Cloning, Expression Of M Gene Of Porcine Transmissible Gastroenteritis Virus And An Indirect ELISA For The Detection Of Viral Antibodies With The Expression Produce

Transmissible gastroenteritis of pigs (TGE), characterized by vomit and severe diarrhea, is an acute, highly contagious disease induced by transmissible gastroenteritis virus of pigs (TGEV). In this study, first, a pair of specific primer was designed and synthesized according to the sequence of membrane (M) protein gene of TGEV published in GenBank. A fragment of M gene was amplified from RNA of TGEV strain HB06 by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into pMD18-T Vector, and then the recombinant plasmid was transformed into competent cells of Escherichia coli DH5α. The recombinant plasmid was indentified by PCR and restriction endonuclease analysis. The results showed that the gene fragment of M at length of 789 bp was amplified and cloned into the vector pMD18-T successfully. Sequence analysis indicated that the M gene complete sequence of TGEV strain HB06 shares more than 94% nucleotide homology with that reported strains of TGEV in GenBank. The amino acid sequence homology was not less than 92% which indicated that the M gene conservatism was very high between different TGEV isolated strains.The gene fragment of M in pMD18-T was subcloned into corresponding sites of prokaryotic expression vector pGEX-6P-1 after digestion with EcoRI and XhoI to construct a recombinant expression plasmid pGEX-6P-M, and then the recombinant expression plasmid was identified by restriction endonuclease analysis and PCR method. The recombinant expression plasmid was transformed into competent cells of Escherichia coli Rossetta (DE3) and induced by 0.7mmol/L IPTG as inducer, then analyzed by SDS-PAGE. The results showed that the recombinant membrane protein appeared a molecular mass of approximately 56 ku, which was the same as the expected results, and the recombinant membrane protein was highly expressed in Escherichia coli Rossetta (DE3) in the form of inclusion bodies. Western-blotting showed that the recombinant membrane protein could be recognized by rabbit anti-serum to TGEV, so the expressed fusion protein shared good antigenicity.Using the purified recombinant membrane protein as coating antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibody in serum to TGEV. The optimal reaction conditions for the assay were determined. The results indicated that the threshold value of ELISA was 0.34; optimal reaction condition and working concentration were as follows: incubating the coating 100μL of recombinant antigen (0.7μg/mL) at 37°C for 1 h and at 4°C for overnight; blocking the uncoated part at 37°C for 1 h with PBST containing fetal bovine serum of 5%; diluting serum sample or the HRP conjugated to secodary antibody (goat anti-pig IgG-HRP) with blocking buffers at 1:80 or 1:400 and incubating it at 37°C for 1 h or 45 min; reacting with HRP protected from light at room temperature for 15 min after adding substrate.Moreover,the recombinant membrane protein showed no cross-reaction with positive sera of other 4 swine diseases (PEDV, CSFV, PRV and PRRSV infection). It was shown that the indirect ELISA had good specificity.In summary, it chould be concluded the expressed recombinant membrane protein had high antigenicity. Using it as antigen, indirect ELISA shared good sensitive and specific, and provided an available technique for immune detection and serological survey of TGE.