猪肺炎支原体病亚单位疫苗组合物及其制备方法和应用

摘要

本发明公开了一种猪肺炎支原体病亚单位疫苗组合物，包括PEA(ΔⅢ)‑P42重组蛋白、PEA(ΔⅢ)‑P97重组蛋白、PEA(ΔⅢ)‑P95重组蛋白、PEA(ΔⅢ)‑P46重组蛋白、PEA(ΔⅢ)‑NrdF重组蛋白。本发明还公开了该猪肺炎支原体病亚单位疫苗组合物的制备方法和应用。本发明的猪肺炎支原体病亚单位疫苗，免疫效果良好，免疫剂量小，能有效预防猪肺炎支原体病相关疾病和由猪肺炎支原体病造成感染的相关疾病。本发明的制备方法简单，能大量制备猪肺炎支原体病的抗原蛋白，耗时短，表达量高，大大降低了生产成本，有利于大规模生产。

说明书

技术领域

本发明属于兽用生物制品领域，特别涉及一种猪肺炎支原体病亚单位疫苗组合物及其制备方法和应用。

背景技术

猪支原体肺炎(Mycoplasmal pneumonia of swine，MPS)是由猪肺炎支原体(Mycoplasma hyopneumoniae，Mhp)引起的，是以猪咳嗽和气喘为主要症状的慢性呼吸道传染病，具有慢性、接触性、高传染性、高发病率和低死亡率的特征。该病广泛分布于世界各地，国外常被称为猪地方流行性肺炎，而在我国常被称为猪气喘病(喘气病)。猪肺炎支原体感染导致猪的生长率下降12％～16％，饲料转化率降低13％～22％，体重相对减少10～25kg，商品猪的出栏时间延迟10～30天，同时还破坏猪的支气管和细支气管纤毛，进而损伤阻挡病原微生物入侵的物理屏障，破坏猪的巨唾细胞，降低机体的免疫清除能力，导致免疫抑制。因此，在临床上猪肺炎支原体常常与猪蓝耳病病毒、猪2型圆环病毒、猪流感病毒、胸膜肺炎放线杆菌、多杀性巴氏杆菌等病原混合感染，增加了当前猪病诊断和预防控制的难度。此外，由于猪肺炎支原体是缺少细胞壁的原核微生物，对常用的作用于细胞壁的抗生素(如β-内酰胺酵类抗生素等)不敏感，猪群一旦被猪肺炎支原体感染，控制和净化将十分困难。据初步统计，全球每年因猪肺炎支原体感染造成的直接经济损失高达2亿美元。1999年的统计数据表明，当年全世界仅用于疫苗防治猪支原体肺炎这一项的费用就高达1.1亿英镑，约占年全世界在猪疫苗上花费的四分之一。可以说，猪肺炎支原体已成为当前严重影响养猪业经济效益的重大疫病，被喻为影响养猪业经济效益的“隐性杀手”。

目前猪肺炎支原体的防控主要依赖灭活疫苗和弱毒疫苗，然而疫苗只能减弱疾病的危害程度，并不能彻底的根除猪肺炎支原体。此外，猪肺炎支原体的培养条件苟刻，对营养需求较高，培养周期较长，这些因素加大了猪肺炎支原体疫苗的生产成本。因此，急需研制安全、高效、低成本的新型疫苗。

发明内容

为解决上述技术问题，本发明的目的在于提供一种猪肺炎支原体病亚单位疫苗组合物及其制备方法和应用。该疫苗组合物生产成本低、生产效率高且免疫效果好。

为实现上述技术目的，达到上述技术效果，本发明通过以下技术方案实现：

本发明首先提供一种猪肺炎支原体病亚单位疫苗组合物，其包括PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白；所述PEA(ΔⅢ)-P42重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P42蛋白片段的融合；所述PEA(ΔⅢ)-P97重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P97蛋白片段的融合；所述PEA(ΔⅢ)-P95重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P95蛋白片段的融合；所述PEA(ΔⅢ)-P46重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P46蛋白片段的融合、PEA(ΔⅢ)-NrdF重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体NrdF蛋白的融合。

其中的猪肺炎支原体P42蛋白片段的氨基酸序列如SEQ ID NO.1所示，其核苷酸序列如SEQ ID NO.2所示。

其中的猪肺炎支原体P97蛋白片段的氨基酸序列如SEQ ID NO.3所示，其核苷酸序列如SEQ ID NO.4所示。

其中的猪肺炎支原体P95蛋白片段的氨基酸序列如SEQ ID NO.5所示，其核苷酸序列如SEQ ID NO.6所示。

其中的猪肺炎支原体P46蛋白片段的氨基酸序列如SEQ ID NO.7所示，其核苷酸序列如SEQ ID NO.8所示。

其中的猪肺炎支原体NrdF蛋白的氨基酸序列如SEQ ID NO.9所示，其核苷酸序列如SEQ ID NO.10所示。

其中的绿脓杆菌外毒素PEA(ΔⅢ)蛋白由SEQ ID NO.11所示的核苷酸序列编码，其是通过人工合成。

进一步的，该疫苗组合物还包括佐剂。

更进一步的，疫苗组合物中的佐剂为SEPPIC Montanide IMS 1313VG NST佐剂。

在上述疫苗组合物中，PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白的比例为1:1:1:1:1。

该疫苗组合物中，PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白的总含量为200～300μg/mL。

本发明还提供了一种上述猪肺炎支原体病亚单位疫苗组合物的制备方法，包括如下步骤：

(1)克隆、扩增P42蛋白片段基因、P97蛋白片段基因、P95蛋白片段基因、P46蛋白片段基因及NrdF蛋白基因，并分别与PEA(ΔⅢ)蛋白基因连接，构建PEA(ΔⅢ)-P42重组蛋白基因片段、PEA(ΔⅢ)-P97重组蛋白基因片段、PEA(ΔⅢ)-P95重组蛋白基因片段、PEA(ΔⅢ)-P46重组蛋白基因片段、PEA(ΔⅢ)-NrdF重组蛋白基因片段；

(2)构建表达PEA(ΔⅢ)-P42重组蛋白基因片段、PEA(ΔⅢ)-P97重组蛋白基因片段、PEA(ΔⅢ)-P95重组蛋白基因片段、PEA(ΔⅢ)-P46重组蛋白基因片段、PEA(ΔⅢ)-NrdF重组蛋白基因片段的重组质粒；

(3)将步骤(2)得到的重组质粒转化DH10 Bac感受态细胞，通过转座，获得重组杆粒；

(4)将步骤(3)获得的重组杆粒转染昆虫细胞，获得重组杆状病毒；

(5)将获得的重组杆状病毒接种HF细胞进行大量培养，离心收集培养液上清，得到PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白，再经纯化，加入佐剂，乳化，最后混匀。

本发明进一步提供了上述猪肺炎支原体病亚单位疫苗组合物在制备预防和/或治疗猪肺炎支原体病相关疾病或由猪肺炎支原体病感染的相关疾病的药物中的应用。

本发明的有益效果是：

本发明的猪肺炎支原体病亚单位疫苗，采用了PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白，其免疫效果良好，免疫剂量小，能有效预防猪肺炎支原体病相关疾病和由猪肺炎支原体病造成感染的相关疾病。

