本文選題：肉毒毒素 + 中和抗體��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年碩士論文

【摘要】：肉毒神經(jīng)毒素(botulinum neurotoxins,BoNT)是一類(lèi)烈性的蛋白類(lèi)神經(jīng)毒素,由厭氧的肉毒梭菌產(chǎn)生,是目前已知毒性最強(qiáng)的毒素。近年來(lái),肉毒毒素在民間不時(shí)引發(fā)群體中毒事件,在軍事上也可能作為恐怖劑和生物戰(zhàn)劑被謬用,因此肉毒毒素的研究受到了各國(guó)研究者的關(guān)注。BoNT由一條相對(duì)分子質(zhì)量為100kDa的重鏈和一條相對(duì)分子質(zhì)量為50kDa的輕鏈通過(guò)二硫鍵連接而成。輕鏈(BoNT/AL)是毒素的毒性成分,具有Zn2+依賴性的內(nèi)肽酶活性。重鏈(BoNT/AH)包含兩個(gè)區(qū)域,N端(BoNT/AHn)主要通過(guò)在細(xì)胞膜上形成離子通道在內(nèi)化過(guò)程中起作用,重鏈C端(BoNT/AHc)是受體結(jié)合區(qū)域,也是主要中和抗原的決定區(qū),具有完全保護(hù)效應(yīng),位于BoNT/AHc的抗原表位為中和抗體的篩選提供依據(jù)。已知的BoNT/A受體包括神經(jīng)節(jié)苷脂(ganglioside,GT1b)、突觸囊泡蛋白2(Synaptic Vesicle protein 2,SV2)及2013年新發(fā)現(xiàn)的成纖維細(xì)胞生長(zhǎng)因子受體(Fibroblast Growth Factor Receptor 3,FGFR3),其中GT1b和SV2與BoNT/A的結(jié)合位點(diǎn)已被闡明,新受體FGFR3與BoNT/A的結(jié)合位點(diǎn)還未被闡明。一般認(rèn)為中和抗體的解毒機(jī)制是通過(guò)與BoNT/AHc結(jié)合從而封閉毒素與受體的結(jié)合起到解毒作用,因此受體的結(jié)合位點(diǎn)與中和抗體的結(jié)合位點(diǎn)在空間結(jié)構(gòu)上存在一定的關(guān)聯(lián)性,明確新受體FGFR3與BoNT/AHc的結(jié)合位點(diǎn)以及三個(gè)受體的相互作用關(guān)系可為肉毒毒素中和抗體研究提供有用信息。目前治療肉毒中毒沒(méi)有有效的化學(xué)藥物,主要采用馬源性血清進(jìn)行治療,但馬血清存在病毒污染風(fēng)險(xiǎn)、過(guò)敏反應(yīng)等弊端。近幾年,隨著基因工程生產(chǎn)人源化工程抗體的發(fā)展,發(fā)現(xiàn)單個(gè)抗體使用效果不好,幾個(gè)單抗聯(lián)用可以起到很好的中和保護(hù)效果。加利福尼亞大學(xué)、本所孫志偉教授課題組以及美國(guó)的XOMA公司分別篩選到了針對(duì)BoNT/A不同抗原表位的單抗組合。本研究的篩選工作是將抗原表位分析和抗體篩選結(jié)合起來(lái),由表位分析指導(dǎo)抗體篩選,通過(guò)構(gòu)建突變體庫(kù)和抗體庫(kù)對(duì)抗體進(jìn)行篩選,明確單抗對(duì)應(yīng)的抗原表位及受體結(jié)合表位,為抗體藥物的開(kāi)發(fā)及抗體解毒機(jī)制奠定基礎(chǔ)。根據(jù)BoNT/AHc晶體結(jié)構(gòu)利用Discovery Studio軟件進(jìn)行分析,并參照其二級(jí)結(jié)構(gòu)和空間位置,預(yù)測(cè)了毒素150個(gè)可能參與到抗原表位的氨基酸序列,設(shè)計(jì)突變引物將這些氨基酸每3-5個(gè)為一組進(jìn)行突變,利用快速定點(diǎn)突變技術(shù)構(gòu)建了30個(gè)BoNT/AH多聯(lián)氨基酸突變體,并以BoNT/AH突變體為模板,利用分子克隆技術(shù)構(gòu)建了22個(gè)BoNT/AHc多聯(lián)氨基酸突變體。BoNT/AHc突變體用SDS-PAGE分析均為可溶性表達(dá),經(jīng)Ni-chelating sepharose螯合介質(zhì)純化蛋白純度均在85%以上。構(gòu)建了bont/ahc表達(dá)載體,大規(guī)模發(fā)酵制備并純化了bont/ahc,以此為抗原免疫小鼠篩選單抗陽(yáng)性細(xì)胞株,擬獲得50個(gè)以上單抗,目前已獲得1a4、3h3、3h7、5h8等4個(gè)單抗。經(jīng)鑒定細(xì)胞上清效價(jià)均高于3.0×103,均能與bont/ahc特異性結(jié)合,且4株單抗抗原識(shí)別表位相近,單抗亞型鑒定結(jié)果為1a4、3h7屬于igg1(Κ),3h3屬于igm(Κ),5h8屬于igg2b(Κ)。