iASPP真核表达载体的构建及其对NIH3T3细胞增殖的影响

目的：构建iASPP真核表达载体,稳定转染小鼠成纤维细胞（NIH3T3）并检测其表达,观察其对NIH3T3细胞增殖的影响。方法：设计引物以源克隆PCMV-iASPP为模板PCR扩增其中的CDS区,亚克隆入FLAG-pcDNA3.0,将该重组质粒转化DH5α感受态大肠埃希菌,筛选阳性克隆,经酶切、测序鉴定后,用脂质体介导转染NIH3T3细胞,G418加压筛选28 d,RT-PCR、West-ern blotting检测基因、蛋白表达;MTT法观察细胞增殖能力。结果：重组质粒鉴定正确,筛选所得阳性克隆中可以检测到iASPP基因的转录和表达,且该iASPP阳性表达细胞株增殖能力明显增强。结论：成功构建iASPP真核表达载体并成功构建稳定表达iASPP的NIH3T3细胞株,证明该基因的稳定表达在体外能显著增强NIH3T3细胞增殖能力。

Construction of FLAG-pcDNA3.0-iASPP plasmid and its effect on the proliferation of NIH3T3 cells

Objective: To construct FLAG-pcDNA3.0-iASPP eukaryotic expression plasmid and establish NIH3T3 cell line with stable expression of iASPP gene,explore the effect of iASPP on NIH3T3 cell lines in vitro.Methods: PCR was applied to amplify the CDS of iASPP and then subcloned it to the FLAG-pcDNA3.0 vector and DH5α.E.coli was transformed with the recombinant plasmid,and the ampicyllin-resistant clones were identified by double digestion with EcoR I-Xho I and DNA sequencing.NIH3T3 cells were transfected with the identified FLAG-pcDNA3.0-iASPP by lipofectin and G418-resistant colonies of NIH3T3 cells were obtained,RT-PCR and Western blotting were used to test the gene expression;MTT was used to explore the effect of iASPP on NIH3T3 cell lines.Results: Enzyme digestion and sequencing confirmed the successful construction of the recombinant plasmid,which was expressed in NIH3T3 cells.MTT assay indicated that the growth ability of NIH3T3-FLAG-pcDNA3.0-iASPP cells was enhanced.Conclusion:The recombinant plasmid FLAG-pcDNA3.0-iASPP is successfully constructed and expressed in the NIH3T3 cells.The iASPP can effectively enhance the growth and proliferation of NIH3T3 cells in vitro.