筛选Fab抗体库的细菌展示技术的建立

Establishment of bacteria display technology for Fab antibody library screening

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。

AIM： To establish bacterial display technology for the purpose of Fab antibody library screening, by using six amino acids （CDQSSS） of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane. METHODS： The NlpA Leader sequences （encoding CDQSSS） was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene fragments and the light chains of the anti-human IL-1β （hIL-1β） antibody were inserted downstream of the NlpA leader and pelB leader respectively to construct the pBFD-Fab for Fab antibody display. Then pBFD-Fab transformed E.coli DH5α was induced by IPTG to express the Fab antibodies, as detected by flow cytometry （FCM）, and positive populations were sorted. Instead of PCR, plasmids were extracted for rescue purpose. The rescue plasmids were retransformed to E.coli DH5α and FCM was performed again. RESULTS： The pBFD-Fab-transformed bacteria were incubated with antigen and antigen specific FITC-antibody, and showed strong fluorescence as detected by FCM in a dose-dependent manner. The rescued pBFD-Fab displayed similar fluorescence intensity, indicating the reliability of this technology. CONCLUSION： The Fab expressed by the bacterial display system folds efficiently and binds to hIL-1β specifically. The plasmid rescue works well and it can avoid mutation and mis-pairing chains. This bacterial display technology has the stability of antibody expression. This study has used the technology to screen anti-hIL-1β Fab antibody Library successfully.