基因,病毒

摘要： Objective To investigate the relationship between HBV -DNA load and serum markers in chronic hepatitis B( CHB) patients in Hohhot,Inner Mongolia,and to explore the mutation of HBV genotype and nucleoside analogue.Methods From January 2015 to December 2017,one hundred and ninety-three CHB patients hospitalized in the People＇s Hospital of Inner Mongolia were selected randomly.The clinical diagnostic criteria for all admitted patients were based on the " Guidelines for the Prevention and Treatment of Chronic Hepatitis B" jointly formulated by the Infectious Diseases Society of 2010. The HBV -DNA load of HBV was detected by real -time quantitative PCR,and the correlation between HBV -DNA load and serum markers was analyzed. Seventy -nine patients were selected from 193 hospitalized patients,PCR-reverse dot blot hybridization was used to analyze HBV genotyping and the drug resistance mutations of different genotypes.Results The differences of HBeAb level and HBV-DNA load between HBeAg positive patients and negative patients were statistically significant(all P<0.001). Of 79 serum specimens of HBV infected people,9 cases(11.4% ) were B genotypes,and 70 cases of C genotype (88.6% ).Of them,25 cases had different loci variation,the rate of variation was 31.6% (25/79),with the unit point rtS213T mutation dominated,accounting for about 24.0% (6/25).Conclusion In Hohhot Inner Mongolia patients with CHB,HBV-DNA load with HBeAg and HBe Ab level are correlated;genotype in patients including B type and C type,which is mainly genotype C;patients with CHB mainly had drug resistance to lamivudine and adefovir dipivoxil, mutations including rtS213T,and hybrid mutation.%目的 探讨内蒙古自治区呼和浩特地区慢性乙型肝炎患者病毒脱氧核糖核酸( HBV-DNA)载量与血清标志物水平间的关系,基因分型与核苷(酸)类似物耐药突变情况.方法 选取内蒙古自治区人民医院2015年1月至2017年12月确诊的慢性乙型肝炎患者193例为研究对象,采用实时荧光定量聚合酶链式反应(PCR)法检测HBV-DNA载量,分析其与血清学标志物的相关性.并从193例患者中选取79例采用PCR-反向点杂交法检测HBV基因分型和耐药突变,分析不同基因型患者的耐药突变情况.结果 e抗原阳性和阴性患者的e抗体水平与HBV-DNA载量比较差异均有统计学意义(均P<0.001). 79例HBV感染者中,B基因型9例(11.4% ),C基因型70 例(88.6% ).其中有25 例发生不同位点变异,变异率为31.6% (25/79),以单位点rtS213T突变为主,约占24.0% (6/25).结论 呼和浩特地区慢性乙型肝炎患者的HBV-DNA载量与e抗原、e抗体水平相关;基因型主要为B和C型,以C基因型为多;慢性乙型肝炎患者主要对拉米夫定、阿德福韦酯耐药,突变以rtS213T为主,也有混合位点的突变.

摘要： [目的]探讨上海地区98例手足口病患儿肠道病毒71型(EV71)的基因型的分布情况.[方法]选取2017年1月至2018年5月于本院诊断为手足口病患儿的98份粪便标本,采用反转录半巢式PCR(RT-nPCR)方法对标本中EV71进行检测.[结果]98份标本中28份为阳性EV71 RNA,检出率为28.6％;测序结果表明此28份阳性标本为EV71 C4型.[结论]上海地区98例手足口病病原体为C型EV71病毒;不同患儿间的EV71病毒有一定的变异.

