组织因子途径抑制物2基因真核表达载体的构建及鉴定  

Construction and identification of eukaryotic expression vector of tissue factor pathway inhibitor-2 gene

作　　者：汤志刚[1] 孙振阳[1] 胡何节[1] 许戈良[1] 陈炯[1] 李建生[1] 

机构地区：[1]安徽医科大学附属安徽省立医院普通外科,安徽省合肥市230001

出　　处：《中国组织工程研究与临床康复》2007年第45期9112-9115,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：安徽省自然科学基金资助项目(050430702)~~

摘　　要：目的:构建人组织因子途径抑制物2(tissuefactorpathwayinhibitor2,TFPI-2)基因的真核表达载体pEGFP-C1-TFPI-2,并对其进行酶切鉴定。方法:实验于2006-09/12在安徽省立医院中心实验室完成。实验方法:①从人胎盘组织中提取总RNA,反转录合成cDNA,以特异性引物扩增TFPI-2基因全长mRNA。②扩增产物回收纯化后用限制性内切酶酶切,与经同样处理的载体pEGFP-C1进行连接反应,连接产物转化感受态大肠杆菌DH5α。③碱变性法提取质粒进行限制性内切酶酶切分析,并进行DNA序列测定。结果:①成功克隆了708bp的TFPI-2基因片段。②构建了人TFPI-2基因的真核表达载体pEGFP-C1-TFPI-2,经限制性内切酶酶切,电泳分析后得到了大小分别为708bp的目的基因片段和4.6kb的载体片段,测序结果与TFPI-2mRNA的cDNA序列吻合。结论:通过基因重组技术,构建了稳定表达TFPI-2的人TFPI-2的真核表达载体pEGFP-C1-TFPI-2。AIM： To construct the eukaryotic expression vector of human tissue factor pathway inhibitor 2 （TFPI-2）, pEGFP-C1-TFPI-2, and identify it by enzymatic digestion assay. METHODS： The experiment was conducted at the Central Laboratory of Anhui Provincial Hospital from September to December in 2006. ~）Total RNA was extracted from human placenta tissue, and the desired cDNA fragment was obtained by RT-PCR with two specific primers.②The amplified production was purified and enzymatic analysis by restriction enzyme. The fragment was inserted into mammalian expression vector pEGFP-C1 dealt with the same method as the amplified production, and the resulted plasmid was transformed into E coli DH5α. ③The recombinant plasmid was extracted by alkaline denaturation and analyzed by enzymatic assay and DNA sequencing. RESULTS： ① A fragment of 708 bp of TFPI-2 gene was cloned successfully. ② The recombinant vector pEGFP-C1-TFPI-2 was constructed. The enzymatic assay proved that two independent fragment of 708 bp and 4.6 kb were obtained. DNA sequencing showed that it was consistent with cDNA sequence of TFPI-2 mRNA. CONCLUSION： By the technology of gene recombination, TFPI-2 gene was successfully constructed to the vector pEGFp-C1, and pEGFP-C1-TFPI-2 was constructed successfully.

关 键 词：TFPI-2基因 绿色荧光蛋白质类 克隆 分子 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]