Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray
Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray
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DOI：
10.1128/cvi.00300-08
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发表时间：
2008-12-01
影响因子：
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通讯作者：
Heinzen, Robert A.
中科院分区：
文献类型：
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作者：
Beare, Paul A.;Chen, Chen;Heinzen, Robert A.
Q热是由贝氏柯克斯体引起的一种广泛传播的人畜共患病。Q热的诊断通常基于对患者血清的血清学检测。检测试剂盒的诊断抗原为Nine Mile参考菌株的福尔马林固定I相和II相微生物。这种抗原的缺陷包括（i）与其他病原体交叉反应的可能性;（ii）无法区分C。burnetii菌株;和（iii）需要繁殖和纯化C. burnetii，一个困难的和潜在的危险的过程。因此，需要利用确定的抗原，如重组C. burnetii蛋白。在这里，我们描述了一个C的使用。贝氏梭菌蛋白质芯片，以全面鉴定免疫显性抗原的抗体识别的背景下，人类C。贝氏体感染或接种疫苗。对应于1，988个C的转录活性PCR产物。产生贝氏体开放阅读框（ORF）。全长蛋白的成功合成从75%的ORF通过使用大肠杆菌为基础的体外转录和翻译系统（IVTT）。用粗制IVTT裂解物点样硝酸纤维素微阵列，并用来自急性Q热患者和用Q-Vax接种的个体的血清探测。免疫血清与约50 ℃反应强烈。Burnetii蛋白，包括先前鉴定的免疫原、含锚蛋白重复结构域的蛋白和多种假设蛋白。产生与所选阵列反应性抗原相对应的重组蛋白，并通过酶联免疫吸附测定确认免疫反应性。建立了一种灵敏、高通量的免疫反应性C。贝氏体蛋白将有助于开发基于重组抗原的Q热血清诊断测试。
Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.