团头鲂补体因子Bf/C2的克隆和表达分析
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摘要
为探索团头鲂(Megalobrama amblycephala)补体因子Bf和C2(complement factor B/C2,Bf/C2)的可能功能,在转录组数据基础上,采用RT-PCR克隆得到Bf/C2A和Bf/C2B基因的cDNA序列;采用荧光实时定量PCR技术检测了两基因在团头鲂早期发育过程、健康成鱼及感染嗜水气单胞菌后各组织中的表达变化。结果显示,Bf/C2AcDNA全长2 520bp,包含5′UTR 42bp、ORF 2 298bp、3′UTR 180bp,编码765个氨基酸。Bf/C2B基因ORF全长2 130bp,编码710个氨基酸。两基因的氨基酸序列分别与鲤B/C2-A2及草鱼Bf/C2B相似性最高。在早期发育阶段Bf/C2A和Bf/C2B均在在出膜后1d表达量最高,肠管形成期次之,其他时期的表达量相对较低;两基因在健康成鱼10个组织中均有表达,肝脏中表达量最高,肾脏、头肾、脾脏次之,其他组织表达量相对较低;在嗜水气单胞菌感染后,两基因在免疫相关组织如肝脏和脾脏中的表达均显著上升。上述结果表明Bf/C2在应对细菌感染的免疫过程中发挥着重要的作用。
To explore the possible function of the complement factor B and C2(Bf/C2)gene in blunt snout bream(Megalobrama amblycephala),the cDNA sequences were cloned with RT-PCR and the expression of the Bf/C2 mRNA in healthy fish tissues and after Aeromonas hydrophila infection during early development was investigated with quantitative real-time PCR.The results showed that the sequence length of Bf/C2 AcDNA was 2 520 bp containing a 42 bp 5′UTR,a 2 298 bp ORF and a 180 bp 3′UTR,encoding 765 amino acid.The Bf/C2 B cDNA had an 2 131 bp ORF,encoding 710 amino acid.The mRNA of both Bf/C2 Aand Bf/C2 B were highly expressed at 1 dpf,followed by the intestinal formation stage during early development.The highest expression of both Bf/C2 A and Bf/C2 Bin healthy adults appeared in the liver,followed by the kidney,head kidney and spleen.The expression of Bf/C2 Aand Bf/C2 Bexhibited a significant change after infection of A.hydrophilain the inmmunityrelated tissues including the liver and kidney,indicating that Bf/C2 plays important role in the immune response against bacterial infection.
To explore the possible function of the complement factor B and C2(Bf/C2)gene in blunt snout bream(Megalobrama amblycephala),the cDNA sequences were cloned with RT-PCR and the expression of the Bf/C2 mRNA in healthy fish tissues and after Aeromonas hydrophila infection during early development was investigated with quantitative real-time PCR.The results showed that the sequence length of Bf/C2 AcDNA was 2 520 bp containing a 42 bp 5′UTR,a 2 298 bp ORF and a 180 bp 3′UTR,encoding 765 amino acid.The Bf/C2 B cDNA had an 2 131 bp ORF,encoding 710 amino acid.The mRNA of both Bf/C2 Aand Bf/C2 B were highly expressed at 1 dpf,followed by the intestinal formation stage during early development.The highest expression of both Bf/C2 A and Bf/C2 Bin healthy adults appeared in the liver,followed by the kidney,head kidney and spleen.The expression of Bf/C2 Aand Bf/C2 Bexhibited a significant change after infection of A.hydrophilain the inmmunityrelated tissues including the liver and kidney,indicating that Bf/C2 plays important role in the immune response against bacterial infection.
引文
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[20]周凤娟.团头鲂NLRC3-like基因的克隆、表达及其SNP的开发和鉴定[D].武汉:华中农业大学,2015.
[21]柴欣,胡晓坤,马徐发,等.团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析[J].华中农业大学学报,2017,36(4):76-82.
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[23]GONZALEZ S F,BUCHMANN K,NIELSEN M E.Complement expression in common carp(Cyprinus carpio L.)during infection with Ichthyophthirius multifiliis[J].Developmental&comparative immunology,2007,31:576-586.
[24]WEI W,WU H Z,XU H Y,et al.Cloning and molecular characterization of two complement Bf/C2genes in large yellow croaker(Pseudosciaena crocea)[J].Fish&shellfish immunology,2009,27:285-295.
[25]万成炎,林永泰,黄道明.鲂胚后发育[J].湖泊科学,1999(11):357-362.
[26]LAUFER J,KATZ Y,PASSWELL J H.Extrahepatic synthesis of complement proteins in inflammation[J].Molecular immunology,2001,38:221-229.
[2]SARMA J V,WARD P A.The complement system[J].Cell tissue research,2011,343:227-235.
