猪细小病毒结构蛋白VP2主要抗原表位区基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Major Epitope Domain of Porcine Parvovirus Structural Protein VP2 in Escherichia Coli

作　　者：周洁[1,2] 陈义平[1] 楚电峰[2] 朱吕昌[1,2] 罗飞[1,2] 张秀娟[1] 

机构地区：[1]中国动物卫生与流行病学中心诊断试剂室,山东青岛266032 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国动物检疫》2009年第2期42-44,共3页China Animal Health Inspection

基　　金："十一五"国家科技支撑项目(2006BAD06A12)

摘　　要：本文参照GenBank发表的猪细小病毒结构蛋白VP2基因序列,设计一对引物,通过PCR方法扩增出一段包含VP2主要抗原表位编码区的片段,产物克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32(a)的T7启动子下游,构建的重组质粒pET-VP2经IPTG诱导,在大肠杆菌BL21(DE3)中获得了高效表达。SDS-PAGE结果显示,表达产物分子量为39.1KDa,主要以包涵体形式存在。BandScan分析,表达量约占菌体蛋白的65.2%。表达产物用His亲和层析柱纯化。Western blotting结果显示,该种蛋白能与阳性血清发生特异性反应。结果说明,该重组蛋白具有抗原性,可以作为鉴别诊断用抗原。The major eptiope domain of porcine parvovirus structural protein VP2 was amplified by polymerase chain reaction （PCR） and cloned into pGEM-T vector, then inserted into the downstream of T7 promoter to construct an expression plasmid, pET-32 （a）. After induction by IPTG, the fusion protein was highly expressed in Escherichia Coli BL21 （DE3） in the form of inclusion bodies. BandScan analysis showed that the ratio of the protein is 65.2% to the total protein. The recombinant protein was purified with His-Bind affinity chromatography. SDS-PAGE and Western blotting analysis revealed that the recombinant protein with the expected 39.1KDa could react with pig serum containing antibody against PPV. All results indicated that the recombinant fusion protein can be used as an antigen of diagnostic assay to detect the PPV antibody in serum.

关 键 词：猪细小病毒 结构蛋白VP2 主要抗原表位 原核表达 

分 类 号：S852.659.2[农业科学—基础兽医学]