Prokaryotic Expression Of 42 KDa OmpA Of RA Serotype 2 And Study Of It's Immunocompetence

In this study, 1045bp gene fragment of the 42KDa OmpA of RA serotype 2 (OmpA-2) was amplified by PCR, then combined with pGEX-4T-l vector, and as a sequence, the recombinant plasmid containing OmpA-2 gene fragment was obtained. The recombinant fusion protein (GST-OmpA₁-2) was expressed at highest level in E.coli BL21 induced by 1mmol/L IPTG for 5h in the form of inclusion bodies. The molecular weights of GST-OmpA₁-2 is about 65 KDa.The inclusion bodies were washed with Deoxycholic acid Sodium Salt (DOC) for several times, and then dissolved in N-Lauroyl Sarcosine Sodium (SKL) for denaturant. After that, a good renature result was obtained by dialysis.The purification of GST-OmpA₁-2 was done by affinity-chromatography of Glutathione SepHarose 4B, electrodialysis method and abrasion method, while only the last two methods attained perfect results.The GST-OmpA₁-2 was used to immunize duck. The indirect ELISA was established to detect the level of antibody of vaccinated ducks with procedure as follows: reaction plate coating by GST-OmpA₁-2 of 0.85μg/mL, 200 X dilution of RA positive serum and then 5000 X diluted HRP-goat antiduck IgG The results of indirect ELISA and Western-blotting showed that GST-OmpAi-2 had good antigenicity.GST-OmpA₁-2 showed not absolute protection for the cluck when challenged with RA Type 2 to the immune ducks.From the study, it can be concluded that GST-OmpA]-2 had good antigenicity and showed no specificity between vary serotypes of RA by blocking ELISA, so application to the clinical detection of RA infection and serological investigate is possible.