甜菜夜蛾核多角体病毒在离体细胞中连续传代时重复DNA片段的产生和分析

野生型甜菜夜蛾核多角体病毒 (SeMNPV)US1分离株 (SeUS1 )通过空斑法纯化 ,PCR、长片段PCR和限制性内切酶分析筛选和鉴定 ,获得一株基因型较为均一且具完整基因组的克隆株 ,命名为Se 4。Se 4在其宿主细胞系Se3 0 1中无稀释连续传代至 1 0代 ,各代被感染细胞中的病毒DNA经限制性内切酶分析 ,发现在第 7代时病毒基因组中出现了一条新增的 3 .5kb片段 ,随着代数的增加 ,该片段的摩尔量逐渐增加 ,在第 1 0代时已成为超摩尔带 ,推测该片段为SeMNPVDNA复制的顺式作用元件。序列分析表明 ,该片段覆盖了SeMNPV 81 0 1 4～ 845 3 8nt共 3 5 2 5bp的序列 ,包含被预测为杆状病毒的DNA复制原点的non hr区域以及一些SeMNPV特有的ORF。研究结果为核多角体病毒II组的non hr在病毒复制过程中具有重要作用的观点提供了体外实验的证据

Generation and Analysis of a Repeat DNA Fragment of SeMNPV by Serial Undiluted Passage in Se301 Insect Cells

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis acting DNA segments were studied. A strain, named Se 4, was plaque purified from SeMNPV isolate US1. PCR, ELT PCR and REN showed that Se 4 was genetically relatively homogeneous and retained the full length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se 4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb Pst I fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non hr origin (ori ) of DNA replication and some ORFs including partial vlf 1, partial p26, Se 84 as well as Se83, Se85, Se 86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non hr ori by serial undiluted passage in IPLB SF 21 cell culture, our results provided in vitro evidence that the non hr ori may also play an important role in the viral infection cycle of Groups II NPVs.