人源抗狂犬病毒基因工程单链抗体的研究

摘要

Objective To obtain recombinant human anti-rabies antibodies by antibody phage library approach.Methods A combinatorial ScFv antibody phage library was constructed using pHAL14 vector from the peripheral blood of volunteers after rabies immunization.After package by hyperphage,the ScFv phage library was panned and selected by ELISA and IFA with purified rabies virus aG strain.The specific antibody was converted to full human IgG antibody with the VH Express and VK Express cassettes.Affinity and neutralizing test were performed to verify the function of the IgG molecules.Results Six unique human ScFv antibodies specific for the glycoprotein of rabies virus were obtained by ELISA,IFA and DNA sequencing.The full human IgG antibodies with high affinity represented a high level neutralizing activity to rabies virus aG strain,but not CVS strain.Conclusion The obtained human anti-rabies antibodies are important for diagnosis and treatment of rabies virus infection.%目的 研制人源抗狂犬病毒基因工程抗体.方法 从具有高滴度狂犬病毒抗体的疫苗接种者采集外周血淋巴细胞,通过pHAL14载体和高效噬菌体系统构建人源抗狂犬病毒单链基因工程抗体库,经纯化的狂犬病毒aG株富集筛选,通过ELISA和IFA鉴定阳性抗体克隆并进行序列测定.利用IgG表达载体VH/VK双质粒系统瞬时转染293T细胞实现IgG抗体的分泌型表达,通过亲和力测定和中和试验鉴定IgG抗体功能.结果 成功地获得了6株抗狂犬病毒糖蛋白的人源单克隆抗体,非竞争ELISA显示6株抗体具有较高的亲和力,体外中和试验证实这6株人源单抗对狂犬病毒CVS株无中和活性,对aG株具有较强的中和活性.结论 从免疫库中获得了6株人源单克隆抗体,为狂犬病毒的鉴别诊断和治疗奠定了基础.