【摘要】：結(jié)核病是危害人類和動物健康的重大慢性傳染病之一，尤其是近年來隨著多重耐藥分枝桿菌的出現(xiàn)以及與病毒的混合感染，使得對結(jié)核病的治療變得越來越困難。因此，研發(fā)新型高效的抗結(jié)核病疫苗已成為當(dāng)務(wù)之急。然而針對結(jié)核分枝菌相關(guān)保護性抗原基因及其編碼蛋白功能的研究卻進展緩慢，許多未知功能蛋白及基因的挖掘仍依賴于生物信息學(xué)的預(yù)測。結(jié)核分枝桿菌相關(guān)蛋白Rv3802c、Rv0394c（SEC）、Rv0199、E6c10（ESAT6與CFP10融合蛋白）分別是細(xì)菌的胞壁結(jié)構(gòu)蛋白、透明質(zhì)酸酶蛋白、分泌蛋白以及結(jié)核分枝桿菌感染早期產(chǎn)生的小分子蛋白。以往的研究發(fā)現(xiàn)，這些蛋白不僅與結(jié)核分枝桿菌的毒力及其對細(xì)胞的侵襲作用呈現(xiàn)密切的相關(guān)性，而且可作為藥物靶標(biāo)應(yīng)用于抗結(jié)核藥物的開發(fā)。此外，這些蛋白具有良好的免疫原性，將其制備成重組亞單位疫苗也許可以用于結(jié)核病的免疫預(yù)防。 利用基因工程方法制備的重組亞單位疫苗需輔以佐劑才能刺激機體產(chǎn)生較強的免疫應(yīng)答，從而獲得良好的免疫效果。沙門氏菌菌蛻（ghost）是一種不含有核酸和蛋白等內(nèi)容物的菌體空殼，但其仍然保留著細(xì)菌表面抗原的天然構(gòu)象和活性，可作為良好的載體和疫苗，推測其還可能具有佐劑效應(yīng)。但到目前為止，將其作為免疫佐劑應(yīng)用于細(xì)菌保護性抗原免疫效果的評價研究仍未見報道。 本研究應(yīng)用實驗室已成功制備好的4種分枝桿菌重組蛋白Rv3802c、Rv0394c（SEC）、Rv0199和E6c10，將其分別與禽腸炎沙門氏菌菌蛻和弗氏不完全佐劑（FIA）混合后背部皮下注射BAL B/c小鼠（SPF級）。在首免14d及二免7d時分別檢測4種抗原蛋白誘導(dǎo)小鼠產(chǎn)生抗體和免疫球蛋白亞類情況，，分別測定經(jīng)過4種蛋白刺激后小鼠脾淋巴細(xì)胞分泌IFN-γ和IL-4水平；流式細(xì)胞儀分析4種抗原刺激小鼠脾淋巴細(xì)胞產(chǎn)生CD8+和CD4+T細(xì)胞的動態(tài)變化；組織病理學(xué)觀察4種抗原對注射部位及各主要臟器的損傷和炎癥反應(yīng)情況，并于二免1w后的一個月內(nèi)連續(xù)監(jiān)測小鼠抗體持續(xù)情況。 實驗結(jié)果表明，對于分子量較大的蛋白而言，菌蛻的佐劑效應(yīng)與弗氏不完全佐劑相差不大，但對于低分子量的E6c10蛋白而言，菌蛻佐劑在誘導(dǎo)體液和細(xì)胞免疫的水平上要遠(yuǎn)遠(yuǎn)好于弗氏不完全佐劑(FIA)。同時，4種蛋白可以刺激機體產(chǎn)生持久的抗體分泌能力。組織病理學(xué)觀察發(fā)現(xiàn)，所有實驗組小鼠的注射部位并沒有產(chǎn)生較為明顯的外傷感染和免疫不良反應(yīng)，也無炎性細(xì)胞浸潤現(xiàn)象。表明菌蛻作為免疫佐劑應(yīng)用于結(jié)核分枝桿菌等亞單位疫苗的開發(fā)具有廣闊的應(yīng)用前景，其良好的佐劑效應(yīng)及無毒副作用的特點更為多聯(lián)苗的創(chuàng)制提供了一定的思路。
[Abstract]:Tuberculosis is one of the major chronic infectious diseases that endanger human and animal health, especially with the emergence of multidrug resistant Mycobacterium and the mixed infection with virus in recent years, the treatment of tuberculosis becomes more and more difficult. Therefore, the development of new and efficient anti-tuberculosis vaccine has become a top priority. However, the research on the function of protective antigen genes and their encoded proteins of Mycobacterium tuberculosis is slow. Many unknown functional proteins and genes still depend on the prediction of bioinformatics. Mycobacterium tuberculosis associated protein Rv3802c( SEC) / Rv0199E6c10 (ESAT6 and CFP10 fusion protein) are cell wall structural proteins, hyaluronidase proteins, secretory proteins and small molecular proteins produced by Mycobacterium tuberculosis in the early stage of infection. Previous studies have found that these proteins are not only closely related to the virulence of Mycobacterium tuberculosis and its invasion to cells, but also can be used as drug targets in the development of anti-tuberculosis drugs. In addition, these proteins have good immunogenicity, the preparation of