真核表达质粒pcDNA3.1(+)-A1CF的构建及在肺癌中的表达

Construction of eukaryotic expression plasmid pcDNA3.1(+)-A1CF and its expression in lung cancer

目的构建Apobec-1互补因子(A1CF)真核表达质粒,转染肺癌细胞A549和H1299并检测其表达。方法提取肾癌细胞786-o中总RNA,经RT-PCR扩增人A1CF基因,通过酶切法插入pcDNA3.1(+)载体,构建pcDNA3.1(+)-A1CF真核表达质粒,经BamHI和XbaI双酶切及DNA序列测序鉴定。Lipofectamine 2000脂质体转染肺癌细胞,Western blot法检测A1CF在肺癌细胞中的表达情况。划痕和Transwell小室实验观察A1CF对肺癌迁移侵袭能力的影响。结果酶切及测序结果显示pcDNA3.1(+)-A1CF中插入的片段序列测定结果与人A1CF序列一致,重组质粒转染两种肺癌细胞后,转染组的目的蛋白表达量明显增高,且E-cadherin和P-ERK表达水平也明显增高(P<0.05)。结论成功克隆人A1CF基因cDNA,并构建真核表达质粒。A1CF促进肺癌细胞迁移侵袭可能与ERK的磷酸化有关。

Objective To construct an eukaryotic expression plasmid Apobec-1 complementary factor(A1CF)and its expression in lung cancer A549 and H1299 cells after transfection.Methods Total RNA was extracted from renal cancer 786-o cells.Then,human A1CF gene was amplified by RT-PCR and inserted into pcDNA3.1(+)vector by the enzyme digestion method.The reconstructed plasmid was digested with BamHI and Hind Ⅲand confirmed by sequencing analysis,before transfection into lung cancer cells by Lipofectamine 2000.The expression of A1CF was detected by Western blot.The effect of A1CF on the migration and invasion of lung cancer was observed with the wound healing assay and Transwell assay.Results The results of enzyme digestion and DNA sequencing showed that the sequence of fragments inserted in pcDNA3.1(+)-A1CF was consistent with human A1CF sequence.Western blot results showed that the protein expression of A1CF in A549 and H1299 cells significantly increased and the expression of E-cadherin and P-ERK increased(P<0.05).Conclusions The cDNA of human A1CF gene is successfully cloned and the eukaryotic expression plasmid pcDNA3.1(+)-A1CF was constructed.A1CF promotes the migration of lung cancer cells,which may be associated with phosphorylation of ERK.