人源抗EV71病毒中和表位SP70 的Fab抗体研制

目的构建人源抗EV71病毒Fab噬菌体抗体库,筛选具有中和活性的人源单克隆抗体,为EV71感染引起的手足口病临床诊断及治疗奠定基础。方法从成人志愿者抗凝血中分离外周淋巴细胞,提取总RNA并逆转录为cDNA。以cDNA为模板,用人源IgG Fab段基因引物扩增轻链及重链Fd区基因,分步克隆至pComb3载体,构建Fab噬菌体抗体库。以SP70为抗原对Fab噬菌体抗体库进行富集筛选,将获得的重组噬菌体感染XL1-Blue菌并诱导表达,用SP70对诱导表达的菌上清进行筛选。结果共获得3株轻、重链基因组合序列不同的克隆,且这3株Fab抗体可特异性结合SP70多肽。结论成功构建人源抗EV71病毒Fab噬菌体抗体库,并从中筛选出3株针对EV71病毒结构蛋白VP1上中和线性表位SP70的特异性人源Fab抗体,为研发新的手足口病诊断和治疗工具奠定基础。

Development of a humanized Fab antibody against the neutralizing SP70 epitope of EV71

We developed a humanized Fab phage-display library against EV71, and screened humanized monoclonal antibody with a neutralization activity, so as to provide a basis for clinical diagnosis and treatment of hand, foot and mouth disease induced by EV71 infection. Peripheral blood lymphocytes were isolated from anticoagulated blood specimens of adult volunteers infected with EV71. Total RNA was extracted from lymphocytes, and transcribed reversely into cDNA. The light-chain and heavy-chain Fd fragments were amplified using cDNA as a template, and cloned into the vector pComb3 to construct Fab phage-display library. SP70, a neutralizing linear epitope from the VP1 structural protein of EV71, was used as an antigen for enrichment and screening of Fab phage antibody library, and the yielded recombinant phage was used to infect E. coli XL1-Blue. Then, the supernatant of E. coli XL1-Blue following induced expression was screened using SP70. A total of 3 light- and heavy-chain clones with various sequences were yielded, which bound specifically to SP70 polypeptide. A humanized anti-EV71 Fab phage antibody library is successful constructed, and 3 specific humanized Fab antibodies are screened that target the neutralizing linear epitope SP70, which provide new insights into the development of novel diagnostics and therapeutics for hand, foot, and mouth disease.