Abstract：Haptoglobin (Hp), mainly synthesized by the liver, plays an important role in the acute phase response (APR) induced by infection, trauma, and inflammation. In this study, the full-length cDNA sequence of the Hp gene (LjHp) (GenBank No. MK820673) of the Japanese sea bass (Lateolabrax japonicus) was obtained from the transcriptome sequencing data of Japanese sea bass. The cDNA sequence of LjHp consisted of 2 285 nucleotides, which contained a large open reading frame predicted to encode a precursor protein comprising of 309 amino acids with a molecular weight of approximately 33.82 kD. A predicted signal peptide of 22 amino acids was present in the N-terminal of LjHp. Sequence analysis showed that LjHp had the typical characters of fish Hp, and shared the highest amino acid sequence identity (76.8%) with that of European seabream (Dicentrarchus labrax). Phylogenetic tree analysis confirmed that LjHp fell into the fish Hp cluster, and grouped tightly with swamp eel (Monopterus albus) Hp. LjHp mRNA was mainly expressed in the liver of healthy Japanese sea bass, and its expression increased significantly in liver, spleen and head kidney after infection with Vibrio harveyi (P<0.05). Western blot analysis showed that LjHp was modified by N-glycosylation in the serum of the Japanese sea bass. Moreover, the serum content of LjHp up-regulated significantly after infection with V. harveyi (P<0.05). The above results showed that the mRNA and protein expression of LjHp was closely related to V. harveyi infection, revealing that it might be a positive acute phase protein in fish against bacterial infection. The present study provides a theoretical basis for studying the functions of fish Hp and its molecular mechanism in APR induced by pathogens.
[1] 李长红, 杨智景, 李明云, 等. 2015. 大黄鱼Wap65-2 基因的分子鉴定及其与溶藻弧菌感染的相关性分析[J]. 农业生物技术学报, 23(5): 623-633. (Li C H, Yang Z J, Li M Y, et al.2015. Molecular characterization of the large yellow croaker (Larimichthys crocea) Wap65-2 gene and its correlation analysis upon Vibrio alginolyticus infection[J]. Journal of Agricultural Biotechnology, 23(5): 623-633.) [2] 林星. 2013. 花鲈皮肤溃疡病病原菌的分离与鉴定[J]. 中国农学通报, 29(32): 100-104. (Lin X.2013. Isolation and identification of the pathogens of the skin ulceration disease in Lateolabrax japonicus[J]. Chinese Agricultural Science Bulletin, 29(32): 100-104.) [3] 张春丹, 李明云. 2005. 花鲈繁殖生物学及繁育技术研究进展[J]. 宁波大学学报(理工版), 18(3): 400-403. (Zhang C D, Li M Y.2005. Research progress in reproductive biology and breeding techniques of Japanese sea bass, Lateolabrax japonicus[J]. Journal of Ningbo University (Natural Science Edition), 18(3): 400-403.) [4] 张静, 施慧, 谢建军, 等. 2009. 网箱养殖鲈鱼内脏白点病病原的分离与鉴定[J]. 浙江海洋学院学报 (自然科学版), 28(2): 176-182. (Zhang J, Shi H, Xie J, et al.2009. Isolation and identification of causative pathogen for visceral white spot in Lateolabrax japonicus stocked in sea-cage[J]. Journal of Zhejiang Ocean University (Natural Science Edition), 28(2): 176-182.) [5] Abdullah M, Schultz H, Kähler D, et al.2009. Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues[J]. Pathology-Research and Practice, 205(9): 639-647. [6] Andersen C B F, Stødkilde K, Sæderup K L, et al.2017. Haptoglobin[J]. Antioxidants & Redox Signaling, 26(14): 814-831. [7] Bradford M M.1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 72(1-2): 248-254. [8] Cerón J J, Eckersall P D, Martínez-Subiela S.2005. Acute phase proteins in dogs and cats: Current knowledge and future perspectives[J]. Veterinary Clinical Pathology, 34(2): 85-99. [9] Conner J G, Eckersall P D, Wiseman A, et al.1988. Bovine acute phase response following turpentine injection[J]. Research in Veterinary Science, 44(1): 82-88. [10] Conner J G, Eckersall P D, Wiseman A, et al.1989. Acute phase response in calves following infection with Pasteurella haemolytica, Ostertagia