拟除虫菊酯抗体与大肠杆菌抗体的筛选及活性分析

发布时间：2018-04-21 19:33

本文选题：噬菌体展示 + 拟除虫菊酯　；参考：《南京农业大学》2016年博士论文

【摘要】：本文以食品安全问题中常见的农药残留和致病微生物污染为出发点,选取了拟除虫菊酯和大肠杆菌为研究对象,分别筛选获得各自的抗体,并对抗体的活性进行了研究。拟除虫菊酯因其毒性低、易分解等优势,被广泛地用于农业害虫的防治,近期研究表明,长时间低剂量接触会对人体健康造成威胁;食品中有害微生物污染的预防和控制是目前食品安全领域研究的另一个热点,其中大肠杆菌最为典型。而近年来发展起来的噬菌体展示技术为快速高通量筛选提供了强大的工具和方法,已有抗生素、小分子农药、毒素和微生物等的抗体通过噬菌体展示技术筛选获得。目前从人源化抗体库中筛选拟除虫菊酯抗体和抑菌活性蛋白还较为少见。基于以上情况,本研究以拟除虫菊酯通用抗原、广谱性单克隆抗体和大肠杆菌作为抗原,分别对噬菌体展示的人源化抗体库进行筛选,获得各自的抗体,并对其活性进行研究,主要研究内容及结果如下:1.拟除虫菊酯单域抗体的筛选、鉴定及活性分析以拟除虫菊酯的共性结构域(PBA)作为抗原,对人源化的单域抗体库(DAB库)进行四轮筛选,经验证最终获得5个与PBA具有良好结合活性的单域抗体。分别构建基于这五个单域抗体的竞争曲线,其中基于A3建立的竞争ELISA灵敏度最高,能够识别氯氰菊酯、高效氯氰菊酯和氰戊菊酯,对氟氰戊菊酯识别能力较弱,而对甲氰菊酯、溴氰菊酯和氯菊酯无识别。基于A3建立竞争ELISA对氯氰菊酯、高效氯氰菊酯和氰戊菊酯的IC50分别为2.586 μg/mL、1.814 μg/mL和2.251μg/mL。同时研究了大白菜基质对该检测方法的影响,添加回收试验结果显示三种菊酯添加回收率在89.4%到91.5%之间。经分子对接和丙氨酸扫描分析发现Ile 55、Val 74、Pro 76和Lys 125氨基酸残基是PBA与A3结合的关键氨基酸位点。2.拟除虫菊酯抗独特型抗体的筛选、鉴定及活性分析以拟除虫菊酯的广谱单克隆抗体作为抗原,对噬菌体展示的人源化单域抗体库(DAB库)进行筛选,经四轮筛选后,获得3个能够识别单克隆抗体的单域抗体,分别构建基于这3个单域抗体的竞争曲线。其中基于A8建立的ELISA灵敏度最高,可以识别氯氰菊酯、高效氯氰菊酯和氰戊菊酯,对氟氰戊菊酯识别较弱,对甲氰菊酯、溴氰菊酯和氯菊酯无识别。对氯氰菊酯、高效氯氰菊酯和氰戊菊酯的IC50分别为1.775μg/mL、1.624 μg/mL和3.675 μg/mL。研究了大白菜基质对该检测方法的影响,添加回收试验结果显示三种拟除虫菊酯的添加回收率在87.4%到90.2%之间。并对抗独特型抗体A8的抗原结构进行预测。3.大肠杆菌单链抗体的筛选、鉴定及可溶性表达以E.coli全细胞和外膜蛋白交替作为筛选抗原,对噬菌体展示的人源化单链抗体库(Tomlinson I库)进行四轮筛选,获得4个能识别E.coli的单链抗体,并成功地将其在BL21-pET26b表达体系中进行可溶性表达。采用琼脂孔扩散法初步测定单链抗体的抑菌活性,结果显示A1和B4对E.coli具有抑菌活性。对A1、B4诱导表达条件进行优化,最终确定A1最佳诱导表达条件:诱导温度25 ℃、IPTG浓度为1.0 mmol/L、诱导时间15h, B4最佳诱导表达条件:诱导温度30℃、IPTG浓度0.6mmol/L、诱导时间是21 h。利用His-trap FF纯化柱成功地对表达产物进行了纯化。4.单链抗体B4抑菌活性及抑菌机理的初步研究与分析单链抗体B4对大肠杆菌(Escheriolia coli)、小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)、鼠伤寒沙门氏菌(Salmonella typhimurium)、蜡样芽胞杆菌(Bacillus cereus)均有抑制作用,MIC 分别为 128 μg/mL、256 μg/mL、128 μg/mL、256 μg/mL,对金黄色葡萄球菌(Staphylococcus aureus)、酵母酿酒(Saccharomyces cerevisiae)没有抑制活性。以大肠杆菌作为目标菌株,研究单链抗体B4的抑菌活性、稳定性和溶血性,结果表明:单链抗体B4对热处理比较敏感;中性条件下抑菌活力较好,酸、碱条件下抑菌活力下降;随着离子强度的升高单链抗体B4抑菌活性也有所下降;对木瓜蛋白酶,胰蛋白酶,碱性蛋白酶和蛋白酶K不稳定;未发生溶血现象。以大肠杆菌作为目标菌株,初步探讨了单链抗体B4的抑菌机理。利用扫描电镜观察单链抗体B4处理前后大肠杆菌形态上的变化,发现经单链抗体B4处理后大肠杆菌出现坍塌变形,处理时间越长坍塌变形越严重。通过MTT法测定甲瓒生成量,来研究大肠杆菌的氧化代谢活性,发现单链抗体B4处理后大肠杆菌氧化代谢活性受到抑制,浓度越高处理时间越长,抑制越明显。以罗丹明123为荧光指示剂,分析大肠杆菌的膜电位,发现单链抗体B4处理能够使大肠杆菌膜电位降低,浓度越高处理时间越长,膜电位降低越显著。通过测定细胞上清OD260nm分析具有紫外特征吸收的大分子外渗情况,结果显示随着单链抗体B4浓度和处理时间的增加,大分子物质外渗现象加剧。利用流式细胞仪分析单链抗体B4对细胞膜完整性的影响,结果显示单链抗体B4处理后膜完整性受到破坏,浓度越高,处理时间越长,破坏越严重。
[Abstract]:In this paper, we choose pyrethroid and Escherichia coli as the starting point of the food safety problems, select the pyrethroid and Escherichia coli as the research object, select the respective antibodies, and study the activity of the antibody. The pyrethroid is widely used in agricultural pests because of its low toxicity and easy decomposition. Recent studies have shown that long and low dose exposure can pose a threat to human health, and the prevention and control of harmful microbial contamination in food is another hot spot in the field of food safety research, in which Escherichia coli is the most typical. The antibodies and methods of antibiotics, small molecules, pesticides, toxins, and microbes are screened by phage display techniques. The screening of pyrethroid and bacteriostasis proteins from the humanized antibody library is still rare. Based on the above situation, the general antigen of pyrethroids and broad-spectrum monoclonal antibodies are used