基因组学在微生物疫苗发展中的应用.ppt

1、The use of genomics in microbial vaccine（微生物疫苗 ）development,Vaccination is one of the most effective tools for the prevention of infectious diseases. The availability(可用性 )of complete genome sequences(序列), together with the progression of high-throughput(高通量 )technologies such as functional and stru

2、ctural genomics(功能和结构基因组学）, has led to a new paradigm(范式)in vaccine development.,Pan-genomic（全基因组 ） reverse vaccinology（反向疫苗学 ）, with the comparison of sequence data from multiple isolates of the same species of a pathogen（病原体 ）, increases the opportunity of the identification of novel vaccine candi

3、dates. Overall, the conventional empiric（经验 的） approach to vaccine development is being replaced by vaccine design. The recent development of synthetic genomics（合成基因组学 ） may provide a further opportunity to design vaccines.,The basic paradigm of vaccine development established by Louis Pasteur at th

4、e end of the 19th century (i.e. isolation, inactivation and injection of the causative agent即病原体的隔离，失活和注射 ) constitutes the foundation of classical vaccinology and led vaccine development throughout the 20th century 3.,Conventional approaches on the basis of theseempiric principles have provided vac

5、cines fromthree major categories:,inactivated microorganisms（灭活微生物 ） live-modified agents （活体修饰的病原体 ） Subunit（亚基 ） vaccines, composed by purified portions of the infectious agent,Shortcomings,Not all pathogens can be grown in culture. The application of safety procedures may be required for pathogen

6、 manipulation. Insufficient killing or attenuation（衰减） may result in the presence of virulent （恶毒）organisms in the final vaccine. Time-consuming and could take decades,Reverse vaccinology: an in silico approach,The approach referred to as reverse vaccinology uses the genome sequences of viral, bacte

7、rial or parasitic pathogens（寄生病原体 ） of interest rather than the cells as starting material for the identification of novel antigens, whose activity should be subsequently confirmed by experimental biology 11.,The first example of a successful application of the reverse vaccinology approach was provi

8、ded by Pizza and coworkers in collaboration with The Institute for Genomic Research (TIGR)（基因组研究所 ）12,13. They describe the identification of vaccine candidates against Neisseria meningitidis （脑膜炎奈瑟菌） serogroup B（血清B ）or MenB, the major cause of sepsis （败血症）and meningitis（脑膜炎 ） in children and young

9、 adults.,The reverse vaccinology approach started from the determination of the complete genome sequence of a MenB pathogenic（致病性 ） strain, MC58. Several computational tools were used to find in the genome, on the basis of sequence features, the presence of amino acid motifs responsible for targetin

10、g the mature protein to the outer membrane（外膜 ） (signal peptides信号肽 ), to the lipid bilayer（脂双层 ）(lipoproteins脂蛋白 ),to the integral membrane（整体膜 ） (transmembrane domains跨膜区 ) or for recognition and interaction with host structures.,This analysis allowed researchers to identify 600 putative（假定 ） surf

11、ace-exposed or secreted（分泌） proteins. Three hundred and fifty proteins were expressed in a heterologous system（异种系统 ）, Escherichia coli, purified and used to immunize mice. Mice immune sera（ 血清 ） were tested for specificity（特异性 ） by Western blot, accessibility（ 无障碍环境 ） on the surface of bacterium by

12、 flow cytometry（流式细胞仪 ） and for the capacity to induce bactericidal antibodies by serum bactericidal assay.,The future: synthetic genomics,What is synthetic genomics? Synthetic genomics is a new discipline (科目) related to the generation of organisms（生物体，有机体） articially（人工的）using genetic material（遗传材

13、料）. Include: It involves the design and assembly（组装、组配）of genes, gene pathways（路径）, chromosomes（染色体） and even whole genomes（基因组 ）by the combination of methods for the chemical synthesis（合成）of DNA with computational techniques（计算机技术）.,purpose,The goal of synthetic genomics is to make extensive（广泛的）ch

14、anges to the DNA of a chromosome（染色体）, assemble（组装、组配） it and insert it into an organism（生物体，有机体） to obtain new genomes able to code（编码）for new types of cells with desired properties（渴望得到的性质）.,Progression and Outlook,The era（时代）of synthetic genomics has ofcially（正式的）begun and very recently the group

15、 of J. Craig Venter, in a work published by Gibson et al., described a multistage process（多段处理）to construct the complete genome of Mycoplasma genitalium（生殖支原体） . This rst construction of a synthetic genome encoding（编码）a living self-reproducing（自我再生的、自我复制的）organism provides（提供了）many potential（潜在的）pos