recombinant subunit vaccine may be used to prevent tuberculosis. The recombinant subunit vaccine prepared by genetic engineering method needs adjuvant to stimulate the body to produce a strong immune response, thus obtaining a good immune effect. Salmonella slough (ghost) is a kind of empty shell without nucleic acid and protein, but it still retains the natural conformation and activity of bacterial surface antigen, which can be used as a good carrier and vaccine, and it may also have adjuvant effect. So far, however, the evaluation of bacterial protective antigen as an immune adjuvant has not been reported. In this study, four recombinant proteins Rv3802cnrv0394c (SEC) Rv0199 and E6c10 were successfully prepared in our laboratory and were subcutaneously injected with BAL B / c mice (SPF grade) after mixed with Salmonella enteritidis slough and Freund's incomplete adjuvant (FIA). The production of antibodies and immunoglobulin subclasses in mice induced by four kinds of antigenic proteins were detected at the first 14 days and the second 7 days, and the levels of IFN- 緯 and IL-4 in spleen lymphocytes of mice stimulated by four kinds of proteins were measured respectively. The dynamic changes of CD8 and CD4 T cells produced by spleen lymphocytes stimulated by four antigens were analyzed by flow cytometry, and the injury and inflammatory reaction of the four antigens to injection site and main organs were observed by histopathology. The antibody persistence in mice was continuously monitored within one month after the second immunization for 1 week. The results showed that the adjuvant effect of mycelium slough was similar to that of Freund's incomplete adjuvant for protein with high molecular weight, but for E6c10 protein with low molecular weight, Mycelium adjuvant is much better than Freund's incomplete adjuvant (FIA). In inducing somatic fluid and cellular immunity. At the same time, four proteins can stimulate the body to produce persistent antibody secretion. Histopathological observation showed that there were no obvious traumatic infection and immune adverse reactions and no inflammatory cell infiltration in all experimental mice. The results showed that the application of mycobacterium tuberculosis and other subunit vaccines as immune adjuvant had a broad application prospect, and its good adjuvant effect and non-toxic side effect provided some ideas for the creation of multiplex vaccine.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.1

【參考文獻】

相關(guān)期刊論文 前2條

1 劉東旭;時坤;李健明;杜銳;;卡介苗與結(jié)核菌野毒株的基因差異的研究進展[J];吉林畜牧獸醫(yī);2011年04期

2 吳雪瓊;;結(jié)核分枝桿菌保護性抗原的研究進展[J];中華結(jié)核和呼吸雜志;2006年05期

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