Construction And Screening Of Human Anti-nasopharyngeal Carcinoma Single-chain Fv Fusion Phage Libraries

AIM: To construct and screen fully human anti-nasopharyngeal carcinoma single-chain Fv fusion phage libraries.METHODS: Peripheral blood mononuclear cells (PBMCs) of patients with nasopharyngeal carcinoma were sensitized in vitro and transformed by Epstein-Barr virus (EBV). VH and VL genes were reamplified by PCR and combined to single-chain fragment of variable region (ScFv) genes. ScFv genes were cloned into vector fuse5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library. The library was subjected to three rounds of positive and negative cell panning and enrichment, then was screened by phage-ELISA. The binding specificity of phage antibodies with nasopharyngeal carcinoma cells was confirmed by immunohistochemistry with cultured cells and tissue sections.RESUILS: Detection of ELISA showed that 3 nasopharyngeal carcinoma patients'B cells transformed by EBV could produce specific antibodies to nasopharyngeal carcinoma cell. 6 types of VH genes and 9 types of VL genes were obtained by PCR reamplification then connected with (Gly₄Ser)₃ linker to form 54 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fuse5 and transformed into MC1061 via electroporation. Phage antibody library with sink size being 6.5×10⁷ was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 100%. The library was subjected to three rounds of positive and negative cell panning and enrichment,then was selected by phage-ELISA.After primacy test by CNE2 cell ELISA,245 clones of phage antibodies with positive ELISA reaction were picked out of 828 clones.The percentage of positive clones was 29.6%. Further screened by a panel of cultured cells ELISA,the clone F20 was found to react with CNE2,HNE2 and CNE1,but not with other human tumor cell lines and normal cell lines or react weakly. The results of immunohistochemistry with cultured cells were same as the results of ELISA. F20 reacted specifically with the nasopharyngeal carcinoma cell in ten human nasopharyngeal carcinoma tissue sections but not with any of the cells in five human normal membrana mucosa nasi tissue sections. F20 was further analyed after the DNA sequencing. The VDJ regions of F20 belonged to VH3 - 23 - D6 -6 - JH6 - linker - V4 - 2 - JL2.CONCLUSION: Fully human anti-nasopharyngeal carcinoma single-chain Fv fusion phage libraries with sink size being 6.5×10⁷ was constructed by means of phage antibody library technique in combination with in vitro immunization method and EBV transformation technique . By cell ELISA and immunohistochemistry with cultured cells and tissue sections,the clone F20 was confirmed to specific bind with Nasopharyngeal carcinoma cells.The ScFv fragment against nasopharyngeal carcinoma may be further developed and applied to clinical diagnosis and therapy.