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ABSTRACT: The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.
Applied Microbiology and Biotechnology 02/2010; 87(1):289-95. · 3.42 Impact Factor
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ABSTRACT: The low efficiency of the oral infectivity of recombinant polyhedrin-negative baculovirus is a major bottleneck in the application of the baculovirus expression system in the silkworm (Bombyx mori L). In this study, the effects of a fluorescent whitening agent on improving the oral infection for the recombinant Bombyx mori nuclear polyhedrosis virus in silkworm larva and their possible mechanism were investigated. The results showed that the peroral infection can be remarkably enhanced by adding VBL into the larval artificial diet. The maximum infection rate reached as high as 90% with the concentration of VBL (1%), which was then considered as optimal. The total protease activity and pH value of the larval intestinal juice were found to be lower when compared to the control, indicating an abnormal physiological change of the larval digestive system by VBL, which, in turn, resulted in improved peroral infection of recombinant virus.
Current Microbiology 02/2007; 54(1):5-8. · 1.82 Impact Factor
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ABSTRACT: Silkworm larva has a lot of advantages as a "biofactory" to produce recombinant protein. A recombinant baculovirus, carrying cDNA encoding the 165 amino-acid long isoform of human vascular endothelial growth factor (VEGF) was successfully constructed for the large-scale production of this protein using silkworms as an in vivo host. The fifth-instar silkworm larvae were inoculated with the recombinant virus. Time-course expression analysis indicated that the expression level was highest at around 80 h post-infection and the recombinant protein was found mainly in the haemolymph. Therefore, the hemolymph was collected from the infected larvae and the recombinant protein was purified by using Nickel affinity chromatography under native condition. The expression level was estimated to be as high as approximately 426 microg per larva. Furthermore, the recombinant protein was characterized and was found biologically active in inducing endothelial cell proliferation in vitro.
Journal of Biotechnology 09/2004; 111(3):253-61. · 3.05 Impact Factor
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ABSTRACT: The insect baculovirus expression system is one of the most effective eukaryotic expression systems known, and has been widely used to produce numerous recombinant proteins. The current traditional inoculation method consists of injecting recombinant baculovirus directly into insects, thus causing potential contamination to the environment due to virus diffusion during the inoculation process. In the present experiment, we directly introduced baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus. This new method produced the same infection results as the traditional method. A new safe infection technique without direct use of the virus was thus developed.
Research in Microbiology 155(6):462-6. · 2.76 Impact Factor