摘要： Objective To investigate the pathogen spectrum and molecular epidemiological characteristics of hand,foot and mouth disease (HFMD) in Shenzhen from 2010 to 2012 and to provide scientific basis for HFMD control.Methods A total of 1 523 clinical stool specimens or anal swab from the sentinel surveillance systems of HFMD were obtained.Molecular evolutions of VP1 gene of causative agents were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and reverse transcription semi nested PCR(RT-snPCR),which analyzed the molecular evolution of VP1.The data were analyzed by chi-square test.Results Children under two years old were in high incidence of HFMD,followed by 2-3 years old group.The largest number of HFMD cases from 2010 to 2012 was in Baoan and Longgang districts.There were significant differences between male and female among all age groups (x2=12.365,P=0.030).Totally 1 148 of 1 523 (75.4％) HFMDcases from 2010 to 2012 were found positive for human enterovirus and twenty-eight different genotypes were detected,including Enterovirus (EV71)71,Coxsakievirus (Cox) group A and B,and Echovirus.EV71 (57.1％),CoxA6 (18.5％),CoxA16 (14.0％) and CoxA10 (3.7％) were the top four pathogens,enterovirus group A was the most common (96.2％).CoxA6 strains isolated from Shenzhen were genetically related to those from Finland 2008,France 2010 and Japan 2011,Shenzhen CoxA10 strains showed closest genetic relationship to Slovakia 2007 and Spain 2008.Conclusions The pathogen spectrum of Shenzhen HFMD from 2010 to 2012 are EV71,CoxA6,CoxA16 and CoxA10.The prevalence of Shenzhen CoxA6 ranks second major pathogen of HFMD which may be originated from European countries and Japan.Baoan and Longgang district,locate in the north of Shenzhen are the main distribution area.High incidence of HFMD is 1-2 years old group.%目的 了解2010年至2012年深圳市手足口病病原构成、分子特征和流行病学规律,为制订防制措施提供科学依据.方法 收集2010年至2012年深圳市手足口病疑似患者粪便或肛拭子标本1 523份,实时荧光定量PCR和反转录半套式PCR (RT-snPCR)法进行VP1基因系统进化分析,数据分析行x2检验.结果 手足口病发病以2岁以下儿童居多,其次为2～3岁组;宝安和龙岗区患者数最多;各年龄组性别比较差异有统计学意义(x2=12.365,P=0.030).2010年至2012年1 523例手足口病患者病原学分析,肠道病毒阳性1 148例,占75.4％,包括肠道病毒71型(EV71)、柯萨奇病毒(Cox)A组和B组、埃可病毒等28种病原;居前4位分别为EV71 (57.1％)、CoxA6(18.5％)、CoxA16 (14.0％)和CoxA10(3.7％),以A组人肠道病毒(HEV-A)最为普遍(96.2％).深圳市CoxA6毒株与2008年芬兰、2010年法国和2011年日本毒株亲缘性较近,CoxA10与2007年斯洛伐克、2008年西班牙毒株同源性最高.结论 2010年至2012年深圳市手足口病病原谱以HEV-A组的EV71、CoxA6、CoxA16和CoxA10为主,CoxA6是继EV71之后的第2位主要病原体,深圳市CoxA6毒株可能起源于欧洲和日本.位于深圳北部的宝安区和龙岗区是手足口病的主要分布地区;＜2岁是手足口病高发年龄段.

