夹竹桃天蛾质型多角体病毒在Sf9细胞系中的增殖研究  

Study on Proliferation Characteristics of Daphnis nerii cypovirus-23 in Sf9 Cells

作　　者：况文东 占智高[1] 关丽梅[1] 王金昌[1] 闫成花 陈俊晖 李江怀 周溪 靳亮[1] KUANG Wen-Dong;ZHAN Zhi-Gao;GUAN Li-Mei;WANG Jin-Chang;YAN Cheng-Hua;CHEN Jun-Hui;LI Jiang-Huai;ZHOU Xi;JIN Liang(Institute of Microbiology,Jiangxi Academy of Sciences,Nanchang 330096,China;The State Key Laboratory of Virology,Wuhan 430071,China;College of Life Sciences,Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)

机构地区：[1]江西省科学院微生物研究所,南昌330096 [2]病毒学国家重点实验室,武汉430071 [3]江西中医药大学生命科学学院,南昌330004

出　　处：《农业生物技术学报》2021年第4期772-779,共8页Journal of Agricultural Biotechnology

基　　金：江西省科学院引进博士项目(2019-YYB-04);江西省科学院协同创新专项普惠制一类项目(2019-XTPH1-04);病毒学国家重点实验室开放研究基金(2019IOV001);江西省自然科学基金资助项目(20192ACB20008)。

摘　　要：新型夹竹桃天蛾质型多角体病毒(Daphnis nerii cypovirus-23,DnCPV-23)可杀灭多种天蛾科昆虫,具备开发新型昆虫病毒杀虫剂的潜力。目前,主要利用夹竹桃天蛾(Daphnis nerii)对该病毒进行研究,DnCPV-23在细胞中的复制情况尚不清楚。本研究将DnCPV-23病毒粒子接种至草地贪夜蛾(Spodoptera frugiperda)细胞系Sf9中,分析该病毒在细胞中的复制情况。将病毒感染的细胞置于27℃条件下进行培养,利用光学显微镜观察病毒对Sf9细胞状态的影响;分别提取第1、3和7 d感染细胞的RNA,以其为模板进行反转录获得cDNA,利用qRT-PCR分析DnCPV-23 RNA水平;利用qRT-PCR方法分别对细胞培养上清液和病毒连续传代的细胞进行研究。结果显示,DnCPV-23能较好地适应Sf9细胞且成功感染Sf9细胞,并产生细胞病变效应(cytopathic effect,CPE);在细胞培养上清液中能够检测到病毒片段;病毒能够在Sf9细胞上进行传代。该研究为深入研究DnCPV-23入侵、转录机制、评价病毒毒力提供了良好的细胞模型,同时为新型天蛾科害虫病毒杀虫剂的开发提供一定的参考和依据。A new type of Daphnis nerii cypovirus-23(DnCPV-23)can kill a variety of Sphingidae insects and has the potential to be a new insect virus insecticide.At present,Daphnis nerii is the main model to study the virus,the replication of DnCPV-23 in cells is still unclear.In this study,DnCPV-23 virus particles were inoculated into Spodoptera frugiperda cell line Sf9,and the replication of the virus in the cells was analyzed.The virus-infected cells were cultured at 27℃,and the effect of the virus on the Sf9 cells were observed with the optical microscope;RNA of the infected cells on day 1,3 and 7 was extracted and used as a template to obtain the cDNA,and the DnCPV-23 RNA level was analyzed by q RT-PCR;the supernatant of infected Sf9 cells and the serial passage of virus in Sf9 cells were studied by the qRT-PCR method.DnCPV-23 could adapt to Sf9 cells well,could successfully infect Sf9 cells,and produce cytopathic effect(CPE);virus genes could be detected in the cell culture supernatant;the virus could be passaged in Sf9 cells.DnCPV-23 could replicate and passage well on Sf9 cells and make the cells undergo CPE.The above-mentioned research provides a good cell model for in-depth study of DnCPV-23 invasion,transcription and other mechanisms and evaluation of virus virulence.At the same time,it provides a certain reference and basis for the development of new Sphingidae virus insecticides.

关 键 词：新型夹竹桃天蛾质型多角体病毒(DnCPV-23) SF9细胞 病毒离体增殖 

分 类 号：S182[农业科学—农业基础科学] Q93[生物学—微生物学]