实验用荣昌猪SLA-1等位基因鉴定及分子遗传特征分析

doi:10.11843/j.issn.0366-6964.2023.07.037

摘要/Abstract

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1 086 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0.044 9，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。

关键词: 荣昌猪, SLA-1等位基因, 鉴定, 分子特征

Abstract: 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 1 086 bp, with 127 nucleotide polymorphic sites. The number of non-synonymous SNPs was higher than that of synonymous SNPs. The nucleotide diversity was 0.044 9, 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 with β2-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 alleles of Chinese Rongchang pigs were successfully identified, and it was found to have highly rich polymorphism. The results of this study provided a genetic foundation for revealing the SLA genetic background of Rongchang pigs and conducting xenotransplantation research.

Key words: Rongchang pig, SLA-1 allele, identification, molecular characteristics

中图分类号:

S852.4

刘弘毅, 罗霆宇, 李昌文, 于海波, 路小野, 陈洪岩, 夏长友, 高彩霞. 实验用荣昌猪SLA-1等位基因鉴定及分子遗传特征分析[J]. 畜牧兽医学报, 2023, 54(7): 3064-3077.

LIU Hongyi, LUO Tingyu, LI Changwen, YU Haibo, LU Xiaoye, CHEN Hongyan, XIA Changyou, GAO Caixia. Identification of SLA-1 Alleles and Analysis of Molecular Genetic Characteristics in Rongchang Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3064-3077.

