表达HCV-C抗原的重组腺病毒载体疫苗的实验研究

发布时间：2018-04-11 03:42

本文选题：丙型肝炎 + 疫苗　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:丙型肝炎是由丙型肝炎病毒(Hepatitis C virus,HCV)引起的慢性肝病,是一个全球性健康问题。目前,临床上仍无有效的治疗方法。因此,开发有效的预防和治疗性HCV疫苗,就成了一个亟待解决的问题。 HCV属于黄病毒科丙型肝炎病毒属,由于丙型肝炎病毒(hepatitis C virus,HCV)有多种基因型以及基因型的高度变异性,尤其是包膜区基因变异更大,因此按传统方式研制的HCV疫苗面临着很多困难。将编码目的抗原的外源基因导入被接种者的各类型新型疫苗可望成为丙肝疫苗研发的一条新的思路。丙肝病毒核心区(C区)是最为保守及稳定的区域,有报导表明C抗原可诱导针对丙肝病毒的高水平特异性抗体应答及细胞毒性T淋巴细胞( cytotoxic T lymphocyte, CTL )活性细胞免疫,是研制HCV DNA疫苗的主要靶抗原之一。与质粒载体相比,复制缺陷型腺病毒载体系统(replication-deficient adenovirus vector system)具有进入宿主细胞的能力强,宿主范围广,极高的转染效率,外源基因高表达以及病毒滴度高等特点,在疫苗载体选择中表现出优势,成为极具应用前景的基因转移载体。本研究中我们利用以重组复制缺陷型腺病毒为载体以HCV C抗原作为有效的靶抗原构建了可表达HCV核心蛋白(HCV core protein)的重组腺病毒rAd-C,以探索研制丙型肝炎病毒疫苗新的途径。方法:1.目的基因的扩增与鉴定以重组质粒pEGFP-HCV/C为模板,扩增C目的基因。将扩增产物与pGEM-T载体连接,构建质粒pGEM-T/C。转化DH5α大肠杆菌,提取质粒进行酶切鉴定及测序,筛选重组子。 2.构建真核表达质粒pcDNA3.0/C将质粒pGEM-T/C以合适的限制性内切酶进行双酶切,胶回收目的片段,并与经同样的限制性内切酶双酶切的pcDNA3.0质粒载体连接,经转化、筛选和酶切鉴定,构建真核表达质粒pcDNA3.0/C。 3.构建重组穿梭质粒将质粒pGEM-T/C以合适的限制性内切酶进行双酶切,回收目的片段,将目的片段与经合适的限制性内切酶双酶切的腺病毒穿梭载体pAdTrack-CMV连接。经转化、筛选和酶切鉴定,获得重组穿梭质粒pAdTrack-CMV/C。 4.重组腺病毒质粒的构建将质粒用内切酶PmeI线性化,利用AdEasy系统,经两步法分别在大肠杆菌BJ-5183与骨架载体pAdEasy-1进行同源重组,构建重组腺病毒质粒pAd/C。用PacI酶切鉴定,将阳性质粒转化入感受态细胞E.coli DH5α,以碱裂解法大量提取,并用聚乙二醇(polyethylene glycol,PEG)沉淀法进行纯化。 5.重组腺病毒的包装与扩增将重组腺病毒质粒pAd/C用内切酶Pac-I线性化,以阳离子脂质体法转染HEK293(humanembryo kidney 293 derived cell line)细胞。并观察绿色荧光蛋白(green fluorescent protein,GFP)的表达。冻融法收集初代病毒液。取初代病毒液感染HEK293细胞,逐日观察细胞病变(cytopathic effect,CPE),及有无彗星样斑块(FOCI)形成。并经第三、四轮大量扩增,收集第四轮病毒液,用腺病毒纯化试剂盒进行纯化。 6.重组腺病毒滴度测定将293细胞培养在96孔板中细胞生长至70%-80%汇合时,以不同稀释倍数的各代病毒液感染细胞,感染48小时后,以GFP阳性细胞计数法测定病毒滴度。病毒滴度=荧光细胞数×病毒液的稀释倍数/病毒液量。结果:1.成功构建了质粒pGEM-T/C,限制性酶切鉴定及测序结果证明与预期结果相符合。 2.成功构建了真核表达质粒pcDNA3.0/C,酶切分析证明符合预期设计。 3.成功构建重组穿梭质粒pAdTrack-CMV/C,限制性酶切结果符合预期设计。 4.成功构建重组腺病毒质粒pAd/C,用PacI酶切得到约30Kb的大片段和一个4.5Kb的小片段,PCR鉴定目的基因长度与预期值相符。 5.经293细胞转染48小时后,荧光显微镜下观察到报告基因GFP的表达,重组腺病毒rAd/C包装成功。初代病毒再次感染293细胞,在荧光显微镜下观察到了逐日明显变化的细胞病变和荧光聚集,且感染细胞后的各代病毒液均有彗星样荧光斑块形成。 6.重组腺病毒rAd/C大量扩增后病毒滴度达到:6.8×108pfu/ml。结论:本研究利用AdEasy腺病毒载体系统构建并包装重组腺病毒载体疫苗rAd/C,为丙型肝炎疫苗的研制奠定了基础。
[Abstract]:Objective: hepatitis C is by hepatitis C virus (Hepatitis C, virus, HCV) caused by chronic liver disease, is a global health problem. At present, there is still no effective treatment. Therefore, the development of effective prevention and treatment of HCV vaccine, has become an urgent problem to be solved.
HCV belongs to the hepatitis C virus, the hepatitis C virus (hepatitis C, virus, HCV) high variability of different gene types and genotypes, especially the envelope gene variation is greater, so according to the traditional way of HCV vaccines is facing many difficulties. The exogenous gene encoding the target antigen by all a new type of vaccine recipients is expected to become a new way of HCV vaccine development. HCV core area (C area) is the most conservative and stable region, there have been reports that C antigen can induce high levels to hepatitis C virus specific antibody response and cytotoxic T lymphocyte (cytotoxic T, lymphocyte, CTL) activity of cells immunization is one of the main target antigen for HCV DNA vaccine.
Compared with the plasmid vector, replication defective adenovirus vector system (replication-deficient adenovirus vector system) has the ability to enter the host cell, wide host range, high transfection efficiency, gene expression and the characteristics of the high titer of virus showed advantage in vaccine carrier selection, become a promising gene transfer vector. In this study we use the recombinant replication defective adenovirus vector with HCV C antigen as target antigen effectively constructed expression of HCV core protein (HCV core protein) recombinant adenovirus rAd-C, to explore ways to develop new hepatitis C virus vaccine.
Methods: 1.. Amplification and identification of the target gene. The recombinant plasmid pEGFP-HCV/C was used as template to amplify the C target gene. The amplified product was connected with pGEM-T vector to construct plasmid pGEM-T/C. and to transform DH5 alpha E.coli. The plasmid was extracted, digested, identified and sequenced, and the recombinants were screened.
