基于抗原-抗体共表达的细菌展示技术的优化

Optimization of bacterial display technology based on antigen-antibody co-expression

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 co-express antigen and antibody in E. coli periplasm in which the antigen and antibody will fold correctly and bind to each other. Methods Human IL-1β gone and mouse anti-human IL-1α single chain variable fragment （anti-hlL-1β scFv） were inserted into the down stream from the NIpA and pelB leading peptides of pBFD vector respectively. The recombinant plasmid was named pBFD-Ab-Ag. The E. coli DH5α transformed with pBFD-Ab-Ag was induced by IPTG to co-express antigen and antibody. After the outer membrane of E. coli DH5αwas broken （spheroplast formation）, the bacteria were incubated with mouse anti-hlL-1βantibody labeled by FITC. The co-expression and interaction of antigen and antibody was analyzed by flow cytometry （FCM） based on fluorescence signal intensity. Results The FCM detection showed the strong signal in E. coli DHSα transformed with pBFD-Ab-Ag and the positive rate was significantly improved. Conclusion The results showed that the antigen and antibody can fold and bind to each other efficiently in E. coli periplasm. The system can make antibody screening process easier and also provide a novel method for protein-protein interaction.