in this study. And Escherichia coli as antigen, screening the phage displayed human antibody library respectively, obtaining their respective antibodies and studying their activity. The main contents and results are as follows: 1. the screening, identification and activity analysis of pyrethroid single domain antibody are used as antigen for the common structure domain of pyrethroid (PBA) and human derived. The single domain antibody library (DAB Library) was screened by four rounds. After verification, 5 single domain antibodies with good binding activity with PBA were obtained. The competition curves based on these five single domain antibodies were constructed respectively. The competitive ELISA based on A3 was the highest sensitivity, and could identify Cypermethrin, cypermethrin and Fenvalerate, and the fenvalerate identification of cyfluthrin. There was no weak ability, but no identification of fenpromethrin, deltamethrin and permethrin. Based on A3, the IC50 of Cypermethrin, cypermethrin and fenvalerate were 2.586 g/mL, 1.814 g/mL and 2.251 micron respectively, and the influence of Chinese cabbage matrix on the detection method was studied. The results of the addition recovery test showed three kinds of permethrin. The rate of recovery was between 89.4% and 91.5%. Ile 55, Val 74, Pro 76 and Lys 125 amino acid residues were found to be the key amino acid sites of PBA and A3 by molecular docking and alanine scanning. The screening of anti idiotypic antibodies of pyrethroids, the key amino acid site of PBA and A3, was identified and analyzed with the broad-spectrum monoclonal antibody of pyrethroid as antigen. The human derived single domain antibody library (DAB Library) displayed by the bacteria was screened. After four rounds of screening, 3 single domain antibodies that could identify the monoclonal antibodies were obtained, and the competition curves based on the 3 single domain antibodies were constructed respectively. The sensitivity of the ELISA based on the A8 was the highest, and the Cypermethrin, cypermethrin and fenvalerate were identified, and the cyanogen was identified. Valerthrin, deltamethrin, and permethrin were not identified. The IC50 of Cypermethrin, cypermethrin and fenvalerate were 1.775 mu g/mL, 1.624 g/mL and 3.675 micron g/mL. respectively. The effects of Chinese cabbage matrix on the detection method were studied. The addition recovery test results showed the recovery rate of the addition of pyrethroids. Between 87.4% and 90.2%, and against the antigen structure of the idiotypic antibody A8, the screening of.3. Escherichia coli single chain antibody was predicted, the identification and soluble expression were selected as the screening antigen by E.coli whole cell and outer membrane protein alternately, and four rounds of screening for the phage displayed human single chain antibody library (Tomlinson I Library) were carried out, and 4 can identify E.col. The single chain antibody of I was successfully expressed in the BL21-pET26b expression system. The antibacterial activity of single chain antibody was preliminarily determined by the agar pore diffusion method. The results showed that A1 and B4 had antibacterial activity to E.coli. The optimal expression conditions of A1, B4 induced