西尼罗病毒RNA复制子系统的建立与应用  

Construction and identification of West Nile virus Chin-01 strain replicon

作　　者：张富军 李晓峰[2] 曹飞[2] 赵慧[2] 邓永强[2] 朱舜亚[2] 姜涛[2] 秦鄂德[2] 秦成峰[2] 

机构地区：[1]山东省日照市中医院,山东日照276800 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2013年第2期114-117,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31000083)

摘　　要：目的构建具有复制和表达活性的RNA形式的西尼罗病毒(WNV)复制子。方法在DNA形式的WNV复制子pWNrep的基础上,将其CMV启动子替换成SP6启动子,并在3'UTR端引入PacⅠ酶切位点,构建复制子克隆pSP6-WNrep。同时,将增强型绿色荧光蛋白(eGFP)和海肾荧光素酶(R.luc)报告基因分别插入上述复制子质粒中,获得含有报告基因的复制子pSP6-WNrep/eGFP和pSP6-WNrep/R.luc。将上述克隆线性化后进行体外转录,将转录体RNA转染BHK-21细胞,观察不同时间点绿色荧光蛋白(GFP)的活性和R.luc活性。结果与结论获得了WNV复制子pSP6-WNrep、pSP6-WNrep/eGFP和pSP6-WNrep/R.luc,酶切和测序鉴定结果显示,序列与预期一致。将含有报告基因的复制子转染细胞72 h后仍能观察到荧光信号,R.luc的活性随时间延长而增强。本研究成功构建了WNV Chin-01株的RNA复制子,该复制子能够在哺乳动物细胞中有效复制并表达外源蛋白,为下一步构建WNV单轮感染病毒样颗粒奠定了基础。Objective To obtain a West Nile virus （WNV） RNA replicon efficiently expressing foreign proteins in cells. Methods CMV promoter within WNV replicon pWNrep was replaced with SP6 promoter, and Pac I restriction enzyme site was introduced to the immediate downstream of 3＇UTR of WNV genome, yielding pSP6-WNrep. Then, the reporter genes enhanced green fluorescent protein （eGFP） or Renilla luciferase （ R. luc） were inserted into the pSP6- WNrep, resulting in pSP6-WNrep/eGFP and pSP6-WNrep/R, luc respectively. The plasmids were linearized with Pac I and used as templates for in vitro transcription. The transcript RNA was transfected into BHK-21 cells. The expression level of GFP and luciferase activity were evaluated. Results and Conclusion The plasmids pSP6-WNrep, pSP6-WNrep/eGFP and pSP6-WNrep/R, luc were identified by restriction enzyme digestions and nucleotide sequencing. The expression of GFP could be observed in the transfected cells till 72 h. Additionally, luciferase signals continued to increase at 72 h post transfection. This research can facilitate the development of a single-round infectious WNV vaccine candidate.

关 键 词：RNA 西尼罗病毒 复制子 转染 

分 类 号：R373.3[医药卫生—病原生物学]