STUDY ON STRUCTURE AND FUNCTION OF PREINTEGRATION COMPLEX OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

人类免疫缺陷病毒1型整合前复合物的结构和功能研究

• 批准号: 12670289
• 负责人: KITAMURA Yoshihiro
• 金额: $ 2.11万
• 依托单位: The University of Tokyo (2001-2002)National Institute of Infectious Diseases (2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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/grant/KAKENHI-PROJECT-12670289/
• 关键词: human immunodeficiency virus; INTEGRATION; INTEGRACE; VECTOR; ヒト免疫全ウルス; HIV; ini1; SNF5; アポトーシス; G1アレスト; 組み込み酵素; 単クローン抗体

To examine whether vectors based on human immunodeficiency virus type 1 (HIV) with viral central polypurine tract (cPPT) express transgenes more efficiently than those without cPPT, we constructed a series of vectors carrying a Neo'gene as a stable marker, which is driven by an internal synthetic thymidine kinase promoter. Insertion of cPPT, either 282bp or 178bp long, resulted in about 3-8-fold increase of the amount of produced vector particles. Insertion of the longer cPPT. resulted in decrease in transduction efficiency in about eight-fold, whereas insertion of the shorter resulted in increase in transduction efficiency in about two-fold independently of the orientation of the insert. Moreover, DNA-PCR showed that reverse transcription and nuclear import were cnot affected. Alu-PCR assay showed that integration was impaired in the vectors carrying the 282bp-cPPT. Taking these together, we concluded that insertion of the 282bp-cPPT decreased the number of 0418-resistant colonies without affecting nuclear import of vector DNA. We suppose that insertion of the 282bp-cPPT resulted in impairment of events after import, such as integration.

为了检验带有病毒中枢多尿路(CPPT)的人类免疫缺陷病毒1型(HIV)载体是否比没有cPPT的载体更有效地表达转基因，我们构建了一系列携带Neo‘基因作为稳定标志的载体，该载体由内部合成胸苷激酶启动子驱动。插入282bp或178bp的cPPT可使载体颗粒的产量增加约3-8倍。插入较长的cPPT。导致转导效率降低约8倍，而插入较短的短管导致转导效率增加约2倍，而与插入物的取向无关。此外，DNA-PCR结果显示，反转录和核进口受到CNOT的影响。Alu-PCR检测表明，携带282bp-cPPT的载体整合受损。综上所述，我们得出的结论是，插入282bp-cPPT减少了0418抗性克隆的数量，而不影响载体DNA的核进口。我们推测，插入282bp-cPPT导致了进口后事件的损害，如整合。

项目成果

Takahashi T, Endo T, Nakamura T, Sakashitat H, Kimurat K, Ohnishit K, Kitamura Y, Iwamoto A.: "Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f.sp.hominis in Japan."J Med Microbiol.. 51(6). 510-515 (2002)

Takahashi T、Endo T、Nakamura T、Sakashitat H、Kimurat K、Ohnishit K、Kitamura Y、Iwamoto A.：“日本卡氏肺囊虫 f.sp.hominis 中的二氢叶酸还原酶基因多态性。”J Med Microbiol.. 51(6

Takashi Takahashi 他: "Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan"Journal of Medical Microbiology. 51. 510-515 (2002)

Takashi Takahashi 等人：“日本卡氏肺囊虫 f. sp. hominis 中的二氢叶酸还原酶基因多态性”《医学微生物学杂志》51. 510-515 (2002)。

Ae K, Kobayashi N, Sakuma R, Ogata T, Kuroda H, Kawaguchi N, Shinomiya K, Kitamura Y.: "Chromatin remodeling factor encoded by ini1 induces G1 arrest and apoptosis in ini1-deficient cells."Oncogene. 21(20). 3112-3120 (2002)

Ae K、Kobayashi N、Sakuma R、Ogata T、Kuroda H、Kawaguchi N、Shinomiya K、Kitamura Y.：“ini1 编码的染色质重塑因子在 ini1 缺陷细胞中诱导 G1 停滞和凋亡。”癌基因。

北村義浩, 仲嶋一範(編著): "必ず上手くいく遺伝子導入と発現解析プロトコール"羊土社. 228 (2003)

Yoshihiro Kitamura、Kazunori Nakajima（编辑）：“绝对有效的基因导入和表达分析方案”Yodosha 228 (2003)。

Sakuma R, Kobayashi N, Ae K, Kitamura Y.: "Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1."Biochem Biophys Res Commun.. 302(3). 489-495 (2

Sakuma R、Kobayashi N、Ae K、Kitamura Y.：“使用源自人类免疫缺陷病毒 1 型的载体，插入中央多嘌呤束序列对基因表达的抑制和增强作用。”Biochem Biophys Res Commun. 302(3)。