【摘要】 目的构建含人基质金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinases1,TIMP1)的腺相关病毒(adeno associated virus,AAV)表达质粒并观察其在人胚肾293(HEK293)细胞中的表达,为进一步逆转椎间盘退变提供依据。方法以含有人TIMP1基因序列的质粒pBLAST49-hTIMP1为模板,通过PCR的方法扩增出TIMP1基因,再采用分子克隆的方法,把TIMP1克隆到AAV表达质粒pSNAV2.0构建重组质粒pSNAV2-TIMP1。把重组质粒转染HEK293细胞,用免疫荧光和Western blot的方法对重组质粒的表达进行研究,ELISA法检测细胞培养上清液中TIMP1蛋白的含量。结果PCR、酶切、DNA测序等方法均证实成功构建了含有完全正确的TIMP1基因序列的AAV表达质粒pSNAV2-TIMP1。体外成功转染入HEK293细胞,免疫荧光、Western blot及ELISA的结果表明重组质粒能够高表达TIMP1蛋白。结论成功构建了表达TIMP1的重组质粒pSNAV2-TIMP1,并证实在蛋白质水平获得了表达,为进一步逆转椎间盘退变研究奠定了基础。更多还原

【Abstract】 Purpose To construct a recombinant plasmid of adeno associated virus containing human tissue inhibitor of metalloproteinases,and then to study its expression in human embryonic kidney 293 cells（HEK 293）. Methods TIMP1 gene was amplified from pBLAST49-hTIMP1 by PCR,then ligated with pSNAV2.0 to form recombinant plasmid pSNAV₂-TIMP1 and transfected into HEK293 cells.Immunofluorescence,Western blot and ELISA were used to detect the synthesis of TIMP1 protein in the HEK293 cells and its expression level in the supernatant of culture medium. Results TIMP1 gene was ligated to pSNAV2.0,then transfected into HEK293 cells successfully.Strong expression of TIMP1 protein was observed and detected by Immunofluorescence,Western blot and ELISA. Conclusions The recombinant plasmid pSNAV₂-TIMP1 was constructed and a high level of TIMP1 protein expression was detected,which provided a basis for the further study of the reversion of intervertebral disc degeneration.更多还原