摘要： Objective To analyze the association between rtA181S mutation in the reverse-transcriptase (RT) domain of HBV and adefovir dipivoxil (ADV) resistance. Methods Incidence of rtA181S mutation of HBV RT region was screened and analyzed from large samples of chronically HBV-infected patients by direct sequencing. Clonal sequencing was performed and drug-resistance-associated mutations were analyzed from the serum of an ADV-refractory patient with chronic hepatitis B. pGEM-Teasy RT plasmid and pTriEx-HBV (C) plasmid were digested and ligated by the restriction enzyme ( XhoⅠ/SphⅠ), and the 1.1-ploid genome length HBV recombinant plasmids harboring wild-type and three kinds of mutants in RT region were constructed. Then the replication-competent constructs were transiently transfected into the HepG2 cells. Five hours post-transfection, new medium containing different concentrations of ADV (0, 0.033, 0.100, 0.330, 1.000, 3.300 μmol/L) was supplemented every other day for 4 days. To analyze the phenotypic characteristics of the HBV mutants under the drug pressure, the supernatant was collected and HBV DNA production was quantitated using real-time PCR. Results A total of 12 000 serum samples of 9830 patients with chronic HBV infection were screened. rtA181S mutation was detected in 46 nucleos(t)ide analogues-treated patients. By contrast, signature ADV-resistance mutations rtN236T and A181V were detected in 653 patients. The rtA181S emerged either alone or in concomitance with other drug-resistance mutations. A follow-up patient developed virological and biochemical breakthrough after 19-month ADV monotherapy. At the time of sampling, rtA181S+N236T was detected by direct sequencing. Clonal sequence analysis of 24 clones showed that there were 11 (45.83%) for wild-type, 6 (25.00%) for rtN236T, 5 (20.83%) for rtA181S, 1 (4.16%) for rtA181V, and 1 (4.16%) for rtA181S+N236T. The relative viral replication capacity of rtN236T, rtA181S and rtA181S+N236T mutants was 91 . 35%, 29.90% and 68.53% that of the wild-type strain, respectively. Phenotypic resistance analysis showed that ADV suscep-tibility of mutants harboring rtN236T, rtA181S and rtA181S+N236T was 1/4.41, 1/3.05, and 1/5.43 that of the wild-type strain. Conclusions rtA181S is an ADV-resistance-associated mutation, which may cause the drug resistance solely or with other mutations in patients with chronic HBV infection. Compared to signature ADV-resistance mutation, rtA181S mutation is less resistant and less frequently detected in ADV-resistance patients.%目的：分析HBV反转录酶（RT）区rtA181S变异与阿德福韦酯（ADV）耐药的相关性。方法应用直接测序法筛选分析大样本慢性HBV感染者rtA181S变异的检出频率，并对1例接受ADV单药治疗失败的慢性乙型肝炎患者血清中HBV RT区基因进行克隆测序，分析相关变异形式。用XhoⅠ和SphⅠ双酶切pGEM-Teasy RT及pTriEx-HBV(C)载体后再连接，构建1.1倍HBV野生株和耐药株的重组质粒，转染人肝癌细胞系HepG2细胞，5 h后分别加入不同浓度的 ADV（0、0.033、0.100、0.330、1.000、3.300μmol/L）。隔天换药，4 d后收集细胞上清，采用实时荧光定量PCR法检测不同药物浓度作用下细胞培养上清中的HBV DNA载量，并分析其表型耐药特点。结果9830例慢性HBV感染者的12000个血清样本中，有46例样本检出rtA181S变异（单独或与其他耐药变异联合出现），在653例中检出经典的rtN236T/A181V变异。其中随访的1例在接受ADV治疗19个月后出现了病毒学和生化学突破，直接测序检出rtA181S+N236T变异，克隆测序分析显示在24个克隆中11个（45.83%）为野生型，6个（25.00%）为rtN236T变异型，5个（20.83%）为rtA181S变异型，1个（4.16%）为rtA181V变异型，1个（4.16%）为rtA181S+N236T变异型。在体外实验中，rtN236T、rtA181S和rtA181S+N236T变异株的相对复制力分别是野生株的91.35%、29.90%和68.53%。表型耐药分析显示rtN236T、rtA181S和rtA181S+N236T变异株对ADV的灵敏性分别为野生株的1/4.41、1/3.05和1/5.43。结论 rtA181S是一种ADV耐药相关变异，可单独或联合其他变异引起患者耐药。但与经典ADV耐药变异相比，rtA181S变异引起的ADV耐药相对较弱，临床检出率较低。