本发明的制备方法简单，能大量制备猪肺炎支原体病的抗原蛋白，耗时短，表达量高，大大降低了生产成本，有利于大规模生产。

具体实施方式

下面将结合具体实施例对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。

本发明提供一种猪肺炎支原体病亚单位疫苗组合物，其包括免疫量的PEA(ΔⅢ)-P42重组蛋白、免疫量的PEA(ΔⅢ)-P97重组蛋白、免疫量的PEA(ΔⅢ)-P95重组蛋白、免疫量的PEA(ΔⅢ)-P46重组蛋白、免疫量的PEA(ΔⅢ)-NrdF重组蛋白；所述PEA(ΔⅢ)-P42重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P42蛋白片段的融合；所述PEA(ΔⅢ)-P97重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P97蛋白片段的融合；所述PEA(ΔⅢ)-P95重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P95蛋白片段的融合；所述PEA(ΔⅢ)-P46重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体P46蛋白片段的融合、PEA(ΔⅢ)-NrdF重组蛋白是绿脓杆菌外毒素PEA(ΔⅢ)蛋白与猪肺炎支原体NrdF蛋白的融合。

其中的猪肺炎支原体P42蛋白氨基酸序列、猪肺炎支原体P97蛋白氨基酸序列、猪肺炎支原体P95蛋白氨基酸序列、猪肺炎支原体P46蛋白氨基酸序列、猪肺炎支原体NrdF蛋白氨基酸序列均为猪肺炎支原体病毒7448株相应蛋白的氨基酸序列(NCBI登录号为AE017244.1)。

猪肺炎支原体P42蛋白片段的氨基酸序列如SEQ ID NO.1所示，是以SEQ ID NO.12所示的猪肺炎支原体病毒7448株P42蛋白氨基酸序列为基准的第434位至600位氨基酸序列；P42蛋白片段的核苷酸序列如SEQ ID NO.2所示。

猪肺炎支原体P97蛋白片段的氨基酸序列如SEQ ID NO.3所示，是以SEQ ID NO.13所示的猪肺炎支原体病毒7448株P97蛋白氨基酸序列为基准的第788位至915位氨基酸序列；P97蛋白片段的核苷酸序列如SEQ ID NO.4所示。

猪肺炎支原体P95蛋白片段的氨基酸序列如SEQ ID NO.5所示，以SEQ ID NO.14所示的猪肺炎支原体病毒7448株P95蛋白氨基酸序列为基准的第604位至750位氨基酸序列；P95蛋白片段的核苷酸序列如SEQ ID NO.6所示。

猪肺炎支原体P46蛋白片段的氨基酸序列如SEQ ID NO.7所示，是以SEQ ID NO.15所示的猪肺炎支原体病毒7448株P46蛋白氨基酸序列为基准的第323位至419位氨基酸序列；P46蛋白片段的核苷酸序列如SEQ ID NO.8所示。

猪肺炎支原体NrdF蛋白的氨基酸序列如SEQ ID NO.9所示，其核苷酸序列如SEQID NO.10所示。

其中的绿脓杆菌外毒素PEA(ΔⅢ)蛋白由SEQ ID NO.11所示的核苷酸序列编码，其是通过人工合成。

该疫苗组合物还包括佐剂；该佐剂为SEPPIC Montanide IMS 1313VG NST佐剂。

在该疫苗组合物中，PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白的比例为1:1:1:1:1。

该疫苗组合物中，PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白的总含量为200～300μg/mL。

实施例

取200μL猪肺炎支原体培养物，参照TaKaRa公司Mini BEST Universal GenomicDNA Extraction Kit Ver.5.0试剂盒说明书提取猪肺炎支原体DNA。

1.猪肺炎支原体P42蛋白片段基因的扩增

以提取的DNA为模板，利用引物P42F和P42R扩增编码P42蛋白第434位至600位氨基酸片段的基因。引物见表1，PCR反应体系见表2，PCR反应条件见表3。

表1 P42蛋白片段基因扩增引物

表2 P42蛋白片段基因PCR体系

表3 P42蛋白片段基因PCR反应条件

2.猪肺炎支原体NrdF蛋白基因的扩增

以提取的DNA为模板，参照步骤1中的PCR反应体系及条件，利用引物NrdF-F和NrdF-R扩增NrdF蛋白基因。引物见表4。

表4 NrdF蛋白基因扩增引物

3.猪肺炎支原体P97蛋白片段基因的扩增

以提取的DNA为模板，参照步骤1中的PCR反应体系及条件，利用引物P97-F和P97-R扩增编码P97蛋白第788～915氨基酸片段的基因。引物见表5。

表5 P97蛋白片段基因扩增引物

4.猪肺炎支原体P95蛋白片段基因的扩增

以提取的DNA为模板，参照步骤1中的PCR反应体系及条件，利用引物P95-F和P95-R扩增编码P95蛋白第604～750氨基酸片段的基因。引物见表6。

表6 P95蛋白片段基因扩增引物

5.猪肺炎支原体P46蛋白片段基因的扩增

以提取的DNA为模板，参照步骤1中的PCR反应体系及条件，利用引物P46-F和P46-R扩增编码P46蛋白第323～419氨基酸片段的基因。引物见表7。

表7 P46蛋白片段基因扩增引物

6.重组蛋白基因的连接以及重组蛋白表达载体的构建

将步骤1～5中扩增得到的目的基因分别用Kpn I和Not I双酶切，人工合成的PEA(ΔⅢ)基因用BamH I和Kpn I双酶切，pFastBacHT A载体用BamH I和Not I双酶切。分别将5段目的基因产物与PEA(ΔⅢ)基因酶切产物和pFastBacHT A载体酶切产物进行连接，连接后将连接产物转化大肠杆菌DH 5α感受态细胞，获得的阳性重组质粒分别命名为：pFastBac-PEA(ΔⅢ)-P42、pFastBac-PEA(ΔⅢ)-NrdF、pFastBac-PEA(ΔⅢ)-P97、pFastBac-PEA(ΔⅢ)-P95、pFastBac-PEA(ΔⅢ)-P46。

7.重组杆状病毒的获得及传代

将重组的rBacmid-PEA(ΔⅢ)-P42、rBacmid-PEA(ΔⅢ)-NrdF、rBacmid-PEA(ΔⅢ)-P97、rBacmid-PEA(ΔⅢ)-P95、rBacmid-PEA(ΔⅢ)-P46分别转染昆虫细胞sf9，具体参照Cellfectin

将上述收获的F1代重组杆状病毒以1％的接毒量接入处于生长对数期的sf9细胞(2.0×10

8.蛋白表达

将步骤7中获得的F4代的重组杆状病毒按照MOI＝5～10的接毒量接入HF细胞，接种后约7d左右收获细胞，离心获得的上清进行SDS-PAGE确认目的蛋白得到表达。目的蛋白经过镍柱纯化，参照上海生工BCA试剂盒进行蛋白定量，结果显示纯化后的目的蛋白浓度分别为1.21mg/mL、0.85mg/mL、1.66mg/mL、1.02mg/mL、1.53mg/mL。

猪肺炎支原体病亚单位疫苗组合物的制备

取获得的纯化的PEA(ΔⅢ)-P42重组蛋白、PEA(ΔⅢ)-NrdF重组蛋白、PEA(ΔⅢ)-P97重组蛋白、PEA(ΔⅢ)-P95重组蛋白、PEA(ΔⅢ)-P46重组蛋白，加入佐剂进行乳化，混匀，4℃保存。疫苗具体配比见表8。

表8猪肺炎支原体病毒亚单位疫苗成分配比

适用于本发明的组合物的佐剂的量优选地是有效量。所述“有效量”是指佐剂在同本发明抗原联合施用时在宿主中发挥它们的免疫学作用而言必须或足够的而不导致过度副作用所必需量。待施用的佐剂的精确的量将根据因素如所用的成分和治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。

本发明的组合物的成分或组分的量优选地是治疗有效量。所述治疗有效量是指在组合物施用的宿主中发挥它们的免疫学作用而不导致过度副作用所必需量。所用的成分和待施用的组合物的精确的量将根据因素如治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。

