本文關(guān)鍵詞： 烷基苯酚 單克隆抗體 競(jìng)爭(zhēng)ELISA 同源建模 分子對(duì)接　出處：《華東理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：烷基苯酚(APs)是一類環(huán)境內(nèi)分泌激素,具有生殖、免疫和神經(jīng)等多種生物毒性,對(duì)水生動(dòng)物、兩棲類和哺乳類動(dòng)物的性別分化和生殖產(chǎn)生較大危害,同時(shí)對(duì)人類的健康具有潛在危害。由于烷基苯酚的毒性較大、污染范圍較廣,各國(guó)紛紛出臺(tái)相關(guān)法律法規(guī)限制烷基苯酚的使用,故高親和力的APs單抗的制備對(duì)其含量的檢測(cè)顯得尤為重要。APs是一個(gè)結(jié)構(gòu)相似的大家族,具有共同的苯酚結(jié)構(gòu)和不同長(zhǎng)度的碳鏈。本文以戊基苯酚為研究對(duì)象,通過(guò)碳二亞胺法與BSA和OVA偶聯(lián)制備完全抗原,計(jì)算得到耦合率分別為39:1和41:1。三次免疫后,測(cè)得血清效價(jià)皆高于128 000,通過(guò)電融合法將其脾臟細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行融合,ELISA和流式細(xì)胞儀分篩得到四株單克隆抗體細(xì)胞株,選取IC50分別為9.8 μg/mL和185 ng/mL的F10和戊-13兩株抗體用于后續(xù)研究。分型試劑盒測(cè)得F10和戊-13的重鏈分別為IgG1型和IgM型,輕鏈都為Kappa型;兩株抗體都對(duì)烷基苯酚具有不同程度的識(shí)別,對(duì)APA和AP的識(shí)別較好。同源建模實(shí)驗(yàn)表明兩株抗體的可變區(qū)重鏈和輕鏈分別有22和12個(gè)氨基酸種類不同,其中大部分位于CDR區(qū)域,而改變的氨基酸使得可變區(qū)結(jié)合位點(diǎn)的疏水性增強(qiáng)。分子對(duì)接實(shí)驗(yàn)表明,4-AP的酚羥基與重鏈CDR3的Ser-99形成氫鍵,CDR區(qū)域內(nèi)大量的疏水性氨基酸形成空腔將苯環(huán)和烷基鏈包裹,從而實(shí)現(xiàn)抗原抗體的結(jié)合,抗體戊-13結(jié)合位點(diǎn)的疏水作用力增強(qiáng)是其親和力增加的主要原因。本文制備出親和力和特異性良好的APs單克隆抗體,為APs含量檢測(cè)提供一種新方法,通過(guò)可變區(qū)序列的比對(duì)和同源建模實(shí)驗(yàn)找出影響親和力大小的關(guān)鍵性氨基酸和作用力,為后續(xù)scFv的研究和基因工程改造奠定基礎(chǔ)。
[Abstract]:APs (alkylphenol) is a kind of environmental endocrine hormone, which has many biological toxicity, such as reproduction, immunity and neurotoxicity, which is harmful to the sex differentiation and reproduction of aquatic animals, amphibians and mammals. At the same time, it has potential harm to human health. Due to the toxicity of alkyl phenol and the wide range of pollution, many countries have issued laws and regulations to restrict the use of alkyl phenol. Therefore, the preparation of high affinity APs monoclonal antibody is particularly important for the detection of its content. APs is a large family with similar structure, common phenol structure and different length carbon chain. The complete antigen was prepared by coupling carbodiimide with BSA and OVA. The coupling rates were 39: 1 and 41: 1, respectively. The titers of serum were all higher than 128,000. Four monoclonal antibody cell lines were obtained by fusion Elisa and flow cytometry between spleen cells and SP2/0 myeloma cells by electrofusion method. F10 and pent- 13 antibodies with IC50 of 9.8 渭 g / mL and 185 ng/mL were selected for further study. The heavy chains of F10 and pent- 13 were IgG1 type and IgM type, and light chain were Kappa type, respectively. Homology modeling showed that there were 22 and 12 amino acids in the variable region heavy chain and light chain of the two antibodies respectively, most of which were located in the CDR region. The molecular docking experiments showed that the phenolic hydroxyl of 4-AP and the Ser-99 of heavy chain CDR3 formed a large amount of hydrophobic amino acids in the region of hydrogen bond and formed a large number of hydrophobic amino acids to form a cavity that encapsulated the benzene ring and alkyl chain, while the hydrophobic amino acids increased the hydrophobicity of the variable region binding site. In order to achieve the binding of antigen and antibody, the increase of hydrophobic force at the binding site of antibody pentyl-13 was the main reason for the increase of affinity. In this paper, a good affinity and specificity of APs monoclonal antibody was prepared, which provides a new method for the detection of APs content. The key amino acids and forces affecting the affinity were found through the alignment of variable region sequences and homologous modeling experiments, which laid a foundation for the further study of scFv and genetic engineering.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X830;O652.1

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