羊传染性脓疱病病毒胶体金快速诊断试纸条的研制

发布时间：2018-05-31 07:54

  本文选题：羊传染性脓疱病毒（ORFV） + 单克隆抗体　； 参考：《吉林大学》2015年硕士论文

【摘要】：羊传染性脓疱病是由羊传染性脓疱病毒（Orf virus, ORFV）感染引起绵羊、山羊等中小反刍动物的一种急性、高度接触性人兽共患传染病。ORFV具有高度的嗜上皮性，主要侵害绵羊或山羊的口腔黏膜以及乳房、阴部等无毛或少毛区域。此外，ORFV具有较强的抵抗力，，羊群一旦被感染很难进行清除，可持续多年对羊群造成危害。目前，用于ORFV检测的方法主要包括有病毒的分离鉴定、电镜负染观察、ELISA、PCR、Real-time PCR、LAMP、间接免疫荧光等方法，对于基层检测而言，这些方法存在有费时、费力、需要专业的实验技术人员及特殊的仪器设备等缺点，因此导致其在临床上的应用受到一定的限制。鉴于此，急需建立一种操作简单、快捷方便的ORFV检测方法用于基层现场检测。 本研究利用蔗糖密度梯度离心法纯化ORFV，免疫Balb/c小鼠，取其脾细胞与SP2/0细胞进行融合，经间接ELISA方法筛选阳性杂交瘤细胞，成功筛选获得3株杂交瘤细胞，分别命名为5A5、6F2、7A3。经ELISA检测，三株单抗的效价在1:12800～1:51200之间；亚型鉴定证实3株单抗均为IgG1；3株杂交瘤细胞染色体条数为92～103条；经ELISA和Western blot分析，3株单坑主要是针对ORFV-B2L蛋白；经间接免疫荧光（IFA）方法鉴定，3株单克隆抗体均能与ORFV结合，特异性良好。此外，成功制备获得兔抗ORFV多克隆抗体。 在成功制备ORFV单抗和多抗的基础上，结合胶体金免疫层析技术建立了用于ORFV检测的胶体金快速诊断试纸法。采用柠檬酸三钠还原法制备胶体金，电镜下观察金颗粒大小在30nm左右；通过pH梯度测定确定胶体金与5A5单抗结合的最佳pH值为8.2；通过蛋白梯度测定最佳结合浓度为40.6μg/mL；用金标单抗包被玻璃纤维膜，分别喷点兔抗ORFV多抗、羊抗小鼠IgG于硝酸纤维膜检测线、质控线，组装试纸条。用组装好的试纸条对不同浓度的ORFV进行检测，结果显示，最低可检测6.25μg/mL的病毒，且与SPPV、VSV、FPV、FMDV等病毒无交叉反应。稳定性检测结果显示，将组装好的试纸条分别放置在4℃和室温条件下，其存放时间均能达到4个月。在成功制备出用于ORFV抗原检测胶体金快速诊断试纸条的基础上，应用其对本实验室搜集保存的165份疑似ORFV感染病料样本进行检测，阳性率为20.6%；将该方法与实验室建立的PCR及LAMP检测方法进行对比分析，符合率分别为94.4%和89.5%。 上述结果显示，本研究所制备的ORFV胶体金快速诊断试纸法具有特异性好、敏感性高、重复性好、稳定性高等优点，并因其具有操作简便、快捷等优点，可被广泛用于基层现场检测，因此对于ORFV的早期诊断具有重要意义。
[Abstract]:Infectious pustular disease in sheep is an acute, highly contact zoonotic infectious disease caused by the infection of sheep infectious pustular virus (ORFV) in small and medium-sized ruminants, such as sheep, goats and other small and medium-sized ruminants. Mainly affecting the oral mucosa of sheep or goats, as well as the mammary, pubic and other hairless or less hair areas. In addition, ORFV has a strong resistance, once the sheep are infected, it is difficult to clear, which can cause harm to sheep for many years. At present, the methods used for ORFV detection mainly include virus isolation and identification, electron microscope negative staining observation of ELISARRTReal-time PCRLamp, indirect immunofluorescence and so on. For basic level detection, these methods are time-consuming and laborious. It needs professional laboratory technicians and special instruments and equipment, so its clinical application is limited. In view of this, it is urgent to establish a simple, fast and convenient ORFV detection method for grass-roots field detection. In this study, ORFV was purified by sucrose density gradient centrifugation, Balb/c mice were immunized, spleen cells were fused with SP2/0 cells, positive hybridoma cells were screened by indirect ELISA method, and three hybridoma cells were successfully selected and named 5A5A5F2F2O7A3. The titer of the three McAbs was between 1: 12800 and 1: 51200 by ELISA, and the chromosome number of the three McAbs were confirmed to be 92 ~ 103.The results of ELISA and Western blot analysis showed that the three McAbs were mainly directed against ORFV-B2L protein, and the results were as follows: (1) by ELISA and Western blot analysis, the titer of the three McAbs was between 1: 12800 and 1: 51200. The McAbs were identified by indirect immunofluorescence assay (IFA) to bind to ORFV, and the specificity was good. In addition, rabbit polyclonal antibody against ORFV was successfully prepared. Based on the successful preparation of ORFV monoclonal antibodies and polyclonal antibodies, a rapid gold diagnostic test paper method for ORFV detection was established by using colloidal gold immunochromatography. Colloidal gold was prepared by tri-sodium citrate reduction method. The size of gold particles was observed to be about 30nm under electron microscope. The best pH value for the binding of colloidal gold to 5A5 McAb was determined to be 8.2 by pH gradient determination, the optimum binding concentration was 40.6 渭 g / mL by protein gradient determination, and the glass fiber membrane was coated with gold-labeled McAb. Sheep anti-mouse IgG in nitric acid fiber membrane detection line, quality control line, assembly test strip. The results showed that the virus of 6.25 渭 g/mL could be detected at least, and there was no cross reaction with the virus such as SPPVV VSVV, FPVV, FPV-FMDV and so on. The test strip was used to detect the virus of different concentration. The results showed that the virus of 6.25 渭 g/mL could be detected at the lowest level. The stability test results show that the stored time of the assembled test strip can reach 4 months at 4 鈩

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