抗B型肉毒毒素鼠源中和抗体快速制备及其免疫学活性的初步研究

发布时间：2018-01-12 02:19

本文关键词：抗B型肉毒毒素鼠源中和抗体快速制备及其免疫学活性的初步研究　出处：《中国人民解放军军事医学科学院》2005年博士论文　论文类型：学位论文

【摘要】：肉毒毒素是肉毒梭状杆菌产生的外毒素,属神经强毒,人类的肉毒中毒主是由A,B和E引起。是目前已知毒性最强的细菌蛋白质之一,是重要的生物战剂。本论文以定向免疫和噬菌体展示技术为平台,探索了一种快速制备及筛选鼠源抗体的技术路线,将筛选到的高亲和力抗体直接进行中和活性实验,将抗体基因克隆、高亲和力抗体富集和抗体筛选在噬菌体水平一步完成,简便快速,研究周期短。为生物毒素新型抗毒素的制备提供新的研究思路。本研究中首先克隆了B型肉毒毒素重链C端基因,与已知序列同源性达99%。为了获得重组蛋白,构建了pET22重组质粒载体,诱导表达后未得到目的蛋白。随后构建pET32重组质粒,获得了BoNTB/Hc的融合蛋白。对引物进行改构后,最终获得了以包含体形式存在的BoNTB/Hc N端修饰蛋白,并用金属螯和层析法进行了初步纯化和鉴定。进一步对其免疫学活性进行初步研究,结果表明重链Hc免疫后的BALB/c小鼠可以在一定程度上抵抗6×10~3 LD_(50) B型肉毒毒素攻击,为B 型肉毒毒素疫苗的研究奠定了基础。同时制备了卵黄检测抗体(IgY),抗体效价达10~4以上。用重组表达并纯化的BoNTB/Hc免疫小鼠,从其脾淋巴细胞中提取全套抗体可变区基因,分别构建scFv和Fab段噬菌体抗体文库,库容分别为6.72x10~7cfu和5.69x10~6cfu。以纯化的BoNTB/Hc为抗原,对抗体库进行富集筛选,并以BoNT/B为抗原进一步鉴定特异性抗体,最终获得与BoNT/B结合能力较强的scFv抗体2个,分别命名为S3和$24,VH属于VH2家族,VK分别属于VK2和VK1家族。Fab抗体1个,命名为F38,VH属于VH1家族,VK属于VK2家族。重组抗体免疫学保护试验表明,抗B型肉毒毒素的噬菌体抗体S3对BoNT/B致死作用的保护性较强,能够在一定程度上抵抗10LD_(50)(i.p.)的致死作用,S24次之,F38较弱。未观测到S3,S24和F38三者的明显叠加作用。挑选作用较强
[Abstract]:Botulinum toxin is a foreign toxin produced by Clostridium botulinum, which is a neurotoxic toxin. Botulism in humans is mainly caused by AHB and E. Botulinum toxin is one of the most toxic bacterial proteins known at present. It is an important biological warfare agent. Based on the technology of directed immunity and phage display, this paper explored a rapid preparation and screening of murine antibody. The high affinity antibody was directly neutralized. The antibody gene was cloned, the antibody was enriched and the antibody was screened at the phage level, which was simple and fast. The research period is short, which provides a new research idea for the preparation of new biotoxin antitoxin. In this study, the C-terminal gene of botulinum toxin B heavy chain was cloned, and the sequence homology was 99%. In order to obtain the recombinant protein, the recombinant plasmid vector of pET22 was constructed. The recombinant plasmid of pET32 was constructed and the fusion protein of BoNTB/Hc was obtained. The primers were reconstructed. Finally, the N-terminal modified protein of BoNTB/Hc was obtained in the form of inclusion body, and was purified and identified by metal chelating and chromatography, and its immunological activity was studied. The results showed that BALB/c mice immunized with heavy chain Hc could resist the attack of Botulinum toxin B to a certain extent. It laid a foundation for the study of botulinum toxin B vaccine, and prepared egg yolk detection antibody IgYA, the titer of antibody was over 10 ~ 4. The mice were immunized with recombinant and purified BoNTB/Hc. The phage antibody libraries of scFv and Fab segments were constructed by extracting a full set of antibody variable region genes from the spleen lymphocytes of mice. The storage capacity was 6.72 x 1010 CFU and 5.69 x 106cfu. the purified BoNTB/Hc was used as antigen to enrich and screen the antibody library. BoNT/B was used as antigen to further identify the specific antibody. Finally, two scFv antibodies with strong binding ability to BoNT/B were obtained, named S3 and F24, respectively. VH belongs to VH2 family, which belongs to VK2 and VK1 family. Fab antibody, named F38 VH belongs to VH1 family and belongs to VK2 family. The immunological protective test of recombinant antibody showed that the bacteriophage antibody S3 against botulinum toxin B had strong protective effect on the lethal effect of BoNT/B. The lethal effect of S24 was weaker than that of F38, and S3 was not observed. Obvious superposition of S24 and F38. Strong selection
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392.1

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