【作者】 张辉；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2008， 博士

【摘要】 结核病(Tuberculosis, TB)由来已久,自有人类历史记录开始,就有了TB的记录。迄今为止,约2亿人被TB夺去生命,而这个数字仍在上升,世界卫生组织宣布TB是人类目前面临的最大威胁之一。1882年,Robert Koch证实了结核分枝杆菌(Mycobacterium tuberculosis,MTB)是TB的病原。全世界已有1/3人口约20亿人感染了MTB,现有结核病人2千万,每年新发病人数约880万,每年死亡人数高达160万。目前广泛用于预防TB的唯一疫苗是卡介苗(Bacille Calmette Guérin, BCG),但其免疫保护效果对于肺结核及弥散性结核分别约为50%和80%,存在显著差异。TB野毒株的高度耐药以及与人免疫缺陷病毒( Human immunodeficiency virus,HIV)共感染的不断上升,使TB警戒再次升高,并强调要利用免疫手段来控制TB的感染和传播。TB特异性免疫应答在清除MTB感染中发挥重要作用。BCG对青少年TB的预防具有重要的作用,但对于成人肺结核的保护作用有限,因此在设计新型抗结核疫苗时要考虑到如何预防肺结核。基因组学比较分析显示BCG中要比MTB缺失约100多个编码序列,其中的一些序列编码重要的抗原,当这些抗原被重新重组入BCG中都能够增强抗结核的能力。BCG中缺失RD1(Regions of difference 1),而RD1中的编码基因esxB和esxA基因分别编码培养滤液蛋白10(Culture filter protein 10, CFP-10)和早期分泌抗原靶6(Earlier secreted antigen target 6, ESAT-6),它们仅存在于临床分离的致病性MTB及牛分枝杆菌野毒株(M. bovis),能够诱导有效的Th1应答,并在动物实验中显示其对TB的保护效力,因而成为TB免疫诊断以及疫苗研究中最有力的候选抗原。为此,本研究以沙门菌及其鞭毛蛋白为载体,原核表达并运送ESAT-6、CFP-10蛋白,经滴鼻免疫,探讨了MTB保护性抗原ESAT-6和CFP-10蛋白诱导的黏膜免疫机理。1.结核分枝杆菌ESAT-6、CFP-10蛋白的克隆、表达及免疫原性分析为分析结核分枝杆菌分泌性抗原ESAT-6和CFP-10蛋白的免疫原性,利用PCR扩增了ESAT-6、CFP-10编码基因,经克隆筛选和测序鉴定后,构建了原核表达质粒pBCX-esat及pET-cfp。将两质粒分别转化宿主菌BL21(DE3),进行诱导表达。SDS-PAGE分析结果表明,重组的ESAT-6和CFP-10蛋白均获表达,融合蛋白的大小分别约为39kD和19kD。将CFP-10与本室研制的针对CFP-10蛋白的单克隆抗体6E8进行Western-blotting分析。结果显示,CFP-10融合蛋白能与特异性单克隆抗体6E8反应。同时,在ELISA实验中,重组ESAT-6蛋白与医院结核病人血清呈阳性反应。将ESAT-6及CFP-10融合蛋白分别以腹腔注射免疫C57BL/6小鼠,并于第二次免疫后7d,取小鼠脾脏细胞,用胞内细胞因子染色法检测ESAT-6及CFP-10蛋白特异性CD4+ T细胞分泌的IFN-γ及IL-4。结果两组蛋白均能诱导较高水平的IFN-γ,而IL-4水平低于IFN-γ水平。此外,经检测脾脏中CD3+ T细胞的CD69分子表达发现,ESAT-6和CFP-10蛋白免疫组的CD69水平显著上调。从而说明重组ESAT-6和CFP-10蛋白具有良好的免疫原性;免疫小鼠后,能够激活CD3+ T细胞,并诱导以细胞免疫为主的Th1应答,即重组蛋白ESAT-6及CFP-10具有Th1免疫原性。2.重组沙门菌表达的ESAT-6、CFP-10及其特异性免疫应答分析为了分析重组沙门菌诱导ESAT-6及CFP-10蛋白特异性免疫应答,将ESAT-6及CFP-10蛋白编码基因分别插入原核载体pYA3333中,将构建正确的质粒分别命名为pYA33-esat和pYA33-cfp。通过电穿孔法转化减毒鼠伤寒沙门菌X4550,获得重组菌X4550(33-esat)和X4550(33-cfp)。以每只1×108 cfu剂量的重组菌滴鼻免疫C57BL/6小鼠,间隔18d,在进行第2次免疫后10d取免疫小鼠脾脏、肺脏、肠系膜淋巴结(Mesenteric lymph node, MLN)及派伊尔氏结(Peyer’s patches, PP)细胞,以ESAT-6和CFP-10蛋白作为刺激原,检测抗原特异性的IFN-γ分泌细胞和IL-4分泌细胞。结果表明,经沙门菌表达运送的结核分枝杆菌抗原ESAT-6和CFP-10均能诱导特异性的免疫应答。在肺脏及PP细胞中,检测到较高水平的IFN-γ分泌细胞,与IL-4分泌细胞比较,差异显著。在脾脏和MLN中,免疫应答呈现平衡状态,IFN-γ分泌细胞和IL-4分泌细胞数量相当。此外,对脾脏、MLN、PP及支气管肺泡灌洗液(Bronchoalveolar lavage fluid, BAF)细胞中的共刺激分子检测结果表明,两组重组菌均能上调共刺激分子的表达。利用ELISA分别在免疫鼠的血清和BAF /肠黏膜中检测到ESAT-6及CFP-10蛋白特异性的IgG和IgA抗体。从而表明,以滴鼻方式接种重组减毒沙门菌,能够诱导ESAT-6及CFP-10蛋白特异性的细胞免疫应答,在黏膜邻近器官组织中,免疫应答趋于细胞免疫,而在黏膜远端免疫器官和组织中,免疫应答则处于平衡状态。以滴鼻免疫重组菌,不仅能诱导有效的体液免疫还可以激发局部的黏膜免疫,这为呼吸道疾病的防控提供了新的依据。3.沙门菌鞭毛蛋白嵌合表达的ESAT-6及其免疫学特性分析为研究以沙门菌鞭毛系统作为运送载体诱导的ESAT-6特异性免疫应答,利用overlap PCR将ESAT-6蛋白编码基因插入沙门菌鞭毛蛋白基因fliC的高变区域,构建成嵌合鞭毛片段fliC/esat。将fliC/esat片段插入原核表达载体pET,构建的质粒命名为pET-fliC/esat。将质粒pET-fliC/esat通过电穿孔法转化沙门菌LB5000,重组菌命名为LB5000(fliC/esat)。用P22噬菌体介导LB5000(fliC/esat)向都柏林沙门菌SL5928的转导,利用PCR、动力学检测及凝集试验鉴定并获得阳性转导菌SL5928(fliC/esat)。将转导菌SL5928(fliC/esat)感染结肠癌细胞HT-29,证实转导菌具有良好的感染能力;黏附力试验证实,转导菌具有良好的黏附特性。利用RT-PCR技术检测转导菌SL5928(fliC/esat)感染BMDC、F4/80+ MФ和肠上皮细胞(Enterocyte, EC)后的TLR-5 mRNA表达,结果在3h时,三种细胞就能表达TLR-5 mRNA;至6h,TLR-5 mRNA表达量有所升高。