猪肺炎支原体病亚单位疫苗免疫原性试验

选取断奶仔猪35头，随机分成7组，每组5头，其中的6组分别对应免疫表8中制备的6种疫苗，免疫方式为颈部肌肉注射，免疫剂量为2ml/头；第7组作为对照组，颈部肌肉注射同等剂量的无菌PBS；免疫28天后，免疫组和对照组分别用猪肺炎支原体效检用强毒(YN14株，临床分离株)进行攻击，每头猪气管注射0.2g病肺组织(100MID)，各组猪隔离饲养，饲料中无抗生素。攻毒后25～28日剖杀，并计算肺部病变减少率。免疫组肺部病变减少率达到60％及以上判疫苗检验合格。具体试验结果见表9。从攻毒结果判断，疫苗4、5、6均具有较好的免疫保护效果，符合要求。

表9猪肺炎支原体病毒亚单位疫苗免疫仔猪后的攻毒保护结果

以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的修改或等效变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

序列表

<110> 扬州优邦生物药品有限公司

<120> 猪肺炎支原体病亚单位疫苗组合物及其制备方法和应用

<130> 2020

<141> 2020-12-09

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 167

<212> PRT

<213> P42蛋白片段氨基酸序列(Amino acid sequence of P42 protein fragment)

<400> 1

Ile Glu Ala Ala Pro Arg Gly Leu Pro Gln Ile Glu Val Ser Phe Ser

1 5 10 15

Ile Asp Val Asn Gly Ile Thr Thr Val Ser Ala Lys Asp Lys Lys Thr

20 25 30

Gly Lys Glu Gln Thr Ile Thr Ile Lys Asn Thr Ser Thr Leu Ser Glu

35 40 45

Glu Glu Ile Asn Lys Met Ile Gln Glu Ala Glu Glu Asn Arg Glu Ala

50 55 60

Asp Ala Leu Lys Lys Asp Lys Ile Glu Thr Thr Val Arg Ala Glu Gly

65 70 75 80

Leu Ile Asn Gln Leu Glu Lys Ser Ile Thr Asp Gln Gly Glu Lys Ile

85 90 95

Asp Pro Lys Gln Lys Glu Leu Leu Glu Lys Gln Ile Gln Glu Leu Lys

100 105 110

Asp Leu Leu Lys Glu Glu Lys Thr Asp Glu Leu Lys Leu Lys Leu Asp

115 120 125

Gln Ile Glu Ala Ala Ala Gln Ser Phe Ala Gln Ala Thr Ala Gln Gln

130 135 140

Ala Asn Thr Ser Glu Ser Asp Pro Lys Ala Asp Asp Ser Asn Thr Ile

145 150 155 160

Asp Ala Glu Ile Lys Gln Asp

165

<210> 2

<211> 501

<212> DNA

<213> P42蛋白片段核苷酸序列(P42 protein fragment nucleotide sequence)

<400> 2

attgaagctg ctccacgagg tcttccccag attgaagtta gtttttcaat tgatgtcaac 60

gggattacaa cggtttcagc aaaagataaa aaaaccggca aagaacaaac aattacaatt 120

aaaaatactt caactttatc agaagaagaa attaataaga tgattcagga agccgaagaa 180

aatcgtgaag ctgatgctct taaaaaagac aaaatcgaga caacagttcg tgccgaaggg 240

cttattaatc aacttgagaa atcaataact gatcaaggtg aaaaaattga tccaaaacaa 300

aaagaattac ttgaaaaaca aattcaagaa ttaaaagatc ttctaaaaga agaaaaaact 360

gacgaattaa aattaaaatt agaccaaatt gaagcagctg cccaatcttt tgcgcaggca 420

accgcgcagc aagcaaatac atctgaatct gatccaaaag ctgatgattc aaacacaatt 480

gatgctgaaa tcaagcagga t 501

<210> 3

<211> 128

<212> PRT

<213> P97蛋白片段氨基酸序列(Amino acid sequence of P97 protein fragment)

<400> 3

Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys Glu Leu Asp Asn

1 5 10 15

Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala Lys Pro Glu Ala

20 25 30

Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala

35 40 45

Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys

50 55 60

Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala Ala Lys Pro

65 70 75 80

Val Ala Thr Asn Thr Asn Thr Asn Thr Gly Phe Ser Leu Thr Asn Lys

85 90 95

Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr Lys Leu Glu Tyr

100 105 110

Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu Ile Asn Val Phe

115 120 125

<210> 4

<211> 384

<212> DNA

<213> P97蛋白片段核苷酸序列(P97 protein fragment nucleotide sequence)

<400> 4

gaaggtaaaa gagaagaagt agataaaaaa gttaaagaat tagataataa aataaaaggt 60

atattacctc agcccccagc agctaaacct gaagcagcaa aaccagtagc agctaaacct 120

gaagcagcaa aaccagtagc agctaaacct gaagcagcaa aaccagtagc agctaaacct 180

gaagcagcaa aaccagtagc ggctaaacct gaagcagcta aacctgaagc agccaaacca 240

gttgctacta atactaatac taatactggc ttttcactta caaataaacc aaaagaagac 300

tatttcccaa tggcttttag ttataaatta gaatatactg acgaaaataa attaagccta 360

aaaacaccgg aaattaatgt attt 384

<210> 5

<211> 147

<212> PRT

<213> P95蛋白片段氨基酸序列(Amino acid sequence of P95 protein fragment)

<400> 5

Glu Ile Ser Glu Lys Glu Ser Lys Lys Leu Glu Lys Glu Pro Thr Asn

1 5 10 15

Leu Asn Gln Lys Thr Glu Lys Asn Ser Ser Thr Thr Ala Ile Ser Ser

20 25 30

Glu Lys Asn Glu Lys Pro Glu Lys Ser Met Ser Glu Thr Pro Pro Glu

35 40 45

Leu Val Lys Val Gln Gln Asn Gln Pro Lys Thr Glu Glu Glu Pro Lys

50 55 60

Gln Glu Thr Asp Lys Gln Glu Lys Pro Asn Asn Gln Lys Asn Thr Glu

65 70 75 80

Gln Ala Lys Glu Ser Lys Glu Lys Glu Lys Asn Gly Asn Ser Ser Leu

85 90 95

Lys Ser Pro Asn Leu Pro Val Glu Lys Ser Ser Asp Ser Gln Glu Glu

100 105 110

Gly Gln Lys Thr Asp Arg Leu Gln Gln Glu Asn Glu Gln Ala Ala Phe

115 120 125

Asn Asp Lys Ile Val Ser Ile Leu Gln Ser Gln Tyr Glu Ile Ser Leu

130 135 140

Ile Lys Asn

145

<210> 6

<211> 441

<212> DNA

<213> P95蛋白片段核苷酸序列(P95 protein fragment nucleotide sequence)

<400> 6

gagatttccg aaaaagagtc aaaaaaacta gaaaaagaac caacaaattt aaaccaaaag 60

acagagaaaa atagttctac aaccgcaatt tcttctgaaa aaaatgaaaa accagaaaaa 120

tcaatgtctg aaacaccgcc cgagttagta aaggttcagc aaaatcagcc aaaaaccgaa 180

gaagaaccaa agcaagaaac tgataaacaa gaaaaaccca ataaccaaaa aaatacagaa 240

caagctaaag agagcaagga aaaggaaaaa aacggtaatt cttctttaaa aagtccgaat 300

ttacctgtgg aaaaatcctc tgattcccag gaagaaggtc aaaaaactga tagattacag 360

caagaaaatg aacaagccgc ttttaatgat aaaatagttt ctattctgca aagtcagtat 420

gaaatttcat taattaaaaa t 441

<210> 7

<211> 188

<212> PRT

<213> P46蛋白片段氨基酸序列(Amino acid sequence of P46 protein fragment)

