Cloning and expression of the membrane protein gene of TGEV HB06 strain
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Published on: January 7, 2019
Xiaobo Zhang1, Yuzhu Zuo1, Jinghui Fan1
1College of Animal Science and Veterinary Medicine, Agricultural University of Hebei, Baoding, 071001 China.
Abstract:
The membrane protein gene(M) of transmissible gastroenteritis virus (TGEV) strain HB06, isolated from the feces of piglets infected with TGEVon 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.
Insights
Researchers successfully expressed the membrane protein (M) of transmissible gastroenteritis virus (TGEV) in E. coli. This expressed protein shows strong antigenicity, paving the way for developing TGEV vaccines.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Transmissible gastroenteritis virus (TGEV) causes significant economic losses in the swine industry.
- The membrane protein (M) of TGEV plays a crucial role in viral assembly and pathogenesis.
- Effective vaccines are needed to control TGEV outbreaks.
Purpose of the Study:
- To clone, express, and characterize the TGEV HB06 strain's membrane protein (M).
- To assess the antigenicity of the expressed M protein for potential vaccine development.
Main Methods:
- Amplification of the TGEV M gene using RT-PCR.
- Cloning into expression vectors (pMD18-T, pGEX-6P-1) and transformation into E. coli Rossetta (DE3).
- Induction of protein expression using IPTG, followed by SDS-PAGE and Western blotting.
Main Results:
- Successfully amplified and cloned a 789 bp fragment of the TGEV M gene.
- Sequence analysis revealed >94% nucleotide homology with other TGEV strains.
- Expressed M protein showed an approximate molecular mass of 56 kDa and strong reactivity with TGEV-specific antibodies, indicating good antigenicity.
Conclusions:
- The study successfully established a method for expressing the TGEV M protein in a prokaryotic system.
- The expressed M protein demonstrates significant antigenicity, making it a promising candidate for TGEV vaccine development.
- This work provides a foundation for further research into anti-TGEV vaccine production.
