畜牧兽医学报 ›› 2007, Vol. 38 ›› Issue (12): 1414-1420.doi:

• 研究简报 • 上一篇    

5匹马外周血单个核细胞表达MHC-I类分子的差异分析

马建;郭巍;沈楠;孔宪刚;沈荣显;周建华   

  1. 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,哈尔滨 150001;2东北林业大学野生动物资源学院,哈尔滨 150040
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-12-25 发布日期:2007-12-25

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

MA Jian;GUO Wei;SHEN Nan;KONG Xian-gang;SHEN Rong-xian;ZHOU Jian-hua   

  1. 1 National Key Laboratory of Veterinery Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;2 Wildlife Resources College, Northeast Forestry University, Harbin 150040, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2007-12-25 Published:2007-12-25

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。

Abstract: 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.