猪瘟病毒E2基因主要抗原区的克隆及其原核表达

为研究猪瘟病毒E2蛋白的抗原表位在机体免疫应答中的作用和特点,试验参照猪瘟兔化弱毒疫苗株(HCLV)的基因组序列,设计、合成了1对引物,应用RT-PCR方法扩增出长为786 bp的基因片段,命名为zE2,将zE2基因克隆到原核表达质粒pET 32a中,经酶切鉴定后转化大肠杆菌Rosetta(DE3),于37℃、1.0 mmol/L IPTG条件下诱导表达,大肠杆菌菌体裂解产物再经SDS-PAGE和Western-blot分析。结果表明:该基因片段得到较高表达,融合蛋白的分子质量约为48 ku,主要以包涵体形式存在,且具有一定的免疫学活性。

Cloning and prokaryotic expression of a section fragment of Classical swine fever virus E2 gene

A pair of primers were designed and synthesized according to the sequence of HCLV.A fragment about 786 bp(zE2) was amplified from the genome of Classical swine fever virus by RT-PCR.Then the fragment was cloned to the expression vector pET 32a to construct the recombinant plasmid.The recombinant plasmid was proved to be true by restriction endonuclease analysis,transformed to E.coli Rosetta(DE3) competent cells.The expression of the protein was induced with 1.0 mmol/L IPTG at 37 ℃.The products were analyzed by SDS-polyacrylamide gel electrophoresis and western-blot.The results indicated that the gene fragment was more highly expressed,and the recombinant fusion proteins pET 32a-zE2 were expressed highly in E.coli Rosetta(DE3) and it was about 48 ku.The expression product existed mainly in the form of inclusion body and had a good immunogenicity with the antiserum against CSFV.This experiment lays a foundation for diagnostic reagent development.