發(fā)布時間：2018-09-06 12:25

【摘要】：傳染性法氏囊病毒(IBDV)會導致雞發(fā)生嚴重的免疫抑制疾病,從而引起巨大的經(jīng)濟損失。自然條件下,IBDV廣泛存在于周圍環(huán)境中并且可以通過呼吸進行傳播。IBDV感染的靶器官主要是法氏囊,會引起法氏囊腫大和出血等癥狀,使機體的免疫力下降,對其它病原因子的抵抗力下降,從而繼發(fā)感染多種疾病。IBDV抗原結(jié)合表位是病毒分子感染宿主細胞的主要部位,也是藥物治療的靶序列。如果能找到正確的病毒表位,在IBDV感染宿主之前,采取相應措施切斷病毒的感染途徑,就能起到很好的保護作用。 本實驗室根據(jù)已經(jīng)報道的VP2蛋白的三維晶體結(jié)構(gòu),合成了VP2蛋白的五段序列,發(fā)現(xiàn)其中一條多肽P22可能是IBDV的抗原表位。方法本次實驗將P22多肽進行了原核表達,發(fā)現(xiàn)其主要以包涵體形式存在。用鎳柱純化帶His標簽的P22多肽,純化后的P22多肽放入透析袋中透析復性。SDS-PAGE檢驗多肽的純化程度,Western blot檢測P22多肽的特性。用IBD抗原快速檢測試紙條驗證Western blot結(jié)果。15日齡的SPF雞頸部皮下接種P22多肽,二免后取P22抗血清。瓊脂擴散實驗和中和效價測定P22抗血清的特異性和保護細胞能力。結(jié)果SDS-PAGE結(jié)果表明獲得了比較純的P22多肽。Western blot結(jié)果表明P22多肽可以和IBDV單抗上清、IBDV標準陽性血清起特異反應,和IBDV陰性血清不反應。P22多肽可以與IBD快速檢測試紙條發(fā)生特異反應,試紙條的控制線和檢測線均顯示紅色條帶。瓊脂擴散實驗顯示P22抗血清和IBDV強毒株可以形成明顯的免疫沉淀線,IBDV強毒株和IBDV單抗上清之間也有清晰的免疫沉淀線。隨機選擇3份P22抗血清,在Vero細胞上檢測中和效價,結(jié)果分別是1：640,1：650和1：620。結(jié)論P22是IBDV VP2蛋白上的抗原決定簇,P22是VP2的線性B細胞抗原表位。
[Abstract]:Infectious bursal virus (IBDV) can lead to severe immunosuppressive disease in chickens and cause huge economic losses. Under natural conditions, IBDV is widely present in the surrounding environment and can be transmitted through respiration. The target organ of IBDV infection is mainly the bursa of Fabricius, which can cause symptoms such as bursal enlargement and bleeding and decrease the immunity of the body. IBDV antigen-binding epitopes are the main sites of virus molecules infecting host cells and are also the target sequences of drug therapy because of the decrease of resistance to other pathogenic factors and the secondary infection of many diseases with IBDV antigen binding epitopes. If we can find the correct epitope of the virus and take appropriate measures to cut off the infection pathway of the virus before IBDV infects the host, it can play a good protective role. According to the three-dimensional crystal structure of VP2 protein, we synthesized the five-segment sequence of VP2 protein, and found that one of the polypeptides P22 may be the epitope of IBDV. Methods the prokaryotic expression of P22 polypeptide was found to be mainly in the form of inclusion body. P22 polypeptide with His label was purified by nickel column. The purified P22 peptide was dialyzed in dialysis bag. SDS-PAGE was used to detect the purification degree of P22 polypeptide. Western blot was used to detect the characteristics of P22 polypeptide. P22 peptide was subcutaneously inoculated on the neck of 15-day-old SPF chicken with IBD antigen test strip. P22 antiserum was obtained after two immunizations. Agar diffusion assay and neutralization titer were used to determine the specificity and cell protection ability of P22 antiserum. Results the results of SDS-PAGE showed that P22 polypeptide. Western blot showed that P22 peptide could react specifically with IBDV McAb supernatant IBDV standard positive serum and IBDV negative serum. P22 polypeptide could react with IBD rapid test strip. Test strip control line and test line are shown in red strip. Agar diffusion test showed that P22 antiserum and IBDV virulent strain could form a clear immunoprecipitation line between IBDV virulent strain and IBDV monoclonal antibody supernatant. Three samples of P22 antiserum were randomly selected and neutralized in Vero cells. The results were 1: 64040: 650 and 1: 620, respectively. Conclusion P22 is an antigenic determinant of IBDV VP2 protein and a linear B cell epitope of VP2.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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相關期刊論文 前2條

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2 張改平,李學伍,楊艷艷,鄧瑞廣,李青梅;雞傳染性法氏囊病快速診斷試紙條的研制及特性測定[J];畜牧獸醫(yī)學報;2004年01期

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本文編號：2226346