Cloning,bioinformatics analysis and prokaryotic expression of the Lm4b_02327 gene of LIPI-4 from Listeria monocytogenes
Objectives The objective of the study was to obtain the recombinant protein of the LIPI-4 Lm4b_02327 gene in the Listeria monocytogenes LM928 Xinjiang frozen chicken isolate.Methods The specific primers were designed based on the sequence of Lm4b_02327 of strain LM Clip80459(CAS06084.1)in Gen Bank.The Lm4b_02327 gene was amplified by PCR,purified,and cloned into the pMD19-T vector,and the positive clones were screened for sequencing by double digestion.The software was then used to predict the physicochemical properties,secondary structure,and tertiary structure of the protein encoded by the Lm4b_02327 sequence,and the homology of nucleotide sequence and encoded amino acid sequence of the gene fragment was compared.In the meantime,pET32a-Lm4b_02327 recombinant plasmid was constructed,transformed into E.coli BL21(DE3)receptor cells,and the protein was detected by SDS-PAGE,purified,and identified by Western Blot after induced expression.Conclusions The Lm4b_02327 gene of strain LM928 is 315 bp and encodes 105 amino acids.The encoded protein is an acidic,non-signaling peptide,hydrophilic,non-transmembrane-structured,unstable,cytoplasmic protein that is predicted to be a PTSLac EIIA component.The nucleotide and amino acid sequences of the LM928 Lm4b_02327 gene were identical to those of strains CFSAN023463,52854,02-6680,02-6679,ICDC-LM188,LM3,N2306,GTA-L356 and Clip80459 to a degree of 100%;The nucleotide homology with strains 52860,FSL-J1-158,and FSL J1-208 was 99.7%,97.8%,and 97.8%,respectively;and the amino acid homology was 99.0%,94.3%,and 94.3%.The Lm4b_02327 gene of strain LM928 clustered on the same branch as the 4b serotype strain,as shown by the phylogenetic tree.After induction,the pET32a-Lm4b_02327 recombinant plasmid was abundantly expressed in E.coli BL21,and SDS-PAGE and WesternBlot analysis re-vealed that the size of the recombinant protein was approximately 30 kDa,which was consistent with the predicted size.Results In this study,the Lm4b_02327 gene was successfully cloned,and a large quantity of protein expression was obtained,laying foundations for a comprehensive investigation into the function of the protein.
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