摘要： Objective To explore the correlation between serum hepatitis B virus (HBV) X antigen/antibody (HBxAg-wild/HBxAb-wild,and HBxAg-mutant/HBxAb-mutant) and the disease progression in patients with chronic HBV infection.Methods A direct enzyme immunosorbent asssay (ELISA) was performed to detect HBxAb using recombinant antigen,and a double antibody sandwich ELISA assay to detect HBxAg using monoclonal antibody and specific rabbit polyclonal antibody.HBxAg-wild/HBxAb-wild and HBxAg-mutant/HBxAb-mutant were tested in sera from cases at different stages of chronic HBV infection.A chi-square test was employed to examine statistical significance.Results The positive rates of HBxAg-wild and HBxAg-mutant in the chronic asymptomatic HBV carriers,chronic hepatitis,hepatitis B-related cirrhosis and liver cancer were 6.2％ (2/32),10.7％ (3/28),28.6％ (6/21),43.6％ (17/39) and 3.1％ (1/32),10.7％ (3/28),33.3％ (7/21),48.7％ (19/39),respectively.The positive rates of HBxAb-wild and HBxAb-mutant in the above mentioned groups were 6.2％ (2/32),21.4％ (6/28),38.1％ (8/21),53.8％ (21/39)and 6.2％ (2/32),25.0％ (7/28),42.9％ (9/21),61.5％ (24/39) respectively.The positive rates of HBxAg-wild and HBxAg-mutant were not significantly different among the above groups (χ2 =0.871,0.780,0.565 and 0.317,respectively; all P＞0.05) ; The positive rates of HBxAb-wild and HBxAb-mutant were also similar among all the groups (χ2 =0.780,0.709,0.580 and 0.210,respectively; all P＞0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in patients with low viral loads (HBV DNA＜1 × 104 copy/mL) were 36.5％ (23/63),44.4％ (28/63),42.9％ (27/63) and 54.0％ (34/63),respectively,those in patients with high viral loads (HBVDNA≥1×104 copy/mL) were 8.8％ (5/57),15.8％ (9/57),5.3％ (3/57) and 14.0％ (8/57),respectively.The positive rates of HBxAg and HBxAb were significantly higher in cases with low viral loads than those with high viral loads (χ2 =12.869,11.522,22.556 and 20.976,respectively; all P＜0.05).The positive rates of HBxAg-wild,HBxAb-wild,HBxAg-mutant,HBxAb-mutant in the HBeAg positive group were 21.7％ (18/83),30.1％ (25/83),22.9％ (19/83) and 32.5％ (27/83),respectively,while those in the HBeAg negative group were 27.0％ (10/37),32.4％ (12/37),29.7％ (11/37) and 40.5％ (15/37),respectively.No significant difference of HBxAg/HBxAb positive rates between HBeAg positive group and HBeAg negative group was noticed (χ2 =0.408,0.064,0.638 and 0.722,respectively; all P＞0.05).Conclusions The antigenicity and specificity of HBV X protein remains similar after the occurrence of A1762T/G1764A double mutant in X gene.It is also found that the positive rates of HBxAg and HBxAb increase with disease progression.HBxAg/HBxAb might be promoting factors for tumorigenesis in chronic HBV infection.HBxAg and HBxAb might have negative influence on HBV replication.%目的 探讨HBV慢性感染者血清中野生型及A1762T/G1764A双突变型HBV X抗原/抗体(HBxAg-w、HBxAb-w和HBxAg-m、HBxAb m)与HBV慢性感染者疾病进展的相关性.方法 应用重组抗原创建检测HBxAb的直接ELISA法,应用单克隆抗体和特异性兔多克隆抗体创建检测HBxAg的双抗体夹心ELISA法；对不同临床分期HBV慢性感染者血清中的X抗原/抗体进行检测和比较.数据比较采用∥检验.结果 HBxAg-w和HBxAg-m在慢性无症状HBV携带者、慢性乙型肝炎、乙型肝炎相关性肝硬化和肝细胞癌组血清中的阳性率分别为6.2％(2/32)、10.7％(3/28)、28.6％(6/21)、43.6％(17/39)和3.1％(1/32)、10.7％(3/28)、33.3％(7/21)、48.7％(19/39),HBxAb w和HBxAb-m在慢性无症状HBV携带者、慢性乙型肝炎、乙型肝炎相关性肝硬化和肝细胞癌组血清中的阳性率分别为6.2％(2/32)、21.4％(6/28)、38.1％(8/21)、53.8％(21/39)和6.2％(2/32)、25.0％(7/28)、42.9％(9/21)、61.5％(24/39),每组患者HBxAg w与HBxAg m阳性率的差异(x2值分别为0.871、0.780、0.565、0.317)以及HBxAb-w与HBxAb-m阳性率的差异(x2值分别为0.780、0.709、0.580、0.210)均无统计学意义(均P＞0.05).低病毒载量组(HBVDNA＜1×101拷贝/mL)HBxAgw、HBxAbw、HBxAg-m、HBxAb-m阳性率分别为36.5％(23/63)、44.4％(28/63)、12.9％ (27/63)、54.0％ (34/63),高病毒载量组(HBV DNA≥1×104拷贝/mL)HBxAg w、HBxAb w、HBxAg-m、HBxAb-m阳性率分别为8.8％(5/57)、15.8％ (9/57)、5.3％(3/57)、14.0％(8/57),低病毒载量组HBxAg w、HBxAb-w、HBxAg-m、HBxAb-m阳性率显著高于高病毒载量组(x2值分别为12.869、11.522、22.556、20.976,均P＜0.05)；HBeAg阳性组HBxAgw、HBxAb-w、HBxAg m、HBxAb-m阳性率分别为21.7％(18/83)、30.1％(25/83)、22.9％(19/83)、32.5％(27/83),HBeAg阴性组HBxAg w、HBxAb-w、HBxAg-m、HBxAb-m阳性率分别为27.0％(10/37)、32.4％(12/37)、29.7％(11/37)、40.5％ (15/37),HBeAg阳性组与阴性组HBxAg w、HBxAb-w、HBxAg-m、HBxAb-m阳性率的差异均无统计学意义(x2值分别为0.408、0.064、0.638、0.722,均P＞0.05).结论 HBV X基因A1762T/G1764A双突变对HBxAg的抗原性和特异性无明显影响；血清HBxAg和HBxAb随疾病的进展而升高,可能与疾病的发展和乙型肝炎相关性肝细胞癌的形成有关；HBxAg/HBxAb可能具有抑制HBV复制的功能.

