發(fā)布時(shí)間：2018-02-01 18:31

  本文關(guān)鍵詞： 脫氧雪腐鐮刀菌烯醇(DON) 連接臂 分子模擬 直接競爭酶聯(lián)免疫分析　出處：《江南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：脫氧雪腐鐮刀菌烯醇(DON)半抗原的分子設(shè)計(jì)是建立其免疫分析方法的關(guān)鍵步驟,目前,DON半抗原設(shè)計(jì)仍采用經(jīng)驗(yàn)法,造成時(shí)間和資金的浪費(fèi)。本文從半抗原設(shè)計(jì)的最大原則(不改變待分析物的結(jié)構(gòu)性質(zhì))出發(fā),明確DON最佳半抗原的設(shè)計(jì)思想:最大化抗原表位相似度。利用分子模擬技術(shù),通過分析分子的空間構(gòu)型、電子特性、疏水性質(zhì)這幾個(gè)影響抗原-抗體間作用力的因素在DON修飾前后的變化,研究半抗原結(jié)構(gòu)與免疫識別之間的關(guān)系,建立DON半抗原設(shè)計(jì)的理論模擬方法,為小分子半抗原設(shè)計(jì)提供新的思路,并在此基礎(chǔ)上建立了DON的直接競爭酶聯(lián)免疫分析方法。 根據(jù)DON抗原設(shè)計(jì)流程:選擇結(jié)合位點(diǎn)→引入連接臂→偶聯(lián)載體蛋白,首先研究結(jié)合位點(diǎn)對DON免疫識別的影響,在DON分子中的4個(gè)潛在結(jié)合位點(diǎn)(3 ,7 ,15位羥基以及8位酮基)引入相同碳鏈長度的的連接臂,借助分子模擬軟件Hyperchem7.5對DON及半抗原的表位決定參數(shù)(空間結(jié)構(gòu)、原子點(diǎn)電荷、疏水常數(shù))進(jìn)行計(jì)算分析,3位半抗原具有與DON最相似的結(jié)構(gòu)性質(zhì),表明3位是DON引入連接臂的最佳位點(diǎn),這與Casale和Ramesh的免疫實(shí)驗(yàn)結(jié)果一致。 其次,在3位羥基上設(shè)計(jì)不同的連接臂結(jié)構(gòu),通過抗原表位相似度模擬篩選得到最佳連接臂結(jié)構(gòu):丁二酸酐臂。同時(shí)合成不同連接臂的DON完全抗原,并將之進(jìn)行免疫抗體實(shí)驗(yàn),測定抗體的效價(jià)以及抑制率,驗(yàn)證分子模擬結(jié)果,研究半抗原結(jié)構(gòu)對抗體免疫活性的關(guān)系。結(jié)果表明B組(順丁烯二酸酐臂)抗體平均效價(jià):256000,A組(丁二酸酐臂)、C組(鄰苯二甲酸酐臂)平均效價(jià):64000; DON濃度為500 ng/ml時(shí)對抗體的抑制率分別:A組(66.4 %)B組(21.1 %)C組(10.8 %)。抑制率的大小與抗體對DON的特異性相關(guān),A組與DON具有最高的抗原表位相似度,抗體對DON的特異性強(qiáng),抑制率高;抗體的效價(jià)可以反映半抗原分子的抗原性,苯環(huán)結(jié)構(gòu)、不飽和鍵可以增強(qiáng)抗原性,B組連接臂中含有不飽和鍵,增強(qiáng)了半抗原的抗原性,產(chǎn)生的抗體效價(jià)高;C組雖含有苯環(huán)結(jié)構(gòu),確未能產(chǎn)生高效價(jià)的抗體,結(jié)合其空間結(jié)構(gòu)發(fā)現(xiàn)C組半抗原中羧基端與半抗原主體結(jié)構(gòu)非�？拷�,半抗原結(jié)構(gòu)可能被載體蛋白屏蔽而影響免疫識別;綜合考慮,丁二酸酐臂是DON的最佳連接臂,免疫試驗(yàn)結(jié)果與分子模擬具有很好的相關(guān)性。 在最佳半抗原基礎(chǔ)上,比較BSA和KLH兩種載體蛋白的DON完全抗原的免疫原性。通過丁二酸酐衍生法和碳二亞胺法(EDC法)制備不同載體蛋白的DON完全抗原,免疫新西蘭長耳兔獲得DON抗體,并運(yùn)用酶聯(lián)免疫方法(ELISA)對制備的抗體進(jìn)行研究。 在上述研究結(jié)果基礎(chǔ)上合成DON-KLH完全抗原,建立DON的直接競爭酶聯(lián)免疫分析方法,通過一步抗原抗體反應(yīng),快速檢測食品中DON。檢測范圍為1 -100 ng/mL。半數(shù)抑制率IC50為10 ng/mL,最低檢出限為0.56 ng/mL,與T-2毒素等真菌毒素的交叉反應(yīng)率小于12 %;平均批內(nèi)變異系數(shù)為2.82 %,平均批間變異系數(shù)為14.54 %;對玉米粉的加標(biāo)回收率在80.2-91.1 %,準(zhǔn)確性較高;與商品化試劑盒相比具有所需時(shí)間短,精密度高,靈敏度高的優(yōu)點(diǎn)。 本研究表明分子模擬方法對DON半抗原設(shè)計(jì)有積極作用,指導(dǎo)半抗原設(shè)計(jì),減少半抗原設(shè)計(jì)中時(shí)間及資金的投入。不同載體DON免疫抗原的免疫原性不同,DON-KLH免疫原性優(yōu)于DON-BSA。6本研究所建立的DON的直接競爭ELISA方法,快速、方便,具有較好的實(shí)用價(jià)值,為進(jìn)一步研究DON的ELISA檢測試劑盒提供了重要的實(shí)驗(yàn)依據(jù)。
[Abstract]:Deoxynivalenol (DON) molecular design of the hapten is a key step to establish its immune analysis method, at present, DON hapten design still uses empirical method, resulting in the waste of time and money. This article from the principle of maximum hapten design (without changing the structure properties of the analyte) of clear design ideas DON best hapten: maximizing the epitope similarity. By using molecular simulation methods, the spatial configuration, analysis of molecular electronic properties, hydrophobic nature of these effects of antigen antibody interaction factors in the changes of DON before and after modification, the study of the relationship between hapten structure and immune recognition, a simulation method of DON theory antigen design, provide a new way of thinking for hapten design, and established a direct competitive enzyme linked immunosorbent assay DON.
