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目的利用NlpA前导肽诱导抗体锚定细菌内膜建立筛选scFv抗体库的展示技术,为高亲和力抗体的筛选奠定基础。方法从pNAD质粒中克隆出NlpA前导肽基因序列,酶切后将该序列克隆进pHEN1表达载体中。以PEAI质粒为模板,利用PCR的方法克隆得到抗-hIL-1β抗体的重链可变区和轻链可变区基因,然后利用重叠PCR的方法构建得到抗-hIL-1βscFv抗体。将scFv抗体插入到NlpA leader-pHEN1表达载体中构建成展示scFv的重组质粒pBSD-scFv。将pBSD-scFv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。结果所展示的抗-hIL-1βscFv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pBSD-scFv-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scFv抗体能够有效的折叠,与相应的抗原具有很好的特异结合能力。
OBJECTIVE: To establish a screening technology for scFv antibody library by inducing antibody-anchored bacterial intima with NlpA leader peptide, which lays the foundation for the screening of high-affinity antibodies. Methods The NlpA leader peptide sequence was cloned from pNAD plasmid and cloned into pHEN1 expression vector after digestion. Using PEAI plasmid as a template, the heavy chain variable region and light chain variable region genes of anti-hIL-1β antibody were cloned by PCR and then the anti-hIL-1βscFv antibody was constructed by overlapping PCR. The scFv antibody was inserted into the NlpA leader-pHEN1 expression vector to construct a recombinant plasmid pBSD-scFv that showed scFv. The pBSD-scFv was introduced 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 Anti-hIL-1β scFv antibodies were shown to be detected in real-time with FCM after incubation with antigens and FITC-labeled antigen-specific antibodies, showing strong fluorescence signals and exhibiting antigen concentration-dependence. The FCM results of the rescued pBSD-scFv-spheroplasts were in good agreement with those of the first-demonstrated FCM, which demonstrated stable antibody display. Conclusion The scFv antibody displayed by the bacterial display system can fold effectively and has good specific binding ability with the corresponding antigen.