|
|
|
| Expression of Ayu (Plecoglossus altivelis) Wap65-2 in an Insect Baculovirus Vector System and Verification of Its Interaction with Complement 3 |
|
|
|
|
Abstract The warm temperature acclimation related 65 kD protein-2 (Wap65-2) is a glycoprotein and so far only found in teleost fish. The expression pattern of Wap65-2 discloses its function in response to the stresses of temperature, infection and heavy metal contamination, and especially the function in anti-bacterial immunology attracts broad attention in recent years. In this study, the recombinant ayu (Plecoglossus altivelis) Wap65-2 mature peptide (rPaWap65-2m) was expressed in an insect baculovirus vector system (BEVS), by which we sought to explore a new way to produce biologically active rPaWap65-2m. The recombinant Bacmid-PaWap65-2 was obtained by cloning, and then used to transfect Spodoptera frugiperda cells (Sf9) for the viral stock. Sf9 cells in exponential growth phase were infected with high-titer P3 viral stock and then detected the expression of rPaWap65-2m by Western blot. Sf9 cells were cultured in optimized large scale serum-free system for rPaWap65-2m expression. rPaWap65-2m was purified by anion-exchange chromatography and nickel chelate affinity chromatography from the collected culture supernatant. The interaction between rPaWap65-2m and Complement 3 (C3) was identified by His-tag pulldown method. Results showed that after P3 viral stock infected Sf9 cells, rPaWap65-2m was expressed and secreted into the culture supernatant as a non-glycoprotein detected by Western blot. The maximum expression was obtained when Sf9 cells were infected with the multiplicity of infection (MOI) of 5 and collected at 72 h after infection. The purity of rPaWap65-2m was greater than 93%. rPaWap65-2m was verified to interact with full-length nature C3 by pulldown. In summary, the biologically active rPaWap65-2m was obtained for further study on its immunological function.
|
|
Received: 14 March 2018
Published: 26 September 2018
|
|
|
|
| [1]李长红, 陈炯, 史雨红, 等.宁海地区香鱼弧菌病病原菌鉴定[J].微生物学报, 2009, 49(7):931-937
[2]李长红, 杨智景, 李明云, 等.大黄鱼-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, 2015, 23(5):623-633
[3]凌同, 余黎, 白慕群.昆虫杆状病毒表达系统的研究进展与应用[J].微生物学免疫学进展, 2014, 42(2):70-78
[4]史雨红, 陈炯, 高姗姗, 等.花鲈-基因的克隆、理化性质及其表达与哈维氏弧菌感染的相关性研究动物学研究[J].Zoological Research, 2012, 33(5):481-486
[5]王丹妮, 郑春静, 江婷佳, 等.蓝点马鲛 两种基因克隆及蛋白功能的研究海洋与湖沼[J].Oceanologia et Limnologia Sinica, 2015, 46(5):1052-1060
[6]Aliza D, Ismail I S, Kuah M K, et al.Identification of Wap65,a human homologue of hemopexin as a copper-inducible gene in swordtail fish,Xiphophorus helleri[J].Fish Physiology and Biochemistry, 2008, 34(2):129-138
[7]Banerjee S, Mahanty A, Mohanty S, et al.Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita[J[J].Journal of Hazardous Materials, 2017, 336:71-80
[8]Boshra H, Li J, Sunyer J O.Recent advances on the complement system of teleost fish[J].Fish and Shellfish Immunology, 2006, 20(2):239-262
[9]Chang C H, Lin J Y, Lo W Y, et al.Hypothermal stress induced differential expression profiles of the immune response gene, warm-temperature-acclimation associated 65-kDa protein (Wap65), in the liver of fresh water and seawater milkfish, Chanos chanos[J]. [J].Fish and Shellfish Immunology, 2017, 70:174-184.