在体内实验中,将转导菌以每只1×107cfu滴鼻免疫C57BL/6小鼠,并于第二次免疫后10d,检测脾脏、肺脏、MLN、PP及鼻咽相关淋巴结(Nasopharynx associated lymph node,NALT)细胞中的IFN-γ和IL-4分泌细胞数。结果在肺脏、PP及NALT细胞中,IFN-γ分泌细胞与IL-4分泌细胞水平相比较显著升高,免疫应答趋向于Th1型;而在脾脏及MLN中,IFN-γ与IL-4分泌细胞数无显著性差异,免疫应答呈现Th1/Th2混合应答,处于平衡状态。而在体内的CTL实验中发现,与重组菌X4550(33-esat)相比较,转导菌SL5928(fliC/esat)诱导的CTL具有较强的特异性杀伤力。在血清及BAF的抗体检测中显示,转导菌能够诱导一定水平的血清IgG和黏膜IgA抗体。综上所述,鞭毛中嵌合MTB抗原ESAT-6后,不影响鞭毛自身特性,经沙门菌鞭毛系统运送,能够诱导ESAT-6抗原特异的细胞及黏膜免疫应答,这为TB疫苗研究开辟了新的研究方向。4.嵌合鞭毛蛋白fliC/esat与BCG黏膜共免疫诱导的免疫应答分析为研究嵌合鞭毛蛋白在TB免疫的中作用,将原核表达质粒pET-fliC/esat及pET-fliC转化大肠杆菌BL21(DE3),诱导表达嵌合蛋白fliC/esat及鞭毛蛋白fliC。SDS-PAGE分析结果表明,嵌合蛋白及fliC蛋白均以可溶性表达,大小分别约为64kD和57kD。将嵌合蛋白和fliC蛋白分别进行Western-blotting分析,以鼠伤寒沙门菌全菌血清作为一抗,结果显示嵌合蛋白及fliC蛋白均能与多抗血清反应。与ESAT-6蛋白相比较,嵌合蛋白及鞭毛蛋白fliC在体外均能诱导BMDC成熟。体外刺激F4/80+ MФ细胞实验表明,ESAT-6蛋白、fliC蛋白及嵌合蛋白均能上调腹腔MФ共刺激分子CD40、CD80、CD86和CD54。三种蛋白刺激BMDC及分选的F4/80+ MФ细胞分泌IL-12p70的检测结果显示,在刺激BMDC时,与ESAT-6蛋白组相比较,嵌合蛋白能够诱导分泌较高水平的IL-12p70;而在刺激F4/80+ MФ细胞时,与对照相比较,三种蛋白刺激后,F4/80+ MФ细胞均不能分泌有效的IL-12p70。在体内实验中,用嵌合蛋白静脉注射免疫C57BL/6小鼠,结果嵌合蛋白免疫组能够显著增强ESAT-6特异性免疫应答,而且免疫应答以Th1为主。此外,将BCG与嵌合蛋白共滴鼻免疫发现,在肺脏、PP、NALT及脾脏中均能诱导显著的Th1应答,与嵌合蛋白单一免疫组相比较,BCG能够增强ESAT-6特异性的免疫水平。检测CD8+CD69+细胞结果表明,BCG与嵌合蛋白共免疫组的CD8+CD69+细胞百分数均高于单独BCG及嵌合蛋白免疫组。从而说明,鞭毛嵌合表达ESAT-6抗原,有利于增强ESAT-6的特异性应答,鞭毛蛋白对ESAT-6抗原具有佐剂作用。BCG与嵌合蛋白共免疫结果提示,BCG与嵌合ESAT-6抗原的鞭毛蛋白之间具有协同作用,能够提高ESAT-6特异的免疫应答水平。更多还原

【Abstract】 Tuberculosis (TB) has a long history. It was presented before the beginning of recorded history. TB, together with acquired immune deficiency syndrome(AIDS) and malaria, remains today one of the leading infectious diseases. TB causes an estimated of at least 200 millions deaths now, and it was declared a global emergency by the World Health Organization. In 1882, Robert Koch identified Mycobacterium tuberculosis (MTB) as the causative agent of TB. More than one third of people over the world have infected MTB, and there has more than 20 million patients. There were an estimated 8.8 million new TB cases per year. A total of 1.6 million people died of TB, including 195000 patients infected with HIV. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against TB is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. The alarming increase in the incidence of TB , due to emergence of TB strains resistant to all major chemotherapeutic drugs and to co-infection with human immunodeficiency virus (HIV), has emphasized the need to develop immunological tools for TB control.Adaptive immunity against TB plays an immportant role in clearing infectious MTB. BCG vaccine protected efficiently against leprosy as well as childhood manifestations of TB. However, the protective efficacy against adult pulmonary TB was limited. Therefore, the new vaccine against TB should include protective efficacy against pulmonary TB. Comparative genomics identified up to 100 coding sequences absent from BCG but present in MTB. Some of these coding sequences encode potential antigens that could improve immunogenicity if reintroduced into BCG. RD1(Regions of