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

家蚕核型多角体病毒（NPV）在BmN细胞上进行了噬斑纯化。将其中一株命名为T3的分离株增殖，纯化病毒DNA并克隆到质粒pBR 322和pUC 19中。使用几种内切核酸酶构建克隆到质粒中的每个DNA片段的物理图谱。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的基因组大小估计为约130 kb。在病毒基因组中发现了含有EcoRI识别位点的重复序列，用逆转录酶从感染后期的脂肪体中提取mRNA，合成cDNA，通过杂交实验鉴定了BmNPV T3分离物的多角体蛋白基因。将多角体蛋白基因克隆到pBR 322的EcoRI位点上，通过桑格等人的双脱氧链终止法测定了整个核苷酸序列。推导的多角体蛋白的氨基酸序列相对保守，最近在种子岛分离到一个产生立方多角体的突变体。对BT31菌株进行噬斑纯化，并与T3菌株进行限制性内切酶分析。将多角体蛋白基因克隆到pUC 19载体上并测序。在多角体蛋白的编码区发现了四个碱基的变化。结果表明，在BmNPV中插入氯霉素转移酶基因，构建了重组病毒BmNPV，为研究病毒在体内的复制和感染途径奠定了基础。

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.