Serological Identification And Bioinformatics Analysis Of Immunogenic Antigens In Osteosarcoma

Background: So far, osteosarcoma remains an incurable disease, so novel and more effective therapy of osteosarcoma are required. In recent years, much has been discovered about the mechanisms by which the immune system recognizes and responds to tumor cells. It is now possible to manipulate immunological effector cells or antigen-presenting cell ex vivo in order to induce an effective antitumor response. The osteosarcoma antigens involved have now been identified in many cases which showed an effective therapy. Tumor antigen is the coral of tumor immunotherapy. In order to achieve more complete and selective eradication of osteosarcoma cells and the minimal residual disease states, which may ultimately lead to improve disease-free survival and potentially a cure, it is urgent to identify more potent osteosarcoma antigen.Objective:①To construct human osteosarcoma cell cDNA expression library;②To screen the library by immunologic method in order to discover osteosarcoma asscioated antigens which could excite immunologic response.③To analysis the positive clones by bioinformatics as to get a definite orientation for more research.Methods:①Total RNA and purified mRNA were extracted from human cell line 9901. First and second strand cDNA were synthesized through reverse transcription. The uneven termini of the double-stranded cDNA were blunted with cloned Pfu DNA Polymerase, and EcoRⅠadapters were ligated to the blunt ends. After the Ends of EcoR I adapters were phosphorylated. Excess EcoRⅠadapters and small-sized cDNA were eliminated by size fractionated prior to ligation with lambda vector. About 100ng cohesive ended cDNA was ligated with dephophrylatedλgt11 express vector. The ligated DNA was added to the Ready-to-go Lambda Packaging Extract and incubated at 22℃for 2 hours. Appropriate dilutions of the packaging reactions in phage buffer were prepared and 100μL of the diluted phage was incubated in 200μl freshly prepared E. coli XL1-Blue-MRF'for 15 minutes at 37℃. 3mL molten NZY top agar was mixed with the former mixture, poured onto NZY plates. Color Selection was performed byα-complementation using 15μl 0.5M IPTG and 50μl 250mg/mL X-gal. Plaques were visible after incubation for 8 hours at 37℃. Background plaques were blue, colorless recombinant plaques were colorless and the proportion of recombinant clone was assessed. Ten colorless plaques were picked up randomly and the pBK-CMV phagemids were excised from the vector by using ExAssist helper phage with XLOLR strain, and the pBK-CMV phagemids were digested by Xho I and EcoR I. 5μl digestion products were analyzed on 1.4% agarose gel electrophoresis.②A total of 1×106 recombinants were screened per cDNA library. Recombinant pjhage at a concentration of 5×104 per 15 cm plate was amplified for 6 hr, and proteins were then transferred to nitrocellulose membranes for an additional 10hr at 37℃. The membranes were then blocked with 5% non-fat dried milk for 1 hr at room temperature, washed with TBS and incubated in a 1:100 dilution of pre-absorbed autologous sera(in 0.2% NFDM/TBS) for 15hr at room temperature. Following a TBS wash step, the membranes were incubated in a 1:2500 dilution of biotinylated goat anti-human IgG(H+L) and 1:2500 dilution of alkaline phosphatase streptavidin in turn. Then processed for NBT/BCIP color development.③Positive plaques were picked up and put into 0.5mL SM buffer overnight at 4℃to release the phage. The pBK-CMV phagemids were excised from theλgt11 vector by using ExAssist helper phage with XLOLR strain, and the pBK-CMV phagemids were digested by Xho I and EcoR I. As to evaluate the size of inserts, digestion products were analyzed on 1.4% agarose gel electrophoresis. All clones were sent for sequencing by T3 or T7 primer.④Analyse the sequences by bioinformatics.Results:①By calculation, the human osteosarcoma 9901 cell line cDNA library contained 1.5×106 individual clones and the proportion of recombinant phages was 94.3%. The average size of insert cDNAs was about 1.4kb.②By iterative screening, 32 serum reactive clones were obtained.③By BLAST, we got 12 distinct antigens. Among them, 5 were known gene products and 7 were unknown.④Analyzed by bioinformatics and CrElISA, the optical density (OD) values of OSAA-3 and OSAA-5 were significantly higher in OS patients.Conclusions:①The human osteosarcoma cDNA expression library consisting of 1.5×106 recombinant bacteriophages with the recombinant ratio 94.3% was constructed for the first time, which had a better diversity.②We brought some modifications into SEREX as follows, which could lead a better outcome: Construction of cDNA expression library from cell line that would has a better representation; Get more positive clones by screening the library with sera mixture.③The SEREX method of immunoscreening cDNA library with sera was applied and 12 sera reactive clones were obtained in total. The positive cDNA clones were converted to pBK-CMV phagemid forms by in vivo excision. After sequenced, we get 5 known genes and 7 unknown genes.④Analyzed by bioinformatics, the results suggested that OSAA-3, is 87% homologous to papillomavirus binding factor (PBF), was recently reported as a DNA binding transcription factor cooperating with RUNX1. It expresses in osteosarcomas and can be detected by CTL in osteosarcoma. OSAA-5 may be kind of smooth muscle myosin light chain (SMMLC). Both have significantly higher OD value in OS.