<400> 7

Lys Asn Ser Gly Asn Lys Ile Ile Asp Leu Ser Pro Glu Gly Glu Asn

1 5 10 15

Ala Val Tyr Val Pro Gly Trp Asn Tyr Gly Thr Ala Gly Gln Arg Ile

20 25 30

Gln Ser Phe Leu Thr Ile Asn Lys Asp Pro Ala Gly Gly Asn Lys Ile

35 40 45

Lys Ala Ile Gly Ser Lys Pro Ala Ser Ile Phe Lys Gly Phe Leu Ala

50 55 60

Pro Asn Asp Gly Met Ala Glu Gln Ala Ile Thr Lys Leu Lys Leu Glu

65 70 75 80

Gly Phe Asp Thr Gln Lys Ile Phe Val Thr Gly Gln Asp Tyr Asn Asp

85 90 95

Lys Ala Lys Thr Phe Ile Lys Asp Gly Asp Gln Asn Met Thr Ile Tyr

100 105 110

Lys Pro Asp Lys Val Leu Gly Lys Val Ala Val Glu Val Leu Arg Val

115 120 125

Leu Ile Ala Lys Lys Asn Lys Ala Ser Arg Ser Glu Val Glu Asn Glu

130 135 140

Leu Lys Ala Lys Leu Pro Asn Ile Ser Phe Lys Tyr Asp Asn Gln Thr

145 150 155 160

Tyr Lys Val Gln Gly Lys Asn Ile Asn Thr Ile Leu Val Ser Pro Val

165 170 175

Ile Val Thr Lys Ala Asn Val Asp Asn Pro Asp Ala

180 185

<210> 8

<211> 564

<212> DNA

<213> P46蛋白片段核苷酸序列(P46 protein fragment nucleotide sequence)

<400> 8

aaaaattcgg gaaataaaat aattgattta tctcctgaag gcgaaaatgc tgtttatgtc 60

ccaggatgga attatggaac tgccggtcaa agaatccaat cttttctaac aattaacaaa 120

gatccagcag gtggtaataa aatcaaagct attggttcaa aaccagcttc tattttcaaa 180

ggatttcttg caccaaatga tggaatggcc gaacaagcaa tcaccaaatt aaaacttgaa 240

ggatttgata cccaaaaaat ctttgtaact ggtcaagatt ataatgataa agccaaaact 300

tttatcaaag acggcgatca aaatatgaca atttataaac ctgataaagt tttaggaaaa 360

gttgctgttg aagttcttcg ggttttaatt gcaaagaaaa ataaagcatc tagatcagaa 420

gttgaaaacg aactaaaagc aaaactacca aatatttcat ttaaatatga taatcaaaca 480

tataaagtgc aaggtaaaaa tattaataca attttagtaa gtccagtaat tgttacaaaa 540

gctaatgttg ataatcctga tgcc 564

<210> 9

<211> 353

<212> PRT

<213> NrdF蛋白氨基酸序列(NrdF protein amino acid sequence)

<400> 9

Met Ile Asn Ser Lys Leu Asn Ser Asn Ser Asn Asn Asn Lys Leu Lys

1 5 10 15

Ile Lys Asn Asp Tyr Tyr His Gln Ser Val Ser Pro Leu Glu Tyr Ala

20 25 30

Leu Asn Asn Phe Ser Gly Lys Met Arg Ser Ile Asn Trp Asn Ile Ile

35 40 45

Asn Asp Pro Lys Asp Leu Glu Val Trp Thr Arg Ile Val Gln Asn Phe

50 55 60

Trp Ile Pro Glu Lys Ile Pro Leu Ser Asn Asp Leu Glu Ser Trp Arg

65 70 75 80

Thr Leu Ser Pro Thr Trp Lys Lys Val Val Thr Arg Thr Phe Thr Gly

85 90 95

Leu Thr Leu Leu Asp Thr Ile Gln Ala Thr Ile Gly Asp Val Ala Gln

100 105 110

Ile Lys His Ser Leu Thr Asp His Glu Gln Val Ile Tyr Thr Asn Phe

115 120 125

Ala Phe Met Val Gly Val His Ala Arg Ser Tyr Gly Ser Ile Phe Ser

130 135 140

Thr Leu Cys Ser Ser Glu Glu Ile Glu Glu Ala His Asn Trp Val Ile

145 150 155 160

Asn Asn Glu Lys Leu Gln Lys Arg Ala Arg Ile Leu Ile Pro Phe Tyr

165 170 175

Val Gly Glu Asp Pro Leu Lys Ser Lys Val Ala Ala Ala Leu Met Pro

180 185 190

Gly Phe Leu Leu Tyr Gly Gly Phe Tyr Leu Pro Phe Tyr Leu Ser Ala

195 200 205

Arg Ser Lys Leu Pro Asn Thr Ser Asp Ile Ile Arg Leu Ile Leu Arg

210 215 220

Asp Lys Val Ile His Asn Tyr Tyr Ser Gly Tyr Lys Phe Gln Lys Lys

225 230 235 240

Val Glu Lys Leu Ser Pro Glu Lys Gln Asp Glu Ile Lys Lys Phe Thr

245 250 255

Phe Asp Leu Leu Tyr Lys Leu Ile Glu Leu Glu Lys Asp Tyr Leu Tyr

260 265 270

Asp Leu Tyr Ser Glu Val Gly Leu Ala Glu Ser Ala Ile Lys Phe Ser

275 280 285

Ile Tyr Asn Ala Gly Lys Phe Leu Gln Asn Leu Gly Tyr Asp Ser Pro

290 295 300

Phe Ser Lys Glu Glu Thr Glu Ile Glu Pro Glu Ile Phe Ser Gln Leu

305 310 315 320

Ser Ala Arg Ala Asp Glu Asn His Asp Phe Phe Ser Gly Asn Gly Ser

325 330 335

Ser Tyr Val Met Ala Leu Ala Glu Glu Thr Glu Asp Glu Asp Trp Glu

340 345 350

Phe

<210> 10

<211> 942

<212> DNA

<213> NrdF蛋白核苷酸序列(NrdF protein nucleotide sequence)

<400> 10

atgcgatcaa tcaactggaa tatcataaat gatccaaaag atcttgaagt ttggacccga 60

atagtgcaaa atttctggat tccagaaaaa attccgcttt caaatgatct tgaatcctgg 120

agaactttat cacctacctg gaaaaaagta gttactcgca cttttaccgg acttacttta 180

cttgatacaa ttcaggcaac aatcggtgat gtagcccaaa ttaaacactc tttaaccgat 240

catgaacagg taatttatac aaatttcgct tttatggttg gggttcatgc tcgttcttat 300

ggttcaattt tttctaccct ttgctcaagt gaagaaatag aagaggcgca taactgggta 360

atcaataacg aaaaattgca aaaaagggcc agaattctta taccttttta tgtaggtgaa 420

gatcctttaa aatcaaaagt tgctgctgct ttaatgccag gttttctctt atatggtggt 480

ttttatcttc ctttttatct ttcagcgcgt tcaaaactgc caaatacatc cgatattatt 540

cgactgattt tacgcgataa agtgattcat aattattatt caggttataa atttcaaaaa 600

aaagtggaaa aattaagccc ggaaaaacaa gacgaaatta aaaaatttac ctttgatcta 660

ttatataaac taattgaatt agaaaaagat tatctctatg atttatattc tgaagttgga 720

cttgctgaat cagcaataaa atttagcatt tataatgccg ggaaattctt gcaaaatcta 780

ggatatgatt cacctttttc aaaagaggaa accgaaattg aacctgaaat ttttagtcaa 840

ttatcggctc gagctgatga aaatcatgac tttttttcag gaaatggctc ttcttatgta 900

atggcgcttg ctgaagaaac tgaagatgaa gattgggagt tt 942

<210> 11

<211> 1236

<212> DNA

<213> 绿脓杆菌外毒素PEA(ΔⅢ蛋白核苷酸序列)