expression were optimized, and the optimal induction condition of A1 was determined at the temperature of 25, IPTG. The concentration is 1 mmol/L, the induction time 15h, B4 best inducement condition: the induction temperature 30 C, the IPTG concentration 0.6mmol/L, the induction time is 21 h. using His-trap FF purification column, the purified.4. single chain antibody B4 antibacterial activity and the bacteriostasis mechanism of the purified.4. single chain antibody B4 are preliminarily studied and analyzed, and the single chain antibody B4 on Escherichia coli (Escheriolia) Li), enterocolitis Jerson Prand (Yersinia enterocolitica), Salmonella typhimurium (Salmonella typhimurium) and Bacillus cereus (Bacillus cereus) had inhibitory effects. MIC was 128 u g/mL, 256 mu g/mL, 128 mu g/mL, 256 mu g/mL. Visiae) did not inhibit activity. The bacteriostasis, stability and hemolysis of single chain antibody B4 were studied with Escherichia coli as the target strain. The results showed that the single chain antibody B4 was more sensitive to heat treatment, under neutral condition, the bacteriostasis activity was better under the neutral condition, and the inhibitory activity decreased under the acid and alkali conditions, and the antibacterial activity of single chain antibody B4 was also increased with the increase of ionic strength. Decreased; papain, trypsin, alkaline protease and protease K were unstable; no hemolysis occurred. The bacteriostasis mechanism of single chain antibody B4 was preliminarily discussed with Escherichia coli as the target strain. The morphological changes of Escherichia coli before and after the treatment of single chain antibody B4 were observed by scanning electron microscopy, and the results were found after single chain antibody B4 treatment. The greater the collapse and deformation of Enterobacteriaceae, the longer the treatment time, the more serious the collapse and deformation. The MTT method was used to determine the formation of Zan, to study the oxidative metabolic activity of Escherichia coli, and it was found that the oxidative metabolic activity of Escherichia coli was inhibited after the treatment of single chain antibody B4. The longer the concentration was, the longer the treatment was, the more obvious the inhibition was. Luo Danming 123 was the fluorescent indicator, The membrane potential of Escherichia coli was analyzed. It was found that the single chain antibody B4 treatment could reduce the membrane potential of Escherichia coli, the longer the concentration was, the longer the treatment time, the more significant the membrane potential decreased. By measuring the OD260nm analysis of cell supernatant, the macromolecular exudation with ultraviolet characteristic absorption was observed, and the result showed that the concentration of B4 and the time of treatment increased with the result of the single chain antibody. The effect of single chain antibody B4 on the integrity of cell membrane was analyzed by flow cytometry. The results showed that the membrane integrity was damaged after the single chain antibody B4 treatment, the higher the concentration, the longer the treatment time, the more serious the damage.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS201.3

【参考文献】