GGTA1^(-/-)五指山小型猪SLAI类基因分子特征及与HLA相似性分析  

Characterization of swine leukocyte antigen class I genes and homology analysis of the similarity to HLA in GGTA1^(-/-) Wuzhishan minipigs

作　　者：蒋应弟 曾国敏[2,3] 石宁宁[2] 李西睿[2] 纪慧丽 潘登科[2] 滚双宝[1] 

机构地区：[1]甘肃农业大学动物科学技术学院,兰州730070 [2]中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与种质创新重点实验室,北京100193 [3]甘肃农业大学生命科学技术学院,兰州730070

出　　处：《中国实验动物学报》2016年第4期375-380,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点基础研究发展计划(编号:2015CB554103)

摘　　要：目的明晰α-1,3半乳糖基转移酶基因敲除(GGTA1-/-)的五指山小型猪SLA经典I类基因分子结构特征及其与人HLA的相似性,对研究异种器官移植细胞性排斥反应具有重要意义。方法采集6头祖代GGTA1-/-五指山小型猪耳组织,利用RT-PCR对SLA I类基因(SLA-1、SLA-3、SLA-2)扩增、克隆及测序,对序列进行BLAST分析,并采用生物信息学方法对SLA I类基因分子结构特征及其与人HLA的同源性进行分析。结果测序分析表明,共获得6条等位基因序列,其中4条为已公布的等位基因(SLA-1*0703、SLA-2*1102、SLA-3*0401、SLA-3*0403),另2条为新等位基因(SLA-1*0401wz01、SLA-2*11wz01)。五指山小型猪与人HLA同源性为70.5%~72.1%。SLA-1*0401wz01、SLA-1*0703、SLA-2*11wz01、SLA-2*1102和SLA-3*0401的CD8+分子识别的关键结合域与人HLA氨基酸序列比对,均仅在位点225、228处发生了突变(T→S、T→M),其他位点高度保守。SLA-2*11wz01和SLA-2*1102与人HLA I类基因NK细胞抑制性受体(killer inhibitory receptor,KIR)结合区氨基酸同源性较高,在NKTA-1亚型结合域内仅有1个氨基酸差异,而在NKTA-2和NKTA-3亚型结合域内有2个氨基酸差异。结论从免疫细胞介导的异种排斥反应角度出发,GGTA1-/-五指山小型猪SLA I类基因氨基酸序列与人HLA高度相似,可作为未来猪-人异种器官移植的良好供体之一。Objective This study was aimed to characterize the swine leukocyte antigen（ SLA） class I genes of GGTA1-/-Wuzhishan minipigs and compare their similarity to human leukocyte antigen（ HLA）. It has important implications for understanding the cellular rejection in xenotransplantation. Methods Specimens of ear tissue from six founding GGTA1-/-Wuzhishan minipigs were collected,and the SLA class I genes（ SLA-1,SLA-3,SLA-2） were amplified by RTPCR. Purified products were cloned into pE ASY-T1 vectors and sequenced,followed by BLAST alignment and using bioin-formatc analysis to characterize the SLA class I genes and compare with the similarity to HLA. Results A total of six alleles were detected,among them alleles were previously reported（ SLA-1＊ 0703,SLA-2＊ 1102,SLA-3＊ 0401,SLA-3＊0403）,and the other were novel（ SLA-1 ＊ 0401wz01,SLA-2 ＊ 11wz01）. The homology between alleles of SLA class I genes in Wuzhishan minipigs and HLA was from 70. 5% to 72. 1%. The homology analysis of critical amino acid residues on HLA binding with human CD8＋molecules showed that SLA-1＊ 0401wz01,SLA-1＊ 0703,SLA-2＊ 11wz01,SLA-2＊1102 and SLA-3＊ 0401 occurred mutant at amino acid positions 225 and 228（ T→S,T→M）,whereas the other loci were highly conserved. There was a high homology at amino acid level between SLA-2＊ 11wz01,SLA-2＊ 1102 and HLA class I genes which are NK cell KIRs binding sites. Conclusions The amino acid sequences of SLA class I genes of GGTA1-/-Wuzhishan minipigs have a high homology to HLA. From the point of view of cell-mediated xenograft rejection,the amino acid sequences of SLA class I genes of GGTA1-/-Wuzhishan minipigs have a high homology to HLA,therefore,Wzhishan minipigs may become a good potential donor for pig-human xenotransplantation.

关 键 词：GGTA1-/- 五指山小型猪 SLA 异种移植 免疫排斥 

分 类 号：Q95-33[生物学—动物学]