中文摘要：

目的 克隆细胞外基质金属蛋白酶组织抑制因子4（tissue inhibitor-4 of metalloproteinases,TIMP-4）基因,构建包装携带TIMP-4基因的重组腺病毒载体（Ad/T4）,为基因治疗血管再狭窄的实验研究打下基础.方法 以人心脏cDNA文库为模板,应用PCR克隆TIMP-4基因,经DNA测序确定克隆结果.构建具有强启动子CMV,携带TIMP-4和报告基因GFP的穿梭质粒pAdTrack-T4,利用AdEassy系统,在大肠杆菌BJ5183经同源重组产生重组腺病毒质粒pAd/T4.脂质体介导转染293细胞,包装重组腺病毒载体（Ad/T4）.结果 克隆出含TIMP-4全部编码序列的cDNA片断,并通过亚克隆和同源重组等将其重组入腺病毒,构建包装了重组腺病毒载体Ad/T4.结论 Ad/T4中含有TIMP-4和GFP的完整表达盒,可用于基因治疗的实验.

英文摘要：

Objective To clone the gene of tissue inhibitor-4 of metalloproteinases（TIMP- 4） and construct and package the adenoviral vectors carrying TIMP-4 gene for the future gene therapy of vascular restenosis. Methods The cDNA of TIMP-4 was cloned by polymerase chain reaction（PCR） from human heart cDNA library and confirmed by sequencing analysis. To construct the shuttle vector pAdTrack-T4 which caring TIMP-4, GFP and the cytomegalovirus immediate early promoter（CMV）. Using AdEasy system, pAdTrack-T4 and adenoviral backbone vector （pAdEasy-1） were co-transfected into electrocompetent BJ5183 Cells to generate recombinant adenoviral plasmids （pAd/T4） by homologous recombination. The pAd/T4 was tranfected into 293 cells with lipofectamine to yield the recombinant adenoviruses （Ad/T4）. Results Nucleotide sequencing analysis of the PCR product revealed that it corresponded to the cDNA for TIMP-4 containing all coding sequence of TIMP-4. By sub-cloning and homologous recombination, the recombinant adenovirues （ Ad/T4 ） were packaged. Conclusion The recombinant adenoviral vector Ad/T4, which carries the TIMP-4 and GFP expression cassette, can＇ be used in gene therapy experiment.