犬细小病毒VP2基因的克隆及其在大肠杆菌中的表达与检测

Clone,Expression and Detection of CDV VP2 Gene in E.coli

为表达犬细小病毒VP2蛋白(CPVVP2),用于犬细小病毒病的诊断、疫苗研制和VP2蛋白功能研究,根据基因库已发表犬细小病毒序列设计合成了VP2基因的1对特异引物,以细小病毒感染的犬粪样品中提取的总DNA为模板,进行PCR扩增,把扩增产物克隆至pMD18-T载体进行测序、鉴定。将克隆的VP2基因片段克隆至原核表达载体PGEX-6P-1,再将构建成功的原核表达质粒载体PGEX-6P-VP2转化至大肠杆菌BL21中,进行鉴定、测序、表达。结果表明,克隆的VP2基因片段全长1755bp,与6个VP2基因序列的同源性为98.7%～99.7%。Western-blotting分析显示,表达产物约为90kD的融合蛋白,可被犬细小病毒阳性血清所识别,具有良好的反应原性。

To establish a yeast Pichia pastoris expression system expressing canine parvovirus VP2 protein （CPV VP2） to serve the diagnosis of CPV,development of CPV vaccine and research of VP2 protein function.To test the structure and function of VP2 protein in diagnoses and immunization in CDV.A pair of primers was designed and synthesized based on Canine Parvovims （CPV） from GenBank,and the VP2 gene of CPV was cloned from bloody stool of a dead dog by PCR.The results showed that the complete sequence of the VP2 gene was 1755 bp in length.The nucleotide homology respectively was in 98.7%-99.7% .Then the VP2 gene was expressed by expression vector in BL21.The expressed productions were detected by SDS-PAGE and Western-blotting,and the results showed that the VP2 protein was high-efficiently expressed in E.coli.