牛粘膜病病毒E1基因的克隆及原核表达  

Cloning and prokaryotic expression of the E1 gene of bovine viral diarrhea virus

作　　者：何海娟[1] 王君伟[1] 李昭春[1] 杨玉菊[1] 杨志[1] 

机构地区：[1]东北农业大学动物医学学院

出　　处：《东北农业大学学报》2006年第5期656-661,共6页Journal of Northeast Agricultural University

基　　金：黑龙江省科技攻关项目(GA02B501)

摘　　要：参考BVDVVEDEVAC株的基因组序列及E2基因的测序结果设计一对引物,扩增出预期585 bp的目的片段。扩增产物克隆至pMD18-T载体,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列VEDE-VAC株比较,二者的核苷酸同源性为100%,推导氨基酸同源性为100%。测序结果经同源性比较,克隆得到的基因与VEDEVAC株同源性最高。系统发生树分析推测E1基因与VEDEVAC株在进化上比较接近。将E1基因定向亚克隆到表达载体pGEX-6p-1中,对阳性重组质粒转化的大肠杆菌BL21进行诱导,E1基因获得了成功表达。According to the sequence data of BVDV VEDEVAC strain published by GeneBank, one set of primer was designed and used to amplify E1 gene by the method of RT-PCR. A specific 585 bp DNA product was amplified, which was cloned into pMD18-T vector, and the positive recombinant clone was identified by restriction enzyme digestion. The recombinant plasmid was sequenced and compared with E1 gene of VEDEVAC strain. Compared with Blast on line, the E1 gene of VEDEVAC-like strain exhibits the highest homology with VEDEVAC strain, they shares 100% nucleotide sequence identity and 100% amino acid identity. It suggests that the E1 gene was phylogeneticly closer to BVDV VEDEVAC strain after phylogenetic analysis. The E1 gene. was subcloned into pGEX-6p-1 expression vector, and the recombinant expression plasmid were transformed into engineering bacteria of E.coli BL21, and successfully expressed after induced.

关 键 词：牛粘膜病病毒 E1基因 克隆 序列分析 原核表达 

分 类 号：S852.653[农业科学—基础兽医学]