日本沼虾C型凝集素结构域家族3的cDNA克隆、原核表达和定位分析
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摘要
C型凝集素(C-type lectin)是一类能与糖类结合的非抗体的蛋白质或糖蛋白家族,为了研究C型凝集素基因在日本沼虾组织分布、细胞定位和细菌感染过程中的表达情况,本研究应用cDNA末端快速克隆(rapid-amplification of cDNA ends,RACE)技术首次克隆了日本沼虾C型凝集素结构域家族3基因(MnLec3)的全长序列,通过实时荧光定量PCR(qRT-PCR)分析MnLec3基因在不同组织、细菌感染后不同时间的表达水平,Western blot和免疫荧光分别分析蛋白的表达水平和细胞定位。结果显示,MnLec3基因cDNA全长1 357 bp,包括125 bp的5′末端非翻译区(UTR)、1 026 bp的开放阅读框(ORF)和206 bp的3′UTR,其中开放阅读框编码341个氨基酸。氨基酸序列比对显示,日本沼虾MnLec3基因含有保守钙结合点(Met 1-Glu17)和糖识别结构域(CRD)。同源性分析结果显示,MnLec3与罗氏沼虾C型凝集素3相似度较高;邻接法(Neighbor-Joining,NJ)进化树分析结果显示,MnLec3与其他甲壳动物C型凝集素聚为一支。通过构建原核表达载体获得体外重组蛋白rMnLec3,并将纯化重组蛋白免疫大鼠获得抗血清,免疫荧光结果显示,绿色荧光信号主要在肝胰腺细胞核中表达。qRT-PCR结果显示,MnLec3在日本沼虾所检测组织中均表达,其中肝胰腺中表达量最高,血细胞次之;与对照组相比,在嗜水气单胞菌刺激12～48 h时MnLec3表达量显著升高,48 h表达量最高,Western blot分析结果显示,MnLec3蛋白表达丰度与基因表达模式基本相似,提示克隆得到的MnLec3参与日本沼虾抵御细菌入侵的免疫过程。
C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense(MnLec3) was obtained using rapid amplification of cDNA ends method(RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRTPCR. The full-length cDNA sequence of Mn Lec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp,a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame(ORF) encoding 341 amino acids.The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain(CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.
C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense(MnLec3) was obtained using rapid amplification of cDNA ends method(RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRTPCR. The full-length cDNA sequence of Mn Lec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp,a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame(ORF) encoding 341 amino acids.The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain(CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.
引文
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[5]Sun Y D, Fu L D, Jia Y P, et al. A hepatopancreasspecific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity[J].Molecular Immunology, 2008, 45(2):348-361.
[6]Zhao Z Y, Yin Z X, Xu X P, et al. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses antiwhite spot syndrome virus activity[J]. Journal of Virology, 2009, 83(1):347-356.
[7]王显伟.十足目甲壳动物C型凝集素功能研究[D].济南:山东大学, 2012.Wang X W. Functional study of C-type lectins in decapod crustaceans[D]. Jinan:Shandong University,2012(in Chinese)
[8]Zhang X W, Xu W T, Wang X W, et al. A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein[J]. Molecular Immunology, 2009, 46(8-9):1626-1637.
[9]Supungul P, Klinbunga S, Pichyangkura R, et al.Identification of immune-related genes in hemocytes of black tiger shrimp(Penaeus monodon)[J]. Marine Biotechnology, 2002, 4(5):487-894.
[10]Zhang Y, Qiu L M, Song L S, et al. Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2009, 26(1):183-192.
[11]Rattanaporn O, Utarabhand P. Molecular cloning of a Ctype lectin with two CRD domains from the banana shrimp Fenneropenaeus merguiensis:early gene upregulation after Vibrio harveyi infection[J]. Journal of Invertebrate Pathology, 2011, 106(2):196-204.
[12]Ren Q, Li M, Du J, et al. Immune response of four dualCRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii[J]. Fish&Shellfish Immunology, 2012, 33(2):155-167.
[13]Kong H J, Pake E M, Nam B H, et al. A C-type lectin like-domain(CTLD)-containing protein(PtLP)from the swimming crab Portunus trituberculatus[J]. Fish&Shellfish Immunology, 2008, 25(3):311-314.
[14]Jin X K, Li W W, Cheng L, et al. Two novel short Ctype lectin from Chinese mitten crab, Eriocheir sinensis,are induced in response to LPS challenged[J]. Fish&Shellfish Immunology, 2012, 33(5):1149-1158.
