Wang Su, Xu Zhifei, Tang Hua, Wei Lingyun, Zhao Xuewei
Department of Thoracic Surgery, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China.
Zhongguo Fei Ai Za Zhi. 2010 Nov;13(11):1004-8. doi: 10.3779/j.issn.1009-3419.2010.11.02.
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.
The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-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.
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.
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.
TLE1是一种在调节Wnt、Notch和表皮生长因子受体（EGFR）信号通路中起重要作用的蛋白质。TLE1的N端Q结构域通过介导其寡聚化以及与淋巴样增强因子1（LEF1）的相互作用来调节这些信号通路。本研究的目的是在原核表达系统中构建人TLE1 N端Q结构域片段，表达并纯化蛋白TLE1 N端Q结构域，并制备其多克隆抗体。
通过聚合酶链反应（PCR）从人肺腺癌cDNA中获得TLE1 N端Q结构域序列，将其克隆到含有谷胱甘肽S-转移酶（GST）的原核表达载体pGEX-4T-1中。将载体pGEX-4T1-TLE1-Q转化到大肠杆菌BL21密码子优化菌株中。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达谷胱甘肽S-转移酶（GST）-TLE1-Q（1-136）融合蛋白，用凝血酶进行酶切，再用谷胱甘肽琼脂糖凝胶珠和快速蛋白质液相色谱（FPLC）进行纯化，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。然后用纯化的蛋白TLE1-Q（1-136）免疫兔子以获得抗血清。通过酶联免疫吸附测定（ELISA）和蛋白质印迹法（Western blot）检测抗体的效价和特异性。
重组质粒的PCR鉴定和测序表明载体pGEX-4T1-TLE1-Q成功构建。SDS-PAGE显示目标蛋白（14,000道尔顿）即为目的蛋白TLE1-Q（1-136）。已获得TLE1 N端Q结构域片段TLE1-Q（1-136）及其多克隆抗体，抗体效价为1:20,000。
表达载体pGEX-4T1-TLE1-Q构建正确。已获得TLE1 N端Q结构域片段TLE1-Q（1-136）及其多克隆抗体。这些工作为进一步研究TLE1与肺癌之间的生物学关系奠定了基础。
