原癌基因c-myc真核表达载体构建及其生物学作用

Construction and identification of an original oncogene c-myc eukaryotic expression vector

目的利用基因工程技术构建携带原癌基因c-myc的真核表达载体pIRES2-AcGFP1-Nuc-c-myc重组质粒并在Hela细胞中表达。方法以构建好的表达质粒pET28a-c-myc为基础,利用PCR方法扩增c-myc基因,并加入EcoRⅠ和SmaⅠ酶切位点,克隆至pMD19-T Simple载体,双酶切后将其与同样经过双酶切的真核表达载体pIRES2-AcGFP1-Nuc连接,通过PCR、酶切及测序鉴定重组质粒的正确性,再将重组质粒pIRES2-AcGFPl-Nuc-c-myc转染Hela细胞,利用荧光显微镜观察GFP表达,利用MTT和免疫印迹证实c-myc蛋白表达量提高。结果经PCR和酶切鉴定与预期结果相符,测序结果与GenBank中报道的序列完全一致,成功构建了重组表达质粒。免疫印迹证实c-myc基因在Hela细胞中得到表达。MTT结果显示Hela细胞数量显著增加。结论真核表达载体pIRES2-AcGFP1-Nuc-c-myc成功构建,c-myc基因在Hela细胞中成功表达,具有生物学活性。

This study designed to construct and express original oncogene c-myc eukaryotic expression vector plRES2-AcGFP1-Nuc-c-myc using gene engineering technique. The c-myc gene was obtained by PCR amplification from pET28a-c-mye, to constructed pIRES2-AcGFP1-Nuc eukaryotic expression vector, which was then confirmed by PCR method, restriction analysis and DNA sequencing. In addition, the recombinant plasmid pIRES2-AcGFP1-Nuc-c-myc was transfected to Hela cells, and green fluorescent protein was expressed successfully under fluorescence microscopy. Analysis of Western blotting showed that c-myc protein expression was increased in Hela cell, and MTT assay indicated c-myc protein could promote the proliferation of Hela cells. In conclusion, eukaryotie expression vector of c-myc gene was constructed and transfected, which lay foundation for the lamprey cell line research.