Development of methods that facilitate generation of recombinant herpesviruses using BAC system

使用 BAC 系统开发促进重组疱疹病毒产生的方法

• 批准号: 12556049
• 负责人: KAWAGUCHI Yasushi
• 金额: $ 5.18万
• 依托单位: Nagoya University (2002)Tokyo Medical and Dental University (2000-2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556049/
• 关键词: herpesviruses; reverse genetics; BAC system; 組み替え法; BAC system

In recent years, several laboratories have reported on the cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in E. coli and on procedures to manipulate these genomes using the bacterial recombination machinery. However, the herpesvirus-BACs reported so far are either replication-incompetent or infectious with a deletion of one or more viral genes due to the BAC vector insertion. As a multi-purpose clone for use in the research of herpesviruses, we attempted to generate infectious herpes simplex virus (HSV) -BACs containing the full genome of HSV-1 without any loss of^viral genes. Our results were as follows. (i)E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAG, flanked by loxP sites were inserted into the intergenic region between U_L3 and U_L4 was constructed, (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii)' The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by co-infection of Vero cells with YK304 and a recombinant adenovirus AxCANCre expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulency in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics and a recombinant virus carrying amino acid substitutions in both copies of the αO gene was generated. pYEbac102 will be multi-applicable to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

近年来，一些实验室报道了在大肠杆菌中克隆疱疹病毒基因组作为细菌人工染色体（BAC）。大肠杆菌中的基因组，以及利用细菌重组机制操纵这些基因组的程序。然而，迄今为止报道的疱疹病毒-BAC要么是无复制能力的，要么是由于BAC载体插入而缺失一个或多个病毒基因的感染性的。作为用于疱疹病毒研究的多用途克隆，我们试图产生含有HSV-1全基因组的感染性单纯疱疹病毒（HSV）-BAC，而没有任何病毒基因的丢失。我们的结果如下。（i）E. coli（YEbac 102）中构建了HSV-1全长基因组（pYEbac 102），其中在U_L3和U_L4之间的基因间隔区插入了一个BAG，两侧是loxP位点。（ii）pYEbac 102是一个感染性分子克隆，因为它转染兔皮肤细胞导致了感染性病毒（YK 304）的产生。(iii)通过用YK 304和表达Cre重组酶的重组腺病毒AxCANCre共感染Vero细胞，BAC载体序列几乎完全可从重构病毒YK 304的基因组中切除。(iv)就检测而言，来自pYEBac 102的重构病毒在体外和体内不能与野生型病毒表型区分。因此，病毒在Vero细胞中生长良好，与野生型病毒一样，并在脑内接种的小鼠中表现出野生型毒性。(v)感染性分子克隆pYEBac 102实际上可用于通过细菌遗传学诱变HSV-1基因组，并产生了在αO基因的两个拷贝中携带氨基酸取代的重组病毒。pYEbac 102将在HSV-1的基础研究和人类治疗中的HSV载体的开发中用于基因工程HSV-1重组体的快速产生。

项目成果

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前田 健: "αヘルペスウイルス感染症に対するDNAワクチン"日本臨床. 58. 933-928 (2000)

Ken Maeda：“针对 α-疱疹病毒感染的 DNA 疫苗”日本临床杂志 58. 933-928 (2000)。

G.Matsuda et al.: "Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein bel-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1"Microbiology and Immunology. 49. 91-99 (2003)

G.Matsuda 等人：“Epstein-Barr 病毒 (EBV) 核抗原前导蛋白 (EBNA-LP) 通过 HS1 相关蛋白 X-1 与细胞抗凋亡蛋白 bel-2 或其 EBV 对应物 BHRF1 形成复合物”

K.Sonoda, M.Sakaguchi, H.Okamura, K.Yokogawa, E.Tokunaga, S.Tokiyoshi, Y.Kawaguchi, and K.Hirai: "Development of effective polyvalent vaccine against both Marek's and Newcastle diseases based on recombinant Marek's disease virus type in commercial chicken

K.Sonoda、M.Sakaguchi、H.Okamura、K.Yokokawa、E.Tokunaga、S.Tokiyoshi、Y.Kawaguchi 和 K.Hirai：“基于重组马立克氏病开发针对马立克氏病和新城疫的有效多价疫苗

Damiani AM, Matsumura T, Jang HK, Izumiya Y, Mikami T, Takahashi E: "Identification of the products of the equine herpesvirus type 4 gI and gE genes"Arch Virol. 145(7). 1489-1496 (2000)

Damiani AM、Matsumura T、Jang HK、Izumiya Y、Mikami T、Takahashi E：“马疱疹病毒 4 型 gI 和 gE 基因产物的鉴定”Arch Virol。