IHFB蛋白全人源单克隆抗体制备及对铜绿假单胞菌生物被膜形成影响的研究

Effect of the fully human monoclonal antibodies targeting IHFB on biofilm formation by Pseudomonas aeruginosa

目的探讨靶向铜绿假单胞菌IHFB蛋白的单克隆抗体对细菌生物被膜形成的影响。方法以铜绿假单胞菌PAO1基因组为模板,利用PCR技术扩增Ihfb基因,进一步构建重组表达载体p ET28a-Ihfb,并转化至大肠杆菌BL21(DE3)中,通过IPTG诱导表达IHFB蛋白,经Ni2+亲和树脂纯化获得IHFB蛋白;利用噬菌体抗体库技术筛选获得与IHFB蛋白特异性结合的全人源单克隆抗体,PCR扩增所获得抗体的重链VH和轻链VL基因并构建重组表达载体,继而转染至293E细胞中,收集胞外分泌蛋白,并利用Protein A树脂纯化单克隆抗体。利用ELISA实验检测抗体亲和力,利用铜绿假单胞菌生物被膜形成实验检测抗体活性。结果与结论通过噬菌体展示技术筛选并获得了全人源抗IHFB单克隆抗体,命名为YG15; ELISA结果表明,YG15抗体与IHFB蛋白有较高亲和力(EC50为24. 8 ng/ml),实验结果验证全人源单克隆抗体YG15可抑制铜绿假单胞菌生物被膜的形成。

Objective To investigate the effect of monoclonal antibodies targeting Pseudomonas aeruginosa( PAE)IHFB protein on bacterial biofilm formation. Methods The genome of PAE PAO1 was used as the template to amplify the Ihfb gene by PCR. The recombinant expression vector p ET28 a-Ihfb was further constructed and transformed into E. coli BL21( DE3). The expression of recombinant IHFB protein was induced by IPTG,and purified by Ni2 +affinity resins. The phage antibody library technology was used to screen the fully human monoclonal antibody which specifically bound to the IHFB protein,and the heavy chain VHand light chain VLgenes of the obtained antibody were amplified by PCR and the recombinant expression vector was constructed and transfected into 293 E cells. Extracellular secreted proteins were collected and monoclonal antibodies were purified using Protein A resins. The antibody affinity was detected by an ELISA assay,and the antibody activity was detected using the PAE biofilm formation assay. Results and Conclusion The fully human anti-IHFB monoclonal antibody was screened by phage display technology and named YG15. The ELISA results showed that YG15 antibody has higher affinity with IHFB protein( EC50= 24. 8 ng/ml). We have prepared the fully human monoclonal antibody YG15 against IHFB which inhibits the formation of PAE biofilm.