The Preliminary Study On Gp96Associated Peptides Library In Bladder Cancer And Paratuomor Tissue

Objective It has been known heat shock protein gp96derived from cancer cells or tissues can elicit cancer-specific immunity. Recent studies have indicated that gp96molecules, as a chaperon, participate in the presentation of the associated peptides to T cells through histocompatibility complex class I-restricted antigen presentation pathway. On the basis of these features, gp96can be used for cancer immunotherapy. Though the traditional preparation method of gp96had been developed, it had many limitations and shortage including a limited amount of sample material and the complicated purification procedures. The construction of a new rapid and high effective preparation method is necessary. Further Researches had confirmed that the antigenicity derived from not gp96but gp96associated peptides. The whole peptide pool associated with gp96is the molecular basis of the vaccine preparation and the induction of specific immune reaction. So, to further identify and analyze the gp96-associated peptide pool is very necessary for developing effective cancer vaccine and investigating related mechanism of action.Methods The specific gp-96clones were screened from a’naive’human Fab fragment phage diasplay library against the immobilized gp-96antigens, then the clones were transformed to the BL21(DE3)pLysS to give high-performance soluble expression of Fab antibodies by using IPTG as inducing agent. Soluble Fab antibodies were purified and acted as couple ligand to adsorb gp96peptide complexes from tissue protein suspensions of bladder cancer and paratumor tissue, gp-96associated peptides were eluted and identified by mass spectrometry. Using bioinformatics technology was used to obtain bionomics of polypeptidesResults1. Specific anti-gp96Fab antibodies were enriched by screening from a human naive Fab fragment phage antibody library.2. Soluble expressions of specific anti-gp96Fab antibodies were performed.3. gp-96peptide complexes can be obtained from small amounts of bladder tissue by antibody library technology and immuno affinity chromatography technology. The preparation was identified by Western blot. One gram tissue can obtain20～50μg gp-96peptide complexes.4.14peptides were identified,8peptides were from bladder cancer tissue, and others were from paracancerous tissue of bladder. The molecular weight range is997.5281Da～2211.33Da. Protein source and antigenicity were identified.Conclusions1. gp-96peptide complexes can be obtained from small amounts of bladder tissue by antibody library technology and immunoaffinity chromatography technology.2. Peptides in gp-96associated peptide pool are some small molecular peptides, their length is different. Protein sources of some peptides are known, some are unknown. Some peptides have antigenicity. Further analysis must be accomplished before the application in peptide vaccine.