Cloning, Expression of MsIL-21 Gene in Largemouth Bass (Micropterus salmoides) and Its Response to Bacterial Infection
ZHU Xue-Qing1,2, ZHAO Fei2,*, DENG Yu-Ting2, TAN Ai-Ping2, LAI Ying-Tiao2, GONG Hua2, HUANG Zhi-Bin2, JING Peng-Hua2
1 National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China; 2 Pearl River Fisheries Research Institute/Key Laboratory of Fishery Drug Development of Ministry of Agriculture and Rural Affairs/Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province, Chinese Academy of Fishery Sciences, Guangzhou 510380, China
Abstract：Interleukin-21 (IL-21) is a member of the common gamma chain (γc) cytokine family and plays an important role in defense against a variety of inflammatory and immune diseases. In order to explore the immune response function of IL-21 against pathogen infection, in this study, the ORF of largemouth bass (Micropterus salmoides) MsIL-21 (GenBank No. XP_038555572.1) was cloned, and bioinformatics and expression analysis were performed. The results showed that the full length of MsIL-21 ORF was 441 bp and encoding 146 amino acids. Sequence analysis revealed that MsIL-21 had the typical structure of IL-21 protein, including 4 groups of α-helices and 4 cysteine residues. Homology analysis revealed that MsIL-21 was most closely related to IL-21 of European sea bass (Dicentrarchus labrax). qPCR analysis showed that MsIL-21 was commonly expressed in all the healthy largemouth bass tissues tested, with the highest expression in the liver. After infection with Aeromonas veronii or Nocardia seriolae, the up-regulation of expression was detected in the liver, head kidney, and spleen. Similarly, after stimulation with pathogenic analogs lipopolysaccharide (LPS), lipoteichoic acid (LTA), and polyinosinic-polycytidylic acid [Poly(I:C)], the expression of MsIL-21 could be induced in head kidney leukocytes. The recombinant MsIL-21 protein was expressed and purified by prokaryotic expression system, which induced the expression of IL-10, IL-22, IL-1β, and interferon-γ (IFN-γ) in head kidney leukocytes. Taken together, the present study initially suggests that MsIL-21 may be involved in defense against pathogenic bacterial invasion and infection. This study further enriches the functional studies of fish IL-21.
[1] 刘付翠, 赵飞, 谭爱萍, 等. 2019. 杂交鳢CmaZAP-70基因的克隆, 表达及其功能分析[J]. 农业生物技术学报, 27(11): 2052-2062. (Liu F C, Zhao F, Tan A P, et al.2019. Cloning, expression and functional analysis of CmaZAP-70 from hybrid snakehead (Channa maculata ♀×Channa argus ♂)[J]. Journal of Agricultural Biotechnology, 27(11): 2052-2062.) [2] 吕丽丽, 梅飞, 曹守林, 等. 2021. 加州鲈源鰤鱼诺卡氏菌的分离鉴定及致病性[J]. 微生物学通报, 48(12): 4765-4775. (Lv L L, Mei F, Cao S L, et al.2021. Isolation, identification and pathogenicity of Nocardia seriolae from Micropterus salmoides[J]. Microbiology China, 48(12): 4765-4775.) [3] 农业农村部渔业渔政管理局. 2021. 2021中国渔业统计年鉴[M]. 中国农业出版社, 北京. pp. 33. (Administration of Fisheries and Fishery Administration, Ministry of Agriculture and Rural Affairs. 2021. 2021 China Fisheries Statistical Yearbook[M]. China Agriculture Press, Peking. pp. 33.) [4] 徐佳文. 2020. 草鱼白细胞介素21及其受体IL-21Rα的鉴定和功能研究[D]. 硕士学位论文, 上海海洋大学, 导师: 邹钧, pp. 22-53. (Xu J W.2020. Identification and functional study of interleukin 21 and its receptor IL-21Rα in grass carp[D]. Thesis for M.S., Shanghai Ocean University, Supervisor: Zou J, pp. 22-53.) [5] 杨超, 董浚键, 刘志刚, 等. 2021. 