霍乱弧菌宿主整合因子β亚单位的可溶性克隆表达

Cloning and expression of soluble integration host factor β subunit of Vibrio cholerae

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 and express the β subunit of integration host factor（ IHF） of Vibrio cholerae. Methods PCR was conducted to amplify the ihfB gene with the chromosome of C7258 as template,the product was cloned into vector p XTB1 after digested by NheⅠ and SapⅠ. N-terminal of ihfB gene and C-terminal of ihf A gene encoding α subunit of IHF were joined together byBridge-PCR amplifying. The recombined PCR product was cloned as NheⅠ-SapⅠ fragment into the expression vector p XTB1. Target protein expression was induced with 0. 4 mmol / L IPTG,cell pellet was lysed with sonication treatment,and lysis supernatant was purified with CBD affinity column and eluted by DTT. Results We successfully constructed and expressed the wild-type ihfB gene and the chimera N-ihfB-C-ihf A with the expression vector p XTB1 in the host bacteria in ER2566. In p XTB1-ihfB,the target protein was mainly expressed as insoluble inclusion-body and soluble chimera protein was purified from p XTB1-N-ihfB-C-ihf A. Conclusion C-terminal of IHF-α could significantly increase the soluble expression of IHF-β and soluble chimera protein containing the N terminal of β subunit and C terminal of α subunit was successfully expressed and purified,which is the basis for further function study.