[15]张响,乔慧,傅洪拓,等.青虾(Macrobrachium nipponense)Kazal型丝氨酸蛋白酶抑制因子基因全长cDNA的克隆及表达[J].海洋与湖沼, 2012, 43(6):1292-1298.Zhang X, Qiao H, Fu H T, et al. The full length cDNA cloning and expression analysis of kazal-type serine proteinase inhibitor gene from Macrobrachium nipponense[J]. Oceanologia et Limnologia Sinica, 2012,43(6):1292-1298(in Chinese).
[16]金舒博,王宁,乔慧,等.青虾高血糖激素基因全长cDNA序列的克隆及表达分析[J].中国水产科学, 2013,20(1):82-92.Jin S B, Wang N, Qiao H, et al. Molecular cloning and expression of a full length cDNA encoding crustacean hyperglycemic hormone(CHH)in oriental river pawn(Macrobrachium nipponense)[J]. Journal of Fishery Sciences of China, 2013, 20(1):82-92(in Chinese).
[17]Fu H T, Jiang S F, Xiong Y W. Current status and prospects of farming the giant river prawn(Macrobrachium rosenbergii)and the oriental river prawn(Macrobrachium nipponense)in China[J]. Aquaculture Research,2012, 43(7):993-998.
[18]傅洪拓,乔慧,李法君,等.长江不同江段青虾的遗传多样性[J].水产学报, 2010, 34(2):204-212.Fu H T, Wu Y, Li F J, et al. Genetic diversity of Macrobrachium nipponense on a regional scale in the Yangtze River[J]. Journal of Fisheries of China, 2010,34(2):204-212(in Chinese).
[19]乔慧,吴滟,傅洪拓,等.应用SSR和SRAP标记构建青虾遗传连锁图谱[J].中国水产科学, 2012, 19(2):202-210.Qiao H, Wu Y, Fu H T, et al. Construction of a genetic linkage map for oriental river prawn(Macrobrachium nipponense)using SSR and SRAP markers[J]. Journal of Fishery Sciences of China, 2012, 19(2):202-210(in Chinese).
[20]傅洪拓,龚永生,吴滟,等.日本沼虾与海南沼虾的人工种间杂交及其同工酶分析[J].水生生物学报, 2004,28(3):327-329.Fu H T, Gong Y S, Wu Y, et al. Artificial interspecific hybridization between Macrobrachium nipponense Macrobrachium hainanense and their isozyme analysis[J]. Acta Hydrobiologica Sinica, 2004, 28(3):327-329(in Chinese).
[21]孙盛明,傅洪拓,戈贤平,等.青虾冷休克蛋白Y-box编码基因的cDNA全长克隆与表达分析[J].水产学报,2017, 41(19):1345-1354.Sun S M, Fu H T, Ge X P, et al. Molecular cloning and expression analysis of cold shock protein Y-box gene from oriental river pawn(Macrobrachium nipponense)[J]. Journal of Fisheries of China, 2017, 41(19):1345-1354(in Chinese).
[22]Tamura K, Dudley J, Nei M, et al. MEGA4:molecular evolutionary genetics analysis(MEGA)software version4.0[J]. Molecular Biology and Evolution, 2007, 24(8):1596-1599.
[23]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the2-￠￠CTmethod[J]. Methods, 2001, 25(4):402-408.
[24]Sun S M, Xuan F J, Fu H T, et al. Molecular cloning,mRNA expression and characterization of membranebound hemoglobin in oriental river prawn Macrobrachium nipponense[J]. Comparative Biochemistry and Physiology-Part A-Molecular&Integrative Physiology,2017, 207:36-42.
[25]Wang X W, Wang J X. Diversity and multiple functions of lectins in shrimp immunity[J]. Developmental&Comparative Immunology, 2013, 39(1-2):27-38.
[26]Xiu Y J, Wang Y H, Bi J X, et al. A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense[J]. Fish&Shellfish Immunology, 2016, 50:117-126.
[27]金振晓,刘维永,胡军,等.人甘露糖结合凝集素在小鼠肝脏细胞内表达[J].中国免疫学杂志, 2002, 18(12):828-831.Jin Z X, Liu W Y, Hu J, et al. Expression of human mannose-binding lectin in mice liver[J]. Chinese Journal of Immunology, 2002, 18(12):828-831(in Chinese).
[28]Salerno G, Parisi M G, Parrinello D, et al. F-type lectin from the sea bass(Dicentrarchus labrax):purification,cDNA cloning, tissue expression and localization, and opsonic activity[J]. Fish&Shellfish Immunology, 2009,27(2):143-153.
[29]Odom E W, Vasta G R. Characterization of a binary tandem domain F-type lectin from striped bass(Morone saxatilis)[J]. The Journal of Biological Chemistry, 2006,281(3):1698-1713.
[30]Wei X M, Liu X Q, Yang J M, et al. Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus[J].Fish&Shellfish Immunology, 2012, 32(1):132-140.
