【作者】 朱泳璋；

【导师】 褚嘉祐； 郭晓奎； 张庶民；

【作者基本信息】 中国协和医科大学 ， 分子遗传学， 2007， 博士

【摘要】 百日咳是一种由百日咳杆菌传播，侵袭呼吸道并引发急性呼吸道感染的传染病，每年导致35万儿童死亡，感染百日咳的病例数超过4000万，主要都集中在发展中国家。百日咳疫苗是预防百日咳流行和爆发的最有力的手段。自20世纪50年代百日咳全细胞疫苗即菌体疫苗应用以来，已经极大地降低了百日咳的发病率和死亡率。据世界卫生组织报道，实施百日咳菌体疫苗接种已减少了8561。1万个病例与72.6万例儿童死亡。但是由于接种产生的严重副反应，近年来，百日咳全细胞疫苗正逐渐被第二代百日咳疫苗即无细胞疫苗取代。一直到现在，即使百日咳疫苗已经普遍推广，百日咳仍然是公共健康卫生的一个重大威胁，百日咳发病率一直在显著提高。可能的原因包括：1．目前使用的百日咳全细胞疫苗和无细胞疫苗都不能对儿童产生终生免疫，一般3—5年后保护力就逐渐消失；2．副百日咳杆菌也能引起百日咳，不过症状要比百日咳杆菌引起的要轻，而且病症耐受期也要短。当前使用的百日咳疫苗对越来越来多的副百日咳感染者没有保护力或保护力不足。因此需要进一步提高现有百日咳疫苗的保护效力。由于人是百日咳杆菌的唯一宿主，缺乏合适的动物研究模型，百日咳杆菌的致病机理以及导致宿主产生的免疫机制一直还不是完全清楚，极大地限制了发展百日咳新疫苗。当前对百日咳疫苗的研究主要是融合表达多抗原或DNA疫苗等基因工程疫苗，但这些新疫苗的免疫效果都不令人满意。除了疫苗预防外，药物的预防和治疗也是控制百日咳流行的必不可少的手段。到目前为止，百日咳治疗常用的抗生素(主要是大环内酯类抗生素)都或多或少产生一些严重的副作用，尤其对婴幼儿，而且没有一个大环内酯类抗生素被美国FDA批准应用到6个月以下的婴儿身上。因此，现在急需找到针对百日咳杆菌和副百日咳杆菌的疫苗候选抗原和药物靶点。免疫蛋白质组学技术，融合了抗体识别的特异性和高分辨率能力的2D双向电泳和精确的质谱分析，广泛应用于筛选疫苗抗原、诊断抗原、新型药物和临床标志物等研究。在本研究中，免疫蛋白质组学技术被首次应用鉴定百日咳杆菌的免疫原性蛋白。百日咳杆菌的膜蛋白和胞外分泌蛋白分别用经典的TritonX-114和TCA—丙酮沉淀法制备。免疫印迹使用免疫百日咳全细胞疫苗的儿童血清。MADLI-TOF／MS质谱—共鉴定出了32个免疫原性蛋白，包括27个膜蛋白和11个胞外蛋白。在这些免疫原性蛋白中，69KDa蛋白或粘附素、血清抗性蛋白和外膜孔蛋白是已知的百日咳杆菌重要的毒力因子，其中粘附素还是百日咳无细胞疫苗的重要成分之一；血清抗性蛋白也是百日咳杆菌抵抗宿主机体内补体杀伤作用的决定因素。而编码外膜配体结合蛋白的基因是唯一一个被鉴定出的是Bvg—中间相的基因，Bvg—中间相被认为是百日咳杆菌从体外传播进入到人体内起始粘附过程的一个重要的中间环节。蛋白合成延伸因子Tu／Ts、3-磷酸甘油醛和分子伴侣蛋白GroEL是在许多病原微生物免疫蛋白质组学研究中都被鉴定出具有强免疫原性的标志性蛋白，在本研究中，也被鉴定出来了。乳酸脱氢酶、琥珀酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶、谷氨酸脱氢酶、异丙基异构酶、青霉素结合蛋白和ATP结合蛋白虽然是首次在百日咳杆菌中被证实具有免疫原性，但是这些免疫原性蛋白已经在其他一个或多个病原微生物的免疫蛋白质组学研究中被鉴定出来了。其他被发现具有免疫原性的蛋白还包括硫酸盐结合蛋白、2-羟基酸脱氢酶、氨基酸周质结合蛋白和假定蛋白BP3575和BP2818等，尤其是假定蛋白BP3575和BP2818，虽然功能未知，但是却具有良好的免疫原性，更值得深入去研究。通过对这些免疫原性蛋白的深入研究有助于更好地了解百日咳杆菌的致病机制以及与宿主的相互作用。为研究新的百日咳疫苗提供一定的理论基础。除了免疫蛋白质组学研究外，本研究还整合了生物信息学分析、比较基因组杂交数据、基因表达谱和蛋白质相互作用网络来筛选针对百日咳杆菌和副百日咳杆菌的疫苗候选抗原和药物靶点。在百日咳杆菌3436个ORFs中，总共731个基因，包括671个在百日咳杆菌GMT-1菌株和副百日咳杆菌12822菌株都高度表达基因和81个百日咳杆菌GMT-1菌株的37℃表达上调基因，这37℃表达上调基因同时也存在于副百日咳杆菌基因组，这731个基因被筛选出来作为初步的候选抗原。在去掉胞质蛋白、跨膜结构超过4的内膜蛋白、在137株百日咳杆菌和28株副百日咳杆菌中不保守的蛋白以及与宿主高度同源的蛋白后，最后191个抗原被筛选出来作为针对百日咳杆菌和副百日咳杆菌的候选抗原。这191个候选抗原或者具有高度转录水平，或者和细菌毒力与致病性高度相关，而且这些抗原不仅暴露于细菌表面，还在165株百日咳杆菌和副百日咳杆菌中高度保守，在宿主体内没有高度同源蛋白，不会产生交叉反应，损害宿主。在百日咳杆菌和副百日咳杆菌的蛋白质相互作用网络中，从191个候选抗原进一步鉴定出22个高度连接蛋白，这22个蛋白都参与细菌能量或物质代谢等重要过程，为百日咳杆菌和副百日咳杆菌维持基本的生命活动所必需，能作为合适的百日咳治疗药物靶点。对这191个疫苗候选抗原包括22个潜在的药物靶点的研究能加速发展更有效的百日咳新疫苗和药物。更多还原

【Abstract】 Whooping cough or pertussis has led to 350,000 children deaths mainly in developing countries and has afflicted up to 40 million children worldwide per year. The introduction of pertussis vaccines in the 1950s has significantly reduced the morbidity and mortality of the disease. In view of their serious side effects in infants and children, the second-generation acellular vaccines have gradually replaced whole-cell pertussis vaccines during recent years. However, despite