本文關(guān)鍵詞： 布魯氏菌 外膜蛋白 DNA疫苗 重組蛋白苗 細(xì)胞免疫 體液免疫　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 尋找具有免疫原性的外膜蛋白是構(gòu)建布魯氏菌病亞單位疫苗、DNA疫苗和細(xì)菌活載體疫苗的關(guān)鍵。 本研究利用在線生物學(xué)軟件PSORT和CELLO對(duì)布魯氏菌16M菌株基因組序列進(jìn)行分析,預(yù)測(cè)其外膜蛋白基因序列。選取17個(gè)外膜蛋白基因及核蛋白L7/L12進(jìn)行PCR擴(kuò)增并與原核表達(dá)載體pET32a(+)連接,轉(zhuǎn)入E.coli BL21(DE3)感受態(tài)細(xì)胞;IPTG誘導(dǎo)蛋白表達(dá),通過SDS-PAGE分析及Western blot鑒定目的蛋白的表達(dá);經(jīng)HisTrapTMHP純化,制備重組蛋白疫苗。同時(shí)將上述18個(gè)基因片段和hGM-CSF基因,與真核表達(dá)質(zhì)粒PVAX1連接,轉(zhuǎn)染MDCK細(xì)胞,通過間接免疫熒光證實(shí)各目的蛋白在細(xì)胞中的表達(dá),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(shù)對(duì)其免疫效果進(jìn)行評(píng)價(jià)。 結(jié)果表明,利用PSORT和CELLO進(jìn)行預(yù)測(cè)共得到31個(gè)外膜蛋白。利用原核表達(dá)系統(tǒng),成功構(gòu)建了18個(gè)重組質(zhì)粒,其中13個(gè)分子獲得大量表達(dá),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(dá)的BMEII0983、BMEI0653、BMEI1777、BMEI1980、BMEII0381、BMEII0472及少量表達(dá)的BMEI1829進(jìn)行純化,純化后蛋白純度達(dá)90%以上。成功構(gòu)建了18個(gè)真核表達(dá)重組質(zhì)粒,轉(zhuǎn)染MDCK細(xì)胞,有12個(gè)獲得了表達(dá)。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(chǎn)生了較強(qiáng)的免疫應(yīng)答。DNA疫苗激發(fā)以細(xì)胞免疫為主的免疫應(yīng)答,ELISPOT分析顯示特異性IFN-γ分泌細(xì)胞的數(shù)量要高于IL-4分泌細(xì)胞的數(shù)量,抗體亞型分類結(jié)果表明小鼠IgG2a與IgG1之比1,FCM分析CD4+/CD8+值較陰性對(duì)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(xì)胞的數(shù)量要低于IL-4分泌細(xì)胞的數(shù)量,小鼠的IgG1水平遠(yuǎn)遠(yuǎn)高于IgG2a水平,小鼠CD4+/CD8+值較陰性對(duì)照組增加。制備的候選疫苗能夠刺激機(jī)體產(chǎn)生較強(qiáng)的細(xì)胞和體液免疫應(yīng)答,其中BMEI1980和BMEI1777較其他蛋白免疫原性強(qiáng)。 本研究的實(shí)驗(yàn)結(jié)果為基于疫苗研究策略的有效預(yù)防布魯氏菌病的深入研究奠定了基礎(chǔ)。
[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(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 馬長(zhǎng)勝;布魯氏菌三個(gè)外膜脂蛋白的原核表達(dá)、純化及免疫原性實(shí)驗(yàn)[D];山東農(nóng)業(yè)大學(xué);2012年

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本文編號(hào)：1536682