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1 2016 年 9 月, 23(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 半滑舌鳎 CD40 基因克隆及其免疫功能分析 1, 位战飞 2 1,, 郭华 2, 李海龙 1 1,, 郑卫卫 2, 王文文 1, 戴欢 1, 朱颖 1, 刘洋 1, 董忠典 1 1, 3, 陈松林 1.,, ; 2., ; 3.,, 摘要 : RACE (Cynoglossus semilaevis) 1 CD40 cdna 2098 bp, 1011 bp, 336 CD N- 4, 28%~47%,, CD40 CD40, CD40 PCR, CD40,,, (Vibrio harveyi), PBS, mrna 3 ( ), 12 h, 6 h CD40 关键词 : ; CD40 ; ; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) [1] (tumor necrosis factor receptor superfamily, TNFRSF), [2] CD40(TNFR superfamily member 5) TNFRSF [3], I, Stamenkovic [4] (Homo sapiens) CD40,, CD40, B B [5 6], T [7 9], CD40 T, T, T [10] CD40 CD40L CD40 [11 12], CD40L [13 14], CD40- CD40L T [15 16], CD40-CD40L B [17 19], CD40 (Paralichthys olivaceus) [20] (Danio 收稿日期 : ; 修订日期 : 基金项目 : ( );. 作者简介 : (1989 ),,, DNA. zhanfeiwei@126.com 通信作者 :,,,,. chensl@ysfri.ac.cn

2 rerio) [21] (Salmo salar) [22] Park [20],, CD40, CD40 Gong [21] CD40-CD40L, Lagos [22], CD40- CD40L Shao [23] SNP QTL, 12 (Vibrio anguillarum), CD40 (MHC class II) (Cynoglossus semilaevis) (Pleuronectiformes) (Cynoglossidae) (Cynoglossus),,, [24],, (Vibrio harveyi),, [25],,,, CD40,,, 1 CD40, PCR (qrt-pcr), CD40 3, CD40, 1 材料与方法 1.1 实验用鱼 (1.5 ), (145.01±60.02) g, (27.68±5.53) cm, 7 d, : (21±2),, 1, 1, 1/3 1.2 哈维氏弧菌培养与感染, 3, 9, 80, CD40,, h 8000 r/min 15 min, PBS, CFU/mL, 30 μl/g, 3 PBS 0 h 6 h 12 h 24 h 48 h 72 h 96 h 7, 3, 80, CD RNA 提取及 cdna 合成 TRIZOL(Invitrogen) RNA, RNA, NanoVue TM Plus Spectrophotometer (GE Healthcare, Piscataway, NJ, USA) RNA PrimeScript RT reagent Kit with gdna Eraser (TaKaRa, ) cdna, Smart TM RACE cdna Amplification Kit (Ta- KaRa, ) RACE-Ready- cdna 1.4 CD40 基因的克隆 Chen [26], CD40 cdna, Primer Premier 5.0 (CD40-A/CD40-S, 1), cdna PCR,,

3 5 : CD Tab. 1 表 1 本研究所用到的引物及其序列 Names and sequences of the primers used in this study primer (5 3 ) sequence(5 3 ) purpose CD40-A CD40-S CD40-5 R CD40-3 R CD40-5 N CD40-3 N CD40-qRT-A CD40-qRT-S β-actin-a β-actin-s GAAGCCCAAACTCCAGTCAA CCAGGTTTCTACCCAGGACA ACATTCGTGTCATTCGCTCGCAG AAGGTGTGTAACTGCTGTTGACGGG CGGGACATCTTTGGCACA TAGATTGAAGCGGAGCACG ATTCGTGTCATTCGCTCGC ACTGTGTGCCAAAGATGTCCC GCATCACACCTTCTACAACGA ACCAGAGGCATACAGGGACA verifying the internal fragment cloning the 5 and 3 ends 5 3 real-time PCR PCR CD40 cdna cdna, RACE (CD40-5 R/5 N CD40-3 R/3 N, 1), CD40-5 R CD40-3 R RACE-Ready-cDNA RACE Touchdown PCR 15 μl: 10 LA Taq Buffer 1.5 μl; dntp (2.5 mmol/l) 1.2 μl; UPM 1.6 μl; Gsp (CD40-5 R CD40-3 R) 0.3 μl; LA Taq DNA (TaKaRa) 0.2 μl; RACE-Ready-cDNA 1 μl; ddh 2 O 9.2 μl Touchdown PCR : 94 7 min; 10 : s, s( 0.5 ), 72 3 min; 30 : s, s, 72 3 min; min; 4 PCR 50 NEST PCR, CD40-5 N CD40-3 N : 10 Buffer 5 μl; dntp (2.5 mmol/l) 4 μl; NUP NGsp (CD40-5 N CD40-3 N) 1.5 μl; Taq DNA (TaKaRa) 0.5 μl; Touchdown PCR 50 2 μl; ddh 2 O 50 μl : 94 7 min; 35 : s, s, 72 3 min; min; 4 NEST PCR 2%, TianGen, pmd-18t, Top10, 37 12~16 h, 生物学分析及进化树构建 DNAstar 5.0, (ORF), SingalP 4.0 Soft Berry-Psite TMpred InterProScan SequenceSearch CD40 NCBI Blastp( cgi) Clustal W CD40 Mega 6.0 (Neighbor-Joining, NJ) 1.6 CD40 基因表达模式检测 PCR(qRT-PCR) CD40, 7 3 CD40 β-actin (β-actina/β-actins, 1), ORF (CD40-qRT-A CD40-qRT-S, 1), SYBR Premix Ex Taq TM II (TaKaRa, ), ABI 7500 Fast Real-time (Applied Biosystems, USA) CD40 ± ( x ±SE), SPSS.17.0 Duncan, P< 结果与分析 2.1 半滑舌鳎 CD40 基因序列特征 CD40 cdna 2098 bp,

