Cloning and expression of the membrane protein gene of TGEV HB06 strain

Abstract

The membrane protein gene(M) of transmissible gastroenteritis virus (TGEV) strain HB06, isolated from the feces of piglets infected with TGEV on a pig farm in Hebei province, was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified PCR products of TGEV HB06 were cloned, sequenced, and compared with other TGEV strains genes selected from the GenBank. Then, the recombinant fragment 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 fusion expression vector pGEX-6P-M. Then, the verified recombinant plasmid was transformed into Escherichia coli Rossetta (DE3), and the expression of M fusion protein was induced by using isopropylthio-beta-D-galactoside (IPTG) as inducer. The results showed that the gene fragment of M at a length of 789 bp was amplified and cloned into the vector pMD18-T successfully, and sequence comparison with that reported in GenBank revealed that the M gene complete sequence shares more than 94% homology in nucleotide. The result of SDS-PAGE showed that the recombinant membrane protein had a molecular mass of approximately 56 kDa, which was the same as the expected results. It was proven by Western blotting that the recombinant membrane protein had strong positive reactions with TGEV-specific antibody. Therefore, the expressed fusion protein has a good antigenicity. This work established a good foundation for further studies on the production of anti-TGEV vaccines.

Keywords

transmissible gastroenteritis virus / M gene / cloning / sequence / expression