Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)
Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)
复制标题
家蚕微孢子虫孢子壁蛋白的蛋白质组学分析和两种孢子壁蛋白的鉴定
DOI：
10.1002/pmic.200700584
复制
发表时间：
2008-06-01
期刊：
影响因子：
3.4
通讯作者：
Xiang, Zhonghuai
中科院分区：
文献类型：
--
作者：
Wu, Zhengli;Li, Yanhong;Xiang, Zhonghuai
微孢子虫是真菌样单细胞真核生物，发育为专性细胞内寄生虫。它们分化成抗性孢子，受到厚孢子壁的保护，孢子壁由富含糖蛋白的外层或外孢子和富含几丁质的内层或内生孢子组成。在这项针对家蚕病原体家蚕微孢子虫的研究中，我们通过基于蛋白质组学的方法、MALDI-TOF MS 和 LC-MS/MS 分析了孢子壁蛋白（SWP），总共获得了 14 种假设的孢子壁蛋白（HSWP）或肽。此外，我们通过 SDS-PAGE 检查了 SWP，检测到三种主要孢子壁肽，分子量分别为 32.7 kDa (SWP32)、30.4 kDa (SWP30) 和 25.3 kDa (SWP25)。通过N端氨基酸残基测序，并检索家蚕基因组DNA鸟枪数据库，获得了SWP30和SWP32的完整ORF，分别编码278个和316个氨基酸的肽。针对大肠杆菌中产生的 SWP30 和 SWP32 重组蛋白产生了小鼠多克隆抗体，间接免疫荧光测定 (IFA) 和免疫电子显微镜 (IEM) 分析的结果表明 SWP30 是内孢子蛋白，而 SWP32 是外孢子蛋白。 SWP30 和 SWP32 均包含在 MS 鉴定的 14 个 HSWP 中，证实了基于蛋白质组学方法的结果。 （GenBank (TM)、EMBL 和 DDBJ 登录号：NbHSWP1-NbHSWP12，登录号 EF683101-EF683112。NbHSWP13 和 NbHSWP14，登录号 EU179719 和 EU179720）。
Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches. (GenBank (TM), EMBL and DDBJ accession numbers: NbHSWP1-NbHSWP12, accession no. EF683101-EF683112. NbHSWP13 and NbHSWP14, accession no. EU179719 and EU179720).