DOC-1R基因表达载体的构建及其重组蛋白的表达纯化  

Construction of DOC-1R Gene Expression Vector and Purification of DOC-1R Protein

作　　者：刘杏[1,2] 刘琦[1] 王述森[1] 高锦兰[1] 罗阳[1] 

机构地区：[1]中国医科大学医学基因组学教研室卫生部细胞生物学重点实验室,沈阳市110001 [2]中国医科大学生物科学与生物技术(医学)系,沈阳市110001

出　　处：《医学分子生物学杂志》2009年第6期521-525,共5页Journal of Medical Molecular Biology

基　　金：辽宁省教育厅重点实验室项目（No：20060908）,创新团队项目（No：2008T194）

摘　　要：目的构建DOC-1R（deleted in oral cancer-1related）的原核表达载体并且纯化其重组蛋白，用于进-步研究DOC-1R蛋白的结构与功能。方法采用基因重组技术构建原核表达载体pGEX—DOC-1R，经DNA测序鉴定后，转化E．coliB[21，IFFG诱导表达并纯化融合蛋白。结果以菌落PCR及限制性内切酶酶切鉴定得到候选阳性克隆，测序结果表明所得到的克隆序列及开放阅读框架完全正确，IPTG诱导后SDS—PAGE电泳分析表明在40kD处出现特征蛋白表达带，经磁珠亲合层析后Western印迹检测证实得到较高纯度的GST—p14 DOC-1R融合蛋白。结论成功构建了pGEX—DOC-1R的原核表达载体，表达并纯化出GST—p14 DOC-1R融合蛋白，为DOC，1R抗体制备及研究DOC-1R蛋白结合蛋白及其在细胞周期调控中的生物学功能奠定了基础。Objective To construct prokaryotie expression vector of DOC-1R （deleted in oral cancer-1 related） gene, express and purify its recombinant protein for further structural and func- tional study. Methods Prokaryotic expression vector pGEX-DOC-1R was constructed by means of gene recombinant technique and verified by DNA formed into E. coli BL21 under IPTG induction. sequencing. Recombinant vector was then traMs- Expression of DOC-1R was confirmed by SDS- PAGE and Western Blot. Results The positive recombinant clone candidates were identified by col- ony PCR and digestion analysis. Sequencing analysis revealed the complete nucleotide insert and correct open reading frame. After induced under IPTG and purified by affinity chromatography, a protein band about Mr 40 000 was detected in SDS-PAGE and Western-Blot analysis, proving the purified GST-p14 DOC-1R fusion protein. Conclusion The prokaryotic expression vector of DOC-1R was successfully constructed. Furthermore, purified recombinant protein was obtained through affin- ity chromatography, by which preparation of DOC-I R antibody and research on DOC-1 and its asso- ciating proteins in cell cycle are hopeful to be performed.

关 键 词：DOC-1R 基因表达 基因克隆 融合蛋白 

分 类 号：R730.231[医药卫生—肿瘤] R392-33[医药卫生—临床医学]