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目的建立细菌周质内抗原抗体共表达系统。方法将人白介素-1β(hIL-1β)基因和抗人白介素1β单链抗体(anti-hIL-1βscFv)基因分别插入到细菌展示载体pBFD的pelB前导肽(游离型前导肽)和NlpA前导肽(锚定型前导肽)下游构建出重组载体pBFD-Ab-Ag。将pBFD-Ab-Ag转入到E.coli DH5α中诱导表达,抗原和抗体蛋白相互结合而被锚定到细菌内膜外侧,破除细菌外壁(原生质球制备)后与适当浓度的FITC标记的小鼠抗hIL-1β抗体进行孵育,最后经流式细胞仪检测荧光信号,根据荧光信号强弱分析抗原抗体表达和相互作用情况。结果 pBFD-Ab-Ag E.coli DH5α具有很强的荧光信号,阳性率获得了大大的提高。结论成功建立了建立细菌周质内抗原抗体共表达系统,实现了抗体和抗原细菌周质内的正确折叠和相互识别。简化了现有的抗体筛选的流程,为蛋白质相互作用的研究提供了新的技术手段。
Objective To establish a bacterial periplasmic antigen-antibody co-expression system. Methods The human interleukin-1β (hIL-1β) and anti-hIL-1β scFv genes were inserted into the pelB leader (free leader) and NlpA leader of the bacterial display vector pBFD Anchored leader peptide) to construct the recombinant vector pBFD-Ab-Ag. The pBFD-Ab-Ag was introduced into E. coli DH5α to induce expression. The antigen and the antibody were bound to each other and anchored to the outside of the bacterial endometrium. After removing the outer wall of the bacteria (preparation of the spheroplasts), the FITC-labeled small Mouse anti-hIL-1β antibody was incubated. Finally, fluorescence signal was detected by flow cytometry. Antigen-antibody expression and interaction were analyzed according to the signal intensity of fluorescence signal. Results pBFD-Ab-Ag E.coli DH5α had a strong fluorescence signal, and the positive rate was greatly improved. Conclusion The establishment of a system for the co-expression of antigen and antibody in the periplasm of bacteria has been successfully established and the correct folding and mutual recognition of the antibody and antigen within the bacterial periplasm has been achieved. Which simplifies the current screening process of antibodies and provides a new technical means for the study of protein interactions.