FMDV各型表位嵌入型蛋白SLP-Multi VP1的表位预测及不同种小鼠IgG亚型变化的影响

发布时间：2019-03-08 17:59

【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的偶蹄动物易得的一种烈性疾病,对养殖业造成了巨大的经济损失。随着生物技术的发展,FMD疫苗的研制也从传统疫苗转向新型疫苗,其中表位疫苗因其呈现出诸多优势而成为疫苗研究的热点,如能被多种遗传背景的MHC分子识别并结合,使抗原得到高效的递呈,因此在有效的应对病原微生物的变异以及免疫反应中的诸多不利因素等免疫方面具有独特的优势。本研究的具体实验内容如下:1、本研究通过对已获得的嵌入型蛋白SLP-Multi VP1的氨基酸序列,应用在线软件Swiss-Model和Swiss-Pbd Viewer软件成功对该蛋白三维结构进行预测,并标注了三型FMDV不同表位的位置。2、用已获得的嵌入型蛋白SLP-Multi VP1与重组蛋白SLP免疫6周龄的昆明小鼠,以PBS作为阴性对照组。小鼠经三次免疫后,摘取其脾脏组织分离总淋巴细胞,按既定方法处理并通过流式细胞仪分析各免疫组小鼠CD4+T和CD8+T细胞的变化,试验结果显示:嵌入型蛋白SLP-Multi VP1免疫组的CD4+T与CD8+T数量高于SLP免疫组及PBS对照组,差异显著(P0.05)。该结果提示嵌入型蛋白SLP-Multi VP1可诱导机体产生免疫反应。3、将嵌入型蛋白SLP-Multi VP1与重组蛋白SLP免疫6周龄的BALB/c小鼠和昆明小鼠,用间接ELISA方法检测了三次免疫后各组小鼠的总Ig G水平,试验结果显示,2种小鼠均能产生较高滴度的Ig G(P0.05),该结果表明嵌入型蛋白SLP-Multi VP1具有良好的抗原性,并且2种小鼠的Ig G水平差异不显著(P0.05),表明不同种小鼠间无差异性。4、经第三次免疫后,采集BALB/c和昆明小鼠的血清,采用Ig G亚型ELISA试剂盒(e Bioscience公司)进行检测Ig G1和Ig G2a水平,获得的实验数据通过SPSS统计学分析,试验结果显示:在两种免疫动物小鼠模型中,嵌入型蛋白SLP-Multi VP1组Ig G1和Ig G2a水平均高于SLP免疫组,差异显著(P0.05)。另外,在嵌入型蛋白SLP-Multi VP1免疫组中,Ig G2a的水平高于Ig G1,差异显著(P0.05)。而在SLP免疫组中,Ig G1和Ig G2a水平差异不显著(P0.05),该结果提示嵌入型蛋白SLP-Multi VP1可能倾向于激发Th1类免疫应答。本试验对各型FMDV VP1基因表位嵌入型蛋白SLP-Multi VP1免疫特性更深入的研究奠定了基础。
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is a severe disease of cloven-hoofed animals caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV). With the development of biotechnology, the development of FMD vaccine has changed from traditional vaccine to new vaccine. Epitope vaccine has become a hot spot for vaccine research because of its many advantages, for example, it can be recognized and combined by MHC molecules from a variety of genetic backgrounds. The antigen can be presented efficiently, so it has unique advantages in effectively dealing with the variation of pathogenic microorganisms and many unfavorable factors in immune response. The main contents of this study are as follows: 1. The three-dimensional structure of the embedded protein SLP-Multi VP1 was predicted successfully by using the on-line software Swiss-Model and Swiss-Pbd Viewer, and the amino acid sequence of the embedded protein was successfully predicted by using the on-line software Swiss-Model and Swiss-Pbd Viewer. The positions of different epitopes of three types of FMDV were labeled. 2. Kunming mice were immunized with the obtained chimeric protein SLP-Multi VP1 and recombinant protein SLP at the age of 6 weeks, and PBS was used as the negative control group. After three times of immunization, the total lymphocytes were isolated from the spleen tissue of the mice, and the changes of CD4 T and CD8 T cells were analyzed by flow cytometry according to the established method. The results showed that the number of CD4-T and CD8-T in SLP-Multi VP1-immunized group was higher than that in SLP-immunized group and PBS-control group, and the difference was significant (P0.05). The results suggested that the chimeric protein SLP-Multi VP1 could induce immune responses. 3. The chimeric protein SLP-Multi VP1 and recombinant protein SLP were immunized in 6-week-old BALB/c mice and Kunming mice. The total Ig G level of three groups of mice was detected by indirect ELISA method. The results showed that both mice could produce high titers of Ig G (P0.05). The results showed that the chimeric protein SLP-Multi VP1 had good antigenicity, and the results showed that the chimeric protein SLP-Multi VP1 had good antigenicity, and the results showed that the chimeric protein SLP-Multi VP1 had good antigenicity. There was no significant difference in the level of Ig G between the two kinds of mice (P0.05). 4. After the third immunization, the serum of BALB/c and Kunming mice were collected. The levels of Ig G1 and Ig G2a were detected by Ig G subtype ELISA kit (e Bioscience (Inc.). The experimental data were analyzed by SPSS statistics. The results showed that in the two immune animal models, the results of the experiment showed that in the two immune animal models, the levels of Ig G1 and Ig G2a were measured. The levels of Ig G1 and Ig G2a in SLP-Multi VP1 group were significantly higher than those in SLP group (P0.05). In addition, the level of Ig G2a in SLP-Multi VP1 immunized group was significantly higher than that in Ig G1 group (P0.05). However, there was no significant difference in Ig G1 and Ig G2a levels between SLP immunized group and Ig G2a group (P0.05). The results suggested that SLP-Multi VP1, an embedded protein, might tend to stimulate Th1-like immune responses. This study laid a foundation for further study on the immune characteristics of the chimeric protein SLP-Multi VP1 of each type of FMDV VP1 gene epitope.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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