中文摘要：

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。

英文摘要：

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.