Abstract:Abstract The cluster of differentiation 63 (CD63) belongs to the tetraspanin superfamily, which plays an important role in immuno-physiological functions including cell adhesion, signal transduction and immune cell activation. In recent years, there are serious disease problems in the aquaculture of Lateolabrax japonicus, in order to strengthen the basic research for fish diseases and immune. In this study, CD63 cDNA sequence of Lateolabrax japonicus was cloned using rapid-amplification of cDNA ends (RACE) method. The protein molecular weight, isoelectric point and structure prediction using online software. To expression analysis of CD63 using qRT-PCR. The experimental results showed that the amplified sequence was 1 219 bp, including 114 bp 5'-UTR, 382 bp 3'-UTR and 723 bp ORF which encoding 240 amino acids. It was predicted that the molecular weight of protein was 26.26 kD and the isoelectric point is 8.43. The results of online software analysis showed that the protein was a 4 transmembrane protein, including 4 transmembrane regions (13~35, 50~72, 85~107 and 207~229 aa), three intracellular domains (1~12, 73~84 and 230~240 aa) and 2 extracellular loops (36~49, 108~206 aa). A conserved Cys-Cys-Gly motif and 3 potential N-glycosylation sites (Asn128, Asn150 and Asn172) were found in the large extracellular loop region. The carboxy terminus of the protein had a tyrosine lysosome target sequence (GYEVM, Gly-Tyr-Glu-Val-Met). Bioinformatics analysis indicated that Lateolabrax japonicus CD63 peptide share 61%~84% similarity with other fish counterparts. Quantitative real-time PCR showed that the mRNA of CD63 was expressed in all tissues examined, including liver, spleen, head kidney, intestines, gill, fat, muscle, brain and blood. The higher expression of CD63 was detected in the liver, muscle and head kidney. The expression level of CD63 in head kidney didn't show any difference at 3 h after intraperitoneal injection with Vibrio harveyi, but up-regulated significantly at 6 h (P<0.05) and down-regulation at 12 h. In vitro experiment, the mRNA expression of CD63 showed significantly up-regulation in head kidney macrophages after stimulation with LPS 12 h (P<0.05). The CD63 expression were also significantly up-regulated in macrophages at 24 h after stimulating by Poly I:C (P<0.05). The results indicated that CD63 may play important roles in pathogen invasion and inflammation reaction. This provides the theoretical basis for the further study of fish immune response molecules regulatory networks.
[1]包晓婧, 冯若飞, 马忠仁.分子的研究进展[J].西北民族大学学报, 2015, 36(3):22-26[2]石刚, 董明.四跨膜蛋白超家族在消化系统恶性肿瘤中的生物学行为的研究进展[J]. 中国普外基础与临床杂志, 2014, (9): 1173-1178. (Shi G, Dong M. Advances in biological behavior of four transmembrane protein superfamily in digestive system malignancies [J]. Chinese Journal of Bases and Clinics in General Surgery, 2014, (9): 1173-1178.)[J].中国普外基础与临床杂志, 2014, (9):1173-1178[3]史雨红, 陈炯, 高珊珊, 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[J].动物学研究, 2012, 33(5):481-486[4]王玉珍, 谢基明, 王宇飞, 等.负调节巨噬细胞中 :诱导的细胞因子的表达[J].分子与细胞免疫学, 2011, 27(10):867-870[5]赵春晖, 袁晓飞, 刘欣, 等.七鳃鳗的分子克隆、生物学特性及表达分析[J].辽宁师范大学学报, 2016, 39(2):247-253[6]Adell T, Gamulin V, Perovic O S, et al.Evolution of metazoan cell junction proteins: the scaffold protein MAGI and the transmembrane receptor tetraspanins in the demosponge Suberites domuncula[J].Molecular Evolution, 2004, 59(1):41-50[7]Buhring H J, Seiffert M.Giesert C,et alThe basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatasephosphodiesterase 3[J].Blood, 2001, 97(10):3303-3305[8]Ebgering A, Pieters J.Association of distinct tetraspanins with MHC class Ⅱ molecules at different subcellular locations in human immature dendritic cells[J].International immunology, 2001, 13(2):127-137[9]Engering A, Kuhn L, Fluitsma D, et al.Differential posttranslational modification of CD63 molecules during maturation of human dendritic cells[J].Europe Journal Biochemistry, 2003, 270(11):2412-2420[10]Fujihara M, Muroi M, Tanamoto K I, et al.Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex [J]. Phamacology and Therapeutics, 2003, 100: 171-194.[J].Phamacology and Therapeutics, 2003, 100(2):171-194[11]Hemler M E.Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of memberane microdomain [J]. Annual Review of Cell and Developmental Biology, 2003, 19: 397-422.[J].Annual Review of Cell and Developmental Biology, 2003, 19(19):397-422[12]Maaike S P, Judith K.Trafficking and function of the tetraspanin CD63 [J]. Science Direct, 2009, 315: 1584-1592.[J].Science Direct, 2009, 315(9):1584-1592[13]Maecker H T, Todd S C, Levy S.The tetraspanin superfamily: molecular facilitators[J].Faseb Journal, 1997, 11(6):428-442[14]Miyamoto H, Homma M, Hotta H.Molecular cloning of the murine homologue of CD63ME491 and detection of its strong express in the kidney and activated macrophages[J].Biochimica Et Biophysica Acta, 1994, 1217(3):312-316[15]Rosario C, Beatriz A, Lucia G, et al.Molecular characterization of CD9 and CD63, two tetraspanin family members expressed in trout B lymphocytes [J]. Developmental and Comparative Immunology, 2015, 15: 116-125.[J].Developmental and Comparative Immunology, 2015, 51(1):116-125[16]Rous B A, Reaves B J, Ihrke G, et al.Role of adaptor complex AP-3 in targeting wild-type and mutated CD63 to lysosomes[J].Molecular Biology of the Cell, 2002, 13(3):1071-1082[17]Stipp C S, Kolesnikova T V, Hemler M E.Functional domains in tetraspanins proteins[J].Trends Biochemistry Science, 2003, 28(2):106-122[18]Todres E, Nardi J B, Robertson H M.The tetraspanins superfamily in insects[J].Insect Molecular Biology, 2000, 9(6):581-590[19]Trikic M Z, Monk P, Roehl H, et al.This is an author produced version of regulation of zebrafish hatching by tetraspanin CD63[J].Molecular Biology and Biotechnology, 2011, 6(5):e19683-[20]Wright M D, Tomlinson M G.The ins and outs of the transmembrane 4 superfamily[J].Immunology Today, 1994, 15(12):588-594[21]Yeh H Y, Klesius P H.Channel catfish (Ictalurus punctatus Ra?nesque, 1818) tetraspanin membrane protein family: Identi?cation, characterization and expression analysis of CD63 CDNA [J]. Veterinary Immunology and Immunopathology, 2010, (15): 302-308.[J].Veterinary Immunology and Immunopathology, 2010, 133(15):302-308[22]Yuk J M, Jo E K.Toll-like Receptors and Innate Immunity[J].Journal of Bacteriology and Virology, 2011, 41(4):225-235