猪瘟病毒E2蛋白的原核表达及抗体间接ELISA检测方法的建立

E2 Protein Expression of Classical Swine Fever Virus and Development of Antibody Indirect ELISA Detection

以猪瘟病毒(Classical swine fever virus,CSFV)标准强毒石门株为模板,RT-PCR扩增CSFV E2基因N端的主要抗原区(ΔE2)。PCR产物定向克隆至原核表达载体pET-32a中,构建了重组表达质粒pET-ΔE2,转化Eacterium coli BL21-Codonplus(DE3),IPTG诱导重组ΔE2蛋白表达,经SDS-PAGE和Western-blot鉴定蛋白的正确表达。KCl预染切胶法纯化ΔE2蛋白,以其为包被抗原,通过反应条件的优化,建立了CSFV抗体ELISA检测方法。用该方法对331份临床血清进行检测,并与IDEXX公司阻断ELISA试剂盒进行了相符性验证,符合率为74%。为研制猪瘟病毒抗体ELISA检测试剂盒奠定了基础。

In order to express and purify the recombinant E2 protein of classical swine fever virus（CSFV）,a truncated E2 gene fragment was amplified by RT-PCR with the CSFV virulent Shimen strain as template.After digestion with BamHI/ HindIII,the PCR product was cloned into pET-32a vector.The recombinant plasmid pET-ΔE2 was analyzed by resctriction enzyme digestion and sequencing,then was transformed into Eacterium coli BL21-Condonplus（DE3）.The recombinant ΔE2 protein was expre indirect ELISA Then, 331 swine △E2-ELISA and direct ELISA Kit ssed by the induction of IPTG and identified by （△E2-EL1SA） was developed and optimized with sera samples were detected with the △E2-ELISA IDEXX ELISA Kit was determined as 74%. Furth for the detection of CSFV antibodies. SDS-PAGE and Western-blot analysis. An CSFV antibody the purified recombinant △E2 protein as coating protein. and IDEXX ELISA Kit, the coincidence rate between the er investigation was focused on the development of an in-