FimH重组蛋白的原核表达及纯化  

Prokaryotic Expression and Purification of Recombinant Protein FimH

作　　者：刘忠芳[1] 陈林[1] 殷晓佳[1] 李晓霞[1] 李宇红[1] 孟柳燕[1] 

机构地区：[1]武汉大学口腔医学院口腔生物医学教育部重点实验室,湖北武汉430079

出　　处：《口腔医学研究》2013年第10期893-896,共4页Journal of Oral Science Research

基　　金：国家自然基金面上项目资助(编号:81070822);大学生科学研究项目(编号:S2013700)

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。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 PET28a vector, 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 20h with 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 nlckel- charged column. Conclusion： This study aims to build three FimH as expression vector of the surface rendering system ,analyse FimH gene and predict protein structure.

关 键 词：FimH 大肠杆菌 沙门氏菌 重组蛋白 表达和纯化 

分 类 号：R392[医药卫生—免疫学]