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Construction of tumor targeting ScFv library and screening of tumor vesselspecific antibody by phage display in vivo
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Abstract:
To obtain phagedisplayed ScFv library targeting tumor tissues and to screen for antibodies specifically binding to tumor vessels using in vivo phage display, so as to lay a foundation for diagnosis and treatment of cancer. Methods: The membrane proteins were extracted from the specimens of esophageal carcinoma, stomach carcinoma, brain cancer, lung cancer, and spinal cord tumor. The recombinant phageantibody system was used to construct a singlechain Fv fragment (ScFv) cDNA library from the total RNA of the BALB/c mice immunized with purified membrane protein. The specific primers of VH and VL were used to amplify the cDNA ofVH and VL, respectively, which were then assembled into ScFv gene with a specially constructed linker DNA. The ScFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated samples were transformed into competent E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage.Using the animal model of human cervical carcinoma (HeLa cells), sepecific phageScFvs were selected by phage displaying and panning in vivo. After four rounds, 24 phageScFvs, which were identified by PCR, were analyzed immunohistochemically. The ScFvs expressed in the tumor tissue slices and negative in control kidney tissue slices were sequenced. Results: Tomorsbearing animal models were established with 7 different kinds of carcinoma cell lines in BALB/c nude mice. It was found that inoculation with HeLa cells resulted in most satisfactory tumorigenesis in nude mice. A ScFv library of 1.6×106 was obtained and a tumor vessel specific phageScFv named ScFvH1 (VHlinkerVL) was selected from the library. Conclusion: A tumor targeting ScFv library has been successfully constructed and a tumor vesselspecifrc antibody has been identified from the library, which provides a new way for the early diagnosis and therapy of cancer.
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Clc Number:
R730
