实验用荣昌猪SLA-1等位基因鉴定及分子遗传特征分析  

Identification of SLA-1 Alleles and Analysis of Molecular Genetic Characteristics in Rongchang Pigs

作　　者：刘弘毅 罗霆宇 李昌文[1] 于海波[1] 路小野[1] 陈洪岩[1] 夏长友[1] 高彩霞[1] LIU Hongyi;LUO Tingyu;LI Changwen;YU Haibo;LU Xiaoye;CHEN Hongyan;XIA Changyou;GAO Caixia(State Key Laboratory for Animal Disease Control and Prevention,Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine,National Poultry Laboratory Animal Resource Center,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室,黑龙江省实验动物与比较医学重点实验室,国家禽类实验动物资源库,哈尔滨150069

出　　处：《畜牧兽医学报》2023年第7期3064-3077,共14页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2021YFF0703000);国家自然科学基金(31872313);中央级公益性科研院所基本科研业务费专项(1610302022018);兽医生物技术国家重点实验室课题(SKLVBP202120;SKLVBP202101)。

摘　　要：旨在明确实验用荣昌猪SLA遗传背景,深入研究SLA-1分子相关的抗原递呈和免疫应答机制,本研究对27头荣昌猪采集抗凝血,分离外周血淋巴细胞,提取总RNA,设计特异性引物对SLA-1基因进行RT-PCR扩增、克隆和测序,对获得序列的分子特征进行分析。结果显示,荣昌猪中共获得11个SLA-1等位基因,其中,9个为新等位基因,全部获得GenBank登录号和ISAG SLA命名委员会官方命名,SLA-1^(*)24:01等位基因在该群体中频率最高。SLA-1基因编码区全长1086 bp,存在127个核苷酸多态位点,非同义单核苷酸多态性位点数高于同义单核苷酸多态性位点数。核苷酸多样度为0.0449,单倍型多样度为1,平均核苷酸差异数为48.782,G+C含量为64.9%。SLA-1基因编码的361个氨基酸中,有75个氨基酸变异位点;第2和第3外显子编码区多态性最高,且第2外显子区域的氨基酸变异程度高于第3外显子区,组成抗原肽结合槽6个口袋的33个关键氨基酸位点中,11个在人与荣昌猪之间高度保守,与β2-微球蛋白结合的19个关键氨基酸位点中,10个保持一致,CD8分子与MHC结合的关键氨基酸位点中,只有225(Thr/Ser)和228(Thr/Met)位点不同。同源性和系统进化树分析表明,SLA-1^(*)10:03和SLA-1^(*)18:03分别与人HLA-A^(*)02:01和HLA-A^(*)11:01等位基因的同源性最高,荣昌猪与亚洲野猪、巴马小型猪和融水小型猪等亚洲猪种具有较近的亲缘关系。本研究成功获得了中国地方猪种荣昌猪SLA-1基因,发现其具有极为丰富的多态性,为揭示荣昌猪的SLA遗传背景和开展异种移植研究奠定了遗传学基础。The study aimed to clarify the SLA genetic background of experimental Rongchang pigs and further study SLA-1 molecule-related antigen presentation and immune response mechanism.In this study,the anticoagulant bloods were collected from 27 Rongchang pigs,and peripheral blood lymphocytes were isolated.Total RNA were extracted and SLA-1 genes were amplified using specific primers with RT-PCR method,cloned and sequenced.Molecular genetic characte-ristics of the obtained sequences were further analyzed.The results showed that a total of 11 SLA-1 alleles were obtained in Rongchang pigs,of which nine were novel alleles.All alleles obtained GenBank accession numbers and official names were assigned by the ISAG SLA Nomenclature Committee.The frequency of SLA-1^(*)24:01 allele was the highest in this population.The full-length of the coding region of SLA-1 gene was 1086 bp,with 127 nucleotide polymorphic sites.The number of non-synonymous SNPs was higher than that of synonymous SNPs.The nucleotide diversity was 0.0449,the haplotype diversity was 1,the average nucleotide difference number was 48.782,and the G+C content was 64.9%.There were 75 amino acid variation sites among 361 amino acids encoded by SLA-1 gene.The coding region of exon 2 and exon 3 had the highest polymorphism,and the degree of amino acid variation in exon 2 region was higher than that in exon 3 region.Among the 33 key amino acid sites that make up the six pockets of the peptide-binding groove,11 of them were highly conserved between human and Rongchang pig,and 10 of the 19 key amino acid sites were consistent withβ2-microglobulin.Only two sites,225(Thr/Ser)and 228(Thr/Met),were different among the key amino acid sites that CD8 molecules bind to MHC.The analysis of homology and phylogenetic tree showed that SLA-1^(*)10:03 and SLA-1^(*)18:03 had the highest homology with human HLA-A^(*)02:01 and HLA-A^(*)11:01 alleles,respectively.Rongchang pig was closely related to Asian boar,Bama miniature pig,Rongshui miniature pig and other Asian pig breeds.The SLA-1

关 键 词：荣昌猪 SLA-1等位基因 鉴定 分子特征 

分 类 号：S852.4[农业科学—基础兽医学]