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

发布时间：2018-06-19 05:02

本文选题：狂犬病病毒G蛋白 + 噬菌体抗体库　；参考：《南京医科大学学报(自然科学版)》2013年07期

【摘要】：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。
[Abstract]:Aim: to construct a full-human scFv phage antibody library against rabies virus and to screen scFv antibody against different epitopes of rabies virus G protein. Methods: peripheral blood lymphocytes of 130 volunteers immunized with rabies vaccine were isolated, total RNAs were extracted, and cDNAs were prepared by reverse transcription. The whole human anti-rabies virus scFv phage antibody library was constructed. The purified rabies virus G protein envelope was screened, the positive clones were expressed in a soluble way, and neutralization activity was identified. Results: the whole human phage antibody library against rabies virus scFv was constructed. The library capacity was 5.0 脳 10 7. The inserted fragment was confirmed as scFv by nucleic acid sequence analysis. After five rounds of screening, 140 clones were randomly selected from the enriched secondary antibody library. Four scFv antibodies with different nucleic acid sequences were identified by phage-ELISA. After being purified by His-Trap, a neutralizing scFvv strain was obtained. The neutralization activity of scFvwas 0.13 IU / mg. Conclusion: the whole human anti-rabies virus scFv phage antibody library was constructed, and a neutralizing antibody against rabies virus G protein scFv was obtained, which laid a foundation for further research and development of rabies therapeutic antibody drugs.
【作者单位】：南京医科大学卫生部抗体技术重点实验室;南京医科大学病理学系;杭州市红十字会医院结核科;江阴力博医药生物技术有限公司;南京军区军事医学研究所;
【基金】：国家自然科学基金资助(81273325) 江苏省科技支撑计划资助(BE2011842)
【分类号】：R392.12

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