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      Construction and identification of dengue recombinatant subviral particles carrying luciferase

      1 , 1 , , 1

      China Tropical Medicine

      China Tropical Medicine

      Dengue virus , RSPs, luciferase, ADE, envelop protein

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          Abstract

          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.

          Abstract

          摘要: 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。

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          Author and article information

          Journal
          CTM
          China Tropical Medicine
          China Tropical Medicine (China )
          1009-9727
          01 November 2019
          01 January 2020
          : 19
          : 11
          : 1009-1013
          Affiliations
          1Department of General Surgery, China-Japan Friendship Hospital, Beijing 100029, China
          Author notes
          *Corresponding author: WANG Peigang, E-mail: pgwang@ 123456ccmu.edu.cn
          Article
          j.cnki.46-1064/r.2019.11.01
          10.13604/j.cnki.46-1064/r.2019.11.01
          © 2019 Editorial Department of China Tropical Medicine

          This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 Unported License (CC BY-NC 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See https://creativecommons.org/licenses/by-nc/4.0/.

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