scholarly journals Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

1984 ◽  
Vol 4 (3) ◽  
pp. 399-406 ◽  
Author(s):  
G D Pennock ◽  
C Shoemaker ◽  
L K Miller
Keyword(s):  
Escherichia Coli ◽  
Insect Cells ◽  
Dna Encoding ◽  
Gene Encoding ◽  
Recombinant Viruses ◽  
Virus Particles ◽  
Nuclear Polyhedrosis ◽  
High Level ◽  
Fused Gene ◽  
Beta Galactosidase

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector

1984 ◽  
Vol 4 (3) ◽  
pp. 399-406
Author(s):  
G D Pennock ◽  
C Shoemaker ◽  
L K Miller
Keyword(s):  
Escherichia Coli ◽  
Insect Cells ◽  
Dna Encoding ◽  
Gene Encoding ◽  
Recombinant Viruses ◽  
Virus Particles ◽  
Nuclear Polyhedrosis ◽  
High Level ◽  
Fused Gene ◽  
Beta Galactosidase

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.

A novel way to purify recombinant baculoviruses by using bacmid

2009 ◽  
Vol 29 (2) ◽  
pp. 71-75 ◽  
Author(s):  
Wu-Jie Su ◽  
Wei-De Shen ◽  
Bing Li ◽  
Yan Wu ◽  
Guang Gao ◽  
...  
Keyword(s):  
Insect Cells ◽  
Nuclear Polyhedrosis Virus ◽  
Essential Gene ◽  
Essential Genes ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Recombinant Bacmid ◽  
Bmnpv Bacmid ◽  
Nuclear Polyhedrosis ◽  
Recombinant Bacmids

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

scholarly journals Translational Regulation of the Escherichia coli Stress Factor RpoS: a Role for SsrA and Lon

2007 ◽  
Vol 189 (13) ◽  
pp. 4872-4879 ◽  
Author(s):  
Caroline Ranquet ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Translational Regulation ◽  
Regulatory Rna ◽  
Gene Encoding ◽  
Ribosome Stalling ◽  
High Level ◽  
Sigma Subunit

ABSTRACT Escherichia coli cell viability during starvation is strongly dependent on the expression of the rpoS gene, encoding the RpoS sigma subunit of RNA polymerase. RpoS abundance has been reported to be regulated at many levels, including transcription initiation, translation, and protein stability. The regulatory RNA SsrA (or tmRNA) has both tRNA and mRNA activities, relieving ribosome stalling and cotranslationally tagging proteins. We report here that SsrA is needed for the correct high-level translation of RpoS. The ATP-dependent protease Lon was also found to negatively affect RpoS translation, but only at low temperature. We suggest that SsrA may indirectly improve RpoS translation by limiting ribosome stalling and depletion of some component of the translation machinery.

scholarly journals Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabriela Gregova ◽  
Vladimir Kmet
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Virulence Genes ◽  
Gene Encoding ◽  
Antibiotic Resistant ◽  
Beta Lactamases ◽  
E Coli ◽  
Animal Wastes ◽  
Extended Spectrum Beta Lactamases ◽  
High Level

Abstract Processing of animal carcasses and other animal wastes in rendering plants is a significant source of antibiotic resistant microorganisms. The main goal of this study was to investigate the resistance to 18 antibacterial agents including β-lactams, fluoroquinolones, colistin and virulence factors (iss, tsh, cvaC, iutA, papC, kps and ibeA genes) in 88 Escherichia coli strains isolated from a rendering plant over 1 year period. ESBL (Extended-spectrum beta-lactamases) and plasmid-mediated Amp were screened by interpretative reading of MIC. ESBL phenotype was detected in 20.4% of samples and high level of resistance to fluoroquinolone was found in 27.2% of strains. Cephalosporinase CTX-M1, cephamycinase CMY-2, integrase 1 and transposon 3 genes were detected by PCR. Furthermore, there were found three CMY-2 producing E. coli with O25b-ST131, resistant to the high level of enrofloxacin and containing the gene encoding the ferric aerobactin receptor (iutA). One enrofloxacin resistant E. coli strain possessed iss, ibeA, kps and papC virulence genes also with CMY-2, integrase1 and Tn3. ST131 E. coli with CMY-2 has a zoonotic potential and presents a serious health risk to humans.

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli

Gene ◽  
1994 ◽  
Vol 151 (1-2) ◽  
pp. 37-43 ◽  
Author(s):  
Joseph Zock ◽  
Cathleen Cantwell ◽  
James Swartling ◽  
Roland Hodges ◽  
Tonya Pohl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
High Level Expression ◽  
Gene Encoding ◽  
Expression In Escherichia Coli ◽  
High Level

scholarly journals High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

2003 ◽  
Vol 10 (4) ◽  
pp. 596-601 ◽  
Author(s):  
Shinya Fukumoto ◽  
Xuenan Xuan ◽  
Kimie Kadota ◽  
Ikuo Igarashi ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Babesia Gibsoni ◽  
Gene Encoding ◽  
Hydrophobic Region ◽  
Immunodominant Antigen ◽  
Diagnostic Potential ◽  
Highly Hydrophobic ◽  
High Level

ABSTRACT Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 108 cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.

scholarly journals Production of human beta interferon in insect cells infected with a baculovirus expression vector.

1983 ◽  
Vol 3 (12) ◽  
pp. 2156-2165 ◽  
Author(s):  
G E Smith ◽  
M D Summers ◽  
M J Fraser
Keyword(s):  
Expression Vector ◽  
Insect Cells ◽  
Biologically Active ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Protein Coding ◽  
Beta Interferon ◽  
Baculovirus Expression Vector ◽  
Nuclear Polyhedrosis ◽  
Hybrid Genes

Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 X 10(6) U of interferon activity was produced by 10(6) infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

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