新型布鲁氏菌疫苗的构建及免疫效果的初步研究

发布时间：2018-02-26 05:21

本文关键词： 布鲁氏菌外膜蛋白 DNA疫苗重组蛋白苗细胞免疫体液免疫　出处：《内蒙古农业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 寻找具有免疫原性的外膜蛋白是构建布鲁氏菌病亚单位疫苗、DNA疫苗和细菌活载体疫苗的关键。本研究利用在线生物学软件PSORT和CELLO对布鲁氏菌16M菌株基因组序列进行分析,预测其外膜蛋白基因序列。选取17个外膜蛋白基因及核蛋白L7/L12进行PCR扩增并与原核表达载体pET32a(+)连接,转入E.coli BL21(DE3)感受态细胞;IPTG诱导蛋白表达,通过SDS-PAGE分析及Western blot鉴定目的蛋白的表达;经HisTrapTMHP纯化,制备重组蛋白疫苗。同时将上述18个基因片段和hGM-CSF基因,与真核表达质粒PVAX1连接,转染MDCK细胞,通过间接免疫荧光证实各目的蛋白在细胞中的表达,制备DNA疫苗。用候选疫苗免疫Balb/c小鼠,通过间接ELISA、ELISPOT、FCM技术对其免疫效果进行评价。结果表明,利用PSORT和CELLO进行预测共得到31个外膜蛋白。利用原核表达系统,成功构建了18个重组质粒,其中13个分子获得大量表达,以可溶形式存在的是BMEI0402、BMEI0454、BMEII0983、BMEI0653、BMEI1777、BMEI1980、BMEII0381、BMEII0472,包涵体形式存在的包括BMEI1830、BMEI1305、BMEI1306、BMEII1120。选取在上清中获得大量表达的BMEII0983、BMEI0653、BMEI1777、BMEI1980、BMEII0381、BMEII0472及少量表达的BMEI1829进行纯化,纯化后蛋白纯度达90%以上。成功构建了18个真核表达重组质粒,转染MDCK细胞,有12个获得了表达。用制备的BMEII0381、BMEII0472、BMEI0653、BMEI0748、BMEI1777、BMEI1980的DNA疫苗和重组蛋白苗,免疫Balb/c小鼠,产生了较强的免疫应答。DNA疫苗激发以细胞免疫为主的免疫应答,ELISPOT分析显示特异性IFN-γ分泌细胞的数量要高于IL-4分泌细胞的数量,抗体亚型分类结果表明小鼠IgG2a与IgG1之比1,FCM分析CD4+/CD8+值较阴性对照组减少。而重组蛋白及DNA蛋白混合疫苗则以体液免疫为主,特异性IFN-γ分泌细胞的数量要低于IL-4分泌细胞的数量,小鼠的IgG1水平远远高于IgG2a水平,小鼠CD4+/CD8+值较阴性对照组增加。制备的候选疫苗能够刺激机体产生较强的细胞和体液免疫应答,其中BMEI1980和BMEI1777较其他蛋白免疫原性强。本研究的实验结果为基于疫苗研究策略的有效预防布鲁氏菌病的深入研究奠定了基础。
[Abstract]:Searching for immunogenicity outer membrane protein is the key to construct brucellosis subunit vaccine DNA vaccine and bacterial live vector vaccine. In this study, PSORT and CELLO were used to analyze the genomic sequence of Brucella 16M strain. 17 outer membrane protein genes and nucleoprotein L7 / L12 were amplified by PCR and ligated with prokaryotic expression vector pET32a (). The expression of the target protein was identified by SDS-PAGE analysis and Western blot, and the recombinant protein vaccine was prepared by HisTrapTMHP purification. At the same time, the 18 gene fragments and hGM-CSF gene were ligated with the eukaryotic expression plasmid PVAX1 and transfected into MDCK cells. DNA vaccine was prepared by indirect immunofluorescence assay, and Balb/c mice were immunized with candidate vaccine, and the immunological effect was evaluated by indirect ELISA-ELISPOTC-FCM technique. The results showed that 31 outer membrane proteins were obtained by using PSORT and CELLO, and 18 recombinant plasmids were successfully constructed using prokaryotic expression system, 13 of which were expressed in large quantities. BMEI0402BMEI0454, BMEI0983BMEI0653BMEI17777 BMEI1980 BMEII0381BMEII04772, including BMEI1830BMEI1305BMEI1306BMEII1120. BMEII0983BMEI1777BMEI1980BMEI1777BMEI1980BME0381BMEII040472 and a small amount of BMEIIII0472 were purified, and 18 recombinant eukaryotic expression plasmids were constructed successfully. After transfection of MDCK cells, 12 of them were expressed. Balb/c mice were immunized with the DNA vaccine and recombinant protein vaccine BMEI1777, BMEI1777 and BMEI1980. The results of Elispot analysis showed that the number of specific IFN- 纬 secreting cells was higher than that of IL-4 secreting cells. The results of antibody subtype classification showed that the ratio of IgG2a to IgG1 decreased in CD4 / CD8 ratio compared with that in negative control group, while the recombinant protein and DNA protein mixed vaccine was mainly humoral immunity, and the number of specific IFN- 纬 secreting cells was lower than that of IL-4 secreting cells. The IgG1 level of mice was much higher than that of IgG2a, and the CD4 / CD8 ratio of mice was higher than that of the negative control group. The candidate vaccine could stimulate the cellular and humoral immune response, and the immunogenicity of BMEI1980 and BMEI1777 was stronger than that of other proteins. The results of this study laid a foundation for further research on the effective prevention of brucellosis based on vaccine research strategy.
【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】：2010
【分类号】：R392

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