The Expression Of α-Enolase Gene Of Mycoplasma Suis And The Esterblishment Of Indirect Elisa Test Method

Mycoplasma suis is a kind of uncultural hemotrophic mycoplasma, and cause a terrible economic loss to pig industury all over the world. The main character of this desease is the fierese infection, which could cause the piggies grow slower, has lower fertilizer ability, and it also could cross infecte with other dieases and make the diagnosis more difficult.According to the whole genome of Mycoplasma suis in GeneBank, designed a pair of specibility primers using the software Primer5.0. The products were double digested by BamH I and Xho I enzymes, subcloned to the prokaryotic expression vector pGEX-4T-1, then constructed the recombinated expression plasmid pGEX-4T-Eno. Then induced by IPTG which concentration is1mmol/L, tested the Mycoplasma suis recombinated a-enolase gene through the SDS-PAGE method, and it succeeded expressing the recombinated protein which molecular weight was about69ku, it was soluble in the plasma. Analysed by the Western blot, the recombinated protein had good antigenicity.Meanwhile, after the recombinated expression plasmid pVAX-4T-Eno was constructed, transfected into the vero cells by the liposome2000. When cultured to70%monolayer cells on the culture vessels, binded into the pig sera and the HRP respectively. Identified by the IF AT, there are many whole green fluorecence which meant this recombinated protein succeeded in expressing.Purificated the Mycoplasma suis a-enolase gene recombinated protein, then used this purified protein as the antigen, established the indirected ELISA method. By screened the conditions, definated the best envelope concentration is5g/mL; The best dilution of the tested sera is1:200; The best dilusion of HRP-rabbit against pig is1:4000; The best funcional time of substract is15min; Through the specibility, repeatability and sencitivity test and the sera sambles test, indicated that the establiehed indirect ELISA test had the strong specibility, good repeatability and the high sencitivity.This test provided the fundimental materials for doing research on the Mycoplasma suis a-enolase gene, lay the fundimention for the further study of Vaccine and animal immunity, and provided a more economical and effectable test method.