Screening And Primary Identification Of Anti-CCR7 ScFv From Large Phage Library

Objective: To screen and identify anti-CCR7 single chain variable fragments (scFv) from lager phage library, and to detect the scFv efficiency.Methods: The insert ratio of ScFv antibodies library was identified by PCR. The products of Sfi I / Not I double enzyme digest were identified by 1% agarose gel electrophoresis. Panning against breast cancer cell line MDA-MB-435s and CCR7 were performed four and three rounds respectively. Positive clones were transformed to E.coli HB2151, and their dissolvability was observed. The soluble scFv was purified by affinity chromatography and its relative molecular mass was determined by Western blotting. The ELISA assay was used to identify the immunocompetence of the antibody. Immunocytochemical staining and radioimmunoimaging were employed to determinate the affinities of scFv binding with CCR7 in cell line and nude mice. The influence of anti-CCR7 scFv on the invasive potential of cells was investigated by using Transwell methods.Results: The insert ratio of ScFv gene was 90% (18/20), enzyme digest reaction showed the aim products on 1% agarose gel. ScFvs were obviously enriched by 7 round panning. Western blotting result showed soluble scFv' molecular mass was about 34 kd. ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435s cells. Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435s cells which expressed CCR7. Transwell methods showed that anti-CCR7 scFv decrease the invasive potential of cell line MDA-MB-435s which expressed CCR7.Conclusion: ScFvs against CCR7 were successfully acquired by screening the phage antibody library. The soluble ScFvs has avidity specifically to human breast cancer cell MDA-MB-435s which expressed CCR7, and decrease the invasive potential.