摘要
采用快速扩增cDNA末端方法,获得1007bp草鱼MHCⅡα基因全长cDNA片段,序列包括717bp的开放阅读框、34bp的5′端非编码区以及256bp的3′端非编码区.氨基酸序列比对和同源性比较分析结果表明,草鱼MHCⅡα与鲤鱼MHCⅡα相似性最高,为79%,与斑马鱼的相似性为75%,与人的相似性最低,为34%.RT-PCR组织表达分析,MHCⅡα基因在正常草鱼组织中除了肌肉中没有扩增出MHCⅡα基因转录本外,在所检测的其他组织中都有不同程度的表达,其中脾脏、头肾有较强的表达,血液有中等程度的表达,肝脏、眼与肌肉相对表达较弱.
The full-length eDNA encoding grass carp MHC class Ⅱα was cloned from grass carp using eDNA library and RACE PCR approach. The MHC classⅡα was 1007 bp in length, including 34 bp 5＇ terminal UTR,717 bp encoding region and 256 bp 3＇ terminal UTR with a AU motif （ATTTA） that associated with mRNA instability and one canonical polyadenylation signals （AATAAA）, followed by an additional 29 bp poly （A） tail. The 717 bp ORF was found to code a protein with 238 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. Three protein kinase C phosphorylation sites （S/T-X-R/K） were identified in alpha 2 domain region and four 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 180-186）. Alignment of deduced amino acid sequences of the MHC class Ⅱα 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 Ⅱα and that of common carp were close to each other in similarity of 79%. For other animals, the similarity was 75%, 56%, 48%, 46%, 45%, 44% and 34% from zebrafish, rainbow trout, red sea bream, striped sea bass, Turbot and human, respectively. Semi-quantitative RT-PCR analysis demonstrated that the MHC Ⅱα transcript was detected at a high level in spleen, head kidney, moderate in blood, and low in liver, eyes and muscle.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2008年第6期673-679,共7页
Journal of Hunan Agricultural University(Natural Sciences)
基金
湖南省科学技术厅科技专项(湘[03]NKY-1001)
湖南农业大学校青年基金项目(SP-0354)
关键词
草鱼
组织相容性复合体
克隆
组织特异性表达
grass carp
major histocompatibility complex （MHC）
cloning： mRNA expression
