CAJ | 학술논문

目的构建BAG4(Bcl-2-associated athanogene)基因过表达重组质粒,转染结肠癌细胞株,并对其是否转染成功进行鉴定.方法 Real-time聚合酶链反应(PCR)检测和筛选6株结肠癌细胞BAG4mRNA表达.采用全基因合成法合成BAG4编码区序列,克隆入pEGFP-C1载体末端,构建重组质粒pEGFP-Cl-BAG4并DNA测序,以500ul脂质体质粒复合物瞬时转染SW480细胞,Western blot(鼠抗人BAG4单抗1:500)、RT-PCR等检测目的基因表达效率.结果 6株细胞中,HT29和SW480细胞BAG4 mRNA及蛋白水平最低.测序结果表明BAG4重组质粒构建成功.瞬时转染SW480细胞后,BAG4 mRNA分别升高约100倍,并BAG4蛋白表达也明显升高.结论成功构建BAG4基因过表达重组质粒,并有较高的转染水平,为进一步的研究提供了条件.
목적 구건BAG4(Bcl-2-associated athanogene)기인과표체중조질립,전염결장암세포주,병대기시부전염성공진행감정.방법 Real-time취합매련반응(PCR)검측화사선6주결장암세포BAG4mRNA표체.채용전기인합성법합성BAG4편마구서렬,극륭입pEGFP-C1재체말단,구건중조질립pEGFP-Cl-BAG4병DNA측서,이500ul지질체질립복합물순시전염SW480세포,Western blot(서항인BAG4단항1:500)、RT-PCR등검측목적기인표체효솔.결과 6주세포중,HT29화SW480세포BAG4 mRNA급단백수평최저.측서결과표명BAG4중조질립구건성공.순시전염SW480세포후,BAG4 mRNA분별승고약100배,병BAG4단백표체야명현승고.결론 성공구건BAG4기인과표체중조질립,병유교고적전염수평,위진일보적연구제공료조건.
Objective To construct the recombinant BAG4 (Bcl-2-associatedathanogene) overexpressed plasmids and transfect them in SW480 colon cancer cells.Methods The expression of BAG4 mRNA in six colon cancer cell lines was detected by using re-al-time polymerase chain reaction (PCR); BAG4 coding regions were synthesized to splice whole gene and then cloned into Cl terminus of pEGFP vector to construct recombinant plasmids; the plasmids with 500 μl Lipofectamin were transfected into SW480 cells after DNA sequencing; the efficiencies of BAG4 expression were validated by Western bl0tting (mouse anti-hu-man BAG4 mAb 1: 500) and RT-PCR.Results The lowest expression of BAG4 mRNA was seen in HT29 and SW480 cells.The sequencing results validated the well recombinant plasmids.BAG4 mRNA in transient transfeted SW480 cells was upreg-ulated about to100 fold,and BAG4 protein expression increased obviously as well.Conclusions The recombinant BAG4 over-expressed plasmids were successfully constructed with high transfection efficiency,which offers a good condition for our fur-ther research.