A型肉毒毒素抗原表位及抗体研究

发布时间：2018-05-27 15:09

本文选题：肉毒毒素 + 中和抗体　；参考：《中国人民解放军军事医学科学院》2016年硕士论文

【摘要】：肉毒神经毒素(botulinum neurotoxins,BoNT)是一类烈性的蛋白类神经毒素,由厌氧的肉毒梭菌产生,是目前已知毒性最强的毒素。近年来,肉毒毒素在民间不时引发群体中毒事件,在军事上也可能作为恐怖剂和生物战剂被谬用,因此肉毒毒素的研究受到了各国研究者的关注。BoNT由一条相对分子质量为100kDa的重链和一条相对分子质量为50kDa的轻链通过二硫键连接而成。轻链(BoNT/AL)是毒素的毒性成分,具有Zn2+依赖性的内肽酶活性。重链(BoNT/AH)包含两个区域,N端(BoNT/AHn)主要通过在细胞膜上形成离子通道在内化过程中起作用,重链C端(BoNT/AHc)是受体结合区域,也是主要中和抗原的决定区,具有完全保护效应,位于BoNT/AHc的抗原表位为中和抗体的筛选提供依据。已知的BoNT/A受体包括神经节苷脂(ganglioside,GT1b)、突触囊泡蛋白2(Synaptic Vesicle protein 2,SV2)及2013年新发现的成纤维细胞生长因子受体(Fibroblast Growth Factor Receptor 3,FGFR3),其中GT1b和SV2与BoNT/A的结合位点已被阐明,新受体FGFR3与BoNT/A的结合位点还未被阐明。一般认为中和抗体的解毒机制是通过与BoNT/AHc结合从而封闭毒素与受体的结合起到解毒作用,因此受体的结合位点与中和抗体的结合位点在空间结构上存在一定的关联性,明确新受体FGFR3与BoNT/AHc的结合位点以及三个受体的相互作用关系可为肉毒毒素中和抗体研究提供有用信息。目前治疗肉毒中毒没有有效的化学药物,主要采用马源性血清进行治疗,但马血清存在病毒污染风险、过敏反应等弊端。近几年,随着基因工程生产人源化工程抗体的发展,发现单个抗体使用效果不好,几个单抗联用可以起到很好的中和保护效果。加利福尼亚大学、本所孙志伟教授课题组以及美国的XOMA公司分别筛选到了针对BoNT/A不同抗原表位的单抗组合。本研究的筛选工作是将抗原表位分析和抗体筛选结合起来,由表位分析指导抗体筛选,通过构建突变体库和抗体库对抗体进行筛选,明确单抗对应的抗原表位及受体结合表位,为抗体药物的开发及抗体解毒机制奠定基础。根据BoNT/AHc晶体结构利用Discovery Studio软件进行分析,并参照其二级结构和空间位置,预测了毒素150个可能参与到抗原表位的氨基酸序列,设计突变引物将这些氨基酸每3-5个为一组进行突变,利用快速定点突变技术构建了30个BoNT/AH多联氨基酸突变体,并以BoNT/AH突变体为模板,利用分子克隆技术构建了22个BoNT/AHc多联氨基酸突变体。BoNT/AHc突变体用SDS-PAGE分析均为可溶性表达,经Ni-chelating sepharose螯合介质纯化蛋白纯度均在85%以上。构建了bont/ahc表达载体,大规模发酵制备并纯化了bont/ahc,以此为抗原免疫小鼠筛选单抗阳性细胞株,拟获得50个以上单抗,目前已获得1a4、3h3、3h7、5h8等4个单抗。经鉴定细胞上清效价均高于3.0×103,均能与bont/ahc特异性结合,且4株单抗抗原识别表位相近,单抗亚型鉴定结果为1a4、3h7属于igg1(Κ),3h3属于igm(Κ),5h8属于igg2b(Κ)。另制备了文献报道的3个位点明确的单抗c25、s25和3d12,其中单抗3d12是在原来序列基础上进行几个位点突变制备的抗体,经鉴定3d12抗体效价为1.22×106,能与bont/ahc特异性结合,亲和力常数k=10.6×108l/mol。此外还对本实验室筛选到的3个人源单抗ml01、ml02和ml03进行了制备和分析,单抗ml01针对bont/ahc效价为5.12×105,单抗ml02针对bont/ahc效价为1.28×105,ml01亲和力常数kd=2.80×10-9mol/l,ml02的亲和力常数kd=6.89×10-9mol/l。两个单抗均能与bont/ahc特异性结合,动物实验表明,单抗ml01对bont/a的中和效价为16ld50/μg,单抗ml02对bont/a的中和效价为4ld50/μg,两个单抗的中和保护效果很好,具有进一步开发的前景。通过elisa的方法考察了突变体与受体和单抗的相互作用情况,分析突变体与受体、单抗结合能力的变化来确定受体、单抗与bont/ahc的结合表位,进而通过组合和中和实验筛选有效抗体组合。在检测突变体蛋白与受体fgfr3的结合情况前,首先通过检测bont/ahc突变体与已明确位点的受体sv2c的结合情况对elisa的方法进行了验证,实验结果表明与受体sv2c结合力下降的突变体突变位点覆盖了受体sv2c已明确的位点,说明了用elisa的方法检测突变体蛋白与受体的结合情况是有效可靠的,进而用elisa的方法考察了bont/ahc突变体与受体fgfr3的结合情况,并将fgfr3与bont/ahc的结合表位进行了总结,初步确定受体fgfr3与bont/ahc的结合表位为1256-1258fhq、1255-1257fnn、1270-1272wyn、1273-1276rqie、1277-1281rssrt。在检测突变体蛋白及突变体诱导菌液与抗体ml01、ml02的结合情况前,同样对elisa的方法进行了验证,采用的是检测bont/ahc突变体与已明确位点的抗体3d12的结合情况,实验结果表明与抗体3d12结合力下降的突变体突变位点覆盖了抗体3d12的已明确位点,说明了用elisa的方法检测突变体蛋白与抗体的相互作用是有效可靠的,进而用elisa的方法检测了纯化好的22个bont/ahc突变体蛋白及30个bont/ah突变体诱导菌液与抗体ml01、ml02相互作用结果,并将抗体ml01、ml02与bont/ahc的结合表位进行了总结,单抗ml01与bont/ahc的结合表位为1127-1131nvgir、1163-1167kkyas、1179-1181rvy、1240-1242qdn、1255-1257fnn,单抗ml02与bont/ahc的结合表位为917-921 FNLES、958-961 LNNE、1064-1066 HRY。综上所述,本研究构建的突变体库为肉毒毒素的中和表位分析奠定基础,筛选的单克隆抗体具有一定的开发前景,通过突变体库和抗体库表位分析初步确定了受体FGFR3及抗体ML01、ML02与BoNT/AHc结合的可能的表位,这些表位有待于通过其他蛋白相互作用的方法进行验证。
[Abstract]:Botulinum neurotoxins (BoNT) is a kind of strong protein neurotoxin, which is produced by the anaerobic Clostridium botulinum. It is the most toxic toxin known at present. In recent years, botulinum toxin has caused the group poisoning from time to time in the folk. It may also be used as a terrorist and biological warfare agent in military, so botulinum toxin The research has attracted the attention of researchers in various countries.BoNT, a heavy chain with a relative molecular mass of 100kDa and a light chain with a relative molecular mass of 50kDa connected by a two sulfur bond. The light chain (BoNT/AL) is a toxic component of the toxin and has a Zn2+ dependent endopeptidase activity. The heavy chain (BoNT/AH) contains two regions, and the N end (BoNT/AHn) is mainly passed. The formation of ion channels