Chinese Journal of Zoology 2010 45 4 118 ~ 128 MHC-Ⅱβ cdna 12 1* 2* 1 1 1 1 510300 2 510642 RACE 1 161 bp Rachycentron canadum MHC-Ⅱβ cdna 20 bp 5 UTR 394 bp 3 UTR 747 bp ORF 248 27. 99 ku 6. 21 MHC-Ⅱβ Mus musculus Gallus gallus Homo sapiens MHC-Ⅱ β 28. 5% ~ 77. 5% β1 β2 CP / TM / CYT Real-time PCR MHC-Ⅱβ MHC-Ⅱβ cdna Q781 A 0250-3263 2010 04-118-11 Full Length cdna Cloning and Tissue Expression of Major Histocompatibility Complex MHC -Ⅱβ in Cobia Rachycentron canadum MAO Li-Na 1 2 FENG Juan 1 * LI Yu-Gu 2 * HOU Yue-E 1 GUO Zhi-Xun 1 XU Hai-Dong 1 1 The South China Sea Fisheries Research InstituteChinese Academy of Fishery ScienceGuangzhou 2 South China Agriculture UniversityGuangzhou 510642China 510300 Abstract The homology cloning and RACE PCR were employed to clone the MHC-Ⅱ β gene from cobia Rachycentron canadium. The full length cdna of MHC-Ⅱβ comprises 1 161 bp with a 20 bp 5 untranslated region UTR a 394 bp 3 UTR and a 747 bp open reading frame ORF encoding a polypeptide of 248 amino acid residues with a predicted molecular weight of 27. 99 ku and theoretical isoelectric point of 6. 21. The putative protein showed homology varying from 28. 5% to 77. 5% when compared with other fishesmouse Mus musculus chicken Gallus gallus and human Homo sapiens. The protein sequence showed all the important features including leader peptideβ1β2cp / TM / CYT regions and conserved cysteines. The MHC-Ⅱ β expression was detected in all tested tissues with different expression levels. The high expression was detected in head kidneymoderate expression was detected in gillspleen and intestinewhile low expression was No. 2007ZD10 * E-mail juanf@ 21cn. com liyugu@ scau. edu. cn E-mail maolina2003@ 163. com 2010-01-08 2010-05-20
4 MHC-Ⅱβ cdna 119 detected in heartbrain and muscle. Key words Cobia Rachycentron canadum MHC-Ⅱβ Full length cdna mrna expression MHC major histocompatibility complex Perciformes Percoidei Rachycentridae MHC 8 MHC MHC MHC 9 MHC 10 - MHC 12 MHC-Ⅱβ MHC MHC-Ⅱβ cdna cdna MHC 1950 George Snell 1 MHC-Ⅱβ MHC Homo MHC-Ⅱβ sapiens HLA Mus musculus H-2 4 MHC-Ⅱ β Gallus gallus B MHC MHC- MHC Ⅱ MHC-Ⅱ MHC MHC 1 1990 Hashimoto 2 Ⅰα3 Ⅱβ 2 1 d Cyprinus carpio MHC MHC 1. 2 RNA cdna 30 0. 1 g Trizol Invitrogen RNA RNA Danio rerio 3-80 RNA 5 μg Oncorhynchus mykiss 4 Salmo salar 5 Ictalurus punctatus 6 Chrysophrys major 7 MHC-Ⅱβ Rachycentron 1. 1-80 oligo-dt 1 1 μl 10 pmol / L RT-PCR cdna - 80 1. 3 MHC-Ⅱβ canadum MHC CLUSTAL W2
120 Chinese Journal of Zoology 45 MEGA4. 1 RCMC2BF 1. 6 5 UTR 3 UTR RCMC2BR 1 MHC-Ⅱβ PCR 1% MHC-Ⅱ β ORF pmd18-t 1. 4 cdna 3 RCMC32BOUT RCMC32BIN RCMC52BOUT RCMC52BIN 1 3 qpcr semi-nested PCR cdna rapid amplification of cdna ends RACE 3 5 PCR PCR PCR PCR 1% pmd18-t TOP10 SYBR Green Ⅰ Mastercyclerep realplex DNAstar M5 M3 1 4 TOP10 1. 7 MHC-Ⅱ β Real- time PCR 3 200 g 1 106 bp cdna β-actinf β-actinr PCR PCR Eppendorf AGHamburgGer- 1. 