high vaccination coverage, whooping cough caused by both B. pertussis (B.p) and B. parapertussis (B.pp) is still a serious public health threat and the incidence of pertussis has continued to rise. the main reasons include: 1. the currently used pertussis vaccines does not produce life-long protection, after 3-5 years, the vaccine-induced immunity would be too weak to protect children; 2. Pertussis vaccines have low efficiency against increasing numbers of B.pp infections. So the protective effectiveness of the currently used pertussis vaccines needs to be significantly improved. Because the pathogenicity and immunological mechanism have been not completely clarified, the current researches focusing on developing novel pertussis vaccines, such as fusion expressin of several protective antigens and pertussis DNA vaccine, which could not be satisfactory. In addition to vaccine prevention, drug therapy is also necessary for controlling whooping cough. Macrolide antibiotics have been the antimicrobial of main choice for treatment or postexposure prophylaxis of pertussis, however, macrolide antibiotics frequently produce a lot of serious reverse reactions especially against infants, so no recent macrolide antibiotics have been licensed for use in infants aged＜6 months by the FDA of the USA. Therefore, there is an urgent need to develop novel vaccine candidates and drug targets against both B.p and B.pp. The immunoproteomics techniques, combining specificity of antibody with high resolving power of 2D and precision of mass spectral analysis, has globally applied to search for vaccine candidates、novel drug targets、diagnostic antigens and clinical biomarker. In this article immunoproteomics techniques were firstly used to screen immunogenic proteins from membrane proteins and total extracellular proteins of Bordetella pertussis. The membrane proteins were enriched using Triton X-114 extraction protocol. The total extracellular proteins were isolated using classic TCA method. By using the sera of immunized infants with whole-cell pertussis vaccine, Western blotting was performed with these proteins. Total 32 unique immunogenic proteins including 11 extracellular proteins and 27 membrane proteins were identified successfully by MADLI-TOF/MS. In total immunogenic proteins, 69KDa protein or peractin、serum resistantance protein and outer membrane porin protein were known pertussis virulence factors; putative outer membrane ligand binding protein was important Bvg-intermediate phase protein; elongation factor Tu/Ts、glyceraldehyde-3-phosphate dehydrogenase and chaperonin GroEL which were identified as globally immunogenic proteins in several pathogens such as H.p、Group A streptococcus and