|
|
Prokaryotic Expression of Recombinant Interferon IFNγ of Grass Carp (Ctenopharyngodon idella) and Its Anti-GCRV Effects |
YUAN Xue-Mei1, XU Ling-Wei2, HUANG Lei1, HAO Gui-Jie1, LIN Ling-Yun1, PAN Xiao-Yi1, YAO Jia-Yun1, SHEN Jin-Yu1* |
1 Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs/Key Laboratory of Fish Health and Nutrition of Zhejiang Province/Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;
2 Hangzhou Xiaoshan Aquatic Station of Agricultural and Forestry Technology Extension Center, Hangzhou 311225, China |
|
|
Abstract In recent years, grass carp hemorrhagic disease caused by Grass carp reovirus (GCRV) has caused serious losses to the grass carp industry. In order to explore the immune protective effects of recombinant interferon on grass carp and its mechanism, the prokaryotic expression vector PET-30a-rCiIFNγ of Ctenopharyngodon idella interferon γ (IFNγ) was constructed and transferred into Transetta (DE3). The recombinant protein rCiIFNγ inducted by IPTG and purified by nickel column chromatography. The antiviral activity of rCiIFNγ in vitro was detected by cytopathic effect (CPE) observation. The protective efficacy of rCiIFNγ in vivo was evaluated by challenge experiment. The results showed that the molecular weight of rCiIFNγ is about 23kD, and the antiviral titer of purified rCiIFNγ was 2.8×103 U/μg. rCiIFNγ could not only inhibit cytopathic effect induced by GCRV in vitro, but also induced the expression of IFN-stimulated genes (IFN, IRF1, IRF7, Mx, PKR, STAT3 and IFNr) in liver, spleen, kidney and intestine, and significantly reduced the mortality of C. idellus challenged with GCRV. These results indicated that the recombinant interferon IFNγ fusion protein rCiIFNγ was effective in protecting grass carp against GCRV infection. This study provides a theoretical basis for the antiviral application of grass carp interferon γ.
|
Received: 26 August 2021
|
|
Corresponding Authors:
*sjinyu@126.com.
|
|
|
|
[1] 常恒祯, 常江, 战俊澎, 等 . 2021. 包涵体重组蛋白不同纯化方法的比较[J]. 中国生物制品学杂志 , 34(07): 862-867.
(Chang H Z, Chang J, Zhan J P, et al.2021. Com‐ parison of various methods for purification of recombi‐ nant inclusion body protein[J]. Chinese Journal of Biologicals, 34(07): 862-867.)
[2] 刘洋, 刘永亮, 葛胜祥 . 2018. 干扰素刺激基因及临床意义研究进展[J]. 中国免疫学杂志, 34(03): 454-459.
(Liu Y, Liu Y L, Ge S X.2018. Research progress towards in‐ terferon stimulated genes and its clinical significance[J]. Chinese Journal of Immunology, 34(03): 454-459.)
[3] 罗凯 .2018. 鱼类抗病毒基因 TRIM 和 IFN 基因克隆及功能研究[D]. 硕士学位论文, 长江大学, 导师: 许巧情, pp.61-72.
(Luo K.2018. Cloning and functional study of antiviral TRIM and IFN genes in fish[D]. Thesis for M. S., Changjiang University, Supervisor: Xu Q Q, pp. 61-72.)
[4] 马普 .2015. 红鳍东方鲀 IFN-γ 基因和 CCL19 基因的原核表达及重组 IL15 的活性鉴定[D]. 硕士学位论文, 大连海洋大学, 导师: 王秀利, pp. 1-30.
(Ma P.2015. Prokaryotic expression of IFNγ gene and CCL19 gene and the activity identification of recombinaton IL15 of Takifugu rubripes[D]. Thesis for M. S., Dalian Ocean University, Supervisor: Wang X L, pp. 1-30.)
[5] 徐洋, 郝贵杰, 沈锦玉, 等 . 2010. 两株草鱼呼肠孤病毒江西株的分离与鉴定[J]. 淡水渔业 , 40(03): 44-49.
