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Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles.

Yao L, Wang S, Su S, Yao N, He J, Peng L, Sun J - PLoS ONE (2012)

Bottom Line: Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae.The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva.For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.

View Article: PubMed Central - PubMed

Affiliation: China-United Kingdom Nanyang Normal University-Rothamsted Research Joint Laboratory of Insect Biology, Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang, People's Republic of China. lunguangyao@163.com

ABSTRACT
A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.

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Related in: MedlinePlus

SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.
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pone-0032510-g003: SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.

Mentions: About 150 ml hemolymph was collected from 500 red larvae. The rotavirus VLPs were purified from the larval hemolymph by traditional ultracentrifugation using CsCl gradients. Two major bands were recovered for VLPs detection by TEM. One band was composed of baculoviral particles in rod shape, the other was made of round rotavirus VLPs. The result of SDS-PAGE proved the VLPs were constructed by three viral coat proteins including VP2, VP6 and VP7 as expected (Fig. 3). The total protein content in the purified VLPs from the hemolymph collected from 500 larvae was 6.35 mg, which meaned the yield of VLPs was 12.7 µg per larval hemolymph. Obviously this BmNPV-silkworm multigene expression system provides an economic and efficient solution for producing antiviral vaccine derived from VLPs.


Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles.

Yao L, Wang S, Su S, Yao N, He J, Peng L, Sun J - PLoS ONE (2012)

SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293821&req=5

pone-0032510-g003: SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.5 µg VLPs was loaded on 12% SDS-PAGE gel. Lane Marker: standard protein marker. lane VLPs: purified VLPs from silkworm hemolymph. The three bands VP2, VP6 and VP7 were labelled.
Mentions: About 150 ml hemolymph was collected from 500 red larvae. The rotavirus VLPs were purified from the larval hemolymph by traditional ultracentrifugation using CsCl gradients. Two major bands were recovered for VLPs detection by TEM. One band was composed of baculoviral particles in rod shape, the other was made of round rotavirus VLPs. The result of SDS-PAGE proved the VLPs were constructed by three viral coat proteins including VP2, VP6 and VP7 as expected (Fig. 3). The total protein content in the purified VLPs from the hemolymph collected from 500 larvae was 6.35 mg, which meaned the yield of VLPs was 12.7 µg per larval hemolymph. Obviously this BmNPV-silkworm multigene expression system provides an economic and efficient solution for producing antiviral vaccine derived from VLPs.

Bottom Line: Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae.The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva.For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.

View Article: PubMed Central - PubMed

Affiliation: China-United Kingdom Nanyang Normal University-Rothamsted Research Joint Laboratory of Insect Biology, Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang, People's Republic of China. lunguangyao@163.com

ABSTRACT
A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.

Show MeSH
Related in: MedlinePlus