【摘要】 目的构建RV-G/LTB双基因融合真核表达质粒,并在Vero细胞中表达。方法应用重叠延伸PCR方法,采用一定的linker序列(Gly4Ser)2将已克隆的狂犬病病毒糖蛋白(Rabies virus glycoprotein,RV-G)基因片段和大肠杆菌不耐热肠毒素B亚单位(E.coli heat-labile enterotoxin subunit B,LTB)基因片段进行融合,扩增融合基因G-linker-LTB,并插入真核表达质粒pVAX1中,构建重组真核表达质粒pVAX1-G-linker-LTB,转染Vero细胞,间接免疫荧光试验和Western blot法检测融合蛋白的表达。结果重组真核表达质粒pVAX1-G-linker-LTB经双酶切及测序鉴定证明构建正确,转染Vero细胞后,可检测到融合蛋白的表达。结论已成功构建了RV-G/LTB双基因融合真核表达质粒,并在Vero细胞中表达了融合蛋白。更多还原

【Abstract】 Objective To construct a eukaryotic expression vector for fusion gene RV-G / LTB and express in Vero cells.Methods The cloned gene fragments encoding rabies virus(RV) glycoprotein(G) and E.coli heat-labile enterotoxin subunit B(LTB) were fused by SOE-PCR using a linker sequence(Gly4Ser)2 and inserted into eukaryotic expression vector pVAX1.The constructed recombinant plasmid pVAX1-G-linker-LTB was transfected to Vero cells,and the expressed fusion protein was identified by indirect IFA and Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pVAX1-G-linker-LTB was constructed correctly,and the expression of fusion protein was proved in transfected Vero cells.Conclusion A eukaryotic expression vector for fusion gene RV-G / LTB was successfully constructed and expressed in Vero cells.更多还原