单增李斯特菌lmo0331基因的克隆、序列分析及原核表达

doi:10.16431/j.cnki.1671-7236.2018.05.006

Abstract

Abstract:

The experiment was aimed to clone lmo0331 gene of LPXTG motif from Listeria monocytogenes clinical isolate (LM90SB2),analyze the sequence and express it in prokaryotic system.Specific primers were designed according to the sequence of lmo0331 in GenBank.LM90SB2 lmo0331 gene was amplified by PCR,its amplified product was cloned into pMD19-T vector and sequenced for nucleotide sequence and protein domain analysis.The expression plasmid pET32a-0331 was transformed into E.coli BL21 (DE3) competent cells.After induced by IPTG,the recombinant proteins were detected by SDS-PAGE and Western blotting.The results showed that the homologies of LM90SB2 lmo0331 gene with NTSN,F2365,LL195 and WSLC 1033 strains were 100.0%,and the homologies with N2306,02-1792 and H34 strains were 99.9%.LM90SB2 lmo0331 gene was 1 956 bp in length and 1 857 bp in ORF,encoding a total of 618 amino acids.Protein domain prediction showed that lmo0331 protein had NEL,LRR,IR,PulA and LPXTG domains.SDS-PAGE results showed about 88 ku recombinant protein band,and Western blotting result showed that the protein could specifically bind with mouse anti-His-tag monoclonal antibody.In this study,the prokaryotic expression vector pET32a-0331 was successfully constructed,and the expression of lmo0331 fusion protein was achieved,which laid the foundation for further study on the function of lmo0331 gene and the preparation of monoclonal antibodies.

Key words: Listeria monocytogenes; lmo0331 gene; domains; prokaryotic expression

CLC Number:

S852.61

DU Dongdong, ZHANG Qiwen, LI Honghuan, QIAN Lingxiao, MA Xun. Cloning, Sequence Analysis and Prokaryotic Expression of lmo0331 Gene from Listeria monocytogenes[J]. , 2018, 45(5): 1170-1176.

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Cloning, Sequence Analysis and Prokaryotic Expression of lmo0331 Gene from Listeria monocytogenes

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