Genetic engineering approach to develop next-generation reagents for endotoxin quantification

Autor: Norihiko Ogura, Shun Ichiro Kawabata, Hikaru Mizumura, Jun Aketagawa, Yuki Kobayashi, Maki Aizawa, Toshio Oda
Rok vydání: 2016
Předmět:
Cell Extracts
Amoebocyte lysate reagent
0301 basic medicine
Amebocyte
endotoxin
Lysis
Glycosylation
medicine.medical_treatment
Immunology
Biology
Sensitivity and Specificity
Microbiology
Complement factor B
mammalian cell
law.invention
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
law
horseshoe crab
Endopeptidases
Horseshoe Crabs
medicine
Animals
recombinant cascade reagent
Molecular Biology
Limulus Test
Enzyme Precursors
Protease
Serine Endopeptidases
Original Articles
Cell Biology
Reference Standards
biology.organism_classification
Molecular biology
Recombinant Proteins
Horseshoe crab
Endotoxins
030104 developmental biology
Infectious Diseases
chemistry
Reagent
Recombinant DNA
Insect Proteins
Indicators and Reagents
Genetic Engineering
Complement Factor B
030215 immunology
Zdroj: Innate Immunity
ISSN: 1753-4267
1753-4259
DOI: 10.1177/1753425916681074
Popis: The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.
Databáze: OpenAIRE
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