Difference 1) is one of the regions absent from BCG. Among the missing genes are the coding sequences esxB and esxA, which respectively encode CFP-10 (Culture filter protein 10, CFP-10) and ESAT-6 (Earlier secreted antigen target 6, ESAT-6), low-molecular-weight proteins that induce potent Th1 responses. Both M.bovis and some clinical pathogenic MTB share the two proteins. ESAT-6 and CFP-10 protein elicit protection against TB in animal models and become two of the most potential candidates in diagnosis and vaccine research. The purpose of this study were to: (1) express ESAT-6 and CFP-10 antigens using attenuated Salmonella and/or chimeric flagellin vector; (2) analyze the mucosal immune responses and their mechanisms induced by recombinant ESAT-6 and CFP-10 proteins through intranasally immunization; (3) study the immune responses initiated by chimeric flagellin fliC/esat co-immunized with BCG.1. Cloning, expression and immunogenic analysis of Mycobacterium tuberculosis ESAT-6 and CFP-10 proteinTo analyze immunogenic characteristics of ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis. ESAT-6 and CFP-10 coding genes were cloned and identified by PCR. Prokaryotic exprssion plasmids pBCX-esat and pET-cfp were constructed harboring the coding gene respectively and transformed into E.coli BL21(DE3). Recombinant bacteria were identified and induced by IPTG. SDS-PAGE results showed that both ESAT-6 and CFP-10 proteins were expressed and the sizes of them were 34kD and 19kD, respectively. Western-blotting results showed that fusion protein CFP-10 could react with the specific mAb 6E8. And, fusion protein ESAT-6 can bind to the serum of TB patients in ELISA assay. C57BL/6 mice were immunized intraperitoneally with ESAT-6 and CFP-10 fusion proteins respectively, which were emulsified by Freund’s adjuvant. At the 7th day of second immunization in three-week intervels, splenocytes were collected from the immunized mice. IFN-γ-producing and IL-4-producing CD4~+ T cells were detected by intracellular cytokine staining assay. The results showed that both ESAT-6 and CFP-10 proteins can induce higher level of IFN-γcompared with that of IL-4. Furthermore, both ESAT-6 and CFP-10 proteins can significantly up-regulate CD69 molecule expression on CD3~+ T cells. Overall, both ESAT-6 and CFP-10 fusion proteins are capable of up-regulating CD69 molecule of CD3~+ T cells and inducing Th1 immune responses.2. Immune responses specific for ESAT-6 and CFP-10 antigen expressed by recombinant Salmonella typhimuriumTo determine mucosal immune responses induced by recombinant Salmonella harboring ESAT-6 and CFP-10 antigens, prokaryotic exprssion plasmids pYA33-esat and pYA33-cfp carrying the ESAT-6 and CFP-10 coding genes were constructed firstly and eletro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria were named as X4550(33-esat) and X4550(33-cfp), respectively. C57BL/6 mice were immunized intranasally with 1×108 cfu recombinant bacteria in 18d intervals. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer’s patches (PP) were collected from mice after second immunization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected using ESAT-6 and CFP-10 proteins as stimuli. The results showed that the immune responses specific for ESAT-6 and CFP-10 proteins could be detected by the ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 and CFP-10 proteins both were biased toward Th1 response, the frequency of IFN-γ-secreting cells was much higher than that of IL-4 -secreting cells. However, in splenocytes and MLN cells, the antigen specific immune responses acted as Th1 and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. Also, costimulatory molecules CD80 and CD86 from splenocytes, MLN, PP and bronchoalveolar lavage fluid (BAF) cells were up-regulated after the second immunization. Furthermore, IgG and IgA antibodies from serum, BAF and fecal extracts were detected by ELISA respectively, and the levels of antibodies in immunized group had significantly increased compared with those of the negative control. These results suggested that intranasal immunization of recombinant Salmonella not only can induce cellular immune responses, but also can elicit humoral and mucosal immune responses. It provided the bases for the development of mocusal vaccine against infectious disease of respiratory tract.3. Immunologic characteristics of ESAT-6 antigen expessed by Salmonella chimeric flagellin To understand the mucosal immune responses against ESAT-6 in the Salmonella chimeric flagellin system, which act as a vector to deliver ESAT-6 antigen of Mycobacterium tuberculosis. Chimeric flagellin gene fliC/esat were constructed by overlap PCR technique. The ESAT-6 coding gene was inserted to the hypervariable region of Salmonella flagellin gene fliC. Prokaryotic exprssion plasmid pET-fliC/esat carrying the chimeric fragment was constructed firstly and eletro-transformed into the strain LB5000 of Salmonella typhimurium, the recombinant bacteria were named as LB5000(fliC/esat). And then, the transduction was conducted between LB5000(fliC/esat) and Salmonella dublin strain SL5928 using bacteriophage P22HT int. The positive transductants were further identified by PCR, motolity observation and serological agglutination assay. In order to determine the invasion and adhesion capacities of SL5928(f/e). The colon carcinoma cells HT-29 were infected with SL5928(f/e). The results showed that SL5928(f/e) had favourable invasion and adhesion. It was detected TLR-5 mRNA from BMDC, F4/80+ MФand enterocytes infected with SL5928(f/e) by RT-PCR. All three type cells expressed TLR-5 mRNA at 3h post infection and the quantity of TLR-5 mRNA up-regulated at 6h. Furthermore, C57BL/6 mice were immunized intranasally with 1×107 cfu SL5928(f/e) in 18d intervals. Cells from spleen, lung, MLN, PP and nasopharynx associated lymph node (NALT) were harvested