Abstract:

【Objective】 To study the protein expression effect and immunogenicity of recombinant plasmids of rabies virus G gene, Echinococcus granulosus EgM123 gene and EGFP gene in eukaryotic cells in the hope of constructing the recombinant rabies SRV9 virus vaccine strain with EgM123 gene by reverse geneticstechnology,which can save EgM123 gene recombinant rabies by reverse genetics, it also provides the research basis for the development of rabies and hydatid disease combined gene recombinant vaccine.【Methods】 In this study, we successfully constructed the recombinant plasmid(3033bp) of G gene of rabies virus carrying EGFP enhanced green fluorescent protein EgM123 gene of Echinococcus granulosus and transfected it into BHK-21 cells by liposome transfection. The fusion protein was expressed in BHK-21 cells and observed by fluorescence microscopy, SDS-PAGE polyacrylamide gel electrophoresis. The fluorescent protein expression, molecular weight and immunogenicity of the fusion protein were identified by Western blotting test. 【Results】 The results of recombinant plasmid transfection showed that the recombinant plasmid transfection was effective, the expression of green fluorescent protein could be seen in 48 hours after transfection, and SDS-PAGE polyacrylamide gel electrophoresis showed that the recombinant plasmid was expressed in BHK-21 cells with fusion protein with molecular weight of about 120KDa; Western Blotting results showed that the protein gel transferred PVDF membrane was incubated with rabies virus G protein monoclonal antibody, EgM123 polyclonal antibody and GFP monoclonal antibody respectively, and antigen antibody binding bands were seen at 120KDa. 【Conclusion】 The fusion protein is successfully expressed in eukaryotic cells with EGFP labeled recombinant egm123 gene of rabies virus, and the recombinant plasmid has immunogenicity.

Key words: EGFP gene; EgM123 gene; Rabies virus G gene; fusion protein

摘要：

【目的】反向遗传学技术构建EgM123基因重组狂犬病SRV₉病毒疫苗株,研究狂犬病病毒G基因、细粒棘球绦虫EgM123基因、eGFP基因重组质粒在真核细胞中的蛋白表达效果与蛋白免疫原性,为通过反向遗传学拯救eGFP标记EgM123基因重组狂犬病病毒,制备狂犬病-包虫病二联基因重组疫苗提供研究基础。【方法】将已构建的携带eGFP增强型绿色荧光蛋白的狂犬病病毒G基因重组细粒棘球绦虫EgM123基因重组质粒(3033 bp)利用脂质体转染方法转染BHK-21细胞,使其在BHK-21细胞中表达出融合蛋白,并通过荧光显微镜观察、SDS-PAGE聚丙烯酰氨凝胶电泳、Western blotting试验鉴定融合蛋白的荧光蛋白表达、融合蛋白分子量,鉴定其免疫原性。【结果】在转然后48 h可见绿色荧光蛋白的表达;通过SDS-PAGE聚丙烯酰氨凝胶电泳结果显示,重组质粒在BHK-21细胞中表达获得分子量约为120KDa的融合蛋白;Western blotting结果显示,将蛋白凝胶转移PVDF膜分别经狂犬病病毒G蛋白单克隆抗体、EgM123多克隆抗体、GFP单克隆抗体分别孵育,均在120KDa处可见抗原抗体结合条带。【结论】eGFP标记的狂犬病病毒G基因重组细粒棘球绦虫EgM123基因重组质粒在真核细胞中成功表达出融合性蛋白,且具有免疫原性,

关键词: eGFP基因, EgM123基因, 狂犬病病毒G基因, 融合蛋白