Construction and evaluation of anti-gastrin immunogen based on P64K protein

Abstract

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 plasmi d, 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 bacte ria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84th 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. © 2006 The WJG Press. All rights reserved.