| Objective: The purpose of this study was to construct the fullyhumanized anti-hepatoma Fab fragment phage library. And then byscreening the library, we tried to pick out several colonies that expressantibodies against the antigens of the hepatoma cells specifically.METHODS: Peripheral blood mononuclear cells (PBMCs)isolated from liver cancer patients were immunized in vitro withhepatoma carcinoma cell HpeG2 that treated by mitomycin C and thenwere transformed by Epstein-Barr virus (EBV). The hepatoma carcinomacell antigen positive B lymphocytes were selected and amplified. Thetotal RNA was extracted from the lymphocytes. Genes of light chain andFd of antidodies were amplified by RT-PCR. Then the genes wereinserted into phagemid vector pComb3 and transformed into E.coliXLI-Blue by electroporation to construct the Fab-displaying phagelibrary. The phage antibody library was rescued by helper phageVCSM13 firstly, then was subjected to negative selection by hepatocytechang's liver and positive selection by hepatoma carcinoma cell HepG2.The screening repeated three cycles. After the third round, 540 clonesthat were randomly selected from this enrichment library to performPhage-ELISA using hepatoma carcinoma cells HepG2. The phageantibodies against HepG2 then screened by chang's liver, other normalcell lines and malignant cell lines. The positive clones that reacted withHepG2 specially but not with other cell lines were amplified and thenthe cultivate supernatant were precipitated by PEG/NaC1 and resus-pened in PBS(pH7.2). The concentrated antibodies were performimmunocytochemistry using cultured cells HepG2 and chang's liver tofind its location at cellular structure and then were perform immunohisto-chemistry using hepatoma carcinoma tissue sections to confirm theresults.RESUILS: Detection of ELISA showed that 4 liver cancer patients'B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell. 13 types of light chain genes and 28 types ofFd genes were obtained by PCR reamplification. The genes were clonedinto vector pComb3 and transformed into XLI-BLue via electroporation.The capacity of the phage antibody library being 1.7×10~7 was obtainedthrough ampicillin -resistant secreening. The percentage of Fd and lightchain genes inserted into phage DNA was about 100%. The librarywas subjected to three rounds of positive and negative cell panning andenrichment, then was selected by phage-ELISA. After primacy test byHepG2 cell ELISA, 163 clones of phage antibody with positive ELISAreaction were picked out of 540 clones. The percentage of positive cloneswas 30.2%. Through further screened by a panel of cultured cells ELISA,the clone E35 was found to react strongly with HepG2, HEK293, but notwith other human tumor cell lines. It also reacted weakly with humanhepatic cell line chang's liver. The results of immunohistochemistry withcultured cells were same as the results of ELISA. E35 reacted specifi-cally with the hepatoma cells in human hepatoma tissue section butalmost not with the cells in human liver tissue section. E35 was furtheranalyed after the DNA sequencing. The sequence of E35 was identical.CONCLUSION: Fully humanized anti-hepatoma Fab phage anti-body libraries with sink size being 1.7×10~7 were constructed by meansof phage antibody library technique in combination with in vitroimmunization method and EBV transformation technique. By ELISA andimmunocytochemistry with cultured cells and immunohistochemistrywith tissue sections, the clone E35 was confirmed to specific bind withhepatoma carcinoma cells. The Fab fragment against hepatoma may befurther developed and applied to clinical diagnosis and therapy. |
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