<400> 11

atgcacctga cccctcactg gattcctctg gtggcctccc tgggactgct ggccggtggc 60

tcattcgctt ccgctgccga ggaggctttc gacctgtgga acgagtgcgc caaggcttgc 120

gtgctggacc tgaaggacgg agtcaggtcc agcagaatgt cagtggaccc tgccatcgct 180

gacactaacg gccagggagt gctgcactac tccatggtcc tggaaggagg taacgacgct 240

ctgaagctgg ccatcgacaa cgctctgagc atcacctctg acggtctgac tatccgcctg 300

gagggcggag tggaaccaaa caagcctgtc cgttactctt acactaggca ggctaggggc 360

agctggtctc tgaactggct ggtcccaatc ggacacgaga agccttctaa catcaaggtg 420

ttcatccacg aactgaacgc tggtaaccag ctgtcacaca tgtcccctat ctacactatc 480

gagatgggcg acgaactgct ggccaagctg gctagggacg ccaccttctt cgtcagagct 540

cacgagagca acgaaatgca gcctactctg gccatcagcc acgctggcgt gtctgtggtc 600

atggctcaag ctcagccaag gagggagaag aggtggtcag aatgggcttc tggcaaggtc 660

ctgtgcctgc tggaccccct ggacggcgtg tacaactacc tggctcagca gaggtgcaac 720

ctggacgaca cctgggaggg caagatctac agagtcctgg ccggcaaccc tgctaagcac 780

gacctggaca tcaagcccac cgtgatctca cacaggctgc acttccctga gggtggctcc 840

ctggctgctc tgactgctca ccaggcctgc cacctgccac tggaaacctt cactaggcac 900

agacagcctc gtggatggga gcagctggaa cagtgcggtt acccagtgca gaggctggtc 960

gccctgtacc tggctgccag actgagctgg aaccaggtgg accaggtcat caggaacgct 1020

ctggcttctc ctggctctgg aggtgacctg ggagaggcta tccgcgagca gccagaacaa 1080

gctaggctcg ctctgaccct ggctgctgct gagtctgaaa ggttcgtcag acagggtact 1140

ggcaacgacg aagccggagc cgcttcagct gacgtggtct ccctgacttg ccctgtggct 1200

gctggagagt gcgctggtcc tgctgactca ggtgac 1236

<210> 12

<211> 599

<212> PRT

<213> Mycoplasma hyopneumoniae 7448株P42蛋白氨基酸序列(Amino acidsequence of P42 protein of Mycoplasma hyopneumoniae 7448 strain)

<400> 12

Met Ala Lys Glu Ile Ile Leu Gly Ile Asp Leu Gly Thr Thr Asn Ser

1 5 10 15

Val Val Ala Ile Ile Glu Asn Gln Lys Pro Val Val Leu Glu Asn Pro

20 25 30

Asn Gly Lys Arg Thr Thr Pro Ser Val Val Ala Phe Lys Asn Asn Glu

35 40 45

Glu Ile Val Gly Asp Ala Ala Lys Arg Gln Leu Glu Thr Asn Pro Glu

50 55 60

Ala Ile Ala Ser Ile Lys Arg Leu Met Gly Thr Asp Lys Thr Val Arg

65 70 75 80

Ala Asn Glu Arg Asp Tyr Lys Pro Glu Glu Ile Ser Ala Lys Ile Leu

85 90 95

Ala Tyr Leu Lys Glu Tyr Ala Glu Lys Lys Ile Gly His Lys Val Thr

100 105 110

Lys Ala Val Ile Thr Val Pro Ala Tyr Phe Asp Asn Ala Gln Arg Glu

115 120 125

Ala Thr Lys Asn Ala Gly Lys Ile Ala Gly Leu Gln Val Glu Arg Ile

130 135 140

Ile Asn Glu Pro Thr Ala Ala Ala Leu Ala Phe Gly Leu Asp Lys Thr

145 150 155 160

Glu Lys Glu Met Lys Val Leu Val Tyr Asp Leu Gly Gly Gly Thr Phe

165 170 175

Asp Val Ser Val Leu Glu Leu Ser Gly Gly Thr Phe Glu Val Leu Ser

180 185 190

Thr Ser Gly Asp Asn His Leu Gly Gly Asp Asp Trp Asp Asn Glu Ile

195 200 205

Val Asn Leu Val Lys Lys Ile Lys Glu Glu Tyr Asp Phe Asp Pro Lys

210 215 220

Ser Asp Lys Met Ala Leu Thr Arg Leu Lys Glu Glu Ala Glu Lys Thr

225 230 235 240

Lys Ile Asn Leu Ser Asn Gln Ser Val Ser Thr Val Ser Leu Pro Phe

245 250 255

Leu Gly Met Gly Lys Asn Gly Pro Ile Asn Val Glu Leu Glu Leu Lys

260 265 270

Arg Ser Glu Phe Glu Lys Met Thr Ala His Leu Ile Asp Arg Thr Arg

275 280 285

Lys Pro Ile Val Asp Ala Leu Lys Gln Ala Lys Ile Glu Ala Ser Asp

290 295 300

Leu Asp Glu Val Leu Leu Val Gly Gly Ser Thr Arg Met Pro Ala Val

305 310 315 320

Gln Ser Met Ile Glu His Thr Leu Asn Lys Lys Pro Asn Arg Ser Ile

325 330 335

Asn Pro Asp Glu Val Val Ala Ile Gly Ala Ala Ile Gln Gly Gly Val

340 345 350

Leu Ala Gly Glu Ile Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu

355 360 365

Thr Leu Gly Ile Glu Thr Leu Gly Gly Ile Ala Thr Pro Leu Ile Pro

370 375 380

Arg Asn Thr Thr Ile Pro Val Thr Lys Ser Gln Ile Phe Ser Thr Ala

385 390 395 400

Glu Asp Asn Gln Thr Glu Val Thr Ile Ser Val Val Gln Gly Glu Arg

405 410 415

Gln Leu Ala Ala Asp Asn Lys Met Leu Gly Arg Phe Asn Leu Ser Gly

420 425 430

Ile Glu Ala Ala Pro Arg Gly Leu Pro Gln Ile Glu Val Ser Phe Ser

435 440 445

Ile Asp Val Asn Gly Ile Thr Thr Val Ser Ala Lys Asp Lys Lys Thr

450 455 460

Gly Lys Glu Gln Thr Ile Thr Ile Lys Asn Thr Ser Thr Leu Ser Glu

465 470 475 480

Glu Glu Ile Asn Lys Met Ile Gln Glu Ala Glu Glu Asn Arg Glu Ala

485 490 495

Asp Ala Leu Lys Lys Asp Lys Ile Glu Thr Thr Val Arg Ala Glu Gly

500 505 510

Leu Ile Asn Gln Leu Glu Lys Ser Ile Thr Asp Gln Gly Glu Lys Ile

515 520 525

Asp Pro Lys Gln Lys Glu Leu Leu Glu Lys Gln Ile Gln Glu Leu Lys

530 535 540

Asp Leu Leu Lys Glu Glu Lys Thr Asp Glu Leu Lys Leu Lys Leu Asp

545 550 555 560

Gln Ile Glu Ala Ala Ala Gln Ser Phe Ala Gln Ala Thr Ala Gln Gln

565 570 575

Ala Asn Thr Ser Glu Ser Asp Pro Lys Ala Asp Asp Ser Asn Thr Ile

580 585 590

Asp Ala Glu Ile Lys Gln Asp

595

<210> 13

<211> 1082

<212> PRT

<213> Mycoplasma hyopneumoniae 7448株P97蛋白氨基酸序列(Amino acidsequence of P97 protein of Mycoplasma hyopneumoniae 7448 strain)

