Expression and Identification of Glycoprotein of Siniperca chuatsirhabdovirus in Baculovirus Expression System
ZHANG Yanbing1,2, YE Jiaxin2, SUN Wei2, LIU Xiaodan2, LIN Qiang1
1. Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China; 2. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
Abstract：Siniperca chuatsi rhabdovirus (SCRV) genome encodes five structural proteins, in which glycoprotein G is the main antigen on the surface of the virus, In order to achieve large expression of the antigen protein, the glycoprotein G of SCRV was inserted into Baculovirus shuttle vector pFastBac1 by molecular cloning technology and transformed into Escherichia coli DH5α competent cells. Then it was transformed into DH10Bac E. coli competent cells, and the recombinant plasmid rBacmid-G was obtained by blue-white spot and resistance screening. The recombinant bacmid was transfected into Sf9 insect cells by liposome transfection method to obtain recombinant Baculovirus expressing glycoprotein. The obtained P3 generation recombinant virus was infected with Sf9 insect cells for SDS-PAGE detection of the recombinant protein. After successful expression, the target protein was purified by nickel ion affinity chromatography column, and then the recombinant protein was identified by Western blot. There was a specific band at 50 ku, and no band in the control group, indicating that the prepared antigen can specifically bind to the antibody. Therefore, the SCRV glycoprotein G was successfully expressed in the Baculovirus expression system. The findings provide technical support for the development and large-scale production of SCRV glycoprotein G vaccine.
张彦冰, 叶加鑫, 孙威, 刘晓丹, 林强. 鳜弹状病毒糖蛋白在杆状病毒表达系统中的表达与鉴定[J]. 水产科学, 2024, 43(2): 281-288.
ZHANG Yanbing, YE Jiaxin, SUN Wei, LIU Xiaodan, LIN Qiang. Expression and Identification of Glycoprotein of Siniperca chuatsirhabdovirus in Baculovirus Expression System. 水产科学, 2024, 43(2): 281-288.
[1] ZHANG Q Y, LI Z Q. Three different viruses observed from the tissues of diseased mandarin fish Siniperca chuatsi[J].Chinese Science Bulletin,1999,44(5):437-441. [2] 吴淑勤,李新辉,潘厚军,等.鳜暴发性传染病病原研究[J].水产学报,1997,21(增刊):56-60. [3] 黄志斌,吴淑勤,石存斌,等.珠江三角洲地区鳜鱼病害现状及综合防治对策[J].淡水渔业,1999,29(7):12-14. [4] TAO J J, GUI J F, ZHANG Q Y. Isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish[J].Aquaculture,2007,262(1):1-9. [5] LIN Q, FU X Z, LI N Q, et al. Co-infections of infectious spleen and kidney necrosis virus and Siniperca chuatsi rhabdovirus in Chinese perch (Siniperca chuatsi)[J].Microbial Pathogenesis,2017,111:422-430. [6] TAO J J, ZHOU G Z, GUI J F, et al. Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF[J].Virus Research,2008,132(1/2):86-96. [7] JAYAKAR H R, JEETENDRA E, WHITT M A. Rhabdovirus assembly and budding[J].Virus Research,2004,106(2):117-132. [8] ALBERTINI A A V, BAQUERO E, FERLIN A, et al. Molecular and cellular aspects of rhabdovirus entry[J].Viruses,2012,4(1):117-139. [9] BELOT L, ALBERTINI A, GAUDIN Y. Structural and cellular biology of rhabdovirus entry[J].Advances in Virus Research,2019,104:147-183. [10] COLL J M. The glycoprotein G of rhabdoviruses[J].Archives of Virology,1995,140(5):827-851. [11] DA POIAN A T, CARNEIRO F A, STAUFFER F. Viral membrane fusion:is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?[J].Brazilian Journal of Medical and Biological Research,2005,38(6):813-823. [12] 张逸驰,李媛媛.昆虫细胞-杆状病毒表达系统的研究进展[J].中国生物制品学杂志,2020,33(12):1454-1459. [13] NISHIZONO A, YAMADA K. Rhabdoviruses[J].Uirusu,2012,62(2):183-196. [14] PURCELL M K, LAING K J, WINTON J R. Immunity to fish rhabdoviruses[J].Viruses,2012,4(1):140-166. [15] ZHANG Q Y, GUI J F. Virus genomes and virus-host interactions in aquaculture animals[J].Science China Life Sciences,2015,58(2):156-169. [16] OGINO T, BANERJEE A K. An unconventional path-way of mRNA cap formation by vesiculoviruses[J].Virus Research,2011,162(1/2):100-109. [17] GAUDIN Y, RUIGROK R W H, TUFFEREAU C, et al. Rabies virus glycoprotein is a trimer[J].Virology,1992,187(2):627-632. [18] 王树云,李江宇,高志强,等.鲤春病毒血症病毒糖蛋白基因在毕赤酵母中的分泌表达[J].淡水渔业,2015,45(4):31-35. [19] LYU S, YUAN X, ZHANG H, et al. Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system[J].Journal of Zhejiang University-Science B (Biomedicine ＆ Biotechnology),2019,20(9):728-739. [20] 徐黎明,刘红柏,尹家胜,等.传染性造血器官坏死病毒糖蛋白原核表达及免疫原性分析[J].病毒学报,2013,29(5):529-534. [21] 赵景壮,徐黎明,刘淼,等.鱼类传染性造血器官坏死病病毒糖蛋白的截短表达及免疫原性检测[J].细胞与分子免疫学杂志,2014,30(12):1238-1242. [22] 陶健,徐黎明,刘淼,等.传染性造血器官坏死病毒抗原表位富集区的原核表达及应用[J].淡水渔业,2015,45(2):49-55. [23] 林婧楠,赵景壮,徐黎明,等.鲤春病毒血症病毒截短糖蛋白的原核表达及抗体制备[J].淡水渔业,2019,49(5):44-50. [24] 徐进,周勇,陈倩,等.鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析[J].中国水产科学,2016,23(2):352-358. [25] 陈中元,雷晓颖,张奇亚. 鳜鱼弹状病毒糖蛋白(G)基因的克隆与表达[C] //湖北省暨武汉微生物学会,四川省微生物学会. “环境与健康”学术研讨会论文摘要集. 武汉:暨武汉微生物学会,四川省微生物学会,湖北省科学技术协会,2007:1. [26] 刘丽琴,陈婷婷,李少伟,等.大肠杆菌表达系统在基因工程疫苗研发中的应用与策略优化[J].中国新药杂志,2020,29(21):2434-2442. [27] CHEN Z Y, LEI X Y, ZHANG Q Y. The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus[J].Veterinary Microbiology,2012,157(3/4):264-275. [28] 刘微,罗晓华,陈文婧,等.HSV-2糖蛋白D在昆虫-杆状病毒表达系统中的表达及免疫原性分析[J].吉林大学学报(医学版),2017,43(4):720-724. [29] 谢三磊,温书香,罗琳,等.传染性造血器官坏死病病毒糖蛋白杆状病毒表达系统的构建及其抗原性分析[J].中国畜牧兽医,2017,44(3):854-860. [30] 唐琦,邱立鹏,李东,等.杆状病毒表达载体的应用现状[J].微生物学通报,2018,45(2):442-450. [31] CHAMBERS A C, AKSULAR M, GRAVES L P, et al. Overview of the baculovirus expression system[J].Current Protocols in Protein Science,2018,91(1):5.4.1-5.4.6. [32] ONO C, OKAMOTO T, ABE T, et al. Baculovirus as a tool for gene delivery and gene therapy[J].Viruses,2018,10(9):510. [33] SAXENA A, BYRAM P K, SINGH S K, et al. A structured review of baculovirus infection process:integration of mathematical models and biomolecular information on cell-virus interaction[J].The Journal of General Virology,2018,99(9):1151-1171.
2024, Vol. 43 
