抗庆大霉素禽源单链抗体的筛选及间接竞争ELISA检测方法的建立

发布时间：2018-03-20 18:01

本文选题：庆大霉素　切入点：人工抗原　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：庆大霉素(Gentamicin,Gent)属于氨基糖苷类抗生素药物,对革兰阴性菌和革兰阳性菌有极好的抑制作用,因此,广泛的应用于医药和兽药领域。然而,庆大霉素对人类具有明显的毒害作用,对肾脏、听觉以及脑神经的损害严重。随着庆大霉素在国内外医疗机构、畜牧养殖业中越来越广泛地应用,它所带来的负面问题日益严重,其残留检测也渐渐被人们所重视,我国及欧盟均对庆大霉素的残留制订了严格的检测标准。因此,开发一种高通量、快速、灵敏的检测技术对庆大霉素的残留监控及保证动物源性食品安全具有重要意义。本研究在总结前人工作的基础上,探讨了禽源单抗用于抗生素残留检测的可行性并建立了ic-ELISA快速检测方法。主要研究内容如下:1.庆大霉素(Gent)人工抗原的合成与鉴定根据庆大霉素的结构特点,采用戊二醛法将庆大霉素与载体蛋白(牛血清白蛋白、卵清蛋白)偶联。通过紫外可见分光光度法、SDS-PAGE鉴定偶联产物,初步认为Gent人工抗原合成成功。2.抗-Gent特异性单链抗体的筛选及原核表达使用免疫原Gent-BSA免疫青年白来航产蛋鸡,待第四次加强免疫后第7天安乐死产蛋鸡,取脾脏并分离脾脏B淋巴细胞,提取总RNA并反转录成cDNA,再以cDNA为模板分别扩增抗体的轻链与重链可变区基因,并经Overlap PCR组装为单链抗体(scFv)基因。分别通过Sfi I/Not I酶切scFv与pCANTAB5E后,将scFv基因片段连接入pCANTAB5E噬菌粒载体,并将重组载体经热激法转化入TG1宿主菌中,构建噬菌体展示抗体库。经4轮固相生物淘选后,使特异性抗体得到有效富集,库容分别为:3.7×105、6.7×106、3.71×109和1.65×109。第4轮淘选后,随机挑选33个克隆,进行phage-ELISA检测。选取2株(S-1和S-5)反应性良好的phage-scFv,并将这两株抗体基因克隆入pET-30a载体,然后将重组载体转化入BL21(DE3)中,诱导表达,发现重组scFv蛋白主要以包涵体形式存在,将其变性复性后用于后续实验。3.间接竞争ELISA(ic-ELISA)检测方法的建立通过优化ic-ELISA的工作条件,建立了快速检测Gent的ic-ELISA检测方法,并通过交叉反应实验与添加回收实验评估该检测方法的可行性。结果显示:该方法的相关回归方程分别为:S-1:y=0.74407-0.23033x(R2=0.9849),S-5:y=0.76066-0.23479x(R2=0.97475),IC50分别为:12.418ng/mL和14.674ng/mL,与Gent类似物(卡那霉素和阿米卡星)的交叉反应不超过0.04%。添加回收检测显示组内回收率在70.70%-118.09%之间,组间回收率在60.91%-118.04%之间。以上表明该检测方法可行。本研究成功制备了Gent人工抗原,建立了抗Gent的鸡源scFv噬菌体抗体库,并制备了高特异性scFv抗体;并进一步建立了ic-ELISA检测方法,为开发动物源可食性组织中Gent快速检测ELISA试剂盒奠定了基础。同时证明了禽源单链抗体可用于抗生素残留检测。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.84

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