Cloning, expression and identification of truncated spike gene of IBV (Sf200) and chicken granulocyte- macrophage colony stimulating factor (GM-CSF)
Thai Journal of Veterinary Medicine
; 52(3):583-590, 2022.
Article
in English
| CAB Abstracts | ID: covidwho-2323611
ABSTRACT
The aim of this study was to clone, express and identify the truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken. Firstly, two genes were amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The truncated S1 gene designated as Sf200 containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa) 291-398 and (aa) 497-543 and GM-CSF were then amplified from the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis. The in vitro expression of truncated Sf200 and GM-CSF constructs were later expressed in E. coli BL21 with a molecular mass of approximately 38 kDa and 29 kDa respectively as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polyclonal antibodies were developed by injecting E. coli expressed Sf200 and GM-CSF into the SPF mice and were used to identify the recombinant proteins by Western blot analysis. These findings indicated that the polyclonal antibodies produced in mice could be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.
Prion; Viral; Bacterial and Fungal Pathogens of Animals [LL821]; Animal Immunology [LL650]; Host Resistance and Immunity [HH600]; Molecular Biology and Molecular Genetics [ZZ360]; colony stimulating factor; immunogenetics; DNA cloning; polymerase chain reaction; antigens; amino acid sequences; recombinant proteins; antibodies; poultry; avian infectious bronchitis; Infectious bronchitis virus; fowls; Avian coronavirus; Gammacoronavirus; Coronavirinae; Coronaviridae; Nidovirales; positive-sense ssRNA Viruses; ssRNA Viruses; RNA Viruses; viruses; Gallus gallus; Gallus; Phasianidae; Galliformes; birds; vertebrates; Chordata; animals; eukaryotes; recombinant plasmid; IBV infection; chickens; PCR; antigenicity; immunogens; protein sequences; domesticated birds
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Database:
CAB Abstracts
Language:
English
Journal:
Thai Journal of Veterinary Medicine
Year:
2022
Document Type:
Article
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