论文部分内容阅读
目的研究开发人源抗huIFN-α的基因工程抗体,为系统性红斑狼疮(Systemic lupus erythematosus,SLE)的治疗提供有效的抗体药物。方法本研究利用噬菌体表面展示技术,以纯化的人干扰素α1b(huIFN-α1b)和人干扰素α2b(huIFN-α2b)蛋白为抗原从人源全合成抗体库中筛选抗huIFN-α的基因工程单链(single chain variable fragment,scFv)抗体,通过phage-ELISA对噬菌体单链抗体的结合特异性和稳定性进行验证。将单链抗体基因克隆到原核分泌表达载体pET22b上在大肠杆菌BL21(DE3)中表达,通过Westernblot检测单链抗体的分泌表达,并通过ELISA对单链抗体的结合特异性进行验证。通过金属螯合亲和层析纯化单链抗体,并用WesternBlot对单链抗体的结合特异性进行鉴定。结果经过3轮富集筛选,获得100株对huIFN-α1b有特异性结合的噬菌体单链抗体。对得到的86株克隆的测序分析表明,共有9株带有不同的抗体轻重链可变区序列及其组合的抗体,有7株抗体轻重链可变区序列分类在VH3和VL3家族,有2株抗体轻重链可变区序列分类在VH3和VL1家族。获得了5株稳定且特异针对huIFN-α1b而与huIFN-α2b和huIFN-γ无交叉反应的人源单抗。这5株抗体在BL21(DE3)中分泌表达,有3株单链抗体能特异性结合huIFN-α1b而与无关抗原无交叉反应。结论通过噬菌体表面展示技术,从人源全合成抗体库中筛选得到3株稳定且特异结合huIFN-α1b的人源治疗用基因工程单克隆抗体。
Objective To study the genetically engineered human anti-huIFN-α antibody and to provide an effective antibody for the treatment of systemic lupus erythematosus (SLE). Methods In this study, phage display technique was used to screen the human anti-huIFN-α gene from human total synthetic antibody library using purified human IFNα1b (huIFN-α1b) and human interferon α2b (huIFN-α2b) The single-chain variable fragment (scFv) antibody was used to verify the binding specificity and stability of the phage scFv by phage-ELISA. The single-chain antibody gene was cloned into the prokaryotic expression vector pET22b and expressed in E. coli BL21 (DE3). The secretion of single-chain antibody was detected by Western blot and the binding specificity of the single-chain antibody was verified by ELISA. Single-chain antibodies were purified by metal chelation affinity chromatography and the binding specificity of the single-chain antibodies was identified by WesternBlot. Results After three rounds of enrichment screening, 100 phage scFv antibodies with specific binding to huIFN-α1b were obtained. The sequencing analysis of the obtained 86 clones showed that there were 9 antibodies with different antibody light and heavy chain variable region sequences and their combinations, 7 antibody light and heavy chain variable region sequences were classified in the VH3 and VL3 families, and 2 Strains of antibody heavy and light chain variable regions are classified in the VH3 and VL1 families. Five human mAbs that were stable and specific for huIFN-alb without cross-reaction with huIFN-alpha 2b and huIFN-gamma were obtained. These five antibodies were secreted in BL21 (DE3), and three single-chain antibodies specifically bound to huIFN-α1b without cross-reaction with unrelated antigens. Conclusion Three phage display monoclonal antibodies against human IFN-α1b were successfully screened from human total antibody library by phage display technique.