(Xu Y, Hao G J, Shen J Y, et al.2010. Isolation and identifica‐ tion of two grass carp reovrius strain in Jiangxi province[J]. Freshwater Fisheries, 40(03): 44-49.)
[6] 余新建, 李东明, 马梅生, 等 . 2013. 重组干扰素上调草鱼干扰素系统基因的表达[J]. 水生生物学报, 37(06):1184-1188.
(Xu X J, Li D M, Ma M S, et al.2013. Recombinant IFN upregulates the expression of interferon system genes in grass carp(Ctenopharyngodon idelus)[J]. Acta Hydrobiologica Sinica, 37(06): 1184-1188.)
[7] Gattoni A, Parlato A, Vangieri B, et al.2006. Interferon-gam‐ ma: Biologic functions and HCV therapy (type Ⅰ/Ⅱ) (1 of 2 parts)[J]. Clinical Therapeutics, 157(5): 377-386.
[8] Igawa D, Sakai M, Savan R.2006. An unexpected discovery of two interferon gamma-like genes along with interleu‐ kin (IL) -22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals[J]. Molecular Immunology, 43(7): 999-1009.
[9] Li D M, Lin G, Yu X j, et al.2013. Immunoprotection of grass carp (Ctenopharyngodon idella ) with recombinant inter‐ feron (rCiIFN) against GCHV infection[J]. Aquaculture,388: 42-48.
[10] Pestka S, Krause C D, Walter M R, 2004. Interferons, interfer‐ on-like cytokines, and their receptors[J]. Immunological Reviews, 202(1): 8-32
[11] Platanias L C, 2005. Mechanisms of type-Ⅰ- and type-Ⅱ-in‐terferon-mediated signalling[J]. Nature Reviews Immunology, 5(5): 375-386.
[12] Pyo S, Gangemi J D, Ghaffar A, et al.1993. Poly I:C-induced antiviral and cytotoxic activities are mediated by differ‐ ent mechanisms[J]. International Journal of Immuno‐pharmacology, 15(4): 477-486
[13] Samuel C E.2001. Antiviral actions of interferons[J]. Clinical Microbiology Reviews, 14(4): 778-809.
[14] Schroder K, Hertzog P J, Ravasi T, et al.2003. Interferon- γ: An overview of signals, mechanisms and functions[J].Journal of Leukocyte Biology, 75(2): 163-189.
[15] Sun B, Skjaeveland I, Svingerud T, et al.2011. Antiviral activ‐ ity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type Ⅰ interferon[J]. Journal of Virolo‐ gy, 85(17): 9188-9198.
[16] Verhelst J, Hulpiau P, Saelens X.2013. Mx proteins: Antiviral gatekeepers that restrain the uninvited[J]. Microbiology and Molecular Biology Reviews, 77(4): 551-566.
[17] Wang Q, Zeng W W, Liu C, et al.2012. Complete genome sequence of a reovirus isolated from grass carp, indicating different genotypes of GCRV in China[J]. Journal of Virology, 86(22): 12466.
[18] Xu C, Guo T C, Mutoloki S, et al.2010. Alpha interferon and not gamma interferon inhibits Salmonid alphavirus subtype 3 replication in vitro[J]. Journal of Virology, 84(17): 8903-8912.
[19] Xiang Y X, Liu W, Jia P, et al.2017. Molecular characterization and expression analysis of interferon-gamma in black seabream Acanthopagrus schlegelii[J]. Fish & Shellfish Immunology, 70(1): 140-148.
[20] Yuan X M, Yao J Y, Liu L Y, et al.2019. Preparation of liposome-encapsulated interferon alpha (IFN-α) vaccine and antigrass carp reovirus effect[J]. Aquaculture Research,50(9): 2600-2607.
[21] Zhang Y B, Gui J F.2012. Molecular regulation of interferon antiviral response in fish[J]. Developmental & Compara‐ tive Immunology, 38(2): 193-202. |
|
|
|