Utility of the Trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening
Utility of the Trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening
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DOI：
10.1128/iai.72.11.6245-6254.2004
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发表时间：
2004-11-01
影响因子：
3.1
通讯作者：
Garg, N
中科院分区：
文献类型：
--
作者：
Bhatia, V;Sinha, M;Garg, N
糖基磷脂酰肌醇（GPI）锚定蛋白在克氏锥虫的感染和细胞内阶段大量表达，并且被免疫系统的体液和细胞臂识别为抗原靶标。以前，我们证明了编码GPI锚定蛋白的基因在引发对T. Cruzi感染和疾病，表明它们作为候选疫苗的效用。为了鉴定额外的疫苗靶点，在本研究中，我们筛选了T。cruzi表达序列标签（EST）和基因组序列调查（GSS）数据库。通过应用各种基于网络的基因组挖掘工具对2,500个相似序列进行分析，我们鉴定了348个（37.6%）EST和260个（17.4%）GSS序列编码新的寄生虫特异性蛋白。其中，19个序列表现出分泌和/或膜相关GPI蛋白的特征。从所选序列中扩增出8个基因TcG 1、TcG 2、TcG 3、TcG 4、TcG 5、TcG 6、TcG 7和TcG 8（TcG 1-TcG 8），这些基因在寄生虫的不同发育阶段中表达，并在多种T. cruzi菌株。流式细胞术证实克隆基因编码的抗原在锥鞭毛体和/或无鞭毛体阶段的T.克鲁兹当作为DNA疫苗递送时，基因TcG 1-TcG 6在小鼠中引发寄生虫特异性抗体应答。除TcG 5外，TcG 1-TcG 6基因的抗血清对T. cruzi，一种已知与T.克鲁兹总而言之，我们的结果验证了生物信息学在基因组挖掘中的适用性，从而鉴定了T。cruzi膜相关蛋白是潜在的候选疫苗。
Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genomemining tools to the analysis of similar to2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasite-specific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.