以HBc为载体美洲型PRRSV颗粒候选疫苗构建及免疫原性分析

发布时间：2018-12-11 00:39

【摘要】：目前,猪繁殖与呼吸综合征(PRRS)在全球范围内流行,基因型表现出高度可变性,对疾病的防控产生了巨大的阻碍。现阶段对PRRS的防控主要采取疫苗接种的方式,但存在一定的局限性,现有疫苗不能对猪群提供全面持久的保护,因此积极发展新型疫苗对控制PRRSV感染与流行有着重要的意义。自成功采用乙肝病毒核心蛋白(HBc)作为疫苗载体表达了口蹄疫病毒抗原表位以来,HBc被越来越多的应用于病毒样颗粒(VLPs)疫苗的研制。作为VLPs疫苗载体,HBc具有以下特点:天然条件下能够自我组装成颗粒样结构,插入位点灵活,装配效率高(95%),可以提呈抗原,能够在多种表达系统中稳定表达等。本研究以HBc为载体表达PRRSV GP5保护性抗原,构建了HBc-GP5表位VLPs候选疫苗,并对其进行小鼠免疫评价。首先根据Escherichia coli(strain K12)密码子偏好性对HBc序列进行密码子优化,并将其克隆至改造的pET-28a原核表达载体中,获得重组质粒pEO-NEW。经SDS-PAGE、Western blot及透射电镜等方法对表达获得的VLPs进行鉴定,结果表明成功诱导表达了HBc蛋白且表达后蛋白能够自主装配成VLPs,为下一步重组疫苗制备提供研究基础。基于所得到的pEO-NEW表达载体,将优化后的GP5基因插入至pEO-NEW载体的免疫显性区(MIR),获得重组质粒pEO-LEORF。通过SDS-PAGE和Western blot方法鉴定重组目的蛋白,结果显示,成功诱导表达GP5蛋白。经透射电镜对其形态进行观察,结果显示,诱导后的重组蛋白能够自主装配成VLPs。以小鼠为模型分析该HBc-GP5 VLPs候选疫苗的免疫原性,利用ELISA进行抗体检测,结果表明其能够诱导机体产生较高的特异抗体水平;对T细胞亚群及细胞因子进行检测结果表明,疫苗免疫组小鼠CD3+CD4+T细胞淋巴细胞亚型数量及IL-4水平显著高于对照组(P0.05),淋巴细胞刺激指数(SI)及第三次免疫后IFN-γ含量与对照组相比显著提高(P0.05),说明HBc-GP5表位VLPs候选疫苗能够模拟病毒样颗粒,有效提呈GP5并诱导机体产生体液和细胞免疫,为下一步实验研究提供数据基础。
[Abstract]:At present, porcine reproductive and respiratory syndrome (PRRS) is prevalent all over the world, and the genotypes exhibit high variability, which is a great hindrance to the prevention and control of swine reproductive and respiratory syndrome (RRS). At present, vaccination is mainly used to prevent and control PRRS, but there are some limitations. The existing vaccine can not provide comprehensive and lasting protection for swine population. Therefore, it is of great significance to develop new vaccine to control PRRSV infection and epidemic. Since hepatitis B virus core protein (HBc) was successfully used as vaccine vector to express the antigen epitopes of foot-and-mouth disease virus, HBc has been used more and more in the development of virus-like granular (VLPs) vaccine. As a vector of VLPs vaccine, HBc has the following characteristics: it can self-assemble into granular structure under natural conditions, the insertion site is flexible, the assembly efficiency is high (95%), it can present antigen, and can be expressed stably in many expression systems. In this study, HBc was used as vector to express PRRSV GP5 protective antigen, and the candidate vaccine of HBc-GP5 epitope VLPs was constructed and evaluated in mice. Firstly, according to the Escherichia coli (strain K12) codon preference, the HBc sequence was optimized and cloned into the modified pET-28a prokaryotic expression vector to obtain the recombinant plasmid pEO-NEW.. The expression of VLPs was identified by SDS-PAGE,Western blot and transmission electron microscopy. The results showed that the expression of HBc protein was successfully induced and the expressed protein could be assembled into VLPs, by itself to provide the basis for further preparation of recombinant vaccine. Based on the obtained pEO-NEW expression vector, the optimized GP5 gene was inserted into the immune dominant region (MIR), of the pEO-NEW vector to obtain the recombinant plasmid pEO-LEORF.. The recombinant target protein was identified by SDS-PAGE and Western blot. The results showed that the expression of GP5 protein was successfully induced. The morphology of the recombinant protein was observed by transmission electron microscope. The results showed that the induced recombinant protein could assemble into VLPs. by itself. The immunogenicity of the HBc-GP5 VLPs vaccine was analyzed by using the mouse model and the antibody was detected by ELISA. The results showed that the vaccine could induce the body to produce a higher level of specific antibody. The detection of T cell subsets and cytokines showed that the number of CD3 CD4 T lymphocyte subtypes and the level of IL-4 in the vaccine immunized mice were significantly higher than those in the control group (P0.05). The lymphocyte stimulating index (SI) and the content of IFN- 纬 after the third immunization were significantly higher than those in the control group (P0.05), which indicated that the candidate vaccine of HBc-GP5 epitope VLPs could mimic virus-like particles. Effectively presenting GP5 and inducing humoral and cellular immunity provide data basis for further experimental research.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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