Immunogenicity Of Riemerella Antipestifer OmpA Protein

Riemerella anatipestifer (RA) is the causative agent of septicaemic and exudative diseases in a variety of bird species. In this study, what I did is the OmpA gene(Genbank accession No.:CP003388.1) from RA DSM 15868 strain(which Avian Disease Research Center,College of Veterinary Medicine of Sichuan Agricultural University was registered) focuses researches on bioinformatics analysis, cloing, expression, protein purification and polyclonal antibody preparation of RA OmpA gene, development of an ELISA using recombinant outer membrane proteins A in order to detect the serotype 6 Riemerella anatipestifer infections in ducks, immugenicity of RA recombination proteins. The results are as follows:1. Bioinformatic analysis of OmpA gene of Riemerella antipestiferThe whole RA OmpA gene length is 1194bp, encoding 397 amino acid protein with theoretical molecular weight of 43.52kDa and isoelectric point pI4.81, which contained four N-glycosylation sites, nine Protein kinase C phosphorylation sites, seven Casein kinase II phosphorylation sites and three N-cardamom acylation site, but without signal peptide and transmembrand domain. The subcelluar localization analysis showed that the protein is mainly distributed in the outer membrane and the cytoplasm. Phylogenetic analysis showed that amino acids of the protein with other members of Flavobacterium OmpA protein have a low similarity. Analysis of the antigenic protein indicates that the 185-337 amino acids has a continuous strong antigenicity.2. Cloing, expression, protein purification and polyclonal antibody preparation of RA OmpA geneWe designed a pair of primers and used to amplify the OmpA gene which fragment is 1194bp length. A pJET1.2/OmpA cloning vector and pET32a (+) OmpA /expression vector were constructed, and expressed in E.coli BL21 system. The best expression conditions is:37℃,0.4mmol/L IPTG final concentration induced expression of the 4h, under optimal conditions can be a lot of recombinant protein expression. The results of Western blot and SDS-PAGE analysis show that the recombination protein has a better reactogenicity. Finally, the purified recombination protein was used to prepare polyclonal antibody and the agar gel diffusion test results indicate that the titer is 1:16.3. Development of an ELISA using recombinant outer membrane proteins AAn indirect-ELISA was developed and its optimal reactions were determined by using the purified recombinant protein. The optimized conditions were as following: concerntration of antigen 3.6125μg/mL, dilution of sera were respectively 1:100 and 1:400, optimal reaction time were respectively 1.5h and 2h, the confining solution was 1% BSA, the substrate time is 0.5h. According to the 26 negative sera, the threshold value of the ELISA was conformed. When the value of OD450>0.379, its definite positive, when the value of OD450≤0.279, it was negative. The results of repeatability showed that the coefficient of variation is less than 11%, indicating the method has good repeatability. There had no cross-reactivity with the other five kinds of common duck diease, indicating that it was highly specific.4. Immugenicity of RA recombination proteinsUsing purified recombinant proteins as immunogens to immune ducklings. There are three groups:OmpA protein group, OmpA protein with adjuvant and negative groups. To monitor humoral and cellular immune, results show that after the second immunization all the results has significantly increased. The results between OmpA protein group and OmpA protein with adjuvant group after the second immunization have a more significant differences. Showing that if there is only OmpA without adjuvant could not cause strong immunogenicity.