Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Apr 7;12(13):2040-6.
doi: 10.3748/wjg.v12.i13.2040.

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

Xiang-Hua Xiong  1 , Hong-Liang Zhao, Chong Xue, Wei Zhang, Bing-Fen Yang, Xue-Qin Yao, Zhi-Min Liu
Affiliations

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

Xiang-Hua Xiong et al. World J Gastroenterol. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
PCR products on agarose electrophoresis. Lane1: DL2000 marker; Lanes 2, 3: PCR products.
Figure 2
Figure 2
15 % SDS-PAGE analysis of expression products. Lane 1: Non-recombinant bacteria; Lane 2: Low molecular protein marker; Lane 3: Recombinant bacteria; Lane 4: Deposition after sonication; Lane 5: Supernatant after sonication.
Figure 3
Figure 3
The expression of G17P64K protein in bioreactor. Lane 1: Low molecular protein marker; Lane 2: Sample before IPTG induction; Lanes 3-9: Sample after induction 1-7 h.
Figure 4
Figure 4
15 % SDS-PAGE analysis of sample after ammonium sulfate deposition. Lane 1: Low molecular protein marker; Lanes 2, 4, 6, 8: Supernatant after ammol/Lonium sulfate deposition; Lanes 3, 5, 7, 9: Deposition after 20 %, 30 %, 40 %, 50 % ammonium sulfate deposition.
Figure 5
Figure 5
15 % SDS-PAGE analysis of purified G17P64K fusion protein. Lane 1: Low molecular protein marker; Lane 2: Total protein after induction; Lane 3: Supernatant after sonication; Lane 4: Sample after hydrophobic chromatography; Lane 5: Sample after gel filtration chromatography; Lane 6: Sample after anion exchanger chromatography.
Figure 6
Figure 6
N-terminal amino acid sequence of G17P64K protein.
Figure 7
Figure 7
HPLC identification of the purity of synthetic polypeptide.
Figure 8
Figure 8
Effects of rabbit anti-gastrin 17 antibody on the in vitro growth of SW480. ◆: anti-gastrin 17(9): P64; ■Red: rabbit anti-gastrin 17(9): DT; ▲: rabbit control IgG
See this image and copyright information in PMC

References

    1. Modlin IM, Kidd M, Marks IN, Tang LH. The pivotal role of John S. Edkins in the discovery of gastrin. World J Surg. 1997;21:226–234. - PubMed
    1. Urushidani T, Forte JG. Signal transduction and activation of acid secretion in the parietal cell. J Membr Biol. 1997;159:99–111. - PubMed
    1. Wang Z, Chen WW, Li RL, Wen B, Sun JB. Effect of gastrin on differentiation of rat intestinal epithelial cells in vitro. World J Gastroenterol. 2003;9:1786–1790. - PMC - PubMed
    1. Smith JP, Verderame MF, Ballard EN, Zagon IS. Functional significance of gastrin gene expression in human cancer cells. Regul Pept. 2004;117:167–173. - PubMed
    1. Zhou JJ, Chen ML, Zhang QZ, Hu JK, Wang WL. Coexpression of cholecystokinin-B/gastrin receptor and gastrin gene in human gastric tissues and gastric cancer cell line. World J Gastroenterol. 2004;10:791–794. - PMC - PubMed

Publication types