猪圆环病毒2型ORF2基因原核表达蛋白间接ELISA检测方法的建立

Establishment of an Indirect ELISA for PCV2 Recombinant Capsid Protein Expressed in E. coli

[目的]建立一种检测PCV2血清抗体的间接ELISA方法。[方法]从已构建的克隆质粒pMD-ORF2上酶切回收ORF2基因的羧基端部分ORFc片段,克隆到pGEX-6P-1表达载体上,构建原核表达质粒pGEX-ORFc,转化入大肠杆菌BL21进行原核表达,用Western-blot法检测纯化蛋白,将其作为抗原包被酶标板,优化间接ELISA试验条件,从而建立检测PCV2血清抗体的间接ELISA方法。[结果]重组表达质粒的酶切鉴定证明已成功构建重组表达质粒pGEX-ORFc,其在E.coliBL21中诱导5、6 h时表达效果最好。纯化后的蛋白条带与原重组菌位置一致均在40.0 kD处,证明重组蛋白得到良好纯化。试验确定抗原最佳包被浓度为12.85μg/m l,血清稀释度为1∶40。3次重复检测表明,变异系数最大为7.12%。[结论]建立的ELISA方法对检测PCV2感染具有良好的特异性及重复性。

[Objective] The study aimed to establish an indirect ELISA for determining the serum antibody against PCV2.[Method] The ORFc fragment from ORF2 gene recovered through the enzyme digestion on the constructed cloning plasmid pMD-ORF2 was cloned and inserted into the prokaryotic expression vector pGEX-6P-1 so as to construct the recombinant plasmid pGEX-ORFc which was transferred into E.coli BL21 for the prokaryotic expression,the inclusion body of recombinant protein was purified and its purified protein was detected by western-blot assay,later,the purified protein was used as the coating antigen of ELISA plate and the indirect ELISA test conditions were optimized,thus,an indirect ELISA for detecting the serum antibody against PCV2 was established.[Result] The identification through the enzyme digestion of recombinant expressed plasmid proved that the recombinant expressed plasmid pGEX-ORFc was constructed successfully and it got best expressing effect when it was induced in E.coli BL21 for 5 and 6 h.The purified protein band was consistent with the original recombinant bacterium at 40.0 kD,showing that the recombinant protein was well purified.The test confirmed that the best coating concn.of the antigen was 12.85 μg/ml and the diluted serum ratio was 1∶40.The repeating test for 3 times showed that the maximum variation coefficient was 7.12%.[Conclusion] The established ELISA method showed the good specificity and repeatability for detecting the PCV2 infection.