摘要
目的构建钙整合素结合蛋白(calcium-andintegrin-bindingprotein,CIB)的真核表达载体,了解它在真核细胞中的表达及定位。方法从人脑组织分离总RNA,采用RT-PCR获得CIB开放读框序列,克隆至真核表达载体pCMV-myc,构建表达带myc表达标签的真核表达载体pCMV-myc-CIB,将其转染至NIH3T3细胞中,免疫组化鉴定其在细胞中的表达及定位。结果序列分析表明克隆的CIB序列与GeneBank报道的序列一致,脂质体转染入NIH3T3细胞中,用myc抗体检测到目的蛋白myc-CIB的表达。结论pCMV-myc-CIB表达载体构建成功,并在真核细胞中得到了正确表达,CIB蛋白主要定位于细胞质中。
Objective To construct the expression vector of calcium-and integrin-binding protein （ CIB）, and study its localization as a fusion protein in NIH3T3 cell. Methods Total RNA was extracted from human brain tissue for CIB sequence with RT-PCR, and then the obtained fragment was ligated into pCMV-myc vector. After confirmation with sequence analysis, the expression vector pCMV-myc-CIB was transformed into the NIH3T3 cell by Lipofectamin 2000. The protein expression was observed by immunofluorescent staining. Re- suits DNA sequence analysis showed that the CIB cDNA had been successfully inserted into pCMV-myc expression vector, myc-CIB was expressed in the NIH3T3 cells transfected with pCMV-myc-CIB. Conclusion The eukaryotic expression vector containing CIB is successfully constructed and expressed in the cytoplasma of NIH3T3 cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第3期216-218,共3页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目青年基金(30400125)~~
关键词
CIB
基因克隆
真核表达
calcium- and integrin-binding protein
gene cloning
eukaryotic expression
