pcDNA3.1/DLC-1重组质粒的构建及其在HT-29细胞中的表达  

Construction and Expression of Recombinant Plasmid pcDNA3.1/DLC-1

作　　者：吴平平[1] 金治[2] 吴鹏[2] 金月玲[2] 黄培林[2] 

机构地区：[1]东南大学附属中大医院肿瘤科,南京210009 [2]东南大学医学院病理学系

出　　处：《肿瘤防治研究》2010年第4期417-420,共4页Cancer Research on Prevention and Treatment

基　　金：国家自然科学基金资助项目(30471937)

摘　　要：目的构建和表达DLC-1(deleted in liver cancer)基因重组质粒。方法用PCR法得到DLC-1基因片段,此基因片段带有XbaⅠ和BamHⅠ两个酶切位点,然后将此片段和真核表达载体pcDNA3.1连接,转化大肠杆菌DH5a,利用脂质体介导将pcDNA3.1/DLC-1重组质粒转染到结肠癌HT-29细胞中,再用RT-PCR法检测重组质粒的表达。结果重组质粒经XbaⅠ和BamHⅠ双酶切和测序与DLC-1基因序列一致;利用脂质体介导将pcDNA3.1/DLC-1重组质粒导入HT-29细胞,获得了DLC-1基因的表达。结论成功构建pcDNA3.1/DLC-1重组质粒并在HT-29细胞内表达。Objective To construct and express the recombinant plasmid of DLC-1（deleted in liver cancer）.Methods DLC-1 gene fragment was obtained by PCR. DLC-1DNA fragment was digested with Xba Ⅰ and BamH Ⅰ together, and then ligation reaction to eukaryotic expression vector pcDNA3.1 which was also digested under the same condition. The product of ligation reaction was transformed into Escherichia coli DH5a. Recombinant plasmid pcDNA3.1/DLC-1 was transfected into HT-29 cells with liposome. Then, the product of recombinant plasmid was obtained by RT-PCR.Results Double restriction enzyme digestion and subsequent sequencing of recombinant plasmids confirmed the correctness of the recombinant plasmids. Recombinant plasmid pcDNA3.1/DLC-1 was transfected into HT-29 cells with liposome, and the product of recombinant plasmid was obtained.Conclusion The pcDNA3.1/DLC-1 was expressed in HT-29. This method provides a basis for studying colorectal cancer.

关 键 词：DLC-1 重组质粒 HT-29细胞株 结直肠癌 基因转染 

分 类 号：R735.3[医药卫生—肿瘤]