[10]Chen M, Huang X, Yuen D S, et al.A study on the functional interaction between the GHPRL family of polypeptides with their receptors in zebrafish: Evidence against GHR1 being the receptor for somatolactin[J].Molecular and Cellular Endocrinology, 2011, 337(1):114-121
[11]Clark M S, Burns G.Characterisation of the warm acclimated protein gene (wap65) in the Antarctic plunderfish (Harpagifer antarcticus)[J].DNA Sequence, 2008, 19(1):50-55
[12]Degn S E, Thiel S.Humoral pattern recognition and the complement system[J].Scandinavian Journal of Immunology, 2013, 78(2):181-193
[13]Diaz-Rosales P, Pereiro P, Figueras A, et al.The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus)[J].Fish and Shellfish Immunology, 2014, 41(1):80-92
[14]Dong M, Liang Y, Ramalingam R, et al.Proteomic characterization of the interactions between fish serum proteins and waterborne bacteria reveals the suppression of anti-oxidative defense as a serum-mediated antimicrobial mechanism[J][J].Fish and Shellfish Immunology, 2017, 62:96-106
[15]Hang B T, Milla S, Gillardin V, et al.In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus)[J].Fish and Shellfish Immunology, 2013, 34(1):339-347
[16]Hirayama M, Kobiyama A, Kinoshita S, et al.The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme[J].Journal of Experimental Biology, 2004, 207(8):1387-1398
[17]Hirayama M, Nakaniwa M, Ikeda D, et al.Primary structures and gene organizations of two types of Wap65 from the pufferfish Takifugu rubripes[J].Fish Physiology and Biochemistry, 2003, 29(3):211-224
[18]Kikuchi K, Watabe S, Aida K.The Wap65 gene expression of goldfish (Carassius auratus) in association with warm water temperature as well as bacterial lipopolysaccharide (LPS)[J].Fish Physiology and Biochemistry, 1997, 17(1):423-432
[19]Kikuchi K, Watabe S, Suzuki Y, et al.The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus[J].Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 1993, 163(5):349-354
[20]Li C H, Chen J.Molecular cloning,characterization and expression analysis of a novel wap65-1 gene from Plecoglossus altivelis[J].Comparative Biochemistry and Physiology ? Part B: Biochemistry & Molecular Biology, 2013, 165(2):144-152
[21]Li C H, Chen J, Shi Y H, et al.Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection[J].Fish and Shellfish Immunology, 2011, 31(3):500-506
[22]Mastellos D, Morikis D, Isaacs S N, et al.Complement: structure,functions,evolution,and viral molecular mimicry[J].Immunologic Research, 2003, 27(2):367-386
[23]Nishimori E, Kita-Tsukamoto K, Wakabayashi H.Pseudomonas plecoglossicida spnov.,the causative agent of bacterial haemorrhagic ascites of ayu,Plecoglossus altivelis[J].International Journal of Systematic and Evolutionary Microbiology, 2000, 50(1):83-89
[24]Patel D M, Brinchmann M F.Skin mucus proteins of lumpsucker (Cyclopterus lumpus) [J]. [J].Biochemistry and Biophysics Reports, 2017, 9:217-225
[25]Peatman E, Baoprasertkul P, Terhune J et al.Expression analysis of the acute phase response in channel cat?sh (Ictalurus punctatus) after infection with a Gram-negative bacterium[J].Developmental and Comparative Immunology, 2007, 31(11):1183-1196
[26]Peatman E, Terhune J, Baoprasertkul P, et al.Microarray analysis of gene expression in the blue catfish liver reveals early activation other MHC class I pathway after infection with Edwardsiella ictaluri[J].Molecular Immunology, 2008, 45(2):553-566
[27]Ramos A D, Concei??o K, Silva P I, et al.Specialization of the sting venom and skin mucus of Cathorops spixii reveals functional diversification of the toxins[J].Toxicon, 2012, 59(6):651-665
[28]Sakai D K.Repertoire of complement in immunological defense mechanisms of fish[J]. [J].Annual Review of Fish Diseases, 1992, 2:233-247
[29]Sha Z, Xu P, Takano T, et al.The warm temperature acclimation protein Wap65 as an immune response gene: its duplicates are differentially regulated by temperature and bacteria infections[J].Molecular Immunology, 2008, 45(5):1458-1469
[30]Shi Y H, Chen J, Li C H, et al.Molecular cloning of liver Wap65 cDNA in ayu (Plecoglossus altivelis) and mRNA expression changes following Listonella anguillarum infection[J].Molecular Biology Reports, 2010, 37(3):1523-1529
[31]Shi Y H, Chen J, Li C H, et al.The establishment of a library screening method based on yeast two-hybrid system and its use to determine the potential interactions of liver proteins in ayu,Plecoglossus altivelis[J].Fish and Shellfish Immunology, 2011, 30(4):1184-1187
[32]Shi Y H, Chen K, Ma WJ, et al.Ayu C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/macrophages[J]. Fish and Shellfish Immunology, 2018, 76: 58-67.[J].Fish and Shellfish Immunology, 2018, 76:58-67
[33]Steele K H, Stone B J, Franklin K M, et al.Improving the baculovirus expression vector system with vankyrin-enhanced technology[J]. [J].Biotechnology Progress,, 2017, 33(6):1496-1507
[34]Watabe S, Kikuchi K, Aida K.Cold-and warm-temperature acclimation induced specific cytosolic protein in goldfish and carp[J].Nippon Suisan Gakkaishi, 1993, 59(1):151-156
|
|
|
|
