YFV17D非感染性报告复制子及假病毒包装系统的建立

doi:10.13560/j.cnki.biotech.bull.1985.2022-1402

摘要/Abstract

摘要：

构建YFV17D非感染性复制子及假病毒包装系统，旨在进一步解析黄热病毒的复制、组装分子机制。该研究利用反向遗传学技术，将基于SP6启动子的单拷贝YFV17D报告复制子改造成由CMV启动子驱动的低拷贝YFV17D报告复制子，同时将表达YFV17D结构蛋白的包装质粒与复制子质粒共同转染BHK-21细胞而建立YFV17D的反式假病毒包装系统。通过检测NanoLuc萤光素酶活性（Nluc）、oxGFP和mCherry荧光蛋白表达、间接免疫荧光实验监测病毒双链RNA来确定复制子在BHK-21细胞中复制情况和包装效果。结果表明，与复制缺陷型复制子相比，野生型复制子转染至BHK-21细胞中，随着转染时间的增加，Nluc活性、oxGFP和mCherry荧光蛋白表达也随之增加，同时dsRNA也随时间延长而增多。将复制子质粒和包装质粒共同转染至BHK-21细胞后，收集上清再次感染BHK-21细胞，随后通过检测Nluc活性和oxGFP与mCherry蛋白的表达而确定假病毒包装成功。综上，成功构建了携带不同报告基因的YFV17D复制子，用于监控病毒基因组RNA的复制；成功搭建由包装质粒提供的结构蛋白，将不具备感染性的YFV17D亚基因复制子打包成具有单轮感染能力的YFV17D假病毒系统。

关键词: YFV17D报告复制子, 反向遗传学, 反式互补, 单轮感染病毒颗粒

Abstract:

The YFV17D noninfectious replicon and pseudovirus packaging system was constructed to further elucidate the molecular mechanism of replication and assembly of yellow fever virus. In this study, the single-copy YFV17D reporter replicon based on the SP6 promoter was transformed into a low-copy YFV17D reporter replicon driven by the CMV promoter using reverse genetics. At the same time, the packaging plasmid expressing YFV17D structural protein and reporter replicon plasmid were co-transfected into BHK-21 cells to establish a YFV17D trans-pseudovirus packaging system. NanoLuc luciferase activity（Nluc）, oxGFP and mCherry fluorescent protein expression, and indirect immunofluorescence assay were used to monitor viral double-stranded RNA to determine the replication and packaging effect of replicon in BHK-21 cells. The results showed that Nluc activity, oxGFP and mCherry fluorescent protein expression increased steadily in wild-type replicon compared with replication-deficient replicon, while dsRNA also increased. After co-transfection of the replicon plasmids and packaging plasmids into BHK-21 cells, the supernatant was collected to re-infect BHK-21 cells. The success of pseudovirus packaging was confirmed by detecting Nluc activity and oxGFP and mCherry protein expression. In conclusion, the YFV17D replicon carrying different reporter genes was successfully constructed in this study to monitor the replication of viral genomic RNA. The structural proteins provided by the packaging plasmid can successfully package the non-infectious YFV17D subgene replicons into a YFV17D pseudovirus system with single round infection ability.

Key words: YFV17D reporter replicon, reverse genetics, trans-complementation, single-round infection virions

何宇航, 胡涛, 吴震, 贺煜, 程安春, 陈舜. YFV17D非感染性报告复制子及假病毒包装系统的建立[J]. 生物技术通报, 2023, 39(8): 165-172.

HE Yu-hang, HU Tao, WU Zhen, HE Yu, CHENG An-chun, CHEN Shun. Establishment of YFV17D Non-infectious Reporter Replicon and Pseudovirus Packaging System[J]. Biotechnology Bulletin, 2023, 39(8): 165-172.

图/表 4

图1 YFV17D报告复制子构建示意图

Fig. 1 Schematic diagram of the construction of YFV17D reporter replicon

图2 YFV17D报告复制子在BHK-21细胞中的荧光蛋白表达 A：YFV17D-Nluc-rep转染BHK-21后Nluc萤光素酶活性检测， **** P<0.000 1； B：荧光蛋白表达；（a）YFV17D-mCherry-rep 转染组；（b）YFV17D-oxGFP-rep转染组；复制缺陷型复制子用作阴性对照

Fig. 2 Fluorescent protein expression of the YFV17D reporter replicon in BHK-21 cells A: Nluc luciferase activity assay, ****P<0.000 1. B: Fluorescent protein expression; （a）YFV17D-mCherry-rep transfection panel.（b）YFV17D-oxGFP-rep transfection panel; replication-defective replicons were used as negative controls

图3 YFV17D复制子在BHK-21细胞中的RNA复制情况 A：YFV17D-mCherry-rep转染组；B ：YFV17D-oxGFP-rep 转染组；C：YFV17D-Nluc-rep转染组；复制缺陷型复制子用作阴性对照

Fig. 3 RNA replication of YFV17D replicon in BHK-21 cells A: YFV17D-mCherry-rep transfection panel; B: YFV17D-oxGFP-rep transfection panel; C: YFV17D-mCherry-rep transfection panel; replication-defective replicons were used as negative controls

图4 YFV17D包装系统的建立 A：包装质粒构建示意图；B：产生 SRIPs 的实验程序示意图；C：SRIPs感染BHK-21细胞后Nluc萤光素酶活性检测，**** P<0.000 1；D：SRIPs感染BHK-21细胞后荧光蛋白表达（a）YFV17D-oxGFP-rep 转染组；（b）YFV17D-mCherry-rep转染组（C：pcDNA3.1-CprME；C28：pcDNA3.1-C28prME）

Fig. 4 Establishment of the YFV17D packaging system A: Schematic diagram of packaging plasmid construction.B: Schematic presentation of the experimental procedure used for the production of SRIPs. C: Detection of Nluc luciferase activity after SRIPs infection of BHK-21 cells, ****P<0.000 1. D: Fluorescent protein expression after SRIPs infection of BHK-21 cells（a）YFV17D-oxGFP-rep transfection panel;（b）YFV17D-mCherry-rep transfection panel（C: pcDNA3.1-CprME; C28: pcDNA3.1-C28prME）

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