以CⅡTA-pⅠ作为树突状细胞特异启动子构建腺病毒双表达载体  

Construction and expression of murine recombinant adenovirus dual expression vector using CⅡTA-pⅠ as DCs specific promotor

作　　者：张燕[1] 徐玉莲[1] 沈茜[1] 

机构地区：[1]第二军医大学附属长海医院实验诊断科,上海200433

出　　处：《中华微生物学和免疫学杂志》2003年第7期562-566,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 3 9970 2 70 )

摘　　要：目的　构建CⅡTA pⅠ启动子特异调控的鼠TGF β1和PLP Ig重组双表达载体 ,以期达到在树突状细胞中特异表达。方法　从鼠外周血单个核细胞克隆CⅡTA pⅠ启动子 ,从ConA刺激的小鼠脾单个核细胞和WT 1杂交瘤细胞克隆TGF β1基因和Ig重链Fc基因 ;体外经IRES序列连接后 ,通过穿梭载体与腺病毒载体骨架连接 ,鉴定后在HEK2 93包装。从小鼠骨髓造血细胞培养树突状细胞。流式细胞仪检测树突状细胞CD80、CD5 4和Ⅰ Ad 的表达 ,鉴定树突状细胞的成熟度和纯度。病毒转染后 ,采用ELISA方法鉴定该病毒在树突状细胞中TGF β1基因的表达水平 ;Westernblot鉴定PLP Ig的表达。结果　该双表达重组腺病毒载体经测序、限制性内切酶分析、PCR等鉴定 ,与预期结果一致 ;病毒感染树突状细胞的培养上清液和细胞分别经ELISA和Westernblot检测证实TGF β1和PLP Ig得到了独立表达 ,在HEK2 93细胞中则无表达 ,并随树突状细胞的成熟 ,表达量逐渐增高。结论以CⅡTA pⅠ作为树突状细胞特异启动子构建的重组腺病毒双表达载体 ,在树突状细胞中得到了特异、独立的表达 ,为靶向树突状细胞表达功能基因进行基因治疗奠定了基础。Objective Construction and expression of murine recombinant adenovirus dual expression vector using CⅡTA-pⅠ as a DCs specific promotor. Methods CⅡTA-pⅠ, TGF-β1 and Ig Fc fragment were cloned from murine blood, BALB/c mouse spleen was pre-treated with ConA and WT-1 hybridoma respectively and ligated by IRES sequence. pTRE-shuttle vector was used as a mediator to ligate the backbone of the adenoviral vector. The identified adenovirus was packaged in HEK293 cells. Supernatant of high titer adenovirus was collected to detect the TGF-β1 gene expression by ELISA kit and PLP-Ig by Western blot in DCs cultured from murine bone marrow. Results The constructed adenovirus has been demonstrated to express the products specifically and independently in DCs. Conclusion The constructed recombinant adenovirus using CⅡTA-pⅠ as a DCs specific promotor is a potential approach for targeting DCs to express foreign genes in gene therapy.

关 键 词：树突状细胞 CⅡTA-pⅠ 转化生长因子Β 基因治疗 腺病毒载体 

分 类 号：R392[医药卫生—免疫学]