16、itive commercial（商业）applications（应用）.,Application,In vaccine development, the chemical synthesis（化学合成）of genetic material could involve the creation（创造） of new proteins（蛋白质）, the reduction in cost for protein engineering（蛋白质工程） and structural analysis（结构分析）or the possibility（可能性）of rapidly（快速） gener

17、ating（产生） recombinant（重组） vaccines （疫苗）against emerging microbial diseases（新兴的由微生物引起的疾病）。,Genome-based technologies: functional and structural genomics,Significance and classification The study of bacterial gene （细菌基因）expression and function is essential for understanding pathogenesis（发病机理） and the

18、interaction between pathogen（病原体） and host（宿主）. For this kind of investigation, there are two main subelds（子域） in genomics: functional genomics and structural genomics.,The definition and the basic mechanism,Functional genomics has emerged as a scientic eld from molecular（分子生物学） biology characterize

19、d by the development of large-scale（大规模） technologies such as transcriptomics（转录子） and proteomics（蛋白质组学）. They contribute to vaccinology（疫苗接种学） in the selection of appropriate vaccine candidates （候选疫苗）not by examining directly the genetic content but by the transcription（转录）and expression proles（蛋白质

20、组表达模式）.,Transcriptomics（转录子）provides an overview（概观）of the overall transcriptional activity of a given pathogen（病原体） and allows the comparison（比照） of gene expression under different growth and environmental conditions.,Brief Introduction About Proteomics,Proteomics（蛋白组学） is the study of proteins tha

21、t are expressed in a cell. It has the advantage of dening（定义） proteins that are differentially expressed and differentially located, for example those situated（处于） outside the cell, the so-called surfaceome 39, important in inducing （产生诱导作用的）immune responses.,Discovery Approaches,By this approach, t

22、he protein mixture is rst resolved（分解） into individual components（个别的组分） using separation procedures like 2D gel electrophoresis（凝胶电泳）or liquid chromatography（液相色谱法）, digested（消化） with specic proteases （蛋白酶）and the molecular mass of each peptide fragment(肽的片段) is then measured using matrix-assisted

23、laser desorption（基质辅助激光解析电离）/ionization time-of-ight（电离飞行时间质谱） (MALDI-TOF) and tandem mass spectrometry（串联质谱法）(MS/MS).,The peptide-mass ngerprint(质谱分析,并获得相应蛋白质点的肽指纹图谱) is used for a database search of predicted masses（预测质量） that result from the digestion of known proteins. Classical proteomics（蛋白组学）

24、 approaches provide an experimental support to the in silico（硅） prediction of protein localization（定位） and have already been used to identify additional proteins suitable as vaccine components.,The significance of those approaches,This approach allowed the identication of 28 surface-exposed proteins

25、 suitable as vaccine candidates 17. Additionally, immunoproteomics（免疫蛋白质组学）approaches such as serological proteome analysis（血清蛋白分析） (SERPA), which is a combination of proteomics with serological analysis, have been used for identifying potential vaccine candidates(潜在的候选疫苗).,The impact of Gene sequen

26、ces towards functional genomics,The increase of genome sequence（基因序列） data has also led to the development of structural genomics, a high-throughput application of the traditional structural biology, on the basis of experimental methods such as X-ray crystallography（结晶学）, nuclear magnetic resonance

27、(NMR)（核磁共振） spectroscopy（光谱学） or molecular electron microscopy（分子的电子显微镜法）42. Structural genomics projects are likely to solve and provide high-quality 3D structures for the macromolecules（高分子） encoded by complete genomes and to furnish a library of protein structures（蛋白质结构库）.,The ultimate goal is to

28、 have a complete view of protein folds that may help in assigning function（分配蛋白） for hypothetical proteins（假设蛋白）, with the prediction of the protein structure by computational（计算） approaches such as homology modeling（同源建模法）, founded on similarities with known protein structures for constructing atom

29、ic-resolutionmodels（原子模型解析） from amino acid sequences（氨基酸序列）.,The ultimate goal is to have a complete view of protein folds that may help in assigning function（分配蛋白） for hypothetical proteins（假设蛋白）, with the prediction of the protein structure by computational（计算） approaches such as homology modeling（同源建模法）, founded on similarities with known protein structures for constructing atomic-resolutionmodels（原子模型解析） from amino acid sequences（氨基酸序列）.,History of its Application,A co