【摘要】 目的探讨小反刍兽疫病毒(Peste des Petits Ruminants Virus,PPRV)N蛋白原核表达、条件优化及反应活性。方法参照GenBank中PPRV (Nigeria/75/1)N基因序列,人工合成该基因,构建重组表达质粒pET-30a (+)-N,转化至BL21 (DE3)感受态细胞,IPTG诱导表达,进行SDS-PAGE电泳、Western-blot分析。在不同温度、时间、IPTG浓度条件下诱导表达N蛋白,确定最佳表达条件。结果 PPRV N蛋白(64.4kD)成功表达,能被羊PPRV免疫血清所识别。N蛋白主要以包涵体存在,其最佳诱导条件:诱导温度28℃、IPTG终浓度2.0mmol/L、诱导时间16h。结论原核表达的PPRV N蛋白具有良好的特异性和反应活性,为单克隆抗体的制备及快速诊断方法的建立提供了技术资料。更多还原

【Abstract】 Objective To explore the prokaryotic expression of N protein of Peste des Petits Ruminants Virus,the optimal conditions and reactivity.Methods According to the sequence of PPRV(Nigeria/75/1)N gene published in GenBank,the gene was synthesized and cloned into the prokaryotic expression vector pET-30a(+).After the recombinant plasmid was obtained,it was transformed into BL21(DE3)competent cells.The expression of the recombinant protein was induced by IPTG,and identified by using SDS-PAGE electrophoresis and Western-blot.The expression of N protein was induced under different temperatures and IPTG concentrations and at different times to determine the optimal expression conditions.Results The PPRV N protein was successfully expressed.The fusion protein had a relative molecular mass of about64.4kD and could be identified by immunized sera from sheep PPRV.The induced expression of N protein was mainly in inclusion bodies.The optimal inducing conditions were a temperature at 18℃and a final concentration of IPTG at 2.0mmol/L with 16 hours of induction.Conclusion The prokaryotic expression of PPRV N protein has good specificity and reactivity,which lays a foundation for the preparation of monoclonal antibodies and the establishment of a rapid diagnostic method.更多还原