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。
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 BI21 （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 vires 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%. Conehmion The recombinant His-Pn protein expressed in E. coli cells could be used for specific serodiag- nesis 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.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2013年第8期595-599,共5页
Chinese Journal of Microbiology and Immunology
