Hyper-IL-6重组腺病毒的制备  

Generating of recombinant adenovirus vector containing Hyper-IL-6 (AdHIL-6)

作　　者：王传敏[1] 郄春花[2] 秦波[1] 

机构地区：[1]重庆医科大学附属第二医院感染病科,重庆400010 [2]天津市传染病医院检验科,天津300192

出　　处：《中国现代医学杂志》2009年第7期1003-1006,共4页China Journal of Modern Medicine

基　　金：重庆市自然科学基金(渝科发计字[2004]54号)

摘　　要：目的探讨大量制备高滴度重组腺病毒的方法。方法以Pac-I酶切Hyper-IL-6重组腺病毒(AdHIL-6)质粒,乙醇沉淀回收DNA,将线性化的AdHIL-6质粒经脂质体转染293细胞进行病毒的包装扩增,以氯化铯密度梯度离心法纯化重组腺病毒,根据GFP(绿色荧光蛋白)计数法和有限稀释法原理测定病毒滴度及感染293细胞、L02细胞的MOI(Multiplicity of infection,感染复数),并以免疫细胞化学检测感染L02细胞后目的蛋白Hyper-IL-6的表达。结果收集的病毒液经PCR扩增出目的条带,测得病毒滴度为1.6×109pfu/mL,AdHIL-6感染293细胞、L02细胞的MOI分别是7和54,感染L02细胞后Hyper-IL-6强阳性表达。结论成功地完成了Hyper-IL-6重组腺病毒的制备,为后续的腺病毒载体介导的基因治疗奠定了基础。[ Objective] An approach to method of generating recombinant adenoviruses. [Methods] The recombinant adenoviral plasmid was digested with Pac-Ⅰ. To reclaim DNA precipitated by alcohol, and transfect the lined AdHIL-6 plasmid to 293 cells with liposome for generating recombinant adenoviruses. To purify the viruses through CsCl density-rgradient centrifugalizaton, then detemline the viruses titer and the efficiency of infecting target ceils （293 ,L02）. The expression of object protein HIL-6 was detected by immunocytochemistry. [Results] The viruses liquid can be amplified object genes. The titer of AdHIL-6 is 1.6×10^9 pfu/mL. MOI equals to 7 in 293 cell and to 54 in L02.The expression of HIL-6 was markedly detected in L02 cell. [Conclusions] The success in generating recombinant adenoviruses may be a preparation to sequent study on gene therapy mediated by adenovirus.

关 键 词：重组腺病毒 293细胞 超级白细胞介素6 基因治疗 

分 类 号：R511.8[医药卫生—内科学]