Prokaryotic Expression, Subcloning Of Ge Gene Of PRV And Development Of ELISA For The Detection Of Viral Antibodies With The Expression Produce

porcine Pseudorabies（PR）is a highly contagious and infections disease.PRV infeetioncould cause its natural and main infectious origination,bring important economic losses to the pigindustry.It could prevent this diease to take appropriate control measures.and takeappropriate differential diagnosis. gE glycoprotein is one of the major virulence factors.Anindirect ELISAassay was constructed with gE recombinant protein as coating antigen.In this study, a pair of specific primer was designed according to the sequence of gEcomplete gene of PRV published in GenBank.The specific gE complete gene obtained byPCR was cloned into pMD19-T vector.The recombinant plasmid was indentified byrestriction endonuclease analysis.1pairs of primers were designed which were inserted intothe restriction endonuclease BamHI and HindIII sites,A gene fragment encoding the mainantigtic domain of PRVgE was cloned into prokaryotic expression vector pET32a and therecombinant plasmid named pET32a-gE，was transformed into E.coli cell BL21, after theanalysis of gene fragment was proved exactly.The recombinant plasmid was induced byIPTG, then SDS-PAGE indicatesd that the recombinant protein was about45ku, whichwas the same as the expected results and expressed in the form of Solubilities.After purification,the protein pET32a-gE，showed a strong immunological reaction to thePRV positive sera in Western-blotting analysis.Using the purified recombinant protein pET32a-gE，expressed in E.coli cell as coating antigen. anindirect enzyme-linked immunosorbent assay (ELISA) was constructed for detection ofantibody in serum to PRV.The optimal concentration of antigen was6.0μg/mL, the sealingbuffer was PBST with5%fetal bovine serum. The serum sample was diluted by1:40, andincubated at37℃for1h. The dilution of secondary antibody was1:200, and reaction timewas at37℃for1h.The TMB substrate was add for15min before terminated with stoppingsolution. Compared with IDEXX company, the specificity was84.0%, sensitivity was90.0%, and coincidence rate was87%. Moreover, pET32a-gE，protein had no cross-reactionwith CSFV,PRRSV and PPV. The difference value among wells in a plate and among platesfor ELISA was both less than10%.The results showed that the indirect ELISA assay hasgood specificity and repetition.