ostertagi and endotoxin administration[J]. Research in Veterinary Science, 47(2): 203-207. [11] Cordero H, Li C H, Chaves-Pozo E, et al.2017. Molecular identification and characterization of haptoglobin in teleosts revealed an important role on fish viral infections[J]. Developmental & Comparative Immunology, 76: 189-199. [12] Gabay C, Kushner I.1999. Acute-phase proteins and other systemic responses to inflammation[J]. New England Journal of Medicine, 340(6): 448-454. [13] Gerwick L, Corley-Smith G, Bayne C J.2007. Gene transcript changes in individual rainbow trout livers following an inflammatory stimulus[J]. Fish & Shellfish Immunology, 22(3): 157-171. [14] Gerwick L, Steinhauer R, Lapatra S, et al.2002. The acute phase response of rainbow trout (Oncorhynchus mykiss) plasma proteins to viral, bacterial and fungal inflammatory agents[J]. Fish & Shellfish Immunology, 12(3): 229-242. [15] Jain S, Gautam V, Naseem S.2011. Acute-phase proteins: As diagnostic tool[J]. Journal of Pharmacy and Bioallied Sciences, 3(1): 118-127. [16] Jayasinghe J, Elvitigala D A S, Whang I, et al.2015. Molecular characterization of two immunity-related acute-phase proteins: Haptoglobin and serum amyloid A from black rockfish (Sebastes schlegeli)[J]. Fish & Shellfish Immunology, 45(2): 680-688. [17] Joshi V, Gupta V K, Bhanuprakash A G, et al.2018. Haptoglobin and serum amyloid A as putative biomarker candidates of naturally occurring bovine respiratory disease in dairy calves[J]. Microbial Pathogenesis, 116: 33-37. [18] Kalmus P, Simojoki H, Pyörälä S, et al.2013. Milk haptoglobin, milk amyloid A, and N-acetyl-β-D-glucosaminidase activity in bovines with naturally occurring clinical mastitis diagnosed with a quantitative PCR test[J]. Journal of Dairy Science, 96(6): 3662-3670. [19] Li C H, Chen J, Shi Y H, et al.2011. Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection[J]. Fish & Shellfish Immunology, 31(3): 500-506. [20] Lin B, Chen S, Cao Z, et al.2007. Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: Striking similarities and obvious differences with mammals[J]. Molecular Immunology, 44(4): 295-301. [21] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 25(4): 402-408. [22] Murata H, Shimada N, Yoshioka M.2004. Current research on acute phase proteins in veterinary diagnosis: An overview[J]. The Veterinary Journal, 168(1): 28-40. [23] Peatman E, Baoprasertkul P, Terhune J, et al.2007. Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) after infection with a Gram-negative bacterium[J]. Developmental & Comparative Immunology, 31(11): 1183-1196. [24] Ren Y, Liu S F, Nie L, et al.2019. Involvement of ayu NOD2 in NF-κB and MAPK signaling pathways: Insights into functional conservation of NOD2 in antibacterial innate immunity[J]. Zoological Research, 40(2): 77-88. [25] Shrimal S, Cherepanova N A, Gilmore R.2015. Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum[J]. Seminars in Cell & Developmental Biology, 41: 71-78. [26] Tamura K, Peterson D, Peterson N, et al.2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 28(10): 2731-2739. [27] Thomas F C, Waterston M, Hastie P, et al.2015. The major acute phase proteins of bovine milk in a commercial dairy herd[J]. BMC Veterinary Research, 11(1): 207-216. [28] Tian F J, Zhang Y Y, Liu L Q, et al.2016. Haptoglobin protein and mRNA expression in psoriasis and its clinical significance[J]. Molecular Medicine Reports, 14(4): 3735-3742. [29] Upadhyaya I, Thanislass J, Veerapandyan A, et al.2016. Characterization of haptoglobin isotype in milk of mastitis-affected cows[J]. Veterinary Sciences, 3(4): 29. [30] Xu F, Shi Y H, Chen J.2019. Characterization and immunologic functions of the macrophage migration inhibitory factor from Japanese sea bass, Lateolabrax japonicus[J]. Fish & Shellfish Immunology, 86: 947-955.
