结核分枝杆菌融合蛋白疫苗（Ag85B-ESAT6）的构建及免疫学特性研究
发布时间：2018-12-14 13:07
【摘要】： 结核病(Tuberculosis,TB),是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的以呼吸道系统感染为主的慢性传染病,是目前世界死亡人数最多、造成危害性最大的疾病之一。 卡介苗(BCG)是当前预防结核病的唯一疫苗,但它的预防保护效果欠佳,对儿童具有很好的保护力,但对成人无保护作用,主要是由于BCG的不断变异,导致免疫效果不稳定;同时在BCG的制备和传代过程中丢失了部分与保护力相关的基因,使BCG保护性免疫应答减弱;近些年来,伴随着艾滋病的流行和MTB自身耐药性的增强,进一步加剧了结核病对人类的威胁,因此迫切需要研制出更有效的TB疫苗。 研究发现在MTB培养上清滤液(CFP)中存在许多具有保护力的分泌蛋白,其中Ag85B和ESAT6具有很好的免疫原性,Ag85B具有分支杆菌酸转移酶活性,可以介导吞噬细胞对MTB进行吞噬,并且能够诱导很强的Th1免疫反应,ESAT6是MTB短期培养滤液中纯化分离出的一种低分子量分泌性蛋白,不存在于BCG中,在MTB感染早期阶段即可被机体识别,具有较强的细胞免疫活性,它们均能刺激机体产生保护性免疫反应,是机体抗结核感染的主要保护性抗原。研究还表明,Ag85B和EAST6的融合蛋白形式进行免疫可提高机体的免疫应答水平。本研究构建了原核表达载体pET-20b- Ag85B- EAST6,制备了融合蛋白Ag85B- EAST6。实验选用了BALB/c小鼠作为实验模型,重组卡介苗(rBCG)进行初免,Ag85B-ESAT6融合蛋白分别联合三种佐剂DDA/MPL,AD11.sm,ADO-1进行加强,分别检测了抗体效价,Th1/Th2细胞因子含量,流式细胞亚型分选,IFN-r的ELISPOT实验,CTL实验等。结果显示: Ag85B-ESAT6融合蛋白联合佐剂疫苗形式优于rBCG,引起了较强的细胞免疫,但以CD4+细胞反应为主,CD8+细胞反应相较弱;同时也初步评价了三种佐剂在提高Ag85B-ESAT6融合蛋白免疫水平中的效果。
[Abstract]:Tuberculosis (Tuberculosis,TB), a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), is one of the most harmful diseases in the world. BCG (BCG) is the only vaccine to prevent tuberculosis at present, but its preventive and protective effect is not good for children, but it has no protective effect on adults, mainly because of the continuous variation of BCG, which results in unstable immune effect. At the same time, some protective genes were lost during the preparation and passage of BCG, which weakened the protective immune response of BCG. In recent years, with the AIDS epidemic and the increase of MTB resistance, the threat of tuberculosis to human beings is further aggravated, so it is urgent to develop a more effective TB vaccine. It was found that there were many protective secretory proteins in the supernatant of MTB culture filtrate (CFP), among which Ag85B and ESAT6 had good immunogenicity, Ag85B had mycoic acid transferase activity, which could mediate phagocytic phagocytosis of MTB. ESAT6 is a kind of low molecular weight secretory protein isolated from the short term culture filtrate of MTB. It does not exist in BCG and can be recognized by the body at the early stage of MTB infection. Both of them can stimulate the body to produce protective immune response and are the main protective antigens against tuberculosis infection. The results also showed that the immune response of Ag85B and EAST6 could be improved by immunizing with the fusion protein. In this study, the prokaryotic expression vector pET-20b- Ag85B- EAST6, was constructed to prepare the fusion protein Ag85B- EAST6.. BALB/c mice were selected as the experimental model, the recombinant BCG (rBCG) was first immunized, the Ag85B-ESAT6 fusion protein was strengthened with three adjuvant DDA/MPL,AD11.sm,ADO-1, and the antibody titers were detected. Th1/Th2 cytokine content, flow cytometry subtype sorting, IFN-r ELISPOT test, CTL test, etc. The results showed that Ag85B-ESAT6 fusion protein combined with adjuvant vaccine was superior to rBCG, in inducing strong cellular immunity, but CD4 cell reaction was dominant, and CD8 cell reaction was weak. At the same time, the effects of three adjuvants on improving the immune level of Ag85B-ESAT6 fusion protein were also preliminarily evaluated.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11
本文编号：2378663
[Abstract]:Tuberculosis (Tuberculosis,TB), a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), is one of the most harmful diseases in the world. BCG (BCG) is the only vaccine to prevent tuberculosis at present, but its preventive and protective effect is not good for children, but it has no protective effect on adults, mainly because of the continuous variation of BCG, which results in unstable immune effect. At the same time, some protective genes were lost during the preparation and passage of BCG, which weakened the protective immune response of BCG. In recent years, with the AIDS epidemic and the increase of MTB resistance, the threat of tuberculosis to human beings is further aggravated, so it is urgent to develop a more effective TB vaccine. It was found that there were many protective secretory proteins in the supernatant of MTB culture filtrate (CFP), among which Ag85B and ESAT6 had good immunogenicity, Ag85B had mycoic acid transferase activity, which could mediate phagocytic phagocytosis of MTB. ESAT6 is a kind of low molecular weight secretory protein isolated from the short term culture filtrate of MTB. It does not exist in BCG and can be recognized by the body at the early stage of MTB infection. Both of them can stimulate the body to produce protective immune response and are the main protective antigens against tuberculosis infection. The results also showed that the immune response of Ag85B and EAST6 could be improved by immunizing with the fusion protein. In this study, the prokaryotic expression vector pET-20b- Ag85B- EAST6, was constructed to prepare the fusion protein Ag85B- EAST6.. BALB/c mice were selected as the experimental model, the recombinant BCG (rBCG) was first immunized, the Ag85B-ESAT6 fusion protein was strengthened with three adjuvant DDA/MPL,AD11.sm,ADO-1, and the antibody titers were detected. Th1/Th2 cytokine content, flow cytometry subtype sorting, IFN-r ELISPOT test, CTL test, etc. The results showed that Ag85B-ESAT6 fusion protein combined with adjuvant vaccine was superior to rBCG, in inducing strong cellular immunity, but CD4 cell reaction was dominant, and CD8 cell reaction was weak. At the same time, the effects of three adjuvants on improving the immune level of Ag85B-ESAT6 fusion protein were also preliminarily evaluated.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11
【引证文献】
相关期刊论文 前1条
1 尹文东;于庭;梁艳;阳幼荣;肖漓;王兰;王莹;史迎昌;张俊仙;吴雪琼;;结核分枝杆菌重组Ag85A、Ag85B蛋白免疫原性的研究[J];中国实验诊断学;2011年10期
相关硕士学位论文 前1条
1 尹文东;结核分枝杆菌重组Ag85A、Ag85B蛋白联合母牛分枝杆菌免疫原性的研究[D];吉林大学;2012年
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