携带荧光素酶登革亚病毒颗粒的构建和鉴定

目的 拟将荧光素酶基因整合入登革病毒（DENV）亚病毒颗粒（RSPs）中,构建能用于抗体介导的感染增强（ADE）高通量筛选的研究工具。方法 将荧光素酶基因luciferase插入DENV包膜蛋白基因prME的3'端,取代prME第二个跨膜区,构建prME-luc融合基因。用prME-luc融合基因转染293T细胞,在上清中检测携带荧光素酶的登革亚病毒颗粒（RSP-luc）。结果 转染prME-luc的293T细胞可以将RSP-luc释放至培养上清中,共转染野生型prME能够提高RSP-luc的产量。Western-blot技术确认了RSP-luc中含有DENV包膜蛋白和荧光素酶形成的融合蛋白。RSP-luc易于制备并可通过超速离心分离纯化,且可通过测定荧光素酶活性快速定量。体外实验显示RSP-luc能够与Vero细胞、U937细胞以及小鼠腹腔巨噬细胞等DENV靶细胞结合,结合过程可被受体类似物硫酸肝素竞争性抑制,也可被DENV特异性抗体4E11增强。结论 RSP-luc 能够模拟DENV 感染过程,有望成为深入研究DENV 与宿主相互作用及ADE 发生机制的有效工具。

Construction and identification of dengue recombinatant subviral particles carrying luciferase

Objective Luciferase gene was integrated into DENV subvirus particle (RSPs) to construct a research tool that can be used for antibody dependent enhancement (ADE) high throughput screening. Methods To develop a screening tool for ADE research, we used DENV envelope gene prME and luciferase gene to generate a prME-luciferase recombinatant gene (prME-luc). In this construct, the luciferase replaced the second transmembrane domain of DENV envelop protein and thus cells expressing prME-luc produced dengue recombinatant subvirus particles carrying luciferase (RSP-luc). Results 293T cells transfected with prME-luc produced RSP-luc into supernatant, which could be enhanced by co-exressing wild type of prME. The presence of E-luciferase fusion protein in RSP-luc was verified by Western-blot. RSP-luc is easy to prepare and can be purified using ultra-centrifugation. Their amount could be quickly quantified by measureing luciferase activity. In vitro experiments showed that RSP-luc could bind to DENV target cells, such as Vero cells, U937 cells and mouse peritoneal macrophages, and the binding process was competitively inhibited by heparin sulfate, a receptor analogue of DENV, and enhanced by DENV specific antibodies 4E11. Conclusion These results suggested that RSP-luc might simulate DENV infection, and could be an effective tool for viral-host interaction and ADE screening.