多杀性巴氏杆菌OmpA基因的原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2019.07.006

Abstract

Abstract:

The aim of this study was to investigate the prokaryotic expression and bioinformatics character of Pasteurella multocida OmpA gene.Using the genome of Pasteurella multocida HN-01 strain as a template,specific primers were designed to amplify OmpA gene.The recombinant plasmid pET-28a(+)-OmpA was constructed and transferred into E.coli BL21(DE3) competent cells,and the correct recombinant plasmid was identified and expressed by IPTG.The expressed proteins were analyzed by SDS-PAGE and Western blotting,and the OmpA gene sequence was analyzed by bioinformatics softwares.The results showed that the OmpA fragment of Pasteurella multocida was about 1 044 bp,and its coding region sequence was 89.72% homologous to HN-06 strain.After induction,the optimal induction condition of pET-28a(+)-OmpA recombinant strain was 1 mmol/L IPTG at 37℃ for 6 h,and the expressed recombinant protein was about 40 ku in the form of inclusion bodies.Western blotting results showed that about 40 ku of recombinant protein carried His-tag.Bioinformatics analysis found that the OmpA had the formula C₁₆₈₄H₂₆₁₉N₄₅₇O₅₀₅S₃ and was a basic hydrophobin.The amino acids 1 to 21 of the polypeptide chain was signal peptide regions and had various structures.In summary,OmpA might had a special structure,consistent with the structural characteristics of many outer membrane proteins.In this study,the prokaryotic expression system of Pasteurella multocida OmpA gene was constructed,and the OmpA recombinant protein was stably obtained after optimized induction conditions,which provided a theoretical basis for further exploring the pathogenic mechanism of Pasteurella.

Key words: Pasteurella multocida; OmpA gene; prokaryotic expression

CLC Number:

S852.61

ZHENG Yiying, LI Baobao, HUANG Haifeng, ZHANG Zhenxing, ZHANG Luyin, ZHANG Mengmeng, AN Qi, WANG Chengqiang, CHEN Jie, LI Bin, CHEN Zhen, DU Li, WANG Fengyang. Prokaryotic Expression and Bioinformatics Analysis of Pasteurella multocida OmpA Gene[J]. , 2019, 46(7): 1926-1934.

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Prokaryotic Expression and Bioinformatics Analysis of Pasteurella multocida OmpA Gene

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