MULTIPLICATION MECHANISMS OF BOMBYX MORI NUCLEAR POLYHEDROSIS VIRUS

家蚕核多角体病毒的增殖机制

• 批准号: 61480052
• 负责人: KOBAYASHI Michihiro
• 金额: $ 4.22万
• 依托单位: NAGOYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61480052/
• 关键词: Silkworm; Nuclear polyhedrosis virus; Protein; DNA; Monoclonal antibody; Electrophoresis; Fluorography; 蛍光抗体法; カイコ; 培養細胞; プラーク・アツセイ; たんぱく質合成

Genomic DNA of BmNPV was digested with several different restriction endonucleases and the DNA fragments generated were analyzed by an agarose gel electrophoresis. The sum of the sizes of the DNA fragments indicated that BmNPV contained a genome of about 115 kilobase pairs of DNA. In an attempt to construct physical map of BmNPV DNA, DNA fragments generated after partial digestion with Sau3AI endonuclease were cloned into <lambda> phage vector EMBL3.Structural polypeptides of BmNPV were analyzed by two different two-dimensinal gel systems. Isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis (PAGE) detected 41 acidic polypeptides ranging in molecular weights from 68,000 (68K) to 12.5K, and nonequilibrium pH gel electrophoresis followed by SDS-PAGE resolved 45 basic polyepetides ranging in molecular weights from 125K to 13.5K.Hybridomas raisedagainst BmNPV structural protein were prepared using conventional mouse hybridoma technology, and six hybridomas which produced … More monoclonal antibodies positive to BmNPV proteins in both an enzyme-linked immunosorbent assay and an immunofluorescent analysis were screened. After examining the specificity of the monoclonal antibodies by Western blot analysis, these six monoclonal antibodies were classified into three types. When the BM-N cells infected with BmNPV were subjected to an immunoflourescent analysis, it was found that antigens specific to different monolonal antibodies showed different localization in the infected cells.BM-N cells infected with BmPNV were labelled with (^<35>S)methionine at 3 hr intervals postinfection and synthesis of virus-induced polypeptides was analyzed by SDS-PAGE followed by fluorography. The flouorogram showed that 27 polypeptides with molecular weights ranging from 130K to 14K appeared sequentially in the infected cells. Immunoprecipitation experiments revealed that 23 polypeptides from the virus-induced polypeptides reacted with anti-BmNPV serum. Syntheses of virus-induced glycoproteins and lipoproteins were also examined employing (^<14>C)N-acetylglucosamine and (^<32>P)phosphate as the tracers. Less

用几种不同的限制性内切酶消化BmNPV的基因组DNA，并通过琼脂糖凝胶电泳分析所产生的DNA片段。DNA片段大小之和表明BmNPV基因组含有约115个DNA内切酶对。为构建家蚕核型多角体病毒（BmNPV）DNA物理图谱，将Sau 3AI酶切后的DNA片段克隆到<lambda>噬菌体载体EMBL 3中，用两种不同的双向凝胶电泳系统分析BmNPV的结构多肽。等电聚焦-SDS-聚丙烯酰胺凝胶电泳（PAGE）共检测到41个酸性多肽，分子量范围为68，000 ~ 12.5K;非平衡pH凝胶电泳-SDS-PAGE共检测到45个碱性多肽，分子量范围为125 ~ 13.5K。和六个杂交瘤， ...更多信息 筛选在酶联免疫吸附测定和免疫荧光分析中对BmNPV蛋白呈阳性的单克隆抗体。通过Western blot分析检测单克隆抗体的特异性后，将这六种单克隆抗体分为三种类型。BmNPV感染BM-N细胞后，<35>每隔3小时用（^ S）蛋氨酸标记BmNPV-N细胞，用SDS-PAGE分析病毒诱导多肽的合成。荧光图谱显示，感染细胞中依次出现27条多肽，分子量在130 ~ 14 K之间。免疫沉淀实验表明，23个多肽从病毒诱导的多肽与抗BmNPV血清反应。病毒诱导的糖蛋白和脂蛋白的合成也采用（^<14>C）N-乙酰葡糖胺和（^<32>P）磷酸作为示踪剂进行了检查。少

项目成果

Mori,H.: "Difference of proteins from inclusion bodies in the nucleus and cytoplasm of the CPV-infected midgut in the silkworm, Bombyx mori." Journal of Invertebrate Pathology. 50. 1987 26-32

Mori,H.：“家蚕 Bombyx mori 感染 CPV 的中肠的细胞核和细胞质中包涵体的蛋白质差异。”

Hashimoto, Y.: Archeves of Virology. 90. 301-312 (1986)

Hashimoto, Y.：病毒学档案。

Bando, H.: Journal of Virology. 61. 553-560 (1987)

Bando，H.：病毒学杂志。

Nagamine,T.: Applied Entomology and Zoology.

Nagamine,T.：应用昆虫学和动物学。

Bando, H.: Archieves of Virology. 93. 139-146 (1987)

Bando, H.：病毒学档案。