犬细小病毒南京株VP2蛋白免疫原性片段的克隆表达

以Protean软件对犬细小病毒南京株(CPV-GN)VP2蛋白的分子结构进行分析,从中筛选出具有良好免疫原性潜能的部分片段(VP2′片段),利用PrimerPremier5.0软件设计一对引物,用以该片段的扩增。将PCR扩增产物克隆入pMD18-T载体,构建了pMD-VP2′重组质粒。双酶切回收目的条带,将之亚克隆入表达载体pET32a(+),构建了重组表达质粒pET32-VP2′。转化宿主菌BL21,经IPTG诱导后成功表达了分子量约为34ku的融合蛋白。免疫转印显示,目的蛋白可被CPV-2b抗血清所识别,证明其具有与特异性抗体结合的抗原性,为CPV亚单位疫苗和免疫学诊断方法的研究打下基础。

Cloning and expression of the immunogenic fragment of VP2 protein of canine parvovirus isolated from Nanjing area

The molecular structure of VP2 protein of canine parvovirus isolated from Nanjing area(CPV-GN)was predicted by bioinformatic software,from which a highly immunogenic fragment VP2'was screened and amplified by PCR.The PCR product was inserted into pMD 18-T vector and the resulting recombinant plasmid was double digested with EcoR I and Sal I.The expression vector pET32-VP2',designed to propagate and express the fusion protein in Escherichia coli BL21,was constructed by subcloning VP2' into pET32a( )vector.The desired fusion protein with a molecular weight of 34 ku was detected by SDS-PAGE after induction by IPTG.Subsequent Western bloting assay showed that the recombinant protein could be recognized by the antibody against CPV-2b,and thus has retained the major epitopes of the native protein VP2.