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目的构建抗人IL-1β单链抗体(anti-hIL-1βscFv)的原核表达载体,并对其表达的蛋白进行生物学活性检测。方法使用PCR的方法从含有抗人IL-1β抗体基因的质粒中获得抗人IL-1β的单链抗体基因,将其插入原核表达载体pET-27b中,构建重组表达载体pET-27b-hIL-1βscFv。将该载体转化大肠杆菌Rosetta(DE3)后诱导表达,经凝胶过滤色谱柱上复性得到可溶的抗人IL-1β的scFv蛋白。使用ELISA方法鉴定scFv抗体的特异性结合活性,使用Real time-PCR的方法检测scFv抗体的中和活性。结果成功地对抗人IL-1β单链抗体进行诱导、表达和复性,蛋白相对分子质量约为28 000。ELISA结果证实scFv蛋白对hIL-1β具有特异性结合能力。Real time-PCR实验结果证实该scFv抗体可以有效阻断人IL-1β刺激人T细胞表达细胞因子IL-18及IL-1β的作用。结论通过原核表达系统得到的抗人IL-1β单链抗体经复性后,与人IL-1β蛋白有特异性结合能力,并且表现出中和活性,为进一步研究人IL-1β相关疾病及其治疗性抗体奠定了基础。
Objective To construct a prokaryotic expression vector of anti-hIL-1β scFv and test the biological activity of the expressed protein. Methods The single-chain anti-human IL-1β antibody gene was obtained from the plasmid containing anti-human IL-1β antibody by PCR and inserted into the prokaryotic expression vector pET-27b to construct the recombinant expression vector pET-27b-hIL- 1β scFv. The vector was transformed into Escherichia coli Rosetta (DE3) and then induced to express, screened on gel filtration column to obtain soluble anti-human IL-1β scFv protein. The specific binding activity of the scFv antibody was identified using the ELISA method and the neutralizing activity of the scFv antibody was detected using the Real time-PCR method. Results The human IL-1β single chain antibody was successfully induced, expressed and renatured. The relative molecular mass of the protein was about 28,000. ELISA results confirmed that scFv protein has specific binding ability to hIL-1β. Real time-PCR results confirmed that the scFv antibody can effectively block the human IL-1β-stimulated human T cells to express cytokines IL-18 and IL-1β role. CONCLUSION: The anti-human IL-1β single-chain antibody obtained by prokaryotic expression system has the ability to bind specifically with human IL-1β protein after refolding and shows neutralizing activity. To further study the human IL-1β-related diseases and their Therapeutic antibodies laid the foundation.