5 NCBI BLAST many 4 DNAstar MHC-Ⅱβ cdna Mastercyclerep realplex System PHDsec program 2 - ΔΔCT CLUSTAL W2 Table 1 1 Oligonuleotide primers used in this experiment Prime Nucleotide sequence 5-3 Applied range RCMC2BF AGCAGCAGTGTGGGGAAGT RCMC2BR ACTCAGGCATGGACGGATC oligo-dt AAGCAGTGGTATCAACGCAGAGTACT 30 VN SMART 3a CAGAGTACTTTTTTTTTTTTTTTT 3 RACE RCMC32BOUT GAACGCTGGAACAAAGGTC 3 RACE RCMC32BIN CCGAGATGGACAGGAAGTG 3 RACE Olido-dG GGGGGGGGGGGGGGG A / T / C 5 RACE RCMC52BOUT TACCAGTCGCCGTCAGCC 5 RACE RCMC52BIN TTGTTCCAGCGTTCTGCGTT 5 RACE M5 AGAGCTCTCAACTCACCATC M3 GGACCAGACTGAGGTGTTC β-actinf TGAGACCACCTACAACAGC RT-PCR β-actinr CTGCATCCTGTCAGCGAT RT-PCR J2BF ACCTCTGGTTATTGACTGGG RT-PCR J2BR TCCAGCCAGAGATAAGATCAGAC RT-PCR
4 MHC-Ⅱβ cdna 121 2 2. 1 460 bp 5 UTR 394 bp 3 UTR 3 RACE PCR 5 RACE PCR 704 bp 325 bp MHC-Ⅱβ MHC-Ⅱβ cdna 2 MHC-Ⅱβ cdna 747 bp 20 bp AATAAA 16 bp Poly A 3 UTR RNA 1 DNAstar ATTTA RNA instability motifs 1 161 bp Fig. 1 1 RACE PCR Electrophoresis pictures of homology cloning and RACE PCR A. 1. PCR B. 3 RACE 1. 2. C. 5 RACE 1. 2. M. DL 2000 DNA A. Product of homology cloning1. Homology cloning product B. 3 RACE PCR product1. The first round PCR product 2. The second round PCR product C. 5 RACE PCR product1. The first round PCR product 2. The second round PCR product. M. DL 2000 DNA ladder. 2. 2 MHC-Ⅱ β N- 69 GVKNAE 98 MHC-Ⅱ β cdna 248 GLDYTA 149 GQEVAS 220 GLILGL 2 DNAstar CP / TM / CYT 27. 99 ku 6. 21 leader peptide β1 β2 CP / TM / CYT 17 92 94 45 β1 32 N- NSSD β2 4 C- 112 SVR 178 TPR 193 SLK 209 SER 5 Ⅱ 132 SVYD 157 SSTE 173 SHLE 193 SLKE 205 GXXGXXXGXXXXXXG CLUSTAL W2 MHC-Ⅱβ MHC-Ⅱβ 3 β2 MEGA4. 1 4 Cyphotilapia frontosa SMPE β1 β2 4 DNA star
122 Chinese Journal of Zoology 45 Fig. 2 2 MHC-Ⅱβ cdna cdna sequence of cobia MHC-Ⅱβ and deduced amino acid sequence atg tga aataaa RNA attta < > β1 β2 The start and stop codons or the putative polyadenylation signal aataaa are shown in bold print. RNA instability motif attta in the 3 UTR is represented by box. The start amino acid of different region is underlined. Lowercase letters show nucleotide sequencecapital letters show amino acid sequence < indicates the starting position of the region > indicates the termination position of the regionthese regions include the leader peptideβ1 regionβ2 regioncp / TM / CYT region connecting peptidetransmembrane and cytoplasm. Epinephelus akaara 77. 5% 77. 1% 77. 1% 75. 5% 75. 1% 73. 5% Dicentrarchus labrax Morone 70. 3% 69. 9% 69. 9% 68. 7% saxatil Stizostedion vitreum Larimichthys crocea 63. 1% 61. 4% 51. 8% 51. 8% Gasterosteus aculeatus Scophthalmus MHC-Ⅱ β maximus Paralichthys olivaceus 28. 5% 33. 7% 30. 1%
4 MHC-Ⅱβ cdna 123 2. 3 MCH-Ⅱβ 25 22 ORF PCR 4 β1 β1 23. 9% β1 MHC-Ⅱβ 5 81. 5% β1
