本文關鍵詞： 布魯氏菌 外膜蛋白 DNA疫苗 重組蛋白苗 細胞免疫 體液免疫　出處：《內蒙古農業(yè)大學》2010年碩士論文　論文類型：學位論文

【摘要】： 尋找具有免疫原性的外膜蛋白是構建布魯氏菌病亞單位疫苗、DNA疫苗和細菌活載體疫苗的關鍵。 本研究利用在線生物學軟件PSORT和CELLO對布魯氏菌16M菌株基因組序列進行分析,預測其外膜蛋白基因序列。選取17個外膜蛋白基因及核蛋白L7/L12進行PCR擴增并與原核表達載體pET32a(+)連接,轉入E.coli BL21(DE3)感受態(tài)細胞;IPTG誘導蛋白表達,通過SDS-PAGE分析及Western blot鑒定目的蛋白的表達;經HisTrapTMHP純化,制備重組蛋白疫苗。同時將上述18個基因片段和hGM-CSF基因,與真核表達質粒PVAX1連接,轉染MDCK細胞,通過間接免疫熒光證實各目的蛋白在細胞中的表達,制備DNA疫苗。用候選疫苗免疫Balb/c小鼠,通過間接ELISA、ELISPOT、FCM技術對其免疫效果進行評價。 結果表明,利用PSORT和CELLO進行預測共得到31個外膜蛋白。利用原核表達系統(tǒng),成功構建了18個重組質粒,其中13個分子獲得大量表達,以可溶形式存在的是BMEI0402、BMEI0454、BMEII0983、BMEI0653、BMEI1777、BMEI1980、BMEII0381、BMEII0472,包涵體形式存在的包括BMEI1830、BMEI1305、BMEI1306、BMEII1120。選取在上清中獲得大量表達的BMEII0983、BMEI0653、BMEI1777、BMEI1980、BMEII0381、BMEII0472及少量表達的BMEI1829進行純化,純化后蛋白純度達90%以上。成功構建了18個真核表達重組質粒,轉染MDCK細胞,有12個獲得了表達。用制備的BMEII0381、BMEII0472、BMEI0653、BMEI0748、BMEI1777、BMEI1980的DNA疫苗和重組蛋白苗,免疫Balb/c小鼠,產生了較強的免疫應答。DNA疫苗激發(fā)以細胞免疫為主的免疫應答,ELISPOT分析顯示特異性IFN-γ分泌細胞的數(shù)量要高于IL-4分泌細胞的數(shù)量,抗體亞型分類結果表明小鼠IgG2a與IgG1之比1,FCM分析CD4+/CD8+值較陰性對照組減少。而重組蛋白及DNA蛋白混合疫苗則以體液免疫為主,特異性IFN-γ分泌細胞的數(shù)量要低于IL-4分泌細胞的數(shù)量,小鼠的IgG1水平遠遠高于IgG2a水平,小鼠CD4+/CD8+值較陰性對照組增加。制備的候選疫苗能夠刺激機體產生較強的細胞和體液免疫應答,其中BMEI1980和BMEI1777較其他蛋白免疫原性強。 本研究的實驗結果為基于疫苗研究策略的有效預防布魯氏菌病的深入研究奠定了基礎。
[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- 緯 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- 緯 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.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

【引證文獻】

相關碩士學位論文 前1條

1 馬長勝;布魯氏菌三個外膜脂蛋白的原核表達、純化及免疫原性實驗[D];山東農業(yè)大學;2012年

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本文編號：1536682