An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas
An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas
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DOI：
10.1099/00221287-147-3-571
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发表时间：
2001-03-01
期刊：
影响因子：
2.8
通讯作者：
Kirkpatrick, BC
中科院分区：
文献类型：
--
作者：
Blomquist, CL;Barbara, DJ;Kirkpatrick, BC
膜蛋白介导几个重要的过程，包括附着，在几个柔膜菌属物种。植原体是一种不可培养的植物病原性柔膜菌，通过某些取食韧皮部的昆虫载体以特定方式传播。由于植原体膜蛋白很可能参与传播过程的某些方面，因此它们的鉴定、分离和表征是理解植原体传播的重要的第一步。一个32 kDa的免疫显性蛋白（IDP）从西方X-疾病（WX）植原体纯化感染的植物通过免疫沉淀使用单克隆抗体，和两个肽从胰蛋白酶消化进行测序。根据这些序列设计的PCR引物扩增出145 bp的产物，该产物与WX相关植原体在Southern印迹中杂交。该PCR产物用于鉴定克隆和测序的2.5kbp EcoRI-HindIII片段。鉴定了一个完整的864 bp ORF（idpA），其推定的翻译产物含有用于设计PCR引物的两个胰蛋白酶消化肽序列。预测的IdpA序列的分析表明两个跨膜结构域，但没有切割点。氨基酸序列与已知植原体IDP基因没有明显的同源性。将idpA ORF克隆到大肠杆菌表达载体中，并使用WX特异性抗血清在Western印迹中鉴定预测大小的融合蛋白。制备了纯化的表达蛋白的兔多克隆抗血清，并与E.大肠杆菌表达的和天然的WX植原体蛋白。这种新鉴定的WX IDP（IdpA）不同于其他已知的柔膜蛋白。
Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2.5 kbp EcoRI-HindIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.