摘要
背景与目的 TLE1是参与Wnt、Notch以及EGFR信号通路调控的一种重要蛋白。TLE1N端Q结构域片段通过介导自身寡聚化及与LEF1结合发挥调控作用。本实验旨在构建人TLE1N端Q区基因在大肠杆菌的融合表达载体,制备并纯化人TLE1N端Q结构域蛋白片段,制备TLE1Q结构域多克隆抗体。方法以人肺腺癌cDNA库为模板聚合酶链反应(polymerase chain reaction,PCR)特异性扩增TLE1-Q(1-136)基因序列,与pGEX-4T-1质粒连接后转化感受态大肠杆菌E.coli。以异丙基-β-D-硫代半乳糖苷(isopropyl-β-D thiogalactoside,IPTG)诱导表达产生融合蛋白GST-TLE1-Q(1-136)。经亲和层析,rombin酶切,FPLC纯化,SDS-PAGE鉴定目的蛋白TLE1-Q(1-136)。免疫家兔,制备多克隆抗体。结果测序证实重组表达质粒中的人TLE1N端Q结构域基因序列正确,成功构建表达型重组质粒pGEX-4T1-TLE1-Q。重组质粒转化大肠杆菌C+后,经诱导,重组蛋白GST-TLE1-Q(1-136)得到表达。SDS-PAGE鉴定示纯化蛋白为目的蛋白人TLE1N端Q结构域片段TLE1-Q(1-136)。免疫家兔后收获抗血清,ELISA显示抗体效价为1:20000,具有高度特异性。免疫印迹结果显示,制备的多抗可与TLE1-Q(1-136)蛋白特异性结合。结论成功构建了人TLE1N端Q结构域重组融合蛋白表达质粒pGEX-4T1-TLE1-Q,表达纯化了稳定可溶TLE1N端Q结构域蛋白TLE1-Q(1-136),制备了人TLE1N端Q结构域蛋白片段多克隆抗体,为进一步研究TLE1在肺癌生成中的作用奠定了基础。
Background and objective TLE1 is an important protein in regulating Wnt,Notch and EGFR signaling pathways.The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1.The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system,express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody.Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA,was cloned into the prokaryotic expression vector pGEX4T-1 containing Glutathione S-transferase （GST）.Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus.The GST-TLE1-Q（1-136） fusion protein was induced by IPTG,digested by Thrombin,purified with glutathione-sepharose beads and FPLC,identified by SDS-PAGE.Then rabbits were immunized with the purified protein TLE1-Q（1-136） for obtaining the antiserum.The titers and specificity of antibodies were measured by ELISA and Western blot.Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed.The SDS-PAGE shows target protein （14 000 Da） is the interest protein TLE1-Q（1-136）.The TLE1 N-terminal Q domain fragment TLE1-Q（1-136） and its polyclonal antibody have been acquired,with an antibody titer of 1：20 000.Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed.The TLE1 N-terminal Q domain fragment TLE1-Q（1-136） and its polyclonal antibody have been acquired.These work established the foundation for further biological study between TLE1 and lung cancers.
出处
《中国肺癌杂志》
CAS
2010年第11期1004-1008,共5页
Chinese Journal of Lung Cancer
基金
国家自然科学基金项目(No.30972965)资助~~
关键词
肺肿瘤
TLE1
基因克隆
原核表达
蛋白纯化
多克隆抗体
Lung neoplasms
TLE1
Gene cloning
Prokaryotic expression and purification
Polyclonal antibody
