人CD40L胞外区和人CTLA4Ig双基因共表达腺病毒载体的克隆和鉴定

目的：构建人CD40L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CD40L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pAdshCD40L-IRES2-CTLA4Ig，经293细胞包装，获共表达双基因的腺病毒pAdvshCD40L-IRES2-CTLAg，并行凝胶电泳、PCR、共聚焦显微镜检测。结果：电泳、PCR扩增出了600bp和400bp的特异性片段、共聚焦显微镜观察到CD40L绿色荧光和CTLA4Ig红色荧光的共表达。结论：成功构建了人CD40L．胞外区和CTLA4Ig共表达双基因重组腺病毒载体。

Gene cloning and identification of shCD40L- IRES2-CTLA4Ig recombination adenovirus vector and exprssion in vitro

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.