甜菜夜蛾核多角体病毒BAC-TO-BAC外源基因表达系统的建立

Establishment of Spodoptera exigua Multicapsid Nucleopolyhedrovirus BAC-TO-BAC Expression System

用直接克隆法将miniF lacZ attTn7 kan片段插入甜菜夜蛾核多角体病毒 (Spodopteraexiguamulticapsidnucleopolyhe drovirus,SeMNPV)美国分离株 (SeUS1)基因组的多角体蛋白基因框内 ,miniF是大肠杆菌F因子复制子 ,携带miniF的重组病毒能够在大肠杆菌中低拷贝稳定复制 ,称为bacmid。由于SeUS1由不同的SeMNPV基因型组成 ,每个bacmid携带了一种病毒基因型 ,所有bacmid构成了SeUS1分离株的BAC文库。REN对 111个bacmid分析表明 ,SeUS1分离株中除了包含具有完整SeMNPV遗传信息的基因型外 ,还包括不同类型的缺失基因型。将具有完整SeMNPV基因组的基因型SeBAC10转染昆虫细胞 ,可产生子代病毒 ,故SeBAC10是一种在真核细胞和原核细胞中均能复制的穿梭质粒。因为SeBAC10中多角体蛋白基因 (Seph)被插入失活 ,将Seph作为报告基因通过位点特异性重组方式插入位于LacZ框内转座子Tn7的附着靶位点attTn7,得到重组SeBAC10 (即SeBAC10ph)转染甜菜夜蛾培养细胞Se3 0 1后 ,细胞出现典型的病理变化 ,核中出现多角体 ,证明SeMNPVBAC TO

Present studies describe the successful establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) BAC-TO-BAC expression system. The mini-F-lacZ-attTn7-kan fragment (Luckow et al, 1993) was inserted into SeMNPV US1 isolate (SeUS1) at polyhedrin gene locus by directly cloning. The recombinant virus containing low-copy-number mini-F replication, which named bacmid, can propagate in Escherichia coli. Because SeUS1 isolate is make up of several genotypes and one bacmid carries one SeMNPV genotype, the SeUS1 BAC library is established by all SeMNPV bacmids (SeBAC). REN analysis for 111 SeBAC shows that SeUS1 consists of the genotype with whole SeMNPV genetic information and several genotypes with various different deletions. Progeny virus can be produced in insect cell line after transfection with SeBAC10, which carries the whole SeMNPV genome. So SeBAC10 is a shuttle vector that can replicate in eukaryocyte as well as prokaryocyte. Considering the insert mutation of SeMNPV polyhedrin gene (Seph) in SeBAC10, Seph was reintroduced into the bacmid by site-specific transposon-mediated insertion at attTn7, the target site for the bacterial transposon Tn7. The derived recombinant SeBAC10 was named SeBAC10ph. After SeBAC10ph was transfected into Se301 cells (a susceptible insect cell line to SeMNPV), cytopathogenic effect was shown and polyhedra appeared, which indicate that the foreign gene (Seph) is expressed.