口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。

High Expression of the Foot-and-mouth Disease's Structural Protein P1 in Escherichia coli and Analysis of its Biology Activity

Foot and mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX KG, resulting in the fusion expression plasmid pKG P1. After transformed into E.coli BL21(DE3)and induced by IPTG, the results of SDS PAGE showed that the GST P1 fusion protein was expressed in high level. The molecular weight of the fusion protein was 110kD and the expressed products were soluble. Western blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1 ELISA) based on the purified proteins was developed. Comparison between P1 ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1 ELISA.