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首页> 外文期刊>Journal of Clinical Microbiology >Enhanced Patient Serum Immunoreactivity to Recombinant Mycobacterium tuberculosis CFP32 Produced in the Yeast Pichia pastoris Compared to Escherichia coli and Its Potential for Serodiagnosis of Tuberculosis
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Enhanced Patient Serum Immunoreactivity to Recombinant Mycobacterium tuberculosis CFP32 Produced in the Yeast Pichia pastoris Compared to Escherichia coli and Its Potential for Serodiagnosis of Tuberculosis

机译：与大肠杆菌相比，提高了对酵母巴斯德毕赤酵母中产生的重组结核分枝杆菌CFP32的患者血清免疫反应性及其对结核病的血清学诊断的潜力

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CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.
机译：CFP32是一种结核分枝杆菌复合物限制性分泌蛋白，以前据报道存在于患有活动性结核病（TB）患者的大部分痰液样本中，并能刺激血清抗体产生。因此，CFP32（最初标注为Rv0577，也称为TB27.3）被认为是快速诊断TB的良好候选靶抗原。但是，CFP32血清识别的最大敏感性可能在早期研究中受到了限制，因为在大肠杆菌中产生的重组CFP32（rCFP32）被用作测试抗体捕获抗原，潜在的缺点是由于细菌蛋白质翻译后修饰的差异所致。为了进一步研究在不同异源宿主中合成的rCFP32的血清诊断潜力，我们在酵母 Pichia pastoris 中表达了rCFP32。与 E相比。大肠杆菌rCFP32，酵母rCFP32在Western blot研究中显示出更高的捕获多克隆抗血清的能力。同样，通过酶联免疫吸附测定（ELISA），来自结核病患者和健康的牛分枝杆菌卡麦特-圭兰（BCG）接种疫苗的个体的血清中，酵母rCFP32的识别效果明显优于 E．大肠杆菌 rCFP32。在随后的测试中，基于酵母rCFP32的抗体捕获ELISA对BCG疫苗（ n = 40）中活动性结核病例（ n = 40）的区分灵敏度为85％，特异性为98％ > n = 39）。与使用 E的测试相比，单抗原TB血清学诊断测试的灵敏度高得惊人。大肠杆菌表达的抗原。总体而言， P中的rCFP32的 trans 生产。 pastoris 显着改善了患者血清中CFP32特异性抗体的血清学检测，从而提供了一种新的，可能更好的方法，可用于生产具有诊断潜力的抗原，用于开发针对结核病和其他传染病的免疫测定方法。

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