【作者】 张彦明；

【导师】 李明；

【作者基本信息】 第一军医大学 ， 免疫学， 2004， 博士

【摘要】 丙型肝炎病毒(HCV)是输血后非甲非乙肝炎的主要病因，全世界约有1.7亿人感染HCV，中国有大约4000万的HCV携带者。超过一半的HCV感染者会转化成慢性感染，其中10-20％的慢性感染者会发展成为肝脏疾病如肝硬化，并且最终1-5％的人会发展成为肝细胞癌。目前，对HCV感染的治疗尚无有效的方法，干扰素治疗只对10-20％的病人有效，低于50％的病人对干扰素和病毒唑的联合治疗有反应。由于HCV难以在体外培养，使得HCV疫苗和抗HCV药物的研究进展缓慢。 质粒BRT703’X(NIHJ1)含有HCV全长基因组DNA，将其用限制性内切酶EcoRⅠ和HindⅢ不完全酶切后，回收HCV基因组序列，插入到质粒载体pCOUS-T7的EcoRⅠ和HindⅢ之间，使HCV基因组置于载体的T7启动子和T7终止子控制下，构建质粒pT7HCV。 质粒0.pMKC1A(HCV)含有HCV基因组前体蛋白的开放阅读框(ORF)序列，将其用EcoRⅠ和HindⅢ双酶切以后，插入到质粒PSC59的EcoRⅠ和Stu Ⅰ位点之间，使HCV的ORF置于载体的痘苗病毒晚期启动子控制之下，构建质粒pVHCV。 在本研究中，两个质粒被引入一个体外HCV细胞培养系统中：质粒pT7HCV携带有HCV全长基因组cDNA，并将HCV基因组置于载体的T7启动子和T7终止子的控制下。质粒pVHCV含有HCV前体蛋白开放阅读框(ORF)序列，并且HCV的ORF在载体的痘苗病毒晚期启动子控制下。将两个质粒pT7HCV和pVHCV共转染真核细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒感染此细胞，在一定条件下培养后得到重组张彦明:丙型肝炎病毒体外大规模培养技术研究及其在抗丙肝病毒药物筛选中的初步应用一定条件下培养后得到重组HCV。在此基础上，对一系列的培养条件进行了优化，以期得到最高产量的重组HCV。随后将培养得到的重组HCV利用蔗糖密度梯度离心进行纯化和鉴定。并且对基因重组干扰素一alb和病毒哇的抗HCV作用进行了初步的研究，探索本系统应用于筛选抗HCv药物的可能性。 构建的重组质粒用酶切和核普酸序列测定的方法进行鉴定，结果证明外源基因序列正确地插入到了载体的相应位置。 将质粒pT7HCv和pvHCv用脂质体LipofeCtaminTM2000共转染BHK21细胞，然后用携带有T7RNA聚合酶基因的重组痘苗病毒vTF7一3感染此细胞，病毒吸附细胞2小时后，换新鲜的10%DMEM培养基在30℃、5%COZ培养箱中培养2一5天后得到重组HCV。用引物特异性RT一PCR检测培养上清中HCV的病毒基因组RNA;荧光定量PCR方法测定培养上清中HCV的拷贝数;Western blotting和间接免疫荧光检测细胞中HCV蛋白的表达;免疫电镜观察病毒的亚细胞定位。 试验结果显示，在六孔细胞培养板中，培养96小时以后可以在培养上清中检测到每毫升7xl护个拷贝的重组HCV;RT一PCR结果显示，重组HCV中含有HCV的基因组RNA;在细胞中检测到了HCV蛋白的表达;免疫电镜在胞质中观察到直径约SOnm左右的深染色病毒样颗粒。 为了适应HCV疫苗研究的需要，我们对体外培养HCV的条件进行了优化。以六孔细胞培养板为基础，我们对培养HCV所用的细胞系、转染细胞所用的质粒量、重组痘苗病毒感染时痘苗病毒和细胞数的比例以及培养后收获病毒的时间等条件进行了优化。 试验结果显示，选用BHKZ，细胞做为宿主细胞时;细胞用5 pg质粒pT7HCV和5 pg质粒pVHCV共转染，转染后的细胞以每个细胞5个PFU的比例用重组痘苗病毒感染:感染后培养96小时的条件下，在细胞培养上清中可以以相对最少的消耗和最短时间得到最高浓度的HCV。确定了最终用于HCV生产的优化条件。 将培养得到的重组HCV进行纯化和鉴定。培养结束后，收获细胞和培养上清，将细胞裂解，低速离心去除细胞碎片，然后用35%的PEG6O00一NaCI溶液将离心上清进行浓缩，继之以不连续的蔗糖密度梯度离心。HCV基因组引物特异性RT一PCR鉴定HCV的存在;SDS一PAGE、第一军医大学博士学位论文western blotting和免疫荧光抗原片鉴定HCV蛋白的表达;磷钨酸负染后在电子显微镜下观察纯化后病毒的形态。 结果显示，重组HCV的密度梯度大都集中在约1.159/ml，但在1.09g/ml处有一小的吸收峰。以重组HCV为模板进行的引物特异性RT一PCR可以扩增出HCV的5’一UTR、核心蛋白、NS。蛋白和3’UTR基因片段，显示在重组HCV中包含有全长的HCV基因组;SnS一PAGE、Western blotting和免疫荧光抗原片检测到了HCV结构蛋白的表达。纯化的病毒用磷钨酸负染后在电镜下观察可以看到直径为6Onm的带有6nm棘突的HCV病毒样颗粒。 以干扰素a(IFN一Q)和病毒哇(RBV)为对象，初步研究了本系统用于筛选抗丙型肝炎病毒药物的可行性。探索了二者对于体外培养的HCV早期复制过程的影响。 在BHKZ，细胞经过质粒pT7HCV和pVHCv转染并且被重组痘苗病毒vTF7一3感染后，加入药物基因重组干扰素Q一lb(rIFN一Q lb)和RBV，培养过程中每隔24小时取样检测上清中HCV的拷贝数。结果显示，rl FN-Qlb直接抑制HCV在体外的早期复制，并且抑制效应呈剂量依赖性。RBV在最初的48小时对HCV复制有明显的抑制作用，但是后期的效果不明显，并且药物浓度对于HCV复制的影响关系很大。说明rIFN一alb对于HCV的作用除调节机更多还原

