两种人乳腺珠蛋白基因亚型cDNA的克隆及其原核表达  

Cloning and prokaryotic expression of two human mammaglobin cDNA subtypes

作　　者：潘自铁[1] 李鹏[1] 刘秀娜[1] 陈安[1] 赵力军[2] 胡川闽[1] 

机构地区：[1]第三军医大学临床生物化学教研室,重庆400038 [2]美国疾病预防控制中心,艾滋病,性传播疾病和结核病预防中心,亚特兰大ga30333

出　　处：《免疫学杂志》2005年第3期252-255,共4页Immunological Journal

基　　金：重庆市科委应用基础研究(2004);校归国人员启动资金(2004)资助项目

摘　　要：目的克隆hMAM编码全长cDNA,建立hMAM蛋白原核表达系统及其纯化方法,为进一步研究hMAM的结构、功能及其在乳腺癌免疫诊断中的应用奠定工作基础。方法自乳腺癌组织和乳腺癌细胞株MD-MB453提取总RNA,通过RT-PCR克隆hMAMcDNA,构建pGEX-KG-hMAM表达质粒,在大肠杆菌BL21中表达,鉴定表达产物的形式后建立目的蛋白的纯化方法。结果乳腺癌组织和乳腺癌细胞株中发现两种hMAMcDNA亚型:hMAM和hMAM(Isoform),长度分别为279和270bp,二者相差9个连续碱基,其翻译产物相差3个连续氨基酸残基;GST-hMAM蛋白在本原核表达系统中以不溶性包涵体形式存在,通过改良的纯化方案可以获得纯化的复性蛋白。结论获得hMAMcDNA并发现一个新的hMAM突变体;hMAM可在pGEX-KG中与GST融合,实现高效表达;通过本法建立的改良的纯化流程,可获得纯度达96.5%的hMAM的两种融合蛋白,为进一步研究两种亚型hMAM的结构、功能及其在乳腺癌免疫诊断中的应用奠定了工作基础。Objective To clone the full-length cDNA of human mammaglobin (hMAM) and to establish a purification method and a prokaryotic expression system for hMAM as a basis for further study of the hMAM structure and function, as well as for application of hMAM in immunological diagnosis of breast cancer.Methods hMAM cDNA was amplified by RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453, and then the prokaryotic expression vector pGEX-KG-hMAM was constructed. The purification procedure was set up after the pGEX-KG-hMAM was transfected into Escherichia coli BL21 and the location of the interest protein was identified. Results Two subtypes of hMAM cDNA, hMAM (isoform) cDNA and wildtype hMAM cDNA, were found. The hMAM (isoform) cDNA was consisted of 270 bp while the wildtype hMAM cDNA was consisted of 279 bp. The fusion protein GST-hMAM formed inclusion body in prokaryotic expression system and the renatured GST-hMAM was purified by our modified method. Conclusion The cDNA of hMAM is cloned successfully and the fusion protein GST-hMAM is expressed in Escherichia coli BL21. Purified GST-hMAM (96.5%) is obtained by using our modified purification procedure, which provides a foundation for studying the structure and function of two hMAM subtypes, as well as for its application in immunological diagnosis for breast cancer.

关 键 词：乳腺癌 人乳腺珠蛋白 原核表达系统 蛋白纯化 

分 类 号：R737.9[医药卫生—肿瘤]