<400> 13

Met Ser Lys Lys Ser Lys Thr Phe Lys Ile Gly Leu Thr Ala Gly Ile

1 5 10 15

Val Gly Leu Gly Val Phe Gly Leu Thr Val Gly Leu Ser Ser Leu Ala

20 25 30

Lys Tyr Arg Ser Glu Ser Pro Arg Lys Ile Ala Asn Asp Phe Ala Ala

35 40 45

Lys Val Ser Thr Leu Ala Phe Ser Pro Tyr Ala Phe Glu Thr Asp Ser

50 55 60

Asp Tyr Lys Ile Val Lys Arg Met Leu Val Asp Ser Asn Asn Asn Ile

65 70 75 80

Arg Asn Lys Glu Lys Val Ile Asp Ser Phe Ser Phe Phe Thr Lys Asn

85 90 95

Gly Asp Gln Leu Glu Lys Ile Asn Phe Gln Asp Pro Glu Tyr Thr Lys

100 105 110

Ala Lys Ile Thr Phe Glu Ile Leu Glu Ile Ile Pro Asp Asp Val Asn

115 120 125

Gln Asn Phe Lys Val Lys Phe Gln Ala Leu Gln Lys Leu His Asn Gly

130 135 140

Asp Ile Ala Lys Ser Asp Ile Tyr Glu Gln Thr Val Ala Phe Ala Lys

145 150 155 160

Gln Ser Asn Leu Leu Val Ala Glu Phe Asn Phe Ser Leu Lys Lys Ile

165 170 175

Thr Glu Lys Leu Asn Gln Gln Ile Glu Asn Leu Ser Thr Lys Ile Thr

180 185 190

Asn Phe Ala Asp Glu Lys Thr Ser Ser Gln Lys Asp Pro Ser Thr Leu

195 200 205

Arg Ala Ile Asp Phe Gln Tyr Asp Leu Asn Thr Ala Arg Asn Pro Glu

210 215 220

Asp Leu Asp Ile Lys Leu Ala Asn Tyr Phe Pro Val Leu Lys Asn Leu

225 230 235 240

Ile Asn Arg Leu Asn Asn Ala Pro Glu Asn Lys Leu Pro Asn Asn Leu

245 250 255

Gly Asn Ile Phe Glu Phe Ser Phe Ala Lys Asp Ser Ser Thr Asn Gln

260 265 270

Tyr Val Ser Ile Gln Asn Gln Ile Pro Ser Leu Phe Leu Lys Ala Asp

275 280 285

Leu Ser Gln Ser Ala Arg Glu Ile Leu Ala Ser Pro Asp Glu Val Gln

290 295 300

Pro Val Ile Asn Ile Leu Arg Leu Met Lys Lys Asp Asn Ser Ser Tyr

305 310 315 320

Phe Leu Asn Phe Glu Asp Phe Val Asn Asn Leu Thr Leu Lys Asn Met

325 330 335

Gln Lys Glu Asp Leu Asn Ala Lys Gly Gln Asn Leu Ser Ala Tyr Glu

340 345 350

Phe Leu Ala Asp Ile Lys Ser Gly Phe Phe Pro Gly Asp Lys Arg Ser

355 360 365

Ser His Thr Lys Ala Glu Ile Ser Asn Leu Leu Asn Lys Lys Glu Asn

370 375 380

Ile Tyr Asp Phe Gly Lys Tyr Asn Gly Lys Phe Asn Asp Arg Leu Asn

385 390 395 400

Ser Pro Asn Leu Glu Tyr Ser Leu Asp Ala Ala Ser Ala Ser Leu Asp

405 410 415

Lys Lys Asp Lys Ser Ile Ile Leu Ile Pro Tyr Arg Leu Glu Ile Lys

420 425 430

Asp Lys Phe Phe Ala Asp Asp Leu Tyr Pro Asp Thr Lys Asp Asn Ile

435 440 445

Leu Val Lys Glu Gly Ile Leu Lys Leu Thr Gly Phe Lys Lys Gly Pro

450 455 460

Lys Ile Asp Leu Pro Asn Ile Asn Gln Gln Ile Phe Lys Thr Glu Tyr

465 470 475 480

Leu Pro Phe Phe Glu Lys Gly Lys Glu Glu Gln Ala Lys Leu Asp Tyr

485 490 495

Gly Asn Ile Leu Asn Pro Tyr Asn Thr Gln Leu Ala Lys Val Glu Val

500 505 510

Glu Ala Leu Phe Lys Gly Asn Lys Asn Gln Glu Ile Tyr Gln Ala Leu

515 520 525

Asp Gly Asn Tyr Ala Tyr Glu Phe Gly Ala Phe Lys Ser Val Leu Asn

530 535 540

Ser Met Thr Gly Lys Ile Gln His Pro Glu Lys Ala Asp Ile Gln Arg

545 550 555 560

Phe Thr Arg His Leu Glu Gln Val Lys Ile Ser Ser Asn Ser Val Leu

565 570 575

Asn Gln Pro Gln Thr Thr Lys Glu Gln Val Ile Ser Ser Leu Lys Ser

580 585 590

Asn Asn Phe Phe Lys Asn Gly His Gln Val Ala Ser Tyr Phe Gln Asp

595 600 605

Leu Leu Thr Lys Asp Lys Leu Thr Val Leu Glu Thr Leu Tyr Asp Leu

610 615 620

Ala Lys Lys Trp Gly Leu Glu Thr Asn Ser Ala Gln Phe Pro Lys Gly

625 630 635 640

Ala Phe Gln Tyr Thr Lys Asp Ile Phe Ala Glu Ala Asp Lys Leu Glu

645 650 655

Phe Leu Glu Trp Lys Lys Lys Asp Pro Tyr Asn Gln Ile Lys Glu Ile

660 665 670

His Gln Leu Ser Phe Asn Ile Leu Ala Arg Asn Asp Val Ile Lys Ser

675 680 685

Asp Gly Phe Tyr Gly Val Leu Leu Leu Pro Gln Ser Val Lys Thr Glu

690 695 700

Leu Glu Gly Lys Asn Glu Ala Gln Ile Phe Glu Ala Leu Lys Lys Tyr

705 710 715 720

Ser Leu Ile Glu Asn Ser Ala Phe Lys Thr Thr Ile Leu Asp Lys Asn

725 730 735

Leu Leu Glu Gly Thr Asp Phe Lys Thr Phe Gly Asp Phe Leu Lys Ala

740 745 750

Phe Phe Leu Lys Ala Ala Gln Phe Asn Asn Phe Ala Pro Met Ala Lys

755 760 765

Leu Asp Asp Asn Leu Gln Tyr Ser Phe Glu Ala Ile Lys Lys Gly Glu

770 775 780

Thr Thr Lys Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys Glu

785 790 795 800

Leu Asp Asn Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala Lys

805 810 815

Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro

820 825 830

Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu

835 840 845

Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala

850 855 860

Ala Lys Pro Val Ala Thr Asn Thr Asn Thr Asn Thr Gly Phe Ser Leu

865 870 875 880

Thr Asn Lys Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr Lys

885 890 895

Leu Glu Tyr Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu Ile

900 905 910

Asn Val Phe Leu Glu Leu Val His Gln Ser Glu Tyr Glu Glu Gln Lys

915 920 925

Ile Ile Lys Glu Leu Asp Lys Thr Val Leu Asn Leu Gln Tyr Gln Phe

930 935 940

Gln Glu Val Lys Val Ala Ser Asp Gln Tyr Gln Lys Leu Ser His Pro

945 950 955 960

Met Met Thr Glu Gly Ser Ser Asn Gln Gly Lys Lys Gly Glu Gly Thr

965 970 975

Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro Ser Gln Gly Lys Lys

980 985 990

Ala Glu Gly Thr Ser Asn Gln Gln Asn Thr Thr Thr Glu Leu Thr Asn

995 1000 1005

Tyr Leu Pro Glu Leu Gly Lys Lys Ile Asp Glu Ile Ile Lys Lys Gln

1010 1015 1020

Gly Lys Asn Trp Lys Thr Glu Val Glu Leu Ile Glu Asp Asn Ile Ala

1025 1030 1035 1040

Gly Asp Ala Lys Leu Leu Tyr Phe Val Leu Arg Asp Asp Ser Lys Ser

1045 1050 1055

Gly Asp Pro Lys Lys Ser Ser Leu Lys Val Lys Ile Thr Val Lys Gln

1060 1065 1070

Ser Asn Asn Asn Gln Glu Leu Lys Ser Lys

1075 1080

<210> 14

<211> 1147

<212> PRT

<213> Mycoplasma hyopneumoniae 7448株P95蛋白氨基酸序列(Amino acidsequence of P95 protein of Mycoplasma hyopneumoniae 7448 strain)

