A Way To Identify Breast Cancer Cell Surface Antigen Single-chain Antibody

scFv-507 is a single-chain antibody selected from phage display antibody library, which specifically recognizes MCF-7 breast tumor cell, but not nomal cell lines. The basic purposes for this research are to analyze the tumor specificity of scFv-507,and to identify the scFv-507 antigen by MS or other technologies.To obtain pure scFv-507 antibody, scFv-507 gene and the encoding sequence of signal peptide of M13 phage pIII protein in its 5' end were cloned into pET24a vector to construct the secretary expression vector. This recombinant expression vector could express and secret scFv-507 into the periplasm of E.coli. The expressed scFv-507 was soluble, not in inclusion body, this avoids the refolding of scFv-507 antibody. The His-tag was also retained in the C-terminus of scFv-507. Being a small tag, His-tag does not affect the function of the target protein. Using this tag, scFv-507 was purified by Ni²⁺ chalating chromatography, its purity is great than 90 percent.The purified scFv-507 was used to analyze the expression profiles of scFv-507 antigen in different tumor cells by cellular ELISA and FACS technologies. The results of cellular ELISA indicated that scFv-507 antigen was highly expressed in MCF-7 and HepG2 cells, but not in normal cell lines. The FACS analysis demonstrated that there were different expression levels of scFv-507 antigen in three different breast tumor cell lines. In MCF-7 cells, 90 percent of the cells expressed scFv-507 antigen, while in MD-MAB-231 cells only about half of the cells expressed this antigen. In SK-BR-3 cells, this rate was decreased to 13.6 percent. scFv-507 antigen was also expressed in 76.8 percent of SK-OV-3 ovarian adenocarcinoma cells,and in 59 percent of HepG2 heptacellar carcinoma cells. But low expression level was observed in GLC82 lung adenocarcinoma cells, only 9.98 percent of the cells were scFv-507 antigen positive, this rate approach the one in L02 normal hepatocellular cells. In normal lung cells, this rate was further reduced to 3 percent. It is not clear wheather the expression of scFv-507 antigen is related to the mutation in cells.To identify scFv-507 antigen, the proteins in MCF-7 cells were extracted. Because scFv-507 antigen is membrane-associated antigen, high concentration of detergent was added into the cell lysis buffer in order to extract the membrane proteins thoroughly. The extracted proteins included soluble fraction and insoluble fraction, they were all used as the starting materials for identification of scFv-507 antigen. The extracted soluble proteins were analyzed by SDS-PAGE, and transferred to PVDF membrane. After Western blotting, one 10kD positive band recognized by scFv-507 was found. This band was cut away, and then the binding antibodies on it were removed using acidic dissociation buffer. Afer that the absorbed proteins on the membrane were digested with trypsin, and used to analyze by MALDI-TOF-MS. The data demonstrated that the candidate protein was G protein-regulated inducer of neurite outgrowth 1(GRIN1), its molecular weight was 102308 dalton. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. It functions as a downstream target for Galphao.No evidence exhibits its relationship with the genesis and development of tumor. The identified protein from protein band or dot by MALDI-TOF-MS only meant the protein existed in the band or dot, not meant that this protein was very target protein recognized by the antibody, so other tests are needed to further identify the scFv-507 antigen.Phage display peptide library has the great advantage in identification of the epitope recognized by antibody. Fortunately, the identified epitope by panning exhibits highly homologous to the corresponding epitope in target protein. In order to further identify scFv-507 antigen, the phage display peptide library was used to select the scFv-507 recognizing epitope. Because the binding to the solid media directly will cause protein denaturation, proteinA/G media was used to capture scFv-507 protein through anti-His tag antibody. After four rounds of panning, the phage recovery rose from 6.7×10^-5 in the first round to 1.6×10^-2 in the fourth round, this meant the specific scFv-507 binding clones were enriched. Ten phage clones were picked up randomly and sequenced. Two consitency sequences were found, they were AQALHHH and QHHLWPE, respectively. The sequence AQALHHH existed in five clones, while the sequence QHHLWPE existed in two clones. They all contains the core sequence LHH( or HHL),but no corresponding homologous sequences were found in GRIN1 protein. After similarity searching in human protein database, no possible target antigen was found.In summary, in this work, the secretary scFv-507 was successfully expressed in E.coli, and purified. The scFv-507 antigen expression profile was also analysis by cellular ELISA and FACS. The results indicated that scFv-507 antigen exhibits tumor cell specificity in some extent, and one candidate antigen GRIN1 was identified by MALDI-TOF-MS. On the other hands, two epitopes recognized by scFv-507 were selected from phage display peptide library.But the real scFv-507 antigen still needs be further investigated.