A型口蹄疫病毒结构蛋白VP1单克隆抗体的制备及初步应用

发布时间：2018-05-16 11:53

  本文选题：A型口蹄疫病毒 + 结构蛋白VP1　； 参考：《中国农业科学院》2010年硕士论文

【摘要】： 口蹄疫(Foot-and-mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus, FMDV)所引起的一种偶蹄动物的急性高度接触性传染病,被世界动物卫生组织(OIE)列为A类传染病之首,给流行国家和地区的政治、经济和社会问题带来了不利影响,而单克隆抗体以其特异性强、重复性好、稳定性高的优点已成为口蹄疫诊断和研究的焦点。 本研究利用重组菌pGEM-VP1为模板成功克隆出VP1基因,将其连接到表达载体上诱导表达出融合蛋白。可溶性分析表明表达的融合蛋白是以包涵体的形式存在,超声裂解菌体后利用镍离子亲和层析的方法进行蛋白纯化,SDS-PAGE分析显示得到了较纯的融合蛋白,并以此作为免疫原免疫6周龄的雌性BALB/c小鼠,加强免疫后取其脾脏和骨髓瘤细胞(Sp2/0)进行融合,通过间接ELISA法初步筛选,有限稀释法进行亚克隆,最终获得了4株能稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为7B11、7H11、8G2、8H4。利用SDS-PAGE、间接ELISA法、Western blot分析和间接免疫荧光法进行了生物学特性的分析和鉴定。实验结果表明获得的4株单抗具有较高的抗体效价和特异性,抗体亚类鉴定结果显示,7B11和7H11均属于IgG1,8H4和8G2属于IgG2a,杂交瘤细胞的染色体数目均在98-108条之间,辛酸-饱和硫酸铵法纯化的单抗只有抗体的重链和轻链两条链,大小分别为50ku和25ku,相对亲和力为8G28H47H117B11。 用纯化的单抗与乳胶致敏,初步建立了快速检测A型FMDV抗原的方法,对致敏抗体浓度、致敏时间和温度等条件进行优化。确定包被单抗(7B11,7H11)的稀释倍数为1:40,37℃致敏4h能达到较好的实验效果。特异性试验显示,该方法与O型、Asia1型FMDV病毒的反应均呈阴性。 本研究成功制备了针对A型FMDV的单克隆抗体,并对其生物学特性进行了分析和鉴定,在此基础上建立了反向乳胶凝集法检测A型口蹄疫病毒抗原的方法,这为口蹄疫的诊断和研究提供了平台。
[Abstract]:Foot-and-mouth disease (FMD) is an acute highly contact infectious disease of cloven-hoofed animals caused by foot-and-mouth Disease Virus, FMDV). It is listed as the first class A infectious disease by the World Organization of Animal Health (OIE), and it is given to the politics of endemic countries and regions. Economic and social problems have brought adverse effects, and monoclonal antibodies have become the focus of diagnosis and research of foot-and-mouth disease because of their strong specificity, good reproducibility and high stability. In this study, the recombinant strain pGEM-VP1 was used as the template to clone the VP1 gene and ligated to the expression vector to induce the expression of the fusion protein. Soluble analysis showed that the expressed fusion protein existed in the form of inclusion body. The purified protein was purified by nickel ion affinity chromatography after ultrasonic lysis, and a pure fusion protein was obtained by SDS-PAGE analysis. Female BALB/c mice of 6 weeks old were immunized by this method. The spleen and myeloma cell line Sp2 / 0 were harvested after enhanced immunization and then subcloned by indirect ELISA method, and then subcloned by limited dilution method. Finally, four hybridoma cell lines which can secrete monoclonal antibody stably were obtained and named as 7B117H117H118G2O8H4. The biological characteristics were analyzed and identified by SDS-PAGE, indirect ELISA blot analysis and indirect immunofluorescence assay. The results showed that the antibody titers and specificity of the four McAbs obtained were higher. The results of antibody subclass identification showed that both IgG1B11 and 8G2 belonged to IgG2a, and the chromosome number of hybridoma cells ranged from 98-108. The monoclonal antibody purified by octanoic acid-saturated ammonium sulfate method had only two chains of antibody, heavy chain and light chain, with the size of 50ku and 25ku. the relative affinity was 8G28H47H117B11. A method for rapid detection of type A FMDV antigen was established by sensitizing the purified McAb with latex. The sensitized antibody concentration, sensitizing time and temperature were optimized. It was determined that the dilution ratio of the coated McAb was 1: 4037 鈩，

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