摘要： 目的 研究人乳头瘤病毒(HPV)16型E2基因和长控制区(LCR)序列变异以及其与子宫颈病变的相关性.方法 成都地区HPV16型感染者标本50份,包括38份HPV携带者的子宫颈脱落细胞,8份生殖器疣患者的疣体组织,2份子宫颈上皮内瘤变(CIN)Ⅱ级和2份子宫颈CINⅢ级的病变组织.PCR扩增E2基因与LCR序列并测序,构建进化树.结果 50份标本中,E2基因有12个突变位点,其中1份生殖器疣标本3683有C→A突变；其他48份标本均存在≥2个位点的突变.所有标本的LCR序列都有突变,共有28个突变位点.选择10份标本测序并构建进化树,8份为Asian变异株；E2突变在各种子宫颈病变中均存在,而LCR 7867 G→A为4份子宫颈CIN所特有.结论 LCR 7867 G→A突变是成都地区子宫颈病变相关的一个突变位点.%Objective To explore the relevance between sequence variation of human papillomavirus (HPV)16 subtypes E2 gene or long control region (LCR) and cervical lesions.Methods Fifty specimens from HPV16 infected people in Chengdu were collected,including cervical exfoliated cells from 38 HPV carriers,papilloma tissues from 8 cases of genital warts,2 with cervical intraepithelial neoplasia (CIN) Ⅱ and 2 with CIN Ⅲ in this study.Polymerase chain reaction was used to amplify E2 gene and LCR,then an evolutionary tree was constructed.Results In all the 50 specimens,there were 12 mutation sites in E2 gene,among which,C→A existed in one specimen of genital warts,and ≥2 mutation sites existed in all the other 48 specimens.There were 28 mutation sites of LCR sequence of all the specimens.Ten specimens were chosen to construct evolutionary tree and were sequenced.The data showed that 8 specimens were Asian variants,E2 gene mutation existed in all the specimens while the LCR 7867 G→A only existed in the four CIN.Conclusion LCR 7867G→A is a correlative mutation site of cervical lesions in Chengdu.

