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SOS反应不仅能够促使细菌本身产生耐药性,而且与细菌耐药基因的水平转移密切相关。rec A是细菌SOS反应的重要基因,影响SOS反应的开启和关闭。本研究以水产养殖经济动物的条件致病菌哈氏弧菌ZJ0603基因组DNA为模板,设计同源引物克隆出SOS基因rec A(recombinase A),并对rec A基因进行了生物信息学分析。并构建了rec A基因的原核表达载体PGEX-rec A,在大肠杆菌BL21(DE3)中成功诱导表达,对Rec A蛋白的诱导温度、时间、IPTG浓度等条件进行了优化。结果表明,rec A基因的开放阅读框为693 bp,编码230个氨基酸,Rec A蛋白理论分子量为25.29 k D。重组蛋白表达的优化条件为37℃、0.04 mmol/L IPTG诱导6 h。
Abstract：SOS response can make the bacteria produce resistance,and it is closely related to horizontal transfer of bacterial drug resistance genes.rec A is an important gene for bacterial SOS reaction,which controls the opening and closing of SOS reaction.In this study,the homologous primers was designed to clone the SOS gene rec A basing on the genomic DNA of Vibrio harveyi ZJ0603,which was the conditional pathogen of the economic animals of aquaculture.And the bioinformatics analysis on rec A was performed.The prokaryotic expression vector PGEX-rec A of rec A gene was constructed,which was successfully induced and expressed in coli BL21(DE3).The conditions of the induction temperature,time and IPTG concentrations for Rec A protein were optimized as well.The results showed that the open reading frame of rec A gene was 693 bp,encoding 230 amino acids,and the theoretical molecular weight of Rec A protein was 25.29 k D.The optimal conditions for the expression of recombinant protein were inducing with 0.04 mmol/L IPTG for 6 hours at 37℃.
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基本信息:
DOI：10.13417/j.gab.037.001947
中图分类号:Q78;S941.42
引用信息:
[1]常云胜,周维,高增鸿,等.哈氏弧菌(Vibrio harveyi)recA基因的克隆与原核表达[J].基因组学与应用生物学,2018,37(05):1947-1954.DOI:10.13417/j.gab.037.001947.
基金信息:
广东省教育厅高等学校高层次人才项目(“谷胱甘肽及其合成酶系在哈氏弧菌耐药中的作用机制研究”);; 农业部行业专项(No.201203085);; 广东省自然科学基金(2014A030313604)共同资助
2017-09-21
2017-09-21
2017-09-21