本文選題：結(jié)核分枝桿菌 + 疫苗�。� 參考：《復(fù)旦大學(xué)》2007年博士論文

【摘要】： 結(jié)核病正對(duì)人類構(gòu)成巨大的威脅。目前全球接近32%的人口已感染了結(jié)核桿菌。每年約有300萬人死于結(jié)核病,平均每天死亡超過8000人。更為嚴(yán)重的是近十幾年來由于結(jié)核耐藥株增多和HIV/AIDS的蔓延,結(jié)核病發(fā)病率增高,已經(jīng)成為傳染病中的第二大殺手。目前全球廣泛使用的唯一的結(jié)核病疫苗是卡介苗(BCG)。至今已有30億以上的人接種了BCG,并且每年有1億左右的新生兒接種此疫苗。它可以較好的阻止年幼兒粟粒型結(jié)核及結(jié)核性腦膜炎的發(fā)生。但是,它不能保護(hù)最常見的成人肺結(jié)核的發(fā)生。所以研制新型的結(jié)核疫苗刻不容緩,這對(duì)于控制結(jié)核傳染具有非常重大的意義。當(dāng)前疫苗研究的主要有兩個(gè)方向;(1)研制重組卡介苗,使其比親代卡介苗具有更強(qiáng)的抗原性和免疫性,以代替現(xiàn)在使用的卡介苗;(2)研制亞單位疫苗,用于BCG免疫后的加強(qiáng)免疫。因此本研究工作包括兩個(gè)方面;重組BCG疫苗的研究和嵌合蛋白亞單位疫苗的研究。 1.重組BCG疫苗的研究 BCG是很好的活的疫苗載體,可以誘導(dǎo)較強(qiáng)的細(xì)胞免疫和體液免疫反應(yīng)。將一些重要的結(jié)核桿菌抗原基因?qū)隑CG,讓BCG大量表達(dá)保護(hù)性抗原,有望誘導(dǎo)比BCG強(qiáng)大而又持久的免疫保護(hù)力。本研究選擇結(jié)核分枝桿菌最重要的保護(hù)性抗原Ag85B,BCG在傳代過程中丟失的保護(hù)性抗原ESAT-6及重要的細(xì)胞因子IFN-γ,重組入BCG,構(gòu)建了三株重組BCG菌株,分別是rBCG-Ag85B(簡(jiǎn)稱rBCG-A)、rBCG-Ag85B-ESAT6(簡(jiǎn)稱rBCG-AE)、rBCG-Ag85B-ESAT6-IFNγ(簡(jiǎn)稱rBCG-AEI),其中rBCG-AEI的構(gòu)建是國內(nèi)外首次報(bào)道。 我們?cè)贐CG和三株重組BCG菌株免疫C57BL/6小鼠后,通過分析抗體IgG水平、IgG2b/IgG1和IFN-γ的分泌,比較了它們的免疫原性。研究結(jié)果表明,三株重組BCG針對(duì)特異抗原均可以引起特異性的較BCG強(qiáng)的體液免疫和細(xì)胞免疫反應(yīng)。其中rBCG-AEI的免疫原性較高,而且還能誘導(dǎo)較其它重組BCG高的Th1反應(yīng),而后者是成為成功疫苗的重要因素。 而且我們進(jìn)一步觀察了重組BCG的免疫保護(hù)效果。皮下免疫C57BL/6小鼠和豚鼠(劑量分別為5×10~6和5×10~4 cfu),8周后給予結(jié)核桿菌毒力標(biāo)準(zhǔn)株H37Rv攻擊,觀察攻擊后菌落計(jì)數(shù)、體重變化和組織病理變化。研究結(jié)果顯示攻擊后9周rBCG-AEI免疫的小鼠肺部菌落數(shù)較少,凈體重增加。雖然肺部組織的病理改變與BCG和其它重組BCG無明顯差別,但抗酸染色顯示rBCG-AEI免疫的小鼠肺部組織的結(jié)核桿菌數(shù)最少。因此rBCG-AEI能夠產(chǎn)生與BCG,rBCG-A和rBCG-AE相當(dāng)甚至更好的保護(hù)作用。當(dāng)然為了進(jìn)一步區(qū)分重組BCG與BCG疫苗的保護(hù)效率差異,還需要延長(zhǎng)毒力株攻擊后觀察時(shí)間。 2.嵌合蛋白亞單位疫苗的研究 Ag85B,ESAT-6是結(jié)核桿菌重要的保護(hù)性抗原。其抗原表位已得到廣泛研究,其中ESAT-6的T細(xì)胞抗原表位位于蛋白分子的N端及第51～60氨基酸之間;Ag85B的T細(xì)胞抗原表位位于蛋白分子C端的第241～260氨基酸之間和第261～280氨基酸之間。我們依據(jù)Ag85B,ESAT-6的T細(xì)胞抗原表位,將ESAT-6插入到Ag85B第169～182氨基酸之間形成嵌合蛋白;Ag85B_N-ESAT-6-Ag85B_C。研究發(fā)現(xiàn)這種新的嵌合蛋白疫苗與佐劑MPL-TDM共免疫小鼠后,IgG2b/IgG1的比例和IFN-γ的分泌都比Ag85B-ESAT-6融合蛋白高,后者已被報(bào)道在結(jié)核桿菌感染后可以產(chǎn)生與BCG相似的保護(hù)效果。因此,Ag85B,ESAT-6嵌合亞單位疫苗有可能成為一種有效的新的蛋白多肽疫苗而用于結(jié)核分枝桿菌的預(yù)防。該研究還提出了一種根據(jù)T細(xì)胞表位設(shè)計(jì)亞單位疫苗的新策略。
[Abstract]:TB is human pose a huge threat. Currently close to 32% of the population have been infected with tuberculosis. There are about 3 million people died of TB each year, the average daily death of more than 8000 people. The more serious is in recent years due to the increase of tuberculosis drug resistant strain HIV/AIDS and the spread of tuberculosis incidence, has become infectious the second biggest killer disease. Currently widely used global TB vaccine is the only BCG (BCG). It has been more than 3 billion people were BCG, and about 100 million of the newborns vaccinated each year. It can be used to prevent children in miliary tuberculosis and tuberculous meningitis occurred. However, it can not protect the most common adult pulmonary tuberculosis. So the development of new TB vaccine urgent, this is of great significance for the control of TB vaccine research are the main current. Two directions; (1) the development of recombinant BCG, which are more than the parental BCG antigenicity and immunity, to replace the currently used BCG; (2) subunitvaccine, used to strengthen the immune BCG after immunization. The research work includes two aspects; study and chimeric protein subunit vaccine a group of BCG vaccine.
Study on 1. recombinant BCG vaccine
