小反刍兽疫病毒重组N蛋白抗原制备及间接ELISA检测方法的建立

发布时间：2018-07-02 22:26

本文选题：小反刍兽疫病毒 + N蛋白　；参考：《畜牧与兽医》2017年12期

【摘要】：将编码小反刍兽疫病毒(PPRV)N蛋白的基因全长克隆到pBacPAK9载体中,并在昆虫细胞中进行表达。对表达产物进行SDS-PAGE分析,并用PPRV阳性血清进行了Western blot鉴定。用Ni-IDA亲和柱对组氨酸融合表达的N蛋白进行了纯化,并对纯化产物进行了理化性质和抗原活性分析。最后以表达的N蛋白作为包被抗原,建立间接ELISA检测方法,临床检测137份山羊血清并与IDvet公司的商品化PPRV抗体检测试剂盒进行比较。结果表明,制备的PPRV N蛋白抗原在溶液中以单体形式存在,具有非常好的抗原活性。用该蛋白建立的间接ELISA检测方法与IDvet试剂盒符合率为956%。本研究为小反刍兽疫病毒重组N蛋白抗原制备相关质量标准的建立奠定了基础,同时建立的ELISA抗体检测方法可以很好地用于小反刍兽疫的临床诊断。
[Abstract]:The gene encoding the N protein of small ruminant virus (PPRV) was cloned into pBacPAK9 vector and expressed in insect cells. The expressed product was analyzed by SDS-PAGE and identified by Western blot with PPRV-positive serum. The N protein expressed by histidine fusion was purified by Ni-IDA affinity column, and the physicochemical properties and antigen activity of the purified product were analyzed. In the end, the expressed N protein was used as the coated antigen, and an indirect Elisa method was established. 137 goat serum samples were detected and compared with the commercial PPRV antibody detection kit of IDvet Company. The results showed that the prepared PPRV N protein antigen existed in the form of monomer in the solution and had very good antigenic activity. The coincidence rate between the indirect Elisa method and IDvet kit was 956. This study laid a foundation for the establishment of quality standard for the preparation of recombinant N protein antigen of small ruminant disease virus, and the Elisa antibody detection method can be used in the clinical diagnosis of small ruminant disease.
【作者单位】：北京世纪元亨动物防疫技术有限公司;中国动物疫病预防控制中心;
【基金】：公益性行业科研专项经费(201510017-03)
【分类号】：S852.65

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