Cloning and expression of soluble integration host factor subunit of Vibrio cholerae
摘要: 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)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pXTB1;将ihfB的N端和亚单位基因ihfA的C端通过搭桥PCR重组, 重组片段N-ihfB-C-ihfA经NheⅠ和SapⅠ双酶切后克隆入表达载体pXTB1。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-ihfA的重组表达质粒pXTB1-ihfB和pXTB1-N-ihfB-C-ihfA, 并在宿主菌ER2566中诱导表达。pXTB1-ihfB为不溶性的包涵体表达, pXTB1-N-ihfB-C-ihfA为可溶性表达, 并获得高纯度的表达产物。结论 克隆表达了IHF 亚单位基因ihfB, IHF-的C端可显著提高IHF-的可溶性, 获得可溶性表达的纯化IHF-嵌合体蛋白, 为后续调控功能研究奠定了基础。Abstract: Objective To clone and express the 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 pXTB1 after digested by NheⅠ and SapⅠ. N-terminal of ihfB gene and C-terminal of ihfA gene encoding subunit of IHF were joined together by Bridge-PCR amplifying. The recombined PCR product was cloned as NheⅠ-SapⅠ fragment into the expression vector pXTB1. 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-ihfA with the expression vector pXTB1 in the host bacteria in ER2566. In pXTB1-ihfB, the target protein was mainly expressed as insoluble inclusion-body and soluble chimera protein was purified from pXTB1-N-ihfB-C-ihfA. Conclusion C-terminal of IHF- could significantly increase the soluble expression of IHF- and soluble chimera protein containing the N terminal of subunit and C terminal of subunit was successfully expressed and purified, which is the basis for further function study.
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