Expression of outer membrane protein p5 gene of haemophilus parasuis and establishment of an indirect elisa based on the omp5 protein | 副猪嗜血杆菌OMP5基因的克隆、表达及间接ELISA检测方法的建立

Chinese. 【目的】利用表达纯化的副猪嗜血杆菌（Haemophilus Parasuis，HPS）外膜蛋白P5（outer-membrane protein P5，OMP5），建立检测副猪嗜血杆菌抗体的间接ELISA 检测方法。【方法】克隆扩增HPS OMP5基因，并将OMP5基因与原核表达载体 pET-32a(+)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BL21（DE3），经IPTG诱导表达。亲和层析法纯化蛋白，尿素梯度透析复性，Western blot鉴定表达产物。将超声破碎抗原和复性蛋白分别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，并对其它条件进行优化，最终建立检测副猪嗜血杆菌抗体的间接ELISA方法。【结果】利用克隆表达的HPS OMP5蛋白做抗原，通过方阵滴定法确定蛋白最佳包被浓度为 1:400，血清的最佳稀释度为1:160。用建立的间接ELISA 方法检测317份未进行HPS免疫的健康猪血清，结果检出72份阳性，检出率为22.71%，与广东地区发病猪中的HPS分离率24%接近。同时，用该方法与用超声破碎抗原建立的ELISA方法同时检测了78份血清样品，结果，该法检出27份阳性，检出率为34.62%，后者则检出31份阳性，检出率为39.74%，二者符合率为71.79%。【结论】本研究利用重组表达的OMP5蛋白作为抗原建立的检测副猪嗜血杆菌抗体的间接ELISA 方法，特异性好、重复性好，检出率与HPS临床分离率接近，可用于副猪嗜血杆菌的临床检测、流行病学调查和免疫监控。

English. 【Objective】 In the present study，an indirect ELISA was developed using the purified OMP5 protein to detect the antibody of HPS．【Method】Using gene recombination technology，the OMP5 gene was cloned and inserted into prokaryotic expression vector pET-32a (+) and the recombinant expression vector was identified with PCR，restriction enzyme digestion and sequence analysis．The positive recombinant plasmid was then transformed into E.coli BL21 (DE3) and induced by IPTG．The expressed protein was purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE．The purified protein was refolded by urea gradient dialysis，and its specificity was tested by Western blot．An indirect ELISA was developed with the purified protein and supersonic bacterial antigen at different concentrations and the optimal antigen concentration and serum dilution were determined by phalanx titration．The other assay conditions were also optimized．【Result】The optimal coating concentration of OMP5 and serum dilution were 1s400 and 1s160，respectively. Three-hundred and seventeen swine sera collected from healthy pigs which were never immunized with HPS were assayed by the ELISA with urea treated antigen. Seventy-two samples were positive，and the positive rate was 22.71%，which was closed to the isolating rate of 24% from HPS naturally infected pigs. Seventy-eight serum samples were assayed using the ELISA coated with the supersonic HPS antigen. Twenty-seven samples were detected as positive with the ELISA coated with the purified OMP5 protein and the detection rate was 34.62%，and 31 positive was detected with the supersonic bacterial antigen coated ELISA and the detection rate was 39.74%．Coincidence of the two methods was 71.79%．【Conclusion】The developed ELISA had good specificity and reproducibility．It could provide a reliable method for the clinical detection of HPS，epidemiological investigations，and immunization surveillance．