鸭瘟病毒dUTPase基因克隆、原核表达及在病毒感染宿主亚细胞中的定位分析

Clone,Prokaryotic Expression of DPV dUTPase Gene and Its Subcellular Localization in Virus-infected Host Cells

根据笔者实验室新发现的鸭瘟病毒(DPV)dUTPase基因(GenBank登录号DQ486149)序列设计1对引物,PCR扩增后将产物亚克隆至pET32a(+)原核表达载体,获得表达质粒pET32a-DU。然后将其转化至E.coliBL21(DE3)进行IPTG诱导表达。SDS-PAGE检测显示诱导表达蛋白约66.8 ku,与预期表达蛋白大小一致。薄层扫描分析表明重组蛋白占菌体总蛋白的36.2%,主要以包涵体的形式存在。重组蛋白纯化后制备兔抗血清,间接ELISA效价为1∶409 600。通过免疫荧光技术进行DPV dUTPase亚细胞定位检测,结果显示最早可在感染后4 h的胞质中检测到特异性荧光,12 h大量点状绿色荧光聚集于胞核;24 h后胞核内点状荧光减弱,而胞质点状荧光增强;48 h胞核和胞质荧光均显著减弱。本研究为DPV dUTPase基因功能和DPV致病机理的研究提供了重要数据。

A pair of primers was designed based on the DPV dUTPase gene sequence that our laboratory newly discovered （GenBank accession number DQ486149）. Amplified PCR fragments were subcloned into the prokaryotic expression vector pET-32a （＋）. Then the recombinant plasmid pET32a-DU was transformed into E. coli BL21 （DE3）strain and expressed under IPTG induction. SDS-PAGE analysis showed that the induced expressed protein was about 66.8 ku and accounted for 36.2% of total bacterial protein by gel scanning. The protein was purified and used to immunize rabbit for the dUTPase anti-serum production. Its ELISA antibody titer was up to 1 ＂ 409 600. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that specific fluorescence appeared in cytoplasm as early as 4 hours post infection and a great deal of specific fluorescence concentrated in the cell nucleus by 12 hours. But after 24 hours, the fluorescence in nucleus was dispersed while that in cytoplasm increased. Forty-eight hours later, the fluorescence weakened significantly in both cytoplasm and nucleus. Above all, the results afforded significant data for the study on function of DPV dUT- Pase gene and the pathogeny of DPV.