东篱科研大数据发现系统（DRDS）

位置：成果数据库 > 期刊 > 期刊详情页

Raf全长及氨基端、羧基端真核表达载体的构建和表达

期刊名称：中国肺癌杂,2008,11(3):306-310
时间：0
分类：R734.2[医药卫生—肿瘤;医药卫生—临床医学]
作者机构：[1]四川大学华西医院肺癌分子生物学重点实验室,成都610041, [2]天津市肺癌转移与肿瘤微环境重点实验室,天津市肺癌研究所,天津医科大学总医院,天津300052
相关基金：本研究受国家自然科学基金重点项目（No.30430300）,天津市科技职称计划重点项目（No.06YFSZSF05300）和国家自然科学基金（No.30500221;No.30500496）资助
相关项目：nm23基因逆转肺癌转移的分子机理和信号传导调节

关键词： RAF, 基因重组, 真核表达, Raf Gene recombination Eukaryotic expression

中文摘要：

背景与目的Raf是Ras—Raf—MEK—ERK信号转导通路中的关键分子，在多种人类肿瘤中存在高度活化，然而其生物学功能和详细调节机理目前尚未完全明了。本研究旨在构建人Raf基因全长（Raf-1），氨基端（N—Raf）及羧基端（C—Raf）真核表达载体，并观察其在293T细胞中的表达情况。方法通过PCR方法扩增Raf-1，N—Raf和C—RgH的片段，利用基因重组技术构建pCMV-Tag2b—Raf-1，pCMV—Tag2b—N—Raf和pCMV-Tag2b—C—R填核表达载体，并进行酶切和测序鉴定。鉴定正确的克隆瞬时转染293T细胞，Westem blot检测目的蛋白的表达。结果酶切和测序结果均证实pCMV-Tag2b—Raf-1，pCMV-Tag2b—N—Raf和pCMV-Tag2b—C—Raf真核表达载体的序列和编码框均正确无误，转染后的293T细胞经Western Blot检测可正确表达目的蛋白。结论本研究成功构建了pCMV-Tag2b—Raf-1，pCMV—Tag2b—N—Raf和pCMV-Tag2b—C—Raf真核表达载体并可在293T细胞中表达，为今后进一步研究Raf基因的生物学机理奠定了基础。

英文摘要：

Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf- 1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-CRaf were successfully constructed and can be expressed in 293T cells.