Research of Therapeutic Vaccine Against Gastrin17 Based on P64K Protein
|School||PLA Military Academy of Medical Sciences|
|Keywords||Gastrointestinal tumors Gastrin Meningococcal P64K protein Therapeutic vaccine|
Gastrin (gastrin, GAS) antral and duodenal mucosa G cells and the secretion of peptide hormones, and the major physiological role is to promote the growth of gastric acid secretion and gastrointestinal mucosa. Studies have shown that the gastrin autocrine growth factor for gastrointestinal tumors, stimulate the infiltration and the expression of COX-2 synergy to promote tumor growth by promoting tumor cell proliferation and inhibition of apoptosis. Anti-gastrin treatment of gastrointestinal tumors into secretion inhibition, receptor antagonist, antisense oligonucleotide inhibition and anti-gastrin antibody treatment. The purpose of this subject is the developing a meningococcal P64K protein for gastrointestinal tumors gastrin G17 therapeutic Phytophthora song. We chose gastrin G17N terminal 9 amino acids of the B-cell epitope (Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu) as a target, this epitope the gastrin G17 and glycine-extended gastrin G17 unique, resulting in anti-gastrin G17 antibody does not cross-react with the G34 and CCK. The P64K protein is one of the outer membrane protein from Neisseria meningitidis. P64K protein as a carrier protein can help to activate the specific humoral immune response in recent years, studies have shown that the P64K than conventional carrier protein such as BSA, TT have better immune enhancement effect. We choose the P64K protein, the cross-linking bridge, through a period of polypeptide (Gly-Gly-Gly-Gly-Ser) and a B cell epitope of the G17 crosslinked or build G17P64K fusion protein as a therapeutic vaccine to induce anti-gastrin G17 antibodies neutralize and clear the endocrine or from tumor cell autocrine gastrin, to achieve the purpose of the treatment of tumors of the gastrointestinal tract. Was cloned by PCR from the meningococcal P64K gene was constructed the P64K gene and G17P64K gene pET28a expression vector, and the high expression in E. coli. The target protein after ammonium sulfate precipitation, hydrophobic chromatography, molecular sieve chromatography, and anion exchange chromatography to obtain a sample with a purity of over 90%. Fmoc-phase method chemically synthesized polypeptide Pyro-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Gly-Gly-Gly-Gly-Ser-Cys. The MBS Law crosslinked polypeptide carrier protein P64K and DT. Preparation of anti-gastrin the G17 antibody test is divided into five groups: the synthetic peptide Group, P64K protein group, DT polypeptide crosslinking group, P64K polypeptide crosslinked group and G17P64K fusion protein group, respectively, plus Freund's adjuvant immune rabbit. Strengthen immunity after two DT polypeptide cross-linked group and P64K polypeptide cross-linked group of animal serum high titers of anti-gastrin G17 antibody. The in vitro activity of the cell level evaluation test antibody preferably the biological activity of inhibition of tumor growth.