摘要： Objective To analyze the VP1 and VP4 genetic region of enterovirus 71 (EV71)isolated from severe cases and mild cases with hanD-foot-mouth disease (HFMD) in Shanghai in 2011.Methods Five EV71 strains isolated from severe cases and five EV71 strains from mild cases in 2011 were included.Reverse transcription-polymerase chain reaction (RT-PCR) method was used to amplify and sequence the VP1 and VP4 genes of EV71,and then the sequencing results were compared with those of A,B,C genotype reference EV71 strains from GenBank by nucleotide alignment,amino acid alignment and phylogenetic tree analyses.Results The homogeneity between EV71 strains from severe cases and mild cases was 96.0％-98.1％ and 93.7％-99.5％ for VP1 and VP4 nucleotide sequences,respectively.The VP1 nucleotide sequences of 5 strains isolated from severe cases and 5 strains from mild cases in Shanghai shared 86.9％-98.2％ and 87.4％ 98.5％ identity with genotype C,respectively,while the homogeneity of VP4 nucleotide sequence was 85.5％-100.0％and 84.5％-99.5％,respectively.In addition,compared with the Fuyang EV71 strains (representative of C4 subtype),the strains from mild and severe cases shared homogeneity of 97.0％-98.2％ and 97.9％-98.5％ for VP1 gene,respectively,96.1％-100.0％ and 97.1％-99.5％ for VP4 gene,respectively.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein (T→A) were discovered simultaneously.Conclusions The 10 EV71 strains isolated from severe and mild cases in Shanghai belong to subgenogroup C4.Among 3 strains isolated from severe cases,mutations at the residue 282 in the VP1 protein (N→S) and residue 7 in the VP4 protein(T→A) are discovered simultaneously.%目的 了解2011年上海地区手足口病重症和轻症患儿中肠道病毒71型(EV71)分离株VP1、VP4区的基因特征.方法 对来自2011年重症与轻症手足口病患儿的各5株EV71分离株进行VP1、VP4全序列的RT-PCR扩增测序,并与美国国立生物技术信息中心公布的EV71 A、B、C基因型代表株进行核苷酸、氨基酸比对分析和系统进化分析.结果 轻、重症患儿的EV71分离株之间VP1基因的核苷酸同源性为96.0％～98.1％;VP4基因的核苷酸同源性为93.7％～99.5％.轻、重症患儿的EV71分离株与C基因型代表株比较接近,VP1区核苷酸同源性分别为86.9％～98.2％、87.4％o～98.5％,VP4区核苷酸同源性分别为85.5％～100.0％、84.5％～99.5％,其中与2008年安徽省阜阳市的EV71流行株(C4亚型)VP1区核苷酸同源性分别可达97.0％～98.2％、97.9％～98.5％,VP4区核苷酸同源性分别可达96.1％～100.0％、97.1％～99.5％.3例重症患儿分离株在VP1和VP4的天冬酰胺(N)282丝氨酸(S)、苏氨酸(T)7丙氨酸(A)同时发生变异.结论 2011年上海地区10例轻、重症手足口病患儿中分离的EV71流行株均属C基因型的C4亚型；3例重症患儿分离株在VP1和VP4的N282S、T7A同时发生变异.

摘要： Objective To investigate the effects of hepatitis Bvirus X (HBVX ) gene expression in HL-7702 live cells on cell apoptosis and the expression of HSP70 gene ,and explore their association . Methods The eukaryotic expression vector of HBVX gene (pcDNA3-X) was transfected into human liv-er cell HL-7702 and selected with G418 to construct a new cell line L02/HBx ,which was confirmed to ex-press HBVX gene steadily by RT-PCR and western-blot analysis . Compared with HL-7702 cells trans-fected with empty vector (L02/pcDNA3) ,apoptosis of L02/HBx were detected by flow cytometric analy-sis ,TUNEL analysis and DNA ladder . Then gene arrays were used to select out the different apoptosis gene expressions between the two cells . Then the differential expression of HSP70 was further confirmed by Western-blot analysis . Results RT-PCR and Western-blot analysis showed L02/HBx cells canex-press mRNA and protein of HBVX gene steadily . Flow cytometric and TUNEL analysis showed that the apoptosis rates of the L02/HBx cells were much higher than those of L02/pcDNA3 . And apoptosis phenomena in L02/HBx were observed by DNA ladder ,but not in L02/pcDNA3 . HSP70 ,as a differen-tial expression gene in the two cells ,was selected out by gene array . Western-blot analysis further con-firmed that the protein expression of HSP70 in L02/HBx cells was much higher than that in HL-7702 cells without expressing X gene . Conclusion The expression of HBVX gene in HL-7702 cells can promote the apoptosis of live cells and up-regulate the expression of HSP70 gene ,which play important roles in the process of HBV-induced HCC .%目的研究乙型肝炎病毒X（HBVX）基因在肝细胞HL-7702中的稳定表达对其细胞凋亡和表达HSP70的影响，并探讨二者之间的关联。方法将已构建好的HBVX基因真核表达载体pcDNA3-X转染人肝细胞HL-7702，48h后经G418筛选出稳定表达HBVX基因的肝细胞株（L02/HBx）。RT-PCR、蛋白印迹实验鉴定L02/HBx细胞HBVX基因的稳定表达。以转染空质粒肝细胞（L02/pcDNA3）为对照，运用流式细胞分析、TUNEL分析和DNA ladder观察检测L02/HBx细胞凋亡情况。基因芯片技术筛查L02/HBx和L02/pcDNA3两组肝细胞差异表达的凋亡相关基因，并运用蛋白印迹实验技术对差异表达的HSP70基因进行蛋白水平验证。　　结果RT-PCR和蛋白印迹实验显示，L02/HBx细胞中有HBVX基因mRNA和蛋白的表达。流式细胞和TUNEL分析显示，L02/HBx细胞组的凋亡率显著高于对照组L02/pcDNA3，DNA ladder可观测到L02/HBx的凋亡现象，而对照组未观测到。两次基因芯片结果筛查出L02/HBx细胞组与对照组差异表达基因HSP70，蛋白印迹实验进一步证实HSP70在L02/HBx细胞中的蛋白表达显著高于未表达X基因的肝细胞组。结论HBVX基因在肝细胞HL-7702中的表达促进了肝细胞的凋亡，上调了肝细胞中HSP70基因的表达，从而在乙型肝炎病毒致肝细胞癌变的过程中起重要作用。

