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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 pcDNA3-TIMP-2 of TIMP-2 gene and to investigate its expression in vitro. Methods pcDNA3-TIMP-2 was constructed by cloned human TIMP-2 gene and its molecular cloning technology. The recombinant plasmid was identified by restriction enzyme digestion and transfected into pcDNA3-TIMP-2 cells by RT-PCR and Western blot blotting were used to detect the effect of exogenous gene transfection, Zymography detection of culture supernatant MMPs activity. Results The constructed pcDNA3-TIMP-2 was identified by restriction enzyme digestion and sequencing. After electroporation into human THP-1 cells, the mRNA and protein expression of TIMP-2 increased about 2.8-fold and TIMP-2, And MMP-9 activity were about 32% and 28% of the control group, respectively. Conclusion The pcDNA3-TIMP-2 was successfully constructed and proved to have good transfection and expression in vitro, which laid the foundation for the relationship between the MMPs of TIMPs gene transfer and the various pathological processes such as atherosclerosis.