IDENTIFICATION AND CLONING OF A 27-KDA COXIELLA-BURNETII IMMUNOREACTIVE PROTEIN
IDENTIFICATION AND CLONING OF A 27-KDA COXIELLA-BURNETII IMMUNOREACTIVE PROTEIN
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DOI：
10.1111/j.1749-6632.1990.tb42263.x
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发表时间：
1990-06-26
影响因子：
5.2
通讯作者：
MALLAVIA, LP
中科院分区：
文献类型：
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作者：
HENDRIX, LR;SAMUEL, JE;MALLAVIA, LP
急性Q热患者会产生伯氏柯克斯体特异性抗体，慢性病患者通常会产生极高水平的伯氏柯克斯体特异性抗体。抗体反应性内毒素已被很好地描述，但仅有少数免疫反应蛋白被鉴定出来。一个约62 kDa的免疫活性蛋白已被克隆、测序，并被证明与一种大肠杆菌热休克蛋白有关。还鉴定了一个约27 kDa的免疫活性外膜蛋白(S)。我们已经开始用基因克隆的方法来鉴定伯氏梭菌免疫反应蛋白。以lambda载体EMBL3为载体，构建了伯氏狸殖吸虫9英里总1期(急性株)DNA基因库。这个库筛选出与大肠杆菌预吸收的兔抗血清发生反应的斑块，这些抗血清是针对纯化的9英里伯氏隐孢子虫全细胞的。从3000个斑块中筛选出23个免疫反应斑块。22个斑块产生了一个约60-65 kDa的免疫反应蛋白。其中一个斑块产生了一个约27 kDa的免疫反应蛋白，以及一个约60-65 kDa的免疫反应蛋白。噬菌体提取物被用来浓缩抗血清，以产生约27 kDa的特异性抗血清。这种浓缩的血清与所有被测试的伯氏梭菌分离物中约27 kDa的蛋白质发生反应，包括来自慢性和急性分离株的分离物。DNA杂交结果表明，免疫活性克隆中的DNA是伯氏梭菌特异的。我们目前正在亚克隆约27 kDa的蛋白编码区，以进行测序，以确定该基因是否编码先前确定的约27 kDa的免疫优势蛋白。这种蛋白最终可能同时具有诊断和疫苗生成的潜力。
Patients with acute Q fever develop Coxiella burnetii-specific antibody, and chronic disease patients often develop extremely high levels of C. burnetii-specific antibody. Antibody-reactive LPS has been well characterized, but only a few immunoreactive proteins have been identified. An immunoreactive ca. 62-kDa protein has been cloned, sequenced, and shown to be related to an Escherichia coli heat-shock protein. A ca. 27-kDa immunoreactive outer membrane protein(s) has also been identified. We have begun characterizing C. burnetii immunoreactive proteins by gene-cloning methods. A gene bank of total C. burnetii Nine Mile phase 1 (acute strain) DNA was created using the lambda vector EMBL3. This bank was screened for plaques which reacted with E. coli-pre-absorbed rabbit antisera specific for purified, Nine Mile C. burnetii whole cells. Twenty-three immunoreactive plaques were identified from a screening of 3000 plaques. Twenty-two plaques produced a ca. 60-65-kDa immunoreactive protein. One plaque produced a ca. 27-kDa immunoreactive protein, as well as a ca. 60-65-kDa immunoreactive protein. Phage extracts of this plaque were used to enrich the antisera to produce a ca. 27-kDa-specific antisera. This enriched sera reacted with a ca. 27-kDa protein in all C. burnetii isolates tested, comprising isolates from both chronic and acute strains. The DNA contained in the immunoreactive clone was C. burnetii-specific, as shown by DNA hybridization. We are currently subcloning the ca. 27-kDa protein-coding region for sequencing to determine if this gene encodes the previously identified ca. 27-kDa immunodominant protein. This protein may ultimately have both diagnostic and vaccinogenic potential.