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1 2011 3, 18(2): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J MHC IIB 李宏俊 1, 王娟 1,2, 高祥刚 1, 苏浩 1, 刑坤 3, 傅立元 1,2, 赫崇波 1.,, ; 2.,, ; 3., 摘要 : cdna (RACE), (Verasper variegatus) (MHC) IIB cdna, cdna bp, 5'UTR (untranslated region) 7 bp, 3'UTR 450 bp, (ORF) 687 bp, 228, (β1) IGC (β2) 5, MHC IIB 49%~79%,, 34% 33% 31% 30% MHC IIB 5,, β bp MHC IIB, bp DNA, PCR 270 bp 23, PCR, MHC IIB 9,,,, MHC, [, 2011, 18(2): ] : ; MHC IIB; cdna; ; ; 中图分类号 : Q959 文献标识码 : A 文章编号 : (2011) (major histocompatibility complex, MHC),, [1] MHC II, CD4 + T, [2] MHC,, [3] MHC,, MHC [4-6] MHC 1990, Hashimoto [7] MHC I, (Danio rerio) [8] (Morone saxatilis) [9] (Scophthalmus maximus) [10] (Pagrus major) [11] (Cynoglossus semilaevis) [2] 30 MHC, MHC (Verasper variegatus) 收稿日期 : ; 修订日期 : 基金项目 : ( ); (nycytx-50-s08); ( ). 作者简介 : (1982-),,,. oceanlhj@163.com 通讯作者 :,,. Tel: ; hechongbo@hotmail.com

2 300 18,,,, [12],,,, [13] [14-15] [16], EST RACE MHC IIB cdna, PCR, MHC, 1 1.1, 1, o C,, Takara Trizol RNA 50~100 mg, - DNA [17] cdna Clontech SMART cdna Library Constrution Kit, cdna, 1 058, NCBI tblastx (singleton) (contig) GenBank MHC IIB cdna, EST 1 singleton MHC IIB,, primer premier 5.0 MHCIIB3'( 1), MHCIIB3' T7, cdna MHC IIB 3' PCR : 94 5 min; s, 5 1 min, 72 1 min, 35 ; min MHC IIB, MHC IIB, 2 ( 1) MHC IIB PCR : 94 5 min; s, 30 s, 72 1 min, 35 ; 72 10min PCR pmd18-t simple, E. coli Top10, Takara, EST MHC IIB 表 1 PCR 扩增引物序列 Tab. 1 Sequences of primers used in this study primer (5' 3') sequence(5' 3') RACE PCR MHCIIB3 GGATTCATCTACTACAAGAGGAAGG T7 TAATACGACTCACTATAGGG Intron PCR MHCIIB-intron1F ACATGGCTTCATTCTTCCTCAG MHCIIB-intron1R TTTACTCAGGCTGGCGTGTTC MHCIIB-intron2F AACACGCCAGCCTGAGTAAAC MHCIIB-intron2R TTTACTCAGGCTGGCGTGTTC polymorphism MHCIIBPF TCCCTCTTCTTCATCACTGTCTGC MHCIIBPR ACCTGACTTCGTCAGAGCATTTTC realtime PCR MHCIIB-RTF GCTGCTTCAACCTGCTTTCTGT MHCIIB-RTR GTCGGGATCAGACCAGCACAG β-actinf AGCCACACTGTTCCCATCTAC β-actinr CTCGGTGAGGATCTTCATCAG MHC IIB cdna GenBank tblast, DNAMAN cdna,, SMART, PHDsec program Clustalx 1.83, MEGA 4.0 (neighborjoining, NJ)

