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  本文關(guān)鍵詞：抗B型肉毒毒素鼠源中和抗體快速制備及其免疫學(xué)活性的初步研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肉毒毒素 表達(dá)純化 免疫保護(hù) 噬菌體抗體 scFv Fab中和作用 IgY

【摘要】：肉毒毒素是肉毒梭狀桿菌產(chǎn)生的外毒素,屬神經(jīng)強(qiáng)毒,人類的肉毒中毒主是由A,B和E引起。是目前已知毒性最強(qiáng)的細(xì)菌蛋白質(zhì)之一,是重要的生物戰(zhàn)劑。本論文以定向免疫和噬菌體展示技術(shù)為平臺,探索了一種快速制備及篩選鼠源抗體的技術(shù)路線,將篩選到的高親和力抗體直接進(jìn)行中和活性實驗,將抗體基因克隆、高親和力抗體富集和抗體篩選在噬菌體水平一步完成,簡便快速,研究周期短。為生物毒素新型抗毒素的制備提供新的研究思路。 本研究中首先克隆了B型肉毒毒素重鏈C端基因,與已知序列同源性達(dá)99%。為了獲得重組蛋白,構(gòu)建了pET22重組質(zhì)粒載體,誘導(dǎo)表達(dá)后未得到目的蛋白。隨后構(gòu)建pET32重組質(zhì)粒,獲得了BoNTB/Hc的融合蛋白。對引物進(jìn)行改構(gòu)后,最終獲得了以包含體形式存在的BoNTB/Hc N端修飾蛋白,并用金屬螯和層析法進(jìn)行了初步純化和鑒定。進(jìn)一步對其免疫學(xué)活性進(jìn)行初步研究,結(jié)果表明重鏈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型肉毒毒素攻擊,為B 型肉毒毒素疫苗的研究奠定了基礎(chǔ)。同時制備了卵黃檢測抗體(IgY),抗體效價達(dá)10~4以上。 用重組表達(dá)并純化的BoNTB/Hc免疫小鼠,從其脾淋巴細(xì)胞中提取全套抗體可變區(qū)基因,分別構(gòu)建scFv和Fab段噬菌體抗體文庫,庫容分別為6.72x10~7cfu和5.69x10~6cfu。以純化的BoNTB/Hc為抗原,對抗體庫進(jìn)行富集篩選,并以BoNT/B為抗原進(jìn)一步鑒定特異性抗體,最終獲得與BoNT/B結(jié)合能力較強(qiáng)的scFv抗體2個,分別命名為S3和$24,VH屬于VH2家族,VK分別屬于VK2和VK1家族。Fab抗體1個,命名為F38,VH屬于VH1家族,VK屬于VK2家族。 重組抗體免疫學(xué)保護(hù)試驗表明,抗B型肉毒毒素的噬菌體抗體S3對BoNT/B致死作用的保護(hù)性較強(qiáng),能夠在一定程度上抵抗10LD_(50)(i.p.)的致死作用,S24次之,F38較弱。未觀測到S3,S24和F38三者的明顯疊加作用。挑選作用較強(qiáng)
[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
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 張志超,胡學(xué)軍,包永明,安利佳;大容量人源單鏈抗體庫構(gòu)建中輕鏈重鏈可變區(qū)的連接[J];免疫學(xué)雜志;2001年06期

2 張達(dá)矜,王琰,化冰,劉曉琳;用噬菌體蛋白Ⅸ展示抗HBsAg分子Fab段[J];中華微生物學(xué)和免疫學(xué)雜志;2004年01期

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本文編號：1412264