O型口蹄疫病毒VP1基因的原核表达及间接ELISA检测方法的建立

Prokaryotic Expression of O Type FMDV VP1 Gene and Establishment of Indirect ELISA

表达O型口蹄疫病毒VP1蛋白,用于O型口蹄疫病毒的检测。以质粒T234/FMDV为模板,PCR扩增VP1基因片段,构建重组质粒pET-41b/VP1,转化表达菌BL21(DE3),IPTG诱导,SDS-PAGE凝胶电泳和Western blot进行分析检测,目的蛋白经镍离子亲和层析纯化,以纯化的目的蛋白包被,建立了间接VP1-ELISA检测方法。VP1基因表达的蛋白主要以包涵体形式存在,复性后具有免疫反应性,方阵滴定法确定了包被抗原的最佳工作浓度(1 mg/L),最佳的酶结合稀释度为1∶8 000,检测结果特异性好。O型口蹄疫病毒VP1基因可在表达载体pET-41b中表达,并建立了O型口蹄疫病毒检测方法。

To express O type Foot-and-mouth disease virus VP1 protein for O type FMDV detection.VP1 gene was amplified by PCR from plasmid T234/FMDV as a template,and the recombinant plasmid pET-41b/VP1 was constructed,then transformed into the expression bacteria BL21（DE3）.After IPTG induction,the protein was analyzed by SDS-PAGE and Western blot.The interest protein was purified by Ni-NTA affinity chromatography and the indirect ELISA was established with the purified recombinant protein as coating antigen.The protein was expressed from VP1 gene in the form of inclusion bodies,with immunoreactivity after refolding.The indirect ELISA assay was performed,and the optimal concentration of antigen coating was 1 mg/L,and the optimal dilution of conjugated horseradish peroxidase was 1∶8 000,and test results had higher specificity.O Type FMDV VP1 gene could be expressed in the expression vector pET-41b,and expressed products can be used for establishing the detection of O type FMDV.