Candida albicans, were also identified in our study. Putative L-lactate dehydrogenas、glutamate dehydrogenase、succinate dehydrogenase、isocitrate dehydrogenas、putative penicillin-binding protein precursor firstly identified as immunogenic proteins of B.pertussis have been validated to have immunogenity in other pathogen immunoproteomic researches. Other novel immunogenic proteins included sulfate-binding protein precursor、putative 2-hydroxyacid dehydrogenase、amino acid-binding periplasmic protein、hypothetical protein BP3575 and BP2818. BP3575 and BP2818 with unknown function and strong immunoreactivity should be paid more attention. Further reseach focusing on these immunogenic proteins will devote to known pathogenicity and immunological mechanism of B.pertussis.In addition to immunoproteomic analysis, this preliminary work also integrates results from in silico analysis, comparative genomic hybridization, global transcriptional profiling and protein-protein interaction (PPI) networks to screen potential vaccine candidates and drug targets from B.p against both B.p and B.pp. Of 3436 open reading frames (ORFs) in B.p, 671 had high transcriptional levels in the B.p GMT-1 and B.pp 12822 strain, and 81 Bvg-activated (up-regulated) B.p genes were also found in the genome of B.pp. Seven hundred and thirty-three unique genes, 671 highly expressed and 81 Bvg-activated, were regarded as the preliminary vaccine candidates. When cytoplasmic proteins, inner membrane proteins (IMPs) with transmembrane topologies＞4, highly variable B.p genes among the 165 B.p and B.pp strains, and human homologs in B.p were subtracted from these 733 candidates, 191 genes remained. These 191 were thus identified as the final vaccine candidates against both B.p and B.pp. They had high transcriptional levels in both species, or were associated with virulence and pathogenesis. Moreover, the proteins encoded by these genes were not only potentially surface-exposed in the bacteria, but also well-conserved among the 165 B.p and B.pp strains. We also constructed two PPI networks of B.p and B.pp. Among the 191 vaccine candidates, 22 proteins that were highly essential in both these networks were regarded as suitable drug targets against the two species.Using the theory of reverse vaccinology, 191 potential cross-protective vaccine candidates against both B.p and B.pp were identified among the 3436 ORFs in the B.p genome. Of these 191 vaccine candidates, 22 with high essentiality can be used as suitable drug targets for both species. Further research focusing on the 191 vaccine candidates could accelerate the development of more effective pertussis vaccines and drug therapy against B.p and B.pp infection in the future.更多还原