抑癌基因MTS1真核表达载体的构建及其在卵巢癌细胞系HO-8910中的表达

Construction of recombinant vector of multiple tumor suppressor 1 and its expression in human ovarian cancer cell line HO 8910

目的：构建多肿瘤抑制基因１（ＭＴＳ１）的真核表达载体，将其转入人源性卵巢癌细胞株ＨＯ－８９１０中，并得到稳定表达，为进一步研究其对卵巢癌细胞的影响奠定实验基础．方法：利用ＢａｍＨⅠ与ＥｃｏＲⅠ从ｐＵＣ１９－ｐ１６质粒上切下ＭＴＳ１ｃＤＮＡ片段并克隆到真核表达载体ｐｃＤＮＡ３上，构建成ＭＴＳ１真核表达载体ｐｃＤＮＡ３－ｐ１６；利用脂质体（Ｆｕ－ＧＥＮＥＴＭ６）介导的基因导入法将ｐｃＤＮＡ３－ｐ１６导入人源性卵巢癌细胞株ＨＯ－８９１０中；采用ＡＢＣ法对转染后的不同代数细胞进行免疫细胞化学检测，观察ｐ１６蛋白的表达．结果：成功构建了ＭＴＳ１真核表达载体ｐｃＤＮＡ３－ｐ１６，并进行酶切鉴定；转染后经Ｇ４１８筛选２ｗｋ出现阳性克隆８９１０－ｐ１６，并检测到其中有ｐ１６蛋白的稳定表达．结论：成功构建ＭＴＳ１真核表达载体ｐｃＤＮＡ３－ｐ１６，并可在人源性卵巢癌细胞株ＨＯ－８９１０中得到稳定表达．

AIM: To construct an recombinant vector consisting of pcDNA3 with MTS1 cDNA, transfect the wild type p 16 gene into human ovarian cancer cell line HO 8910 and prove its expression. METHODS: Having constructed an recombinant vector (pcDNA3 p 16) of pcDNA3 with MTS1 cDNA at its Bam H Ⅰ and Eco R Ⅰ sites,we proved the existence of p 16 in pcDNA3 by restriction pattern. Then we transfected the wild type p 16 gene into human ovarian cancer cell line HO 8910 using Lipofectamin(FuGENE TM 6), selected the positive clone 8910 p 16 and 8910 pcDNA3 by G418 and proved the expressions of MTS1 in different generations of 8910 p 16 by immunocytochemical method. RESULTS: We successfully constructed the recombinant vector pcDNA3 p 16. After transfecting it into HO 8910, we have got the positive clone by selecting the cell with G418 for 2 weeks and detected the steady expression of protein p 16 in 8910 p16. CONCLUSION: The recombinant vector pcDNA3 p 16 that we have constructed can be expressed steadily in human ovarian cancer cell line HO 8910.