猪博卡病毒VP2基因的克隆与原核表达  

Cloning and prokaryotic expression of VP2 gene of porcine bocavirus

作　　者：李彬[1] 杜露平[1] 何孔旺[1] 温立斌[1] 茅爱华[1] 张雪寒[1] 倪艳秀[1] 郭容利[1] 王小敏[1] 周俊明[1] 吕立新[1] 俞正玉[1] 胡屹屹[1] 于洋[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《江苏农业学报》2013年第4期796-799,共4页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(11)2060]

摘　　要：为研究猪博卡病毒VP2蛋白的功能及其在疫病诊断中的作用,对其基因进行了克隆和原核表达。根据猪博卡病毒基因组序列(GenBank登录号GU902967-GU902971)设计了1对引物,采用PCR方法扩增猪博卡病毒的VP2基因,并将其克隆到表达载体pET32a(+)中,构建重组质粒pET32a-VP2,经测序鉴定正确后,将其转化感受态细胞BL21(DE3),并进行IPTG诱导表达。结果表明,重组菌可表达分子量为49 000的融合蛋白,蛋白质表达量较高,且以可溶性形式存在于菌体中。表达产物经纯化后,目的蛋白纯度较好。Western blot结果显示,表达的VP2蛋白能与His抗体发生特异性免疫反应。说明该试验成功克隆了猪博卡病毒的VP2基因并进行了原核表达,为研究该蛋白的功能及建立血清学诊断方法奠定了基础。According to the published sequences of genome of porcine bocaviruses （GenBank accession number GU902967-GU902971）, one pair of primers was designed to amplify VP2 gene by PCR, and then the gene was cloned into the prokaryotic expression vector pET32a （＋） to construct a recombinant pET32a-VP2. After sequencing, the recombinant was transformed into Escherichia coli BL21 （DE3） competent cells. The transformed bacterium was induced by IPTG to produce a recombinant protein of 49 000 of molecular weight. The recombinant protein was in high expression and was soluble in the bacterium. The recombinant protein could react with the antibody of His in a Western blot test. The above indica-ted that the VP2 gene of porcine bocavirus was cloned and expressed in E. coli successfully, which would be helpful to study the function of the protein.

关 键 词：猪博卡病毒 VP2 基因 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]