Screening and primary identification of anti-CCR7 scFv from large phage library / 中国药理学通报

ABSTRACT

Aim To screen and identify anti-CCR7 single chain fragments variable(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 digested by Sfi I/Not I double enzyme.Panning against breast cancer cell and CCR7 were performed four and three rounds respectively.Positive clones were transformed to E.coli HB2151, and their dissolvability was assayed.The soluble scFv was purified by affinity chromatography, and its relative molecular mass was determined by Western blot.The ELISA assay was used to identify the immunocompetence of the antibody.Immunocytochemical staining and radioimmunoimaging were employed to determine the affinities of scFv binding with CCR7 in cell line and in nude mice.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 after 7 round panning.Western blot result showed soluble scFv's molecular mass was about 34 KD.ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435 s cells.Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435 s cells which expressed CCR7.Conclusions ScFvs against CCR7 are successfully acquired by screening the phage antibody library.The soluble ScFvs have high affinity and specifical binding to human breast cancer cells.