HCV湖北株全基因组克隆

发布时间：2018-04-13 12:59

本文选题：HCV + 全长基因　；参考：《中国科学院研究生院（武汉病毒研究所）》2005年硕士论文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)是输血后及散在性的非甲非乙型肝炎(Non-A, Non-B Hepatitis, NANBH) 主要病因。缺乏可以在实验室内稳定扩增病毒的细胞系统和动物模型严重阻碍了HCV 病毒本身的研究发展和由其感染引起的疾病的治疗方法的研究进步。各国科学家一方面致力于扩增病毒的细胞系和动物模型的发展,另一方面利用分子生物学的手段对HCV 开展了基础性的研究。在如今分子生物学研究发展迅猛的背景下,HCV 的研究水平也很快得到了很大提高。本研究通过分段RT-PCR 的方法分段得到了HCV 的全基因组克隆,分析得到所获得的克隆为1b亚型并且与HCV-Shanghai最为接近;通过对所采集的HCV阳性血清UTR 进行测序分析,得到目前在武汉地区流行的主要HCV 基因型为1 型。另外采用杆状病毒系统在昆虫细胞中表达HCV 结构蛋白E1,并采用Confocal 显微镜观察E1 在昆虫细胞中的表达分布。本论文包括以下三个章节: 第一章对HCV 的发现、其基因组结构和基因型的特点和分布、检测手段、治疗方法和E1 蛋白做了简要综述,对利用杆状病毒表达系统表达外源蛋白的广泛应用作了简要介绍。第二章描述了采用分段RT-PCR的方法从湖北省HCV 感染阳性病人血清中克隆得到了HCV 的全基因组,通过软件对其进行基因型和进化树的分析。最终认为所克隆的HCV 基因组为1b 亚型,与目前在NCBI 上发布的HCV-Shanghai株亲缘关系最为接近。但是在比较中发现在湖北株内有363 nt 的缺失,而同时又替换了285nt。在基因组内这285nt 替换了NS5b 的C 端和3’UTR 的前端。而替换的285nt 中有153nt 位与NS5b 内而剩余132nt 位于3’UTR 内。通过分析发现替换序列在结构上与原序列相似,但是功能分析有待进一步的实验证实。第三章通过克隆得到HCV UTR 和core 的部分基因,测序分析和软件对比
[Abstract]:Hepatitis C virus (HCV) is the main cause of non-A, Non-B hepatitis B after blood transfusion.The lack of cell systems and animal models that can stably amplify the virus in the laboratory seriously impedes the development of HCV itself and advances in the treatment of diseases caused by its infection.On the one hand, scientists from all over the world are committed to the development of cell lines and animal models for the amplification of viruses; on the other hand, molecular biology is used to carry out basic research on HCV.With the rapid development of molecular biology, the research level of HCV has been greatly improved.In this study, the whole genome clone of HCV was obtained by segmenting RT-PCR, the clone obtained was 1b subtype and closest to HCV-Shanghai, and the HCV positive serum UTR was sequenced.The main genotype of HCV in Wuhan is genotype 1.In addition, baculovirus system was used to express HCV structural protein E1 in insect cells, and Confocal microscope was used to observe the expression distribution of E1 in insect cells.This thesis includes the following three chapters:In the first chapter, the discovery of HCV, the characteristics and distribution of its genome structure and genotypes, the methods of detection, treatment and E1 protein were briefly reviewed, and the wide application of the expression of exogenous proteins by baculovirus expression system was briefly introduced.In the second chapter, the whole genome of HCV was cloned from the sera of HCV positive patients in Hubei province by using the method of segmented RT-PCR. The genotypes and evolutionary trees of HCV were analyzed by software.It was concluded that the cloned HCV genome was subtype 1b, which was most closely related to the HCV-Shanghai strain published on NCBI.However, 363 NT deletion was found in Hubei strain, and 285 NT was replaced at the same time.Within the genome, the 285nt replaces the C end of NS5b and the front end of 3'UTR.In the alternative 285nt, there are 153nt bits and NS5b bits, while the remaining 132nt is located in 3'UTR.It is found that the substitution sequence is similar to the original sequence in structure, but the functional analysis needs further experimental verification.In chapter 3, some genes of HCV UTR and core were cloned, sequenced and compared with software.
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【引证文献】