论文部分内容阅读
应用重组技术构建野生型及缺失型CDK2基因的真核表达载体 ,分别使野生型及缺失型CDK2蛋白与增强型绿色荧光蛋白 (Enhanced greenFluorescentProtein ,EGFP)形成融合蛋白。通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO ,经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达。结果表明 ,野生型CDK2基因的表达产物定位于细胞核 ,而两种缺失型CDK2基因分别编码的CDK2蛋白N 端 1～ 2 0 1及 98～ 2 98多肽均主要定位于细胞质。以上结果提示 ,CDK2蛋白序列中不含有与核定位直接相关的信号 ,其入核过程可能是由其N 端 1～ 97及 2 0 2～ 2 98多肽范围内的部分氨基酸共同形成高级结构 ,并依赖此高级结构与其他含有入核信号的蛋白形成复合物 ,从而被带动进入细胞核的。
The eukaryotic expression vector of wild type and deletion type CDK2 gene was constructed by using recombinant technology, and the wild type and deletion type CDK2 protein were respectively fused with Enhanced green fluorescent protein (EGFP). The vector was transfected into human cervical cancer cell line HeLa and Chinese hamster ovary cell line CHO by liposome-mediated method. After cell cycle synchronization, subcellular localization of EGFP was observed under a fluorescence microscope to trace wild type and deletion CDK2 gene expression. The results showed that the expression product of wild-type CDK2 gene was located in the nucleus, whereas the two N-terminal CDK2 protein encoded by the two deletion type CDK2 proteins mainly localized in the cytoplasm. The above results suggest that the CDK2 protein sequence does not contain a signal directly related to nuclear localization. The process of its nuclear entry may be due to the formation of a higher-order structure of some amino acids within the range of 1-97 and from 202-298 of the N-terminus Rely on this advanced structure with other proteins containing nuclear signal into a complex, which is driven into the nucleus.