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


CONCLUSIONS
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.
Xiong
Xiang-Hua
XH
Department of Microbiologic Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
Zhao
Hong-Liang
HL
Xue
Chong
C
Zhang
Wei
W
Yang
Bing-Fen
BF
Yao
Xue-Qin
XQ
Liu
Zhi-Min
ZM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology*; D005755:Gastrins / Q000235:genetics / Q000276:immunology*; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification; D013274:Stomach Neoplasms / Q000628:therapy*
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D019496:Cancer Vaccines; D005755:Gastrins; D011993:Recombinant Fusion Proteins; C112623:lpdA protein, Neisseria meningitidis; C013550:gastrin 17