Production of a human single-chain variable fragment antibody against esophageal carcinoma.

Abstract

To construct a phage display library of human single-chain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer. Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography. Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing. The size of scFv gene library was approximately 9X10(6) clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the gamma chain subgroup IV of immunoglobulin, and variable light (VL) gene from the kappa chain subgroup I of immunoglobulin. A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.