人重型肝炎相关基因Fas、TNFR1真核表达载体和microRNA表达载体的构建及其体外干预效应的初步研究  

Construction of the Eukaryotic Expression Vectors and the microRNA Expression Plasmids of Human Fas and TNFR1 Gene and Their Biological Effects in vitro

作　　者：高随[1] 习东[1] 郭健文[1] 严伟明[1] 罗小平[1] 宁琴[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院1感染科2儿科,武汉430030

出　　处：《华中科技大学学报（医学版）》2010年第1期50-54,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家“973计划”重要传染病基础研究重大项目(No.2005CB522901,No.2007CB512900);国家自然科学基金青年基金资助项目(No.NSFC30700702)

摘　　要：目的构建人Fas(hFas)和人TNFR1(hTNFR1)基因的真核表达质粒pcDNA3.0-hFas、pcDNA3.0-hTN-FR1和microRNA(miRNA)干扰质粒p-hFasmiRNA、p-hTNFR1miRNA,初步验证其在体外细胞系对Fas和TNFR1基因表达的干预效应。方法从人肝癌细胞HepG2细胞中获得模板cDNA,通过PCR扩增出hFas和hTNFR1全长片段,将目的片段通过T载体过渡克隆至表达载体pcDNA3.0,得到重组质粒pcDNA3.0-hFas和pcDNA3.0-hTNFR1。利用miRNA设计软件,针对Fas和TNFR1基因分别设计3对pre-microRNA(pre-miRNA)序列,通过T4连接酶将pre-miRNA克隆至pcDNA6.2-GW/EmGFP-miRNA表达载体,同时构建非相关干扰质粒。构建成功的p-hFasmiRNA1、p-hFasmiRNA2、p-hFasmiRNA3和p-hTNFR1miRNA1、p-hTNFR1miRNA2、p-hTNFR1miRNA3分别与pcDNA3.0-hFas和pcDNA3.0-hTNFR1共转染至人293T细胞,通过Real-time PCR和Western blot检测转染48 h后对基因表达的干预效应。结果通过测序鉴定表明Fas和TNFR1基因的真核表达载体和miRNA干扰质粒均构建成功。Real-time PCR结果显示干预组的Fas和TNFR1基因的表达与对照组比较明显减少,抑制效率分别可达到87%和80%。Western blot结果也同样证实干预组的Fas和TNFR1基因的蛋白表达量与对照组比较明显减少。结论成功构建了hFas和hTN-FR1的真核表达载体和microRNA干扰质粒,并初步证实构建的microRNA干扰质粒在细胞水平对hFas和hTNFR1的表达具有特异性的抑制效应。Objective To construct the eukaryotic expression plasmids of human Fas and TNFR1 gene（pcDNA3.0-hFas and pcDNA3.0-hTNFR1）and microRNA（miRNA）expression pIasmid of hFas and hTNFR1 named p-hFasmiRNA and p-hTN-FRlmiRNA,and to investigate their inhibitory effects in vitro. Methods The eukaryotic expression plasmids of human Fas and TNFR1 gene were constructed（pcDNA3.0-hFas and pcDNA3.0-hTNFR1）and have been shown successfully to express hFas and hTNFR1 protein, miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFRlmiRNA complimentary to the sequence responsible for hFas and hTNFR1 respective were constructed,meanwhile irrelevant miRNA plasmid was used as a control. By respective co-transfection of p-hFasmiRNA and pcDNA3.0-hFas, p-hTNFR1 miRNA and pcDNA3.0- hTNFR1 expression construct into 293T cells, the inhibition of hFas and hTNFR1 expression was analyzed by real-time PCR and Western blot. Results The experiments showed the significant inhibitory effect of p hFasmiRNA on hFas and p hTN-FRlmiRNA on hTNFR1 expression at 48 h post-transfection both at RNA level and protein level as well in 293T cell lines with the inhibitory efficiency being as high as 87 % for hFas and 80 % for hTNFR1, respectively. Conclusion The p-hFasmiRNA and p-hTNFR1miRNA were constructed successfully,and it was verified that they could specifically inhibit the hFas and hTNFR1 expression at the cellular level.

关 键 词：FAS TNFR1 MICRORNA 细胞凋亡 重型肝炎 

分 类 号：R575.1[医药卫生—消化系统]