本文選題：噬菌體展示 + 擬除蟲菊酯��； 參考：《南京農(nóng)業(yè)大學》2016年博士論文

【摘要】：本文以食品安全問題中常見的農(nóng)藥殘留和致病微生物污染為出發(fā)點,選取了擬除蟲菊酯和大腸桿菌為研究對象,分別篩選獲得各自的抗體,并對抗體的活性進行了研究。擬除蟲菊酯因其毒性低、易分解等優(yōu)勢,被廣泛地用于農(nóng)業(yè)害蟲的防治,近期研究表明,長時間低劑量接觸會對人體健康造成威脅;食品中有害微生物污染的預防和控制是目前食品安全領域研究的另一個熱點,其中大腸桿菌最為典型。而近年來發(fā)展起來的噬菌體展示技術(shù)為快速高通量篩選提供了強大的工具和方法,已有抗生素、小分子農(nóng)藥、毒素和微生物等的抗體通過噬菌體展示技術(shù)篩選獲得。目前從人源化抗體庫中篩選擬除蟲菊酯抗體和抑菌活性蛋白還較為少見�；谝陨锨闆r,本研究以擬除蟲菊酯通用抗原、廣譜性單克隆抗體和大腸桿菌作為抗原,分別對噬菌體展示的人源化抗體庫進行篩選,獲得各自的抗體,并對其活性進行研究,主要研究內(nèi)容及結(jié)果如下:1.擬除蟲菊酯單域抗體的篩選、鑒定及活性分析以擬除蟲菊酯的共性結(jié)構(gòu)域(PBA)作為抗原,對人源化的單域抗體庫(DAB庫)進行四輪篩選,經(jīng)驗證最終獲得5個與PBA具有良好結(jié)合活性的單域抗體。分別構(gòu)建基于這五個單域抗體的競爭曲線,其中基于A3建立的競爭ELISA靈敏度最高,能夠識別氯氰菊酯、高效氯氰菊酯和氰戊菊酯,對氟氰戊菊酯識別能力較弱,而對甲氰菊酯、溴氰菊酯和氯菊酯無識別。基于A3建立競爭ELISA對氯氰菊酯、高效氯氰菊酯和氰戊菊酯的IC50分別為2.586 μg/mL、1.814 μg/mL和2.251μg/mL。同時研究了大白菜基質(zhì)對該檢測方法的影響,添加回收試驗結(jié)果顯示三種菊酯添加回收率在89.4%到91.5%之間。經(jīng)分子對接和丙氨酸掃描分析發(fā)現(xiàn)Ile 55、Val 74、Pro 76和Lys 125氨基酸殘基是PBA與A3結(jié)合的關(guān)鍵氨基酸位點。2.擬除蟲菊酯抗獨特型抗體的篩選、鑒定及活性分析以擬除蟲菊酯的廣譜單克隆抗體作為抗原,對噬菌體展示的人源化單域抗體庫(DAB庫)進行篩選,經(jīng)四輪篩選后,獲得3個能夠識別單克隆抗體的單域抗體,分別構(gòu)建基于這3個單域抗體的競爭曲線。其中基于A8建立的ELISA靈敏度最高,可以識別氯氰菊酯、高效氯氰菊酯和氰戊菊酯,對氟氰戊菊酯識別較弱,對甲氰菊酯、溴氰菊酯和氯菊酯無識別。對氯氰菊酯、高效氯氰菊酯和氰戊菊酯的IC50分別為1.775μg/mL、1.624 μg/mL和3.675 μg/mL。研究了大白菜基質(zhì)對該檢測方法的影響,添加回收試驗結(jié)果顯示三種擬除蟲菊酯的添加回收率在87.4%到90.2%之間。并對抗獨特型抗體A8的抗原結(jié)構(gòu)進行預測。3.大腸桿菌單鏈抗體的篩選、鑒定及可溶性表達以E.coli全細胞和外膜蛋白交替作為篩選抗原,對噬菌體展示的人源化單鏈抗體庫(Tomlinson I庫)進行四輪篩選,獲得4個能識別E.coli的單鏈抗體,并成功地將其在BL21-pET26b表達體系中進行可溶性表達。采用瓊脂孔擴散法初步測定單鏈抗體的抑菌活性,結(jié)果顯示A1和B4對E.coli具有抑菌活性。對A1、B4誘導表達條件進行優(yōu)化,最終確定A1最佳誘導表達條件:誘導溫度25 ℃、IPTG濃度為1.0 mmol/L、誘導時間15h, B4最佳誘導表達條件:誘導溫度30℃、IPTG濃度0.6mmol/L、誘導時間是21 h。利用His-trap FF純化柱成功地對表達產(chǎn)物進行了純化。4.單鏈抗體B4抑菌活性及抑菌機理的初步研究與分析單鏈抗體B4對大腸桿菌(Escheriolia coli)、小腸結(jié)腸炎耶爾森氏菌(Yersinia enterocolitica)、鼠傷寒沙 門 氏菌(Salmonella typhimurium)、蠟樣芽胞桿菌(Bacillus cereus)均有抑制作用,MIC 分別為 128 μg/mL、256 μg/mL、128 μg/mL、256 μg/mL,對金黃色葡萄球菌(Staphylococcus aureus)、酵母釀酒(Saccharomyces cerevisiae)沒有抑制活性。以大腸桿菌作為目標菌株,研究單鏈抗體B4的抑菌活性、穩(wěn)定性和溶血性,結(jié)果表明:單鏈抗體B4對熱處理比較敏感;中性條件下抑菌活力較好,酸、堿條件下抑菌活力下降;隨著離子強度的升高單鏈抗體B4抑菌活性也有所下降;對木瓜蛋白酶,胰蛋白酶,堿性蛋白酶和蛋白酶K不穩(wěn)定;未發(fā)生溶血現(xiàn)象。以大腸桿菌作為目標菌株,初步探討了單鏈抗體B4的抑菌機理。利用掃描電鏡觀察單鏈抗體B4處理前后大腸桿菌形態(tài)上的變化,發(fā)現(xiàn)經(jīng)單鏈抗體B4處理后大腸桿菌出現(xiàn)坍塌變形,處理時間越長坍塌變形越嚴重。通過MTT法測定甲瓚生成量,來研究大腸桿菌的氧化代謝活性,發(fā)現(xiàn)單鏈抗體B4處理后大腸桿菌氧化代謝活性受到抑制,濃度越高處理時間越長,抑制越明顯。以羅丹明123為熒光指示劑,分析大腸桿菌的膜電位,發(fā)現(xiàn)單鏈抗體B4處理能夠使大腸桿菌膜電位降低,濃度越高處理時間越長,膜電位降低越顯著。通過測定細胞上清OD260nm分析具有紫外特征吸收的大分子外滲情況,結(jié)果顯示隨著單鏈抗體B4濃度和處理時間的增加,大分子物質(zhì)外滲現(xiàn)象加劇。利用流式細胞儀分析單鏈抗體B4對細胞膜完整性的影響,結(jié)果顯示單鏈抗體B4處理后膜完整性受到破壞,濃度越高,處理時間越長,破壞越嚴重。
[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.

【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：TS201.3

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