摘要
目的构建TIMP-2基因的真核表达载体pcDNA3-TIMP-2,并探讨其体外表达效果。方法应用已克隆得到的人TIMP-2基因和分子克隆技术构建pcDNA3-TIMP-2,酶切鉴定,并用体外培养人THP-1细胞,电穿孔转入pcDNA3-TIMP-2,RT-PCR和Western blotting分别检测外源基因转染效果,Zymography检测培养上清MMPs活性。结果构建pcDNA3-TIMP-2经酶切和测序鉴定正确,电穿孔法导入体外培养人THP-1细胞后,TIMP-2 mRNA约增加2.8倍,TIMP-2蛋白表达增加约2.7倍,MMP-2和MMP-9的活性分别约为对照组的32％和28％。结论成功构建了pcDNA3-TIMP-2,且证实有良好的体外转染和表达效果,为进行转移TIMPs基因抑制MMPs及探讨其与动脉粥样硬化等多种病理过程的关系奠定基础。
Objective To construct the eukaryotic expression vector of TIMP-2 gene ( pcDNA3-TIMP-2 ) and evaluate the effect of expression in vitro. Methods We cloned the human TIMP-2 gene, constructed and verified pcDNA3-TIMP-2 , using standard molecular cloning technology and enzyme digestion. After pcDNA3-TIMP-2 vector was transfected into human cultured THP-1 cells, RT-PCR and Western blotting were performed to evaluate the effect of expression. Furthermore, MMPs activity in the cell culture liquid was measured with Zymography. Results The constructed vector pcDNA3-TIMP-2 was proved correct by enzyme digeston and sequencing. After pcDNA3-TIMP-2 was transfected into human THP-1 cells by electroperforation, the expression of TIMP-2 mRNA was elevated 2.8 times, TIMP-2 protein was expression was increased 2. 7 times but the activity of MMP-2 and MMP-9 was only 32% and 28% , respectively, compared the with control group. Conclusion pcDNA3-TIMP-2 was constructed successfully and was proved having good effect in transfection and expression in vitro. This study laid dawn the foundation for further research on suppressing MMPs by transfecting TIMPs gene and investigating the relation of MMPs with atherosclerosis and other pathophysiological processes.
出处
《上海第二医科大学学报》
CAS
CSCD
2004年第6期413-416,420,共5页
Acta Universitatis Medicinalis Secondae Shanghai
基金
上海市青年科技启明星计划资助项目(98QB14014)
