Chen Liang, Li Gang, Tang Lei, Wang Jue, Ge Xi Rui
Laboratory of Cellular Immunology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Cell Res. 2002 Mar;12(1):47-54. doi: 10.1038/sj.cr.7290109.
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-A-1-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.
作者实验室获得了一种识别肺癌相关共同抗原的单克隆抗体LC-1。基于重组噬菌体展示技术构建了其单链Fv片段（ScFv），命名为LC-1 ScFv。为了在细胞膜上表达，将LC-1 ScFv克隆到pDisplay载体中，该载体指导克隆基因表达为细胞膜结合蛋白。对所得质粒进行测序，然后通过脂质体转染法将其导入肺腺癌细胞系SPC-A-1。通过G418筛选获得G418抗性细胞。转染后，通过蛋白质免疫印迹分析观察LC-1 ScFv的表达，酶联免疫吸附测定显示同源抗原的表达下调。SPC-A-1-pDisplay-ScFv细胞在体外的生长速度低于对照完整细胞和转染空载体的细胞。流式细胞术分析检测到，与SPC-A-1和SPC-A-1-pDisplay细胞相比，SPC-A-1-pDisplay-ScFv细胞群体中G1期显著增加，S期减少。半定量逆转录-聚合酶链反应分析表明，SPC-A-1-pDisplay-ScFv细胞中c-myc表达下调。似乎LC-1识别的抗原可能以某种方式参与生长刺激途径，抗体阻断抗原功能可关闭该途径，从而下调c-myc的表达和细胞生长。
