小鼠Ras激酶抑制剂(KSR)基因氨基端和羧基端真核表达载体的构建及其在293T细胞中的表达  

Construction of eukaryotic expression vectors of carboxyl terminus and amino terminus of kinase suppressor of Ras (KSR) and their expression in 293T cell line

作　　者：杨雪琴[1] 周清华[1] 朱文[1] 朱大兴[1] 马力[1] 李昌林[1] 王艳萍[1] 陈小禾[1] 马家宝[1] 

机构地区：[1]四川大学华西医院四川省肺癌分子重点实验室,肿瘤中心,成都610041

出　　处：《中国肺癌杂志》2006年第2期123-126,共4页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金重点项目(No.30430300);国家自然科学基金(No.30070333);高等学校博士学科点专项基金(No.20040610060)资助~~

摘　　要：背景与目的已有的实验证据表明,Ras激酶抑制剂(kinasesuppressorofRas,KSR)的功能主要是作为一个支架蛋白组装MAPK及其上游调控子形成多蛋白的复合体。但KSR是否存在激酶活性,一直是争论的焦点。本研究的目的是构建小鼠KSR基因氨基端(NKSR)和羧基端(CKSR)的真核表达载体,并观察其在293T细胞中的表达。方法通过PCR方法扩增NKSR和CKSR目的片段,利用基因重组技术构建pCMVTag2bNKSR和pCMVTag2bCKSR真核表达载体,并进行酶切和测序鉴定。鉴定正确的克隆瞬时转染293T细胞,通过Westernblot方法检测目的蛋白的表达。结果通过酶切和测序鉴定,pCMVTag2bNKSR和pCMVTag2bCKSR真核表达载体序列正确,编码框正确。转染后的293T细胞经Westernblot检测,能够表达目的蛋白。结论成功构建了pCMVTag2bNKSR和pCMVTag2bCKSR真核表达载体并可在293T细胞中表达,为以后的研究奠定了基础。Background and objective The present experimental data have showed that the function of kinase suppressor of Ras （KSR） is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line. Methods N-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot. Results The sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.

关 键 词：KSR 真核表达 基因重组 

分 类 号：Q78[生物学—分子生物学]