Résumé
Objective: To investigate the targeting and anti-tumor ability of the tumor vessel-specific antibody ScFvH1 selected from phage-ScFv library, and to discuss the application of the antibody in clinical diagnosis and therapy of cancer. Methods: The ScFvH1 gene was inserted into pET-28a(+)/EGFP vector containing green fluorescent protein(GFP) gene and pTIG-Trx vector containing thioredoxin gene; the products were then expressed in E.coli and purified by using Ni-NTA. Tumor-bearing mice model was established by subcutanuous injection of cervical cancer cell line HeLa. The mice were injected with purified ScFv-EGFP fusion protein through vena caudalis and the GFP signals were observed by fluorescent microscope to evaluate the targeting ability of the antibody. Meanwhile, the mice model also received intratumoral injection of purified ScFv-EGFP fusion protein to evaluate the anti-tumor effect of the antibody. Results: Soluble ScFvH1 gene and ScFvH1-EGFP protein were successfully expressed in E.coli; a single band was showed in SDS-PAGE after the purification by Ni-NTA. We found that ScFvH1-EGFP fusion protein was enriched to tumor tissues, but there was only weak fluorescent signal when EGFP protein was injected. No EGFP signal was observed in the lung of tumor-bearing mice. Tumor inhibition experiment showed that the tumor growth in the antibody treatment group was similar to that of the PBS control group. Conclusion: The tumor vessel-specific antibody ScFvH1 selected from phage-ScFv library can specifically target tumor vessels, but it has no obvious inhibitory effect on tumor growth. Our findings pave a way for antibody in cancer diagnosis and treatment.
Résumé
Objective:To obtain phage-displayed 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 phage-antibody system was used to construct a single-chain 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 of VH 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 phage-ScFvs were selected by phage displaying and panning in vivo.After four rounds,24 phage-ScFvs,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:Tomors-bearing 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 phage-ScFv named ScFvH1(VH-linker-VL)was selected from the library.Conclusion:A tumor targeting ScFv library has been successfully constructed and a tumor vessel-specifrc antibody has been identified from the library,which provides a new way for the early diagnosis and therapy of cancer.