Clone Of Eae Gene From Enteropathogenic Escherich Coli And Its Prokaryotic Expression

According to the eae gene sequence of the EPEC.Desine the primers.Amplified the eae gene directly from Enteropathogenic Escherichia coli(EPEC) chromosome DNA by PCR.Then,we could obtain the conservative parts of entire open reading frame of eae gene.It was a unique segment about 1800bp.PCR production was ligated into pMD18-T Vector and transferred into competent cell DH5αthrough TA cloning technology.The method of plasmid extraction,restriction enzyme analysis,PCR,sequencing were used to identify the recombinant cloning plasmid pMD18T-eae.The nucleotide sequence was analyzed by DNASTAR software,the results showed that compared with the Genbank record,this gene has 99.0%homology with the eae protein gene in nucleotides,and amino acids homology was 99.5%.Applying gene in vitro recombnation technology,the PCR production which was retrieved and purified was ligated into an E.coli-Lactobacillus shuttle secretion vector pW425et.The method of plasmid extraction,restriction enzyme analysis,PCR analysis, sequencing were used to screen masccline plasmid pW425et-eae.SDS-PAGE analysis were used to detect the expression condition of eae protein in E.coli DH5α.Results shows that eae protein of EPEC was expressed by pW425et-S in recombinant E.coli DH5α.