Baculovirus vectors for multiple gene expression and for occluded virus production

Wang, Xunzhang; Ooi, Beng Guat; Miller, Lois K.

doi:10.1016/0378-1119(91)90358-i

Cited by 216 publications

(155 citation statements)

References 18 publications

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“…Lepidopteran cells from Trichoplusia ni (BTITn5B1-4) were maintained at 27ºC in TC-100 medium supplemented with 10% fetal bovine serum (Gibco-BRL) and served as host for the wild type virus AcMNPV, the recombinant viruses vSynVI-gal (Wang et al, 1991), and the viruses constructed in this work (vAcCry4Aa,vSynCry4Aa and vSynCry4Ba). The Brazilian Bacillus thuringiensis subsp.…”

Section: Insect Cells Viruses and Bacillus Thuringiensis Strainsmentioning

confidence: 99%

“…Fragments of approximately 3,500 bp, corresponding to the cry4Aa and cry4Ba genes were purified using the GFX TM PCR DNA and Gel Band Purification Kit (GE Healthcare). The cry4Aa gene was either cloned into the pSynXIVVI+X3 transfer vector (Wang et al, 1991), previously digested with BglII, or into the pFastBac®1 vector previously digested with BamHI. The cry4Ba gene was cloned into the pSynXIV+X3/3, which had previously been linearized with EcoRI and BglII.…”

Section: Recombinant Viruses Constructionmentioning

confidence: 99%

“…The purpose of this was to verify if differences in the heterologous expression of Cry4Aa or its toxicity would be detected in any of the two methods. In the first strategy, cells infected with recombinant baculoviruses express large amounts of not only the exogenous protein, but also another baculovirus polypeptide, polyhedrin (Wang et al, 1991). This latter protein is responsible for the occlusion of viral particles, making baculovirus more resistant to adverse environmental conditions (Slack & Arif, 2007;Rohrmann, 2011).…”

Section: Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp Israelementioning

confidence: 99%

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Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Corrêa¹,

Aguiar²,

Monnerat³

et al. 2013

VR&R

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Bacillus thuringiensis subsp. israelensis (Bti) is highly toxic to mosquito larvae. Most of its toxicity relies on δ-endotoxins (Cry and Cyt) crystalline inclusions produced at the sporulation phase of growth. Obtention of individual Bti mosquitocidal toxins is crucial for the understanding of Bti´s potential activity against dipteran larvae. A Cry toxin expression system based on baculovirus was developed to produce two of the Cry toxins comprised in the crystals of Bti. Th e cry4Aa and cry4Ba genes from two Brazilian strains of Bacillus thuringiensis were amplifi ed by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis of the cry4Aa and cry4Ba genes showed high identity to previous known cry genes. Both cry4Aa and cry4Ba genes were further cloned into transfer vectors for construction of recombinant baculoviruses.. Aft er isolation of the recombinant viruses, they were used to infect insect cells (BTI-TN5B1-4) that were analyzed by light microscopy at diff erent times post infection. Putative crystals consisting of either Cry4Aa or Cry4Ba were observed in the cytoplasm of infected insect cells. RT-PCR was performed with mRNA from insect cell extracts (72 h.p.i.) in order to confi rm the presence of the genes specifi c transcripts. Recombinant virus-infected insect extracts (120 h.p.i.) were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) showing the presence of polypeptide bands of around 128 and 130 kDa, corresponding, respectively to the sizes of the proteins Cry4Aa and Cry4Ba. Bioassays with virus-infected insect extracts were shown to be toxic to second instar Aedes aegypti larvae, confi rming the usefulness of this expression system for the study of Cry proteins.

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Section: Insect Cells Viruses and Bacillus Thuringiensis Strainsmentioning

confidence: 99%

Section: Recombinant Viruses Constructionmentioning

confidence: 99%

Section: Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp Israelementioning

confidence: 99%

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Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Corrêa¹,

Aguiar²,

Monnerat³

et al. 2013

VR&R

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“…Furthermore, the recombinant Cry10Aa protein had the capacity of crystallization into cuboidal crystals in insect cells. Since the cry10Aa gene is under transcriptional control of two promoters in tandem, pSyn and pXIV (Wang et al, 1991), the high expression level of the Cry10Aa protein may have contributed to the crystallization of the protein. SDS-PAGE of purified crystals produced by vSyncry10Aa recombinant viruses showed the presence of a protein of molecular mass of approximately 85 kD (Figure 3).…”

Section: )mentioning

confidence: 99%

“…Trichoplusia ni (BTI-Tn5B1-4) cells were maintained at 27℃ in TC-100 medium supplemented with 10% fetal bovine serum (Gibco-BRL) and served as host for the wild type virus AcMNPV, the recombinant viruses vSynVI -gal (Wang et al, 1991), and vSyncry10Aa (constructed in this work). Bacillus thuringiensis subsp.…”

Section: Cells Viruses and Bacteriamentioning

confidence: 99%

Cry10Aa Protein is Highly Toxic to <i>Anthonomus grandis</i> Boheman (Coleoptera: Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields

Souza¹,

Martins²,

Ribeiro

et al. 2012

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The cry10Aa gene from the Brazilian Bacillus thuringiensis subsp israelensis S1804 strain was introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate its expression in insect cells and its toxicity to A. grandis. The recombinant virus (vSyncry10Aa) was amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analyzed by SDS-PAGE and showed the presence of a polypeptide around 85 kD. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the recombinant virus infected-insect extracts, showed high toxicity to A. grandis larvae, with a LC 50 of 7.12 µg/mL. Furthermore, a competition binding assay with the recombinant biotin-labeled Cry10A protein and brush border membrane vesicles (BBMV) from A. grandis indicated that the toxin binds specifically to BBMVs. Therefore, the Cry10A protein has a potential to be used in transgenic cotton plants for the control of this important insect pest.

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Desmoglein 3-ELISA

Lenz¹,

Amagai²,

Volc‐Platzer³

et al. 1999

Arch Dermatol

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Baculovirus vectors for multiple gene expression and for occluded virus production

Cited by 216 publications

References 18 publications

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

Cry10Aa Protein is Highly Toxic to <i>Anthonomus grandis</i> Boheman (Coleoptera: Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields

Desmoglein 3-ELISA

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