[3]SCHREIBER R D,PANGBURN M K,LESAVRE P H,et al.Initiation of the alternative pathway of complement:recognition of activators by bound C3band assembly of the entire pathway from six isolated proteins[J].Proceedings of the national academy of sciences of the United States of America,1978,75:3948-3952.
[4]ISHIKAWA N,NONAKA M,WETSELRA,et al.Murine complement C2and factor B genomic and cDNA cloning reveals different mechanisms for multiple transcripts of C2and B[J].Journal of biological chemistry,1990,265:19040-19046.
[5]BHATTACHARYA A A,LUPHER M L,STAUNTON D E,et al.Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation[J].Structure,2004,12:371-378.
[6]OSTERGAARD J R,BRUUN P G,LAMML U.HLA antigens and complement types in patients with intracranial saccular aneurysms[J].Tissue antigens,1986,28(3):176-181.
[7]BEN M S,GHANEM N,KNEISSIT,etal.Immunogenetic markers(BF,C2,C4,21-OH,TNF alpha,TCR beta,Ig)and insulin-dependent diabetes in the Tunisian population:serological and molecular study[J].Annales de biologie clinique,1991,49(7):389-396.
[8]SUNYER J O,LAMBRIS J D.Encyclopedia of life sciences[M].[S.l.:s.n.],1999.
[9]SEEGER A,MAYER W E,KLEIN J.A complement factor B-like cDNA clone from the zebrafish(Brachydanio rerio)[J].Molecular immunology,1996,33(6):511-520.
[10]KURODA N,SASAKI M,NONAKA M,et al.Molecular cloning and linkage analysis of the Japanese medaka fish complement Bf/C2gene[J].Immunogenetics,1996,44(6):459-467.
[11]SUNYER J O,ZARKADIS I,SARRIAS M R,et al.Cloning,structure,and function of two rainbow trout Bf molecules[J].Molecular immunology,1998,161(8):4106-4114.
[12]NAKAO M,FUSHITANI Y,FUJIKI K,et al.Two diverged complement factor B/C2-like cDNA sequences from a teleost,the common carp(Cyprinus carpio)[J].Journal of immunology,1998,161(9):4811-4818.
[13]ZHOU Z C,LIU H,LIU S K,et al.Alternative complement pathway of channel catfish(Ictalurus punctatus):molecular characterization,mapping and expression analysis of factors Bf/C2and Df[J].Fish&shellfish immunology,2012,32:186-195.
[14]SHEN Y B,ZHANG J B,XU X Y,et al.Molecular cloning,characterization and expression of the complement Bf/C2gene in grass carp[J].Fish&shellfish immunology,2012,32:789-795.
[15]KIMURA A,SAKAGUCHI E,NONAKA M.Multi-component complement system of Cnidaria3,Bf and MASP genes expressed in the endodermal tissues of a sea anemone,Nematostella vectensis[J].Immunobiology,2009,214(3):165-178.
[16]ZHONG L,ZHANG F,CHANG Y.Gene cloning and function analysis of complement B factor-2of Apostichopus japonicus[J].Fish&shellfish immunology,2012,33:504-513.
[17]SHIN D H,WEBB B,NAKAO M,et al.Molecular cloning,structural analysis and expression of complement component Bf/C2genes in the nurse shark,Ginglymostoma cirratum[J].Developmental&comparative immunology,2007,31(11):1168-1182.
[18]王卫民.团头鲂养殖产业现状[J].科学养鱼,2009(4):44-45.
[19]蔡丽娟,许宝青,林启存.水产致病性嗜水气单胞菌耐药性比较与分析[J].水产科学,2011,30(1):42-45.
[20]周凤娟.团头鲂NLRC3-like基因的克隆、表达及其SNP的开发和鉴定[D].武汉:华中农业大学,2015.
[21]柴欣,胡晓坤,马徐发,等.团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析[J].华中农业大学学报,2017,36(4):76-82.
[22]VOLANAKIS J E,YAMAUCHI Y,ISHII Y.Structure,polymorphism and regulation of expression of the C2gene[M]//CRUSE J N,LEWIS J R.Complement today.Basel:Karger,1993.
[23]GONZALEZ S F,BUCHMANN K,NIELSEN M E.Complement expression in common carp(Cyprinus carpio L.)during infection with Ichthyophthirius multifiliis[J].Developmental&comparative immunology,2007,31:576-586.
[24]WEI W,WU H Z,XU H Y,et al.Cloning and molecular characterization of two complement Bf/C2genes in large yellow croaker(Pseudosciaena crocea)[J].Fish&shellfish immunology,2009,27:285-295.
[25]万成炎,林永泰,黄道明.鲂胚后发育[J].湖泊科学,1999(11):357-362.
[26]LAUFER J,KATZ Y,PASSWELL J H.Extrahepatic synthesis of complement proteins in inflammation[J].Molecular immunology,2001,38:221-229.