from mice after second immunization, and the specific IFN-γ-secreting cells and IL-4 secreting cells were detected using ESAT-6 protein or peptide of ESAT-6(1-20aa) as stimuli. The results showed that in lung, PP cells and NALT cells, immune responses against ESAT-6 protein or ESAT-6 peptide (1-20aa) both were biased toward Th1 response, the frequency of IFN-γ-secreting cells was markedly higher than that of IL-4 -secreting cells. However, in spleen and MLN cells, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. Then, the percentage of specific lysis of SL5928(f/e) was higher than that of X4550(33-esat). Finally, the levels of IgG and IgA antibodies from serum and BAF in immunized group were significantly higher compared with those of the control group. These results suggested that Salmonella chimeric flagellin delivery system was effective after intranasal immunization. The cellular and mucosal immune responses specific for ESAT-6 can be induced by tranductant SL5928(f/e). This system could develop a new pathway for TB vaccine.4. Immune responses initiated by intranasal co-immunization of chimeric flagellin fliC/esat and BCGTo define the role of ESAT-6 chimeric flagellin in TB immunology, prokaryotic expression plasmids pET-fliC/esat and pET-fliC were transformed into E.coli and induced by IPTG. SDS-PAGE results showed the ESAT-6 chimeric flagellin and fliC protein both are soluble. The sizes of the two proteins were 64kD and 57kD respectively. Western-blotting analysis showed that chimeric flagellin and fliC protein both could be bind to serum of Salmonella typhymurium. BMDC maturation was triggered by both ESAT-6 chimeric flagellin and fliC protein. In contrast, ESAT-6 protein alone didn’t activate BMDC. There were up-regulated the expression level of CD40, CD80, CD86 and CD54 costimulatory molecules on F4/80+ MФafter the stimulation of ESAT-6, ESAT-6 chimeric flagellin and fliC protein, respectively. IL-12p70 was detected in the superantants of BMDC and F4/80+ MФ. The results showed that chimeric flagellin was able to induce higher level of IL-12p70 than that of ESAT-6 protein. However, there weren’t any IL-12p70 from F4/80+ MФafter the three proteins stimulating. In order to further demonstrate the adjuvant activity of flagellin, C57BL/6 mice were immunized intravenously with ESAT-6 chimeric flagellin. The results showed that ESAT-6 chimeric flagellin could enhance the immunogenic activity of ESAT-6 protein. Furthermore, mice were co-immunized with BCG and ESAT-6 chimeric flagellin. This immune strategy increased the level of immune responses and biased toward Th1 response in lung, PP, NALT and spleen. Percentage of CD8+CD69+ cells of co-immunization group were higher than that of BCG or ESAT-6 chimeric protein group. Overall, flagellin can present adjuvant activity for ESAT-6 protein, and the novel co-immunization of Chimeric flagellin and BCG could enhance the specific Th1 immune responses.更多还原