用体外致敏联合EB病毒转化技术构建抗肝癌人源Fab噬菌体抗体库.docVIP

用体外致敏联合EB病毒转化技术构建抗肝癌人源Fab噬菌体抗体库 　 作者：水漩，李官成，李跃辉，黄建，赵艳 【摘要】 目的 构建抗肝癌的人源Fab噬菌体抗体库。方法 体外致敏并用EB病毒(EBV)转化肝癌患者的外周血单个核细胞(PBMC)。RTPCR扩增人抗体轻链和重链Fd段基因，将抗体基因分别与载体pComb3连接后，电转化大肠杆菌XLIBlue，构建噬菌体呈现型Fab抗体库。结果 经EBV转化的4例肝癌患者PBMC，ELISA检测均有抗肝癌抗体产生；经PCR扩增出13条轻链基因片段(κ、λ)和28条重链Fd基因片段(γ)；电转化构建的噬菌体抗体库库容为1.7×107puf/mL；Fab基因的重组率约为100%。结论 用体外致敏法结合噬菌体抗体库技术，成功构建了全人源抗肝癌Fab组合抗体文库。 【关键词】 EB病毒转化；噬菌体抗体库；肝癌；Fab抗体 　　Construction of phage antibody library using sensitized in vitro　Blymphocytes of liver cancer patients 　　ABSTRACT: Objective To construct human phage antibody library against hepatoma carcinoma. Methods Peripheral blood mononuclear cells (PBMCs) of patients with liver cancer were sensitized in vitro and transformed by EpsteinBarr virus (EBV). The genes of light chain and Fd of antibodies were amplified by RTPCR. Fab genes were cloned into vector pComb3 and transformed into E.coli XLIBlue by electroporation to construct the Fabdisplaying phage antibody library. Results ELISA detection showed that 4 liver cancer patients B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell. Totally 13 types of light chain genes and 28 types of Fd genes were obtained by RTPCR. The capacity of the primary phage library was 1.7×107pfu/mL. The percentage of recombinant clones was about 100%. Conclusion A human phage antibody library has been constructed successfully by means of EBV transformation technique. 　　KEY WORDS: EBV immortalization; phage antibody library; liver cancer; Fab fragment 　　肝癌是一种恶性程度极高的肿瘤。目前，其治疗手段仍以手术切除为首选，但复发率极高，且常用的化疗药物对肿瘤的杀伤作用缺乏特异性，常导致明显的副作用。近年来以抗体为基础的免疫疗法、肿瘤靶向治疗和基因治疗成为肿瘤治疗研究的热点，部分已经在实验室甚至临床上取得了初步的效果[12]。利用噬菌体抗体库技术，可克服杂交瘤技术制备人源性单克隆抗体(monoclonal antibody, mAb)的复杂过程，而且可筛选出多种特异性抗体[34]。本研究以肝癌患者外周血淋巴细胞作为基因来源，联合体外致敏和EB病毒(Epstein Barr virus, EBV)转化技术构建全人源的抗肝癌Fab段噬菌体抗体库，为进一步筛选获得高亲和力、高特异性的Fab抗体奠定了基础。 　　1 材料与方法 　　1.1 材料 肝癌细胞株HepG2、绒猴淋巴母细胞样细胞系B95.8，由本室保存。大肠杆菌XL1Blue、噬菌粒载体pComb3及辅助噬菌体VCSM13均由本室保存。胎牛血清(fetal calf serum，FCS)及新生牛血清(newborn calf serum, NBS