Construction And Characterization Of Human Osteosarcoma 9901 Cell CDNA Library

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: (1)To construct human osteosarcoma cell cDNA expression library; (2)To screen the library by immunologic method in order to discover osteosarcoma asscioated antigens which could excite immunologic response. (3) To analysis the positive clones by bioinformatics as to get a definite orientation for more research.Methods: (1)Total RNA and purified mRNA were extracted from human cell line U937. First and second strand cDNA were synthesizedthrough reverse transcription. The uneven termini of the double-stranded cDNA were blunted with cloned Pfu DNA Polymerase, and EcoR I adapters were ligated to the blunt ends. After the Ends of EcoR I adapters were phosphorylated. Excess EcoR I adapters and small-sized cDNA were eliminated by size fractionated prior to ligation with lambda vector. About 100ng cohesive ended cDNA was ligated with dephophrylated γ gt11 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. coll XLl-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 by a -complementation using 15μl 0.5M EPTG 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.(2)A total of 1 X 106 recombinants were screened per cDNA library. Recombinant pjhage at a concentration of 5 X 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 forNBT/BCIP color development. (3)Positive plaques were picked up and put into 0.5mLSM buffer overnight at 4 to release the phage. The pBK-CMV phagemids were excised from the gt11 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. (4)Analyse the sequences by bioinformatics.Results: By calculation, the human osteosarcoma 9901 cell line cDNA library contained 1.5 X106 individual clones and the proportion of recombinant phages was 94.3%. The average size of insert cDNAswas about 1...