人大肠癌线粒体DNA重组质粒的构建

Construction of recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA of human colorectal carcinoma cells

目的了解大肠癌细胞株(SW480,LoVo,HT29)线粒体DNA的突变,克隆突变的大肠癌线粒体DNA(mtDNA)基因,构建pcDNA3.I(+)-mtDNA真核表达重组体,并导入NIH3T3细胞,以探讨线粒体基因突变与肿瘤发生的关系。方法提取大肠癌细胞株(SW480,LoVo,HT29)mtDNA,扩增D-LOOP区,产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插人真核表达质粒pcDNA3.1(+),并用脂质体法导人NIH3T3细胞。结果检测出大肠癌细胞株SW480、LoVo、HT29细胞mtDNAD-LOOP分别有10、9、8个突变位点。成功克隆1119bp的mtDNAD-LOOP区至表达质粒pcDNA3.1(+),并导入NIH3T3细胞中。结论线粒体DNAD-LOOP区是一个具有高度多态性和突变性的区域,在大肠癌细胞株中突变率较高。

Objective To construct recombinant eukaryotic expression plasmid pcDNA3.1 （＋）-mtDNA for investigation of mutations in the D-loop region of mitochondrial DNA in human colorectal carcinoma. Methods The D-loop region of 3 colorectal carcinoma cell lines （SW480, LoVo, and HT29） were amplified by PCR and sequenced. The mtDNA fi＇agment was recombined in the eukaryotic expression plasmid pcDNA3. 1 （＋）, and the resultant pcDNA3.1 （＋）-mtDNA recombinant was used to infect murine fibroblast cell line NIH3T3. Results Among the 3 colorectal carcinoma cell lines （SW480, LoVo, HT29）, 10, 9, 8 mutations, were identified, respectively. The 1119-bp fragment of mtDNA was successfully cloned. DNA sequencing analysis demonstrate total agreement of the sequence with that in GenBank. The mtDNA fi＇agments were cloned into the multiple cloning sites of vector pcDNA3.1 （＋） properly and the recombinant was introduced into NIH3T3 cells. Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region with high mutation rate in human colorectal carcinoma cells. The recombinant eukaryotic expression plasmid pcDNA3.1 （＋）-mtDNA is successfully constructed.