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  

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

作　　者：霍晓伟[1] 金宁一[2] 鲁会军[2] 马鸣潇[3] 牟伟峰[4] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062 [3]辽宁医学院畜牧兽医学院,锦州121001 [4]延边大学农学院,延边133002

出　　处：《中国生物制品学杂志》2008年第5期371-373,377,共4页Chinese Journal of Biologicals

基　　金："863"重大项目(2006AA10A204)

摘　　要：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。Objective To construct a eukaxyotic 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 pMDI8-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 pVAX I -P1-2A-3C in mediation of liposomo,and identify the expressed product by IFA.Results Restriction analysis proved that eukaryotic expression vector pVAX I -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. Cone.lusion pVAX I -PI-2A-3C might be used as a candidate FMDV DNA vaccine.

关 键 词：口蹄疫病毒 P1-2A-3C基因 真核表达 核酸疫苗 

分 类 号：Q786[生物学—分子生物学]