發(fā)布時(shí)間：2018-03-21 07:30

  本文選題：T-2毒素　切入點(diǎn)：人工抗原　出處：《河南科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：T-2毒素是一種毒性較強(qiáng)的霉菌毒素,可通過(guò)污染農(nóng)作物及飼料導(dǎo)致人和動(dòng)物中毒。目前,檢測(cè)T-2毒素最主要的是儀器分析方法。儀器檢測(cè)方法雖然靈敏度高,但由于技術(shù)要求高且不適于現(xiàn)場(chǎng)快速檢測(cè),因此限制了其使用范圍,由于免疫分析技術(shù)具有速度快、操作簡(jiǎn)單等顯著優(yōu)點(diǎn),在獸藥殘留、農(nóng)藥殘留、違禁添加物監(jiān)控檢測(cè)領(lǐng)域得到廣泛應(yīng)用。本研究擬通過(guò)T-2毒素抗原結(jié)構(gòu)分析,制備出人工抗原;并通過(guò)動(dòng)物免疫、細(xì)胞融合及陽(yáng)性雜交瘤篩選制備T-2毒素單克隆抗體;并在此基礎(chǔ)上研制出T-2毒素快速檢測(cè)ELISA試劑盒。本研究的主要內(nèi)容及結(jié)果如下:1.T-2毒素人工抗原的合成與鑒定對(duì)T-2毒素化學(xué)結(jié)構(gòu)進(jìn)行分析,針對(duì)T-2毒素的化學(xué)結(jié)構(gòu)中含有的羥基活性基團(tuán),選取牛血清蛋白(BSA)和雞卵清蛋白(OVA)作為載體蛋白,利用N,N’-羰基二咪唑(CDI)法合成人工抗原T-2-BSA和T-2-OVA,并采用紫外掃描、SDS-PAGE電泳及動(dòng)物免疫對(duì)人工抗原進(jìn)行了鑒定。結(jié)果顯示,偶聯(lián)蛋白的吸收峰與載體蛋白吸收峰沒(méi)有重疊,但略有偏移,且載體蛋白的遷移速率比偶聯(lián)后的蛋白要快,由此初步推斷人工抗原偶聯(lián)成功;T-2-BSA所免疫小鼠的多抗血清效價(jià)高達(dá)1:1×104,半數(shù)抑制濃度(IC_(50))為8.32 ng/mL,說(shuō)明所制備的人工抗原具有良好的免疫原性,且能誘導(dǎo)動(dòng)物機(jī)體產(chǎn)生針對(duì)T-2毒素的特異性抗體。2.T-2毒素單克隆抗體的制備及鑒定選取T-2-BSA免疫小鼠中血清效價(jià)高、抑制效價(jià)好的小鼠作為融合對(duì)象。通過(guò)細(xì)胞融合技術(shù)將小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行細(xì)胞融合,利用ELISA篩選體系對(duì)融合后的細(xì)胞進(jìn)行篩選,選取陽(yáng)性雜交瘤細(xì)胞進(jìn)行擴(kuò)大培養(yǎng)亞克隆,最終篩選出1株可以穩(wěn)定產(chǎn)生抗T-2毒素單克隆抗體的雜交瘤細(xì)胞株,命名為4F7,經(jīng)測(cè)定細(xì)胞上清效價(jià)在1:1×10~3以上,采用體內(nèi)誘生腹水法制備腹水,采集的腹水效價(jià)高達(dá)1:6.4×105,且對(duì)T-2毒素的半數(shù)抑制IC_(50)可以達(dá)到2.28ng/mL,該單克隆抗體的亞型為Ig G1型,親和常數(shù)Ka為1.49×1011 L/mol,與其它霉菌毒素交叉反應(yīng)率均低于0.01%。3.T-2毒素間接競(jìng)爭(zhēng)ELISA快速檢測(cè)試劑盒的制備采用間接競(jìng)爭(zhēng)ELISA棋盤法方法確定包被原包被濃度及抗體工作濃度,通過(guò)對(duì)反應(yīng)時(shí)間的優(yōu)化,建立間接競(jìng)爭(zhēng)ELISA檢測(cè)方法從而制備出T-2毒素間接競(jìng)爭(zhēng)ELISA快速檢測(cè)試劑盒。在包被原濃度為1:4000稀釋,抗體工作濃度為1:32000稀釋時(shí)所制備的T-2毒素快速檢測(cè)試劑盒的標(biāo)準(zhǔn)曲線的線性回歸方程為y=-0.2742x+0.5939,R~2=0.9911,根據(jù)方程計(jì)算出IC_(50)為2.19 ng/mL,靈敏度可以達(dá)到0.129 ng/m L,添加回收率在83.8%~94.3%之間,變異系數(shù)小于15%且交叉反應(yīng)率均小于0.01%,在4°C存放180 d未出現(xiàn)質(zhì)量問(wèn)題。
[Abstract]:T-2 toxin is a highly toxic mycotoxin, which can lead to human and animal poisoning by contaminating crops and feed. At present, the most important method for detecting T-2 toxin is instrument analysis. However, due to the high technical requirements and unsuitable for rapid detection in the field, the scope of its application is limited. Due to the remarkable advantages of the immunoassay technology, such as high speed and simple operation, the residues of veterinary drugs and pesticides are found in the veterinary medicine residues, pesticide residues, and so on. In this study, artificial antigens were prepared by analyzing the structure of T-2 toxin antigen, and monoclonal antibodies against T-2 toxin were prepared by animal immunity, cell fusion and positive hybridoma screening. On this basis, the ELISA kit for rapid detection of T-2 toxin was developed. The