3 2 : MHC IIB MHC IIB MHC IIB MHCIIBPF MHCIIBPR( 1), 10 DNA, 1 2 DNA, MHC IIB PCR 25 µl, 2.5 µl 10 PCR buffer, 2.0 µl dntp(2.5 mmol/l), (10 µmol/l) 1.0 µl, 1 µl, 0.4 µl Pfu DNA polymerase(2.5 U/µL, ) 17.1 µl PCR Takara MHC IIB PCR MHC IIB 9 RNAfast200 ( ) RNA, M-MLV (Takara) cdna, Realtime PCR PCR 25 µl, 2 SYBR Premix Ex Taq (Takara) 12.5 µl, ROX II 0.5 µl, ( 1) 1.0 µl, 2 µl 8 µl : s; 95 5 s, s, 40 ;, (MHC IIB) (β-actin), ΔCT, 2 ΔΔCT [18], SPSS 12.0 t-test MHC IIB cdna EST bp, tblastx MHC IIB MHCIIB3 3, bp MHC IIB cdna (GenBank accession No. GU253881) cdna 7 bp 5'UTR, 450 bp 3'UTR, 3'UTR AATAAA Poly A ; 687 bp, 228 cdna, MHC IIB, 2, PCR, 6 5 MHC IIB ( 1) 7 bp 5'UTR, β1, β2, MHC IIB 5 85~356 bp, 2 1 (GT) n (CTGT) n ;, MHC IIB β bp ( 2) 2.2 MHC IIB MHC IIB 228, kd, 7.00, SMART,,, MHC II, (β1) IGC(Immuno Globulin C-Type) (β2) 5 PHDsec program MHC IIB, β N- (N-X-S/T), β1 β2 4 C- (S/T-X-R/K), β1 β2 7 II (S/T-X-X-D/E), β2 2 N- (G-E/D/R/K/H/P/F/Y/W-X-X-S/T/A/G/C/N) Clustalx MHC IIB, MHC IIB, 30%~79%( 2), 79%, 75% 70% 69% 67% 57% 55% 51%,, 34% 33% 31% 30% MEGA,, MHC IIB ( 3),, MHC IIB, 1, 1,

4 MHC IIB 5'UTR 3'UTR, ; ATG,, AATAAA polya ; N-, C-, II, (GT) n (CTGT) n. Fig.1 Genomic sequence of spotted halibut MHC IIB gneome Exons, 5'UTR and 3'UTR are in uppercase and introns are in lowercase; the start (ATG) codon is underlined and * represents the stop codon; N-linked glycosylation site is underlined with single straight line; protein kinase C phosphorylation sites are shadowed; casein kinase II phosphorylation sites are shown by curved line; (GT) n (CTGT) n microsatellite loci is indicated by double straight line. 图 2 MHC IIB CP: ; CT: ; LP: ; TM:. Fig.2 Schematic illustration of spotted halibut MHC IIB genomic structure CP: connecting peptide; CT: cytoplasmic domain; LP: leader peptide; TM: transmembrane region.

5 2 : MHC IIB 303 表 2 圆斑星鲽 MHC IIB 与其他鱼类和高等脊椎动物的氨基酸相似度 Tab.2 Identity of spotted halibut MHC IIB amino acid sequence with those of other fishes and high vertebrates species GenBank /% GenBank accession no. identity with spotted halibut Paralichthys olivaceus AY Morone saxatilis AAA Larimichthys crocea ABV Pagrus major AAX Cynoglossus semilaevis ACI Salmo salar CAA Cyprinus carpio CAA Danio rerio CAD Mus musculus P Homo sapiens M Gallus gallus AAS Ginglymostoma cirratum AAF Fig.3 3 MHC IIB Neighbor-joining phylogenetic tree constructed with the amino acid sequences of MHC IIB ; MHC IIB 2.3 MHC IIB 1 2 MHCIIBPF MHCIIBPR MHC II 1 2, PCR, 388 bp, 1 85 bp, bp, 90 1 ; 2 23 ( 3), , 9 4, MHC IIB 3 9 RNA, cdna, cdna Realtime PCR,, 9 MHC IIB

