Basic Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Apr 7, 2006; 12(13): 2040-2046
Published online Apr 7, 2006. doi: 10.3748/wjg.v12.i13.2040
Construction and evaluation of anti-gastrin immunogen based on P64K protein
Xiang-Hua Xiong, Hong-Liang Zhao, Chong Xue, Wei Zhang, Bing-Fen Yang, Xue-Qin Yao, Zhi-Min Liu
Xiang-Hua Xiong, Hong-Liang Zhao, Chong Xue, Wei Zhang, Bing-Fen Yang, Xue-Qin Yao, Zhi-Min Liu, Department of Microbiologic Engineering, Beijing Institute of Biotechnology, Beijing 100071, China
Co-first-author: Hong-Liang Zhao and Chong Xue
Supported by Grants from National High Technology Research and Development Program, No.2002AA2Z345B and No.2004AA2Z3803 of the Ministry of Science and Technology of China
Correspondence to: Dr. Zhi-Min Liu, Department of Microbiologic Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. liuzhm@vip.sina.com
Telephone: +86-10-66948825 Fax: +86-10-63833524
Received: April 14, 2005
Revised: October 25, 2005
Accepted: November 10, 2005
Published online: April 7, 2006
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 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.

Keywords: Gastrin; P64K protein; therapeutic vaccine