本文選題：慶大霉素　切入點(diǎn)：人工抗原　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：慶大霉素(Gentamicin,Gent)屬于氨基糖苷類抗生素藥物,對(duì)革蘭陰性菌和革蘭陽(yáng)性菌有極好的抑制作用,因此,廣泛的應(yīng)用于醫(yī)藥和獸藥領(lǐng)域。然而,慶大霉素對(duì)人類具有明顯的毒害作用,對(duì)腎臟、聽(tīng)覺(jué)以及腦神經(jīng)的損害嚴(yán)重。隨著慶大霉素在國(guó)內(nèi)外醫(yī)療機(jī)構(gòu)、畜牧養(yǎng)殖業(yè)中越來(lái)越廣泛地應(yīng)用,它所帶來(lái)的負(fù)面問(wèn)題日益嚴(yán)重,其殘留檢測(cè)也漸漸被人們所重視,我國(guó)及歐盟均對(duì)慶大霉素的殘留制訂了嚴(yán)格的檢測(cè)標(biāo)準(zhǔn)。因此,開(kāi)發(fā)一種高通量、快速、靈敏的檢測(cè)技術(shù)對(duì)慶大霉素的殘留監(jiān)控及保證動(dòng)物源性食品安全具有重要意義。本研究在總結(jié)前人工作的基礎(chǔ)上,探討了禽源單抗用于抗生素殘留檢測(cè)的可行性并建立了ic-ELISA快速檢測(cè)方法。主要研究?jī)?nèi)容如下:1.慶大霉素(Gent)人工抗原的合成與鑒定根據(jù)慶大霉素的結(jié)構(gòu)特點(diǎn),采用戊二醛法將慶大霉素與載體蛋白(牛血清白蛋白、卵清蛋白)偶聯(lián)。通過(guò)紫外可見(jiàn)分光光度法、SDS-PAGE鑒定偶聯(lián)產(chǎn)物,初步認(rèn)為Gent人工抗原合成成功。2.抗-Gent特異性單鏈抗體的篩選及原核表達(dá)使用免疫原Gent-BSA免疫青年白來(lái)航產(chǎn)蛋雞,待第四次加強(qiáng)免疫后第7天安樂(lè)死產(chǎn)蛋雞,取脾臟并分離脾臟B淋巴細(xì)胞,提取總RNA并反轉(zhuǎn)錄成cDNA,再以cDNA為模板分別擴(kuò)增抗體的輕鏈與重鏈可變區(qū)基因,并經(jīng)Overlap PCR組裝為單鏈抗體(scFv)基因。分別通過(guò)Sfi I/Not I酶切scFv與pCANTAB5E后,將scFv基因片段連接入pCANTAB5E噬菌粒載體,并將重組載體經(jīng)熱激法轉(zhuǎn)化入TG1宿主菌中,構(gòu)建噬菌體展示抗體庫(kù)。經(jīng)4輪固相生物淘選后,使特異性抗體得到有效富集,庫(kù)容分別為:3.7×105、6.7×106、3.71×109和1.65×109。第4輪淘選后,隨機(jī)挑選33個(gè)克隆,進(jìn)行phage-ELISA檢測(cè)。選取2株(S-1和S-5)反應(yīng)性良好的phage-scFv,并將這兩株抗體基因克隆入pET-30a載體,然后將重組載體轉(zhuǎn)化入BL21(DE3)中,誘導(dǎo)表達(dá),發(fā)現(xiàn)重組scFv蛋白主要以包涵體形式存在,將其變性復(fù)性后用于后續(xù)實(shí)驗(yàn)。3.間接競(jìng)爭(zhēng)ELISA(ic-ELISA)檢測(cè)方法的建立通過(guò)優(yōu)化ic-ELISA的工作條件,建立了快速檢測(cè)Gent的ic-ELISA檢測(cè)方法,并通過(guò)交叉反應(yīng)實(shí)驗(yàn)與添加回收實(shí)驗(yàn)評(píng)估該檢測(cè)方法的可行性。結(jié)果顯示:該方法的相關(guān)回歸方程分別為:S-1:y=0.74407-0.23033x(R2=0.9849),S-5:y=0.76066-0.23479x(R2=0.97475),IC50分別為:12.418ng/mL和14.674ng/mL,與Gent類似物(卡那霉素和阿米卡星)的交叉反應(yīng)不超過(guò)0.04%。添加回收檢測(cè)顯示組內(nèi)回收率在70.70%-118.09%之間,組間回收率在60.91%-118.04%之間。以上表明該檢測(cè)方法可行。本研究成功制備了Gent人工抗原,建立了抗Gent的雞源scFv噬菌體抗體庫(kù),并制備了高特異性scFv抗體;并進(jìn)一步建立了ic-ELISA檢測(cè)方法,為開(kāi)發(fā)動(dòng)物源可食性組織中Gent快速檢測(cè)ELISA試劑盒奠定了基礎(chǔ)。同時(shí)證明了禽源單鏈抗體可用于抗生素殘留檢測(cè)。
[Abstract]:Gentamicinine is a kind of aminoglycoside antibiotic, which has excellent inhibitory effect on gram-negative bacteria and gram-positive bacteria, so it is widely used in medicine and veterinary medicine. However, gentamicin has obvious toxic effect on human beings. The damage to kidney, hearing and brain nerve is serious. With the more and more extensive application of gentamicin in domestic and foreign medical institutions and animal husbandry, the negative problems brought by gentamicin are becoming more and more serious, and its residue detection is gradually being paid more and more attention by people. China and the European Union have established strict standards for the detection of gentamicin residues. The sensitive detection technique is of great significance to the residue monitoring of gentamicin and the safety of animal origin food. The feasibility of using avian monoclonal antibody for the detection of antibiotic residues was discussed and a rapid ic-ELISA detection method was established. The main contents of the study were as follows: 1. Synthesis and identification of gentamicin artificial antigen according to the structural characteristics of gentamicin. Gentamycin was coupled with carrier protein (bovine serum albumin, ovalbumin) by glutaraldehyde method. The coupling product was identified by UV-Vis spectrophotometry and SDS-PAGE. The results showed that the synthesis of Gent artificial antigen was successful. 