CONSTRUCTION OF NONDEFECTIVE ADENOVIRUS TYPE-5 BEARING A 2.8-KILOBASE HEPATITIS-B VIRUS-DNA NEAR THE RIGHT END OF ITS GENOME
CONSTRUCTION OF NONDEFECTIVE ADENOVIRUS TYPE-5 BEARING A 2.8-KILOBASE HEPATITIS-B VIRUS-DNA NEAR THE RIGHT END OF ITS GENOME
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DOI：
10.1128/jvi.54.3.711-719.1985
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发表时间：
1985-01-01
影响因子：
5.4
通讯作者：
SHIMOJO, H
中科院分区：
文献类型：
--
作者：
SAITO, I;OYA, Y;SHIMOJO, H
开发了一种新的无辅助基因的5型腺病毒（Ad 5）载体系统，该系统利用了距离基因组右端0.2 kb的克隆位点。为了构建在该位点携带有B型肝炎病毒（HBV）2.8-kb DNA片段的无缺陷Ad 5，从克隆的DNA中获得覆盖Ad 5基因组右1/4的2.1-kb非必需E3片段，（76-100个图单位）缺失，将HBV DNA插入该位点，将重组DNA连接到Ad 5基因组的其余部分，并将连接的DNA转染入人胚肾细胞。大多数回收的病毒克隆只有E3缺失而没有HBV插入，这表明在这些细胞中转染的DNA种类之间发生同源重组。携带HBV DNA的分离的Ad 5病毒（Ad 5-HBL）在没有辅助病毒的情况下在HeLa细胞中与野生型Ad 5一样有效地生长，尽管在Ad 5-HBL感染的细胞中仅在早期阶段检测到1.9 kb的主要E4转录物，这表明插入E4启动子上游的HBV DNA减少了E4转录物。从插入的DNA转录的HBV mRNA种类在Ad 5-HBL感染的晚期至少与Ad 5早期mRNA种类一样丰富，但在感染的细胞裂解物和培养基中几乎检测不到HBV表面抗原。这一结果表明，HBV mRNA种类可以从插入的基因转录，但没有蛋白质可以从HBV mRNA种类翻译，推测是因为腺病毒在其晚期引起的细胞mRNA种类的翻译抑制。
A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, was developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, the 2.1-kb nonessential E3 fragment from cloned DNA covering the right 1/4 of the Ad5 genome (76-100 map units) was deleted, the HBV DNA was inserted into this site, the recombinant DNA was ligated to the rest of the Ad5 genome, and the ligated DNA was transfected into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNA species in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNA species transcribed from the inserted DNA were at least as abundant as Ad5 early mRNA species in the late phase of Ad5-HBL infected, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNA species can be transcribed from the inserted genes but no protein can be translated from the HBV mRNA species, presumably because of the translation suppression of cellular mRNA species caused by adenovirus in its late phase.