Studies on the viral DNA of the nuclear polyhedrosis virus of the silkworm, Bombyx mori

对家蚕核型多角体病毒在BmN细胞上进行了空斑纯化。对其中一株T3进行繁殖，纯化病毒DNA并克隆到质粒pBR322和pUC19中。克隆到质粒中的每个DNA片段的物理图谱使用几种内切酶构建。通过克隆质粒的杂交实验，构建了病毒DNA的物理图谱。病毒DNA的基因组大小估计约为130 kb。在病毒基因组中发现含有EcoRI识别位点的重复序列。采用逆转录酶合成的cDNA，从感染后期的感染脂肪体中分离得到BmNPV T3分离物的多面蛋白基因进行杂交鉴定。在EcoRI位点将多面蛋白基因克隆到pBR322中，Sanger等采用双脱氧链终止法确定了整个核苷酸序列。比较加州签名蝇的序列，在多面蛋白基因的编码区发现了几个核苷酸的变化。推导出的多面蛋白氨基酸序列相对保守。最近在种子岛分离出一种产生立方多面体的突变体。对BT31分离物进行空斑纯化，并与T3分离物进行限制性内切酶分析。将多面蛋白基因克隆到pUC19中并测序。在编码区发现了4个碱基变化。四种碱基变化中的两种引起氨基酸的变化，表明这些变化对多面体包涵体的三维结构很重要。构建了一种插入氯霉素转移酶基因的重组BmNPV，用于研究病毒在体内的复制和感染途径。

A nuclear polyhedrosis virus of the silkworm, Bombyx mori, was plaque purified on BmN cells. One of the isolates, named T3, were propagated and viral DNA was purified and cloned into plasmids, pBR322 and pUC19. Physical maps of each DNA fragments cloned into plasmids were constructed using several endonucleases. By hybridization experiments of cloned plasmids, a physical map of the viral DNA was constructed. The genome size of the viral DNA was estimated as about 130 kb. Repeated sequences containing EcoRI recognition site were found in the viral genome.The polyhedrin gene of the BmNPV T3 isolate was identified by hybridization experiments using cDNA synthesized by a reverse transcriptase from mRNA isolated from infected fat bodies at late stage of infection. The polyhedrin gene was cloned into pBR322 at an EcoRI site and the entire nucleotide sequence was determined by dideoxy chain termination procedure by Sanger et al. Comparing the sequences of Autographa californica, several nucleotide changes were found in the coding region of the polyhedrin gene. Deduced amino acid sequences of the polyhedrins were relatively conserved.A mutant, which produces a cubic polyhedra, was recently isolated in Tanegashima. BT31 isolate was plaque purified and compared with T3 isolate by restriction endonuclease analysis. The polyhedrin gene was molecular cloned into pUC19 and sequenced. Four base changes were found in the coding region of the polyhedrin. Two of the four base changes causes amino acid changes indicated that these changes are important for the 3-D structure of the polyhedral inclusion body.A recombinant BmNPV with insertion of the gene of chloramphenicol transferase was constructed for the studies on viral replication and infectious path way in in vivo system.