天津医药

• 论著 •    

增强型绿色荧光蛋白基因与人组织型金属蛋白酶抑制剂1（hTIMP1）基因融合表达载体的构建及鉴定

徐昭 1,周丽娟2,李广平3   

  1. 1. 天津医科大学第二医院
    2. 天津医科大学第二医院 心脏科
    3. 天津医科大学第二医院心脏科
  • 收稿日期:2009-12-03 修回日期:2010-04-04 出版日期:2010-06-15 发布日期:2010-06-15
  • 通讯作者: 徐昭

Construction and Identification of fusion expression vector of the genes of EGFP and hTIMP1

  • Received:2009-12-03 Revised:2010-04-04 Published:2010-06-15 Online:2010-06-15

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摘要： 摘 要：目的 构建增强型绿色荧光蛋白基因与人组织型金属蛋白酶抑制剂1（hTIMP1）基因融合表达载体；方法 自正常人心肌组织提取总RNA，通过套式PCR获得hTIMP1开放读码框（ORF），将扩增产物与pEASY-Blunt Simple载体连接，经序列分析后双酶切，定向克隆入载体pEGFP-C1，继而将重组质粒转化入E.coliTOP10，筛选正确重组子进行鉴定；结果 hTIMP1 ORF约为624bp，与pEASY-Blunt Simple载体相连测序结果与Genbank相应序列比较，核苷酸序列无改变。定向克隆获得重组质粒pEGFP-hTIMP1经PCR及双酶切鉴定与预期相符；结论 成功构建增强型绿色荧光蛋白基因与hTIMP1基因融合表达载体，为进一步研究奠定基础。

关键词: 增强型绿色荧光蛋白, 人组织型金属蛋白酶抑制剂

Abstract: Abstract: Objective To construct the fusion expression vector of the genes of enhancement green fluorescent protein (EGFP) and human tissue inhibitor of metalloproteinases1 (hTIMP1). Methord After extracting mRNA from normal human myocardial tissue, open reading frame of hTIMP1 was obtained by nested-PCR. Then the PCR products was cloned into the pEASY-Blunt Simple vector. After identified by sequence analysis, the hTIMP1 ORF gene was cutted by two enzymes and inserted into pEGFP-C1. Then the recombinant plasmid was transformed into the competent cell of E.coliTOP10, and the correct transforments was screened to be identified. Results The PCR products was about 624bp. We compared the sequence of the hTIMP1 ORF gene which inserted into the pEASY-Blunt Simple vector with the ones reported in Genbank, the sequence was not changed. The recombinant plasmid pEGFP-hTIMP1 was also confirmed correct by identification of PCR and double enzymes’ digestion. Conclusion: We constructed fusion expression vector of the genes of EGFP and hTIMP1 successfully and laid primary basis on further reserches.

Key words: EGFP, TIMP1