[1]刘龙,孙丽英,王敦.RBSDV P7-2基因重组AcMNPV的构建及其对sf9细胞的致死作用[J].南京农业大学学报,2019,42(1):81-87.[doi:10.7685/jnau.201803035]
 LIU Long,SUN Liying,WANG Dun.Construction of recombinant AcMNPV with P7-2 gene of RBSDV and its lethal effect on sf9 cells[J].Journal of Nanjing Agricultural University,2019,42(1):81-87.[doi:10.7685/jnau.201803035]
点击复制

RBSDV P7-2基因重组AcMNPV的构建及其对sf9细胞的致死作用()
分享到:

《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
42卷
期数:
2019年1期
页码:
81-87
栏目:
植物科学
出版日期:
2019-01-09

文章信息/Info

Title:
Construction of recombinant AcMNPV with P7-2 gene of RBSDV and its lethal effect on sf9 cells
作者:
刘龙 孙丽英 王敦
西北农林科技大学植物保护学院, 陕西 杨凌 712100
Author(s):
LIU Long SUN Liying WANG Dun
College of Plant Protection, Northwest A & F University, Yangling 712100, China
关键词:
RBSDV P7-2基因重组AcMNPV多角体形成速度致死作用
Keywords:
RBSDV P7-2 generecombinant AcMNPVspeed of polyhedra formationlethal effect
分类号:
S433.4;Q789;Q965
DOI:
10.7685/jnau.201803035
摘要:
[目的]为克服杆状病毒杀虫速度慢等固有缺点,首次尝试将异源病毒细胞凋亡诱导基因重组到昆虫杆状病毒中,以期为遗传修饰杆状病毒、提高杀虫效果提供新思路。[方法]将带有SV40终止子的苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)多角体基因ph、AcMNPV ie-1启动子、在C端融合Flag标签的水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)的P7-2基因依次插入到供体质粒pFastBac-HTB的多克隆位点。使用供体质粒pFastBac-HTB自身的启动子及ie-1启动子分别启动phP7-2基因的表达。通过转座的方法将phP7-2基因及启动子插入到bacmid bMON14272的多角体位置,获得重组bacmid Ac-P7-2,并通过Western blot检测P7-2基因的表达。转染sf9细胞后通过光学显微镜观察重组病毒多角体的形成并统计不同时间细胞死亡数量。通过TCID50的方法绘制病毒生长曲线。[结果]成功构建了P7-2基因重组AcMNPV bacmid Ac-P7-2,Western blot检测到P7-2蛋白可以正确表达。在转染sf9细胞48、72、96和120 h后,重组病毒vAc-P7-2细胞死亡数量显著高于野生型病毒vAc-WT,并发现重组病毒vAc-P7-2多角体形成并释放的速度也快于野生型病毒vAc-WT。病毒的生长曲线显示,在转染sf9细胞48、72、96和120 h后,vAc-P7-2的增殖速度显著快于vAc-WT。[结论]成功构建了多角体形成迅速的重组病毒vAc-P7-2,与野生型病毒vAc-WT相比其具有较快的增殖速度,对寄主细胞有较强致死作用。
Abstract:
[Objectives]To overcome the disadvantages of baculovirus,eg. slow killing speed to host insects,a new idea was developed to construct recombinant baculovirus using apoptosis inducing gene from heterogametic virus to improve its insecticidal effect. [Methods]The polyhedrin gene(ph),ie-1 promoter and Rice black-streaked dwarf virus(RBSDV)P7-2 gene fused to a Flag-tag at the C-terminus were inserted into the multiple cloning site(MCS) of the donor plasmid pFastBac-HTB. The ph gene was initiated by the promoter of the donor plasmid while P7-2 gene was driven by ie-1 promoter. ph,P7-2 and promoters were inserted into the polyhedrin locus of bacmid bMON14272 by transposition to get the recombinant bacmid named Ac-P7-2. The expression of P7-2 gene was confirmed by Western blot. The polyhedra formation of the recombinant virus and the amount of dead cells were observed by light microscope post transfection. The viral growth curve was measured by TCID50. [Results]P7-2 recombinant AcMNPV bacmid Ac-P7-2 was constructed successfully and P7-2 protein expressed correctly. The amount of dead cells caused by recombinant virus vAc-P7-2 was significantly more than those caused by wild type virus vAc-WT at 48,72,96 and 120 h post transfection and speed of polyhedra formation of the recombinant virus was faster than wild type virus. The viral growth curve indicated that the multiplication rate of vAc-P7-2 was notably faster than vAc-WT at 48,72,96 and 120 h post transfection. [Conclusions]The recombinant virus was constructed successfully and it was characterized with rapid-polyhedra formation,faster multiplication rate and a short lethal time against host cells.

