Sequence Analyzing, Prokaryotic Expressing And Constructing Eukaryotic Expression Vector Of TGEV M Gene Of SC-Y Strain

The research was to amplify and clone the M gene of Porcine Transmissible Gastroenteritis virus of SC-Y strain. Then this sequence was analyzed by bioinformatics methods, and the study was carried out on Prokaryotic Expression and Constructing Eukaryotic expression Vector. The following was the main experimental results:Firstly, One pair of primer was designed to amplify the cDNA sequence of the M gene by Reverse-Transcription-Polymerase-Chain-Reaction (RT-PCR) and a TA cloning recombination plasmid (pMD18-TrM) was constructed. Sequence analysis indicated that there is more than 94% nucleotide homology with TS,HN2002,TFI,96-133,Purdue, Purdue-115,PUR46-MAD, FS772/70 and TOM strains of TGEV. The amino acid sequence homology was not less than 91% which indicated that the M gene conservatism was very high between different TGEV isolated strains. The SC-Y strain M precursor protein have four transmembrane-domains by bioinformatics software, and there has a signal peptide sequence locating in amino-acid residues 1-16 of N termination, and the mature M protein has three transmembrane-domains most possibly. Structural prediction of the SC-Y strain M protein demonstrates that functional structural-domains mainly centralized in amino-acid residues 8-21, which were closely related with its biological function. The utility epitopes domains mainly collected in amino-acid residues 4-30 of N termination or its vicinity by predicting analysis.Secondly, the TA cloning recombination plasmid (pMD18-TAM) that contains the M protein de-signal-peptide gene was constructed, then the objective gene was cloned into pET32a(+) and the recombination prokaryotic expression vector (pET-32a(+)-AM) was obtained. The objective M gene in the pMD18-TrM was transferred and cloned into pET32a(+) and the pET32a(+)-rM was obtained. The Trx-M fusion protein was not...