关键词: 副猪嗜血杆菌, 外膜蛋白P5, 克隆, 表达, 间接ELISA

Abstract: 【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 1﹕400 and 1﹕160，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．

Key words: Haemophilus parasuis, outer membrane protein P5, clone, expression, indirect ELISA