Expression Of B And C Antigenic Sites Of TGEV Spike Gene In Eucaryotae And Antigenicity Comparison With The Protein Expressed In Prokaryosyte

Porcine transmissible gastroenteritis virus(TGEV) belongs to the family coronaviridae. The virus causes transmissible gastroenteritis (TGE) in all breeds and ages of pigs. Particularly its mortality in the pigs under 2 weeks old may reach 100%. TGEV has been spread worldwide after the domestic TGEV was isolated in 1973. Spike protein on the surface of the virus encoded by S gene can stimulated the protective antibody, S gene consist of A, B, C and D antigenic site are located in the N terminal. Site B and C locating in amino acid 97-144 and 48-52 respectively are deleted in PRCV genome which was considered a variant of TGEV.In order to disclose the molecular differences between them and to investigate the molecular epidemiology and diagnosis, the DNA fragment of the B and C antigen sites was cloned, sequencing analyzed and expressed expecting to gain the expressed protein in prokaryosyte and eucaryotae. Then, the proteins were subjected to Western Blotting assay and Dot-ELISA to testify the immunological ability.A pair of primers for specially multiplication fragment of site B and C by RT-PCR was designed according to the DNA sequence published in GeneBank with using the Oligo 4.01 and Primer 5.0. The recombinant pBlueBacHis2-TG was identified with RE, PCR and sequence analysis. Co-transfection of Sf9 insect cells with pBlueBacHis2-TG and baculovirus linear DNA (Bac-N-Blue DNA) was performed. After three times purification by plaque assay, a recombinant baculovirus was obtained. We generate a high-titer viral stock(-1.06×10~8pfu/mL) which can be used to initiate expression studies in insect cells. Recombinant protein expression levels were optimized. Finally, we gain the soluble recombinant protein.The E. coli with recombinant plasmid pGEX-6P-TS was grown at 28℃ with isopropylthio-β-D-galactoside (IPTG) when optical density of the culture was 0.6. After growing for another four hours, the cultured E. coli was harvested by centrifuge. The way to purification recombinant protein followed the procedures offered by description of GST Gene Fusion System (amersham pharmacia biotech, third edition, revision 2).