Establishment And Application Of An Indirect ELISA With The Major Epitope Domain Of Apxâ…¡ Of Actinobacillus Pleuropneumoniae Expressed In Prokaryotic Expression System

Actinobacillus pleuropneumoniae is known to be a causative agent of a porcine pleuropneumonia that characterized as necrotizing pneumonia and pleurisy cellulose. It has been classified 15 serotypes based on capsular polysaccharide antigens. Apx toxin was the major virulence factor of Actinobacillus pleuropneumoniae, as well a major immunogen. It produced four toxins:Apx Ⅰ, Apx Ⅱ, ApxⅢ, ApxIV. C, A, B, D genes was required for four toxins expressed, and Gene A was structural gene of Apx. In addition to serum 10 type, and the remaining serotypes are produced Apx Ⅱ. Apx Ⅱ-based ELISA method could be established as a method for detecting obstacles across serotypes, which could detect all serotypes except serotypes 10. In this study, it was obtained the recombinant protein of the major epitope domain of Apx Ⅱ from Actinobacillus pleuropneumoniae by prokaryotic expression, and used it as a coating antigen to established an indirect Enzyme-linked immunosorbent assay to detect antibody against Actinobacillus pleuropneumoniae. It was suitable for development of commercial ELISA kit and epidemiological surveys.Firstly, The major epitope domain of Apx Ⅱ of Actinobacillus pleuropneumoniae was cloned into the prokaryotic expression vector pET-28a(+) to obtain the expressed plasmid pET-Apx IIAl. It was transformed into E.coli BL21(DE3) for expression, then the target protein was highly expressed. The best induce time was 5h after added IPTG to 1mmol/mL, and the fusion protein was present as precipitation. Western-blotting analysis showed that the recombinant protein was recognized specifically by rabbit positive serum of Actinobacillus pleuropneumoniae, the recombinant protein had a favourable immunogenicity.Secondly, An indirect Enzyme-linked immunosorbent assay was established to detect antibody against Actinobacillus pleuropneumoniae, it was used the recombinant protein of the major epitope domain of Apx Ⅱ from Actinobacillus pleuropneumoniae as a coating antigen.Then the best reaction condition was confirmed by test optimization. The result showed that the concentration of coated antigen was 1.23μg/mL; the dilution of serum was 1:100; it was coated at 4℃ overnight; it was blocking with 1% BSA at 37℃ for 1h; it was 60min of reaction time between serum and antigen; the optimal dilutions of enzyme labeled antibody was 1:10000. At last, the cut-off value was determined, it was evaluated the stability and speciality of this indirect ELISA, and compared with commercial ELISA kit detecting antibody against ApxIV, the coincidence rate of this indirect ELISA was 90.4%. The results indicated that the ELISA had well speciality, reliability.Finally, We had used the developed indirect ELISA to investigate 552 serum samples which were collected from Jiang Su and An Hui two province in 2013, the total positive rate was 41.67%.460 serum samples were collected from 12 farms of Jiang Su, the total positive rate was 40.65%; 92 serum samples were collected from 5 farms of An Hui, the total positive rate was 46.74%.98 serum samples from pig farm of Nantong and XU zhou, the positive rate of Pregnant sows, fattening pigs, nursery pigs, feeding pigs were 100%、 25%、14.29%、84.21%, respectively.The results showed Actinobacillus pleuropneumoniae were present in most of the pigs farm from two province, and infected in different grown stage pigs。In conclusion, the present study had successfully obtained the recombinant protein of the major epitope domain of Apx Ⅱ from Actinobacillus pleuropneumoniae.It was used as a coating antigen to established an indirect Enzyme-linked immunosorbent assay to detect antibody against Actinobacillus pleuropneumoniae. Then it was used for Epidemiological Surveys of Actinobacillus pleuropneumoniae in Jiang Su and An Hui...