从人源全合成抗体库中筛选到抗人干扰素α1b单链抗体

Antibody against human interferon α1b from human synthetic recombinant antibody library

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 obtain human antibody against human interferon aipha（huIFN-α） from human synthetic recombinant antibody library for the therapy of systemic lupus erythematosus. Methods In our study, using phage display technology, antibodies against purified huIFN-α1b and huIFN-α2b were screening from a full synthetic human phage display library. Antibody genes were identified by sequence analysis and their stability and specificity of antibodies confirmed several times by phage ELISA. The scFv antibody genes were cloned into vector pET22b and expressed in E. coli strain BL21（DE3）, and secreted antibodies were further detected by Western blot assay. After purified by metal chelate affinity chromatography （MCAC）, the scFv antibodies were further detected by Western blot assay. Results After three rounds of panning, 100 human scFv clones with specific reactivity with huIFN-α1b but not huIFN-α2b and huIFN-γ were selected by phage ELISA. Nine different antibody genes were identified by sequence analysis of 86 clones. And 7 antibody genes were derived from VH3 and VL3 germ/ine family and 2 antibody genes derived from VH3 and VL1 germline family. Five of 9 phage antibodies were found to bind huIFN-α1b with a high stability and specificity by phage ELISA. Three of 5 secreted single chain antibodies were found to bind huIFN-α1b with a high specificity by ELISA test. The purified scFv antibodies were detected to bind huIFN-α1b without cross-reaction by Western blot assay. Conclusion Three single chain antibodies against human interferon alpha （huIFN-α） with a high stability and specificity were obtained from human synthetic recombinant antibody library.