MT1F基因过表达重组质粒构建及转染结肠癌RKO细胞的鉴定

Construction of recombinant MT1F overexpressed plasmids and validation of their transfection to RKO colon cancer cells

目的构建MT1F基因过表达重组质粒，并转染结肠癌细胞株。方法Real—time聚合酶链反应（PCR）检测和筛选6株结肠癌细胞MT1FmRNA表达。采用全基因合成法合成MT1F编码区序列，分别克隆人pEGFP和pcDNA3．1（＋）载体的C1末端，构建重组质粒pEGFP-C1-MT1F和pcDNA3．1-MT1F，DNA测序，以500ul脂质体质粒复合物转染RKO细胞，G418（400mg／L）维持筛选，Westernblot（鼠抗人MT单抗1：500、鼠抗GFP抗体1：1000）等检测目的基因表达效率。结果6株细胞中，RKO细胞MT1FmRNA水平最低。测序表明MT1F重组质粒构建成功。瞬时和稳定转染RKO细胞后，MT1FmRNA分别升高约600和2000倍，并表达15×10^3的MT蛋白。结论成功构建MT1F基因过表达重组质粒．获得外源性MT1F稳定转染的结肠痛RKO细胞株。

Objective To construct the recombinant MT1F overexpressed plasmids and transfect them in RKO colon cancer cells. Methods The expression of MT1F mRNA in six colon cancer cell lines was detected by using real-time polymerase chain reaction （PCR）. MT1F coding regions were synthesized to splice whole gene and then cloned into C1 terminus of pEGFP or pcDNA3. 1 （ ＋ ） vector to construct recombinant plasmids. The plasmids with 500 ul Lipofectamine were transfected into RKO cells after DNA sequencing, then screened by G418 （400 mg/L）. The efficiencies of MT1F expression were validated by Western blotting （ mouse anti-human MT mAb 1： 500, mouse anti-GFP mAb 1 ： 1000）. Results The lowest expression of MT1F mRNA was seen in RKO cells. The sequencing results validated the well recombinant plasmids. MT1F mRNA in transient and stable transfeted RKO cells was upregulated about to 600 and 2000 fold, respectivly, and the 15 kD metallothionein proteins were detected. Conclusion The recombinant MT1F overexpressed plasmids were successfully constructed, and the stable RKO cells with ectopic MT1F transfection were obtained.