BCG is a good live vaccine vector, can induce strong cellular and humoral immune response. Some of the important Mycobacterium tuberculosis antigen gene BCG, BCG expression for protective antigen, is expected to induce protective immunity than BCG. Strong and lasting protective antigen Ag85B of Mycobacterium tuberculosis most the protective antigen ESAT-6 BCG lost during subculture and important cytokines IFN-, recombinant BCG, constructed three recombinant BCG strains were rBCG-Ag85B (rBCG-A), rBCG-Ag85B-ESAT6 (rBCG-AE), rBCG-Ag85B-ESAT6-IFN gamma (rBCG-AEI), where rBCG-AEI is the construction of the first report.
We are BCG and three strains of recombinant BCG strains after immunization of C57BL/6 mice, through the analysis of the level of antibody IgG, the secretion of IgG2b/IgG1 and IFN- gamma, their immunogenicity was compared. Results showed that three strains of recombinant BCG specific antigen can cause a strong BCG specific humoral immune and cellular immune responses. The immunogenicity of rBCG-AEI is higher than other, but also induced by recombinant BCG Th1 reaction, and the latter is to become an important factor in the success of the vaccine.
And we further observed the effect of immune protection of recombinant BCG. Subcutaneous immunization of C57BL/6 mice and guinea pigs (doses were 5 * 10~6 and 5 * 10~4 CFU), after 8 weeks for Mycobacterium tuberculosis virulence standard strains of H37Rv attack, attack after observation of colony counting, weight changes and pathological changes. The results show that the lungs of mice colonies small number of 9 weeks after the attack of immune rBCG-AEI, net weight gain. Although there was no significant difference between the pathological changes of lung tissue with BCG and other recombinant BCG, but the number of acid fast staining showed that Mycobacterium tuberculosis rBCG-AEI immunized mice lung tissue. It can produce at least rBCG-AEI and BCG, the protective effect of rBCG-A and rBCG-AE is even better. Of course in order to investigate the protective efficacy difference between recombinant BCG and BCG vaccines, but also need to observe the time of virulent attacks.
Study on 2. chimeric protein subunit vaccine
Ag85B ESAT-6 is a protective antigen of Mycobacterium tuberculosis. The important epitope has been widely studied, among which ESAT-6 T cell epitopes in the protein molecules of N end and 51~60 amino acids; between Ag85B T cell epitopes in C protein molecule end 241st ~ 260 and 261st ~ 280 amino acid our amino acids. According to Ag85B, the T cell epitopes of ESAT-6, insert the ESAT-6 into the Ag85B chimeric protein formed between 169th ~ 182 amino acid; Ag85B_N-ESAT-6-Ag85B_C. study found that the new chimeric protein vaccine with adjuvant MPL-TDM were immunized mice, the secretion of IgG2b/IgG1 and the proportion of IFN- gamma than Ag85B-ESAT-6 fusion protein, which has been in the reports of tuberculosis infection can produce protective effect similar to that of BCG. Therefore, Ag85B and ESAT-6 chimeric subunit vaccine has the potential to become a new effective protein peptide vaccine The vaccine is used for the prevention of Mycobacterium tuberculosis. The study also proposed a new strategy for the design of subunit vaccines based on the epitopes of T cells.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 潘怡,蔡宏,李淑霞,田霞,李唐,朱玉賢;結(jié)核分枝桿菌組合DNA疫苗的免疫效果[J];生物化學(xué)與生物物理學(xué)報(bào);2003年01期

2 謝建平,王洪海,陳永青;結(jié)核分枝桿菌的后基因組研究和新型疫苗[J];微生物學(xué)報(bào);2001年02期

，

本文編號(hào)：1763265