NT4-Apoptin-HA2-TAT融合基因重组腺相关病毒的构建及鉴定  

Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT

作　　者：王健生[1] 张明鑫[1] 刘德纯[2] 段小艺[3] 周苏娜[1] 张广健[4] 杨广笑 王全颖 

机构地区：[1]西安交通大学医学院第一附属医院肿瘤外科,陕西西安710061 [2]河南科技大学第一附属医院普外科,河南洛阳471003 [3]西安交通大学医学院第一附属医院核医学科,陕西西安710061 [4]西安交通大学医学院第一附属医院胸外科,陕西西安710061 [5]西安华广生物工程公司,陕西西安710025

出　　处：《细胞与分子免疫学杂志》2008年第8期754-756,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：2006年教育部博士点专项科研基金(20060698055)

摘　　要：目的:构建编码融合基因NT4-Apoptin-HA2-TAT的重组腺相关病毒表达载体。方法:利用限制性内切酶切相应载体后将Apoptin和HA2-TAT连入pUC19/NT4质粒,再将融合基因NT4-Apoptin-HA2-TAT亚克隆至腺相关病毒的穿梭质粒内,与辅助质粒pAAV/Ad、腺病毒质粒pFG140共同转染HEK-293细胞,通过同源重组获得NT4-Apoptin-HA2-TAT重组腺相关病毒载体,收集病毒上清,Dot blot法测定其滴度。MTT比色法观察NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体,对HepG2细胞存活率的影响。结果:经酶切及测序证实克隆出NT4-Apoptin-HA2-TAT融合基因;得到高滴度的(3.14×1015pfu/L)重组腺相关病毒表达载体。NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体,对HepG2细胞有强烈的诱导凋亡作用,与对照组比较,处理组细胞的存活率明显降低。结论:通过分子克隆体外重组技术成功制备了NT4-Ap-optin-HA2-TAT重组腺相关病毒载体,为下一步的Apoptin应用于基因治疗奠定了基础。AIM： To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT,laying a foundation for gene therapy research of malignant tumors.METHODS： The Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus;the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno-plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization.The effect of AAV-NT4-Apoptin-HA2-TAT on HepG2 cell line was measured by a colorimetric 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide（MTT） assay.RESULTS： The NT4-Apoptin-HA2-TAT was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells（3.14×10^15 pfu/L）.AAV-NT4-Apoptin-HA2-TAT had strong deduce apoptosis effect on HepG2 cells.Compared with Adeno-associated mock virus and in normal cell line NIH3T3,Aden-associated virus NT4-Apoptin-HA2-TAT significantly decreased the survival rate of HepG2 cells.CONCLUSION： The recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.

关 键 词：NT4-Apoptin-HA2-TAT 重组腺相关病毒 肿瘤 融合基因 

分 类 号：Q754[生物学—分子生物学]