用体外致敏联合EB病毒转化技术构建抗肝癌的人源Fab噬菌体抗体库_医学论文.docVIP

用体外致敏联合EB病毒转化技术构建抗肝癌的人源Fab噬菌体抗体库_医学论文 用体外致敏联合EB病毒转化技术构建抗肝癌的人源Fab噬菌体抗体库_医学论文 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，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 　　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 EpsteinFd 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)为杭州四季青公司产品；rhIL2、谷氨酰胺、丙酮酸钠及环孢菌素A均为Sigma公司产品；辣根过氧化物酶(h