MULTIPLICATION MECHANISMS OF BOMBYX MORI NUCLEAR POLYHEDROSIS VIRUS

用几种不同的限制性内切酶酶切BmNPV基因组DNA，并用琼脂糖凝胶电泳分析所产生的DNA片段。DNA片段的大小总和表明，BmNPV包含约115千碱基对的DNA基因组。为了构建BmNPV DNA的物理图谱，我们将Sau3AI内切酶部分酶切产生的DNA片段克隆到<lambda>噬菌体载体EMBL3中。采用两种不同的二维凝胶体系对BmNPV的结构多肽进行了分析。sds -聚丙烯酰胺凝胶电泳（PAGE）等电聚焦检测到41个分子量在68,000 （68K）至12.5K之间的酸性多肽，非平衡pH凝胶电泳（SDS-PAGE）检测到45个分子量在125K至13.5K之间的碱性多肽。采用常规小鼠杂杂瘤技术制备了针对BmNPV结构蛋白的杂杂瘤，筛选了6个对BmNPV结构蛋白产生单克隆抗体的杂杂瘤。经Western blot检测单克隆抗体特异性后，将这6种单克隆抗体分为3种类型。对感染BmNPV的BM-N细胞进行免疫荧光分析，发现不同单克隆抗体特异性抗原在感染细胞中表现出不同的定位。感染BmPNV的BM-N细胞在感染后每隔3小时用（^<35>S）蛋氨酸标记，用SDS-PAGE分析病毒诱导多肽的合成，然后用荧光成像分析。流式图显示，感染细胞中依次出现27个分子量为130K ~ 14K的多肽。免疫沉淀实验显示，病毒诱导的多肽中有23个多肽与抗bmnpv血清发生反应。用（^<14>C） n -乙酰氨基葡萄糖和（^<32>P）磷酸作为示踪剂，研究了病毒诱导的糖蛋白和脂蛋白的合成。少

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