【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的偶蹄動物易得的一種烈性疾病,對養(yǎng)殖業(yè)造成了巨大的經(jīng)濟損失。隨著生物技術(shù)的發(fā)展,FMD疫苗的研制也從傳統(tǒng)疫苗轉(zhuǎn)向新型疫苗,其中表位疫苗因其呈現(xiàn)出諸多優(yōu)勢而成為疫苗研究的熱點,如能被多種遺傳背景的MHC分子識別并結(jié)合,使抗原得到高效的遞呈,因此在有效的應(yīng)對病原微生物的變異以及免疫反應(yīng)中的諸多不利因素等免疫方面具有獨特的優(yōu)勢。本研究的具體實驗內(nèi)容如下:1、本研究通過對已獲得的嵌入型蛋白SLP-Multi VP1的氨基酸序列,應(yīng)用在線軟件Swiss-Model和Swiss-Pbd Viewer軟件成功對該蛋白三維結(jié)構(gòu)進行預(yù)測,并標(biāo)注了三型FMDV不同表位的位置。2、用已獲得的嵌入型蛋白SLP-Multi VP1與重組蛋白SLP免疫6周齡的昆明小鼠,以PBS作為陰性對照組。小鼠經(jīng)三次免疫后,摘取其脾臟組織分離總淋巴細(xì)胞,按既定方法處理并通過流式細(xì)胞儀分析各免疫組小鼠CD4+T和CD8+T細(xì)胞的變化,試驗結(jié)果顯示:嵌入型蛋白SLP-Multi VP1免疫組的CD4+T與CD8+T數(shù)量高于SLP免疫組及PBS對照組,差異顯著(P0.05)。該結(jié)果提示嵌入型蛋白SLP-Multi VP1可誘導(dǎo)機體產(chǎn)生免疫反應(yīng)。3、將嵌入型蛋白SLP-Multi VP1與重組蛋白SLP免疫6周齡的BALB/c小鼠和昆明小鼠,用間接ELISA方法檢測了三次免疫后各組小鼠的總Ig G水平,試驗結(jié)果顯示,2種小鼠均能產(chǎn)生較高滴度的Ig G(P0.05),該結(jié)果表明嵌入型蛋白SLP-Multi VP1具有良好的抗原性,并且2種小鼠的Ig G水平差異不顯著(P0.05),表明不同種小鼠間無差異性。4、經(jīng)第三次免疫后,采集BALB/c和昆明小鼠的血清,采用Ig G亞型ELISA試劑盒(e Bioscience公司)進行檢測Ig G1和Ig G2a水平,獲得的實驗數(shù)據(jù)通過SPSS統(tǒng)計學(xué)分析,試驗結(jié)果顯示:在兩種免疫動物小鼠模型中,嵌入型蛋白SLP-Multi VP1組Ig G1和Ig G2a水平均高于SLP免疫組,差異顯著(P0.05)。另外,在嵌入型蛋白SLP-Multi VP1免疫組中,Ig G2a的水平高于Ig G1,差異顯著(P0.05)。而在SLP免疫組中,Ig G1和Ig G2a水平差異不顯著(P0.05),該結(jié)果提示嵌入型蛋白SLP-Multi VP1可能傾向于激發(fā)Th1類免疫應(yīng)答。本試驗對各型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礎(chǔ)。
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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