生长抑制肽表达载体的构建及鉴定
|
摘要
目的构建带有Flag标签的生长抑制肽(34p)的真核表达载体,并验证其表达与功能。方法抽提人肝癌细胞系HepG2的基因组DNA为模板进行PCR扩增目的片段,通过酶切连接,将DNA片段连接到p3XFLAGCMVTM-9表达载体上,进行测序验证;随后进行瞬时转染,通过免疫荧光实验验证该载体的表达情况,用流式细胞术实验证实其功能。结果测序结果显示载体构建成功,序列正确;免疫荧光结果证实该载体可以正常表达目的肽段;流式细胞术结果过表达34p组相较于转染空载组,细胞凋亡明显增加。结论成功构建了34p的真核表达载体,并验证其促凋亡的生物学功能,为后续的机制研究提供了研究手段和工具。
Objective To construct the eukaryotic expression vectors of Flag-labeled growth inhibited peptide,and verify its expression and function. Methods Using PCR amplification growth inhibited peptide DNA fragments from template of HepG2 genome DNA,by enzyme digestion and connection,the DNA fragments connected on the p3XFLAG-CMVTM-9 expression vector,then validated by sequencing. The vectors transient transfect to HLE cells,immunofluorescence assay to detect the expressions,and flow cytometry(FCM) using to detect apoptosis. Results The sequencing result shows that the sequence is correct; Immunofluorescence exhibited that growth inhibited peptide was corrected expression and located in the HLE cells cytoplasm; FCM results verified that overexpress of 34p induced cell apoptosis increased. Conclusion Successful construct 34p truncated eukaryotic expression vectors which can express in the HLE cells and induce HLE cell apoptosis. This plasmid provides an important tool for further studying.
Objective To construct the eukaryotic expression vectors of Flag-labeled growth inhibited peptide,and verify its expression and function. Methods Using PCR amplification growth inhibited peptide DNA fragments from template of HepG2 genome DNA,by enzyme digestion and connection,the DNA fragments connected on the p3XFLAG-CMVTM-9 expression vector,then validated by sequencing. The vectors transient transfect to HLE cells,immunofluorescence assay to detect the expressions,and flow cytometry(FCM) using to detect apoptosis. Results The sequencing result shows that the sequence is correct; Immunofluorescence exhibited that growth inhibited peptide was corrected expression and located in the HLE cells cytoplasm; FCM results verified that overexpress of 34p induced cell apoptosis increased. Conclusion Successful construct 34p truncated eukaryotic expression vectors which can express in the HLE cells and induce HLE cell apoptosis. This plasmid provides an important tool for further studying.
引文
[1]王珊珊,李慧,李刚.甲胎蛋白的分子结构及生物学功能[J].世界华人消化杂志,2014,(11):1487-1494.
[2]Li P,Wang SS,Liu H,et al.Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma[J].World J Gastroenterol,2011,17(41):4563-4571.
[3]王珊珊,寇卜心,欧阳雅博,等.外源性afp对肝癌细胞增殖和抗凋亡的作用研究[J].河北医药,2014,36(22):3369-3371.
[4]Alisa A,Ives A,Pathan AA,et al.Analysis of CD4+T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients[J].Clin Cancer Res,2005,11(18):6686-6694.
[5]Mizejewski GJ.Therapeutic use of human alpha-fetoprotein in clinical patients:is a cancer risk involved[J]?Int J Cancer,2011,128(1):239-242.
[6]Mizejewskia GJ,Butterstein G.Survey of functional activities of alpha-fetoprotein derived growth inhibitory peptides:Review and prospects[J].Curr Protein Pept Sci,2006,7(11):73-100.
[7]王珊珊,陈宁,庞丽君,等.Afp抑制肝癌细胞中ra-rar信号通路的传递[J].河北医药,2014,36(24):3693-3695.
[8]李焱,程朋.中晚期肝癌临床治疗进展[J].临床肝胆病杂志,2014,30(3):233-236.
[9]王军,刘德慧,郑江涛,等.原发性肝癌临床治疗研究进展[J].中国老年学杂志,2010,30(5):423-429.
[10]胡斌,俞小琴.肝癌分子信号通路及靶向治疗现状及进展[J].铜陵职业技术学院学报,2013,12:29-32.
[11]曹雪涛.树突状细胞与肝癌的免疫治疗[J].中华肝脏病杂志,2003,11(3):133-134.
[2]Li P,Wang SS,Liu H,et al.Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma[J].World J Gastroenterol,2011,17(41):4563-4571.
[3]王珊珊,寇卜心,欧阳雅博,等.外源性afp对肝癌细胞增殖和抗凋亡的作用研究[J].河北医药,2014,36(22):3369-3371.
[4]Alisa A,Ives A,Pathan AA,et al.Analysis of CD4+T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients[J].Clin Cancer Res,2005,11(18):6686-6694.
[5]Mizejewski GJ.Therapeutic use of human alpha-fetoprotein in clinical patients:is a cancer risk involved[J]?Int J Cancer,2011,128(1):239-242.
[6]Mizejewskia GJ,Butterstein G.Survey of functional activities of alpha-fetoprotein derived growth inhibitory peptides:Review and prospects[J].Curr Protein Pept Sci,2006,7(11):73-100.
[7]王珊珊,陈宁,庞丽君,等.Afp抑制肝癌细胞中ra-rar信号通路的传递[J].河北医药,2014,36(24):3693-3695.
[8]李焱,程朋.中晚期肝癌临床治疗进展[J].临床肝胆病杂志,2014,30(3):233-236.
[9]王军,刘德慧,郑江涛,等.原发性肝癌临床治疗研究进展[J].中国老年学杂志,2010,30(5):423-429.
[10]胡斌,俞小琴.肝癌分子信号通路及靶向治疗现状及进展[J].铜陵职业技术学院学报,2013,12:29-32.
[11]曹雪涛.树突状细胞与肝癌的免疫治疗[J].中华肝脏病杂志,2003,11(3):133-134.