摘要
目的:分析人癌症高表达蛋白1(HEC1)在有丝分裂期和分裂间期的定位和表达调控相关蛋白基序。方法:用逆转录聚合酶链反应法从HeLa细胞中克隆HEC1基因cDNA,并构建其真核表达载体;对HEC1 cDNA测序序列进行生物信息学分析;HEC1真核表达载体转染293T细胞后,用Western blot进行表达分析;并用有丝分裂抑制剂(nocodazole)对细胞进行同步化和释放,观察有丝分裂期和分裂间期时HEC1定位。结果:电泳和酶切鉴定,得到含有HEC1的真核表达载体,Westernblot证实表达载体可在转染细胞中表达重组HEC1蛋白;HEC1测序序列分析发现多个与定位和调控相关基序,提示HEC1可能受APC/C蛋白酶体调控;HEC1在有丝分裂期定位于染色体,在分裂间期定位于细胞质靠近细胞核部位。结论:获得HEC1基因cDNA及表达载体,HEC1定位随不同细胞周期而变化。
Objective：To analyze the localization and regulation motifs of human highly expressed in cancer 1（HEC1）in mitosis and interphase.Methods：The human HEC1 cDNA was cloned by RT-PCR from HeLa cells,and inserted into expression vectors.The localization and regulation motifs of HEC1 were analyzed by bioinformation software.The HEC1 protein in transfected 293T cell was detected by Western blot.The transfected cells were synchronized by nocodazole and released by fresh medium.The HEC1 localization in transfected cell was observed in mitosis and interphase,respectively. Results：The HEC1 recombinant plasmids were identified by DNA sequencing.The expression of HEC1 protein was detected in transfected cells.Some motifs associated localization and regulation of HEC1 were found,that indicated anaphase promoting complex/cyclosome regulate the degradation of HEC1.The localization of HEC1 was in chromosome at mitosis,and in cytoplasm at interphase.Conclusion：HEC1 cDNA has been cloned and expressed.The localization of HEC1 can change with different cell cycle.
出处
《汕头大学医学院学报》
2010年第2期78-80,F0003,共4页
Journal of Shantou University Medical College
基金
国家自然科学基金资助项目(30771988
30972766)
广东省自然科学基金资助项目(9451503102003499)
高校博士点专项科研基金资助项目(20094402110004)
广东高校优秀青年创新人才培育资助项目(LYM08056)
农业微生物学重点实验室开放课题
汕头大学医学院科研基金资助项目
关键词
人癌症高表达蛋白
细胞周期
定位
human highly expressed in cancer 1
cell cycle
localization