<400> 14

Met Lys Ile Val Lys Lys Ile Lys Lys Thr Lys Tyr Leu Thr Arg Lys

1 5 10 15

Ser Lys Ile Leu Leu Gly Leu Gly Leu Ser Ser Ser Ile Leu Gly Ile

20 25 30

Leu Gly Ile Ser Val Ala Val Ser Tyr Gly Leu Ala Val Ile Lys Lys

35 40 45

Asn Ser Tyr Asn Thr Thr Ile Ala Asp Leu Asn Arg Leu Ala Gln Lys

50 55 60

Ile Asn Gly Leu Ser Phe Asn Ala Gln Lys Ile Ser Pro Phe Ser Thr

65 70 75 80

Tyr Ala Ser Leu Lys Lys Glu Trp Lys Asn Leu Glu Ser Ser Glu Lys

85 90 95

Ser Gly Glu Phe Phe Asp Phe Tyr Thr Leu Asn Tyr Lys Arg Leu Gln

100 105 110

Pro Tyr Lys Leu Pro Asn Gly Ile Trp Ala Glu Phe Leu Lys Val Glu

115 120 125

Pro Asp Asp Ala Asn Gln Gln Phe Asn Val Glu Phe Val Leu Lys Ser

130 135 140

Phe Asn Gly Ser Arg Val Ile Lys Ser Asp Ile Lys Ser Glu Lys Val

145 150 155 160

Ala Ile Ser Pro Asn Ser Thr Phe Phe Leu Glu Asn Phe Tyr Gln Ala

165 170 175

Leu Glu Ile Asp Leu Lys Asn Ile Ser Pro Tyr Ser Asn Gly Asp Lys

180 185 190

Gly Lys Lys Asn Pro Lys Asn Trp Leu Ala Ser Asp Phe Leu Thr Glu

195 200 205

Ile Asn Ser Arg Glu Thr Ala Gln Asp Ala Ile Lys Arg Ile Arg Asp

210 215 220

Phe Phe Asn Phe Asp Phe Asp Ser Val Leu Lys Asn Lys Asn Phe Ala

225 230 235 240

Ile Lys Tyr Lys Asp Asn Leu Ile Phe Pro Tyr Lys Ile Glu Ile Leu

245 250 255

Lys Asn Asp Asp Asp Ser Trp Ile Lys Pro Ser Gln Leu Asn Pro Asp

260 265 270

Phe Leu Glu Ile Gln Gly Arg Val Ser Phe Thr Asp Gln Ala Lys Lys

275 280 285

Leu Phe Pro Lys Ser Phe Asn Thr Asn Ile Thr Lys Asn Phe Ser Phe

290 295 300

Leu Leu Phe Asp Leu Ala Leu Asn Lys Ser Ala Phe Ala Asp Pro Lys

305 310 315 320

Val Phe Ile Arg Ile Pro Lys Leu Val Glu Pro Lys Ile Asp Glu Phe

325 330 335

Ser Ser Glu Asn Pro Gln Glu Lys Ile Asp Leu Ser Gln Lys Ser Ile

340 345 350

Phe Trp Ile Tyr Asn Phe Leu Lys Tyr Lys Glu Asp Asn Ile Ser Leu

355 360 365

Lys Thr Pro Glu Glu Ala Lys Asn Ala Leu Asn Ser Leu Leu Asn Asn

370 375 380

Asp Leu Glu Leu Asp Phe Ser Gln Asn Asn Asn Leu Glu Pro Asn Ile

385 390 395 400

Lys Glu Lys Phe Glu Tyr Gln Leu Leu Val Asp Lys Ile Asn Phe Asn

405 410 415

Ser Asp Gln Asn Ser Ser Phe Ile Asn Ile Pro Phe Glu Ile Ser Tyr

420 425 430

Pro Leu Asp Ser Glu Lys Lys Asn Lys Leu Lys Thr Glu Thr Gln Val

435 440 445

Leu Leu Arg Lys Phe Lys Asn Ser Ser Ser Leu Asp Ser Thr Val Phe

450 455 460

Asp Pro Lys Asn Phe Ser Ser Leu Pro Ile Val Asn Leu Lys Phe Ile

465 470 475 480

Ser Glu Lys Asn Arg Asp Lys Asp Asp Ile Phe Glu Ala Phe Glu Thr

485 490 495

Val Ser Lys Phe Glu Leu Glu Arg Leu Leu Asn Leu Asn Gln Gln Gln

500 505 510

Gln Ile Tyr Gln Ile Leu Thr Asp Pro Thr Lys Phe Asn Leu Thr Phe

515 520 525

Pro Glu Lys Glu Ile Leu Asp Ala Trp Ile Ser Ser Tyr Asn Phe Pro

530 535 540

Ser Val Gln Glu Phe Ser Arg Arg Thr Leu Val Ala Lys Lys Leu Glu

545 550 555 560

Asp Gly Ser Gln Ser Arg Pro Phe Phe Glu Asn Ser Arg Glu Phe Ile

565 570 575

Ala Phe Thr Lys Lys Ile Leu Ser Leu Glu Lys Ala Glu Ala Lys Lys

580 585 590

Tyr Ile Glu Pro Phe Phe Glu Ala Leu Lys Thr Glu Ile Ser Glu Lys

595 600 605

Glu Ser Lys Lys Leu Glu Lys Glu Pro Thr Asn Leu Asn Gln Lys Thr

610 615 620

Glu Lys Asn Ser Ser Thr Thr Ala Ile Ser Ser Glu Lys Asn Glu Lys

625 630 635 640

Pro Glu Lys Ser Met Ser Glu Thr Pro Pro Glu Leu Val Lys Val Gln

645 650 655

Gln Asn Gln Pro Lys Thr Glu Glu Glu Pro Lys Gln Glu Thr Asp Lys

660 665 670

Gln Glu Lys Pro Asn Asn Gln Lys Asn Thr Glu Gln Ala Lys Glu Ser

675 680 685

Lys Glu Lys Glu Lys Asn Gly Asn Ser Ser Leu Lys Ser Pro Asn Leu

690 695 700

Pro Val Glu Lys Ser Ser Asp Ser Gln Glu Glu Gly Gln Lys Thr Asp

705 710 715 720

Arg Leu Gln Gln Glu Asn Glu Gln Ala Ala Phe Asn Asp Lys Ile Val

725 730 735

Ser Ile Leu Gln Ser Gln Tyr Glu Ile Ser Leu Ile Lys Asn Leu Thr

740 745 750

Asn Gln Ile Glu Ala Leu Lys Asn Ser Pro Glu Lys Gln Lys Ala Thr

755 760 765

Leu Asp Ala Ala Ser Phe Thr Asp Leu Phe Ile Glu Thr Tyr Ile Asn

770 775 780

Asn Asp Leu Val Ser Gln Phe Asp Ser Phe Ala Ala Gly Leu Asn Tyr

785 790 795 800

