坦布蘇病毒NS5蛋白單克隆抗體的制備與鑒定及快速檢測膠體金試紙條方法的建立
發(fā)布時間：2018-11-25 11:12
【摘要】：2010年春夏之交,我國江浙和山東等地區(qū)均報道了以一種引起鴨產(chǎn)蛋率大幅下降、生長發(fā)育遲緩、卵巢出血為主要癥狀的急性傳染病,之后廣泛傳播到我國的大部分地區(qū),并給蛋鴨的養(yǎng)殖業(yè)造成了很大的經(jīng)濟(jì)損失。感染鴨群主要表現(xiàn)為采食量和產(chǎn)蛋量的急速下降,依照發(fā)病的日齡不同,死亡率為5%~10%之間。經(jīng)過國內(nèi)多個試驗室的診斷和病毒的分離,可以確定該病是由黃病毒引起的,隸屬黃病毒屬(Flavivirus)、恩塔亞病毒群(Ntaya virus group)的鴨坦布蘇病毒(duck Tembusu virus,DTMUV)。研究坦布蘇病毒蛋白的結(jié)構(gòu)功能及建立快速檢測方法,是控制該病的有效措施。本研究建立了快速檢測坦布蘇病毒的膠體金檢測方法,成功地制備了抗坦布蘇病毒NS5蛋白的單克隆抗體,為防治該病提供了技術(shù)支持。本研究內(nèi)容分為以下兩個部分:1.坦布蘇病毒NS5蛋白單克隆抗體的制備本研究利用本試驗室構(gòu)建的PET-28a+NS5原核表達(dá)載體,成功轉(zhuǎn)化入大腸桿菌BL21細(xì)胞,并純化產(chǎn)生的坦布蘇病毒NS5蛋白。利用純化的NS5蛋白免疫6-8周齡的BALB/c小鼠,經(jīng)過細(xì)胞融合、間接ELISA篩選和有限稀釋亞克隆法獲得了3株穩(wěn)定分泌針對坦布蘇病毒NS5蛋白的單克隆抗體A4D1、B4B8和C12D2,并對A4D1和B4B8進(jìn)行相關(guān)特性鑒定。經(jīng)ELISA效價測定,兩株單抗的細(xì)胞上清效價均為1:100,誘導(dǎo)產(chǎn)生腹水的效價分別為1:128000和1:64000。間接ELISA結(jié)果表明,兩株單抗只與DTMUV反應(yīng),無交叉反應(yīng)性。Western blot和IFA結(jié)果表明,兩株單抗均可與體外純化的NS5蛋白以及坦布蘇病毒發(fā)生特異性反應(yīng)。抗坦布蘇病毒NS5蛋白單克隆抗體的成功研制,為坦布蘇病毒的免疫學(xué)診斷及致病機理的研究奠定基礎(chǔ)。2.坦布蘇病毒膠體金檢測試紙條方法的建立利用試驗室保存的抗坦布蘇病毒E蛋白單克隆抗體A12D3為金標(biāo)抗體,制備的鼠抗E蛋白多克隆抗體為包被抗體,建立了雙抗體夾心膠體金試紙條檢測坦布蘇病毒的方法。用該試紙條可特異性檢測坦布蘇病毒,在10~15 min即可觀察檢測結(jié)果。利用建立的膠體金試紙條的方法和RT-PCR對50份臨床樣品進(jìn)行檢測比較,結(jié)果顯示兩者符合率為93.8%。該試紙條重復(fù)性好,特異性高,可用于坦布蘇病毒的臨床快速診斷。
[Abstract]:At the turn of spring and summer in 2010, Jiangsu, Zhejiang, Shandong and other regions in China reported an acute infectious disease characterized by a sharp drop in duck laying rate, stunted growth and ovarian hemorrhage, and then spread widely to most areas of China. And to the egg duck breeding industry caused great economic losses. The main characteristics of infected ducks were the rapid decrease of feed intake and egg production. According to the age of the infected ducks, the mortality rate was between 510% and 10%. It was confirmed that the disease was caused by yellow virus, which belonged to (Ntaya virus group) of (Flavivirus), Entavirus group (duck Tembusu virus,DTMUV). To study the structure and function of Tambusuvirus protein and to establish a rapid detection method are effective measures to control the disease. In this study, a colloidal gold assay for rapid detection of TBV was established, and monoclonal antibodies against TBV NS5 protein were successfully prepared, which provided technical support for the prevention and treatment of TBV. The content of this study is divided into the following two parts: 1. Preparation of Monoclonal Antibody against Tambusuvirus NS5 protein in this study the PET-28a NS5 prokaryotic expression vector constructed in our laboratory was successfully transformed into Escherichia coli BL21 cells and the resulting NS5 protein was purified. The purified NS5 protein was used to immunize 6-8 week-old BALB/c mice. Through cell fusion, indirect ELISA screening and limited dilution subcloning, three strains of monoclonal antibodies A4D1B4B8 and C12D2 secreted against NS5 protein of TBV were obtained. The related characteristics of A4D1 and B4B8 were identified. The titer of supernatant of the two McAbs was 1: 100, and the titer of induced ascites was 1: 128000 and 1: 64000, respectively. Indirect ELISA results showed that the two McAbs reacted only with DTMUV, while the non-cross-reactive. Western blot and IFA showed that the two McAbs could react specifically with the purified NS5 protein and Tambusuvirus in vitro. The successful development of monoclonal antibody against NS5 protein of TBV lays a foundation for the immunological diagnosis and pathogenesis of TBV. 2. The Establishment of TBV Colloidal Gold Test Strip the monoclonal antibody (A12D3) against tambusuvirus E protein was used as gold labeled antibody and the polyclonal antibody of mouse anti-E protein was coated antibody. A double antibody sandwich colloidal gold test strip for detection of tambusuvirus was established. The test strip can be used to detect tambusuvirus specifically, and the results can be observed at 10 ~ 15 min. 50 clinical samples were detected and compared by using the method of colloidal gold test strip and RT-PCR. The results showed that the coincidence rate of the two methods was 93.884%. The test strip has good reproducibility and specificity and can be used for rapid clinical diagnosis of TBV.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：2355898
