1987 Fiscal Year Final Research Report Summary
MULTIPLICATION MECHANISMS OF BOMBYX MORI NUCLEAR POLYHEDROSIS VIRUS
| Project/Area Number |
61480052
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
蚕糸学
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| Research Institution | NAGOYA UNIVERSITY |
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Principal Investigator |
KOBAYASHI Michihiro Assistant, School of Agriculture, Nagoya University, 農学部, 助手 (60111837)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASE Shigemi Professor, School of Agriculture, Nagoya University, 農学部, 教授 (90023382)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Silkworm / Nuclear polyhedrosis virus / Protein / DNA / Monoclonal antibody / Electrophoresis / Fluorography / 蛍光抗体法 |
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| Research Abstract |
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
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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
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