烷基苯酚单克隆抗体制备及其可变区序列的同源建模

发布时间：2018-02-15 06:42

本文关键词： 烷基苯酚单克隆抗体竞争ELISA 同源建模分子对接　出处：《华东理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：烷基苯酚(APs)是一类环境内分泌激素,具有生殖、免疫和神经等多种生物毒性,对水生动物、两栖类和哺乳类动物的性别分化和生殖产生较大危害,同时对人类的健康具有潜在危害。由于烷基苯酚的毒性较大、污染范围较广,各国纷纷出台相关法律法规限制烷基苯酚的使用,故高亲和力的APs单抗的制备对其含量的检测显得尤为重要。APs是一个结构相似的大家族,具有共同的苯酚结构和不同长度的碳链。本文以戊基苯酚为研究对象,通过碳二亚胺法与BSA和OVA偶联制备完全抗原,计算得到耦合率分别为39:1和41:1。三次免疫后,测得血清效价皆高于128 000,通过电融合法将其脾脏细胞与SP2/0骨髓瘤细胞进行融合,ELISA和流式细胞仪分筛得到四株单克隆抗体细胞株,选取IC50分别为9.8 μg/mL和185 ng/mL的F10和戊-13两株抗体用于后续研究。分型试剂盒测得F10和戊-13的重链分别为IgG1型和IgM型,轻链都为Kappa型;两株抗体都对烷基苯酚具有不同程度的识别,对APA和AP的识别较好。同源建模实验表明两株抗体的可变区重链和轻链分别有22和12个氨基酸种类不同,其中大部分位于CDR区域,而改变的氨基酸使得可变区结合位点的疏水性增强。分子对接实验表明,4-AP的酚羟基与重链CDR3的Ser-99形成氢键,CDR区域内大量的疏水性氨基酸形成空腔将苯环和烷基链包裹,从而实现抗原抗体的结合,抗体戊-13结合位点的疏水作用力增强是其亲和力增加的主要原因。本文制备出亲和力和特异性良好的APs单克隆抗体,为APs含量检测提供一种新方法,通过可变区序列的比对和同源建模实验找出影响亲和力大小的关键性氨基酸和作用力,为后续scFv的研究和基因工程改造奠定基础。
[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.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X830;O652.1

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