EXPRESSION OF A PLASMID GENE OF CHLAMYDIA-TRACHOMATIS ENCODING A NOVEL 28-KDA ANTIGEN
EXPRESSION OF A PLASMID GENE OF CHLAMYDIA-TRACHOMATIS ENCODING A NOVEL 28-KDA ANTIGEN
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DOI：
10.1099/00221287-139-5-1083
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发表时间：
1993-05-01
期刊：
影响因子：
--
通讯作者：
RATTI, G
中科院分区：
文献类型：
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作者：
COMANDUCCI, M;CEVENINI, R;RATTI, G
质粒 pCT 基本上存在于所有沙眼衣原体分离株中，并且可能编码对于在自然环境中生存重要的因子。然而，迄今为止尚未描述与 pCT 相关的表型。为了研究 pCT 在沙眼衣原体致病性中的作用的可能性，我们检查了可能编码 28 kDa 多肽 (pgp3) 的 ORF (ORF3) 的表达。从感染后 20 小时起，对从衣原体感染的 Vero 细胞中提取的 RNA 进行分析，检测到 ORF3 特异性转录物，主要为包含下游 ORF4 序列的 1390 个核苷酸的离散 RNA 种类。 ORF3 DNA 在大肠杆菌中克隆并表达为 39 kDa 融合蛋白 (MS2/pgp3)。针对纯化的 MS2/pgp3 产生的抗体，可在纯化的衣原体基本体 (EB) 蛋白质的蛋白质印迹上特异性识别 28 kDa 的蛋白质。相同的抗体通过免疫荧光检测甲醇固定的感染细胞中的衣原体包涵体。对用 2% Sarkosyl 提取的 EB 进行蛋白质印迹分析，结果表明大部分 28 kDa 抗原与去污剂不溶性（“膜”）部分相关。在衣原体感染患者的血清中检测到识别 pgp3 表位的抗体，但在血清阴性对照血清中未检测到。这些发现支持这样的假设：pCT 可能提供与衣原体细胞生理学相关的功能。
Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in C. trachomatis pathogenicity, we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2 % Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The findings support the hy thesis that pCT may provide a function related to chlamydial cell physiology.