Expression Of Transmissible Gastroenteritis Virus Spike Protein In Insect Cells And Development Of An Indirect ELISA For Detecting Antibodies

Transmissible gastroenteritis(TGE) is a sever infectious disease which is caused by Transmissible gastroenteritis coronavirus (TGEV). It infects the enteric and respiratory tissues of newborn piglets and can cause severe and often fatal diarrhoea in young pigs resulting in mortality of nearly 100%. It has to be affirmed by the laboratory diagnosis because it's clinical symptom and the epidemic characteristics are much the same as that of PED, PRV and PRC.The study of the induction of protective immunity to TGEV has focused on S protein because it is the major inducer of TGEV-neutralizing antibodies and it mediates binding of TGEV to its cellular receptor. The four major antigenic sites (A, B, C and D) of S protein have been described on the amino-terminal domain. And earlier studies indicated that the intact globular N-terminal half of the protein is sufficient to achieve a protective immune response equivalent to that induced by the full S protein. Sites A,B and D are highly conservative. In this study, The main sites of transmissible gastroenteritis virus spike protein A and D was expressed in insect cells using the Baculovirus Expressing System and is used as the antigen to develop an indirect ELISA.According to GenBank, a pair of primers was designed aiming at sites A and D which were on the amino-terminal domain. The DNA fragment was amplified by RT-PCR and was cloned into the contribution vector pFastBac1 to get the recombinant plasmid pFastBac1-TS1. Plasmid pFastBac1-TS1 was then introduced into E.coli DH10Bac, which included a shuttle vector, Bacmid. By site-specific transposition, S gene sites A and D was integrated into bacmid, and a recombinant shuttle vector was constructed, named Bacmid-TS1. The recombinant bacmid, which was identified by PCR, was transfected into Sf9 insect cells with Transfast Transfection Reagent Kit. The cell CPE could be observed within 5 days after transfection. And the pure recombinant baculovirus was obtained by plaque. Expression of TS1 protein (about 43KD) was examined and identified by RT-PCR, IFA, SDS-PAGE and Western-blotting.The recombinant transmissible gastroenteritis virus spike protein expressed in the Baculovirus Expressing System was used as antigen for developing an indirect enzyme-linked immunosorbent assays(ELISA). This indirect ELISA method was used to detect 240 sera. The results indicated that this diagnose method had good sensitivity and differentiation. It provided a quick and easy serological diagnose method which can surveil the changes of antibody level of the immuned swinery and the investigate the epidemiology of TGE.