| Full text | |
| Author(s): |
A.M. Gibertoni
[1]
;
M.C.M. Gonçalves
[2]
;
M.F.S. Montassier
[3]
;
C.C. Fernandes
[4]
;
H.J. Montassier
[5]
Total Authors: 5
|
| Affiliation: | [1] Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária - Brasil
[2] Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária - Brasil
[3] Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária - Brasil
[4] Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária - Brasil
[5] Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária - Brasil
Total Affiliations: 5
|
| Document type: | Journal article |
| Source: | Arq. Inst. Biol.; v. 77, n. 1, p. 1-9, 2020-10-19. |
| Abstract | |
ABSTRACT The nucleocapsid protein (N) gene (1,230 bp) of the M41 strain of infectious bronchitis virus (IBV) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned in two systems; pET28a Escherichia coli and pFLD Pichia pastoris. The recombinant expression constructs (pET28a-N or pFLD-N) were identified by PCR and sequencing analysis. The transformant clones of BL21 strain of E. coli or GS115 of P. pastoris were submitted to appropriate inducting protocols. Expression of histidine-tagged fusion N proteins with a molecular mass of 54 kDa was determined by SDS-PAGE and Western blotting analysis, confirming that both recombinant N proteins were comparable in size and antigenicity to native IBV N protein. The E. coli system overexpressed the recombinant N protein, while the P. pastoris system produced a low yield of this recombinant protein. The bacteria expressed N protein was purified by chromatography on nickel-sepharose resin. These results indicated that the pET28a E. coli expression system is more effective to generate N recombinant protein for using as an antigen to detect anti-IBV antibodies in immuno-assays for this viral infection. (AU) | |