检测猪丹毒杆菌抗体重组SpaA ELISA的建立

Development of recombinant SpaA-based ELISA for detection of antibodies against swine erysipelas

摘要:

【目的】利用表达纯化的猪丹毒杆菌表面保护性蛋白SpaA，建立检测猪丹毒杆菌抗体的间接ELISA方法。【方法】克隆扩增猪丹毒杆菌SpaA基因，并将SpaA基因与原核表达载体pGEX-6P-1连接，通过PCR、双酶切及测序鉴定后，将阳性重组质粒转化入受体菌E. coli Rosetta (DE3)，并利用IPTG进行诱导表达，SDS-PAGE和Western blot鉴定表达产物。将SpaA重组蛋白按不同浓度包被酶标板，通过方阵滴定法确定最佳抗原包被浓度及血清稀释度，并对其他条件进行优化，最终建立检测猪丹毒杆菌抗体的间接ELISA方法。【结果】利用克隆表达的猪丹毒杆菌SpaA蛋白作抗原，通过方阵滴定法确定蛋白最佳包被浓度为1.0 mg/L，血清的最佳稀释度为1:100，建立了检测猪丹毒杆菌抗体的间接ELISA方法，批内及批间变异系数均小于10%，具有较好的重复性及特异性。用建立的间接ELISA方法检测猪丹毒疫苗免疫后的健康猪血清样品，检测结果与美国TSZ公司猪丹毒杆菌抗体检测试剂盒和western blot鉴定结果进行对比，两者总符合率分别为92.20%、92.59%。【结论】本试验利用原核表达的SpaA重组蛋白作抗原建立的检测猪丹毒杆菌抗体的间接ELISA方法，特异性强、重复性好、敏感性高，可用于猪丹毒杆菌的抗体检测及流行病学调查。

Abstract:

[Objective] In the present study, an indirect ELISA was developed using the purified recombinant SpaA protein for detection of the antibodies against Erysipelothrix rhusiopathiae. [Methods] Using gene recombination technology, the SpaA gene was cloned and inserted into prokaryotic expression vector pGEX-6P-1 and the recombinant expression vector was identified with PCR, restriction enzyme digestion and sequencing. The positive recombinant plasmid was then transformed into E. coli Rosetta(DE3) and induced by IPTG. The recombinant protein was detected by SDS-PAGE and Western-blot. An indirect ELISA was developed with the purified protein at different concentrations and the optimal antigen concentration and serum dilution were determined by phalanx titration. The other assay conditions were also optimized. [Results] The optimal coating concentration of SpaA and serum dilution were 1.0 mg/L and 1:100, respectively. Both the intra- and inter-coefficient of variation were lower than 10%, suggesting its reproducibility and repeatability. The developed indirect ELISA method was compared with the ELISA kit supplied by TSZ and Western blot, the coincidence rate was 92.20% and 92.59% respectively. [Conclusion] The developed ELISA had good specificity, reproducibility and sensitivity. It could provide a reliable method for the clinical detection of Erysipelothrix rhusiopathiae and epidemiological investigations．