2. construction of eukaryotic expression plasmid pcDNA3.0/C pGEM-T/C plasmid with suitable restriction endonuclease digested gel fragment, and pcDNA3.0 plasmid by restriction endonuclease digestion of the same connection, through the transformation, screening and enzyme digestion, to construct eukaryotic expression plasmid pcDNA3.0/C.
3. construction of recombinant shuttle plasmid pGEM-T/C plasmid with suitable restriction endonuclease digested fragment recycling, the fragment with suitable restriction endonuclease digested Adenovirus Shuttle Vector pAdTrack-CMV. After connection transformation, screening and enzyme digestion, the recombinant shuttle plasmid pAdTrack-CMV/C.
4. recombinant adenovirus plasmid plasmid by restriction endonuclease PmeI linearized by AdEasy system, the two step of homologous recombination in Escherichia coli BJ-5183 with backbone plasmid pAdEasy-1, recombinant adenovirus plasmid pAd/C. by PacI enzyme digestion, recombinant plasmid was transformed into competent cells of E.coli DH5 alpha, a large number of alkaline lysis extraction method and using polyethylene glycol (polyethylene, glycol, PEG) was purified by precipitation method.
5. recombinant adenovirus packaging and amplification of recombinant adenovirus plasmid pAd/C by restriction endonuclease Pac-I linearized by cationic liposome transfection (HEK293 humanembryo kidney 293 derived cell line) cells. And observed the green fluorescent protein (green fluorescent, protein, GFP). The expression of freeze thawing method. The collected primary virus liquid primary virus liquid the infection of HEK293 cells, by observing the cytopathic effect (cytopathic effect, CPE), and there is no comet like (FOCI) and plaque formation. After third, fourth rounds of amplification, collecting fourth round virus liquid was purified with adenovirus purification kit.
6. the recombinant adenovirus titer of 293 cells were cultured in 96 well plate cell growth to 70%-80% confluence, with each generation of virus infected cells in liquid dilution, 48 h after infection, the virus titer was determined by counting the number of GFP positive cells. The virus titer = number of fluorescent cells * virus liquid dilution / virus liquid.
Results: 1. the plasmid pGEM-T/C was successfully constructed. Restriction endonuclease identification and sequencing results proved to be consistent with the expected results.
2. the eukaryotic expression plasmid pcDNA3.0/C was successfully constructed, and the enzyme cutting analysis proved to be in conformity with the expected design.
3. the recombinant shuttle plasmid pAdTrack-CMV/C was successfully constructed, and the restrictive enzyme cutting results were in conformity with the expected design.
4., the recombinant adenovirus plasmid pAd/C was successfully constructed, and a large fragment of 30Kb and a small fragment of 4.5Kb were obtained by PacI enzyme digestion. The length of the target gene was consistent with the expected value by PCR.
5. after 48 hours of 293 cells after transfection, fluorescence microscopy showed that expression of GFP reporter gene, recombinant adenovirus rAd/C packaged successfully. Primary virus re infection of 293 cells under the fluorescence microscope observed daily obvious change cytopathic and fluorescence aggregation, and the generation of virus infection have comet like fluorescent plaque cells after the formation.
The virus titer of 6. recombinant adenovirus rAd/C was reached: 6.8 x 108pfu/ml.
Conclusion: the recombinant adenovirus vector vaccine (rAd/C) was constructed and packaged by the AdEasy adenovirus vector system, which laid the foundation for the development of hepatitis C vaccine.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

【参考文献】