Ariel N, Zvi A, Grosfeld H, Gat O, Inbar Y, Velan B, Cohen S, Shafferman A
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona 74100, Israel.
Infect Immun. 2002 Dec;70(12):6817-27. doi: 10.1128/IAI.70.12.6817-6827.2002.
A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.
开展了炭疽芽孢杆菌毒力质粒pXO1的基因组分析，旨在鉴定潜在的疫苗候选物和毒力相关基因。对143个先前定义的开放阅读框（ORF）（R.T. 冲中、K. 克劳德、O. 汉普顿、A.R. 霍夫马斯特、K.K. 希尔、P. 凯姆、T.M. 克勒、G. 拉姆克、S. 熊野、J. 马希隆、D. 曼特、Y. 马丁内斯、D. 里克、R. 斯文森和P.J. 杰克逊，《细菌学杂志》181:6509 - 6515，1999年）进行了广泛的序列相似性搜索（使用非冗余和未完成的微生物基因组数据库），以及基序、细胞定位和结构域分析。对枯草芽孢杆菌、嗜碱芽孢杆菌和蜡样芽孢杆菌的相关基因组以及苏云金芽孢杆菌以色列变种的pBtoxis质粒进行了比较基因组学分析。结果，有功能线索的ORF的比例从大约30%（如先前报道）增加到了60%以上。生物信息学分析有助于鉴定与发病机制和毒力可能相关的新基因。基于我们的分析，选择了11个推定蛋白作为功能基因组学研究的靶点。开发了一种快速有效的功能筛选方法，其中将所选ORF的PCR扩增全长线性DNA产物进行体外转录和直接翻译，并根据它们与抗炭疽芽孢杆菌超免疫抗血清的反应性评估其免疫原性。在选择进行分析的11个ORF中，9个成功表达为全长多肽，其中3个被发现具有抗原性并有免疫原性潜力。目前正在对后几个ORF进行评估以确定其疫苗潜力。