According to the design process: introduction of DON antigen, connecting arm coupled to carrier protein binding sites, first, study the combined impact of DON sites of immune recognition, 4 potential binding sites in DON molecules (3, 7, 15 and 8 hydroxy ketone) connecting arm uses the same length of carbon chain, with the help of molecular simulation software Hyperchem7.5 of DON and semi epitope decision parameters (spatial structure, atomic charge, hydrophobic constant) were calculated and analyzed, 3 semi antigen has the most similar structural properties with DON, showed that the 3 is DON into the best sites of the connecting arm, and the experimental results of immune Casale and Ramesh.
Secondly, the design of connecting arm structure in different 3 hydroxy group, through epitope screening the optimal similarity simulation connecting arm structure: Ding two anhydride arm connecting arm. At the same time, the synthesis of different DON antigen, and the immune antibody test, determination of antibody titer and inhibition rate, simulation results verify the molecular research. Relationship of hapten structure and immune activity. The results showed that group B (maleic anhydride arm): 256000, the average antibody titer of A group (d two C group (anhydride arm), phthalic anhydride two formic acid arm) average value: 64000; 500 DON concentration of ng/ml to inhibit the antibody respectively. Group A (66.4%) B (21.1%) C group (10.8%). The size and inhibition rate of antibodies specific for DON, A and DON have the highest similarity of antibody epitopes, the specificity of strong DON inhibitory rate; antibody titer can reflect the antigenicity of hapten molecules, The benzene ring structure, unsaturated bond can enhance the antigenicity of B group, the connecting arm containing unsaturated bonds, enhance the antigenicity of hapten, the high antibody titer; although group C containing benzene ring structure, it failed to produce high titer antibody, combined with the spatial structure of C group in the end carboxyl hapten with the hapten subject very close to the structure, structure of hapten carrier protein may be shielded by immune recognition; comprehensive consideration, Ding two anhydride arm is the best connecting arm of DON, simulation and molecular immunity test results have a good correlation.
In the best hapten based on the immunogenicity of two carrier proteins BSA and KLH DON complete antigen. By two D anhydride derivatization and carbon two imide method (EDC method) were prepared with different carrier protein DON antigen, immune New Zealand rabbits received DON antibody, and using ELISA method (ELISA) to study the preparation of antibody.
鍦ㄤ笂榪扮爺絀剁粨鏋滃熀紜，

本文編號：1482560