参考文献 40

[1]	马丽颖, 岳秉飞. 主要组织相容性复合体(MHC)研究进展[J]. 中国比较医学杂志, 2005, 15(3):182-185.MA L Y, YUE B F. Advances in research on major histocompatibility complex[J]. Chinese Journal of Comparative Medicine, 2005, 15(3):182-185. (in Chinese)
[2]	刘子展. 贵州小型猪SLA经典Ⅰ类和Ⅱ类基因克隆和生物信息学分析及PCR-SSP分型方法的建立[D]. 合肥:安徽农业大学, 2012.LIU Z Z. Cloning and bioinformatics analysis of swine leukocyte antigens class I and class II genes in Guizhou minipigs and establishment of PCR-SSP typing method[D]. Hefei:Anhui Agricultural University, 2012. (in Chinese)
[3]	YAMAGUCHI T, DIJKSTRA J M. Major histocompatibility complex (MHC) genes and disease resistance in fish[J]. Cells, 2019, 8(4):378.
[4]	CARRINGTON M, BONTROP R E. Effects of MHC class I on HIV/SIV disease in primates[J]. AIDS, 2002, 16:S105-S114.
[5]	ROCK K L, REITS E, NEEFJES J. Present Yourself! By MHC class I and MHC class II molecules[J]. Trends Immunol, 2016, 37(11):724-737.
[6]	SEETHARAM A, TIRIVEEDHI V, MOHANAKUMAR T. Alloimmunity and autoimmunity in chronic rejection[J]. Curr Opin Organ Transplant, 2010, 15(4):531-536.
[7]	AFZALI B, LOMBARDI G, LECHLER R I. Pathways of major histocompatibility complex allorecognition[J]. Curr Opin Organ Transplant, 2008, 13(4):438-444.
[8]	WEISS R A. Xenotransplantation[J]. BMJ, 1998, 317(7163):931-934.
[9]	COOPER D K C, GASTON R, ECKHOFF D, et al. Xenotransplantation-the current status and prospects[J]. Br Med Bull, 2018, 125(1):5-14.
[10]	CELIS-GIRALDO C T, BOHÓRQUEZ M D, CAMARGO M, et al. A comparative analysis of SLA-DRB1 genetic diversity in Colombian (creoles and commercial line) and worldwide swine populations[J]. Sci Rep, 2021, 11(1):4340.
[11]	权金强, 江新杰, 李昌文, 等. 实验用SPF大白猪和长白猪SLA-1基因特征分析[J]. 农业生物技术学报, 2017, 25(5):770-780.QUAN J Q, JIANG X J, LI C W, et al. Characterization analysis of SLA-1 gene from SPF yorkshire (Sus scrofa) and landrace[J]. Journal of Agricultural Biotechnology, 2017, 25(5):770-780. (in Chinese)
[12]	HAMMER S E, HO C S, ANDO A, et al. Importance of the major histocompatibility complex (Swine leukocyte antigen) in swine health and biomedical research[J]. Annu Rev Anim Biosci, 2020, 8:171-198.
[13]	GAO C X, HE X W, QUAN J Q, et al. Specificity characterization of SLA class I molecules binding to swine-origin viral cytotoxic T lymphocyte epitope peptides in vitro[J]. Front Microbiol, 2017, 8:2524.
[14]	LUNNEY J K. Advances in swine biomedical model genomics[J]. Int J Biol Sci, 2007, 3(3):179-184.
[15]	SWINDLE M M, MAKIN A, HERRON A J, et al. Swine as models in biomedical research and toxicology testing[J]. Vet Pathol, 2012, 49(2):344-356.
[16]	GEISLER K, KVNZEL J, GRUNDTNER P, et al. The perfused swine uterus model:long-term perfusion[J]. Reprod Biol Endocrinol, 2012, 10:110.
[17]	RAMIREZ-MEDINA E, VUONO E, RAI A, et al. Deletion of E184L, a putative DIVA target from the pandemic strain of african swine fever virus, produces a reduction in virulence and protection against virulent challenge[J]. J Virol, 2022, 96(1):e0141921.
[18]	CHEN W, YI H J, ZHANG L, et al. Establishing the standard method of cochlear implant in Rongchang pig[J]. Acta Oto-Laryngol, 2017, 137(5):503-510.
[19]	任丽丽. 白化荣昌猪耳聋的分子病理机制研究[D]. 北京:中国人民解放军医学院, 2013.REN L L. Probing the molecular pathological mechanism underlying the deafness in albino Rongchang swine[D]. Beijing:Chinese PLA General Hospital & Medical School, 2013. (in Chinese)
[20]	陈伟, 陈磊, 杨仕明. 荣昌猪遗传性听力缺陷家系的发掘与应用[J]. 中华耳科学杂志, 2016, 14(1):10-14.CHEN W, CHEN L, YANG S M. Excavation and application of the Rongchang pig hereditary pedigree connected to hearing defects[J]. Chinese Journal of Otology, 2016, 14(1):10-14. (in Chinese)
[21]	周福波. 汉族白癜风患者HLA-A0201限制性CTL表位的筛查与鉴定[D]. 西安:第四军医大学, 2014.ZHOU F B. Screening and identification of HLA-A0201-restricted CTL epitope in Chinese vitiligo patients[D]. Xi'an:The Fourth Military Medical University, 2014. (in Chinese)
[22]	ZHANG N Z, QI J X, FENG S J, et al. Crystal structure of swine major histocompatibility complex class I SLA-1*0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides[J]. J Virol, 2011, 85(22):11709-11724.
[23]	SALTER R D, BENJAMIN R J, WESLEY P K, et al. A binding site for the T-cell co-receptor CD8 on the α3 domain of HLA-A2[J]. Nature, 1990, 345(6270):41-46.
[24]	GAO F S, ZHAI X X, JIANG P, et al. Identification of two novel foot-and-mouth disease virus cytotoxic T lymphocyte epitopes that can bind six SLA-I proteins[J]. Gene, 2018, 653:91-101.
[25]	FAN S H, WU Y N, WANG S, et al. Structural and biochemical analyses of swine major histocompatibility complex class I complexes and prediction of the epitope map of important influenza a virus strains[J]. J Virol, 2016, 90(15):6625-6641.
[26]	PAN X C, ZHANG N Z, WEI X H, et al. Illumination of PRRSV cytotoxic T lymphocyte epitopes by the three-dimensional structure and peptidome of swine lymphocyte antigen class I (SLA-I)[J]. Front Immunol, 2020, 10:2995.
[27]	李婧娜, 疏泽, 吴俊超, 等. 基于重测序的民猪与大白猪SLA Ⅰ类基因多态性及抗病潜能差异分析[J]. 畜牧兽医学报, 2021, 52(7):1820-1830.LI J N, SHU Z, WU J C, et al. Analysis of SLA class I gene polymorphism and potential disease resistance difference between Min pigs and Large White pigs based on resequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7):1820-1830.
[28]	LUNNEY J K, HO C S, WYSOCKI M, et al. Molecular genetics of the swine major histocompatibility complex, the SLA complex[J]. Dev Comp Immunol, 2009, 33(3):362-374.
[29]	GAO C X, JIANG Q, GUO D C, et al. Characterization of swine leukocyte antigen (SLA) polymorphism by sequence-based and PCR-SSP methods in Chinese Bama miniature pigs[J]. Dev Comp Immunol, 2014, 45(1):87-96.
[30]	GAO C X, QUAN J Q, JIANG X J, et al. Swine leukocyte antigen diversity in Canadian specific pathogen-free Yorkshire and landrace pigs[J]. Front Immunol, 2017, 8:282.
[31]	SØRENSEN M R, ILSØE M, STRUBE M L, et al. Sequence-based genotyping of expressed swine leukocyte antigen class I alleles by next-generation sequencing reveal novel swine leukocyte antigen class I haplotypes and alleles in Belgian, Danish, and Kenyan fattening pigs and Göttingen minipigs[J]. Front Immunol, 2017, 8:701.
[32]	SOMMER S. The importance of immune gene variability (MHC) in evolutionary ecology and conservation[J]. Front Zool, 2005, 2:16.
[33]	SPURGIN L G, RICHARDSON D S. How pathogens drive genetic diversity:MHC, mechanisms and misunderstandings[J]. Proc Biol Sci, 2010, 277(1684):979-88.
[34]	MACHUKA E M, MUIGAI A W T, AMIMO J O, et al. Comparative analysis of SLA-1, SLA-2, and DQB1 genetic diversity in locally-adapted Kenyan pigs and their wild relatives, warthogs[J]. Vet Sci, 2021, 8(9):180.
[35]	GAO C X, XIN C, WANG X Y, et al. Molecular genetic characterization and haplotype diversity of swine leukocyte antigen in Chinese Rongshui miniature pigs[J]. Mol Immunol, 2019, 112:215-222.
[36]	FAN S H, WANG Y L, WANG S, et al. Polymorphism and peptide-binding specificities of porcine major histocompatibility complex (MHC) class I molecules[J]. Mol Immunol, 2018, 93:236-245.
[37]	FAN S, WANG Y, WANG X, et al. Analysis of the affinity of influenza A virus protein epitopes for swine MHC I by a modified in vitro refolding method indicated cross-reactivity between swine and human MHC I specificities[J]. Immunogenetics, 2018, 70(10):671-680.
[38]	MADDEN D R. The three-dimensional structure of peptide-MHC complexes[J]. Annu Rev Immunol, 1995, 13:587-622.
[39]	YUE C, XIANG W Z, HUANG X W, et al. Mooring stone-like Arg114 pulls diverse bulged peptides:first insight into African swine fever virus-derived T cell epitopes presented by swine major histocompatibility complex class I[J]. J Virol, 2022, 96(4):e0137821.
[40]	COWAN P J, TECTOR A J. The resurgence of xenotransplantation[J]. Am J Transplant, 2017, 17(10):2531-2536.