Identification Of The Target Antigen Recognized By A Hepatoma Cell Specific Antibody And Research On The Expression Of This Antigen In Hepatoma Tissues

Screening of tumor cell specific antibodies from various kinds of antibody libraries has been performed twenty years ago, and many tumor specific antibodies were successfully selected, but there were obstacles in the identification of the corresponding antigens of these antibodies. Only part of these antigens had been identified, this is mainly because that the micro-protein identification technologies developed more slowly. Accompanied by the emergence of proteomics, many new technologies had been developed. Among them, rapid improved mass spectrometry technologies played indispensable roles in protein analysis and identification. PMF and LC-MS/MS are the most commonly used technologies for protein identification.The single-chain variable fragment N14 (scFv N14) is an antibody directed against HepG2 hepatoma cells, it could specifically recognize HepG2 cells, but not L02 cells. The purpose of this research is to identify the antigen of scFv using proteomic approach, and to analyze the relationship between this antigen and the genesis and progress of hepatocellular carcinoma.Firstly, the expression vector pET-scFv N14 was used to transform E. coli BL 21(DE3)LysS, and the high expression level of scFv N14 was obtained. The scFv N14 expressed as inclusion bodies were dissolved in 8mol/L urea, refolded by dilution and purified by metal chelating chromatography. The purified scFv N14 was subjected to analysis of the expression levels of scFv N14 antigen by Western blot, the results indicated that HepG2 cell had high expression level of scFv N14 antigen, whereas L02 cell had low expression level. The subcellular localization of scFv N14 antigen was also analyzed by immunochemistry, the results showed that scFv N14 antigen mainly distributed in nucleus.Because scFv N14 antigen had nuclear localization, the nucleus of HepG2 cell was separated and used to extract proteins for identification of scFv N14 antigen. Western blot analysis demonstrated that scFv N14 could recognize 37kD and 35 kD protein bands. The proteins in the two bands were identified by Q-TOF, the candidate proteins in 37kD band were annexin A2 isoform 2, glyceraldehyde-3-phosphate dehydrogenase, hnRNP protein...