A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage disp...A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A -1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.展开更多
基金This work was supperted by Sciense and Tech-nology Promoting FOundation of Shanghai No.984419071.
文摘A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A -1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.
