Isolation of a Spodoptera exigua baculovirus recombinant with a 10•6 kbp genome deletion that retains biological activity

被引:30
作者
Dai, XJ
Hajós, JP
Joosten, NN
van Oers, MM
IJkel, WFJ
Zuidema, D
Pang, Y
Vlak, JM
机构
[1] Univ Wageningen & Res Ctr, Virol Lab, NL-6709 PD Wageningen, Netherlands
[2] Zhongshan Univ, State Key Lab Biocontrol, Guangzhou 510275, Peoples R China
[3] Zhongshan Univ, Inst Entomol, Guangzhou 510275, Peoples R China
[4] Hungarian Acad Sci, Inst Enzymol, Biol Res Ctr, H-1518 Budapest, Hungary
关键词
D O I
10.1099/0022-1317-81-10-2545
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
When Spodoptera exigua muliticapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated, These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel, In the present study a novel procedure to isolate SeMNPV recombinants was adapted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV, polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13.7 and 21.6 map units of SeMNPV and including ecdysteroid UDP glucosyl transferase, gp37, chitinase and cathepsin genes, as well as several genes unique to SeMNPV, The result indicated, however, that these genes are dispensable for virus replication both in vitro and in vivo. A mutant with a similar deletion was identified by PCR in the parental wild-type SeMNPV isolate, suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses. The generation of recombinants in vivo, combined with the alternate cloning between insects and insect cells, is likely to be applicable to many baculovirus species in order to obtain biologically active recombinants.
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收藏
页码:2545 / 2554
页数:10
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