【摘要】： 在病毒免疫過程中,不僅需要體液免疫,而且需要細胞免疫。如在新城疫的免疫方面,細胞免疫與體液免疫協(xié)同發(fā)揮作用。Ghumman(1976)等發(fā)現(xiàn),接種新城疫活苗和滅活苗后2-3天,就能檢測到淋巴細胞內(nèi)DNA合成增加。Agrawal(1991)等用白細胞游走抑制試驗來評價接種ND疫苗后細胞免疫反應,發(fā)現(xiàn)接種后確實存在細胞免疫反應。機體針對新城疫的細胞免疫和體液免疫之間究竟是怎么變化的呢?為什么經(jīng)常出現(xiàn)免疫失敗呢?這些問題沒有明確的答案。 酶聯(lián)免疫斑點檢測技術(shù)(ELISPOT)結(jié)合了細胞培養(yǎng)技術(shù)與酶聯(lián)免疫吸附技術(shù)(ELISA)的長處,能夠分析經(jīng)特異抗原活化后分泌細胞因子(如IFN-γ、TNF-α等)的單個效應細胞的頻數(shù),具有敏感、特異、易于重復的優(yōu)點。本研究將ELISPOT技術(shù)應用于新城疫細胞免疫的研究,通過對脾淋巴細胞濃度,培養(yǎng)時間,抗原刺激量進行選擇,建立了檢測NDV誘導特異性IFN-γ、IL-4的ELISPOT方法。結(jié)果顯示,試驗的特異性與培養(yǎng)時間和刺激抗原密切相關,在一定范圍內(nèi),培養(yǎng)時間越長、刺激抗原越多,試驗的特異性越低,當抗原量為5ug/孔,培養(yǎng)時間為7h時,試驗的特異性最強。ELISPOT斑點數(shù),隨細胞濃度的增加而增加,當小鼠的脾淋巴細胞濃度為106/孔時,最有利于斑點的計數(shù),試驗誤差最小。用建立的ELISPOT方法對NDV免疫小鼠的IFN-γ、Il-4水平進行檢測發(fā)現(xiàn),IFN-γ、Il-4與血清抗體的產(chǎn)生存在著一定的時差關系,在血清抗體陽性檢出的前期,IFN-γ的分泌降至最低水平,Il-4升至最高水平。
[Abstract]:In the process of virus immunization, not only humoral immunity, but also cellular immunity is needed. For example, in the immunity aspect of Newcastle disease, the synergistic effect of cellular immunity and humoral immunity on. Ghumman (1976 was found, 2-3 days after inoculation of live and inactivated Newcastle disease vaccine, The leukocyte migration inhibition test was used to evaluate the cellular immune response after inoculation of ND vaccine. It was found that there was a cellular immune response after inoculation. How on earth does the cellular and humoral immunity of the body change in response to Newcastle disease? Why do you often fail to get immunized? There are no clear answers to these questions. (ELISPOT) combines the advantages of cell culture and enzyme linked immunosorbent assay (ELISA) to analyze cytokines (such as IFN- 緯) secreted by specific antigens. The frequency of single effector cells of TNF- 偽 is sensitive, specific and easy to repeat. In this study, ELISPOT technique was applied to the study of cellular immunity of Newcastle disease (NDV). By selecting the concentration of splenic lymphocytes, culture time and the amount of antigen stimulation, a ELISPOT method for detecting the specific IFN- 緯 and IL-4 induced by NDV was established. The results showed that the specificity of the test was closely related to the culture time and the stimulation antigen. In a certain range, the longer the culture time was, the more the stimulating antigen was, the lower the specificity of the test was. When the amount of antigen was 5ug/ pore, the culture time was 7 h. The specificity of the experiment was the strongest. The number of ELISPOT spots increased with the increase of cell concentration. When the concentration of spleen lymphocytes in mice was 106 / well, the number of spots was the most favorable, and the error of the experiment was the least. The levels of IFN- 緯 and Il-4 in mice immunized with NDV were detected by using the established ELISPOT method. It was found that there was a time-difference relationship between IFN- 緯, Il-4 and the production of serum antibodies. The secretion of IFN- 緯 decreased to the lowest level and Il-4 to the highest level.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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