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

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(△E2).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-AE2,转化Eacterium coli BL21-Codonplus(DE3),IPTG诱导重组△E2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△E2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.

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

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 BamHl/ HindIII, the PCR product was cloned into pET-32a vector. The recombinant plasmid pET-△E2 was analyzed by resctriction enzyme digestion and sequencing, then was transformed into Eaeterium coli BL21-Condonplus (DE3). The recombinant △E2 protein was expressed by the induction of IPTG and identified by SDS-PAGE and Westem-blot analysis. An CSFV antibody indirect ELISA (△E2-ELISA) was developed and optimized with the purified recombinant △E2 protein as coating protein. Then, 331 swine sera samples were detected with the △E2-ELISA and IDEXX ELISA Kit, the coincidence rate between the △E2-ELISA and IDEXX ELISA Kit was determined as 74%. Further investigation was focused on the development of an indirect ELISA Kit for the detection of CSFV antibodies.