HLA-DRα*0101、DRβ1*04051和DRβ1*07011的分子克隆、测序及空间结构模建

MOLECULAR CLONING AND THREE DIMENSIONAL STRUCTURE MODELLING OF HLA DRα*0101,DRβ1*04051 AND DRβ1*07011

目的 :为探索HLA(humanleukocyteantigens,HLA)不同分子的空间结构差异与功能之间的关系以及结构差异能否对预测造血干细胞移植GVHD(graft versus hostdisease ,GVHD)的发生奠定基础。方法 :提取人外周血总RNA ,RT PCR扩增HLA DR等位基因全序 ,连接、转化大肠杆菌后并测序。将第二外显子氨基酸序列输入微机 ,通过INTER NET传送至SWISS MODELLING蛋白质空间结构模建服务器进行结构模建。结果 :本文反转录了 2例健康无血缘关系个体的HLA DR位点基因全序 ,测序结果与HLA DRα 0 10 1、DRβ1 0 40 5 1和DRβ1 0 70 11的公布序列完全一致。对后两者的蛋白质空间结构模建发现它们在空间结构上存在显著差异。结论 :对HLA基因位点进行全长测序可以明确判断基因型别 ;空间结构模建可以直观地辨别HLA不同分子在空间结构上的差异 。

Aim: To study the correlation between the differences of three dimensional structures among different HLA loci and their functions, and to investigate the potential usage of HLA conformational differences in predicting the occurrence of graft versus host disease(GVHD). Methods: Total RNA were extracted from human peripheral blood, and reverse transcription PCR (RT PCR)were used to amplify the whole gene sequences of HLA DR loci, and the PCR products were linked to vector and transformed to E.coli, and then sequencing. HLA DR second exon amino acid sequences were inputted to computer, through internet to SWISS MODELLING molecular modelling server to model their three dimensional structure.Results: The whole HLA DR loci sequences were successfully reversibly transcripted from 2 normal unrelated donors. The sequencing results were completely identical to the published sequences of HLA DRα*0101,DRβ1*04051 and DRβ1*07011 . The modelling results of the later two indicated that there were significant differences between their structures.Conclusion: The sequencing analysis of the whole HLA loci could clearly designate HLA allelic types, and three dimensional structure modelling could directly identify the differences among them. Therefore, this study provide experimental foundation to the study of HLA structure and function, and uo the study of predicting GVHD aftier HLA incompletely matched hematopoietic stem cell transplantation (HSCT).