Kallio-Kokko H, Vapalahti O, Hedman K, Brummer-Korvenkontio M, Vaheri A
Department of Virology, University of Helsinki, Finland.
J Clin Microbiol. 1993 Mar;31(3):677-80. doi: 10.1128/jcm.31.3.677-680.1993.
Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.
普马拉病毒是流行性肾病（NE）的病原体，流行性肾病是一种汉坦病毒感染，在北欧和中欧广泛发生，通常通过间接免疫荧光（IF）法进行诊断。我们现在利用pEX2表达载体在大肠杆菌中将普马拉病毒索特卡莫株核衣壳（N）蛋白编码的S基因组片段表达为β-半乳糖苷酶融合蛋白。通过从琼脂糖凝胶上切下蛋白条带、熔化琼脂糖，并通过冷冻、在冰上孵育和离心去除蛋白，对重组蛋白进行纯化。回收率约为1至5mg/200ml细菌悬液，足以包被100至500个酶免疫测定微孔板。在一项对312份来自NE患者的IF阳性血清样本和233份IF阴性血清样本的研究中，与IF检测结果相比，重组N蛋白酶免疫测定检测普马拉病毒免疫球蛋白G抗体的灵敏度为97.8%，特异性为98.5%。此外，还开发了一种免疫球蛋白G亲和力酶免疫测定法，并成功用于从单一血清样本诊断急性NE。结果表明，这种生物工程抗原适用于普马拉病毒免疫和近期感染的常规诊断检测。
