牛源无乳链球菌菌毛岛屿AP2蛋白抗原优势区的原核表达及其间接ELISA检测方法的建立

Prokaryotic expression of the AP2 gene of bovine Streptococcus agalactiae and establishment of an indirect ELISA technique

目的构建牛源无乳链球菌AP2蛋白原核表达质粒,表达AP2蛋白,并以此为包被抗原建立检测无乳链球菌抗体的ELISA方法。方法取pMD18-T-AP2和pET-30a(+)分别进双酶切,酶切片段连接后转染BL21(DE3)细胞,进行AP2的诱导表达及鉴定。以重组AP2为抗原建立检测无乳链球菌抗体的ELISA方法,并用无乳链球菌、化脓性链球菌、大肠埃希菌和金黄色葡萄球菌感染兔血清进行检测。结果双酶切及PCR鉴定重组质粒构建正确。Western blot分析表明重组AP2蛋白具有反应原性,建立的间接ELISA抗原最佳包被浓度为25μg/ml,血清最佳稀释度为1∶160,酶标二抗最适稀释度为1∶70。抗体滴度检测显示重组AP2具有良好的免疫原性,3次免疫(56 d)的兔血清抗体滴度达1∶10 000。用该方法检测化脓性链球菌、大肠埃希菌和金黄色葡萄球菌感染兔血清,均未出现交叉阳性反应。结论成功表达了重组无乳链球菌菌毛岛屿AP2蛋白,建立的间接ELISA方法稳定性高,特异性良好,可用于无乳链球菌感染检测。

Objective To establish an ELISA technique to detect antibodies against Streptococcus agalactiae using an AP2 protein prokaryotic expression plasmid and bovine AP2 protein as an encapsulated antigen. Methods PMD18-T-AP2 and pET-30 a(+) were digested with two enzymes, and the enzymatic fragments were ligated and transformed into BL21(DE3) cells to induce the expression of and identify AP2. An ELISA technique to detect antibodies against S. agalactiae was established using recombinant AP2 as the antigen, and rabbit serum was tested for S. agalactiae, S. pyogenes, Escherichia coli, and Staphylococcus aureus. The recombinant plasmid was successfully constructed using double-enzyme digestion, and it was identified with PCR. Western blot analysis indicated that the recombinant AP2 protein has reactive properties. The indirect ELISA antigen had an optimal coating concentration of 25 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the HRP-conjugated antibody was 1:70. An antibody titration test indicated that recombinant AP2 had good immunogenicity. The serum antibody titer for 3 immunizations(56 d) reached 1:10,000 in rabbits. Conclusion A recombinant AP2 protein from S. agalactiae was successfully expressed. An indirect ELISA technique was established with high stability and good specificity, and it can be used to detect a streptococcal infection.