生长抑制肽表达载体的构建及鉴定  

Construction and identification of growth inhibited peptide eukaryotic expression vector

作　　者：王珊珊[1] 乔录新[1] 庞丽君[1] 欧阳雅博[1] 李刚[2] 陈德喜[1] 

机构地区：[1]首都医科大学附属北京佑安医院(北京市肝病研究所),北京100069 [2]北京大学医学部基础医学院生物化学与分子生物学系,北京100191

出　　处：《医药论坛杂志》2017年第4期1-3,6,共4页Journal of Medical Forum

基　　金：国家自然科学基金资助项目(81401970);北京市肝病研究所所内基金项目(BJIH-01606);首都发展卫生专项(2014-1-2181)

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 ceils, immunofluo- rescence 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.

关 键 词：生长抑制肽 真核表达载体 Flag标签 凋亡 

分 类 号：R446.5[医药卫生—诊断学]