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 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.

• Authors: Xiang-Hua Xiong, Hong-Liang Zhao, Chong Xue, Wei Zhang, Bing-Fen Yang, Xue-Qin Yao, Zhi-Min Liu
• Journal: World journal of gastroenterology (World J Gastroenterol) Vol. 12 Issue 13 Pg. 2040-6 (Apr 07 2006) ISSN: 1007-9327 [Print] United States
• PMID: 16610054 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies
• Bacterial Outer Membrane Proteins
• Cancer Vaccines
• Gastrins
• Recombinant Fusion Proteins
• lpdA protein, Neisseria meningitidis
• gastrin 17

Topics

• Amino Acid Sequence
• Animals
• Antibodies (blood)
• Bacterial Outer Membrane Proteins (genetics, immunology)
• Cancer Vaccines (immunology)
• Gastrins (genetics, immunology)
• Immunization
• Mice
• Mice, Inbred BALB C
• Molecular Sequence Data
• Rabbits
• Recombinant Fusion Proteins (immunology, isolation & purification)
• Stomach Neoplasms (therapy)