【摘要】：雞傳染性支氣管炎（Infectious bronchitis, IB）是由IBV感染引起的急性炎癥疾病，具有高度傳染性。IBV血清型眾多，變異頻率高，，IBV的組織嗜性也在不斷變化，目前國際公認(rèn)的有呼吸型、腎型、產(chǎn)蛋下降型、腺胃型及腸型。最近幾年國內(nèi)外都有對IBV組織嗜性的研究報(bào)道，但結(jié)論各異。本研究嘗試性地通過比較呼吸型與腎型不同致病性毒株S1蛋白的差異，來研究能否篩出有特異性中和活性的單克隆抗體，以探討不同IBV毒株間致病性與組織嗜性的差異性。 本實(shí)驗(yàn)利用濃縮的IBV H120和K136病毒分別免疫6周齡的雌性BALB/c小鼠，采用傳統(tǒng)雜交瘤技術(shù)，建立間接ELISA檢測方法，經(jīng)篩選克隆后共獲得了三株單克隆抗體雜交瘤細(xì)胞系，分別為抗IBV H120的3F6G7株和抗IBV K136的1E9A3株、4F5E8株。單克隆抗體亞型鑒定結(jié)果顯示3F6G7株為IgG2b亞型，1E9A3和4F5E8株為IgM亞型，輕鏈均為Kappa。ELISA結(jié)果表明3F6G7與H120的反應(yīng)比與K136的反應(yīng)強(qiáng)烈，而1E9A3、4F5E8則沒有這種差異；這三株單抗都可與其他IBV毒株發(fā)生反應(yīng)，說明其針對的抗原表位比較保守，具有群特異性。Western blot試驗(yàn)結(jié)果表明這三株單抗都特異性識別IBV S1蛋白。病毒中和試驗(yàn)結(jié)果顯示，僅3F6G7株單抗具有中和活性，但3F6G7沒有表現(xiàn)出ELISA試驗(yàn)中的差異性。 本研究雖未能成功篩選出鑒別性單克隆抗體，仍可為以后IBV組織嗜性的研究提供參考與借鑒。另外，獲得的三株單抗仍可作為防治、檢測與診斷IBV的候選工具以及研究S1蛋白抗原表位的有力素材，具有實(shí)踐與研究價(jià)值。
[Abstract]:Avian infectious bronchitis (Infectious bronchitis, IB) is an acute inflammatory disease caused by IBV infection. Egg-laying type, glandular stomach type and intestinal type. In recent years, there have been reports of IBV tissue tropism at home and abroad, but the conclusions are different. The purpose of this study was to study whether monoclonal antibodies with specific neutralizing activity could be screened by comparing the differences of S1 protein between respiratory and renal virulence strains in order to explore the differences of pathogenicity and tissue tropism among different IBV strains. In this study, female BALB/c mice aged 6 weeks were immunized with concentrated IBV H120 and K136 viruses respectively. Using traditional hybridoma technique, indirect ELISA detection method was established, and three monoclonal antibody hybridoma cell lines were obtained after screening and cloning. 3F6G7 strains resistant to IBV H120 and 1E9A3 strains resistant to IBV K136 were strain 4F5E8, respectively. The results of monoclonal antibody subtype identification showed that 3F6G7 strain was IgG2b subtype 1 E9A3 and 4F5E8 strain was IgM subtype. The results of light chain Kappa.ELISA showed that the reaction ratio of 3F6G7 to H120 was stronger than that of K136, but 1E9A3O4F5E8 had no such difference. All of the three McAbs could react with other IBV strains, indicating that the antigenic epitopes were conserved. The results of group specificity. Western blot test showed that all three McAbs specifically recognized IBV S1 protein. The results of neutralization test showed that only 3F6G7 McAb had neutralization activity, but 3F6G7 showed no difference in ELISA test. Although this study failed to screen the discriminant monoclonal antibody successfully, it can be used as reference for the study of IBV tissue tropism in the future. In addition, the obtained McAbs can be used as a candidate tool for the prevention and treatment, detection and diagnosis of IBV, as well as a powerful material for the study of S1 antigen epitopes, which has practical and research value.
【學(xué)位授予單位】：天津農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31

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