| Purpose Gastric cancer is a highly prevalent neoplasm and the first killer among various malignancies in China. Many conventional approaches, including surgical, chemical and physical treatments, appear palliative in most advanced cases. These conventional approaches cannot be selectively targeted to the tumor cells and completely eradicate them, this may account for the development of recurrence and metastasis of gastric cancer due to the existence of residual tumor cells; In addition, as to some kinds of conventional approaches, such as chemical therapy, their tumoricidal effects also could cause damage to some other normal tissues/cells and undermine the function of immune system in patients with gastric cancer. Thus, introducing a new way of targeting therapy still remains as one of the highlights in the field of gastric cancer therapy. Ascribed to the discoveries of many tumor specific/associated antigens (TSAs/TAAs) and the unique/excellent properties of antibody, the antibody-mediated targeting therapy of tumor persistently attracts worldwide attentions. MG7, a murine mAb against human gastric cancer, was prepared and proved to possess quite high specificity and sensitivity in recognize to an ascertained antigen associated with gastric cancer in the previous studies of our lab. Conditioned on the successful preparation of MG7 mAb, this study was conducted with an attempt to develop the MG7ScFv for its further application in the targeting therapy of gastric cancer.Methods1. Culture of MG7 hybridoma cells and detection of antigen-binding affinity of MG7 mAb by ELISA2. Construction and identification of MG7 recombinant phage antibody librarymRNA was isolated from cultured MG7 hybridoma cells and converted into cDNA; The variable fragments of heavy and light chain were separately amplified and assembled into ScFvs with a specially constructed DNA linker by PCR. The ScFvs DMA was ligated into the phagmid vector pCANTABSE and the ligated sample was transfered into competent E. Co// TG1 to generate a bacterial form of MG7recombinant phage antibody library; Volume and recombinant ratio of the library were evaluated by means of bacterial colony counts and restriction analysis (EcoR I and Hind III) .3. Panning and enrichment ofMG7 recombinant phage antibodyThe transformed cells were infected with M13K07 helper phage to produce a phage form of MGy recombinant phage antibody library; After three rounds panning of the library with the MGrbinding antigen highly expressed gastric cancer cell line KATO III, the MG7 ScFv displayed phage dones were selected from the enriched recombinant phages by ELISA.4. Detection of antigen-binding affinity of MG7 recombinant phage antibodyELISA was repeated to confirm the antigen-binding affinity of positive clones screened out in the former procedure; These positive phages were examined by restriction analysis (EcoR I and Hind III) ; Competitive ELISA was performed to test the inhibitory ratio of these positive clones to the binding affinity of MG7mAb and its relevant antigen, the positive dones possessing apparent inhibitory effect were singled out for later use.5. Expression and detection of soluble MG7 scFvThe positive dones possessing apparent inhibitory effect were individually transfected into Eco// HB2151; After induction by IPTG, perplasmic extracts was prepared from the induced transfectant; The yields of soluble MG7 scFv produced by the transfected Eco// HB2151 were evaluated via immunodoting assay.6. Detection of antigen-binding affinity of soluble MG7 scFvELISA was adopted to examine the antigen-binding affinity of soluble MG7 scFv; Competitive ELISA was performed to test the inhibitory ratio of soluble MG7 scFv to the binding affinity of MG7 mAb with its relevant antigen.7. The molecular mass of MG7 scFv was measured by Western blot.8. Sequencing of the MG7 scFv DNADNA sequendng was performed by ABI PRISM?377 DNA sequencer. Results1. ELISA test suggested that the MG7 mAb had apparent antigen-binding affinity and it...
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