人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

目的:构建全人源抗HER2胞外段(HER2 ECD)噬菌体Fab抗体库,从中筛选出特异性的抗体,并对其进行鉴定。方法:体外致敏并用EB病毒(EBV)转化HER2高表达乳腺癌患者的外周血单核细胞(PBMC),用PCR分别扩增重链Fd和轻链κ/λ基因。经SacI/XbaI和XhoI/SpeI双酶切,依次克隆入噬菌体载体pComb3中,并电转化大肠杆菌XL1-Blue,以辅助噬菌体VCSM13进行超感染,构建抗HER2 ECD人源化Fab噬菌体抗体库。以纯化HER2 ECD蛋白为抗原进行3轮固相淘选,富集抗HER2 ECD的抗体,并随机挑选克隆进行ELISA,获得的阳性克隆进一步以Western blot鉴定其抗原结合活性,对其中活性最高的克隆进行DNA测序。结果:构建了容量为2.5×107的抗HER2 ECD的Fab噬菌体抗体库,并筛选获得了4株与HE2 ECD特异性结合的阳性克隆,Western blot分析显示其与HER2 ECD能较好的结合。选取结合活性最高的阳性克隆进行DNA序列分析,结果显示其重链可变区、轻链可变区分别与人胚系免疫球蛋白基因有高度的同源性。结论:成功构建了全人源抗HER2 ECD噬菌体抗体库,并筛选出抗HER2 ECD特异性较强的噬菌体克隆,为获得新的有临床应用价值的HER2 ECD抗体提供了实验基础。

Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2

AIM: To construct the fully humanized anti-extracellular domain(ECD) of HER2 Fab fragment phage library,select antibodies against HER2 ECD specifically and identify its characteristics.METHODS: Peripheral blood monouclear cells(PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HER2 ECD and were then transformed by Epstein-Barr virus(EBV).After total RNA was extracted,the heavy chain Fd and κ/λ light chain were amplified by RT-PCR.Following restrictive digestion with Sac I/Xba I and Xho I/Spe I,the light chain κ/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue.The humanized Fab phage antibody library against HER2 ECD was constructed by infection of helper phage VCSM13.The libraries were enrich after panned three cycles by purification protein of recombinant HER2 ECD.Then random clones were tested by ELISA to select the positive ones,which were furher identified their antigen binding acticities by Western blot,and the strongest binding to HER2 ECD clone was sequenced.RESULTS: The Fab phage antibody library with 2.5×107 volume was constructed and four positive clones which specifically recognized the HER2 ECD were isolated and further demonstrated by Western blot.Sequence analysis of the positivest clone showed that the variable heavy domains(VH) and variable light domains(VL) were highly homologous with the human embryonal Ig heavy chain V region sequences and kappa light chain sequences,respectively.CONCLUSION: A fully humanized Fab phage antibody library is successfully constructed and specific antibodies against HER2 ECD are obtained,which provides an experimental foundation for new humanized anti-HER2 ECD monoclonal antibodies.