摘要： Objective; To investigate the optimal conditions of tri-expression of CYP3A4 ,POR and cyt b5 in Sf 9 cells. Methods; The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. Results; When the temperature and rotation speed of the shaker were 27 Tl and 90 r/min,the cell density and culture volume were 5 ×10 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic(R) F-68 was 0.1% and the culture time was 72 h,the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km , Fmax, and CLint for testosterone metabolism were 119.6 μmol/L, 0. 52 μmol/( min · g protein) and 4. 34 ml/( min · g protein), respectively. Conclusions; The conditions of tri-expressing of CYP3A4,POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.%目的:研究人CYP3A4与POR和cyt b5用悬浮细胞共表达时的最佳条件.方法:悬浮培养Sf9细胞,分别摸索摇床的温度、转速、摇瓶中细胞液的量、表面活性剂的加量、细胞的培养时间,以及3种病毒的加量比例等条件,并进行优化；然后,将表达产物制成微粒体后用于睾酮的代谢,并分别测其代谢活性.结果:当恒温摇床的温度为27°C,转速为90 r/min,每250 ml摇瓶中加入80～120 ml细胞密度为5×105个/ml的细胞液,并加入0.1％的Pluronic(R) F-68,细胞培养72 h时,最适合细胞的生长和目的蛋白的表达.此时,当3种病毒的加量比例为1∶1∶1时,表达条件最佳,其对底物睾酮代谢的Km值、Vmax和CLint分别为119.6 μmol/L、0.52 μmol/(min·g蛋白)和4.34 ml/(min·g蛋白).结论:通过三重共表达CYP3A4、POR、cyt b5,以及对表达条件的优化,提高了目的蛋白的表达量,得到了较高代谢活性的CYP3A4,从而为新药代谢及药物-药物相互作用研究奠定了基础,为药物代谢酶的大量表达提供了可行的方案.

摘要： 本发明提供有效复制含全长HCV基因组序列的RNA的方法和使用细胞培养系统生产含全长HCV复制子RNA或全长HCV基因组RNA的HCV病毒颗粒的方法。此外，本发明涉及生产丙型肝炎病毒颗粒的方法，其包括培养其中导入复制子RNA的细胞，并在培养基中产生病毒颗粒，其中所述复制子RNA包含的核苷酸序列含有基因型2a丙型肝炎病毒的全长基因组RNA序列、至少一种选择标记基因和/或至少一种报告基因以及至少一种IRES序列，或含有基因型2a丙型肝炎病毒的全长基因组RNA。再进一步，本发明还涉及丙型肝炎疫苗和抗丙型肝炎病毒颗粒的抗体。

摘要： 一种用于生物医学领域的单基因病全基因组永生基因库的构建，其特征在于，构建含新霉素抗性基因的pLXSN载体，进而构建SV40 LT‑pLXSN载体并转染sp2/0细胞，以G418筛选阳性克隆，获得永生化特性改良和新霉素抗性基因标记的sp2/0细胞，经与基因突变细胞融合，获得具有两亲代细胞遗传特性、对G418和HAT联合筛选不敏感、贮藏了基因突变细胞全基因组、能永久冻存并能在体外无限扩增的杂交瘤细胞株，将突变基因收藏于杂交瘤细胞株，又将杂交瘤细胞株收藏于杂交瘤细胞库，以及以杂交瘤细胞的无限繁殖而复制单基因病基因，由此建立单基因病全基因组永生基因库。

