Array-ELISA 技术对肾综合征出血热病毒诊断抗原的评价
Application of an array-ELISA for evaluation of diagnostic antigens of HFRS virus
摘要 目的分段表达并纯化肾综合征出血热病毒核蛋白,应用array-ELISA技术评价重组表达的核蛋白片段的诊断价值。方法构建肾综合征出血热病毒核蛋白表达质粒pET-32a(+)/Pn、pET-32a(+)/Pc,在大肠杆菌中诱导表达并用亲和层析法纯化,运用array-ELISA方法鉴定重组蛋白的检测特异性,检测结果与商品化胶体金试剂盒进行比较。结果在大肠杆菌中正确表达了肾综合征出血热病毒的核蛋白,亲和纯化得到较高纯度的蛋白质;应用array-ELISA技术从16份临床疑似血清样本中检出13份阳性血清,与商品化胶体金试剂盒一致性达到94%。结论获得的重组蛋白His-Pn可作为肾综合征出血热特异性诊断抗原之一;array-ELISA方法可以作为检测病毒抗体的有效手段。
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abstracts Objective To express and purify the recombinant nucleoprotein fragments of hemor-rhagic fever with renal syndrome ( HFRS) virus and to evaluate their diagnostic efficacy by using array-ELISA technology .Methods The target genes encoding nucleoprotein fragments of HFRS virus were amplified by PCR, and then inserted into prokaryotic expression vectors to construct the recombinant plasmids of pET -32a (+)/Pn and pET-32a(+)/Pc.The plasmids were transformed into E.coli BL21 ( DE3) to induce the ex-pression of nucleoprotein fragments by IPTG and the expressed products were purified by affinity chromatog -raphy using Ni-NTA agarose.The specificity and sensitivity of the recombinant antigens were evaluated by the assay of array-ELISA using commercial colloidal gold assay kit as a comparison .Results The recombi-nant nucleoprotein fragments of HFRS virus were correctly expressed in E.coli and highly purified by affinity chromatography .Array-ELISA showed that 13 of 16 suspected serum samples were positive by using the His-Pn protein as diagnostic antigen , consistency with the commercial colloidal gold assay kit reaching 94%. Conclusion The recombinant His-Pn protein expressed in E.coli cells could be used for specific serodiag-nosis of HFRS virus as its high antigenicity and sensitivity .The array-ELISA is an effective assay for the de-tection of virus at protein level .
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