Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.
Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.
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DOI：
10.1371/journal.pntd.0003006
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发表时间：
2014-07
影响因子：
3.8
通讯作者：
Büscher P
中科院分区：
文献类型：
--
作者：
Rogé S;Van Nieuwenhove L;Meul M;Heykers A;Brouwer de Koning A;Bebronne N;Guisez Y;Büscher P
冈比亚昏睡病筛查试验，如CATT/T。B. gambiense和最近开发的侧流试验，迄今为止都是基于天然变体表面糖蛋白（VSG），即LiTat 1.3和LiTat 1.5，从啮齿动物中生长的高毒力锥虫菌株中纯化。我们已经表达了SUMO（小泛素样修饰）融合蛋白的免疫原性N-末端的一部分，这些抗原在酵母毕赤酵母。将分泌的重组蛋白进行亲和纯化，产量高达10 mg/L细胞培养物。在ELISA中对来自88名HAT患者和74名地方性非HAT对照的血清证实了每种单独抗原和两种抗原的混合物的诊断潜力。用重组蛋白替代昏睡病筛查试验中的天然抗原将消除抗原生产期间实验室工作人员的感染风险和对实验室动物的需求。例如在生物发酵罐中重组抗原的规模化生产是直接的，因此导致抗原生产的标准化提高和生产成本降低，这反过来将增加消除冈比亚HAT所需的诊断测试的可用性和可负担性。慢性昏睡病或冈比亚人非洲锥虫病（HAT）的人群筛查仍然基于针对天然变体表面糖蛋白（VSG）LiTat 1.3的抗体检测试验。这种蛋白质是通过大量感染实验室动物的剧毒寄生虫产生的。我们的目标是用在酵母毕赤酵母中表达的重组VSG（LiTat 1.3和LiTat 1.5）取代这种天然抗原。用来自HAT患者和阴性对照的血清在ELISA中证实了这些重组体的诊断潜力。用这些重组体替换天然LiTat 1.3 VSG将防止实验室动物的感染和处死以及与筛选试验生产相关的固有感染风险。例如在生物发酵罐中重组抗原的规模化生产是直接的，因此导致抗原生产的标准化提高和生产成本降低，这反过来将增加消除冈比亚HAT所需的诊断测试的可用性和可负担性。
Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents. We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture. The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT. Population screening for the chronic form of sleeping sickness or gambiense human African trypanosomiasis (HAT) is still based on an antibody detection test against the native variant surface glycoprotein (VSG) LiTat 1.3. This protein is produced through massive infections of lab animals with highly virulent parasites. We aim to replace this native antigen with recombinant VSGs, both LiTat 1.3 and LiTat 1.5, expressed in the yeast Pichia pastoris. The diagnostic potential of these recombinants was confirmed in ELISA with sera from HAT patients and negative controls. Replacement of the native LiTat 1.3 VSG with these recombinants would prevent the infection and sacrifice of lab animals and the inherent infection risk linked to the production of the screening test. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.
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影响因子：
56.3
作者：
Deborggraeve, Stijn;Buscher, Philippe
通讯作者：
Buscher, Philippe
影响因子：
2.7
作者：
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2.1
作者：
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影响因子：
3.3
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影响因子：
3.8
作者：
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通讯作者：
Bieler, Sylvain