[Abstract]:At the turn of spring and summer in 2010, Jiangsu, Zhejiang, Shandong and other regions in China reported an acute infectious disease characterized by a sharp drop in duck laying rate, stunted growth and ovarian hemorrhage, and then spread widely to most areas of China. And to the egg duck breeding industry caused great economic losses. The main characteristics of infected ducks were the rapid decrease of feed intake and egg production. According to the age of the infected ducks, the mortality rate was between 510% and 10%. It was confirmed that the disease was caused by yellow virus, which belonged to (Ntaya virus group) of (Flavivirus), Entavirus group (duck Tembusu virus,DTMUV). To study the structure and function of Tambusuvirus protein and to establish a rapid detection method are effective measures to control the disease. In this study, a colloidal gold assay for rapid detection of TBV was established, and monoclonal antibodies against TBV NS5 protein were successfully prepared, which provided technical support for the prevention and treatment of TBV. The content of this study is divided into the following two parts: 1. Preparation of Monoclonal Antibody against Tambusuvirus NS5 protein in this study the PET-28a NS5 prokaryotic expression vector constructed in our laboratory was successfully transformed into Escherichia coli BL21 cells and the resulting NS5 protein was purified. The purified NS5 protein was used to immunize 6-8 week-old BALB/c mice. Through cell fusion, indirect ELISA screening and limited dilution subcloning, three strains of monoclonal antibodies A4D1B4B8 and C12D2 secreted against NS5 protein of TBV were obtained. The related characteristics of A4D1 and B4B8 were identified. The titer of supernatant of the two McAbs was 1: 100, and the titer of induced ascites was 1: 128000 and 1: 64000, respectively. Indirect ELISA results showed that the two McAbs reacted only with DTMUV, while the non-cross-reactive. Western blot and IFA showed that the two McAbs could react specifically with the purified NS5 protein and Tambusuvirus in vitro. The successful development of monoclonal antibody against NS5 protein of TBV lays a foundation for the immunological diagnosis and pathogenesis of TBV. 2. The Establishment of TBV Colloidal Gold Test Strip the monoclonal antibody (A12D3) against tambusuvirus E protein was used as gold labeled antibody and the polyclonal antibody of mouse anti-E protein was coated antibody. A double antibody sandwich colloidal gold test strip for detection of tambusuvirus was established. The test strip can be used to detect tambusuvirus specifically, and the results can be observed at 10 ~ 15 min. 50 clinical samples were detected and compared by using the method of colloidal gold test strip and RT-PCR. The results showed that the coincidence rate of the two methods was 93.884%. The test strip has good reproducibility and specificity and can be used for rapid clinical diagnosis of TBV.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
【引證文獻(xiàn)】
相關(guān)會議論文 前1條
1 張琳;胡北俠;張秀美;;4株鴨坦布蘇病毒包膜蛋白基因的分子進(jìn)化分析及其表達(dá)[A];中國畜牧獸醫(yī)學(xué)會獸醫(yī)公共衛(wèi)生學(xué)分會第三次學(xué)術(shù)研討會論文集[C];2012年
，本文編號：2355898
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