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.

Figure 1

PCR products on agarose electrophoresis. Lane1: DL2000 marker; Lanes 2, 3: PCR products.

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

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

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

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

N-terminal amino acid sequence of G17P64K protein.

Figure 7

HPLC identification of the purity of synthetic polypeptide.

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