[31]王敏,吴萍,叶元土,等.秀丽白虾4种C型凝集素基因的克隆、组织表达与进化分析[J].水产学报, 2013,37(11):1632-1644.Wang M, Wu P, Ye Y T, et al. Molecular cloning,expression characterization and phylogenetic analysis of four novel C-type lectin genes in white shrimp(Exopalaemon modestus)[J]. Journal of Fisheries of China, 2013,37(11):1632-1644(in Chinese).
[32]Ma T H T, Tiu S H W, He J G, et al. Molecular cloning of a C-type lectin(LvLT)from the shrimp Litopenaeus vannamei:early gene down-regulation after WSSV infection[J]. Fish&Shellfish Immunology, 2007, 23(2):430-437.
[33]Zhang H, Chen L Q, Qin J G, et al. Molecular cloning,characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis[J]. Fish&Shellfish Immunology, 2011, 31(2):358-363.
[2]Sun J, Wang L, Wang B J, et al. Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis[J]. Fish&Shellfish Immunology, 2008, 25(3):290-297.
[3]Luo T, Yang H J, Li F, et al. Purification, characterization and cDNA cloning of a novel lipopolysaccharidebinding lectin from the shrimp Penaeus monodon[J].Developmental&Comparative Immunology, 2006,30(7):607-617.
[4]Drickamer K. Two distinct classes of carbohydraterecognition domains in animal lectins[J]. The Journal of Biological Chemistry, 1988, 263(20):9557-9560.
[5]Sun Y D, Fu L D, Jia Y P, et al. A hepatopancreasspecific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity[J].Molecular Immunology, 2008, 45(2):348-361.
[6]Zhao Z Y, Yin Z X, Xu X P, et al. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses antiwhite spot syndrome virus activity[J]. Journal of Virology, 2009, 83(1):347-356.
[7]王显伟.十足目甲壳动物C型凝集素功能研究[D].济南:山东大学, 2012.Wang X W. Functional study of C-type lectins in decapod crustaceans[D]. Jinan:Shandong University,2012(in Chinese)
[8]Zhang X W, Xu W T, Wang X W, et al. A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein[J]. Molecular Immunology, 2009, 46(8-9):1626-1637.
[9]Supungul P, Klinbunga S, Pichyangkura R, et al.Identification of immune-related genes in hemocytes of black tiger shrimp(Penaeus monodon)[J]. Marine Biotechnology, 2002, 4(5):487-894.
[10]Zhang Y, Qiu L M, Song L S, et al. Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei[J]. Fish&Shellfish Immunology, 2009, 26(1):183-192.
[11]Rattanaporn O, Utarabhand P. Molecular cloning of a Ctype lectin with two CRD domains from the banana shrimp Fenneropenaeus merguiensis:early gene upregulation after Vibrio harveyi infection[J]. Journal of Invertebrate Pathology, 2011, 106(2):196-204.
[12]Ren Q, Li M, Du J, et al. Immune response of four dualCRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii[J]. Fish&Shellfish Immunology, 2012, 33(2):155-167.
[13]Kong H J, Pake E M, Nam B H, et al. A C-type lectin like-domain(CTLD)-containing protein(PtLP)from the swimming crab Portunus trituberculatus[J]. Fish&Shellfish Immunology, 2008, 25(3):311-314.
[14]Jin X K, Li W W, Cheng L, et al. Two novel short Ctype lectin from Chinese mitten crab, Eriocheir sinensis,are induced in response to LPS challenged[J]. Fish&Shellfish Immunology, 2012, 33(5):1149-1158.
[15]张响,乔慧,傅洪拓,等.青虾(Macrobrachium nipponense)Kazal型丝氨酸蛋白酶抑制因子基因全长cDNA的克隆及表达[J].海洋与湖沼, 2012, 43(6):1292-1298.Zhang X, Qiao H, Fu H T, et al. The full length cDNA cloning and expression analysis of kazal-type serine proteinase inhibitor gene from Macrobrachium nipponense[J]. Oceanologia et Limnologia Sinica, 2012,43(6):1292-1298(in Chinese).
[16]金舒博,王宁,乔慧,等.青虾高血糖激素基因全长cDNA序列的克隆及表达分析[J].中国水产科学, 2013,20(1):82-92.Jin S B, Wang N, Qiao H, et al. Molecular cloning and expression of a full length cDNA encoding crustacean hyperglycemic hormone(CHH)in oriental river pawn(Macrobrachium nipponense)[J]. Journal of Fishery Sciences of China, 2013, 20(1):82-92(in Chinese).
[17]Fu H T, Jiang S F, Xiong Y W. Current status and prospects of farming the giant river prawn(Macrobrachium rosenbergii)and the oriental river prawn(Macrobrachium nipponense)in China[J]. Aquaculture Research,2012, 43(7):993-998.
