摘要
携带入ＩＦＮ－γｃＤＮＡ序列的Ｅ１区缺失的腺病毒载体ｐＡｄ１２／ＲＳＶ－ＩＦＮ－ｂｐＡ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．病毒噬斑感染２９３细胞，至出现完全细胞病变效应（Ｃｙｔｏｐａｔｈｉｃｅｆｆｅｃｔ，ＣＰＥ）后，抽提细胞内的病毒ＤＮＡ，经ＸｈｏⅠ酶切后走凝胶电泳，出现重组腺病毒特有的３．５ｋｂ大小的ＤＮＡ条带．３２Ｐ标记的探针ｐＡｄ１２／ＲＳＶ－ＩＦＮ－ｂｐＡ与ＸｈｏⅠ酶切过的病毒ＤＮＡＳｏｕｔｈｅｒｎ杂交，显示出与预期相符的重组腺病毒特有的三条ＤＮＡ片段的杂交信号，大小分别约为３００ｂＰ、５００ｂＰ和３·５ｋｂ；同时，病毒感染人的２９３细胞培养液上清作ＰＣＲ检测，都扩增出ＩＦＮ－γｃＤＮＡ和腺病毒ＤＮＡＥ２Ｂ区特异的两条ＤＮＡ条带，证实这６个病毒噬斑均为带有ＩＦＮ－γ的重组腺病毒．
El-deleted adenoviral vector, pAdl2/RSV-IFN2-bpA,harboring human IFN-γcDNA, and plasmid PJM17 were co-transfected into the human 293 cells by calciumphosphate precipitation-mediated transfection. Six adenovirus recombinants(rAd) were obtained after 11 days' incubation. Viral DNA was extracted from pellets of the 293 cells displaying cytopathic effects after rAd infection. After Xho Ⅰdigestion, a rAdspecific 3. 5 kb DNA band could be seen on eletrophoresis. Southern bloting assay with the probe pAd12/RSV-IFN-bpA resolved three hybridized bands characteristic of homologous recombinants. Two pairs of primers,IFN-γ targeted and adenoviral E2B region targeted,were used to co-amplify the template DNAs prepared from supernatant of 293 cell culture medium following recombinant adenovirus infection. Two expected PCR fragments (570 hp and 860 hp in sizes) were shown. Thus, it was demonstrated that these six adenovirus recombinants were generated by homologous recombination between the two co-transfected plasmids.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1996年第6期607-612,共6页
Journal of Fudan University：Natural Science
基金
利诚-复旦生物高新技术研究发展基金
关键词
腺病毒
同源重组
鉴定
IFN-γ
hornologous recombination
PCR
adenovirus