on the cell membrane plays a role in the internalization process. The heavy chain C terminal (BoNT/AHc) is a receptor binding region and a major Neutralizing Antigen, with a complete protective effect. The antigen epitopes at BoNT/AHc provide basis for the screening of neutralizing antibodies. The known BoNT/A receptors include ganglioside, GT1b, and synapses. Vesicular protein 2 (Synaptic Vesicle protein 2, SV2) and the newly discovered fibroblast growth factor receptor (Fibroblast Growth Factor Receptor 3, FGFR3) in 2013. The binding site of GT1b and SV2 to BoNT/A has been elucidated. The binding site of the new receptor is not yet elucidated. The binding site of the toxin to the receptor is detoxified by binding to BoNT/AHc, so the binding site of the receptor and the binding site of neutralizing antibodies are related to the spatial structure. The interaction between the binding sites of the new receptor FGFR3 and BoNT/AHc and the interaction between the three receptors can be provided for the study of botulinum toxin neutralization antibody. Useful information. There are no effective chemicals in the treatment of botulism at present, mainly using horse derived serum for treatment, but horse serum has the disadvantages of virus pollution risk and allergic reaction. In recent years, with the development of human engineered antibody in genetic engineering, it is found that single anti body use is not good, several McAbs can be used to play a very important role. Good neutralization protection effect. The University of California, Professor Sun Zhiwei of our institute and the XOMA company in the United States have screened the monoclonal antibody combinations for different epitopes of BoNT/A. The screening work of this study is to combine antigen epitope analysis with antibody screening, to guide antibody screening by epitope analysis, and to construct a mutant by constructing a mutant. The library and antibody library screened the antibody, identified the antigen epitopes and receptor binding epitopes corresponding to the monoclonal antibody, and laid the foundation for the development of antibody drugs and the mechanism of antibody detoxification. According to the BoNT/AHc crystal structure, the Discovery Studio software was used to analyze the antibody and the 150 toxins were predicted to be involved in the antigen by reference to the secondary structure and the space position. The amino acid sequence of the epitopes, mutation primers were designed to mutate each 3-5 of these amino acids, and 30 BoNT/AH multiple amino acid mutants were constructed by rapid fixed-point mutation. The BoNT/AH mutant was used as a template, and 22 BoNT/AHc multiple amino acid mutant.BoNT/AHc mutants were constructed by molecular cloning technology with SDS-PAGE The purity of the purified protein purified by Ni-chelating Sepharose chelating medium was above 85%. The bont/ahc expression vector was constructed and bont/ahc was prepared and purified by mass fermentation. In order to screen the monoclonal antibody positive cell lines of the antigen immunized mice, more than 50 monoclonal antibodies were obtained. 