鹿黏膜病免疫胶体金诊断试纸条的研制及应用

发布时间：2018-07-31 15:50

【摘要】：鹿黏膜病(Deer mucosal disease,DMD)是由牛病毒性腹泻/黏膜病病毒(Bovine virus diarrhea/mucosal disease virus,BVD/MDV)引起的一种以怀孕母鹿流产、产死胎或畸形胎、仔鹿严重腹泻为主要特征的多临床症状传染病。目前,该疾病在国内广泛流行,严重影响养鹿业的健康发展。现阶段,对于鹿黏膜病的诊断方法主要包括:病原的分离鉴定、琼脂扩散试验、中和试验、酶联免疫吸附试验、聚合酶链式反应等。以上这些检测方法均存在检出时间长、检测需要专业的实验室条件及相关实验人员和检测仪器设备等条件,不利于基层临床样品的快速检测。因此,急需建立一种简便、快捷、特异、现场应用的鹿黏膜病诊断方法。本实验首先通过Bac-to-Bac表达系统,表达出鹿源BVD/MDV的保护性抗原E2蛋白,经Western Blot分析、直接免疫荧光检测表明该蛋白具有良好的抗原性。将纯化的E2蛋白免疫Balb/c小鼠,经细胞融合,阳性杂交瘤细胞筛选、亚克隆,成功筛选出2株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为1F11和1B11,收集腹水并纯化获得2株单抗1F11和1B11,经Western Blot和间接免疫荧光检测表明所制备的2株单抗均能够与E2蛋白和鹿源BVD/MDV发生特异性的反应。采用柠檬酸钠还原法制备20nm胶体金溶液,通过优化胶体金试纸条的各项参数,最终确定胶体金颗粒与1F11单抗结合的最佳p H为7,最佳的单抗结合浓度为14.4μg/m L,并将1F11单抗标记在20nm胶体金分子上作为金标抗体,1B11单抗作为检测线抗体(浓度1.5mg/m L),羊抗鼠Ig G作为质控线抗体(浓度1mg/m L)。选用聚酯纤维素膜(RB65)和玻璃纤维塑膜(DL68)组合,最终制备了鹿黏膜病免疫胶体金诊断试纸条。实验表明,该试纸条具有良好的特异性,对于BVD/MDV病原的检测灵敏度可达103TCID50/m L。应用免疫胶体金试纸条和传统的RT-PCR法共同检测50份采集于吉林某鹿场仔鹿腹泻的粪便样品,均检测出阳性样品10份,且两种方法检测结果一致。研究结果表明,本实验制备的免疫胶体金诊断试纸条可作为一种快速、灵敏、特异的诊断方法应用于鹿黏膜病的临床现场病原检测,该方法的研制丰富了国内外鹿黏膜病的现场诊断手段,对于该传染病的快速诊断、流行病学调查和种鹿群的净化具有重要的临床应用价值。
[Abstract]:Deer mucosal disease (Deer mucosal) is a multi-clinical disease characterized by pregnant female abortion, stillbirth or abnormal fetus, and severe diarrhea of deer, which is caused by bovine viral diarrhea / mucosal disease virus BVD / MDV). At present, the disease is widespread in China, seriously affecting the healthy development of deer industry. At present, the diagnostic methods of deer mucosal disease mainly include: isolation and identification of pathogen, Agar diffusion test, neutralization test, enzyme-linked immunosorbent assay, polymerase chain reaction and so on. All these methods have long detection time and need professional laboratory conditions and related laboratory personnel and testing instruments and so on, which is not conducive to rapid detection of clinical samples at the grass-roots level. Therefore, it is urgent to establish a simple, rapid, specific and field diagnosis method for deer mucosal disease. In this experiment, the Bac-to-Bac expression system was used to express E2 protein, the protective antigen of venison origin BVD/MDV. The Western Blot analysis and direct immunofluorescence analysis showed that the protein had good antigenicity. After immunizing Balb/c mice with purified E2 protein, two hybridoma cell lines stably secreting monoclonal antibodies were successfully screened by cell fusion, positive hybridoma cell selection and subcloning. They were named as 1F11 and 1B11 respectively. Two McAbs, 1F11 and 1B11, were collected and purified from ascites. Western Blot and indirect immunofluorescence assay showed that the two McAbs could react specifically with E2 protein and Luyuan BVD/MDV. 20nm colloidal gold solution was prepared by sodium citrate reduction method. The parameters of colloidal gold test strip were optimized. Finally, the best binding concentration of colloidal gold particles to 1F11 McAbs was determined to be 7, and the best binding concentration of 1F11 McAbs was 14.4 渭 g / mL. 1F11 McAbs were labeled on the colloidal gold molecules of 20nm as gold labeled monoclonal antibodies (1.5mg/m L),) as detection line antibodies (concentration of 1.5mg/m L),). Sheep anti-mouse IgG as a quality control line antibody (concentration of 1mg/m L).) The combination of polyester cellulose membrane (RB65) and glass fiber plastic film (DL68) was used to prepare the gold diagnostic test strip for animal mucosal disease. The results showed that the test strip had good specificity and the sensitivity of detecting BVD/MDV pathogen was as high as that of 103TCID50/m L. The immune colloidal gold strip and the traditional RT-PCR method were used to detect 50 fecal samples from deer faecal diarrhea in a certain deer farm in Jilin province. 10 positive samples were detected, and the results were consistent with the two methods. The results showed that the immuno-colloidal gold diagnostic strips prepared in this experiment could be used as a rapid, sensitive and specific diagnostic method for the detection of clinical pathogens of deer mucosal diseases. The development of this method enriches the field diagnosis methods of deer mucosal disease at home and abroad, and has important clinical application value for the rapid diagnosis of the infectious disease, epidemiological investigation and purification of breeding deer herd.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.25

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