猪瘟病毒石门株NS3蛋白的原核表达及其抗体检测间接ELISA的建立

为配合猪瘟新型疫苗的研发,建立了猪瘟病毒NS3蛋白抗体检测间接ELISA,以期达到有效区分新型疫苗免疫猪与自然感染猪(包括常规疫苗接种猪)的目的。以接种猪瘟病毒(CSFV)石门株的PK-15细胞为模板提取总RNA,经特异性PCR扩增获得长度为2 049bp的CSFV NS3基因,将其克隆至插入了具有自聚集自切割功能短肽的原核表达载体pET-32a(+),在大肠埃希菌Rosetta(DE3)中优化表达CSFV石门株NS3基因。Western blot分析表明重组蛋白NS3具有反应原性。将纯化的重组蛋白NS3作为包被抗原建立检测CSFV NS3抗体的间接ELISA,以美国爱德士(Idexx)猪瘟病毒抗体检测试剂盒抗体检测结果为标准,对502份血清样品进行检测。结果表明,所建立方法的特异性为96.9%,敏感性为89.7%,总符合率为95.8%,为猪瘟新型疫苗的推广应用提供了血清学检测方法。

Prokaryotic Expression of NS3 Protein in Classical Swine Fever Virus Shimen Strain and Establishment of Indirect ELISA for Antibody Detection

In order to coordinate the new vaccine development,this study established an indirect ELISA for the detection of swine fever virus NS3 protein antibody,to distinguish vaccine immunized pigs and naturally infected pigs(including conventional vaccine vaccination pig)effectively.The total RNA was extracted from PK-15 cells infected with classical swine fever virus(CSFV)Shimen strain,and the CSFV NS3 gene with length of 2 049 bp was obtained by specific PCR amplification.The CSFV NS3 gene was cloned into the prokaryotic expression vector pET-32a(+)with self-immobilized self-cleavage-functional peptide.The expression of NS3 gene was optimized in Escherichia coli Rosetta(DE3).Western blot analysis showed that the recombinant protein NS3 had reactogenicity.The purified recombinant protein NS3 was used as an encapsulated antigen to establish an indirect ELISA for the detection of CSFV NS3 antibody,502 samples were tested on the basis of the antibody test results of Idexx swine fever virus antibody test kit.The result showed that the specificity of this method was 96.9％,the sensitivity was 89.7％,and the total coincidence rate was 95.8％.This research provided the serological diagnosis method for the new vaccine development of classical swine fever.