人CyclinD1基因克隆与原核表达  

Cloning and prokaryotic expression of human CyclinD1 gene

作　　者：谭艳 韩晓芳 贾海琴 白瑞霞 

机构地区：[1]内蒙古自治区人民医院检验科,呼和浩特010017

出　　处：《中国医师杂志》2014年第2期224-226,共3页Journal of Chinese Physician

摘　　要：目的从人肝癌组织中克隆人的CyclinD1基因，并原核表达CyclinD1蛋白。方法从人肝癌组织中提取RNA，逆转录PCR扩增CyclinD1cDNA，PCR产物进行TA克隆和DNA序列分析；阳性TA克隆CyclinD1片段亚克隆入大肠杆菌BL21的表达载体PET32a＋，异丙基-B—D-硫代吡喃半乳糖苷（IsopropylB-D-1-thiogalactopyranoside，IPTG）诱导表达人Cyc—linD1。结果逆转录PCR扩增出约483bp的DNA片段，TIA克隆和DNA序列分析显示重组片段是人CyclinD1基因序列，CyclinD1eDNA亚克隆人大肠杆菌中BL21载体NotI和EcoRI位点之间；异丙基一p-D-硫代吡喃半乳糖苷（Isopropylp—D．1-thio—galactopyranoside，IPTG）诱导出约36KD的蛋白。结论从人肝癌组织中成功地扩增出人CyclinD1基因，并且在大肠杆菌中BL21得到表达。Objective To obtain the Cyclin D1 through cloning and prokaryotic expression of Cyclin D1 gene. Methods The total RNA was extracted from liver cancer tissue. The Cyclin D1 cDNA was obtained by reverse transcriptase polymerase chain reaction （RT-PCR）. The Cyclin D1 cDNA was sequenced, and sub-cloned to the PET32a ＋. The prokaryotic expressed was used to obtain the Cyclin D1. Results The 483 bp Cyclin D1 cDNA was obtained. The sequence of Cyclin D1 was corrected. The 36 KD CyclinD1 was obtained by prokaryotic expression. Conclusions The Cyclin D1 cDNA was obtained. Cyclin D1 was expressed in BL21.

关 键 词：肝肿瘤 遗传学 细胞周期蛋白D1 克隆 分子 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]