本文選題：羊傳染性膿皰病毒（ORFV） + 單克隆抗體��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：羊傳染性膿皰病是由羊傳染性膿皰病毒（Orf virus, ORFV）感染引起綿羊、山羊等中小反芻動(dòng)物的一種急性、高度接觸性人獸共患傳染病。ORFV具有高度的嗜上皮性，主要侵害綿羊或山羊的口腔黏膜以及乳房、陰部等無(wú)毛或少毛區(qū)域。此外，ORFV具有較強(qiáng)的抵抗力，，羊群一旦被感染很難進(jìn)行清除，可持續(xù)多年對(duì)羊群造成危害。目前，用于ORFV檢測(cè)的方法主要包括有病毒的分離鑒定、電鏡負(fù)染觀察、ELISA、PCR、Real-time PCR、LAMP、間接免疫熒光等方法，對(duì)于基層檢測(cè)而言，這些方法存在有費(fèi)時(shí)、費(fèi)力、需要專業(yè)的實(shí)驗(yàn)技術(shù)人員及特殊的儀器設(shè)備等缺點(diǎn)，因此導(dǎo)致其在臨床上的應(yīng)用受到一定的限制。鑒于此，急需建立一種操作簡(jiǎn)單、快捷方便的ORFV檢測(cè)方法用于基層現(xiàn)場(chǎng)檢測(cè)。 本研究利用蔗糖密度梯度離心法純化ORFV，免疫Balb/c小鼠，取其脾細(xì)胞與SP2/0細(xì)胞進(jìn)行融合，經(jīng)間接ELISA方法篩選陽(yáng)性雜交瘤細(xì)胞，成功篩選獲得3株雜交瘤細(xì)胞，分別命名為5A5、6F2、7A3。經(jīng)ELISA檢測(cè)，三株單抗的效價(jià)在1:12800～1:51200之間；亞型鑒定證實(shí)3株單抗均為IgG1；3株雜交瘤細(xì)胞染色體條數(shù)為92～103條；經(jīng)ELISA和Western blot分析，3株單坑主要是針對(duì)ORFV-B2L蛋白；經(jīng)間接免疫熒光（IFA）方法鑒定，3株單克隆抗體均能與ORFV結(jié)合，特異性良好。此外，成功制備獲得兔抗ORFV多克隆抗體。 在成功制備ORFV單抗和多抗的基礎(chǔ)上，結(jié)合膠體金免疫層析技術(shù)建立了用于ORFV檢測(cè)的膠體金快速診斷試紙法。采用檸檬酸三鈉還原法制備膠體金，電鏡下觀察金顆粒大小在30nm左右；通過(guò)pH梯度測(cè)定確定膠體金與5A5單抗結(jié)合的最佳pH值為8.2；通過(guò)蛋白梯度測(cè)定最佳結(jié)合濃度為40.6μg/mL；用金標(biāo)單抗包被玻璃纖維膜，分別噴點(diǎn)兔抗ORFV多抗、羊抗小鼠IgG于硝酸纖維膜檢測(cè)線、質(zhì)控線，組裝試紙條。用組裝好的試紙條對(duì)不同濃度的ORFV進(jìn)行檢測(cè)，結(jié)果顯示，最低可檢測(cè)6.25μg/mL的病毒，且與SPPV、VSV、FPV、FMDV等病毒無(wú)交叉反應(yīng)。穩(wěn)定性檢測(cè)結(jié)果顯示，將組裝好的試紙條分別放置在4℃和室溫條件下，其存放時(shí)間均能達(dá)到4個(gè)月。在成功制備出用于ORFV抗原檢測(cè)膠體金快速診斷試紙條的基礎(chǔ)上，應(yīng)用其對(duì)本實(shí)驗(yàn)室搜集保存的165份疑似ORFV感染病料樣本進(jìn)行檢測(cè)，陽(yáng)性率為20.6%；將該方法與實(shí)驗(yàn)室建立的PCR及LAMP檢測(cè)方法進(jìn)行對(duì)比分析，符合率分別為94.4%和89.5%。 上述結(jié)果顯示，本研究所制備的ORFV膠體金快速診斷試紙法具有特異性好、敏感性高、重復(fù)性好、穩(wěn)定性高等優(yōu)點(diǎn)，并因其具有操作簡(jiǎn)便、快捷等優(yōu)點(diǎn)，可被廣泛用于基層現(xiàn)場(chǎng)檢測(cè)，因此對(duì)于ORFV的早期診斷具有重要意義。
[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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