大口黑鲈源维氏气单胞菌的分离鉴定[J]. 南方水产科学, 17(3): 54-61. (Yang C, Dong J J, Liu Z G, et al.2021. Isolation and identification of Aeromonas veronii from largemouth bass[J]. Southern Aquatic Sciences, 17(3): 54-61.) [6] 杨怀欣. 2021. 基于转录组的大口黑鲈(Micropterus salmoides) piscidins基因鉴定及感染胁迫响应研究[D]. 硕士学位论文, 大连理工大学, 导师: 于海宁, pp. 1. (Yang H X.2021. Transcriptome-based identification of piscidins genes and response to infection stress in largemouth bass (Micropterus salmoides)[D]. Thesis for M.S., Dalian University of Technology, Supervisor: Yu H N, pp. 1.) [7] Abos B, Wang T, Secombes C J, et al.2020. Distinct modes of action of CD40L and adaptive cytokines IL-2, IL-4/13, IL-10 and IL-21 on rainbow trout IgM(+) B cells[J]. Developmental and Comparative Immunology, 111(10): 103752. [8] Adoro S, Cubillos-Ruiz J R, Chen X, et al.2015. IL-21 induces antiviral microRNA-29 in CD4 T cells to limit HIV-1 infection[J]. Nature Communications, 6(6): 7562. [9] Bird S, Zou J, Kono T, et al.2005. Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny[J]. Immunogenetics, 56(12): 909-923. [10] Brenne A T, Ro T B, Waage A, et al.2002. Interleukin-21 is a growth and survival factor for human myeloma cells[J]. Blood, 99(10): 3756-3762. [11] Cui Z W, Zhang X Y, Chen X H, et al.2020. Splicing variants of grass carp (Ctenopharyngodon idellus) IL-21: Functions in IgM(+) B cell proliferation and IgM secretion[J]. Developmental and Comparative Immunology, 110(9): 103728. [12] Cui Z W, Li D Q, Zhao F, et al.2021. Molecular characterization and functional analysis of IL-18 in snakehead (Channa rgus) during Aeromonas schubertii and Nocardia seriolae infections[J]. Molecular Immunology, 137(9): 212-220. [13] Davis M R, Zhu Z, Hansen D M, et al.2015. The role of IL-21 in immunity and cancer[J]. Cancer Letters, 358(2): 107-114. [14] Habib T, Nelson A, Kaushansky K.2003. IL-21: A novel IL-2-family lymphokine that modulates B, T, and natural killer cell responses[J]. Journal of Allergy and Clinical Immunology, 112(6): 1033-1045. [15] Iannello A, Boulassel M R, Samarani S, et al.2010. Dynamics and consequences of IL-21 production in HIV-infected individuals: A longitudinal and cross-sectional study[J]. The Journal of Immunology, 184(1): 114-126. [16] Jin H, Carrio R, Yu A, et al.2004. Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis[J]. The Journal of Immunology, 173(1): 657-665. [17] Kang L, Kent B, Li T, et al.2010. Rational design of interleukin-21 antagonist through selective elimination of the γC binding epitope[J]. The Journal of Biological Chemistry, 285(16): 12223-12231. [18] Lei X, Zhao R, Geng Y, et al.2020. Nocardia seriolae: A serious threat to the largemouth bass Micropterus salmoides industry in southwest China[J]. Diseases of Aquatic Organisms, 142: 13-21. [19] Leonard W J, Wan C K.2016. IL-21 Signaling in immunity[J]. F1000Research, 5(2): 224. [20] Li Q, Wu M, Cui K, et al.2020. Characterization of antiviral immune response induced by poly(I:C) in macrophages of farmed large yellow croaker (Larimichthys crocea)[J]. Fish and Shellfish Immunology, 104(9): 663-672. [21] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the the 2-ΔΔCT method[J]. Methods, 25(4):402-408. [22] Long D, Chen Y J, Wu H J, et al.2019. Clinical significance and immunobiology of IL-21 in autoimmunity[J]. Journal of Autoimmunity, 99(3): 1-14. [23] Muneta Y, Kikuma R.2004. Uenishi H, et al. Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21[J]. Journal of Veterinary Medical Science, 66(3): 269-275. [24] Ozaki K, Kikly K, Michalovich D, et al.2000. Cloning of a type I cytokine receptor most related to the IL-2 receptor β chain[J]. Proceedings of the National Academy of Sciences of the USA, 97(21): 11439-11444. [25] Paidipally P, Tripathi D, Van A, et al.2018. Interleukin-21 regulates natural killer cell responses during Mycobacterium tuberculosis infection[J]. The Journal of Infectious Diseases, 217(8): 1323-1333. [26] Parrish-Novak J, Dillon S R, Nelson A, et al.2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function[J]. Nature, 408(6808): 57-63. [27] Pei C, Song H L, Zhu L, et al.2021. Identification of Aeromonas veronii isolated from largemouth bass Micropterus salmoides and histopathological analysis[J]. Aquaculture, 540(7): 736707. [28] Percy M G, Gründling A.2014. Lipoteichoic acid synthesis and function in gram-positive bacteria[J]. Annual Review of Microbiology, 68(5): 81-100. [29] Qin C B, Zhao W L, Deng D P, et al.2020. Effects of glucose, insulin, glucagon, LPS and poly (I:C) on adiponectin receptors expression in grass carp (Ctenophcuyngodon idellus)[J]. Aquaculture, 528(11): 735501. [30] Ren G, Esser S, Jochum C, et al.2012. Interleukin 21 augments the hepatitis B virus-specific CD8+ T-cell response in vitro in patients coinfected with HIV-1[J]. AIDS, 26(17): 2145-2153. [31] Shi Y L, Chen Z Y, Zhao Z H, et al.2019. IL-21 Induces an imbalance of Th17/Treg cells in moderate-to-severe plaque psoriasis patients[J]. Frontiers in Immunology, 10(8): 1865. [32] Solaymani-Mohammadi S, Berzofsky J A.2019. Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection[J]. PLoS Pathogens, 15(2): 1007614. [33] Spolski R, Leonard W J.2008. Interleukin-21: Basic biology and implications for cancer and autoimmunity[J]. Annual Review of Immunology, 26(1): 57-79. [34] Spolski R, Kim H P, Zhu W, et al.2009. IL-21 mediates suppressive effects via its induction of IL-10[J]. The Journal of Immunology, 182(5): 2859-2867. [35] Strengell M, Matikainen S, Siren J, et al.2003. IL-21 in synergy with IL-15 or IL-18 enhances IFN-γ production in human NK and T cells[J]. The Journal of Immunology, 170(11): 5464-5469. [36] Wan C K, Li P, Spolski R, et al.2015. IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells[J]. Nature Communications, 6(1): 7988. [37] Wang H J, Xiang L X, Shao J Z, et al.2006. Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis[J]. Cytokine, 35(3-4): 126-134. [38] Wang T, Diaz-Rosales P, Costa M M, et al.2011. Functional characterization of a nonmammalian IL-21: Rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-γ, IL-10, and IL-22[J]. The Journal of Immunology, 186(2): 708-721. [39] Yu C, Zhang P, Li X P, et al.2020. Japanese flounder Paralichthys olivaceus interleukin 21 induces inflammatory response and plays a vital role in the immune defense against bacterial pathogen[J]. Fish and Shellfish Immunology, 98(3): 364-373. [40] Zhang A Y, Jian X Y, Wang D, et al.2020. Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation[J]. Fish and Shellfish Immunology, 99(4): 19-26.
