共表达Ag85B、ESAT-6及小鼠IFN-γ重组卡介苗和Ag85B、ESAT-6嵌合蛋白亚单位疫苗的初步研究

发布时间：2018-04-17 10:43

本文选题：结核分枝杆菌 + 疫苗　；参考：《复旦大学》2007年博士论文

【摘要】： 结核病正对人类构成巨大的威胁。目前全球接近32%的人口已感染了结核杆菌。每年约有300万人死于结核病,平均每天死亡超过8000人。更为严重的是近十几年来由于结核耐药株增多和HIV/AIDS的蔓延,结核病发病率增高,已经成为传染病中的第二大杀手。目前全球广泛使用的唯一的结核病疫苗是卡介苗(BCG)。至今已有30亿以上的人接种了BCG,并且每年有1亿左右的新生儿接种此疫苗。它可以较好的阻止年幼儿粟粒型结核及结核性脑膜炎的发生。但是,它不能保护最常见的成人肺结核的发生。所以研制新型的结核疫苗刻不容缓,这对于控制结核传染具有非常重大的意义。当前疫苗研究的主要有两个方向;(1)研制重组卡介苗,使其比亲代卡介苗具有更强的抗原性和免疫性,以代替现在使用的卡介苗;(2)研制亚单位疫苗,用于BCG免疫后的加强免疫。因此本研究工作包括两个方面;重组BCG疫苗的研究和嵌合蛋白亚单位疫苗的研究。 1.重组BCG疫苗的研究 BCG是很好的活的疫苗载体,可以诱导较强的细胞免疫和体液免疫反应。将一些重要的结核杆菌抗原基因导入BCG,让BCG大量表达保护性抗原,有望诱导比BCG强大而又持久的免疫保护力。本研究选择结核分枝杆菌最重要的保护性抗原Ag85B,BCG在传代过程中丢失的保护性抗原ESAT-6及重要的细胞因子IFN-γ,重组入BCG,构建了三株重组BCG菌株,分别是rBCG-Ag85B(简称rBCG-A)、rBCG-Ag85B-ESAT6(简称rBCG-AE)、rBCG-Ag85B-ESAT6-IFNγ(简称rBCG-AEI),其中rBCG-AEI的构建是国内外首次报道。我们在BCG和三株重组BCG菌株免疫C57BL/6小鼠后,通过分析抗体IgG水平、IgG2b/IgG1和IFN-γ的分泌,比较了它们的免疫原性。研究结果表明,三株重组BCG针对特异抗原均可以引起特异性的较BCG强的体液免疫和细胞免疫反应。其中rBCG-AEI的免疫原性较高,而且还能诱导较其它重组BCG高的Th1反应,而后者是成为成功疫苗的重要因素。而且我们进一步观察了重组BCG的免疫保护效果。皮下免疫C57BL/6小鼠和豚鼠(剂量分别为5×10~6和5×10~4 cfu),8周后给予结核杆菌毒力标准株H37Rv攻击,观察攻击后菌落计数、体重变化和组织病理变化。研究结果显示攻击后9周rBCG-AEI免疫的小鼠肺部菌落数较少,净体重增加。虽然肺部组织的病理改变与BCG和其它重组BCG无明显差别,但抗酸染色显示rBCG-AEI免疫的小鼠肺部组织的结核杆菌数最少。因此rBCG-AEI能够产生与BCG,rBCG-A和rBCG-AE相当甚至更好的保护作用。当然为了进一步区分重组BCG与BCG疫苗的保护效率差异,还需要延长毒力株攻击后观察时间。 2.嵌合蛋白亚单位疫苗的研究 Ag85B,ESAT-6是结核杆菌重要的保护性抗原。其抗原表位已得到广泛研究,其中ESAT-6的T细胞抗原表位位于蛋白分子的N端及第51～60氨基酸之间;Ag85B的T细胞抗原表位位于蛋白分子C端的第241～260氨基酸之间和第261～280氨基酸之间。我们依据Ag85B,ESAT-6的T细胞抗原表位,将ESAT-6插入到Ag85B第169～182氨基酸之间形成嵌合蛋白;Ag85B_N-ESAT-6-Ag85B_C。研究发现这种新的嵌合蛋白疫苗与佐剂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,后者已被报道在结核杆菌感染后可以产生与BCG相似的保护效果。因此,Ag85B,ESAT-6嵌合亚单位疫苗有可能成为一种有效的新的蛋白多肽疫苗而用于结核分枝杆菌的预防。该研究还提出了一种根据T细胞表位设计亚单位疫苗的新策略。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【参考文献】