2. Screening and prokaryotic expression of anti-Gent specific single-chain antibodies were used to immunize young Leyahang laying hens with immunogen Gent-BSA, and euthanized laying hens were euthanized on the 7th day after 4th times of intensified immunization. Spleen B lymphocytes were isolated, total RNA was extracted and reversely transcribed into cDNA. then the light chain and heavy chain variable region genes of antibody were amplified using cDNA as template, and the single chain antibody (scFvv) gene was assembled by Overlap PCR. ScFv and pCANTAB5E were digested by Sfi I / Not I enzyme. The scFv gene fragment was ligated into the pCANTAB5E phage vector, and the recombinant vector was transformed into the host strain of TG1 by heat shock method. The phage display antibody library was constructed. After four rounds of solid phase biological panning, the specific antibody was effectively enriched. After the fourth round of panning, 33 clones were randomly selected for phage-ELISA detection. Two strains of phage-scFv with good reactivity were selected and cloned into pET-30a vector, then transformed into BL21DDE3). Induced expression, it was found that the recombinant scFv protein mainly existed in the form of inclusion body. After denaturation and renaturation, the recombinant scFv protein was used in the subsequent experiment .3.The method of indirect competitive ELISA-ELISA-ELISAA detection was established. By optimizing the working conditions of ic-ELISA, the ic-ELISA detection method for rapid detection of Gent was established. The feasibility of the method was evaluated by cross reaction test and adding recovery test. The results showed that the regression equations of the method were: 1: S-1yr 0.74407-0.23033xr2n 0.9849X: S-5: YC 0.76066-0.23479xR2O0.97475IC50 =: 12.418ng/ mL and 14.674ngmL, respectively, with the Gent analogues (kanamycin and amikacin). The cross reaction was not more than 0.04.The recovery rate in the group was between 70.70% and 118.09%, and the recovery rate was between 70.70% and 118.09%. The recoveries between groups ranged from 60.91% to 118.04%, which indicated that the method was feasible. In this study, the artificial antigen of Gent was successfully prepared, the library of scFv phage antibody against Gent was established, and the highly specific scFv antibody was prepared, and the detection method of ic-ELISA was further established. It lays a foundation for the rapid detection of Gent ELISA kit in animal edible tissues and proves that avian scFv can be used for the detection of antibiotic residues.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S859.84

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 LUO Peng Jie;ZHANG Jian Bo;WANG Hua Li;CHEN Xia;WU Nan;ZHAO Yun Feng;WANG Xiao Mei;ZHANG Hong;ZHANG Ji Yue;ZHU Lei;JIANG Wen Xiao;;Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk[J];Biomedical and Environmental Sciences;2016年05期

2 王佳X;孫中遠(yuǎn);劉建新;;酵母細(xì)胞表面展示技術(shù)[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2011年11期

3 職愛(ài)民;李青梅;劉慶堂;劉宣兵;楊繼飛;楊蘇珍;柴書(shū)軍;楊艷艷;鄧瑞廣;張改平;;抗慶大霉素單克隆抗體的制備及其初步應(yīng)用[J];中國(guó)農(nóng)業(yè)科學(xué);2010年12期

4 ;Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen[J];Hepatobiliary & Pancreatic Diseases International;2006年02期

相關(guān)碩士學(xué)位論文 前1條

1 趙津子;氟苯尼考胺IgY抗體的制備及ELISA檢測(cè)方法的建立[D];西北農(nóng)林科技大學(xué);2012年

，

本文編號(hào)：1640194