124 Chinese Journal of Zoology 45 3 Fig. 3 MHC-Ⅱβ MHC-Ⅱβ Alignment of the predicted cobia MHC-Ⅱβ amino acid sequence with other known MHC class Ⅱβ amino acid sequences β1 β2 CP / TM / CYT *. β1 β2 4 GXXGXXXGXXXXXXG Dare AAA87891Icpu U77597 Paol L04805 Scma DQ001730 Dila AM113466 Mosa L33967 Chma AY190711 Lacr ABV48908 Epak ACA63828 Stvi AY158838 Gaac BT027207 Cyfr L13232 Onmy AF115529 Sasa X70167 Homo CAA47028 Mumu AAC05286 Gaga NP-001038144 Regions corresponding to the putative leader peptideβ1 domainβ2 domain connecting peptide transmembrane region and cytoplasmic tail are shown above the sequences. * means that the residues or nucleotides in that column are identical in all sequences in the alignment means that conserved substitutions have been observed. means that semi-conserved substitutions are observed four conserved cysteine is shaded conserved GXXGXXXGXXXXXXG box is represented by box. Sequence accession numbers are as follows Raca Rachycentron canadum Dare Danio rerio AAA87891 Icpu Ictalurus punctatus U77597 Paol Paralichthys olivaceus L04805 Scma Scophthalmus maximus DQ001730 Dila Dicentrarchus labrax AM113466 Mosa Morone saxatilis L33967 Chma Chrysophos major AY190711 Lacr Larimichthys crocea ABV48908 Epak Epinephelus akaara ACA63828 Stvi Stizostedion vitreum AY158838 Gaac Gasterosteus aculeatus BT027207 Cyfr Cyphotilapia frontosa L13232 Onmy Oncorhynchus mykiss AF115529 Sasa Salmo salar X70167 Homo Homo sapiens CAA47028 Mumu Mus musculus AAC05286 Gaga Gallus gallus NP- 001038144.
4 MHC-Ⅱβ cdna 125 4 Clustal W2 MEGA4. 1 MHC-Ⅱβ β2 Fig. 4 Phytogenetic tree of MHC-Ⅱβ gene β2 region amino adds sequences in different groups with program Clustal W2 and MEGA 4. 1 using neighbor-join method 1 000 bootstrap The numbers were the percentages of bootstrap values supporting each node from 1 000 replicas. Rachycentron canadum Danio rerio Ictalurus punctatus Paralichthys olivaceus Scophthalmus maximus Dicentrarchus labrax Morone saxatilis Chrysophos major Larimichthys crocea Epinephelus akaara Stizostedion vitreum Gasterosteus aculeatus Cyphotilapia frontosaoncorhynchus mykiss Salmo salar Homo sapiens Mus musculus Gallus gallus 2. 4 MHC-Ⅱβ MHC-Ⅰ MHC-Ⅱ MHC-Ⅲ ⅡMHC cdna β-actin J2BF J2BR Real-time PCR MHC-Ⅱ β γ ⅡMHC ⅡMHC- MHC-Ⅱβ 61. 70 ± 1. 92 18. 80 ± 1. 38 9. 28 ± 0. 10 7. 62 ± 0. 05 3. 89 ± 0. 34 5. 67 ± 0. 16 1. 03 ± 0. 15 3 α β γ MHC-Ⅱ T ThCD4 + MHC MHC 13 MHC-Ⅱ MHC α1 α2 β1 β2
126 Chinese Journal of Zoology 45 Fig. 5 5 Alignment of amino acid sequences among different Cobias β1 β2 CP / TM / CYT - Regions corresponding to the putative leader peptideβ1 domainβ2 domainconnecting peptidetransmembrane region and cytoplasmic tail are shown above the sequences. The same amino acids is represented by -. MHC-Ⅱ β 33 β1 β2 1 161 bp 20 bp 5 UTR 394 bp 4 3 UTR 747 bp ORF 2 2 248 3 UTR RNA ATTTA RNA instability motifs mrna 14-15 PCR 17 β1 GXXGXXXGXXXXXXG 92 β2 94 CP / Cosson 21 MHC-Ⅱ α TM / CYT 45 β1 β2 4 αβ 2 MHC-Ⅱβ MHC-Ⅱα 3 32 ~ 35 N- 22 NSSD 23 16 17 18 19 4 125 5 20 126 176 MHC-Ⅱ CD4 3 MHC MHC 22 β1 3 β2 156 94 157 158
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