另制備了文獻(xiàn)報(bào)道的3個(gè)位點(diǎn)明確的單抗c25、s25和3d12,其中單抗3d12是在原來(lái)序列基礎(chǔ)上進(jìn)行幾個(gè)位點(diǎn)突變制備的抗體,經(jīng)鑒定3d12抗體效價(jià)為1.22×106,能與bont/ahc特異性結(jié)合,親和力常數(shù)k=10.6×108l/mol。此外還對(duì)本實(shí)驗(yàn)室篩選到的3個(gè)人源單抗ml01、ml02和ml03進(jìn)行了制備和分析,單抗ml01針對(duì)bont/ahc效價(jià)為5.12×105,單抗ml02針對(duì)bont/ahc效價(jià)為1.28×105,ml01親和力常數(shù)kd=2.80×10-9mol/l,ml02的親和力常數(shù)kd=6.89×10-9mol/l。兩個(gè)單抗均能與bont/ahc特異性結(jié)合,動(dòng)物實(shí)驗(yàn)表明,單抗ml01對(duì)bont/a的中和效價(jià)為16ld50/μg,單抗ml02對(duì)bont/a的中和效價(jià)為4ld50/μg,兩個(gè)單抗的中和保護(hù)效果很好,具有進(jìn)一步開(kāi)發(fā)的前景。通過(guò)elisa的方法考察了突變體與受體和單抗的相互作用情況,分析突變體與受體、單抗結(jié)合能力的變化來(lái)確定受體、單抗與bont/ahc的結(jié)合表位,進(jìn)而通過(guò)組合和中和實(shí)驗(yàn)篩選有效抗體組合。在檢測(cè)突變體蛋白與受體fgfr3的結(jié)合情況前,首先通過(guò)檢測(cè)bont/ahc突變體與已明確位點(diǎn)的受體sv2c的結(jié)合情況對(duì)elisa的方法進(jìn)行了驗(yàn)證,實(shí)驗(yàn)結(jié)果表明與受體sv2c結(jié)合力下降的突變體突變位點(diǎn)覆蓋了受體sv2c已明確的位點(diǎn),說(shuō)明了用elisa的方法檢測(cè)突變體蛋白與受體的結(jié)合情況是有效可靠的,進(jìn)而用elisa的方法考察了bont/ahc突變體與受體fgfr3的結(jié)合情況,并將fgfr3與bont/ahc的結(jié)合表位進(jìn)行了總結(jié),初步確定受體fgfr3與bont/ahc的結(jié)合表位為1256-1258fhq、1255-1257fnn、1270-1272wyn、1273-1276rqie、1277-1281rssrt。在檢測(cè)突變體蛋白及突變體誘導(dǎo)菌液與抗體ml01、ml02的結(jié)合情況前,同樣對(duì)elisa的方法進(jìn)行了驗(yàn)證,采用的是檢測(cè)bont/ahc突變體與已明確位點(diǎn)的抗體3d12的結(jié)合情況,實(shí)驗(yàn)結(jié)果表明與抗體3d12結(jié)合力下降的突變體突變位點(diǎn)覆蓋了抗體3d12的已明確位點(diǎn),說(shuō)明了用elisa的方法檢測(cè)突變體蛋白與抗體的相互作用是有效可靠的,進(jìn)而用elisa的方法檢測(cè)了純化好的22個(gè)bont/ahc突變體蛋白及30個(gè)bont/ah突變體誘導(dǎo)菌液與抗體ml01、ml02相互作用結(jié)果,并將抗體ml01、ml02與bont/ahc的結(jié)合表位進(jìn)行了總結(jié),單抗ml01與bont/ahc的結(jié)合表位為1127-1131nvgir、1163-1167kkyas、1179-1181rvy、1240-1242qdn、1255-1257fnn,單抗ml02與bont/ahc的結(jié)合表位為917-921 FNLES、958-961 LNNE、1064-1066 HRY。綜上所述,本研究構(gòu)建的突變體庫(kù)為肉毒毒素的中和表位分析奠定基礎(chǔ),篩選的單克隆抗體具有一定的開(kāi)發(fā)前景,通過(guò)突變體庫(kù)和抗體庫(kù)表位分析初步確定了受體FGFR3及抗體ML01、ML02與BoNT/AHc結(jié)合的可能的表位,這些表位有待于通過(guò)其他蛋白相互作用的方法進(jìn)行驗(yàn)證。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R392.11

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