猪博卡病毒NP1基因的克隆与原核表达  

Cloning and Prokaryotic Expression of NP1 Gene of Porcine Bocavirus

作　　者：李彬[1] 毛立[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] 马俊杰[1] 周俊明[1] 吕立新[1] 俞正玉[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《华北农学报》2011年第3期28-31,共4页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31001066);江苏省农业科技自主创新资金(cx(09)619);国家农业公益性行业专项(200803020)

摘　　要：猪博卡病毒是近年来新发现的一种细小病毒。本研究根据其部分基因组序列(GenBank登录号GU902967-GU902971)设计了一对引物,采用PCR方法扩增出了猪博卡病毒的NP1基因,将其克隆到表达载体pET32a(+)中,构建了重组质粒pET32a-NP1,经测序鉴定正确后,将其转化感受态细胞BL21(DE3)中,并进行IPTG诱导表达。结果表明,重组菌可表达分子量为46 kDa的融合蛋白,蛋白表达量较高,而且蛋白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NP1蛋白能与His抗体发生特异性的免疫反应,表明原核表达的NP1蛋白具有良好的反应原性。Porcine bocavirus（PBoV） was a recently discovered parvovirus from pigs.One pair of primers was designed according to the published sequences of genome of the porcine bocaviruses（GenBank accession number GU902967-GU902971）.The NP1 gene was amplified by PCR and cloned into the prokaryotic expression vector pET32a（＋） to construct a recombinant pET32a-NP1.After sequencing,the recombinant was transformed into Escherichia coli BL21（DE3） competent cells.The transformed bacteria were induced by IPTG to produce a recombinant protein of 46 kDa of molecular weight.The result showed that recombinant protein was higher expression and purity by the Ni-NTA resin,and in soluble form in the bacteria.The recombinant protein could react with the antibody of His in a Western blot test,indicating that the expressed protein possessed strong reactinogenicity.

关 键 词：猪博卡病毒 NP1基因 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学] S855.3[农业科学—临床兽医学]