5匹马外周血单个核细胞表达MHC-I类分子的差异分析  

Analyisis of Classical MHC Class I Alleles Expressed by Peripheral Blood Mononuclear Cells from 5 Horses in Heilongjiang Province

作　　者：马建 郭巍[1] 沈楠[1] 孔宪刚[1] 沈荣显[1] 周建华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处：《畜牧兽医学报》2007年第12期1414-1420,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江省发展高新技术产业专项资金项目(FW05B007);中国农业科学院一级岗位杰出人才基金

摘　　要：基于RT-PCR方法，扩增了用于马传染性贫血病毒（Equine infectious anemia virus，EIAV）减毒疫苗株CTL表位研究的5匹马的编码经典MHC—I类分子的大部分基因，具体包括编码肽结合区大部分区域、α3区、穿膜区及胞浆区蛋白的基因，并进行了PCR产物的克隆和测序。试验马匹外周血单个核细胞（Peripheral blood mononuclear cells，PBMC）的总RNA提取物经RT-PCR扩增，并将所得产物进行T-A克隆。从每匹马的所有克隆中，分别随机挑取鉴定为阳性重组质粒的21～23个克隆用于测序分析。研究结果表明，5匹试验马匹各自具有3～5个等位基因，由于MHC-Ⅰ分子等位基因表达的共显性特征，可以推测马的基因组内可能存在2～3个基因座对应于这些等位基因，这与国外已有研究结论基本一致。另外，不同马匹PBMC表达的MHC-Ⅰ类分子的差别水平不一致。除了6号与7号和10号马之间MHC-Ⅰ类分子的差异较大，无相同或高度相近的经典MHC-Ⅰ类分子序列外，各马匹间均表达具有1个或1个以上的相同或高度同源的对应经典的MHC-Ⅰ类分子的mRNA。以上研究结果既可作为正在开展的EIAV减毒疫苗株CTL表位研究的基础性、支持性数据，又可进一步丰富国内马的经典MHC-Ⅰ分子多态性方面的相关研究。To study CTL epitopes in EIA attenuated vaccine, cDNA sequences encoding majority region of classical MHC class Ⅰ from 5 horses were amplified by RT-PCR and analyzed. The amplified cDNA fragments included the polymorphic peptide binding region, the more conserved regions of alpha 3, transmembrane domain and cytopasmic domain. The RT-PCR products were synthesized from total RNA of PBMC and then cloned into pMD-18T vector. 21-23 clones from each horse were randomly picked up for DNA sequencing. These sequences were further analyzed for their alleles. Our data revealed that each clone contained 3-5 alleles, which indicated there were at least 2-3 loci in MHC class Ⅰ in horse genome. This results were consistent with the published data of other researches. In addition, the expression variation of classical MHC Ⅰ alleles between each individuals were different. There was no identical or highly similar allele among horses No. 6, No. 7 and No. 10. The other animals had at least one allele that shared all or more than 98% of nucleotide sequence when compared each other（data not show）. These results not only provide fundamental information for our on-going study on CTL epitopes and EIAV-induced immune responses, but also served as important data for polymorphic analysis of horse classic MHC class Ⅰ in China.

关 键 词：马 经典主要组织相容性复合体Ⅰ类分子 多态性 反转录一聚合酶链式反应 

分 类 号：S852.4[农业科学—基础兽医学]