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目的: 构建pcDNA3 1( +) mtDNA真核表达重组体,并导入NIH3T3细胞.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( +),并用脂质体法导入NIH3T3细胞.结果: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1119kb的mtDNAD loop区至表达质粒pcDNA3 1( +),并导入NIH3T3细胞中.结论: 成功构建了pcDNA3 1( +) mtDNA真核表达重组体,并成功导入NIH3T3细胞中.
OBJECTIVE: To construct eukaryotic expression recombinant pcDNA3 1 (+) mtDNA and introduce it into NIH3T3 cells.Methods: The mtDNA of colorectal cancer cell lines (SW480, Lovo, HT29) were amplified and the D loop region was amplified.The products were sequenced by DNA sequencing The recombinant plasmid pcDNA3 1 (+) was inserted into eukaryotic expression plasmid pcDNA3 1 (+) by lipofectamine 2000 and transfected into NIH3T3 cells by liposome.Results: The mtDNAD loop of SW480, Lovo and HT29 cells were 10, 9, 8 mutated sites.The 1119kb mtDNAD loop region was successfully cloned into the expression plasmid pcDNA3 1 (+) and introduced into NIH3T3 cells.Conclusion: The eukaryotic recombinant pcDNA3 1 (+) mtDNA was successfully constructed and successfully transfected into NIH3T3 cells in.