水貂阿留申病检测抗原的制备及间接ELISA检测方法的建立

发布时间：2018-04-15 01:16

本文选题：水貂阿留申病 + B细胞表位　；参考：《吉林大学》2015年硕士论文

【摘要】：水貂阿留申病（Aleutian disease，AD）是由水貂阿留申病毒（Aleutian diseasevirus，ADV）引起的自身免疫性、传染性疾病，水貂感染ADV产生的抗体不但不能有效清除病毒，反而通过Fc受体介导的抗体依赖性增强作用（AntibodyDependent Enhancement, ADE）使病毒感染增强，导致水貂免疫系统功能紊乱。该病不仅导致貂群的繁殖能力下降，，而且在一定程度上能引起水貂死亡，给养貂业带来了巨大的经济损失，也严重阻碍我国毛皮动物养殖业发展。目前尚无有效疫苗治疗该病，因此国内外主要通过抗体检测技术如对流免疫电泳（Counterimmune electrophoresis，CIEP）、酶联免疫吸附实验（Enzyme-linked immunosorbentassay，ELISA）筛选并淘汰阳性水貂，从而控制和净化该病。本研究目的在于建立有效的水貂阿留申病间接ELISA检测方法。以水貂阿留申病美国株ADV-G结构蛋白VP2为主要参考，通过B细胞抗原表位预测和保守序列筛选，获得保守性高的优势B细胞抗原表位SD序列。通过大肠杆菌表达系统诱导表达，获得了可溶性SD蛋白，大小为20KDa。表达产物经镍柱纯化、超滤管浓缩后获得高纯度、高浓度的SD蛋白。Western Blot分析结果显示，SD蛋白具有良好的反应原性。将该蛋白作为CIEP检测抗原与商业CIEP抗原蛋白同时检测水貂血清，达到一致的检测结果。将获得的抗原SD蛋白和三氨丙基三乙氧基硅烷（3-Aminopropyltriethoxysilane，APTES）共同包被建立间接ELISA检测方法。通过方阵滴定法，摸索抗原SD蛋白和APTES的包被浓度，并摸索了包被和封闭时间，以及一抗和二抗的稀释度，最终确定包被的抗原SD蛋白和APTES的浓度分别为10ug/mL和1%，一抗稀释度为1:100，二抗稀释度为1:3000，其中与传统间接ELISA比较，包被时间、封闭时间均缩短为30min。特异性试验表明建立的该方法的SD蛋白仅能被水貂阿留申病毒抗体识别，而与犬细小病毒、伪狂犬病毒、犬瘟热病毒、水貂肠炎病毒的阳性血清和水貂阿留申病阴性血清均无交叉反应；敏感性试验表明建立的该方法的敏感性高于CIEP；重复性试验表明批内重复性和批间重复性的变异系数分别为0.612%~5.956%和0.135%~7.614%，均小于10%；临床样品检测结果与CIEP检测结果的符合率为93.2%。通过以上实验得出结论：本实验中通过设计获得的全新的人工抗原SD蛋白具有良好的反应原性和作为检测抗原的能力。将SD蛋白作为包被抗原蛋白建立的间接ELISA检测方法具有特异性好、敏感性高、重复性好、高效快捷的特点，且与CIEP检测方法比较具有较高的符合率。本研究不仅为常用检测方法CIEP提供更合适的基因工程抗原蛋白，也为开发检测水貂阿留申病的ELISA试剂盒及全自动ELISA检测方法提供基础研究。
[Abstract]:Aleutian disease of mink (Aleutian disease ADV) is caused by Aleutian disease virus of mink (Aleutian disease virus ADV).On the contrary, Antibody Dependent Enhancement (ADE), mediated by FC receptor, enhanced the virus infection and resulted in the disorder of the immune system of mink.The disease not only leads to the decline of the breeding ability of mink, but also can cause the death of mink to a certain extent, which brings huge economic loss to mink industry, and seriously hinders the development of fur animal breeding industry in China.At present, there is no effective vaccine for the treatment of the disease, so antibody detection techniques such as counter immune electrophoresis and Enzyme-linked immunosorbent enzyme linked immunosorbent assay (Elisa) are used to screen and eliminate positive minks in order to control and purify the disease.The aim of this study was to establish an effective indirect ELISA method for detection of Aleutian disease in mink.The dominant B cell epitope SD sequence was obtained by predication of B cell epitopes and screening of conserved sequences with reference to the ADV-G structural protein VP2 of Aleutian disease American strain of mink.The expression of soluble SD protein was induced by Escherichia coli expression system and the size of SD protein was 20 K Da.The expressed product was purified by nickel column and concentrated in ultrafiltration tube to obtain high purity. Western Blot analysis of high concentration of SD protein showed that SD protein had good reactivity.Using this protein as CIEP detection antigen and commercial CIEP antigen protein, the mink serum was detected simultaneously, and the results were consistent.An indirect ELISA method was established by co-coating the obtained antigen SD protein with triaminopropyltriethoxysilane trisilane 3-Aminopropyltriethoxysilaneus (APTES).The encapsulation concentration of antigenic SD protein and APTES, the encapsulation and blocking time, and the dilution of the first antibody and the second antibody were studied by the method of square array titration.Finally, the concentration of antigen SD protein and APTES were determined to be 10ug/mL and 1, respectively. The dilution of the first antibody was 1: 100, and the dilution of the second antibody was 1: 3000. Compared with the traditional indirect ELISA, the encapsulation time and the blocking time were shortened to 30 mins.The specific test showed that the SD protein of this method can only be recognized by mink Aleutian virus antibody, but with canine parvovirus, pseudorabies virus, canine distemper virus,There was no cross reaction between the positive serum of mink enteritis virus and the negative serum of mink Aleutin disease.The sensitivity test showed that the sensitivity of this method was higher than that of CIEP, the coefficient of variation of repeatability within and between batches was 0.6125.956% and 0.135% 7.614, respectively, and the coincidence rate between the results of clinical samples and that of CIEP was 93. 2%.It is concluded from the above experiments that the new artificial antigen SD protein obtained in this experiment has good reactivity and ability to detect antigens.The indirect ELISA detection method using SD protein as coated antigen protein has the characteristics of good specificity, high sensitivity, good reproducibility, high efficiency and rapidity, and has a higher coincidence rate compared with CIEP detection method.This study not only provides a more suitable genetic engineering antigen protein for common detection method CIEP, but also provides basic research for the development of ELISA kit and automatic ELISA detection method for Aleutian disease in mink.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92

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