[18]傅洪拓,乔慧,李法君,等.长江不同江段青虾的遗传多样性[J].水产学报, 2010, 34(2):204-212.Fu H T, Wu Y, Li F J, et al. Genetic diversity of Macrobrachium nipponense on a regional scale in the Yangtze River[J]. Journal of Fisheries of China, 2010,34(2):204-212(in Chinese).
[19]乔慧,吴滟,傅洪拓,等.应用SSR和SRAP标记构建青虾遗传连锁图谱[J].中国水产科学, 2012, 19(2):202-210.Qiao H, Wu Y, Fu H T, et al. Construction of a genetic linkage map for oriental river prawn(Macrobrachium nipponense)using SSR and SRAP markers[J]. Journal of Fishery Sciences of China, 2012, 19(2):202-210(in Chinese).
[20]傅洪拓,龚永生,吴滟,等.日本沼虾与海南沼虾的人工种间杂交及其同工酶分析[J].水生生物学报, 2004,28(3):327-329.Fu H T, Gong Y S, Wu Y, et al. Artificial interspecific hybridization between Macrobrachium nipponense Macrobrachium hainanense and their isozyme analysis[J]. Acta Hydrobiologica Sinica, 2004, 28(3):327-329(in Chinese).
[21]孙盛明,傅洪拓,戈贤平,等.青虾冷休克蛋白Y-box编码基因的cDNA全长克隆与表达分析[J].水产学报,2017, 41(19):1345-1354.Sun S M, Fu H T, Ge X P, et al. Molecular cloning and expression analysis of cold shock protein Y-box gene from oriental river pawn(Macrobrachium nipponense)[J]. Journal of Fisheries of China, 2017, 41(19):1345-1354(in Chinese).
[22]Tamura K, Dudley J, Nei M, et al. MEGA4:molecular evolutionary genetics analysis(MEGA)software version4.0[J]. Molecular Biology and Evolution, 2007, 24(8):1596-1599.
[23]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the2-￠￠CTmethod[J]. Methods, 2001, 25(4):402-408.
[24]Sun S M, Xuan F J, Fu H T, et al. Molecular cloning,mRNA expression and characterization of membranebound hemoglobin in oriental river prawn Macrobrachium nipponense[J]. Comparative Biochemistry and Physiology-Part A-Molecular&Integrative Physiology,2017, 207:36-42.
[25]Wang X W, Wang J X. Diversity and multiple functions of lectins in shrimp immunity[J]. Developmental&Comparative Immunology, 2013, 39(1-2):27-38.
[26]Xiu Y J, Wang Y H, Bi J X, et al. A novel C-type lectin is involved in the innate immunity of Macrobrachium nipponense[J]. Fish&Shellfish Immunology, 2016, 50:117-126.
[27]金振晓,刘维永,胡军,等.人甘露糖结合凝集素在小鼠肝脏细胞内表达[J].中国免疫学杂志, 2002, 18(12):828-831.Jin Z X, Liu W Y, Hu J, et al. Expression of human mannose-binding lectin in mice liver[J]. Chinese Journal of Immunology, 2002, 18(12):828-831(in Chinese).
[28]Salerno G, Parisi M G, Parrinello D, et al. F-type lectin from the sea bass(Dicentrarchus labrax):purification,cDNA cloning, tissue expression and localization, and opsonic activity[J]. Fish&Shellfish Immunology, 2009,27(2):143-153.
[29]Odom E W, Vasta G R. Characterization of a binary tandem domain F-type lectin from striped bass(Morone saxatilis)[J]. The Journal of Biological Chemistry, 2006,281(3):1698-1713.
[30]Wei X M, Liu X Q, Yang J M, et al. Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus[J].Fish&Shellfish Immunology, 2012, 32(1):132-140.
[31]王敏,吴萍,叶元土,等.秀丽白虾4种C型凝集素基因的克隆、组织表达与进化分析[J].水产学报, 2013,37(11):1632-1644.Wang M, Wu P, Ye Y T, et al. Molecular cloning,expression characterization and phylogenetic analysis of four novel C-type lectin genes in white shrimp(Exopalaemon modestus)[J]. Journal of Fisheries of China, 2013,37(11):1632-1644(in Chinese).
[32]Ma T H T, Tiu S H W, He J G, et al. Molecular cloning of a C-type lectin(LvLT)from the shrimp Litopenaeus vannamei:early gene down-regulation after WSSV infection[J]. Fish&Shellfish Immunology, 2007, 23(2):430-437.
[33]Zhang H, Chen L Q, Qin J G, et al. Molecular cloning,characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis[J]. Fish&Shellfish Immunology, 2011, 31(2):358-363.
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