北海道型汉坦病毒核蛋白基因的克隆表达及其免疫原性

目的 构建北海道型汉坦病毒(HOKV)核蛋白(NP)基因的重组杆状病毒表达载体,在昆虫细胞中表达出具有免疫原性的抗原,用于HOKV的检测及诊断.方法 应用RT-PCR方法扩增HOKVFusong-Cr-247株的NP基因,并将该基因片段克隆到PCRR]2.1TA载体,转化到One ShortTM TOP10感受态细胞构建TA克隆载体,并与pFastBacTM1转座质粒载体分别用Kpn I和Not I双酶切,用T4连接酶连接,构建pFast PUUV-S重组转座质粒,经双酶切、PCR扩增及插入序列方向鉴定后.转化到DH10BacTM E.coli 感受态细胞,经三抗培养基筛选和PCR扩增鉴定后获得重组Bacmid质粒,将重组Baemid质粒转染Sf9昆虫细胞制备重组杆状病毒毒液,并继续扩增重组杆状病毒,以第三代毒液感染Sf9昆虫细胞,96h后收获细胞.用间接免疫荧光(IFA)、SDS-PAGE和Western blot对表达产物进行鉴定和分析.结果 实验应用Bac-to-BacTM杆状病毒表达系统,成功构建了含普马拉类汉坦病毒核蛋白基因的重组杆状病毒表达载体.并在昆虫细胞中获得表达.IFA结果显示,感染细胞的胞浆中有亮绿色荧光颗粒,SDS-PAGE和Western blot检测细胞表达产物的50×103(M)的蛋白条带,证实重组病毒感染昆虫细胞后表达了相应的重组核蛋白,与预计的结果相符.并能与抗汉坦病毒抗体结合.结论 成功表达具有HOKV免疫原性及反应性的重组核蛋白,为研制HOKV的诊断试剂奠定了基础.

Cloning and expression of the nucleoprotein gene of Puumala-like virus

Objective In order to detect Hokkaido virus(HOKV),a recombinant baculovirus containing the nucleoprotein(NP)gene of HOKV was constructed,and then the NP was expressed in insect cell.Methods The NP gene was cloned into plasmid PCRR]2.1TA vector and then Was ligated into baculovims donor plasmid pFastBacTM1 after cutting by the restriction enzylne Kpn I and Not I.pFastBacTM1 was subsequently transferred into the One ShortTMTOP10 competent cells and then into DH10BacTM E.coli competent cells.which contained the baculovirus shuttle vector(Bacmid)and the helper plasmid to generate a recombinant bacmid.Results The NP gene was successfully expressed jn St9 insect cell.The expressed recombinant nucleoprotein had been identified in the S19 insect cell by indirect immunofluorescence assay,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Westem blot.The results showed that the recombinant nucleoprotein appeared a molecular weight of 50×103M.and could reacmd with anti-recombinant Puumala virus(PUUV)nucleocapsid monoclonal antibodies and polyclonal antibodies against hantavirus.Conclusion Our results indicated that the recombinant nucleoprotein was SUCCESSfully expressed and having the immunogenicity and reactivity of natural nucleoprotein of HOKV.