4 bp, 5 (UTR) 3 44 bp 1043 bp ORF 336, kd, UTR 2 (ATTTA), poly A 28 bp (AATAAA)( 1) NCBI, : KU291170, 1, 1~33, 2 N- ( 146~149 aa 164~167 aa), 3 camp cgmp ( 109~112 aa, 185~188 aa 229~232 aa), 4 C ( 12~14 aa, 134~136 aa, 296~298 aa 306~308 aa) 2 CAAX box ( 115~118 aa 126~129 aa), 202~224 aa ( 1), 4 (cysteine-rich domains, CRDs) ( 2), CD40 28%~47%,, 47%, 45%, 44% ( 3), CD40 CD40, CD40 1 CD40 cdna 5 3 ;., ; ATG TGA AATAAA ; 2 ATTTA ; ; ; 2 N-. Fig. 1 The CD40 cdna sequence and deduced amino acids in Cynoglossus semilaevis Lowercase letters represent the sequence of 5 and 3 unstranslated region. Capital letters represent the coding sequence. The nucleotide sequence is above and the coded amino sequence is below. The initiation codon ATG, the stop codon TGA and the polyadenylation (AATAAA) are characterized in bold. The boxed sequences represent two instability motifs (ATTTA). The signal peptide is shaded. The trans-membrane domain is underlined. Two N-glycosylation sites are double underlined.

5 5 : CD CD40 CD40 GenBank : (KU291170), (BAC ), (ACQ ), (AGM ), 鲀 (ACL ), 鳉 (ACL ), (ACI ), (ADE ), (ACO ), (ACL ), (NP ), (AAO ), (NP ). CD40 4. Fig. 2 Multiple alignment of CD40 amino acid sequence between Cynoglossus semilaevis and other species GenBank accession number: Cynoglossus semilaevis (KU291170), Paralichthys olivaceus (BAC ), Anoplopoma fimbria (ACQ ), Lutjanus sanguineus (AGM ), Takifugu rubripes (ACL ), Oryzias latipes (ACL ), Salmo salar (ACI ), Oncorhynchus mykiss (ADE ), Esox Lucius (ACO ), Danio rerio (ACL ), Rattus norvegicus (NP ), Homo sapiens (AAO ) and Xenopus tropicalis (NP ). The boxed sequences are four cysteine-rich domains., CD 半滑舌鳎 CD40 基因表达模式 qrt-pcr, CD40,,,, ( 4), 3 CD40 12 h, 4.3 (P<0.05); 6 h, (P<0.05), 3 48 h, ( 5) 3 讨论 RACE 1 CD40, GenBank : KU291170

6 CD40 Fig. 3 Phylogenetic analysis of Cynoglossus semilaevis CD40 with other reported CD40s 4 CD40 (P<0.05). Fig. 4 Expression levels of CD40 in various tissues of healthy Cynoglossus semilaevis Different letters denotes significant difference between different tissues (P<0.05)., CD40, 1, 1 4 CD40, CD40, CD40 3 -UTR AU (AU-rich element), 1 (AATAAA) 2 mrna (ATTTA),, mrna 3 -UTR AU mrna, [27] CD40 cdna 3 -UTR 2 CD40 CD40 2 : CD40 (membraneanchored CD40, mcd40) CD40 (soluble CD40, scd40) scd40 CD40L, mcd40 CD40L, B [28 29],, CD40 mcd40, scd40 [20 22] CD40, CD40 N 1, C 1 2 N- CD40 mcd40, I, CD40,, CD40,, 4, 4 CD40L,

