Prokarytic Expression Of Oxaa Gene Extramembranous Protein From Eperythrozoon Suis And The Establishment Of Indirect Elisa

Eperythrozoonosis is caused by Eperythrozoon suis, It mainly exists in the surface of erythrocyte, in the interior of bone marrow and plasma of the swine. It can caused anemia, jaundice and fever.There are no methods to prevent the disease. Meanwhile, there are few functional genes for research. Therefore, in this study, based on the latest genome sequence of E.suis in GenBank, we amplified the1561bp fragment of the OxaA gene with whole gene included, and uploaded to NCBI （accession ID:YP₀04249851.1）. Compared with the original sequence KI₃806（accession ID:NC₀15153）, there were21point mutations that are synonymous mutations; the homology of nucleotide sequence was98%; the homology of amino acid sequence was100%.The structure and the physical and chemical characteristics of OxaA protein were analyzed by means of ExPASy、CPH model data base and TMHMM etc. Results show that, the OxaA protein （PI=9.98） was alkaline; A lot of signal peptide points and five transmembrane regions were discovered in OxaA protein; main component of all secondary structures mainly to a spiral, random coil; the tertiary structure has obvious folded layers. The B cells antigen epitopes were located in the position of20to24,28to40,45to53,142to150and260to263of amino acids. There were two extramembranous domain fragments located in the position of9to63,122to176of amino acids. These two fragments are respectively named A and B temporarily.Two pairs of primers were designed for A and B fragments. The genes were connected to prokaryotic expression vector pGEX-4T-1, induced by IPTG, and then expressed in E. coli BL21（DE3）. The analysis results of SDS-PAGE and Western blotting showed that, the molecular weight of these two recombinant protein are respectively33.9kDa and34.6kDa; the both proteins could be identified by positive pig sera of E.suis and GST tag antibody, which suggested that the recombinant protein has good reactionogenicity. Recombinant protein A is inclusion body, and recombinant protein B is soluble.We established the ELISA method of E.suis with recombinant protein B that purified by GST-tag resin as the identified antigen, and compared with the detecting results of recombinant protein A. By the conditions of the ELISA screened, the6.25μg/mL is the best package concentration of the antigen. Sealed3%skimmed milk reacted at37℃for1h. The detected serum was1:100dilution and HRP-rabbit anti-pig IgG was1:5000dilution, There are all at37℃for1h. The optimal reactive time of the OPD was15min at37℃. Compared with that of the indirect ELISA testing of recombinant protein A protein, the method of recombinant protein B was better in specificity and clinical detection rate than A, suitable for the diagnosis of E.suis infection. The research provides foundation for diagnosis, prevention and control of the Eperythrozoonosis.