13860

accaacaaca ttcattggtc ttatttacat ataaagtttg ctgaacctat cagtcttttt 13920

gtatgtgatg ccgaattgcc tgtaacagtc aactggagta aaattataat agaatggagc 13980

aagcatgtaa gaaaatgcaa gtactgttcc tcagttaata aatgtacgtt aatagtaaaa 14040

tatcatgctc aagatgatat tgatttcaaa ttagacaata taactatatt aaaaacttat 14100

gtatgcttag gcagtaagtt aaagggatcg gaggtttact tagtccttac aataggtcct 14160

gcaaatatat ttccagtatt taatgtagta caaaatgcta aattgatact atcaagaacc 14220

aaaaatttca tcatgcctaa gaaagctgat aaagagtcta ttgatgcaaa tattaaaagt 14280

ttgataccct ttctttgtta ccctataaca aaaaaaggaa ttaatactgc attgtcaaaa 14340

ctaaagagtg ttgttagtgg agatatacta tcatattcta tagctggacg gaatgaagtt 14400

ttcagcaata aacttataaa tcataagcat atgaacatct taaagtggtt caatcatgtt 14460

ttaaatttca gatcaacaga actaaactat aaccatttat atatggtaga atctacatat 14520

ccttacctaa gtgaattgtt aaacagcttg acaactaatg aacttaaaaa actgattaaa 14580

atcacaggta gtctgttata caactttcat aatgaataat gaataaagat cttataataa 14640

aaattcctat agctatacac tagcactgta ttcaattata gttattaaaa aattaaaaat 14700

catataattt tttataaaaa taacttttag tgaactaatc ctaaagttat cattttgatc 14760

taggaggaat aaatttaaat cccaatctaa ttggtttata tgtgtattaa ctaaactacg 14820

agatattagt ttttgacact ttttttctcg tgggtcggca tggcatctcc acctcctcgc 14880

ggtccgacct gggcatccga aggaggacgc acgtccactc ggatggctaa gggagagcca 14940

ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 15000

cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 15060

tatccggatt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg 15120

gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc 15180

agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc actggaccgc 15240

tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg 15300

taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg 15360

ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat 15420

tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat 15480

atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc 15540

ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg 15600

ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg 15660

ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta 15720

aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg 15780

atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc 15840

ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg 15900

ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt 15960

catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa 16020

acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct 16080

ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga 16140

ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct 16200

ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 16260

acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca 16320

gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta 16380

ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 16440

atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 16500

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 16560

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 16620

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 16680

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 16740

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 16800

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 16860

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 16920

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 16980

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 17040

aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 17100

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 17160

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 17220

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 17280

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 17340

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 17400

ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 17460

ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 17520

atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 17580

ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 17640

tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 17700

attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 17760

tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 17820

ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 17880

gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 17940

gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 18000

gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 18060

aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 18120

taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 18180

tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 18240

tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 18300

atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 18360

tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 18420

aaaaataggc gtatcacgag gccctttcgt cttcaagaat tctcatgttt gacagcttat 18480

catcg 18485

<210> 4

<211> 4209

<212> DNA

<213> pBRB载体

<400> 4

gaattctcat gtttgacagc ttatcatcga cgcgttacgt ctagaggtca agcttgagcg 60

agctcggcgt tcgaaggcgg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg 120

ttctcggagc actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg 180

gagccactat cgactacgcg atcatggcga ccacacccgt cctgtggatc ctctacgccg 240

gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg 300

acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg 360

tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac 420

cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc 480

aggagtcgca taagggagag cgtcgaccga tgcccttgag agccttcaac ccagtcagct 540

ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 600

tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc 660

gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg 720

ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg 780

ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg cgaggctgga 840

tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg 900

ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg 960

ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt tatgccgcct 1020

cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc 1080

tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg 1140

gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac 1200

tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag 1260

ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct 1320

cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 1380

accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 1440

aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 1500

ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 1560

tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg 1620

catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc 1680

agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag 1740

aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc 1800

ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga 1860

tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg 1920

cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 1980

cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 2040

tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 2100

tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 2160

ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 2220

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2280

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2340

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2400

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2460

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2520

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 2580

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2640

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2700

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2760

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2820

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 2880

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 2940

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3000

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3060

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3120

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3180

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3240

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3300

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3360

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3420

tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 3480

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3540

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3600

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3660

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3720

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 3780

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3840

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 3900

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 3960

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4020

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4080

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 4140

taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 4200

cgtcttcaa 4209

<210> 5

<211> 24

<212> DNA

<213> 实时定量PCR引物 F

<400> 5

agatcaactt ctgtcatcca gcaa 24

<210> 6

<211> 25

<212> DNA

<213> 实时定量PCR引物 R

<400> 6

gcacatcata attaggagta tcaat 25

<210> 7

<211> 21

<212> DNA

<213> N799G/A引物 F

<400> 7

gatctggtct tacagccgtg a 21

<210> 8

<211> 20

<212> DNA

<213> N799G/A引物 R

<400> 8

tgagttgttc agcatatgcc 20

<210> 9

<211> 23

<212> DNA

<213> F653,654AA/ CG引物 F

<400> 9

gcacaacagc agcaaacaat cga 23

<210> 10

<211> 22

<212> DNA

<213> F653,654AA/ CG引物 R

<400> 10

tcacagtacc atcctctgtc ag 22

<210> 11

<211> 22

<212> DNA

<213> F1568,1569CA/TT引物 F

<400> 11

gtggacactg tgtctgtagg ta 22

<210> 12

<211> 22

<212> DNA

<213> F1568,1569CA/TT引物 R

<400> 12

ttgcaaggat tccttcgtga ca 22

<210> 13

<211> 21

<212> DNA

<213> L 956,957GT/AC引物 F

<400> 13

tgcagcatcc tccatcatgg t 21

<210> 14

<211> 22

<212> DNA

<213> L 956,957GT/AC引物 R

<400> 14

gcatctgtga tgttgttgag ca 22

<210> 15

<211> 20

<212> DNA

<213> L5068,5070 C/T,T/C引物 F

<400> 15

agccaagatg caagtttaca 20

<210> 16

<211> 22

<212> DNA

<213> L5068,5070 C/T,T/C引物 R

<400> 16

ttgctcatgg caacacatgc tg 22

<210> 17

<211> 22

<212> DNA

<213> L2491,2492CA/TT引物 F

<400> 17

gtagtgatgt gctggatgaa ct 22

<210> 18

<211> 22

<212> DNA

<213> L2491,2492CA/TT引物 R

<400> 18

aacaagttgg gatcaccacc ac 22