根据 Gen Bank中 TIMP- 1基因的碱基序列 ,用 RT- PCR方法从人的正常肾组织中克隆出包含信号肽在内的 TIMP- 1全长 c DNA序列 .采用 T- A克隆的方法将之插入 p CRR2 .1中间载体 ,DNA测序证实该片段序列与文献报告的完全一致 .利用亚克隆的方法将 TIMP- 1 c DNA片段克隆到 pc DNA3载体上 ,构建出 pc DNA3/ TIMP- 1的真核表达载体 ,通过脂质体 DOTAP转染至 COS-7细胞 ,Northern印迹及原位杂交证实在 COS- 7细胞上获得人 TIMP- 1的高效表达 ,细胞增殖实验表明 TIMP- 1的高产表达可促进 COS- 7细胞的增殖 ,证实了所转染人 TIMP- 1的生物活性
Abstract
TIMP 1 full length cDNA containing the signal peptide was amplified by reverse transcription and polymerase chain reaction (PCR) from total RNA of human normal renal tissues.Confirmed by the DNA sequence analysis,the TIMP 1 cDNA fragments were then inserted to mammalian expression plasmid pcDNA3 respectively.Thus pcDNA3/TIMP 1 recombinant eukaryotic expression vector was constructed.Moreover,this recombinant plasmid was transfected into COS 7 cells with lipofectin DOTAP.Northern blot analysis and in situ hybridization demonstrated that no TIMP 1 mRNA was detected in normal COS 7 cells,while TIMP 1 mRNA was detected in pcDNA3/TIMP 1 transfected COS 7 cells.Further study indicated that the endogenous TIMP 1 overexpression could induce the proliferation of COS 7 cells.
关键词
TIMP-1 /
克隆 /
细胞转染COS-7细胞
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Key words
TIMP 1 /
Cloning /
Transfection /
COS 7 cells
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参考文献
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