Asia 1型口蹄疫病毒P1-2A-3C基因真核表达质粒的构建及鉴定

目的构建Asia 1型口蹄疫病毒(FMDV)P1-2A-3C基因真核表达质粒。方法采集疫区牛的水疱液及水疱皮,经RT-PCR法扩增出Asia 1型FMDV的前体蛋白基因P1-2A片段和蛋白酶基因3C片段,分别克隆至pMD18-T载体上,通过酶切、连接,获得质粒pMD18-T-P1-2A-3C。经酶切获得P1-2A-3C片段,插入真核表达载体pVAXⅠpCMV启动子下游,构建pVAXⅠ-P1-2A-3C真核表达载体,用脂质体法转染HeLa细胞,进行IFA检测。结果Asia 1型FMDV真核表达载体pVAX1PⅠ-2A-3C,经酶切鉴定证明构建正确,转染HeLa细胞后,可见明显的黄绿色荧光,表明P1-2A-3C基因得到了表达。结论pVAXⅠ-P1-2A-3C可作为Asia1型FMD核酸疫苗的候选疫苗。

Construction and Identification of Eukaryotic Expression Vector for P1-2A-3C Gene of Foot and Mouth Disease Virus Type Asia 1

Objective To construct a eukaryotic expression vector for the P1-2A-3C gene of foot and mouth disease virus (FMDV) type Asia 1.Methods Amplify viral structural protein precursor P1-2A gene and non-structural protein 3C (proteinase) gene by RT-PCR from the vesiculae of bovine in epidemic area of foot and mouth disease and clone into vector pMD18-T.Digest the constructed recombinant plasmid pMD18-T-P1-2A-3C with restriction endonuclease,and insert the obtained P1-2A-3C gene fragment downstream to the pCMV promoter of pVAXⅠ.Transfect HeLa cells with the constructed eukaryotic expression vector pVAXⅠ-P1-2A-3C in mediation of liposome,and identify the expressed product by IFA.Results Restriction analysis proved that eukaryotic expression vector pVAXⅠ-P1-2A-3C was correctly constructed.The HeLa cells transfected with the constructed recombinant plasmid showed obvious yellowish-green fluorescence under fluorescent microscope,which indicated the expression of P1-2A-3C gene.Conclusion pVAXⅠ-PI-2A-3C might be used as a candidate FMDV DNA vaccine.