鹿黏膜病免疫膠體金診斷試紙條的研制及應用
發(fā)布時間：2018-07-31 15:50
【摘要】：鹿黏膜病(Deer mucosal disease,DMD)是由牛病毒性腹瀉/黏膜病病毒(Bovine virus diarrhea/mucosal disease virus,BVD/MDV)引起的一種以懷孕母鹿流產(chǎn)、產(chǎn)死胎或畸形胎、仔鹿嚴重腹瀉為主要特征的多臨床癥狀傳染病。目前,該疾病在國內(nèi)廣泛流行,嚴重影響?zhàn)B鹿業(yè)的健康發(fā)展。現(xiàn)階段,對于鹿黏膜病的診斷方法主要包括:病原的分離鑒定、瓊脂擴散試驗、中和試驗、酶聯(lián)免疫吸附試驗、聚合酶鏈式反應等。以上這些檢測方法均存在檢出時間長、檢測需要專業(yè)的實驗室條件及相關實驗人員和檢測儀器設備等條件,不利于基層臨床樣品的快速檢測。因此,急需建立一種簡便、快捷、特異、現(xiàn)場應用的鹿黏膜病診斷方法。本實驗首先通過Bac-to-Bac表達系統(tǒng),表達出鹿源BVD/MDV的保護性抗原E2蛋白,經(jīng)Western Blot分析、直接免疫熒光檢測表明該蛋白具有良好的抗原性。將純化的E2蛋白免疫Balb/c小鼠,經(jīng)細胞融合,陽性雜交瘤細胞篩選、亞克隆,成功篩選出2株穩(wěn)定分泌單克隆抗體的雜交瘤細胞株,分別命名為1F11和1B11,收集腹水并純化獲得2株單抗1F11和1B11,經(jīng)Western Blot和間接免疫熒光檢測表明所制備的2株單抗均能夠與E2蛋白和鹿源BVD/MDV發(fā)生特異性的反應。采用檸檬酸鈉還原法制備20nm膠體金溶液,通過優(yōu)化膠體金試紙條的各項參數(shù),最終確定膠體金顆粒與1F11單抗結(jié)合的最佳p H為7,最佳的單抗結(jié)合濃度為14.4μg/m L,并將1F11單抗標記在20nm膠體金分子上作為金標抗體,1B11單抗作為檢測線抗體(濃度1.5mg/m L),羊抗鼠Ig G作為質(zhì)控線抗體(濃度1mg/m L)。選用聚酯纖維素膜(RB65)和玻璃纖維塑膜(DL68)組合,最終制備了鹿黏膜病免疫膠體金診斷試紙條。實驗表明,該試紙條具有良好的特異性,對于BVD/MDV病原的檢測靈敏度可達103TCID50/m L。應用免疫膠體金試紙條和傳統(tǒng)的RT-PCR法共同檢測50份采集于吉林某鹿場仔鹿腹瀉的糞便樣品,均檢測出陽性樣品10份,且兩種方法檢測結(jié)果一致。研究結(jié)果表明,本實驗制備的免疫膠體金診斷試紙條可作為一種快速、靈敏、特異的診斷方法應用于鹿黏膜病的臨床現(xiàn)場病原檢測,該方法的研制豐富了國內(nèi)外鹿黏膜病的現(xiàn)場診斷手段,對于該傳染病的快速診斷、流行病學調(diào)查和種鹿群的凈化具有重要的臨床應用價值。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.25
本文編號：2156063
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.25
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