6 ( 4),,,, (P< 0.05) 4 MHC IIB Realtime PCR (P<0.05). Fig.4 Tissue distribution of spotted halibut MHC IIB transcript measured by Realtime PCR Different letters on columns mean significant difference (P<0.05) among different tissues. 3 (MHC),,, MHC, MHC, MHC,, MHC [19] [20] [21] [22] [3,23 24], cdna, MHCIIB cdna, cdna (β1) IGC(β2) 5, β1 β2 4, 4 MHC IIB, number position Tab.3 表 3 圆斑星鲽 MHC IIB 外显子 2 的序列多态性 Distribution of SNP sites within exon2 of spotted halibut MHC IIB sequences nucleotide (n=20) allele nos. (n=20) number position nucleotide (n=20) allele nos. T 16 C C 2 T 7 G 2 A T 17 G 2 A 3 A G 13 C 3 A 7 A T 14 G 4 G 6 G A 11 C 3 T 9 G 10 C C 7 A 5 T 3 T 14 G 10 G C 6 C 4 T 4 T 16 A A 14 C 9 T 6 A G 12 G 7 C 8 G A 16 T 8 T 4 A 8 C T 8 G 4 C 4

7 2 : MHC IIB 305 [2,11] MHC IIB β1 1 N-X-S/T,, [10] MHC II αβ MHC II, GXXGXXXGXXX XXXG (X ), Cosson [25] MHC IIB αβ PCR MHC IIB bp, 6 5, RNA (GT/AG), GT-AG MHC IIB [2], β2 ( 3) bp, Figueroa [26] MHC IIB β2 1,, MHC,,,,,,, MHC, MHC T, II, α β, II A B 2 PCR, MHC IIB 2, 270 bp 23, MHC, [27] MHC IIB 61 13, 6 ; [28] MHC IIB 1 2, 263 bp 2, 60 MHC IIB, MHC, B [29] Koppang [30], MHC IIB, ; Xu [2] realtime PCR MHC IIA MHC IIB, 12,,,,, MHC IIB,, MHC II,, MHC IIB [10],, MHC II / 参考文献 : [1],. MHC [J]., 2008, 29(4): [2] Xu T J, Chen S L, Ji X S, et al. Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole (Cynoglossus semilaevis)[j]. Fish Shell Immunol, 2009, 27(2): [3] Grimholt U, Larsen S, Nordmo R, et al. MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar): facing pathogens with single expressed major histocompatibility class I and class II loci[j]. Immunogenetics, 2003,

8 (4): [4] Hughes A L. MHC polymorphism and the design of captive breeding programs[j]. Conserv Biol, 1991, 5(2): [5] Wedekind C, Walker M, Portmann J, et al. MHC-linked susceptibility to a bacterial infection, but no MHC-linked cryptic female choice in whitefish[j]. J Evolut Biol, 2004, 17(1): [6] Traul D L, Li H, Dasgupta N, et al. Resistance to malignant catarrhal fever in American bison (Bison bison) is associated with MHC class IIa polymorphisms[j]. Ani Gen, 2007, 38(2): [7] Hashimoto K, Nakanishi T, Kurosawa Y. Isolation of carp genes encoding major histocompatibility complex antigens [J]. Proceeding of National Academy of Sciences of the USA, 1990, 87(17): [8] Sültmann H, Mayer W, Figueroa F, et a1. Organization of Mhc class IIB genes in the zebrafish (Brachydanio rerio)[j]. Genomics, 1994, 23(1): [9] Hardee J J, Godwin U, Benedetto R, et al. Major histocompatibility complex class IIA gene polymorphism in the striped bass [J]. Immunogenetics, 1995, 41(4): [10],. (Scophthalmus maximus)mhc IIA cdna [J]., 2007, 38(3): [11] Chen S L, Zhang Y X, Xu M Y, et al. Molecular polymorphism and expression analysis of MHC II B gene from red sea bream (Chrysophrys major)[j]. Developm Comp Immunol, 2006, 30(4): [12],,,. [J]., 2006, 28(3): [13],,,. Verasper variegates (Temminck et Schegel) [J]., 2002, 17(10): [14],,,. [J]., 2009, 36(3): [15],. [J]., 2008, 27(7): [16],,,. [J]., 2007, 14(4): [17],,,. [J]., 2009, 33(12): 4 8. [18] Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))[J]. Method, 2001, 25(4): [19] Flores-Villanueva PO, Hendel H, Caillat-Zucman S, et al. Associations of MHC ancestral haplotypes with resistance/ susceptibility to AIDS disease development[j]. J Immunol, 2003, 170(4): [20] Briles W E, Briles R W, Taffs R E, et al. Resistance to a malignant lymphoma in chickens is mapped to subregion of major histocompatibility (B) complex[j]. Science, 1983, 219(4587): [21] Medina E, North R J. Resistance ranking of some common inbred mouse strains to Mycobacterium tuberculosis and relationship to major histocompatibility complex haplotype and Nramp1 genotype[j]. Immunology, 1998, 93(2): [22] Kaufman J, Wallny H J. Chicken MHC molecules, disease resistance and the evolutionary origion of birds[j]. Curr Top Microbiol Immunol, 1996, 212(285): [23] Xu T J, Chen S L, Ji X S, et al. MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families[j]. Fish Shellf Immunol, 2008, 25(3): [24] Zhang Y X, Chen S L, Liu Y G, et al. Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus)[j]. Mar Biotechnol, 2006, 8(6): [25] Cosson P, Bonifacino J S. Role of transmembrane domain interactions in the assembly of class II MHC molecules[j]. Science, 1992, 258(5082): [26] Figueroa F, Ono H, Tichy H, et al. Evidence for insertion of a new intron into an Mhc gene of perch-like fish[j]. Proceedings: Biological Sciences, 1995, 259(1356): [27],. MHC class B [J]., 2006, 30(5): [28],,. MHC IIB 1 2 [J]., 2008, 15(4): [29] Dixon B, van Erp S H, Rodrigues P N, et al. Fish major histocompatibility complex genes: an expansion[j]. Developm Compar Immunol, 1995, 19(2): [30] Koppang E O, Lundin M, Press C M, et al. Differing levels of Mhc class II β chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.)[J]. Fish Shellf Immunol, 1998, 8(3):