摘要： 提供来自新型的基因型1b的HCV的、自主复制能力优异的亚基因组复制子RNA(核酸)、以及自主复制能力和感染性HCV颗粒产生能力优异的全基因组复制子RNA(核酸)。特别是，提供来自从急性重症丙型肝炎患者体内分离的新型的基因型1b的HCV即NC1株的HCV基因组的亚基因组复制子RNA(核酸)和全基因组复制子RNA(核酸)。

摘要： 本发明属生物技术领域。本发明提供了一种抗癌靶向基因病毒药物的制备方法。本发明构建缺失55Kda基因的腺病毒并携带有外源抗癌基因，命名为Ad5-ZD55-基因，克服ONYX-015病毒不能携带外源抗癌基因的缺点，提高了抗癌效果。本发明构建的腺病毒突变体，可以开发出有效的治疗肿瘤的抗癌新药。

摘要： 本发明公开了一种肠道病毒EV71型VP1基因病毒样颗粒及其制备方法和应用，属于免疫技术领域。为了实现此病毒样颗粒的制备，本发明首先构建了一种SIV GP41与EV71 VP1融合基因cVP1的真核表达载体，证实cVP1能够在哺乳动物细胞中表达并能够在细胞膜上表达。本发明进一步构建cVP1基因、gag基因的重组杆状病毒转座载体，制备杆状病毒，以两种杆状病毒共感染细胞的方式成功制备cVP1病毒样颗粒，此新型病毒样颗粒制备过程简单，易纯化，所获病毒样颗粒结构均一并在小鼠体内发挥较强的免疫原性。本发明通过膜锚定型融合基因的设计，利用杆状病毒表达系统制备了一种高效表达膜锚定VP1基因的病毒样颗粒。

摘要： 本发明涉及传染性法氏囊病病毒变异株VP2基因病毒样颗粒重组蛋白，属于生物制药领域。将IBDV变异株(AH1)VP2基因，克隆、转化、转染，获得重组杆状病毒vBac－VP2。感染的Sf9昆虫细胞具有特异性荧光，抗原效价在1.6×10

摘要： 本发明公开了一种缺失凋亡抑制基因病毒的构建及其应用。它是以腺病毒或单纯疱疹病毒为材料,经过PCR扩增基因片段,插入质粒DNA中,进一步用GFP等标记基因和TNF等效应基因,构建出转移载体,插入失活与复制有关的凋亡抑制基因,并与相应的野生型病毒共转染,筛选重组病毒,分别得到E1b55K基因缺失的重组腺病毒和ν134.5基因失活的重组单纯疱疹病毒。本发明的方法简单、快速、直观,所构建的重组病毒安全、稳定,有高度靶向性和选择性。

摘要： 本专利申请属于生物工程技术领域，具体公开了一种核糖体失活蛋白基因病毒载体构建及其在肿瘤细胞中表达活性蛋白的方法，包括如下步骤：步骤一、选取合适的核糖体失活蛋白，对其成熟区基因进行人源化表达的密码子优化；步骤二、野生型及优化后基因的腺病毒载体构建与重组病毒包装；步骤三、重组腺病毒感染肿瘤细胞，并检测使其在肿瘤细胞中蛋白表达及其抗肿瘤效应。本方案将α‑苦瓜素基因进行密码子优化，构建能感染哺乳动物肿瘤细胞的腺病毒表达载体系统，以适合于在肿瘤细胞中表达活性蛋白，克服了现有技术中使用原核细胞表达体系只能在大肠杆菌中表达以及使用植物病毒载体只能在植物细胞中表达而不能在肿瘤细胞中表达和杀伤肿瘤的缺陷。

摘要： 本发明属生物技术领域。本发明提供了一种抗癌靶向基因病毒药物的制备方法。本发明构建缺失55Kda基因的腺病毒并携带有外源抗癌基因，命名为Ad5－ZD55－基因，克服ONYX－015病毒不能携带外源抗癌基因的缺点，提高了抗癌效果。本发明构建的腺病毒突变体，可以开发出有效的治疗肿瘤的抗癌新药。

摘要： 本发明描述的是一种含有昆虫转录因子的转基因病毒。具体地讲,该转基因昆虫病毒含有涉及蜕皮和变态的昆虫转录因子。这样的转基因昆虫病毒可用作生物杀虫剂。