文献类型：期刊文献

中文题名：利用反向遗传技术产生表达外源基因的重组SA11轮状病毒

英文题名：Production of recombinant SA11 rotavirus expressing exogenous genes through reverse genetic technology

作者：柴萨萨[1,2];蔡昊[2,3];刘夏飞[2];赵静[2,3];宋敬东[2];李金松[2];裴银辉[1];李利利[2];段招军[2]

第一作者：柴萨萨

机构：[1]华北理工大学基础医学院河北省慢性疾病基础医学重点实验室,唐山063000;[2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会医学病毒和病毒病重点实验室,北京102206;[3]甘肃中医药大学公共卫生学院,兰州730000

第一机构：华北理工大学基础医学院河北省慢性疾病基础医学重点实验室,唐山063000

年份：2024

卷号：40

期号：2

起止页码：140

中文期刊名：中国人兽共患病学报

外文期刊名：Chinese Journal of Zoonoses

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：国家重点研发计划(No.2021YFC2301000)。

语种：中文

中文关键词：A组轮状病毒;反向遗传;外源基因;NSP1基因

外文关键词：Group A Rotavirus;reverse genetics;foreign gene;NSP1 gene

摘要：目的利用反向遗传技术产生NSP1基因插入和表达外源基因的重组SA11轮状病毒,构建可表达外源基因的轮状病毒载体。方法本研究将轮状病毒SA11株的NSP1基因片段的223位(5端)至1388位的核苷酸删除,并直接插入连接了终止-再启动元件(P2A)的增强绿色荧光蛋白(EGFP)基因,采用已建立的“12质粒”轮状病毒反向遗传系统拯救NSP1基因插入和表达EGFP的重组SA11轮状病毒。通过观察细胞病变效应和插入基因(EGFP)的荧光表达初步确定重组病毒的成功拯救,再结合电镜的形态学观察、RT-PCR的测序结果及病毒基因组的检测结果,进一步证明重组轮状病毒的成功拯救和传代扩增。利用TCID 50法和qRT-PCR法,测定了rSA11和rSA11/NSP1-EGFP的病毒滴度并绘制了基因组复制动力曲线。结果基于“12质粒系统”成功拯救了重组轮状病毒rSA11/NSP1-EGFP,证明NSP1片段截短并插入外源基因后病毒仍可正常复制,且具有良好的遗传稳定性,但病毒滴度低于其亲本株。结论基于NSP1基因插入和表达EGFP的重组轮状病毒能够实现稳定遗传,为利用反向遗传学构建表达外源基因的轮状病毒载体的深入探索提供了基础。
In recent years,an entirely plasmid-based reverse genetic system for rotavirus has been established.The advantages of using reverse genetic technology to manipulate gene sequences enable rotavirus genes to be carried in vectors for expression of foreign genes.In this study,the nucleotides from positions 223(5 ends)to 1388 of the NSP1 gene fragment of the rotavirus SA11 strain were deleted,and an enhanced green fluorescent protein(EGFP)gene connected to a stop restart element(P2A)was directly inserted.The established 12 plasmid rotavirus reverse transmission system was used to rescue the recombinant virus whose NSP1 fragment was replaced by the NSP1 recombinant fragment.The successful rescue of the recombinant virus was determined preliminarily by observation of cytopathic effects and the fluorescence expression of the inserted EGFP gene.Morphological electron microscopy observation,RT-PCR sequencing results and viral genome detection results together validated the successful rescue and passage amplification of the recombinant rotavirus(rSA11 and rSA11/NSP1-EGFP).The viral titers of rSA11 and rSA11/NSP1-EGFP were measured with the TCID 50 and qRT PCR methods,and genome replication dynamics curves were plotted.The viral titer of the truncated NSP1 fragment was lower than that of its parent strain,but viral replication was not affected.Genome detection results indicated that the recombinant virus rSA11/NSP1-EGFP had good genetic stability.This study provides a foundation for the in-depth exploration of the construction of rotavirus vectors for expressing foreign genes through reverse genetics.