重组组蛋白去乙酰化酶2-pEGFP-C2质粒的构建及表达
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摘要
目的将组蛋白去乙酰化酶2(HDAC2)基因克隆入pEGFP-C2载体,探讨构建的质粒转染进非洲绿猴肾成纤维细胞COS-7株相关蛋白和mRNA的表达及蛋白分布部位的情况。方法从人肝癌细胞(HepG2)中获得组蛋白去乙酰化酶2(HDAC2)的cDNA,采用Xho I和BamH I双酶切,用T4连接酶将该cDNA连接pEGFP-C2质粒,将构建成功的pEGFP-C2-HDAC2质粒经PCR、限制性内切酶酶切和测序鉴定后转染非洲绿猴肾成纤维细胞(COS-7),荧光显微镜下观察绿色荧光蛋白表达,RT-PCR和Western blot鉴定HDAC2的表达。结果双酶切鉴定可见HDAC2质粒片段,转染重组质粒后可观察到绿色荧光蛋白的表达,PCR结果可检测到HDAC2 mRNA表达,同时Western blot可检测HDAC2蛋白和GFP蛋白的表达。结论成功构建了重组pEGFP-C2-HDAC2表达质粒,HDAC2蛋白可与绿色荧光蛋白在COS-7细胞中融合表达。
Abstact: Aims HDAC2 gene was cloned into pEGFPC2 vector to explore the efficiency of the plasmid transfection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to observe the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I,then inserted into the eukaryotic expression vector pEGFP-C2 with T4 enzyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the expression of pEGFP-C2-HDAC2 was monitored by fluo-rescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double digestion,and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detected by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed,the fusion expression of HDAC2 and GFP protein can be detected in COS-7 cells.
Abstact: Aims HDAC2 gene was cloned into pEGFPC2 vector to explore the efficiency of the plasmid transfection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to observe the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I,then inserted into the eukaryotic expression vector pEGFP-C2 with T4 enzyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the expression of pEGFP-C2-HDAC2 was monitored by fluo-rescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double digestion,and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detected by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed,the fusion expression of HDAC2 and GFP protein can be detected in COS-7 cells.
引文
[1]Emiliani S,Fischle W,van Lint C,et al.Characterization of a human RPD3 ortholog,HDAC3[J].Proceed Nat Acad Sci United States Amer,1998,95(6):2795-800.
[2]Li J,Staver M J,Curtin M L,et al.Expression and functional characterization of recombinant human HDAC1 and HDAC3[J].Life Sci,2004,74(22):2693-705.
[3]Barnes P J.Histone deacetylase-2 and airway disease[J].Ther Adv Respir Dis,2009,3(5):235-43.
[4]Barneda-Zahonero B,Parra M.Histone deacetylases and cancer[J].Mol Oncol,2012,6(6):579-89.
[5]Glozak M A,Seto E.Histone deacetylases and cancer[J].Oncogene,2007,26(37):5420-32.
[6]Marks P A,Miller T,Richon V M.Histone deacetylases[J].Curr Opin Pharmacol,2003,3(4):344-51.
[7]Yang W M,Inouye C,zeng Y Y,et al.Transcription repression byYY1 mediated hy interaction with a mammalian homoleg of the yeast globalgulator RPD3[J].Proc Natl Acad Sci USA,1996,93:12845-50.
[8]Spiegel S,Milstien S,Grant S.Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer therapy[J].Oncogene,2012,31:537-51.
[9]Ververis K,Karagiannis T C.An atlas of histone deacetylase expression in breast cancer:fluorescence methodology for comparative semi-quantitative analysis[J].Am J Transl Res,2012,4:24-43.
[10]章慧,黄成,卞尔保,等.脂质体转染HDAC2 siRNA对人肝癌细胞HepG2增殖的影响[J].中国药理学通报,2013,29(10):1378-82.[10]Zhang H,Huang C,Bian E B,et al.Effect of liposomal transfection of HDAC2 siRNA on proliferation of human HepG2 cells[J].Chin Pharmacol Bull,2013,29(10):1378-82.
[11]徐涛,黄成,李琳,等.pEGFP-C2-Trpm7重组质粒的构建及表达[J].安徽医科大学学报,2013,48(1):89-93.[11]Xu T,Huang C,Li L,et al.Construction and expression of pEGFP-C2-Trpm7 recombinant plamid[J].Acta Univ Med Anhui,2013,48(1):89-93.
[12]朱梦瑜,高星杰,钱宝鑫,等,重组真核质粒pEGFP-C2-HG3BP domain(1-5)的构建及表达[J].天津医科大学学报,2011,39(11):990-1.[12]Zhu M Y,Gao X J,Qian B X,et al.Construction and expression of pEGFP-C2-HG3BP domain(1-5)recombinant plamid[J].Acta Univ Med Tianjin,2011,39(11):990-1.
[2]Li J,Staver M J,Curtin M L,et al.Expression and functional characterization of recombinant human HDAC1 and HDAC3[J].Life Sci,2004,74(22):2693-705.
[3]Barnes P J.Histone deacetylase-2 and airway disease[J].Ther Adv Respir Dis,2009,3(5):235-43.
[4]Barneda-Zahonero B,Parra M.Histone deacetylases and cancer[J].Mol Oncol,2012,6(6):579-89.
[5]Glozak M A,Seto E.Histone deacetylases and cancer[J].Oncogene,2007,26(37):5420-32.
[6]Marks P A,Miller T,Richon V M.Histone deacetylases[J].Curr Opin Pharmacol,2003,3(4):344-51.
[7]Yang W M,Inouye C,zeng Y Y,et al.Transcription repression byYY1 mediated hy interaction with a mammalian homoleg of the yeast globalgulator RPD3[J].Proc Natl Acad Sci USA,1996,93:12845-50.
[8]Spiegel S,Milstien S,Grant S.Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer therapy[J].Oncogene,2012,31:537-51.
[9]Ververis K,Karagiannis T C.An atlas of histone deacetylase expression in breast cancer:fluorescence methodology for comparative semi-quantitative analysis[J].Am J Transl Res,2012,4:24-43.
[10]章慧,黄成,卞尔保,等.脂质体转染HDAC2 siRNA对人肝癌细胞HepG2增殖的影响[J].中国药理学通报,2013,29(10):1378-82.[10]Zhang H,Huang C,Bian E B,et al.Effect of liposomal transfection of HDAC2 siRNA on proliferation of human HepG2 cells[J].Chin Pharmacol Bull,2013,29(10):1378-82.
[11]徐涛,黄成,李琳,等.pEGFP-C2-Trpm7重组质粒的构建及表达[J].安徽医科大学学报,2013,48(1):89-93.[11]Xu T,Huang C,Li L,et al.Construction and expression of pEGFP-C2-Trpm7 recombinant plamid[J].Acta Univ Med Anhui,2013,48(1):89-93.
[12]朱梦瑜,高星杰,钱宝鑫,等,重组真核质粒pEGFP-C2-HG3BP domain(1-5)的构建及表达[J].天津医科大学学报,2011,39(11):990-1.[12]Zhu M Y,Gao X J,Qian B X,et al.Construction and expression of pEGFP-C2-HG3BP domain(1-5)recombinant plamid[J].Acta Univ Med Tianjin,2011,39(11):990-1.