小鼠SOCS1基因重组腺病毒载体的构建及其在树突状细胞中的表达  

Construction of Recombinant Adenovirus Vector Containing Mouse SOCS1 Gene and Its Expression in Dentritic Cells

作　　者：陈晓波[1] 秦杰[2] 焦洋[1] 钟翠平[2] 谭建明[1] 

机构地区：[1]上海交通大学附属第一人民医院肾移植科上海市器官移植临床医学中心,上海200080 [2]复旦大学上海医学院解剖组胚系,上海200032

出　　处：《现代生物医学进展》2007年第10期1445-1447,1450,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金(30571759)

摘　　要：目的：构建含小鼠细胞因子信号抑制因子-1基因（SOCS1）的重组腺病毒载体（Ad5F35-SOCS1），探讨其介导SOCS1基因在小鼠树突状细胞中的表达。方法：设计含AgeI和NheI酶切位点的SOCS1基因上下游引物，以质粒pEF-FLAG-1／mSOCS1为模版，通过PCR扩增获得SOCS1全部序列，片段回收后经AgeI和NheI酶切，再定向插入到经AgeI和NheI酶切的质粒pDC316-LacZa中，获得重组穿梭质粒pDC316-SOCS1，经AgeI和NheI酶切、PCR及测序等鉴定后，用脂质体将穿梭质粒pDC316-SOCS1与腺病毒骨架质粒pBHGF35共转染293细胞，经位点特异性重组获得重组腺病毒Ad5F35-SOCS1，行PCR鉴定，经293细胞扩增、纯化制备高滴度病毒液，TCID50法测定病毒滴度。用获得的重组腺病毒感染小鼠树突状细胞，以免疫组化检测SOCS1的表达。结果：成功构建了含小鼠SOCS1基因的重组腺病毒载体，病毒感染滴度为1．4×10^9IU／ml，该载体能有效介导SOCS1基因在小鼠树突状细胞中的表达。结论：重组腺病毒载体能将SOCS1基因转入小鼠树突状细胞并有效表达，为基因转染制备耐受性树突状细胞奠定了基础。Objective： To construct recombinant adenovirus vector containing mouse suppressor of cytokine signaling-lgene （Ad5F35-SOCS1）, and to investigate the expression of SOCS1 in mouse dendritic cells transfected by Ad5F35-SOCS1. Methods： Full length SOCS1 cDNA was obtained from plasmid pEF-FLAG-1/mSOCS1 by PCR amplification. The PCR product was digested by restriction endonucleases AgeI and NheI, and then inserted orientationally into plasmid pDC316-LacZa digested by AgeI and NheI. Once constructed, the recombinant shuttle plasmid pDC316-SOCS 1 was identified by PCR and sequencing, and then cotransfected with rescue plasmid pBHGF35 into 293 cells by liposome reagent. Ad5F35-SOCS1 was generated by site-specific recombination and confirmed by PCR. Ad5F35-SOCS1 was propagated in 293 cells and purified. The titer of viral stock was determined by TCID50 assay. Ad5F35-SOCS1 was then used to transfect mouse dendritic cells, and the expresssion of SOCS1 was confirmed by immunohistochemistry. Results： The recombinant adenovirus vector containing mouse SOCS1 gene （Ad5F35-SOCS1） was successfully constructed and the infective titer was 1.4×10^9 IU/ml. The expression of SOCS1 in mouse dendritic cells transfected by Ad5F35-SOCS1 was efficient. Conclusions： Recombinant adenovirus could transmit SOCS1 gene to mouse dendritic cells, and the expression of SOCSlwas efficient. It laid the foundation for producing tolerogenic dendritic cells via gene transfection.

关 键 词：腺病毒载体 细胞因子信号抑制因子-1 树突状细胞 

分 类 号：R394.2[医药卫生—医学遗传学]