基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建

目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析方法纯化重组腺病毒Ad5-TIMP1并测定重组腺病毒感染滴度.结果 经PCR及酶切方法证实pDC316-TIMP1穿梭质粒中存在630 bp大小左右的插入片段,与目的基因TIMP1的cDNA大小一致;经PCR鉴定证实TIMP1 cDNA重组腺病毒载体Ad5-TIMP1构建成功,病毒感染性滴度为1×1010 IU/mL.结论 成功构建TIMP1 cDNA重组腺病毒载体,为应用基因治疗方法逆转或延缓椎间盘退变的研究奠定实验基础.

Construction of Recombinant Adenovirus Vector Carrying the Human Tissue Inhibitor of Metalloproteinase 1 cDNA

Objective To construct recombinant adenovirus vector carrying the human tissue inhibitor of metalloproteinase 1(TIMP1)cDNA and explore the feasibility of reversing or delaying intervertebral disc degeneration by gene therapy.Methods The hTIMP1 cDNA was amplified from plasmid containing human TIMP1 ORF sequence by PCR.hTIMP1 cDNA was subcloned into the adenovirus shuttle plasmid pDC316 of Ad5MaxTM adenovirus vector system,and the products were co-transfected into HEK293 cells with the helper plasmid pBHGlox_E1,3Cre by lipofectin transfection method.The recombinant adenovirus Ad5-TIMP1 was generated by homologous recombination of the two plasmids in HEK 293 cells.After identification by PCR and enzyme digestion,Ad5-TIMP1 was amplified in HEK 293 adenoviral packaging cells,purified by column chromatography,and virus activity was measured by TCID50 assay.Results Recombinant adenoviral vector carrying hTIMP1(Ad5-hTIMP1)was successfully constructed with virus activity of 1×1010 IU/ml,which was confirmed by PCR,restriction enzyme digestion and DNA sequencing.Conclusion The Recombinant adenoviral vector carrying hTIMP1(Ad5-hTIMP1) was successfully constructed,which provided a foundation for further research on reversing or delaying intervertebral disc degeneration by gene therapy.