人CD_(40)L胞外区和人CTLA_4Ig双基因共表达腺病毒载体的克隆和鉴定  

Gene cloning and identification of shCD_(40)L- IRES_2-CTLA_4Ig recombination adenovirus vector and exprssion in vitro

作　　者：田普训[1] 吴军[2] 张寅生[1] 李钊伦[1] 薛武军[1] 贺伟峰[2] 易绍萱[2] 陈希炜[2] 张小容[2] 

机构地区：[1]西安交通大学第一医院肾病中心,陕西西安710061 [2]第三军医大学西南医院烧伤研究所

出　　处：《西北国防医学杂志》2004年第6期401-404,共4页Medical Journal of National Defending Forces in Northwest China

基　　金：国家自然科学基金资助项目 (3 0 3 714 16)

摘　　要：目的 :构建人CD40 L胞外区和CTLA4Ig双基因共表达重组腺病毒载体并鉴定。方法 :RT -PCR扩增人CD40 L胞外区片段 ,与致瘤素信号肽连接后构建穿梭质粒 ,经酶切、插入、同源重组 ,获质粒pAdshCD40 L -IRES2 -CTLA4Ig ,经 2 93细胞包装 ,获共表达双基因的腺病毒pAdvshCD40 L -IRES2 -CTLA4Ig ,并行凝胶电泳、PCR、共聚焦显微镜检测。结果 :电泳、PCR扩增出了 6 0 0bp和 4 0 0bp的特异性片段、共聚焦显微镜观察到CD40 L绿色荧光和CTLA4Ig红色荧光的共表达。结论 :成功构建了人CD40 L胞外区和CTLA4Ig共表达双基因重组腺病毒载体。Objective:To construct the adenovirus vector which co-express the bi- gene of the extracellular domain of secretable human CD 40L (shCD 40L) and cytotox ic T lymphocyte associated molecule-4Ig (CTLA 4Ig), and to acquire its expres sion stably in vitro.Methods: Firstly, we constructe d the pshuttle-CMV-shCD 40L vect or plasmid, then the IRES2-CTLA 4Ig cDNA was inserted into this vector to const ru ct the recombinant plasmid pAdshCD 40L-IRES 2-CTLA 4Ig. The CD 40L a nd CTLA 4Ig gene recombinant replication-deficient adenovirus (AdvCD 40L-IRES 2-CTLA 4Ig) was constr ucted by recombination of the plasmid AdCD 40L- IRES 2-CTLA 4Ig and the p lasmid BJ5 183-Adeasy-1 in 293 cell line. The recombinant adenovirus that could mediate the expression of CD 40L and CTLA 4Ig was identified by electrophoresis, PCR and micr ography of laser fluorescence. Results:We got the sa me significant phase (600 bp and 400 bp) through electrophoresis and PCR. CD 40L and CTLA 4Ig co-expression wer e observed in same site by micrography of laser fluorescence. Conc lusion:We suc cessfully constructed and expressed the CD 40L-IRES 2-CTLA 4Ig of co-expr essive bi-gene recombinant replication-deficient adenovirus in intro.

关 键 词：重组腺病毒 CD40L胞外区 CTLA4IG 基因表达 免疫耐受 

分 类 号：R392[医药卫生—免疫学]