小反刍兽疫病毒N蛋白的原核表达、纯化及其抗体间接ELISA检测方法的建立

Prokaryotic expression and purification of Peste des petits ruminants virus N protein and development of indirect ELISA method for its antibody

目的原核表达小反刍兽疫病毒(Peste des petits of ruminants virus,PPRV)N蛋白,纯化后建立其抗体间接ELISA检测方法。方法人工合成PPRV N蛋白基因序列,并构建重组质粒pSumo-mut-N,转化至大肠埃希菌Arctic Express中,IPTG诱导表达,并对IPTG浓度和诱导时间进行优化。表达的融合蛋白经Ni柱亲和层析纯化后,以其作为包被抗原建立PPRV抗体间接ELISA检测方法,对临床样本进行检测。结果重组表达质粒pSumo-mut-N经双酶切和测序证明构建正确。N蛋白最佳诱导表达条件为0.5 mmol/L IPTG于11℃诱导10 h。表达的重组蛋白相对分子质量约71 900,纯化后纯度在80%以上,浓度可达120μg/ml,与PPRV阳性抗体具有良好的反应原性。基于N蛋白建立的PPRV抗体间接ELISA方法样品检测结果与已知血清之间的符合率为100%。结论原核表达并纯化了PPRV N蛋白,建立的基于N蛋白的PPRV抗体ELISA检测方法为PPRV抗体间接ELISA检测试剂盒的研制和应用奠定了基础,对于流行病学免疫监测及控制PPRV的流行具有重要意义。

Objective To express the N protein of Peste des petits ruminants virus（PPRV） in prokaryotic cells, purify the expressed product and develop an indirect ELISA for its antibody. Methods The N gene sequence of PPRV was synthesized, based on which a recombinant plasmid pSumo-mut-N was constructed and transformed to E. coli Arctic Express for expression under induction of IPTG. The IPTG concentration and time for induction were optimized. The expressed fusion protein was purified by Ni column affinity chromatography, and used as a coating antigen for development of an indirect ELISA method for PPRV antibody. Clinical samples were determined by the developed method. Results Restriction analysis and sequencing proved that recombinant plasmid pSumo-mut-N was constructed correctly. The condition for induction of expression of N protein was optimized as induction with 0. 5 mmol/L IPTG at is 11 ℃ for 10 h.The expressed recombinant protein, with a relative molecular mass of about 71 900, reached a purity of more than 80%after purification and a concentration of 120 μg/ml, which showed good reactogenicity with PPRV positive antibody. The coincidence rate of determination result by the indirect ELISA developed with the N protein and that of known serum samples was 100%. Conclusion The N protein of PPRV was expressed in prokaryotic cells and purified, with which the developed ELISA method laid a foundation of preparation and application of indirect ELISA kit for PPRV antibody, and of an important significance in epidemiological monitoring and control of epidemic of PPRV.