HCV＋表位抗原的恒河猴免疫学效果评价

发布时间：2018-04-20 17:24

本文选题：丙型肝炎病毒 + 包涵体　；参考：《北京协和医学院》2011年硕士论文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)感染导致的慢性肝炎疾病,现在已成为全球性的公共卫生问题。鉴于目前对于HCV慢性感染尚无特异性治疗方法,且副作用较大,因此研制HCV疫苗对于控制HCV流行具有重要意义。HCV的高度异源性是HCV疫苗研究的重要障碍,通过对来自全球的多株HCV序列分析表明,至少存在6种主要基因型和大约100种基因亚型。由于HCV基因序列存在高度变异性、感染力弱,并且缺乏有效的细胞和动物感染模型,因此采用传统方法研制丙肝疫苗难以达到目的。多表位疫苗,作为通过筛选与组合优势抗原表位(包括T细胞、B细胞表位)的新型疫苗,能够产生高效的细胞免疫和体液免疫应答进而清除病毒,其优势在于通过选择最具免疫保护潜力的表位以覆盖尽量多的病毒亚型和诱导机体全面的抗病毒免疫应答,它能有效地应付病原微生物的变异,克服主要组织相容性复合体(MHC)分子的遗传限制。本研究选取多个HCV基因型中的十个保守性表位,构建了重组抗原多肽CB3的表达基因,并将其连接入载体pBV220进行原核表达,经包涵体洗涤、溶解、复性及离子交换柱纯化后,获得纯度较高的目的多肽CB3。在免疫原性评价研究中,以恒河猴作为实验动物,从体液免疫和细胞免疫两个方面对CB3的免疫学效果进行了评价。本研究分别使用15μg和30μg剂量的多肽CB3以0、1、6月的免疫程序免疫恒河猴后,采用ELISA方法检测到免疫动物体内有特异性抗体的产生,ELISPOT检测到抗原特异性IFN-γ细胞因子。与对照组相比,均有明显的统计学差异(P0.05),且30μg剂量组的免疫应答强于15μg组。在保护性抗体评价方面,本研究采用HCV体外细胞培养模型对免疫后动物血清中的中和抗体进行了检测。首先将体外转录得到的HCV JFH-1株基因组RNA转染Huh-7细胞,免疫荧光、Western实验证明有HCV病毒蛋白表达,实时荧光定量PCR证明有HCV病毒基因组的复制。在蛋白水平和RNA水平上证实了HCV病毒的产生后,进行体外中和实验。结果初步证明CB3能诱发机体产生一定的保护性中和抗体,在免疫血清稀释度1：2～1：4时,能够中和HCV JFH-1株感染Huh-7细胞。
[Abstract]:Chronic hepatitis caused by hepatitis C virus (HCV) infection has become a global public health problem. In view of the fact that there is no specific treatment for chronic HCV infection and its side effects are large, the development of HCV vaccine is of great significance in controlling the prevalence of HCV. The highly heterogenous HCV vaccine is an important obstacle in the research of HCV vaccine. According to the analysis of HCV sequences from all over the world, there are at least 6 major genotypes and about 100 gene subtypes. Because of the high variability of HCV gene sequence, weak infectivity, and the lack of effective models of infection in cells and animals, it is difficult to develop hepatitis C vaccine by traditional methods. Multiepitope vaccines, as a novel vaccine for screening and combining dominant antigen epitopes (including T cell B cell epitopes), can produce highly efficient cellular and humoral immune responses to remove viruses. It has the advantage that by selecting the epitopes with the most potential for immune protection to cover as many virus subtypes as possible and to induce a comprehensive antiviral immune response, it can effectively cope with the variation of pathogenic microorganisms. To overcome the genetic limitation of the major histocompatibility complex (MHC). In this study, ten conserved epitopes of multiple HCV genotypes were selected to construct the expression genes of recombinant antigenic polypeptide CB3, and then ligated into vector pBV220 for prokaryotic expression. After purification by inclusion body washing, dissolution, renaturation and ion exchange column, A high purity target peptide CB3 was obtained. In the study of immunogenicity evaluation, rhesus monkey was used as experimental animal to evaluate the immunological effect of CB3 from humoral immunity and cellular immunity. In this study, rhesus monkeys were immunized with 15 渭 g and 30 渭 g of polypeptide CB3 at a dose of 15 渭 g and 30 渭 g, respectively. After immunizing rhesus monkeys by six months, the production of specific antibodies in immunized animals was detected by ELISA method and antigen-specific IFN- 纬 cytokines were detected by Elispot. Compared with the control group, there was significant statistical difference between the two groups, and the immune response of the 30 渭 g group was stronger than that of the 15 渭 g group. In the aspect of protective antibody evaluation, the neutralizing antibody in animal serum after immunization was detected by HCV cell culture model in vitro. Firstly, the genomic RNA of HCV JFH-1 strain was transfected into Huh-7 cells in vitro. The expression of HCV virus protein was confirmed by immunofluorescence assay, and the replication of HCV virus genome was confirmed by real-time fluorescence quantitative PCR. The production of HCV virus was confirmed at the protein level and RNA level, and neutralized in vitro. Results it was preliminarily demonstrated that CB3 could induce the production of protective neutralizing antibody, and when the dilution of immune serum was 1: 2 / 1: 4, it could neutralize the HCV JFH-1 strain and infect Huh-7 cells.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】