Study Of The Indivect ELISA For Detection Of The Antibody Of Porcine Transminssible Gastroenteritis Virus And Construction Of The Expression Recombinants For S1 Gene In Lactococcus Lactis

Transmissible gastroenteritis of swine(TGE),characterized by vomit and severe diarrhea,is an acute and highly contagious disease induced by transmissible gastroenteritis virus of swine(TGEV).ELISA method is used to detect TGEV antibodies and recommended by the International Organization for Animal Health(OIE).Attenuated live vaccine and inactivated vaccine are used to control TGE at present.But the attenuated live vaccine has the risk of virulence enhancement and the inactivated vaccine can not get good immune effect.In this study,based on key technologies of prevention and control of TGE,TGEV N gene expression vector was constructed and expressed in E.Coli.Using the expressed N protein ELISA antibody detection test was established.Simultaneously,the lactic acid bacteria as a carrier was used to construct the expression recombinants for TGEV S1 gene,which would lay the foundation for the studies of TGEV new-type vaccines.1.Expression and purification of TGEV N gene.The N gene was amplified by RT-PCR with primers designed according to TGEV strains in GenBank.The N gene was cloned into pCold-II Vector and the recombinant expression plasmid pCold-N was constructed successfully.After induction with IPTG,The N protein was expressed in a soluble form with high efficiency.The protein was purified by Ni Sepharose affinity chromatography resin and hydrophobic phenyl chromatography.SDS-PAGE analysis showed that the expressed N protein was about 45ku,which was the same as the expected results.Western-blotting demonstrated that the expressed recombinant protein could recognize specifically with anti TGEV serum.The protein can be used as detection antigen to establish antibody detection technology.2.Establishment of TGEV indivect ELISA.Using the purified recombinant soluble protein as coating antigen,an indirect enzyme-linked immunosorbent assay(ELISA)was developed for detecting TGEV serum antibodies.The results indicated that the threshold value of ELISA was 0.445;The optimal amount of coating antigen was 101.5ng/well,at 4? for overnight;The uncoated part was blocking at 37?for 1h with PBS containing BSA of 1%;serum sample and the HRP conjugated to secondary antibody were diluted at 1:100 and 1:10000 respectively and incubated at 37? for 1h.Moreover,the recombinant N protein was no cross-reaction with the other 2 swine diseases antibodies(PoRV and PEDV infection).Compared with INGENASA production of TGEV antibody detection kit,the coincidence rate was 94.1%.The method could detect TGEV antibody quickly and effectively,and had good repeatability and specificity,which laid the foundation for the development of standardized diagnostic kits.3.Constuction and expression of recombinant Lactococcus Lactis containing TGEV S1 protein gene.The codons of S1 gene(including sites A and D of S gene)was optimized.The signal peptide Usp45 of Lactococcus lactis and a short peptide(LEISSTCDA)with negatively charge was added In front of the S1 gene.After synthetic the fragments,cloned the S1 gene into the Lactococcus lactis expression vector system and transformed in Lactococcus lactis NZ9000.Western-blotting showed that the S1 protein was expressed in lactic acid bacteria induced with nisin,and the expression product was secreted extracellularly.