全人源抗狂犬病病毒G蛋白单链抗体制备及中和活性鉴定

目的:构建全人源抗狂犬病病毒scFv噬菌体抗体库,筛选针对狂犬病病毒G蛋白不同表位的单链抗体(scFv)并鉴定其中和活性?方法:分离130例经狂犬病疫苗免疫接种志愿者的外周血淋巴细胞,提取总RNA,逆转录制备cDNA,构建全人源抗狂犬病病毒scFv噬菌体抗体库?以纯化的狂犬病病毒G蛋白包板筛选,对阳性克隆进行可溶性表达,并鉴定中和活性?结果:构建全人源抗狂犬病病毒scFv噬菌体抗体库,库容为5.0 × 107,经核酸序列分析,证实插入片段为scFv?经过5轮筛选,从富集的次级抗体库中随机挑出140个克隆,通过phage-ELISA鉴定得到4株核酸序列不同的scFv抗体?经His-Trap纯化后进行中和活性鉴定,获得1株有中和活性的scFv,其中和效价为0.13 IU/mg?结论:构建全人源抗狂犬病病毒scFv噬菌体抗体库,获得1株具有中和活性的抗狂犬病病毒G蛋白scFv抗体,为进一步研发狂犬病治疗性抗体药物奠定了基础?

Screening and preparation of a human single chain antibody(scFv)against Rabies Virus G protein from a phage display library and identification of the neutralizing activity

Objective:To construct a human scFv phage antibody library against rabies virus G protein,selected the single chain antibody against rabies virus G protein for different epitopes,and identificated its neutralizing activity. Methods:The human scFv phage antibody library against rabies virus G protein was constructed by using peripheral blood lymphocytes from 130 Rabies vaccine immune volunteers. Screening by the purified rabies virus G protein,the positive clones were identified and expressed,identified neutralizing activity by AMMS. Results:Through the prokaryotic expression system and purification of rabies virus G protein,the human anti rabies virus G protein scFv phage antibody library was constructed with about 5.0×107 size. The nucleic acid sequence analysis confirmed the insertion of a fragment is scFv. After 5 rounds of screening,we randomly picked 140 clones by phage-ELISA,and obtained 4 nucleic acid sequences in different scFv antibodies,purified and identified activity. We finally obtained 1 scFv antibody with neutralizing activity. Conclusion:We have successfully constructed a whole human anti-rabies virus G protein scFv antibody library,and obtained a whole human anti-rabies virus G protein scFv antibody with neutralizing activity from the library. It is established foundation of the new type of rabies virus vaccine for human immunization.