基于抗原-抗体共表达的细菌展示技术的优化

发布时间：2018-10-15 13:29

【摘要】：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。
[Abstract]:Objective to establish a co-expression system of antigens and antibodies in periplasmic bacteria. Methods Human interleukin-1 尾 (hIL-1 尾) gene and anti-human interleukin-1 尾 single chain antibody (anti-hIL-1 尾 scFv) gene were inserted into the downstream of pelB prepeptide (free precursor) and NlpA precursor (anchoring prepeptide) of bacterial display vector pBFD, respectively, and the recombinant vector pBFD-Ab-Ag. was constructed by inserting human interleukin-1 尾 (hIL-1 尾) gene and anti-human interleukin 1 尾 single chain antibody (anti-hIL-1 尾 scFv) gene into the downstream of the bacterial display vector pBFD. PBFD-Ab-Ag was transferred into E.coli DH5 伪 to induce expression, the antigen and antibody protein were combined and anchored to the outside of the bacterial intima, then the outer wall of the bacteria (protoplast preparation) was removed and incubated with the appropriate concentration of FITC labeled mouse anti-hIL-1 尾 antibody. Finally, fluorescence signals were detected by flow cytometry, and the expression and interaction of antigens and antibodies were analyzed according to the intensity of fluorescence signals. Results pBFD-Ab-Ag E.coli DH5 伪 had strong fluorescence signal and the positive rate was greatly improved. Conclusion the co-expression system of antigens and antibodies in the periplasm of bacteria was successfully established, and the correct folding and mutual recognition of antibodies and antigens in the periplasm of bacteria were realized. It simplifies the existing antibody screening process and provides a new technique for protein interaction research.
【作者单位】：中国水产科学研究院黑龙江水产研究所;东北农业大学生命科学学院生物制药教研室;
【基金】：国家自然科学基金(31001084,30700591)
【分类号】：R392.1

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