hFOXP3-Δ E251的克隆及在大肠杆菌中的表达  

Cloning and expression of hFOXP3-Δ E251 in E.coli

作　　者：蔺昕[1,2] 许逊[2] 宗扬勇[2] 邵启祥[2] 

机构地区：[1]南京医科大学附属南京第一医院检验科,江苏南京210006 [2]江苏大学基础医学与医学技术学院免疫学与免疫学检验系,江苏镇江212013

出　　处：《现代生物医学进展》2010年第8期1401-1404,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金(30671984);江苏省自然基金(BK2008231);江苏大学科技创新团队(2008-018-02)

摘　　要：目的:构建和表达带HIV-TAT穿膜序列(Protein transduction domain,PTD)的人FOXP3突变体PTD-hFOXP3-Δ E251,为研究人FOXP3的功能奠定基础。方法:采用逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)方法,从人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)总RNA中扩增得到人FOXP3(hFOXP3)的cDNA,然后再利用重叠PCR技术扩增获得hFOXP3-ΔE251基因实变体片段。将该重组突变体插入表达载体pET28a-PTD中,构建表达质粒pET28a-PTD-hFOXP3-ΔE251,然后转化感受态细菌E.coli Rosetta(DE3),最终采用异丙基-β-D-硫代半乳糖苷(isopropyl-β -D-thiogalactopyranoside,IPTG)诱导表达。结果:SDS-PAGE电泳分析发现,经0.5mM IPTG诱导8h后,可高效表达重组的融合蛋白,表达产物以包涵体形式存在。结论:成功制备了带穿膜序列的人FOXP3ΔE251突变体融合蛋白,为更好地研究人FOXP3的功能与特性奠定了实验基础。Objective：To clone and express a deletion mutanted human FOXP3 fusion protein,which contains a protein transduction domain from HIV-TAT （PTD-hFOXP3-ΔE251） and lay the groundwork for future researching on the behavior of human FOXP3 in vitro even in vivo.Methods：hFOXP3 cDNA was amplified from total RNA of human peripheral blood mononuclear cells （PBMCs）by reverse transcription-polymerase chain reaction （RT-PCR）.Then the expression plasmid named pET28a-PTDhFOXP3-ΔE251 was constructed by genetically cloning the hFOXP3-ΔE251 fragment into the vector pET28a-PTD,which was under control of the T7 promotor.In the end it was expressed in E.coli Rosetta （DE3） induced by isopropyl-β-D-thiogalactopyranoside （IPTG）.Results：The result of SDS-PAGE indicated that the fusion protein was highly expressed in E.coli Rosetta （DE3）,after induced by 0.5mM IPTG for 8h and the expression product was performed as inclusion body.Conclusion：The expression of PTD-hFOXP3-ΔE251 protein was well done and it should be a potential useful tool for studying the biological functions of hFOXP3.

关 键 词：PTD-hFOXP3-Δ E251 基因克隆 表达载体 大肠杆菌 

分 类 号：Q75[生物学—分子生物学] Q78