Prokaryotic Expression Of The N Gene Of Transmissible Gastroenteritis Virus And Development Of Indirect ELISA

Transmissible Gastroenteritis of swine(TGE),characterized by vomit and severe diarrhea,is an acute,highly contagious disease induced by transmissible gastroenteritis virus of swine(TGEV),mortality rate of infected piglets is almost 100%.It causes great Economic losses in pig industry.Nucleocapsid is an structure protein and important antigen of TGEV,nucleocapsid protein(N protein)were high level expressed in infection early stage,and N protein antibody could be detected,antigen is ideal for indirect ELISA method.An indirect ELISA method which was established by recombinant TGEV N protein can be detected swine TGEV serum antibody levels in this study.1.A TGEV variation which called JS2012 has been found in Jiangsu province,2012.In this study,the virus's N gene has been gained by RT-PCR,for investigate it's feature.Additional,the N gene of the virus has been linked to pET-28a vector,builded the recombination plasmid.Then,TGEV N protein has been successf?Lly and efficiently expression in E.coli BL21(DE3).The recombinant protein can be expressed in form of fusion protein,and it is easy to large-scale preparation and purification in lab.It has been confirmed that this N protein has Immunologic Activity by Western Blot.2.Indirect ELISA method has been established to detect IgG antibody,and each steps of method has been optimized.The optimal result of ELISA as follow:The antigen concentration and serum samples dilution were set at 0.152ug/ml and 1:80.The coated time was 2h at 37?.The sealing buffer was 5%skim milk,and the sealing time was 30min at 37?.The serum samples were incubated for 30min at 37?.The dilution of the conjugate was defined as 1:10000 and the reaction time was 30min at 37?.The TMB substrate was added and incubated at room temperature for 10min before terminated with 2M H2SO4.Besides,the recombinant N protein is no cross-reaction to PEDV?CSFV?PRRSV?FMDV and PCV2 antibody,indicating that the method has high specificity.The TGEV positive serum was diluted to 1:1280 and it was still tested positive,indicating that the method has good sensitivity;Repetitive tests showed that the CV is less than 10%,indicating that the method has good stability;80 serum samples were tested respectively by indirect ELISA and virus neutralization test,the indirect ELISA test for detection rate was 81.25%,the virus neutralization test detection rate was 82.5%.Results shows,the coincidence rate was 96.3%between the indirect ELISA test and virus neutralization test,which showed that the establishment has good accuracy for the indirect ELISA method.The indirect ELISA established in this study provides a reliable and accurate method for the rapid detection of serum antibodies to TGEV.