7 5 : CD Fig. 5 5 CD40 (P<0.05). The expression profiles of CD40 in liver, spleen, and kidney of Cynoglossus semilaevis after stimulation by Vibrio harveyi for different times Different letters denotes significant difference between time points (P<0.05). CD40-CD40L [20] CD40 qrt-pcr, CD40,,, [20] [21] [22] CD40,, CD40, [20], ; [21], ; [22],, CD40, CD40,,, [30] CD40, qrt-pcr, 3 ( ), CD40,, CD40 3 [20] [22], [21] (KLH) (LPS) CD40 [20]

8 , (Con A) (PMA), CD40 1 h, LPS, 1~6 h CD40, 6 h [22], LPS (Poly I:C), CD40 12 h [21], KLH, CD40 6 ; LPS, 3 CD40,, CD40,, CD h, CD40-CD40L,, CD40, CD40, CD40 参考文献 : [1] Locksley R M, Killeen N, Lenardo M J. The TNF and TNF receptor superfamilies: integrating mammalian biology[j]. Cell, 2001, 104(4): [2] Hehlgans T, Pfeffer K. The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games[j]. Immunology, 2005, 115(1): [3] Smith C A, Farrah T, Goodwin R G. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death[j]. Cell, 1994, 76(6): [4] Stamenkovic I, Clark E A, Seed B. A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinomas[j]. EMBO J, 1989, 8(5): [5] Clark E A, Ledbetter J A. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50[J]. Proc Natl Acad Sci USA, 1986, 83(12): [6] Kooten C V, Bancherean J. Functions of CD40 on B cells, dendritic cells and other cells[j]. Curr Opin Immunol, 1997, 9(3): [7] Kooten C V, Banchereau J. CD40-CD40 ligand[j]. J Leukoc Biol, 2000, 67(1): [8] Schonbeck U, Mach F, Libby P. CD154 (CD40 ligand)[j]. Int J Biochem Cell Biol, 2000, 32(7): [9] Ridge J P, Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4 + T-helper and a T-killer cell[j]. Nature, 1998, 393(6684): [10] Martin S, Agarwal R, Murugaiyan G, et al. CD40 expression levels modulate regulatory T cells in Leishmania donovani infection[j]. J Immunol, 2010, 185(1): [11] Akira Y A, Sayegh M H. The CD154-CD40 costimulatory pathway in transplantation[j]. Transplantation, 2002, 73(1): S36 S39. [12] Du Z L. The role of CD40-CD40L costimulatory pathway in the humoral and cellular immunity[j]. Chinese Journal of Cellular and Molecular Immunology, 2012, 28(12): [. CD40-CD40L [J]., 2012, 28(12): ] [13] Allen R C, Armitage R G, Conley M E, et al. CD40 ligand gene defects responsible for X-linked hyper-igm syndrome[j]. Science, 1993, 259(5097): [14] Fuleihan R, Ramesh N, Loh R, et al. Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiencywith normal or elevated IgM[J]. Proc Natl Acad Sci USA, 1993, 90(6): [15] Kawabe T, Naka T, Tanaka T, et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation[j]. Immunity, 1994, 1(3): [16] Xu J, Foy T M, Laman J D, et al. Mice deficient for the CD40 ligand[j]. Immunity, 1994, 1(5): [17] Grewal I S, Flavell R A. The role of CD40 ligand in costimulation and T-cell activation[j]. Immunol Rev, 1996, 153(1): [18] Schönbeck U, Mach F, Bonnefoy J Y, et al. Ligation of CD40 activates interleukin 1β-converting enzyme (caspase-1) activity in vascular smooth muscle and endothelial cells and promotes elaboration of active interleukin 1β[J]. J Biol Chem, 1997, 272(31): [19] Sempowski G D, Chess P R, Moretti A J, et al. CD40 mediated activation of gingival and periodontal ligament fibroblasts[j]. J Periodontol, 1997, 68(3): [20] Park C I, Hirono I, Hwang J Y, et al. Characterization and

