同时表达活化型IGF1、EGF和FGF2的慢病毒载体的构建

发布时间：2018-05-21 07:07

本文选题：IGF + EGF　；参考：《中国细胞生物学学报》2014年11期

【摘要】：共培养体系及添加外源性生长因子是诱导干细胞向内耳样细胞分化研究中的常用手段。为了将两种方法结合用于研究,该实验设计了一种可同时表达三种活化型生长因子的慢病毒载体。活化型表皮生长因子(epidermal growth factor,EGF)编码序列采用RT-PCR方法从SW620人大肠癌细胞中克隆,活化型胰岛素样生长因子1(insulin-like growth factor 1,IGF1)和成纤维细胞生长因子2(fi broblast growth factor 2,FGF2)编码序列为人工合成。用PCR方法分别从p Sec Tag2A和p IRES2-EGFP质粒中克隆出引导分泌的免疫球蛋白Igκ链信号肽编码序列和引导翻译的内部核糖体进入位点(internal ribosome entry site,IRES)序列。将Igκ链信号肽和三种因子的编码序列分别融合、由IRES间隔并克隆到带绿荧光蛋白报告基因的慢病毒表达质粒p LVX-IRES-Zs Green1上并包装获得慢病毒。将慢病毒感染HEK293T细胞,通过有限稀释法筛选出稳定表达绿荧光蛋白的单细胞克隆。用Western blot检测单细胞克隆的IGF1、EGF和FGF2的表达,并通过促进肺癌A549细胞生长实验验证了分泌的生长因子的活性。该研究成功构建了导入细胞后可同时表达活化型IGF1、EGF和FGF2的慢病毒载体,为今后设计可表达这三种因子的共培养工程细胞用于干细胞诱导分化提供了有力的工具。
[Abstract]:Co-culture system and the addition of exogenous growth factor are common methods in inducing stem cells to differentiate into inner ear like cells. In order to combine the two methods, a lentivirus vector was designed to express three active growth factors simultaneously. The coding sequence of activated epidermal growth factor-1 (EGF1) was cloned from SW620 human colorectal cancer cells by RT-PCR method. The encoding sequence of activated insulin-like growth factor (1(insulin-like growth factor _ 1) and fibroblast growth factor (2(fi broblast growth factor _ (2) FGF2) was artificially synthesized. The coding sequence of Ig 魏 chain signal peptide guided by immunoglobulin was cloned from p Sec Tag2A and p IRES2-EGFP plasmids by PCR method, and the internal ribosome entry site IRESs sequence of internal ribosome entry site guided translation was cloned, respectively. The signal peptide of Ig 魏 chain and the coding sequence of the three factors were fused, and the lentivirus was isolated from IRES and cloned into the lentivirus expression plasmid p LVX-IRES-Zs Green1 with green fluorescent protein reporter gene and packaged to obtain lentivirus. HEK293T cells were infected with lentivirus and single cell clones expressing green fluorescent protein were screened by limited dilution method. The expression of IGF1EGF and FGF2 was detected by Western blot, and the activity of secreted growth factor was verified by promoting the growth of lung cancer A549 cells. In this study, a lentivirus vector expressing both activated IGF1EGF and FGF2 was successfully constructed, which provides a powerful tool for the design of co-cultured engineering cells expressing these three factors for stem cell induction and differentiation.
【作者单位】：浙江大学医学院附属第二医院耳鼻咽喉科;浙江大学医学院附属第二医院临床研究中心;
【基金】：浙江省医药卫生科技计划项目(批准号:2011ZDA012)资助的课题~~
【分类号】：R764.43

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