表达人γ-干扰素的重组腺病毒的制备及检测  

Construction and Identification of Recombinant Adenovirus for Expressing Human Interferon γ

作　　者：徐人尔[1] 应蓓蓓[1] 王义斌[1] 赵寿元[1] 李昌本[1] 

机构地区：[1]复旦大学遗传所遗传工程国家重点实验室

出　　处：《生物工程学报》1997年第1期6-12,共7页Chinese Journal of Biotechnology

基　　金：利诚-复旦生物高新技术基金

摘　　要：Ｅ１区缺失的腺病毒载体插入人γ干扰素ｃＤＮＡ序列，与ｐＪＭ１７质粒通过磷酸钙沉淀法共转染２９３细胞，经同源重组，共获得６个病毒噬斑。经ＰＣＲ共扩增检测，证实这６个噬斑均为带有人γ干扰素ｃＤＮＡ的重组腺病毒；受其感染的２９３细胞的上清中，都能测到γ干扰素的活性，且这种活性可被一定稀释度的兔抗人γ干扰素多克隆抗体所中和。其中的１号重组病毒纯化后，按不同的ＭＯＩ（Ｍｕｌｔｉｐｌｅｏｆｉｎｆｅｃｔｉｏｎ）值感染复制缺陷型腺病毒不能在其中增殖的Ｂ１６Ｆ１０细胞，未出现细胞病变效应（Ｃｙｔｏｐａｔｈｉｃｅｆｅｃｔ，ＣＰＥ），而上清中的γ干扰素活性却随ＭＯＩ值增大而升高。E1 deleted adenoviral vector,pAdl2/RSV IFN bpA,harboring human IFN γ cDNA, and plasmid pJM17 were co transfected into human embryo kidney cell line 293 cells by calcium phosphate precipitation mediated transfection. Six strains of recombinant adenoviruses(rAd) were obtained after 11 day's incubation. PCR analysis indicated that all six rAds contained human IFN γ cDNA. The biological activity of IFN γ was detected in the supernatant of the cultured 293 cells following rAds' infection, and it could be blocked by the antibody for IFN γ . The B16F10 cells infected with the purified rAd(RSV/IFN 01 ) didn't show cytopathic effect (CPE),and the more biological activity of INF γ was detected in the infected B16F10 cells' supernatant with the increased MOI(multiple of infection) values. Therefore,the recombinant adenovirus constructed in this study can express and secrete human IFN γ

关 键 词：基因治疗 腺病毒 Γ-干扰素 同源重组 活性检测 

分 类 号：R394[医药卫生—医学遗传学]