Construction and evaluation of anti-gastrin immunogen based on P64K protein.
World J Gastroenterol
; 12(13): 2040-6, 2006 Apr 07.
Article
en En
| MEDLINE
| ID: mdl-16610054
AIM: To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. METHODS: G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS: G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION: The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Neoplasias Gástricas
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Proteínas de la Membrana Bacteriana Externa
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Proteínas Recombinantes de Fusión
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Gastrinas
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Vacunas contra el Cáncer
Límite:
Animals
Idioma:
En
Revista:
World J Gastroenterol
Asunto de la revista:
GASTROENTEROLOGIA
Año:
2006
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Estados Unidos