论文部分内容阅读
构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.
A ribosomal antibody against human rabies virus single chain antibody (scFv) was constructed and used to screen for stable humanized antibodies against rabies virus glycoprotein (RVGp) .A ribosomal antibody library was used to immunize rabies virus Vero Of the volunteers from peripheral blood lymphocytes to construct ribosomal scFv gene library.After in vitro transcription and translation, RVGp recombinant protein was used as the screening antigen, and the RVGp specific scFv antibody gene was panned by affinity enrichment method.In the prokaryotic system, pET22b (+) / BL21 (DE3), the positive clones were screened by ELISA, then the scFv was screened for stability and the VH-Lc-VK stable antibody was constructed and its biological activity was evaluated Preliminary studies of ribosome-scFv antibody library with a capacity of about 6.2 × 1012.Among the 180 screening clones, clones RB24, RB71, RB109 and RB156 showed higher ELISA values, and the results of gene sequence analysis Showed that they are brand new human anti-RVGp antibodies.The restructured anti-RVGp VH-Lc-VK antibody has significantly improved stability, can specifically recognize RVGp and effectively neutralize rabies virus, and inhibit rabies virus infection on target cells. The results show that human access to anti RVGp specific antibody, provides a new way for effective prevention, diagnosis and treatment of rabies, and the preparation of antibodies will be other sources provide a theoretical basis and technical basis.