[Establishment of bacteria display technology for Fab antibody library screening].
[Establishment of bacteria display technology for Fab antibody library screening].
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发表时间：
2011-10
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通讯作者：
Li-ming Xu;Cheng-kai Yin;G. Ren;H. Tian;Xue-qin Wang;L. Ding;Deshan Li
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作者：
Li-ming Xu;Cheng-kai Yin;G. Ren;H. Tian;Xue-qin Wang;L. Ding;Deshan Li
目的利用NlpA蛋白氨基端的6个氨基酸(CDQSSS)将抗体锚定在细菌内膜周质侧面，建立用于Fab抗体库筛选的细菌展示技术。方法从Pnad质粒中扩增出编码CDQSSS的NlpA前导序列。将扩增产物亚克隆至pComb3表达载体，构建Fab展示载体pBFD。将Fab基因片段的重链和抗人IL-1β(hIL-1β)抗体的轻链分别插入NlpA前导和PelB前导的下游，构建了Fab抗体展示用的pBFD-Fab。将pBFD-Fab转化大肠杆菌DH5，用α诱导表达Fab抗体，并对阳性菌落进行分选。为了挽救目的，不是提取聚合酶链式反应，而是提取质粒。将回收的重组表达载体转化到E.coliDH5α中，再进行流式细胞仪检测。结果PBFD-Fab转化菌与抗原和抗原特异性FITC抗体共同孵育，FCM检测显示出较强的荧光，且呈剂量依赖关系。回收的PBFD-Fab具有相似的荧光强度，表明该技术的可靠性。结论细菌展示系统表达的Fab能有效折叠并与hIL-1β特异性结合。该方法效果良好，避免了突变和错配链的产生。这种细菌展示技术具有抗体表达的稳定性。本研究成功地应用该技术筛选了抗hIL-1βFab抗体库。
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.