重组人TACE的解整合素域对人肺腺癌A549体外增殖、粘附和侵袭的抑制作用  

Inhibition of Proliferation,Adhesion and Invasion Ability of Human Lung Carcinoma Cell A549 by Tumor Necrosis Factor-α Converting Enzyme (TACE)

作　　者：闫媛[1] 李小欧[2] 章杰[1] 黄巍[1] 李凌波[1] 杨渝珍[1] 

机构地区：[1]华中科技大学同济医学院生化与分子生物学系,武汉430030 [2]华中科技大学同济医学院附属同济医院小儿内科,武汉430030

出　　处：《生物工程学报》2007年第4期584-588,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目(No30471963)~~

摘　　要：为了解人TACE的解整合素域对肿瘤细胞粘附和侵袭的影响以及其独立于其他结构域发挥作用的能力,从单核细胞系THP1中提取RNA,RT-PCR扩增出TACE全长基因,构建了pMD18T-TACE载体。对pMD18T-TACE质粒进行PCR,分别扩增T300(300bp,编码解整合素域)、T800(800bp,编码酶催化域)及T1300(1300bp,编码胞外域)三个片段,将其分别亚克隆至表达载体pET28a(+)。把质粒转化大肠杆菌BL21(DE3),表达产物均以不溶性包涵体形式存在。经过溶解包涵体、BBSTNTA树脂柱亲和层析并透析复性,获得高纯度的三种活性蛋白。MTT法、细胞粘附实验、Transwell小室侵袭实验分别显示TACE解整合素域、胞外域蛋白均能明显抑制A549细胞的增殖,且以剂量依赖性的方式抑制A549细胞与纤维粘连蛋白的粘附,并能抑制Transwell小室中A549细胞对Matrigel模拟的天然基底膜的侵袭,而酶催化域蛋白则无相应的抑制能力。表明重组TACE解整合素域蛋白可独立抑制肿瘤细胞的增殖、粘附和侵袭,为深入研究该区域的作用及TACE在肿瘤发病中的机制提供了新的认识。We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro, The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T- TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a （ ＋ ） to construct expression vector pET28a （ ＋ ）- 300, pET28a （ ＋ ）-T800, and pET28a （ ＋ ）-T1300, which respectively transformed into E. coli BL21 （DE3）. The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a（ ＋ ）-T300 and pET28a（ ＋ ）-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET -28a（ ＋ ）- T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.

关 键 词：解整合素 TACE 亚克隆 Transwell小室 

分 类 号：Q789[生物学—分子生物学]