Lys Ile Val Phe Val Ala Asn Gln Asp Asp Glu Ile Ile Asp Gln Val

805 810 815

Gln Leu Pro Glu Pro Glu Lys Ser Lys Lys Thr Glu Lys Thr Pro Asp

820 825 830

Phe Lys Glu Glu Ser Glu Lys Lys Glu Leu Glu Asn Lys Glu Asn Ser

835 840 845

Pro Ala Val Asp Gln Lys Ser Asp Gln Lys Ile Ser Leu Ser Phe Phe

850 855 860

Gln Glu Glu Lys Asn Asn Thr Gln Asn Gln Gly Gln Glu Glu Ser Asp

865 870 875 880

Lys Lys Gln Gln Thr Pro Lys Thr Ala Gln Glu Asn Ser Gln Asn Leu

885 890 895

Val Gln Gln Pro Lys Pro Glu Gln Glu Thr Glu Ile Phe Lys Leu Gly

900 905 910

Tyr Tyr Tyr Ile Phe Thr Ala Pro Asn Ser Glu Lys Ile Val Phe Arg

915 920 925

Thr Pro Ile Lys Ser Leu Lys Leu Lys Val Phe Leu Ala Glu Lys Gly

930 935 940

Gly Ile Thr Leu Glu Lys Leu Ser Tyr Asn Met Leu Asn Phe Pro Gln

945 950 955 960

Ser Leu Leu Lys Leu Glu Leu Ala Asp Ser Asn Phe Thr Ser Ala Glu

965 970 975

Ala Leu Lys Lys Gln Pro Glu Asp Ile Leu Lys Ala Glu Phe Ser Ser

980 985 990

Gln Asp Lys Asp Phe Lys Ala Thr Met Thr Ser Phe Lys Lys Leu Phe

995 1000 1005

Gly Asn Lys Thr Phe Met Lys Ile Tyr Pro Leu Leu Ser Gly Asn Gly

1010 1015 1020

Leu Ile Tyr Lys Ala Asn Ser Val Phe Lys Asp Lys Phe Gly Asn Leu

1025 1030 1035 1040

Lys Ile Arg Phe Ala Val Lys Asp Leu Asp Ala Ser Glu Lys Lys Gln

1045 1050 1055

Ile Val Phe Pro Asn Ile Leu Glu Pro Glu Leu Glu Lys Lys Glu Gln

1060 1065 1070

Asp Val Arg Pro Lys Ala Thr Glu Ala Glu Ser Lys Gln Glu Lys Gln

1075 1080 1085

Glu Lys Gln Glu Lys Ser Gln Leu Asn Ser Glu Ser Ser Gln Thr Ser

1090 1095 1100

Thr Thr Ser Ser Gln Asn Gln Glu Asn Asn Gln Lys Asn Gln Leu Glu

1105 1110 1115 1120

Ile Phe Lys Pro Ala Glu Lys Glu Ala Lys Tyr Pro Leu Val Phe Thr

1125 1130 1135

Val Ile Lys Pro Asn Arg Gln Phe Arg Arg Asn

1140 1145

<210> 15

<211> 419

<212> PRT

<213> Mycoplasma hyopneumoniae 7448株P46蛋白氨基酸序列(Amino acidsequence of P46 protein of Mycoplasma hyopneumoniae 7448 strain)

<400> 15

Met Lys Lys Met Leu Arg Lys Lys Phe Leu Tyr Ser Ser Ala Ile Tyr

1 5 10 15

Ala Thr Ser Leu Ala Ser Ile Ile Ala Phe Val Ala Ala Gly Cys Gly

20 25 30

Gln Thr Glu Ser Gly Ser Thr Ser Asp Ser Lys Pro Gln Ala Glu Thr

35 40 45

Leu Lys His Lys Val Ser Asn Asp Ser Ile Arg Ile Ala Leu Thr Asp

50 55 60

Pro Asp Asn Pro Arg Met Ile Ser Ala Gln Lys Asp Ile Ile Ser Tyr

65 70 75 80

Val Asp Glu Thr Glu Ala Ala Thr Ser Thr Ile Thr Lys Asn Gln Asp

85 90 95

Ala Gln Asn Asn Met Leu Thr Gln Gln Ala Asn Leu Ser Pro Ala Pro

100 105 110

Lys Gly Phe Ile Ile Ala Pro Glu Asn Gly Ser Gly Val Gly Thr Ala

115 120 125

Val Asn Thr Ile Ala Asp Lys Gly Ile Pro Ile Val Ala Tyr Asp Arg

130 135 140

Leu Ile Thr Gly Ser Asp Lys Tyr Asp Trp Tyr Val Ser Phe Asp Asn

145 150 155 160

Glu Lys Val Gly Glu Leu Gln Gly Leu Ser Leu Ala Ala Gly Leu Leu

165 170 175

Gly Lys Glu Asp Gly Ala Phe Asp Ser Ile Asp Gln Met Asn Glu Tyr

180 185 190

Leu Lys Ser His Met Pro Gln Glu Thr Ile Ser Phe Tyr Thr Ile Ala

195 200 205

Gly Ser Gln Asp Asp Asn Asn Ser Gln Tyr Phe Tyr Asn Gly Ala Met

210 215 220

Lys Val Leu Lys Glu Leu Met Lys Asn Ser Gly Asn Lys Ile Ile Asp

225 230 235 240

Leu Ser Pro Glu Gly Glu Asn Ala Val Tyr Val Pro Gly Met Asn Tyr

245 250 255

Gly Thr Ala Gly Gln Arg Ile Gln Ser Phe Leu Thr Ile Asn Lys Asp

260 265 270

Pro Ala Gly Gly Asn Lys Ile Lys Ala Ile Gly Ser Lys Pro Ala Ser

275 280 285

Ile Phe Lys Gly Phe Leu Ala Pro Asn Asp Gly Met Ala Glu Gln Ala

290 295 300

Ile Thr Lys Leu Lys Leu Glu Gly Phe Asp Thr Gln Lys Ile Phe Val

305 310 315 320

Thr Gly Gln Asp Tyr Asn Asp Lys Ala Lys Thr Phe Ile Lys Asp Gly

325 330 335

Asp Gln Asn Met Thr Ile Tyr Lys Pro Asp Lys Val Leu Gly Lys Val

340 345 350

Ala Val Glu Val Leu Arg Val Leu Ile Ala Lys Lys Asn Lys Ala Ser

355 360 365

Arg Ser Glu Val Glu Asn Glu Leu Lys Ala Lys Leu Pro Asn Ile Ser

370 375 380

Phe Lys Tyr Asp Asn Gln Thr Tyr Lys Val Gln Gly Lys Asn Ile Asn

385 390 395 400

Thr Ile Leu Val Ser Pro Val Ile Val Thr Lys Ala Asn Val Asp Asn

405 410 415

Pro Asp Ala