9 5 : CD expression of a CD40 homolog gene in Japanese flounder Paralichthys olivaceus[j]. Immunogenetics, 2005, 57(9): [21] Gong Y F, Xiang L X, Shao J Z. CD40154-CD40 interactions are essential for thymus-dependent antibody production in zebrafish: insights into the origin of costimulatory pathway in helper T cell-regulated adaptive immunity in early vertebrates[j]. J Immunol, 2009, 182(12): [22] Lagos L X, Iliev D B, Helland R, et al. CD40L-a costimulatory molecule involved in the maturation of antigen presenting cells in Atlantic salmon (Salmo salar)[j]. Dev Comp Immunol, 2012, 38(3): [23] Shao C W, Niu Y C, Rastas P, et al. Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis[j]. DNA Res, 2015, 22(2): [24] Liu X Z, Zhuang Z M, Ma A J, et al. Reproductive biology and breeding technology of Cynoglossus semilaveis Gunther[J]. Marine Fisheries Research, 2005, 26(5): [,,,. [J]., 2005, 26(5): 7 14.] [25] Chen Z Q, Yao Z X, Lin M, et al. Study on pathogen of skin ulcer disease of half-smmoth tongue sole (Cyonlossus semilaevis)[j]. Journal of Fisheries of China, 2012, 36(5): [,,,. [J]., 2012, 36(5): ] [26] Chen S L, Zhang G J, Shao C W. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle[j]. Nat Genet, 2014, 46(3): [27] Hel Z, Marco S D, Radzioch D. Characterization of the RNA binding proteins forming complexes with a novel putative regulatory region in the 3-UTR of TNF-a mrna[j]. Nucl Acids Res, 1998, 26(11): [28] Kooten C V, Gaillard C, Galizzi J P, et al. B cells regulate expression of CD40 ligand on activated T cells by lowering the mrna level and through the release of soluble CD40[J]. Eur J Immunol, 1994, 24(4): [29] Fanslow W C, Anderson D M, Grabstein K H, et al. Soluble forms of CD40 inhibit biologic responses of human B cells[j]. J Immunol, 1992, 149(2): [30] Koppang E O, Fischer U, Moore L, et al. Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue[j]. J Anat, 2010, 217(6):

10 Cloning and immunologic function analysis of CD40 from half-smooth tongue sole (Cynoglossus semilaevis) WEI Zhanfei 1, 2, GUO Hua 1, 2, LI Hailong 1, ZHENG Weiwei 1, 2, WANG Wenwen 1, DAI Huan 1, ZHU Ying 1, LIU Yang 1, DONG Zhongdian 1, CHEN Songlin 1, 3 1. Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Qingdao , China; 2. College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai , China; 3. Laboratory for Marine Fisheries, Qingdao National Laboratory for Marine Science and Technology, Qingdao , China Abstract: Cynoglossus semilaevis, which is distributed along the coast of China, is one of the most economically important marine aquaculture species. With the expansion of C. semilaevis breeding, diseases caused by bacteria and viral pathogens have become more widespread and serious. The disease caused by the bacterial pathogen Vibrio harveyi has become an obstacle in the development of half-smooth tongue sole aquaculture. Therefore, it is important to understand the defense mechanisms of the host, C. semilaevis, against this pathogen. The CD40 protein, which is a member of the tumor necrosis factor receptor superfamily 5, is an important receptor molecule that plays a key role in the host s immune system and participates in several immune signaling pathways. In this study, using the whole-genome sequence of half-smooth tongue sole, we obtained one CD40 homolog using the rapid amplification of cdna ends technique. The full-length cdna was 2098 bp long and included an open reading frame (ORF) of 1011 bp, a 5 -untranslated region (UTR) of 44 bp, and a 3 -UTR of 1043 bp. The ORF of CD40 encoded a 336-amino acid protein with a predicted molecular weight of kda and a theoretical isoelectric point of The 3 -UTR included two instability motifs (ATTTA) and one polyadenylation signal (ATTAAA) located 28 bp upstream of the polya tail signal (AATAAA). The putative amino acid sequence contained one signal peptide, one trans-membrane region, two N-glycosylation sites, and four conserved cysteine-rich domains. The deduced amino acid sequence of C. semilaevis CD40 shared 28% 47% identity with the CD40s from other teleost fish, mammals, and amphibians, with higher similarity to CD40s of bony fish. The phylogenetic tree constructed based on the amino acid sequences demonstrated that the C. semilaevis CD40 clustered in one branch with other teleost fish. Real-time quantitative PCR analyses detected C. semilaevis CD40 transcripts in a wide range of tissues in healthy adult fish. There were higher transcript levels of C. semilaevis CD40 in the liver, spleen, and kidney after challenge with V. harveyi. After challenge with the pathogen, the peak transcript levels of CD40 were at 12 h in the liver, and at 6 h in the spleen and the kidney, before a second peak in transcript levels at 48 h in the three tissues. These results indicate that CD40 is involved in the immune response of C. semilaevis to the bacterial pathogen V. harveyi. Key words: Cynoglossus semilaevis; CD40 gene; gene clone; real-time quantitative PCR; Vibrio harveyi Corresponding author: CHEN Songlin. chensl@ysfri.ac.cn