title: Construction of a transposon-mediated baculovirus vector Hanpvid and a new cell line for expressing barnase.
abstract: In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages (LD(50)) and the lethal time(s) (LT(50)) of rHa-Bar were reduced by 20 % and 30 %, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.
authors.affiliation: School of Life Science, Wuhan University, Wuhan 430072, P. R. China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Qin
authors.forename: Ying-le
authors.initials: YL
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Shun-yi
authors.initials: SY
authors.lastname: Li
authors.forename: Yi-peng
authors.initials: YP
authors.lastname: Qi
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line / Q000201:enzymology; D004251:DNA Transposable Elements / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D003368:Gossypium / Q000821:virology; D007814:Larva / Q000821:virology; D007915:Lepidoptera / Q000472:pathogenicity / Q000821:virology; D007928:Lethal Dose 50; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010572:Pest Control, Biological; D010935:Plant Diseases / Q000469:parasitology; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004279:DNA, Viral; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease