沙门氏菌菌蜕对结核分枝杆菌四种蛋白免疫的佐剂效应
发布时间：2018-07-29 17:31
【摘要】：结核病是危害人类和动物健康的重大慢性传染病之一，尤其是近年来随着多重耐药分枝杆菌的出现以及与病毒的混合感染，使得对结核病的治疗变得越来越困难。因此，研发新型高效的抗结核病疫苗已成为当务之急。然而针对结核分枝菌相关保护性抗原基因及其编码蛋白功能的研究却进展缓慢，许多未知功能蛋白及基因的挖掘仍依赖于生物信息学的预测。结核分枝杆菌相关蛋白Rv3802c、Rv0394c（SEC）、Rv0199、E6c10（ESAT6与CFP10融合蛋白）分别是细菌的胞壁结构蛋白、透明质酸酶蛋白、分泌蛋白以及结核分枝杆菌感染早期产生的小分子蛋白。以往的研究发现，这些蛋白不仅与结核分枝杆菌的毒力及其对细胞的侵袭作用呈现密切的相关性，而且可作为药物靶标应用于抗结核药物的开发。此外，这些蛋白具有良好的免疫原性，将其制备成重组亚单位疫苗也许可以用于结核病的免疫预防。 利用基因工程方法制备的重组亚单位疫苗需辅以佐剂才能刺激机体产生较强的免疫应答，从而获得良好的免疫效果。沙门氏菌菌蜕（ghost）是一种不含有核酸和蛋白等内容物的菌体空壳，但其仍然保留着细菌表面抗原的天然构象和活性，可作为良好的载体和疫苗，推测其还可能具有佐剂效应。但到目前为止，将其作为免疫佐剂应用于细菌保护性抗原免疫效果的评价研究仍未见报道。 本研究应用实验室已成功制备好的4种分枝杆菌重组蛋白Rv3802c、Rv0394c（SEC）、Rv0199和E6c10，将其分别与禽肠炎沙门氏菌菌蜕和弗氏不完全佐剂（FIA）混合后背部皮下注射BAL B/c小鼠（SPF级）。在首免14d及二免7d时分别检测4种抗原蛋白诱导小鼠产生抗体和免疫球蛋白亚类情况，，分别测定经过4种蛋白刺激后小鼠脾淋巴细胞分泌IFN-γ和IL-4水平；流式细胞仪分析4种抗原刺激小鼠脾淋巴细胞产生CD8+和CD4+T细胞的动态变化；组织病理学观察4种抗原对注射部位及各主要脏器的损伤和炎症反应情况，并于二免1w后的一个月内连续监测小鼠抗体持续情况。 实验结果表明，对于分子量较大的蛋白而言，菌蜕的佐剂效应与弗氏不完全佐剂相差不大，但对于低分子量的E6c10蛋白而言，菌蜕佐剂在诱导体液和细胞免疫的水平上要远远好于弗氏不完全佐剂(FIA)。同时，4种蛋白可以刺激机体产生持久的抗体分泌能力。组织病理学观察发现，所有实验组小鼠的注射部位并没有产生较为明显的外伤感染和免疫不良反应，也无炎性细胞浸润现象。表明菌蜕作为免疫佐剂应用于结核分枝杆菌等亚单位疫苗的开发具有广阔的应用前景，其良好的佐剂效应及无毒副作用的特点更为多联苗的创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- 纬 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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.1
本文编号：2153466
[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- 纬 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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.1
【参考文献】
相关期刊论文 前2条
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2 吴雪琼;;结核分枝杆菌保护性抗原的研究进展[J];中华结核和呼吸杂志;2006年05期
本文编号：2153466
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