【Abstract】 Ph.D. candidate Zhang Yan-ming Supervisor Li MingThe hepatitis C virus (HCV) is a major causative agent for most posttransfusion non-A, non-B hepatitis case. There are about 170 million carriers of hepatitis C virus in the world today and about 40 million in China alone. More than half of persons infected with HCV can progress to chronic infection, and of the chronically infected 10-20% will develop complications of chronic liver disease such as liver cirrorhosis and 1-5% will develop hepatocellular carcinoma. Unfortunately, only 10-20% of patients infected by HCV are respond to interferon alone, therapy of IFN- a combined with ribavirin is effective in only about 50% infected persons. The lack of tissue-culture system for HCV replication has limited the development of vaccine for HCV and new treatment.We propose a cell culture system to produce HCV in vitro. In this system, we constructed two plasmids: pT7HCV contains the HCV genomic RNA-coding region between an upstream T7 promoter and a downstream T7 terminator; pVHCV contains the HCV polyprotein open reading frame (ORF) that is linked to the vaccinia late promoter of vector. The two plasmids were co-transfected into eucaryotic cells, the cells then were infected by recombinant vaccinia virus vTF7-3 which contain a T7 RNA polymerase gene under the control of a vaccinia promoter. After cultured, recombinant HCV can be produced in the cells. We optimized the culture condition of the cells to get the highest production of recombinant HCV. Then we separate HCV by discontinuous sucrose density-gradient ultra-centrifugation. We also investigated effect of interferon and rebavirinon HCV early replication stage in this HCV culture system.Plasmid pBRT703’X(NIHJl) containing the full-length HCV genomic cDNA, was incompletely digested with HindIII and EcoR I , then HCV cDNA was reclaimed and inserted into vector pOCUS-T7 (plasmid pOCUS containing a T7 promoter and a T7 terminator) between the EcoR I and Hindlll sites, resulting insertion of the HCV sequence between the bacteriophage T7 promoter and T7 terminator sequences to create plasmid pT7HCV.Plasmid O. pMKC1A (HCV) containing the HCV polyprotein open reading frame was cut with EcoR I and Hindlll and inserted into PSC59 between the EcoR I and Stu I sites to generate plasmid pVHCV, which contained the HCV polyprotein open reading frame (ORF) linked to a vaccinia late promoter.Restriction enzyme digestion and DNA sequencing confirm the correct sequences of plasmid pT7HCV and pVHCV.Cells were then transfected with pT7HCV plus pVHCV using the LipofectaminTM2000 Reagent followed by infection with vTF7-3 vaccinia viruses. Then cells were inoculated two hours for absorption of virus. The inoculum was then removed and the cells were cultured in fresh Dulbecco’s modified Eagle’s medium (DMEM)with 10% fetal calf serum(FCS) in a 30 "C and 5%CO2 incubator for 2-5 days. Genomic RNA of HCV in the supernatants was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR), the titer of HCV was examined by Fluorescent Quantitative PCR (FQ-PCR), HCV proteins were tested by Indirect Immunofluorescence and Western blot analysis and the virus was observed under Immunoelectron microscopy to locate the virus in cell.As the result of experiment showed, after 96 hours of incubation in 6-well plates, 7 106 copies of HCV can be produced in the supernatants. HCV genomic RNA was amplified by RT-PCR, and structural and nonstructural proteins of HCV can be detected by immunofluorescence and Western blot. Virus-like particles of about 50nm in diameter were observedunder immuno-electron microscopy.We optimized the culture condition of HCV for the purpose of produce HCV vaccine. We selected appropriate cell line, determine quantity of plasmid for transfection and choose the best concentration of recombinant vaccinia virus to produce the most abundance HCV in supernatant. The copies of HCV genomic RNA in supernatant was detected by FQ-RT-PCR and used as guideline to determine the condition of HCV culture.Th更多还原