非洲马瘟病毒VP7蛋白的原核表达及其间接ELISA方法的建立

Development of an indirect ELISA based on the prokaryotic expression of VP7 gene of African sickness virus

为了建立可特异性地检测血清中非洲马瘟病毒抗体的方法,优化并合成非洲马瘟病毒VP7蛋白基因,将其插入原核表达载体pET-30a(+)中,转化大肠杆菌Rosetta(DE3)中进行表达。SDS-PAGE和Western-blot分析结果显示,目的蛋白获得了高效表达,且有特异的免疫学活性。用纯化的重组蛋白作为检测抗原,建立了检测血清中非洲马瘟病毒抗体的间接ELISA方法,并对该间接ELISA的抗原包被量、血清稀释度、封闭液的选择等反应条件进行了优化。所建立的间接ELISA方法可以鉴别血清中的非洲马瘟病毒抗体,具有良好的特异性和灵敏性,与标准竞争ELISA试剂盒的符合率达100%。表明本研究建立的检测血清中非洲马瘟病毒抗体的间接ELISA方法具有良好的特异性和灵敏性。

To develop a method for specific detection of antibody of African horse sickness virus （AHSV）,AHSV VP7 gene was optimized from GenBank and synthesized, and then transformed into Es- cherichia coli Rosetta（DE3）. The results of SDS-PAGE and Western-blot revealed that the recombinant fu- sion VP7 protein was expressed in a high level and had good reactinogenicity. The purified recombinant VP7 protein was used as detection antigen to establish a ELISA for detection of antibody against AHSV. The reaction conditions in the indirect ELISA such as antigen coating concentration, the dilution of sera and reaction time of sera were optimized. Compared with the standard competitive ELISA,the coincidence rate between the two methods was 100%. The established indirect ELISA was then used to detect antibody against AHSV,which confirmed that the method was sensitive and specific.