赤点石斑鱼MHC IA基因的克隆与表达多态性分析

Cloning and Polymorphism of MHC Class IA Gene from Epinephelus akaara

采用RACE技术,成功克隆获得赤点石斑鱼(Epinephelus akaara)非特异性免疫因子MHC IA基因的4个cDNA全序列,序列全长为1 680bp,含有3′UTR、启动子、多肽结合区(α1)、IGC区(α2)、跨膜区、胞质区和5′UTR区,编码区大小为1 074～1 080bp,共编码357～359个氨基酸,推测蛋白质分子质量约41.66ku.氨基酸序列分析发现赤点石斑鱼MHC IA分子具有经典MHC蛋白分子的空间结构,在多肽结合区则存在极其丰富的变异.利用Real time PCR和SSCP技术研究了赤点石斑鱼MHC IA的组织表达特异性和表达多态性,发现其在头肾、心、肝等12个组织器官中均能有效表达,在头肾,脾,胸腺等免疫组织表达量不仅高,表达多态性也异常丰富,而在与外界环境接触较多的鳃、肌肉、肠等组织表达较弱.此外,根据MHC IIB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.

The major histocompatibility complex molecules （MHC I and MHC If） are fundamental components of imumune response to foreign protein antigens,and occur extensively in all vertebrate organisms. For the first time,4 complete eDNA sequences of MHC class IA chain,one of the important non-specific immune genes, were obtained from Epinephelus akaara by RACE technique. The whole sequences had 1 680 bp, which consist of： a 3＇ UTR, promoters, a peptide-binding region（cd ）, an immunoglobulin-like region （~2） ,a transmembrane region,a cytoplasmic region and 5＇UTR,and the size of their coding region is 1 074 1 080 bp,which encode a 155 amino acid protein and has a molecular weight about 30.25 ku. The tested gene has classic MHC 3D molecular structure, and ex hibits abundant polymorphisms in the peptide-binding region. Using realtime PCR and SSCP technique, the MHC IA gene was ob served to be expressed in all the 12 tissues studied in this paper,and high levels of expression and polymorphism were observed in im mune organs such as head kidney, spleen and thymus,while in organs such as gills and muscles that are more likely to be frequently exposed to bacterial antigens, the expression and polymorphism levels were lower. Furthermore, a phylogenetic tree was also con structed based on Neighbou-joining methods with IGC region of MHC liB gene. The satisfactory result also meaned that the amino acid sequence of the IGC region of MHC IA should be potentially informative for phylogenetie studies in fishes.