Anderson B, Lu E, Jones D, Regnery R
Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
J Clin Microbiol. 1995 Sep;33(9):2358-65. doi: 10.1128/jcm.33.9.2358-2365.1995.
A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay. Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision. All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen. The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes. Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD. The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.
构建了汉赛巴尔通体（罗卡利马体）DNA文库，该文库克隆于载体λZAPII中，并使用间接荧光抗体（IFA）检测确诊为猫抓病（CSD）且对巴尔通体属有抗体的患者血清池，筛选抗原蛋白的表达情况。通过体内切除将10个免疫反应性噬菌体亚克隆为重组质粒。当用人类血清池进行免疫印迹检测时，所有10个重组体均表达一种约17 kDa的蛋白质。对每个重组质粒进行限制性内切酶消化显示出7种图谱，这表明克隆偏差不是重复分离表达17 kDa抗原的克隆的原因。对编码17 kDa抗原的基因进行测序，结果显示其编码一个148个氨基酸的开放阅读框，预测分子量为16,893 Da。推导的氨基酸序列的氨基末端本质上是疏水的，在大小和组成上与革兰氏阴性菌中发现的信号肽相似。推导的氨基酸序列的其余部分更亲水，可能代表表面暴露的表位。将17 kDa抗原进一步亚克隆为表达载体PinPoint Xa-2中的生物素化融合蛋白，得到一种30 kDa的蛋白质，该蛋白质在免疫印迹上与CSD患者的个体血清样本具有高度反应性。免疫印迹分析中与30 kDa融合蛋白的反应性与IFA检测结果之间的一致性，IFA阳性血清为92%，IFA阴性血清为88%。重组表达的17 kDa蛋白作为CSD和巴尔通体感染血清学诊断的抗原应具有价值，值得进一步研究，以尝试开发一种亚单位疫苗，预防猫长期感染巴尔通体以及这些病原体进一步传播给人类的可能性。