参考文献/References:

[1] Gupta S,Dikshit A K. Biopesticides:an ecofriendly approach for pest control[J]. Journal of Biopesticides,2010,3(1):186-188.
[2] Lacey L A,Frutos R,Kaya H K,et al. Insect pathogens as biological control agents:do they have a future?[J]. Biological Control,2001,21(3):230-248.
[3] Chandler D,Bailey A S,Tatchell G M,et al. The development,regulation and use of biopesticides for integrated pest management[J]. Phil Trans R Soc B,2011,366(1573):1987-1998.
[4] Beascatena A,Sánchezmirón A,Garcíacamacho F,et al. Baculovirus biopesticides:an overview[J]. Journal of Animal & Plant Sciences,2014,24(2):362-373.
[5] Inceoglu A B,Kamita S G,Hammock B D. Genetically modified baculoviruses:a historical overview and future outlook[M]//Inceoglu A B,Kamita S G,Hammock B D. Advances in Virus Research. Amsterdam:Elsevier,2006:323-360.
[6] Inceoglu A B,Kamita S G,Hinton A C,et al. Recombinant baculoviruses for insect control[J]. Pest Management Science,2001,57(10):981-987.
[7] McCutchen B F,Choudary P V,Crenshaw R,et al. Development of a recombinant baculovirus expressing an insect-selective neurotoxin:potential for pest control[J]. Bio/technology,1991,9(9):848-852.
[8] 李坚,王敦.生物防治杆状病毒基因工程研究进展[J]. 生物技术通讯,2009,20(4):587-593. Li J,Wang D. Research progress in genetic engineering of baculovirus for biological control[J]. Letters in Biotechnology,2009,20(4):587-593(in Chinese with English abstract).
[9] Bonning B C,Hammock B D. Development of recombinant baculoviruses for insect control[J]. Annual Review of Entomology,1996,41(1):191-210.
[10] Chang J H,Choi J Y,Jin B R,et al. An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin[J]. Journal of Invertebrate Pathology,2003,84(1):30-37.
[11] 王福山,黄永秀,齐义鹏,等. 多角体完整重组病毒的构建及苏芸金杆菌截短δ内毒素基因的表达[J]. 科学通报,1995,40(20):1896-1900. Wang F S,Huang Y X,Qi Y P,et al. The construction of polyhedrin positive recombinant virus and expression of Bt truncated endotoxin gene[J]. Chinese Science Bulletin,1995,40(20):1896-1900(in Chinese).
[12] Stewart L M D,Hirst M,Ferber M L,et al. Construction of an improved baculovirus insecticide containing an insect-specific toxin gene[J]. Nature,1991,352(6330):85-88.
[13] Zlotkin E,Fishman Y,Elazar M. AaIT:from neurotoxin to insecticide[J]. Biochimie,2000,82:869-881.
[14] Isogai M,Uyeda I,Lee B C. Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7,S8,S9 and S10[J]. Journal of General Virology,1998,79(6):1487-1494.
[15] Karab?rklü S,Azizoglu U,Azizoglu Z B. Recombinant entomopathogenic agents:a review of biotechnological approaches to pest insect control[J]. World Journal of Microbiology and Biotechnology,2018,34:14.
[16] Wu C,Deng Z,Long Z,et al. Generating a host range-expanded recombinant baculovirus[J]. Scientific Reports,2016,6:28072.
[17] Weitzman M D,Lilley C E,Chaurushiya M S. Genomes in conflict:maintaining genome integrity during virus infection[J]. Annual Review of Microbiology,2010,64(1):61-81.
[18] Mitchell J K,Friesen P D. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication[J]. Journal of Virology,2012,86(24):13542-13553.
[19] Mitchell J K,Byers N M,Friesen P D. Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication[J]. Journal of Virology,2013,87(23):12592-12599.
[20] Wang Q,Tao T,Han Y H,et al. Nonstructural protein P7-2 encoded by Rice black-streaked dwarf virus,interacts with SKP1,a core subunit of SCF ubiquitin ligase[J]. Virology Journal,2013,10(1):325.

备注/Memo

备注/Memo:
收稿日期:2018-03-19。
基金项目:国家自然科学基金项目(31670659);国家公益性行业(农业)科研专项(201404403-09)
作者简介:刘龙,硕士研究生。
通信作者:王敦,教授,博导,主要从事昆虫与微生物资源研究,E-mail:wanghande@nwuaf.edu.cn,dunwang@foxmail.com。
更新日期/Last Update: 1900-01-01