PPRV H蛋白的单克隆抗体制备与在杆状病毒中的表达

发布时间：2019-01-21 19:17

【摘要】：小反刍兽疫(Peste des Petits Ruminants,PPR),是由麻疹病毒属的小反刍兽疫病毒(Peste des Petits Ruminants Virus,PPRV)感染引起的一种急性、烈性、高度接触性传染病。该病是OIE规定的A类疫病,我国规定的一类动物疫病。H蛋白是PPRV的主要结构蛋白之一,具有血凝素和神经氨酸酶活性,可诱导机体产生中和抗体,是疫苗和诊断试剂研发的重要靶标。本研究制备了PPRV H蛋白的单克隆抗体,构建了表达H蛋白的重组杆状病毒,并通过动物实验对其免疫原性进行了初步评价,为建立PPRV的检测方法和新型疫苗研究奠定了基础。具体研究内容如下:1.PPRV H蛋白单克隆抗体的制备及鉴定将PPRV H基因克隆至原核表达载体pGEX-KG中,通过摸索蛋白最优表达条件获得纯化蛋白pGEX-KG-PPRV-H,将此蛋白免疫BALB/c小鼠,通过细胞融合和间接酶联免疫吸附实验及亚克隆技术,筛选杂交瘤细胞株,得到4株可稳定分泌抗PPRV H蛋白的特异性单克隆抗体,分别命名为:1C4、1D8、2A1和2E1。经鉴定,IgG亚型均为IgG1,轻链均为Kappa链。通过间接免疫荧光和Western Blot分析,证实了获得的4株单抗均具有较好的特异性。2.PPRV H蛋白在杆状病毒中的表达及其免疫原性初步研究将PPRV H基因克隆至供体质粒Pfast Bac HTB中,将此重组供体质粒转染DH10bac,经PCR验证,得到重组bacmid-PPRV-H。将重组bacmid-PPRV-H转染sf9细胞,成功拯救出重组杆状病毒Bacmid-PPRV-H。通过间接免疫荧光和Western Blot分析,PPRV H蛋白在杆状病毒表达系统中成功表达。将该重组蛋白免疫BALB/c小鼠,结果表明,Bacmid-PPRV-H蛋白可以诱导小鼠产生针对PPRV的特异性中和抗体。
[Abstract]:Small ruminant disease (Peste des Petits Ruminants,PPR) is an acute, acute and highly contagious disease caused by (Peste des Petits Ruminants Virus,PPRV of measles virus. H protein is one of the main structural proteins of PPRV and has the activities of hemagglutinin and neuraminidase, which can induce the body to produce neutralizing antibodies. It is an important target for research and development of vaccines and diagnostic reagents. In this study, monoclonal antibodies against PPRV H protein were prepared, recombinant baculovirus expressing H protein was constructed, and its immunogenicity was preliminarily evaluated through animal experiments, which laid a foundation for the establishment of detection methods for PPRV and the study of novel vaccines. The specific research contents are as follows: preparation and identification of monoclonal antibody against 1.PPRV H protein. The PPRV H gene was cloned into prokaryotic expression vector pGEX-KG, and purified protein pGEX-KG-PPRV-H, was obtained by exploring the optimal expression conditions of the protein. BALB/c mice were immunized with this protein. The hybridoma cell lines were screened by cell fusion, indirect enzyme-linked immunosorbent assay (Elisa) and subcloning techniques. The specific monoclonal antibodies against PPRV H protein could be secreted stably by four hybridoma cell lines. They were named as: 1C4D8A1and 2E1. The subtypes of IgG were identified as IgG1, light chain and Kappa chain. The expression of 2.PPRV H protein in baculovirus and its immunogenicity were confirmed by indirect immunofluorescence and Western Blot analysis. PPRV H gene was cloned into donor plasmid Pfast Bac HTB. The recombinant donor plasmid was transfected into DH10bac, and the recombinant bacmid-PPRV-H. was obtained by PCR validation. Recombinant bacmid-PPRV-H was transfected into sf9 cells, and recombinant baculovirus Bacmid-PPRV-H. was successfully saved. , PPRV H protein was successfully expressed in baculovirus expression system by indirect immunofluorescence and Western Blot analysis. The recombinant protein was used to immunize BALB/c mice. The results showed that Bacmid-PPRV-H protein could induce the production of specific neutralizing antibodies against PPRV in mice.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

【相似文献】