【摘要】 目的:在大肠杆菌中构建并表达3种FimH重组蛋白,并对FimH重组蛋白的培养条件进行优化,制备高纯度FimH重组蛋白。方法:采用原核表达载体pET28a构建出能表达FimH的重组质粒,并分别对诱导温度、IPTG浓度、诱导时间等条件进行优化,以提高重组蛋白的产量和增大其可溶性,最后以亲和层析分离纯化3种FimH重组蛋白。结果:成功构建出表达大肠杆菌(K12,BL21)和沙门氏菌(S.T.)FimH的原核表达载体pET28a-K12/BL21/S.T.,经酶切、测序和Western Blot鉴定,目的蛋白表达正确。在适当浓度的IPTG诱导,15℃震荡培养20h,可使FimH的表达量达到最大,分离纯化的蛋白在SDS-PAGE中显示为一条带。结论:本研究构建以3种FimH为表面呈现系统的表达载体,3种FimH基因和预测蛋白结构略有差异。本研究为后续研究3种蛋白生物活性差异及其靶向M细胞的免疫佐剂活性提供物质基础。更多还原

【Abstract】 Objective:To express and purify three recombinant FimH in E.coli BL21(DE3)plysS and optimize the cultivation regulation.Methods:The recombinant FimH was expressed in the PET28 avector,it was detected by SDS-PAGE under different cultivation conditions,including temperature,concentration of IPTG and time.After that,the expressed recombinant protein FimH was purified using immobilized metal ion affinity chromatography and identified by SDS-PAGE.Results:It was found that the expression level of FimH achieved the highest when the BL21(DE3)plysS had been induced for 20 hwith suitable IPTG beginning from strain density of A600=0.6 at 15℃.FimH was purified from the soluble phase by means of His-tag affinity chromatography on a nickelcharged column.Conclusion:This study aims to build three FimH as expression vector of the surface rendering system,analyse FimH gene and predict protein structure.更多还原