9 2 : MHC IIB 307 Genomic structure of MHC IIB from spotted halibut Verasper variegates: genomic structure, polymorphism and tissue expression analysis LI Hongjun 1, WANG Juan 1, 2, GAO Xianggang 1, SU Hao 1, XING Kun 3, FU Liyuan 1,2, HE Chongbo 1 1. Liaoning Ocean and Fishery Science Institute, Liaoning Key Laboratory of Marine Fishery Molecular Biology, Dalian , China; 2. College of Life Sciences, Liaoning Normal University, Dalian , China; 3. College of Ocean Engineering, Dalian Ocean University, Dalian , China Abstract: Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. Its function is to present self and non-self peptide to T-cells receptor. In order to study the function and molecular polymorphism of class II genes in teleost, MHC IIB was identified from Verasper variegates by expressed sequence tags (ESTs) and rapid amplification of cdna ends (RACE) approach. The full length cdna was of bp, consisting of 5 UTR of 7 bp, a 3 UTR of 450 bp with a poly (A) tail, and an open reading frame (ORF) of 687 bp encoding a polypeptide of 228 amino acids, which contained a signal peptide, a peptide-binding region (β1), an immunoglobulin-like region (β2), a transmembrane region and a cytoplasmic region. Phylogenetic analysis demonstrated that the putative V. variegates MHC IIB amino sequence showed 30%-79% identity with those of other teleosts and vertebrates. The genomic MHC IIB contained 6 exons and 5 introns. An important difference between V. variegates MHC IIB and other known class IIB genes was that β2 domain-encoding exon was split by an intron of 109 bp in length. There were 23 single nucleotide variable sites in exon 2. Realtime PCR analysis demonstrated that MHC IIB was ubiquitously expressed in nine tested tissues, but the expression levels were different. The expression levels in kidney, heart, spleen and gill were significantly higher than those in liver, skin, brain, blood and muscle.[journal of Fishery Sciences of China, 2011, 18(2): ] Key words: Verasper variegates; MHC IIB; cdna; genomic structure; polymorphism; mrna expression Corresponding author: HE Chongbo. hechongbo@hotmail.com

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８