目的：从人肝癌组织中克隆人的Cyclin D1基因，并原核表达Cyclin D1蛋白。方法从人肝癌组织中提取RNA，逆转录PCR扩增Cyclin D1 cDNA，PCR产物进行TA克隆和DNA序列分析；阳性TA克隆Cyclin D1片段亚克隆入大肠杆菌BL21的表达载体PET32a＋，异丙基-β-D-硫代吡喃半乳糖苷（Isopropylβ-D-1-thiogalactopyranoside，IPTG）诱导表达人Cyc-lin D1。结果逆转录PCR扩增出约483 bp的DNA片段，TA克隆和DNA序列分析显示重组片段是人Cyclin D1基因序列， Cyclin D1 cDNA亚克隆入大肠杆菌中BL21载体NotI和EcoRI位点之间；异丙基-β-D-硫代吡喃半乳糖苷（ Isopropyl β-D-1-thio-galactopyranoside ，IPTG）诱导出约36 KD的蛋白。结论从人肝癌组织中成功地扩增出人Cyclin D1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 D 1 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.
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