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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。
Objective To construct the eukaryotic expression vector pIRES2-AcGFP1-Nuc-c-myc carrying proto-oncogene c-myc by gene engineering and express it in Hela cells. Methods Based on the constructed expression vector pET28a-c-myc, the c-myc gene was amplified by PCR and cloned into pMD19-T Simple vector after digestion with EcoRI and SmaI. The recombinant plasmid pIRES2-AcGFP1-Nuc-c-myc was transfected into Hela cells by using the same restriction enzyme digestion and sequencing. GFP expression was observed by fluorescence microscope, and the expression of c-myc protein was confirmed by MTT and Western blotting. Results The PCR and restriction enzyme digestion were consistent with the expected results. The sequencing results were exactly the same as those reported in GenBank. The recombinant plasmid was successfully constructed. Immunoblotting confirmed that c-myc gene was expressed in Hela cells. MTT results showed that the number of Hela cells increased significantly. Conclusion The eukaryotic expression vector pIRES2-AcGFP1-Nuc-c-myc was successfully constructed. The c-myc gene was successfully expressed in Hela cells and has biological activity.