本文選題：口蹄疫病毒 + 單克隆抗體　； 參考：《安徽農(nóng)業(yè)大學》2008年碩士論文

【摘要】： 口蹄疫(FMD)是由口蹄疫病毒(FMDV)感染引起的主要侵害偶蹄動物的急性、熱性、高度接觸性傳染病�？谔阋卟《臼切NA病毒科口蹄疫病毒屬的唯一成員,不含囊膜的病毒粒子包含有一個單鏈正股RNA基因組。目前OIE將其劃分為七個血清型,分別命名為O、A、C、Asia1、SAT1、SAT2和SAT3。然而,研究者通過血清學試驗發(fā)現(xiàn),口蹄疫七個血清型的分離株存在廣泛的抗原變異,每個血清型都含有眾多亞型。在發(fā)展中國家,控制口蹄疫的關鍵是快速準確地診斷和使用有效的疫苗。因此,研發(fā)快速診斷試劑和高效疫苗,對口蹄疫的防控具有十分重要的現(xiàn)實意義。 單克隆抗體有著特異性強、重復性好的特點,可開發(fā)成診斷試劑及應用于各項基礎研究。為制備抗O型口蹄疫病毒特異性單克隆抗體,用純化的O型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾細胞與SP2/0骨髓瘤細胞進行融合,經(jīng)間接ELISA篩選和間接免疫熒光(IFA)驗證,三次有限稀釋法克隆,獲得了3株穩(wěn)定分泌單克隆抗體的雜交瘤細胞株,分別命名為289、5F7和8E8,抗體亞類鑒定其均為IgG1/κ類型;間接ELISA測定,3株雜交瘤細胞培養(yǎng)上清效價分別為1:256、1:512和1:512,小鼠腹水效價分別為1:8×10~3、1:1.6×10~4和1:1.6×10~4;型特異性檢測結(jié)果顯示,三株單克隆抗體僅與O型FMDV反應,不與Asia1型FMDV反應,推測它們均為抗O型FMDV的血清型特異性單克隆抗體;Western blot分析表明,單克隆抗體289和5F7與O型FMDV均沒有特異性反應條帶出現(xiàn),表明它們所針對的抗原表位均為構象型表位;單克隆抗體8E8與病毒抗原有特異性反應條帶,所以它識別線性表位。相加ELISA試驗表明,289與5F7兩株單克隆抗體識別的抗原表位相近或相同,但均不同于單克隆抗體8E8的識別表位;中和試驗顯示單克隆抗體8E8具有很強的中和活性,中和效價為1:1024。經(jīng)硫氰酸鹽洗脫法測定,289和5F7的相對親和力指數(shù)均為1.5mol/L,8E8的相對親和力指數(shù)為2.5mol/L。這3株單克隆抗體的獲得,為研究O型口蹄疫病毒提供了重要的實驗材料。 抗原表位,又稱抗原決定簇,是抗原分子抗原性的基礎。正確而詳細地繪制抗原表位圖譜對疾病的診斷、設計無毒副作用的多表位疫苗以及免疫治療試劑等具有積極的意義。為研究Asia1型口蹄疫病毒的抗原表位性質(zhì),對本實驗室制備的一株抗Asia1型口蹄疫病毒單克隆抗體3E11的識別表位進行鑒定。3E11為一株識別構象型表位的具有很強中和活性的單克隆抗體,利用噬菌體隨機十二肽庫對單克隆抗體3E11進行4輪淘選,前三輪采用固相吸附法淘洗,最后一輪淘洗采用蛋白G液相吸附法;經(jīng)噬菌體ELISA鑒定,獲得8個陽性噬菌體克隆,這些克隆與單克隆抗體3E11均發(fā)生特異性反應;序列測定及比對顯示,5個噬菌體克隆的展示肽含有一致基序GSLxxL(x代表任意氨基酸),所以單克隆抗體3E11識別抗原表位的基序為GSLxxL,為單克隆抗體3E11識別的模擬表位;合成含基序的一種十二肽和該十二肽的隨機打亂肽,肽ELISA顯示該十二肽具有結(jié)合單克隆抗體3E11的活性,而隨機打亂肽不能結(jié)合單克隆抗體3E11;分別合成肽對基序的氨基酸殘基進行定點突變顯示,這些氨基酸殘基對于單克隆抗體3E11識別表位的抗原活性均為關鍵性的;肽競爭ELISA顯示,該模擬表位能夠抑制單克隆抗體3E11與病毒抗原的結(jié)合。 總之,本研究成功制備了3株針對O型FMDV的單克隆抗體,并且鑒定了Asia1型FMDV的一個模擬表位,這些研究將為口蹄疫病毒新型疫苗研制和診斷試劑開發(fā)奠定基礎。
[Abstract]:Foot and mouth disease (FMD) is an acute, thermal, and highly contagious disease caused by foot and mouth disease virus (FMDV) infection. Foot and mouth disease virus is the only member of the foot-and-mouth disease virus in the small RNA virus family. The virus free particles containing the capsule contain a single strand positive strand RNA genome. At present, OIE divides it into seven serotypes. Do not name O, A, C, Asia1, SAT1, SAT2 and SAT3., however, the researchers found that the seven serotype isolates of foot-and-mouth disease have extensive antigen variations, each serotype contains a large number of subtypes. In developing countries, the key to the control of foot and mouth disease is fast and accurate diagnosis and use of effective vaccines. Diagnostic reagents and highly effective vaccines play a very important role in the prevention and control of foot and mouth disease.
The monoclonal antibody has the characteristics of strong specificity and good repeatability. It can be developed into a diagnostic reagent and applied to various basic studies. To prepare specific monoclonal antibodies against O type foot-and-mouth disease virus and immunize BALB/c mice with purified O type FMD virus, the immunized mice spleen cells and SP2 /0 myeloma cells are fused by indirect ELISA screening. And indirect immunofluorescence (IFA) verification, three hybridoma cells were cloned by finite dilution method, and 3 hybridoma cells, which secreted monoclonal antibodies, were named 289,5F7 and 8E8 respectively. The antibody subgroups were identified as IgG1/ kappa type, and indirect ELISA assay, 3 hybridoma cells were cultured at 1:256,1:512 and 1:512 respectively, and Mice Ascites titer scores were respectively. 