4 monoclonal antibodies such as 1a4,3h3,3h7,5h8 have been obtained at present. The titer of the cell supernatant was higher than 3 * 103, all of which could be combined with bont/ahc specificity, and the antigen recognition epitopes of 4 McAbs were similar. The results of monoclonal antibody subtype identification were that 1a4,3h7 belonged to IgG1, 3h3 belonged to IgM and 5h8 belonged to IgG2b. Furthermore, the monoclonal antibody C25, S25 and 3d12 of the 3 loci reported in the literature were prepared, in which the monoclonal antibody 3d12 was in the original order. The antibody prepared on the basis of several loci mutations was identified. The titer of 3d12 antibody was 1.22 * 106, and it could be combined with bont/ahc specificity. The affinity constant was k=10.6 x 108l/mol.. In addition, the 3 individual monoclonal antibody ml01, ml02 and ml03 screened in our laboratory were also prepared and analyzed. The McAb ml01 against bont/ahc titer was 5.12 * 105, McAb ml02. The potency of bont/ahc is 1.28 * 105, the affinity constant of ml01 kd=2.80 x 10-9mol/l, and the affinity constant of ml02 kd=6.89 x 10-9mol/l. can be combined with bont/ahc specificity. Animal experiments show that the neutralization titer of the monoclonal antibody ml01 to bont/a is 16ld50/ micron, and the neutralization titer of the monoclonal antibody is the neutralization protection and the neutralization protection of the two McAbs. The interaction between the mutant and the receptor and the monoclonal antibody was examined by ELISA, and the mutants and receptors, the binding ability of the monoclonal antibody were analyzed to determine the binding epitopes of the receptor, the monoclonal antibody and the bont/ahc, and then the combination and neutralization test were used to screen the effective antibody combinations. Before the binding of protein to receptor FGFR3, the method of ELISA was verified by detecting the binding of the bont/ahc mutant with the receptor sv2c of the identified loci. The results showed that the mutant site of the mutant with the decreasing binding force of the receptor sv2c covered the specific loci of the receptor sv2c, indicating that the mutation was detected by the ELISA method. The binding of body protein to receptor is effective and reliable. Then the binding of bont/ahc mutants to receptor FGFR3 is investigated by ELISA, and the binding epitopes of FGFR3 and bont/ahc are summarized, and the binding epitopes of FGFR3 and bont/ahc are preliminarily identified as 1256-1258fhq, 1255-1257fnn, 1270-1272wyn, 1273-1276rqie, 1277-1281r. Ssrt., before detecting the combination of the mutant protein and the mutant induced liquid with the antibody ml01, ml02, was also verified by the method of ELISA, which was used to detect the combination of the bont/ahc mutant and the antibody 3d12 of the identified loci. The experimental results showed that the mutant site of the mutant with the decrease of the binding force of the antibody 3d12 covered the antibody 3D1. 2 of the clear loci showed that the interaction between the mutant protein and the antibody was effective and reliable by ELISA method. Then the ELISA method was used to detect the purified 22 bont/ahc mutant proteins and 30 bont/ah mutants induced by the antibody ml01, ml02 interaction results and the binding table of the antibody ml01, ml02 and bont/ahc. The binding epitopes of mAb ml01 and bont/ahc are 1127-1131nvgir, 1163-1167kkyas, 1179-1181rvy, 1240-1242qdn, 1255-1257fnn, and the binding epitopes of McAb ml02 and bont/ahc are 917-921 FNLES, 958-961 LNNE, 1064-1066 HRY.. The mutant library constructed in this study lays the foundation for the neutralization epitopes analysis of botulinum toxin. The screened monoclonal antibody has a certain development prospect. Through the mutant library and antibody library epitope analysis, the possible epitopes of the receptor FGFR3 and the antibody ML01, ML02 and BoNT/AHc are identified. These epitopes need to be verified by other protein interaction methods.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392.11

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