Construction of human naive Fab library,screening and identification of phage antibody against c-Met
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    Abstract:

    Objective:To construct a large human Na-觙ve Fab antibody library and to screen the anti-cMet phage antibody from the library. Methods:Human Fab genes from bone marrow lymphocytes of 20 healthy donors were amplified by PCR,and the products were ligated into the phagemid vector pComb3XSS to construct a human naive Fab library. Antibodies against c-Met were screened by biopanning with immobilized antigen. After six rounds of panning,sixty randomly selected clones were identified by phage ELISA to select specific ones with high affinity for c-Met. After analyzed by BstOI digest,the positive clone was used for soluble expression in Escherichia coli. and was characterized by ELISA. Results:A large human na-觙ve Fab phage-display library consisting of 1.2 × 109 clones was successfully constructed. From the enriched phage library,a Fab fragment designated AM2-26 with fine activity to c-Met was selected. DNA sequence analysis show the V genes were human variable region immunoglobulin sequences. AM2-26 was functionally expressed,which was identified by Western blot,and its specificity was confirmed by ELISA. Conclusion:The construction of the human phage antibody library and the preparation of the anti-cMet phage antibody provide a promising candidate for the research of antineoplastic agents.

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万佳艺,孙 慧,焦永军,朱晓娟,朱 进,刘 政,冯振卿.人源天然Fab噬菌体抗体库的构建及抗c-Met抗体的筛选?鉴定[J].南京医科大学学报(自然科学版英文版),2008,28(6):697-701.

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  • Received:January 02,2008
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