人肠三叶肽在大肠杆菌中的表达  

Expression of Human TFF3 in Escherichia coli

作　　者：路睿[1] 王昕[1] 陈建平[1] 陈宪[2,3] 徐佳楠[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫学教研室,成都610041 [2]浙江大学药学院 [3]浙江海正药业股份有限公司,杭州310058

出　　处：《四川大学学报（医学版）》2010年第1期114-117,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30771883);四川大学"大学生创新性实验计划"重点项目资助

摘　　要：目的构建三叶肽(TFF)原核重组质粒pET32a-TFF3,实现TFF3融合蛋白在大肠杆菌中的高效表达并鉴定表达产物。方法用TRIzol试剂提取人结肠组织mRNA,逆转录后经PCR扩增得到不含信号肽的TFF3编码序列,插入原核表达载体pET32a,构建pET32a-TFF3重组质粒,转化大肠杆菌BL-21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,用SDS-PAGE和Western blot检测表达蛋白。结果酶切和测序证实pET32a-TFF3重组质粒构建成功。SDS-PAGE分析表明在IPTG浓度为1 mmol/L时,诱导6 h后蛋白表达量最大。Western blot分析表明,TFF3融合蛋白相对分子质量约为24×103,其能与兔源TFF3抗体特异性结合。结论成功构建了pET32a-TFF3重组质粒,实现了该基因在大肠杆菌中的高效表达,为后续深入研究TFF3奠定了基础。Objective To construct recombinant human TFF3 prokaryotic expressing plasmid, express it in E. coli and identify the expressed protein. Methods The cDNA for mature peptide of TFF3 was amplified by RT- PCR from RNA of human colon tissue and inserted into the MCS of the prokaryotic expressing plasmid pET32a （＋）. Then TFF3 was expressed as a fusion protein by IPTG induction. The recombinant protein was determined by SDS-PAGE and Western blot with a rabbit anti-TFF3 polyclonal antibody. Results Sequencing result indicated that the obtained TFF3 fragment was inserted into plasmid pET32a （＋） successfully and the sequence was correct. The expression level of the fusion protein was highest after 6h induction with 1 mmol/L IPTG. The result of Western blot demonstrated that the relative molecular mass of recombinant protein was about 24 × 10^3 and the protein had good antigenicity and specificity. Conclusion The expression plasmid pET32a-TFF3 was consctructed and expressed successfully. This study will provide a substantial basis for further study of human TFF3.

关 键 词：人肠三叶肽TFF3 蛋白质表达 融合蛋白 

分 类 号：Q786[生物学—分子生物学]