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杆状病毒AcMNPV介导BmK IT的表达促进草地夜蛾卵巢Sf9细胞凋亡及病毒在细胞内的复制

付月君, 赵洁, 梁爱华, 胡风云

山西大学生物技术研究所， 太原 030006；山西省人民医院暨山西医科大学附属人民医院，太原 030012

摘要

昆虫杆状病毒是一类寄生于节肢动物的囊膜包裹的双链环状DNA，杆状病毒作为新型生物杀虫剂逐渐应用于害虫防治。BmK IT是一种从东亚钳蝎分离出来的昆虫毒素，可以与昆虫细胞膜的钠离子通道相互作用，引起钠离子通道动作电位重复发放，并增强钠电流峰值，延缓钠传导关闭，使昆虫产生兴奋性麻痹。为了提高杆状病毒苜蓿银纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)杀虫活性，本研究将BmK IT基因插入到AcMNPV基因组中，形成重组杆状病毒AcMNPV-BmK IT。采用MTT法、TUNEL试剂、Western blot、real-time PCR及病毒滴度测定来检测AcMNPV- BmK IT对草地夜蛾卵巢细胞Sf9发生凋亡及病毒在Sf9细胞内的复制情况。结果显示AcMNPV介导BmK IT的表达促进了Sf9细胞凋亡及病毒复制。此结果为揭示重组病毒AcMNPV-BmK IT的抗虫机制提供了实验依据。

关键词： AcMNPV; BmK IT; Sf9细胞; 凋亡; 复制

分类号：Q965.9

AcMNPV-mediated expression of BmK IT promotes the apoptosis of Sf9 cells and replication of AcMNPV

FU Yue-Jun, ZHAO Jie, LIANG Ai-Hua, HU Feng-Yun

1Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China； 2Shanxi Provincial People’s Hospital, Affiliate of Shanxi Medical University, Taiyuan 030012, China

Abstract

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating or channel currents. To promote the insecticidal effects of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the gene encoding an excitatory insect toxin, BmK IT, was inserted into the genome of AcMNPV to construct a recombinant baculovirus, AcMNPV-BmK IT. Spodopter frugiperda 9 (Sf9) cells were infected with AcMNPV and AcMNPV-BmK IT respectively for 24 h. Results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins (c-Myc, cleaved-Caspase3, Bcl-2 and Bax) of Sf9 cells, the transcription level of key genes (38K, C42, P78, F) of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV. The results laid a foundation for further structural and functional analysis of BmK IT.

Key words: Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV); Buthus martensii Karsch insect toxin (BmK IT); Spodopter frugiperda 9 cell; apoptosis; replication

收稿日期：2014-05-19　　录用日期：2014-11-04

通讯作者：付月君,胡风云　　E-mail: yjfu@sxu.edu.cn, fengyun71@163.com

引用本文：

付月君, 赵洁, 梁爱华, 胡风云. 杆状病毒AcMNPV介导BmK IT的表达促进草地夜蛾卵巢Sf9细胞凋亡及病毒在细胞内的复制[J]. 生理学报 2015; 67 (3): 305-311.

FU Yue-Jun, ZHAO Jie, LIANG Ai-Hua, HU Feng-Yun. AcMNPV-mediated expression of BmK IT promotes the apoptosis of Sf9 cells and replication of AcMNPV. Acta Physiol Sin 2015; 67 (3): 305-311 (in Chinese with English abstract).