1:8 * 10~3,1:1.6 x 10~4 and 1:1.6 x 10~4 showed that three monoclonal antibodies were only reactive with O type FMDV, not with Asia1 type FMDV, and they were all anti O FMDV serotype specific monoclonal antibodies. It is shown that the epitopes they aim at are all conformation epitopes; the monoclonal antibody 8E8 has a specific reactive band with the virus antigen, so it recognizes the linear epitope. The addition of ELISA test shows that the epitopes of 289 and two 5F7 monoclonal antibodies are similar or identical, but are different from the identification epitopes of the monoclonal antibody 8E8; neutralization test The monoclonal antibody 8E8 has strong neutralization activity, and the neutralization titer is 1:1024. by thiocyanate elution. The relative affinity index of 289 and 5F7 is 1.5mol/L, and the relative affinity index of 8E8 is 3 monoclonal antibodies of 2.5mol/L., which provides an important experimental material for the study of O type foot-and-mouth disease virus.
Antigen epitope, also known as antigenic determinant, is the basis of antigenicity of antigen molecules. It is of positive significance to correctly and carefully plot antigenic epitopes for diagnosis of disease, to design nontoxic side effects and immunotherapy reagents. For the study of the epitope properties of Asia1 type foot blight, the laboratory is prepared in this laboratory. The identification epitope of monoclonal antibody 3E11 of Asia1 type FMDV was identified as a strong neutralizing monoclonal antibody that identified conformation epitopes of.3E11. The monoclonal antibody 3E11 was selected by the phage random twelve peptide library. The first three rounds were washed by solid phase adsorption and the last round was washed with protein G solution. 8 positive phage clones were identified by phage ELISA, and these clones were all specific to the monoclonal antibody 3E11. Sequence determination and comparison showed that the display peptides of 5 phage clones contained the uniform sequence GSLxxL (x representing arbitrary amino acids), and the sequence of the antigen epitopes identified by the monoclonal antibody 3E11 was GSLxxL, The mimic epitopes identified for the monoclonal antibody 3E11, a twelve peptide containing the basic sequence and the random disorder peptide of the twelve peptide, the peptide ELISA shows that the twelve peptide has the activity of binding to the monoclonal antibody 3E11, and the random disorder peptide can not combine with the monoclonal antibody 3E11; Some amino acid residues are key to the antigen activity of the monoclonal antibody 3E11 recognition epitopes, and the peptide competitive ELISA shows that the mimic epitope can inhibit the combination of monoclonal antibody 3E11 and viral antigen.
In conclusion, 3 monoclonal antibodies against O FMDV were successfully prepared and an analog epitope of Asia1 type FMDV was identified. These studies will lay the foundation for the development of a new type of FMDV vaccine and the development of diagnostic reagents.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【引證文獻】

相關碩士學位論文 前1條

1 姜志剛;FMDV中和表位在HBc上的展示及其嵌合蛋白在重組腺病毒中的表達[D];中國農(nóng)業(yè)科學院;2010年

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本文編號：1974255