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分枝杆菌穿梭表达质粒pJHSP70的构建及鉴定

, PP. 890-893

Keywords: 结核分枝杆菌,热休克蛋白,启动子,基因表达

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Abstract:

目的用结核分枝杆菌热休克蛋白70(heatshockprotein70,HSP70)启动子改建分枝杆菌穿梭质粒pJEM11为分枝杆菌穿梭表达质粒pJHSP70,并对其功能进行鉴定。方法以卡介苗(BCG)基因组DNA为模板,利用聚合酶链反应(polymerasechainreaction,PCR)扩增HSP70启动子,克隆至pMD18-T载体,经鉴定后,再定向克隆入pJEM11的ApaⅠ位点与SnaBⅠ位点之间,构建pJHSP70载体,并对载体进行PCR及酶切鉴定,然后再将幽门螺杆菌(Helicobacterpylori,HP)尿素酶B亚单位(ureaseBsubunit,UreB)基因克隆入pJHSP70载体,评价其在耻垢分枝杆菌(Mycobacteriasmegmatis,M.smegmatis)mc2155中的表达情况。结果从BCG基因组中扩增出160bp的基因片段,所构建的穿梭质粒经酶切和PCR鉴定与预期结果一致。测序结果证实插入片段正确,UreB基因在M.smegmatis中成功表达。结论成功改建穿梭质粒pJEM11为穿梭表达质粒pJHSP70。

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