| 摘要: |
| 采用同源克隆和末端快速扩增(RACE)方法，得到1131bp的大菱鲆MHCⅡA全长cDNA片段。该序列包括720bp的开放阅读框(0RF)，75bp的5′末端非编码区(UTR)以及336bp的3′末端非编码区。利用CLASTAL W1.8软件进行MHCⅡA基因的氨基酸序列比对和同源性比较，结果表明，大菱鲆MHCⅡA与牙鲆MHCⅡA的相似性最高，为69.8%，与真鲷、鲈鱼和丽鱼的相似性次之，分别为65.5%、69.6%和61.4%，与人、鼠和鲨鱼MHCⅡA的相似性仅为19.8%、31.6%和26.9%。应用RT-PCR分析其组织表达发现MHCⅡA基因在正常大菱鲆组织中均表达，但表达量在各个组织中不同，其中在鳃、脾、头肾、心脏、小肠和皮肤中有较强的转录本，中等程度表达于肝和血，在性腺和肌肉中表达最弱。 |
| 关键词: 大菱鲆 MHC 克隆 组织特异性表达 |
| DOI： |
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| 基金项目:国家重点基础研究发展规划(973)项目,2004B117403号;青岛市科技将才计划项目资助,2005—2008 |
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| FULL LENGTH cDNA CLONING AND TISSUE EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) II A FROM TURBOT SCOPHTHALMUS MAXIMUS |
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ZHANG Yu-Xi1,2, CHEN Song-Lin2
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1.College of Marine Life Science, Ocean University of China, Qingdao, 266003;2.Key Laboratory for Sustainable Utiliza-tion of Marine Fisheries Resource, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071
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| Abstract: |
| The full-length cDNA encoding turbot MHC class II A was cloned from turbot using homology cloning and RACE PCR approach. The MHC class II A was 1131bp in length, including 75bp 5′ terminal UTR, 720bp encoding region and 336bp 3' terminal UTR with a AU motif (ATTTA) that associated with mRNA instability and three canonical polyadenylation signals (AATAAA), followed by an additional 26bp poly (A) tail. The 720bp ORF was found to code a protein with 239 amino acid residues, and its secondary structure was analyzed using PHDsec Program. One N-glycosylation site (N-X-S/T) was observed in alpha 1 domain region. Two protein kinase C phosphorylation sites (S/T-X-R/K) were identified in alpha 2 domain region and five casein kinase II phosphorylation sites (S/T-X-X-D/E) were identified in alpha 1 and alpha 2. Four N-myristoylation sites (G-E/D/R/K/H/P/F/Y/W-X-X-S/T/A/G/C/N-G/I/T) were found in alpha 1, alpha 2 and transmembrane region. In addition, signature (F/Y-X-C-X-V/A-X-H) of immunoglobulins and MHC proteins occurred in alpha 2 domain region (residues 182-188). Alignment of deduced amino acid sequences of the MHC class II A and those of other vertebrates demonstrated that they shared many protein features common in those of other fish and mammalians. Phylogenetic analysis using CLASTAL W software showed that the putative MHC II A and that of Japanese flounder were close to each other in the first sister group in similarity of 69.8%. For other animals, the similarity was 65.5%, 69.6%, 44.4%, 61.4%, 47.6%, 46.3%, 43.6%, 19.8%, 31.6% and 26.9% from red sea bream, striped sea bass, carp, cichlid, rainbow trout, Atlantic salmon, zebrafish, human, mouse and nurse shark, respectively. Semi-quantitative RT-PCR analysis demonstrated that MHC class II A was ubiquitously expressed in tissues, but the expression level was locally unique. The MHC II A transcript was detected at a high level in head kidney, spleen, intestine, gill and heart, moderate in liver and blood, and low in gonad and muscle. |
| Key words: Turbot Scophthalmus maximus, Major histocompatibility complex (MHC), Cloning, mRNA expression |
