Prokaryotic Expression Of M Protein Of Porcine Reproductive And Respiratory Syndrome Virus And Construction Of Indirect ELISA Method

Porcine reproductive and respiratory syndrome (PRRS) which is a highly contagious disease. The disease is mainly made in early abortion, stillbirth and mummy foetus of pregnant sow,as well as swine respiratory disease and reproductive disorders.The global swine industry economy is threatened by large by PRRS.Prokaryotic expressing retained antigen sites truncated M protein of PRRSV,and take the purified recombinant protein as antigen coating enzyme label plate, establishing a diagnosis of PRRS indirect ELIS A method.Amplify fragments that is deletion of N-terminal hydrophobic transmembrane region of M gene by RT-PCR using a pair of primers designed according to CH-1a. Clone fragments into plasmid pMD19-T simple for sequencing,then subclone them into the expression plasmid pET-32a(+),building recombinant expression vector of pET-32a-dM which express dM protein of PRRSV.Convert recombinant vectors into Rosetta and induce the expression.SDS-PAGE result out of a target band of27kDa with expectation.Through the expression condition selection,determine4h as the optimal induction time of the protein expression,37℃as the best induction temperature,and0.8mM of IPTG as the best induction concentration.Soluble analysis showed that soluble part of recombinant protein is about50%.Western-blot results show that the recombinant protein reacted with PRRSV positive serum specifically,showing the antigenicity of it.Take the purified recombinant protein as antigen coating enzyme label plate, known PRRSV positive serum as primary antibody,establishing a preliminary diagnosis of PRRS indirect ELISA method.Determine the optimal concentration of antigen13.07μg/mL,the optimal dilution degree for serum1:80,the optimal dilution degree for second antibody1:4000.Criterion of positive for OD450nm is0.3or higher,or sentenced to negative.The result of specificity test shows that there is no cross reaction with PJEV、TGEV、PEDV、APP and HPS,indicating that the indirect ELISA method has a very good specificity.The average coefficient of variation of repeatability between inter-batch and batches test results were3.53%and6.82% respectively,indicating that the repeatability is good.To compare the indirect ELISA method with PRRSV serum antibody ELISA kit,detecting52serums,the specificity is87.5%,the sensitivity is90%,and the coincidence rate is88.5%,showing that the accuracy of the method is good,and could be used for detection of pig PRRS serum antibody.