流行性乙型脑炎病毒结构蛋白编码基因的重组构建

Construction of genes encoding structure proteins derived from Japanese encephalitis virus

目的:建立流行性乙型脑炎病毒(JEV)C、prME,E等结构蛋白编码基因克隆。方法:病毒感染C6/36细胞,RT-PCR法依次获取该病毒结构蛋白基因并进行DNA测序分析,PCR克隆法将PCR产物分别构建于真核表达载体pcDNA 3.1的BamH I与EcoR I酶切位点并依次命名为pJC、pJME与pJE,通过DNA测序、电泳以及限制性内切酶分析加以鉴定。结果:JEV C、prME与E基因片段分别为380、2 001以及1 500 bp,各基因测序结果与发表的JEV JaGAr-01株相对应序列相一致,从重组质粒释出的插入子分别与各基因大小相符合。结论:pJC、pJME与pJE重组质粒分别含有C、prME与E蛋白编码基因。

Objective： To set up clone of genes encoding C, prME, and E structure proteins derived from Japanese encephalitis virus （JEV） JaGAr-01 strains. Methods： C6/36 cells were infected with 2.7 × 10^7pfu/ml of JEV JaGAr-01 strains. The fragments of JEV C, prME, and E gene amplified with RT-PCR method were sequenced by ABI PRSM^TM 310 Genetic Analyzer. Identified RT-PCR products were inserted between the site BamHI and EcoRI of eukaryotic vector pcDNA 3.1. The constructs containing JEV C, prME, and E genes were named after pJC, pJME, and pJE, respectively. Above recombinants were identified by DNA sequencing, and 1% agarose gel electrophoresis and restricted endonuclease enzyme analysis were performed. Results： The sizes of RT-PCR products were 380 （C） ,2 001 （ prME）, and 1 500 （E） bp, and their sequences were corresponded to those of the published JEV JaGAr-01 strains. It was confirmed that inserts released from pJC, pJME, and pJE were the same as those of RT-PCR products. Conclusion： pJC, pJME, and pJE contain genes encoding JEV C,prME, and E proteins, respectively.