結(jié)核分枝桿菌融合蛋白疫苗（Ag85B-ESAT6）的構(gòu)建及免疫學(xué)特性研究
發(fā)布時間：2018-12-14 13:07
【摘要】： 結(jié)核病(Tuberculosis,TB),是由結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB)引起的以呼吸道系統(tǒng)感染為主的慢性傳染病,是目前世界死亡人數(shù)最多、造成危害性最大的疾病之一。 卡介苗(BCG)是當(dāng)前預(yù)防結(jié)核病的唯一疫苗,但它的預(yù)防保護效果欠佳,對兒童具有很好的保護力,但對成人無保護作用,主要是由于BCG的不斷變異,導(dǎo)致免疫效果不穩(wěn)定;同時在BCG的制備和傳代過程中丟失了部分與保護力相關(guān)的基因,使BCG保護性免疫應(yīng)答減弱;近些年來,伴隨著艾滋病的流行和MTB自身耐藥性的增強,進一步加劇了結(jié)核病對人類的威脅,因此迫切需要研制出更有效的TB疫苗。 研究發(fā)現(xiàn)在MTB培養(yǎng)上清濾液(CFP)中存在許多具有保護力的分泌蛋白,其中Ag85B和ESAT6具有很好的免疫原性,Ag85B具有分支桿菌酸轉(zhuǎn)移酶活性,可以介導(dǎo)吞噬細胞對MTB進行吞噬,并且能夠誘導(dǎo)很強的Th1免疫反應(yīng),ESAT6是MTB短期培養(yǎng)濾液中純化分離出的一種低分子量分泌性蛋白,不存在于BCG中,在MTB感染早期階段即可被機體識別,具有較強的細胞免疫活性,它們均能刺激機體產(chǎn)生保護性免疫反應(yīng),是機體抗結(jié)核感染的主要保護性抗原。研究還表明,Ag85B和EAST6的融合蛋白形式進行免疫可提高機體的免疫應(yīng)答水平。本研究構(gòu)建了原核表達載體pET-20b- Ag85B- EAST6,制備了融合蛋白Ag85B- EAST6。實驗選用了BALB/c小鼠作為實驗?zāi)Ｐ?重組卡介苗(rBCG)進行初免,Ag85B-ESAT6融合蛋白分別聯(lián)合三種佐劑DDA/MPL,AD11.sm,ADO-1進行加強,分別檢測了抗體效價,Th1/Th2細胞因子含量,流式細胞亞型分選,IFN-r的ELISPOT實驗,CTL實驗等。結(jié)果顯示: Ag85B-ESAT6融合蛋白聯(lián)合佐劑疫苗形式優(yōu)于rBCG,引起了較強的細胞免疫,但以CD4+細胞反應(yīng)為主,CD8+細胞反應(yīng)相較弱;同時也初步評價了三種佐劑在提高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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.11
本文編號：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.11
【引證文獻】
相關(guān)期刊論文 前1條
1 尹文東;于庭;梁艷;陽幼榮;肖漓;王蘭;王瑩;史迎昌;張俊仙;吳雪瓊;;結(jié)核分枝桿菌重組Ag85A、Ag85B蛋白免疫原性的研究[J];中國實驗診斷學(xué);2011年10期
相關(guān)碩士學(xué)位論文 前1條
1 尹文東;結(jié)核分枝桿菌重組Ag85A、Ag85B蛋白聯(lián)合母牛分枝桿菌免疫原性的研究[D];吉林大學(xué);2012年
，本文編號：2378663
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2378663.html
最近更新
教材專著
