小鼠结缔组织生长因子基因克隆及其真核表达载体构建

Construction of eukaryotic expression vectors following connective tissue growth factor gene cloning in mice

背景:研究证实结缔组织生长因子在动脉粥样硬化血管平滑肌中呈高表达,构建结缔组织生长因子真核表达载体以特异性上调结缔组织生长因子在血管平滑肌内的表达是进一步通过细胞及动物实验研究其作用机制的基础。目的:克隆小鼠结缔组织生长因子基因、构建其真核表达载体,并在血管平滑肌细胞中瞬时表达。设计、时间及地点:单一样本观察,实验于2007-09/2008-04在辽宁医学院省高校分子细胞生物学与新药开发重点实验室完成。材料:1个月龄昆明小鼠10只。真核表达载体pcDNA3.1(+)由辽宁医学院分子生物学实验室保存。方法:以小鼠肾脏总RNA为模板,通过反转录-聚合酶链反应方法获得结缔组织生长因子(CTGF)目的基因,连接到pMD18-T载体上,酶切鉴定测序,克隆入pcDNA3.1(+)真核表达载体。主要观察指标:酶切测序鉴定重组体pcDNA3.1(+)-CTGF。将pcDNA3.1(+)-CTGF转入血管平滑肌细胞,Western观察结缔组织生长因子蛋白表达。结果:酶切、测序鉴定证实成功扩增出小鼠结缔组织生长因子目的基因并构建其真核表达载体,Western检测证实,重组体转入血管平滑肌细胞后,细胞内高表达结缔组织生长因子蛋白。结论:成功克隆小鼠结缔组织生长因子目的基因并构建其真核表达载体,重组体能在血管平滑肌中表达。

BACKGROUND： There is strong evidence demonstrated that connective tissue growth factor （CTGF） showed higher expression in vascular smooth muscle cells （VSMC） with artherosclerosis, therefore, construction eukaryotic expression vector of connective is the basis for research the action mechanism of tissue growth factor expression. OBJECTIVE： To clone mice CTGF gene and construct its eukaryotic expression vector, in addition, to investigate its transient expression in VSMC. DESIGN, TIME AND SETTING： A single sample observation was performed at the High Educational Key Laboratory of Molecular Cell Biology and Drug Research, Liaoning Medical University from September 2007 to April 2008. MATERIALS： A total of 10 Kunming mice, with 1-month-old. The expression vector of pcDNA3.1 （＋）were preserved in Laboratory of Molecular Biology Department of Liaoning Medical University. METHODS： Using total RNA of mus kidney as template, amplified the fragment of mice CTGF gene by RT-PCR, and then cloned into pMD18-T vector. The eukaryotic expression vector pcDNA3.1 （＋）-CTGF was constructed by ligating mice CTGF gene with pcDNA3.1 （＋） vector. MAIN OUTCOME MEASURES： The recombinant pcDNA3.1 （＋）-CTGF was identified by double enzymes digestion. Then tranfected pcDNA3.1 （＋）-CTGF into VSMC. The expression of CTGF was measured by Western blot. RESULTS： The restriction endonuclease digestion and sequencing identified that the eukaryotic expression vector carring CTGF gene was constructed successfully, and CTGF protein was highly expressed following transfected into VSMC. CONCLUSION： The recombinant pcDNA3.1 （＋）-CTGF have been achieved successfully, which can express in VSMC.