登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用

根据登革 2型病毒衣壳蛋白C基因和葡萄球菌核酸酶SN基因序列设计引物 ,从构建的原核表达载体pLEX D2C SN中扩增获得编码登革病毒衣壳蛋白和葡萄球菌核酸酶的融合基因D2C SN ,将其插入到真核表达载体pcDNA6 V5 His中 ,筛选获得重组质粒pcDNA D2C SN .电穿孔转染BHK细胞后 ,5mg Lblasticidin压力筛选 ,通过RT PCR、间接免疫荧光和免疫印迹鉴定表达的蛋白 ,体外DNA消化试验检测核酸酶活性 .结果表明 ,融合蛋白D2C SN在BHK细胞中获得了稳定表达 ,表达的融合蛋白能够被抗登革病毒衣壳蛋白的单克隆抗体特异识别 ,并具有良好的核酸酶活性 ,能够对DNA进行切割 .同时 ,BHK细胞中稳定表达的融合蛋白D2C SN能够有效抑制登革病毒的增殖 ,使其感染性降低 10 3 ～ 10 4倍 .这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于人类抗登革病毒感染奠定了基础

Establishment and Application of Dengue Virus Capsid Targeted Nuclease Expression System

Based on the nucleotide sequence of the dengue 2 virus capsid protein C gene and staphylococcal nuclease(SN)gene,a pair of primers was designed to amplify the desired fusion gene CSN from the constructed plasmid pLEX/D2C-SN.The fusion gene encoding dengue C gene and SN gene was then cloned into eukaryotic vector pcDNA6/V5-His to generate the desired recombinant plasmid pcDNA/D2C-SN. After transfected into the BHK cells by electroporation,the resistant clones were screened by RT-PCR. The transfected cells were selected by 5 mg/L blasticidin and analyzed by indirected immunofluorescence and Western blot. The activity of nuclease of expressed fusion protein D2C-SN was analyzed in an in vitro DNA digestion assay.It was confirmed that a BHK cell line expressing the fusion protein D2C-SN was obtained. The fusion protein has ideal nuclease activity and no cytotoxicity on host cell. The results paved exploring the feasibility of capsid targeted viral inactivation strategy in anti-dengue infection.