人IL-10基因重组复制缺陷型腺病毒DNA的构建  

Construction of replication-deficient human IL-10 recombinant adenovirus

作　　者：陈紫千[1] 汤耀卿[1] 张奕[1] 张轶[1] 张圣道[1] 刘新垣[2] 邹卫国[2] 

机构地区：[1]上海第二医科大学瑞金医院消化外科,上海200025 [2]中国科学院上海生命科学院细胞生化所

出　　处：《上海第二医科大学学报》2004年第9期720-722,共3页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：上海市科委基金(00419019)资助项目.

摘　　要：目的 构建人IL-10基因的重组腺病毒并进行体外表达和检测。方法从质粒pcDNA3IL-10的CMV启动子下游完整切下IL-10 cDNA的片段,将其定向插入Gateway载体,在克隆酶的作用下将阳性克隆质粒转入目的腺病毒载体。PacI酶线性化IL-10腺病毒DNA后阳离子质脂体转染293A细胞,获得人IL-10的复制缺陷型重组腺病毒。体外感染人胰腺癌细胞株Bxpc-3和大鼠胰腺细胞株AR-42J;Western blot检测人IL-10的表达。结果 成功地构建人IL-10重组腺病毒,病毒滴度达2×109PFU/mL。体外感染的细胞株均检测到IL-10的表达。结论 构建复制缺陷型重组腺病毒能够介导IL-10的基因表达,为细胞因子的抗炎冶疗奠定实验基础。Objective To construct recombinant adenovirus containing human IL-10 gene and check IL-10 expression in vitro. Methods The full-length human IL-10 cDNA was removed from pcDNA3IL-10 and linked to Gateway vector. Then the Gateway vector were mixed with the adenovirus desstination vector. The adenorvirus vector containing IL-10 cDNA was produced. Then the recombinant virus DNA was linearized by RE PacI and transfected into the 293A cell line. The replication-incompetent adenovirus containing IL-10 was harvested from the supernatant of 293A cell line. The Bxpc-3 and AR-42J cell lines were infected by the IL-10 adenovirus recombinant virus DNA and the IL-10 protein was extracted and examined with Western blot. Results The recombinant IL-10 replication-incompetent adenovirus was expressed successfully and the titer of the recombinant adenovirus was 2 × 109 PFU/mL. Conclusion The human IL-10 recombinant adenovirus can be used to infect the target cell in vitro.

关 键 词：IL—10 缺陷型 IL-10 重组腺病毒 体外感染 表达 DNA 阳性克隆 质粒 基因重组 

分 类 号：R346[医药卫生—基础医学]