Research Of Therapeutic Vaccine Against Gastrin17 Based On P64K Protein

The polypeptide hormone gastrin is secreted by G cell of gastric antrum and proximal duodenum, whose role in the regulation of gastric acid secretion and growth of gastrointestinal epithelium cell was well established. Research indicates that gastrin is a autocrine growth factor of gastrointestinal tumour cell and it can promote cancer cells through enhancing proliferation, inhabiting apoptosis,stimulating migration and cooperating with COX-2. Antisecretory agents, receptor antagonist,antisense polynucleotide and anti-gastrin antibody have been evaluated as anti-gastrin therapy of gastrointestinal tumour. Our work aimed to develop a new anti-gastrin therapeutic vaccine based on P64K protein against gastrointestinal tumour.We select a 9 N-terminal amino acids of human gastrinl 7 (Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu) as target sequence. The B-cell antigen epitope is unique to G17 and glycine-extended G17, it is not found on G34 nor on CCK and therefore does not cross-react.P64K protein from pathogenic bacterium Neisseria meningitids is found in the outer membrane of the cell. As a carrier protein, P64K protein can help to activate humoural immune system. Recent research indicates that P64K. has a better immune-enhancing effect than conventional carrier protein such as BSA, TT. We construct a therapeutic vaccine against gastrointestinal tumour consisting of the nine terminal amino acids of human G17 linked or crosslinked via a peptide spacer to P64K. The vaccine induces body to produce anti-gastrin antibody, which could neutrilize the gastrin endocrined or autocrined by tumour cell and inhibit the growth of tumour.We cloned P64K gene from Neisseria meningitids and constructed a pET28a expression vector. P64K protein and G17P64K fusion protein was expressed at highlevel in E.coli. After Ammonium sulfate precipitation, hydrophobic interaction chromatography, Gel filtration chromatography and anion exchanger chromatography, protein sample that has the purity above 90% was achieved. Ploypeptide (pyro-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Gly-Gly-Gly-Gly-Ser-Cys) is synthesis-ed by solid phase chemical method. Synthesised polypeptide is crosslinked to carrier protein P64K and DT by MBS method. Rabbits anti-gastrin antibody experiment divides into five groups: synthesed polypeptide, P64K, P64K-polypeptide crosslinked protein, DT-polypeptide crosslinked protein and G17P64K fusion protein. Rabbit serurn with the crosslinked protein achieved a high-titer anti-gastrin antibody after two enhancing immune. In vitro activity of antibody was evaluated and the result manifested that it has a high biological activity.