main contents and results of this study were as follows: 1. The synthesis and identification of T-2 toxin artificial antigen to analyze the chemical structure of T-2 toxin. Bovine serum protein (BSA) and chicken ovalbumin (OVA) were selected as carrier proteins for the hydroxyl groups contained in the chemical structure of T-2 toxin. The artificial antigens T-2-BSA and T-2-OVA were synthesized by the method of NNN-carbonyldiimidazolium (CDI), and the artificial antigens were identified by UV-scanning SDS-PAGE and animal immunity. The results showed that the absorption peaks of conjugated proteins did not overlap with the absorption peaks of carrier proteins, but shifted slightly. The transfer rate of carrier protein was faster than that of coupling protein. It was preliminarily inferred that the polyclonal antibody titer of mice immunized with artificial antigen coupled successfully with T-2-BSA was as high as 1: 1 脳 10 ~ 4 and ICM _ (50) was 8.32 ng / mL, which indicated that the artificial antigen prepared had good immunogenicity. The monoclonal antibody against T-2 toxin was produced in animal body. 2. Preparation and identification of monoclonal antibody against T-2 toxin in mice immunized with T-2-BSA. The spleen cells of mice were fused with SP2/0 myeloma cells by cell fusion technique, and the fused cells were screened by ELISA screening system. A hybridoma cell line, named 4F7, was selected to produce monoclonal antibody against T-2 toxin. The supernatant titer of the cell supernatant was over 1: 1 脳 10 ~ (-3). Ascites were prepared by inducing ascites in vivo. The titer of ascites collected was as high as 1: 6.4 脳 105, and the half inhibition of T-2 toxin was 2.28 ng / mL. the monoclonal antibody subtype was Ig G1. The affinity constant Ka was 1.49 脳 1011 L / mol, and the cross reaction rate with other mycotoxins was lower than 0.01. 3. The preparation of indirect competitive ELISA kit for toxin was determined by indirect competitive ELISA chessboard method. By optimizing the reaction time, an indirect competitive ELISA detection method was established to prepare the T-2 toxin indirect competitive ELISA rapid detection kit, which was diluted at the original concentration of 1: 4000. The linear regression equation of the standard curve of the T-2 toxin rapid detection kit prepared when the working concentration of antibody is 1: 32000 dilution is yap-0.2742x 0.5939 ~ (+) ~ 0.9911. According to the equation, the ICS50 is calculated as 2.19 ng / mL. the sensitivity can reach 0.129 ng / mL, and the recovery rate is between 83.894% and 94.3%. The coefficient of variation was less than 15% and the cross reaction rate was less than 0.01. There was no quality problem at 4 擄C for 180 days.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S859.8

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