携带TIMP-4 cDNA的重组腺病毒载体的构建  

CONSTRUCTION AND PACKAGE OF ADENOVIRAL VECTORS CARRYING TIMP-4 GENE

作　　者：米立国[1] 高英[2] 孟宪敏[3] 丁金凤[3] 

机构地区：[1]河北医科大学基础医学院解剖学教研室,河北石家庄050017 [2]石家庄市第一医院耳鼻咽喉科,河北石家庄050011 [3]阜外心血管病医院分子医学中心,北京100037

出　　处：《河北医科大学学报》2006年第5期321-326,共6页Journal of Hebei Medical University

基　　金：国家自然基金资助:多基因导入预防再狭窄的实验研究(批准号:39970297)

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。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.

关 键 词：金属蛋白酶类组织抑制剂 腺病毒科 基因 

分 类 号：Q74[生物学—分子生物学]