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目的:利用NlpA蛋白的前六个氨基酸(CDQSSS)将抗体锚定在细菌内膜建立筛选Fabs抗体库的展示技术,为今后抗体的研发工作奠定基础。方法:从pNAD质粒中克隆出NlpAleader(含有CDQSSS序列)基因序列,利用相应的酶切位点将该序列插入pComb3表达载体中构建成用于展示Fab的重组质粒pBFD。将从pEAI质粒克隆得到的anti-human IL-1β抗体的重链Fab和全长轻链分别插入到NlpAleader和pelBleader(pComb3载体自带的果胶酶基因前导肽)的下游。将pBFD-Fab转入到E.coli DH5α中诱导表达,原生质球制备后,采用梯度浓度的抗原进行孵育,最后经流式细胞术(FCM)检测抗体展示情况并且分选阳性群体,利用质粒提取的方法来替代PCR方法拯救阳性基因,转化E.coli DH5α,利用FCM再次检测该群体展示的抗体与抗原结合情况。结果:所展示的anti-hIL-1β Fab抗体依次与抗原和FITC标记的抗原特异性抗体孵育后,用FCM实时检测,结果显示出很强的荧光信号并且表现出抗原浓度依赖性。拯救出的pBFD-Fab-原生质球的FCM检测结果与首次展示的FCM结果一致,该系统能够稳定的展示抗体。结论:经过该细菌展示系统展示的Fab抗体能够有效的折叠,与相应的抗原具有很好的特异性结合能力。成功改进该展示技术的基因拯救方法,避免了基因突变和链置换的发生。此外还证明了该展示技术具有很好的稳定性。本实验成功构建了筛选Fab抗体库的细菌展技术。
OBJECTIVE: To establish a screening technology for screening Fabs antibody library by anchoring the first six amino acids (CDQSSS) of NlpA protein in the bacterial endometrium, which lays the foundation for the future development of antibodies. Methods: The NlpAleader (containing CDQSSS sequence) gene sequence was cloned from the pNAD plasmid and inserted into the pComb3 expression vector using the corresponding restriction sites to construct a recombinant plasmid pBFD for displaying the Fab. The heavy chain Fab and the full length light chain of the anti-human IL-1β antibody cloned from the pEAI plasmid were respectively inserted downstream of the NlpAleader and the pelBleader (pectinase leader carried by the pComb3 vector). The pBFD-Fab was transfected into E. coli DH5α to induce the expression. After the preparation of the spheroplasts, the cells were incubated with gradient concentration of antigen. Finally, antibody expression was detected by flow cytometry (FCM) and the positive colonies were sorted. Plasmid extraction Method to replace the PCR method to save the positive gene, transformed into E. coli DH5α, using FCM to detect again the group displayed antibodies and antigen binding. Results: The anti-hIL-1 [beta] Fab antibodies were incubated in sequence with antigens and FITC-labeled antigen-specific antibodies and detected in real time with FCM. The results showed a strong fluorescence signal and showed an antigen concentration-dependent. Rescued pBFD-Fab-spheroplasts FCM results consistent with the first demonstration of the FCM, the system can display antibodies stably. Conclusion: The Fab antibodies displayed by the bacterial display system can fold effectively and have good specific binding ability with the corresponding antigens. Successfully improved the gene rescue method of this display technology, avoiding the occurrence of gene mutation and strand displacement. In addition, it also proves that the display technology has good stability. This experiment successfully constructed a screening of Fab antibody library bacterial exhibition technology.