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目的克隆表达霍乱弧菌全局性调节子宿主整合因子(integration host factor,IHF)β亚单位基因ihfB。方法以C7258染色体为模板,聚合酶链反应(polymerase chain reaction,PCR)扩增ihfB基因,扩增产物经NheⅠ和SapⅠ酶切后克隆入表达载体p XTB1;将ihfB的N端和α亚单位基因ihf A的C端通过搭桥PCR重组,重组片段N-ihfB-C-ihf A经NheⅠ和SapⅠ双酶切后克隆入表达载体p XTB1。重组质粒导入大肠埃希菌ER2566,0.4 mmol/L IPTG诱导表达,超声裂解,上清经CBD柱吸附纯化,二硫苏糖醇(DTT)柱上裂解洗脱。结果成功构建表达野生型ihfB基因和嵌合体N-ihfB-C-ihf A的重组表达质粒p XTB1-ihfB和p XTB1-N-ihfB-C-ihf A,并在宿主菌ER2566中诱导表达。p XTB1-ihfB为不溶性的包涵体表达,p XTB1-N-ihfB-C-ihf A为可溶性表达,并获得高纯度的表达产物。结论克隆表达了IHFβ亚单位基因ihfB,IHF-α的C端可显著提高IHF-β的可溶性,获得可溶性表达的纯化IHF-β嵌合体蛋白,为后续调控功能研究奠定了基础。
Objective To clone the ihfB gene of integrative host factor (IHF) of Vibrio cholerae. Methods The ihfB gene was amplified by polymerase chain reaction (PCR) using the C7258 as a template. The amplified product was digested with Nhe I and Sap I and cloned into the expression vector p XTB1. The N-terminal and α-subunit genes of ihfB Recombinant fragment N-ihfB-C-ihf A was digested with Nhe I and Sap I and cloned into the expression vector p XTB1. Recombinant plasmids were introduced into Escherichia coli ER2566 and induced with 0.4 mmol / L IPTG. The recombinant plasmids were lysed by sonication. The supernatant was purified by CBD column and lyophilized on dithiothreitol (DTT) column. Results The recombinant expression plasmids p XTB1-ihfB and p XTB1-N-ihfB-C-ihf A expressing the wild-type ihfB gene and chimera N-ihfB-C-ihf A were successfully constructed and induced to express in the host strain ER2566. p XTB1-ihfB is expressed in insoluble inclusion bodies, p XTB1-N-ihfB-C-ihf A is soluble in expression, and high purity expression products are obtained. Conclusions The IHFβ subunit gene ihfB was cloned and expressed. The C-terminal of IHF-α significantly increased the solubility of IHF-β, and obtained the soluble expression of IHF-β chimeric protein, which laid the foundation for further studies on regulatory function.