【摘要】 主要组织相容性复合体(Major histocompatibility complex,MHC)是脊椎动物体内重要的非特异性免疫基因之一。为了研究赤点石斑鱼的MHC基因的结构与表达特性,本研究运用RACE,Realtime PCR及SSCP等技术,首次成功克隆获得赤点石斑鱼(Epinephelus akaara)非特异性免疫因子MHC IIB基因的4个cDNA全序列,序列全长为1 195~1 355 bp,含有3′UTR、启动子、多肽结合区(β1)I、GC区(β2)、跨膜区、胞质区和5′UTR区,编码区大小为750 bp。氨基酸序列分析发现赤点石斑鱼MHC IIB分子具有经典MHC蛋白分子的空间结构,在多肽结合区存在极其丰富的变异,β1区由1个α螺旋与4个反向平行的β折叠构成多肽结合区,β2区由多个β折叠构成2个反向平行的三明治结构。利用SSCP技术研究了赤点石斑鱼MHC IIB的表达多态性,发现其在头肾、心、肝等12个组织器官中均能有效表达,表达多态性也异常丰富。此外,根据MHCIIB分子中相对保守的IGC区氨基酸序列构建了脊椎动物的系统进化树,也表明了该分子的IGC区氨基酸序列可以作为研究鱼类物种间进化关系的良好标记。更多还原

【Abstract】 Major histocompatibility complex is one of crucial innate immune factors in vertebrates.In this study,RACE,Realtime PCR and SSCP techniques had been used to study the structure and expression characterization of MHC gene from Epinephelus akaara,4 complete cDNA sequences of MHC class Ⅱβ chain from E.akaara was obtained,the sequences have the length of 1195~1355 bp,and contain a 3′UTR,promoters,a peptide-binding region(β1),an immunoglobulin-like region(β2),a transmembrane region,a cytoplasmic region,and 5′UTR.The size of their coding region is 750 bp,exhibits a classic MHC 3D molecular structure and abundant polymorphisms in the peptide-binding region.Within the peptide-binding region,β1 included an alpha-helical region and a beta sheet of four strands in antiparallel orientation,while β2 forms a sandwhich-like structure made of two antiparallel sheets,with each containing several strands.Using SSCP technique,we observed high levels of expression of polymorphism in 12 tissue such as head-kidney,heart and liver.We also constructed phylogenetic tree based on Neighbour-Joining methods with IGC region of the MHC IIB gene.These results showed that the amino acid sequence of the IGC region of MHC IIB is informative for phylogenetic studies in fishes.更多还原