抗口蹄疫病毒单克隆抗体的制备和抗原模拟表位的筛选

发布时间：2018-06-03 21:37

本文选题：口蹄疫病毒 + 单克隆抗体　；参考：《安徽农业大学》2008年硕士论文

【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动物的急性、热性、高度接触性传染病。口蹄疫病毒是小RNA病毒科口蹄疫病毒属的唯一成员,不含囊膜的病毒粒子包含有一个单链正股RNA基因组。目前OIE将其划分为七个血清型,分别命名为O、A、C、Asia1、SAT1、SAT2和SAT3。然而,研究者通过血清学试验发现,口蹄疫七个血清型的分离株存在广泛的抗原变异,每个血清型都含有众多亚型。在发展中国家,控制口蹄疫的关键是快速准确地诊断和使用有效的疫苗。因此,研发快速诊断试剂和高效疫苗,对口蹄疫的防控具有十分重要的现实意义。单克隆抗体有着特异性强、重复性好的特点,可开发成诊断试剂及应用于各项基础研究。为制备抗O型口蹄疫病毒特异性单克隆抗体,用纯化的O型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA筛选和间接免疫荧光(IFA)验证,三次有限稀释法克隆,获得了3株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为289、5F7和8E8,抗体亚类鉴定其均为IgG1/κ类型;间接ELISA测定,3株杂交瘤细胞培养上清效价分别为1:256、1:512和1:512,小鼠腹水效价分别为1:8×10~3、1:1.6×10~4和1:1.6×10~4;型特异性检测结果显示,三株单克隆抗体仅与O型FMDV反应,不与Asia1型FMDV反应,推测它们均为抗O型FMDV的血清型特异性单克隆抗体;Western blot分析表明,单克隆抗体289和5F7与O型FMDV均没有特异性反应条带出现,表明它们所针对的抗原表位均为构象型表位;单克隆抗体8E8与病毒抗原有特异性反应条带,所以它识别线性表位。相加ELISA试验表明,289与5F7两株单克隆抗体识别的抗原表位相近或相同,但均不同于单克隆抗体8E8的识别表位;中和试验显示单克隆抗体8E8具有很强的中和活性,中和效价为1:1024。经硫氰酸盐洗脱法测定,289和5F7的相对亲和力指数均为1.5mol/L,8E8的相对亲和力指数为2.5mol/L。这3株单克隆抗体的获得,为研究O型口蹄疫病毒提供了重要的实验材料。抗原表位,又称抗原决定簇,是抗原分子抗原性的基础。正确而详细地绘制抗原表位图谱对疾病的诊断、设计无毒副作用的多表位疫苗以及免疫治疗试剂等具有积极的意义。为研究Asia1型口蹄疫病毒的抗原表位性质,对本实验室制备的一株抗Asia1型口蹄疫病毒单克隆抗体3E11的识别表位进行鉴定。3E11为一株识别构象型表位的具有很强中和活性的单克隆抗体,利用噬菌体随机十二肽库对单克隆抗体3E11进行4轮淘选,前三轮采用固相吸附法淘洗,最后一轮淘洗采用蛋白G液相吸附法;经噬菌体ELISA鉴定,获得8个阳性噬菌体克隆,这些克隆与单克隆抗体3E11均发生特异性反应;序列测定及比对显示,5个噬菌体克隆的展示肽含有一致基序GSLxxL(x代表任意氨基酸),所以单克隆抗体3E11识别抗原表位的基序为GSLxxL,为单克隆抗体3E11识别的模拟表位;合成含基序的一种十二肽和该十二肽的随机打乱肽,肽ELISA显示该十二肽具有结合单克隆抗体3E11的活性,而随机打乱肽不能结合单克隆抗体3E11;分别合成肽对基序的氨基酸残基进行定点突变显示,这些氨基酸残基对于单克隆抗体3E11识别表位的抗原活性均为关键性的;肽竞争ELISA显示,该模拟表位能够抑制单克隆抗体3E11与病毒抗原的结合。总之,本研究成功制备了3株针对O型FMDV的单克隆抗体,并且鉴定了Asia1型FMDV的一个模拟表位,这些研究将为口蹄疫病毒新型疫苗研制和诊断试剂开发奠定基础。
[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.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】