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

发布时间：2018-08-09 08:28

【摘要】：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。
[Abstract]:In order to cooperate with the research and development of new swine fever vaccine, an indirect Elisa was established for detection of NS3 protein antibody of swine fever virus (CSFV), in order to effectively distinguish vaccinated pigs from naturally infected pigs (including routine vaccinated pigs). Total RNAs were extracted from PK-15 cells inoculated with Schimen strain of classical swine fever virus (CSFV), and the 2 049bp CSFV NS3 gene was amplified by specific PCR. The recombinant protein NS3 was cloned into the prokaryotic expression vector pET-32a (), with self-aggregation and self-cleavage function and was optimized to express NS3 gene of CSFV Shimen strain in Escherichia coli Rosetta (DE3). Western blot analysis showed that the recombinant protein NS3 was reactive. The purified recombinant protein NS3 was used as the coating antigen to establish indirect Elisa for detection of CSFV NS3 antibody. 502 serum samples were detected according to the results of the antibody detection kit for (Idexx) swine fever virus in the United States. The results showed that the specificity of the method was 96.9.9, the sensitivity was 89.7and the total coincidence rate was 95.8%, which provided a serological method for the popularization and application of the new vaccine against swine fever.
【作者单位】：云南农业大学动物科学技术学院;
【基金】：国家自然科学基金项目(31560704) 云南农业大学自然科学基金项目(2016ZR12)
【分类号】：S852.651

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