目的 构建pcDNA3.1(+)-NPM1真核表达质粒,体外转染人膀胱移行细胞癌pumc-91细胞并观察其表达情况.方法 设计引物,采用PCR扩增核仁磷蛋白1(Nucleophosmin,npm1)基因编码序列;运用基因重组方法对其进行双酶切,插入pcDNA3.1(+)载体,经菌液PCR、测序对重组质粒进行鉴定.将成功构建的质粒经脂质体介导转染至pumc-91细胞,Western blot验证蛋白表达情况.结果 经菌液PCR、测序鉴定表明成功构建pcDNA3.1(+)-NPM1质粒,将成功构建的质粒转染至pumc-91细胞,Western blot证实转染pcDNA3.1(+)-NPM1表达质粒的pumc-91细胞npm1蛋白过表达.结论 成功构建pcDNA3.1(+)-NPM1重组质粒,为进一步研究NPM1蛋白的功能奠定基础.%Objective To construct the Eukaryotic expression plasmids pcDNA3.1 (+)-NPM1,then transfect human bladder cancer pumc-91 cells in vitro,and observe its proteinexpression condition.Methods Nucleophosmin (Npm1)primers were designed,and the gene was amplified by using PCR.The produced Npm1 gene was double enzyme digested and then was inserted into pcDNA3.1 (+) vectorby using the gene recombination technology.Recombined plasmid was identified with bacterial PCR and following sequencing.The successful construction of pcDNA3.1 (+)-NPMlwas transfected into pumc-91 cells.Western blot was used to confirm its over-expression.Results The results of bacterial PCR and sequencing revealed that the recombinant plasmid pcDNA3.1 (+)-NPM1 were successfully constructed.After transfecting pumc-91 cells with pcDNA3.1 (+)-NPM1,western blot results showed the upregulated npm1 protein levels.Conclusion The Eukaryotic expression plasmids pcDNA3.1 (+)-NPM1 was constructed and expressed in pumc-91cells with promising successfully rate,which developesthe good foundation for future research on biological function of NPM1 protein.
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