Abstract：Abstract Swine (Sus scrofa) leukocyte antigen (SLA) gene is one of the genetic factors that cause xenotransplantation cell immune rejection. To enrich the classical experimental animal model and solve the problem of the lack of heterotopic transplantation model, blood samples were obtained from specific pathogen free (SPF) Yorkshire and Landrace for isolation of peripheral blood mononuclear cells, and SLA-1 gene was amplified by PCR after reverse transcription. SLA-1 gene sequences were obtained by analyzing the direct and cloning results. The polymorphism of alleles was analyzed using the bioinformatics software. A total of 16 alleles was identified at SLA-1 gene, containing a full-length 1 086 bp open reading frame. Nucleotide diversity (Pi) was 0.045 59, haplotype diversity (Hd) was 0.000 49, the average nucleotide difference (K) was 49.508, and the content of G+C reached 60.9%. Among 150 nucleotide mutations, the number of nucleotide parsimony informative sites (NPi) were higher than those of the nucleotide singleton variable sites (NS), and 82 of the 361 amino acids were mutated and the numbers of non-synonymous sites (NSS) were obviously higher than those of synonymous sites (SS). The polymorphism of SLA-1 allele was mainly concentrated in exon 2 and 3. Nine of 33 key amino acids were completely conserved, and eight residues showed complete consistence with the corresponding amino acids of human leukocyte antigen (HLA) allele. Eleven of the 19 amino acids, which bind β2-microglobulin (β2m) to HLA-A, showed complete consistency among the alleles identified in this study. The key amino acid binding sites of CD8 to major histocompatibility complex (MHC) were highly consistent in human (Homo sapiens) and SPF Yorkshire and Landrace, except for 225 (Thr/Ser) and 228 (Thr/Met) sites. The result of phylogenetic relationship showed that there was a close relationship among SPF Yorkshire and Landrace pigs, Korean native pigs and Meishan pigs. This study can provide genetic basis for establishment of animal models antigen presentation and immune response.
参考文献[1] Rabin,M.,Fries,R.,Singer,D.,et al., Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization [J]. Cytogenetic and Genome Research .1985.39, 206-209.[2] Smith, T., Rohrer, G.A., Alexander, L.J., et al., Directed integration of the physical and genetic linkage maps of swine chromosome 7 reveals that the SLA spans the centromere [J]. Genome Research.1995. 5,259-271.[3] Gao Caixia, Jiang Qian, GuoDongchun., et al., Characterization of swine leukocyte antigen(SLA) polymorphism bysequence-based and PCR-SSP methods in Chinese Bama miniature pigs[J].Developmental and Comparative Immunology.2014. 45,87-96.[4] Smith, D., Lunney, J., Martens, G., et al., Nomenclature for factors of the SLA class‐I system[J], Tissue Antigens.2005b. 65, 136-149.[5] Renard, C., Vaiman, M., Chiannilkulchai, N., et al., Sequence of the pig major histocompatibilityregion containing the classical class I genes[J].Immunogenetics. 2001.53, 490-500.[6] Satz, M., Wang, L., Singer, D., et al., Structure and expression of two porcine genomic clones encoding class I MHC antigens [J]. The Journal of Immunology.1985. 135, 2167-2175.[7] Chardon, P., Rogel-Gaillard, C., Cattolico, L., et al., Sequence of the swine major histocompatibility complex region containing all non-classical class I genes [J]. Tissue Antigens.2001.57, 55-65.[8] 唐军, 陈福祥, 李宁丽, 等.中国版纳猪MHCⅠ类P1 分子全长的原核表达和纯化. 中国免疫学杂志, 2002, 18: 264-267.TANG Jun,CHENG Fuxiang,LI Ningli,et al.,Expression in E? coli of Chinese bama pigs SLA class I P1protein and its purification[J]. Chinese Journal of Immunology. 2002, 18: 264-267.[9] YewdellJW, Reits E, Neefjes J. Making sense of mass destruction: quantitating MHC class I antigen presentation [J]. Immunology. 2003. 3(12):952-61.[10] 胡文平. 云南地方猪种遗传多样性研究[J]. 家畜生态,1998,02:15-19.HU Wenping. Study on the Genetic Diversity of Yunnan Local pig Breeds[J]. Journal of Domestic Animal Ecology. 1998,02:15-19.[11] 周福波. 汉族白癜风患者HLA-A*0201限制性CTL表位的筛查与鉴定[D].第四军医大学,2014.Zhou Fubo .Screening and Identification of HLA-A*0201-Restricted CTL Epitope in Chinese Vitiligo Patients[D]. The Fourth Military Medical University,2014.[12] 赵生国.亚洲部分鸡种系统发育研究及遗传资源优先保护顺序评估[D]. 甘肃农业大学. 2009.Zhao Shengguo.Phylogeny and Assessment of Conservation Priority for cAsian Native Chicken Genetic Resources[D]. Gansu Agriculture University.2009.[13] Renard, C., Hart, E., Sehra, H., et al., The genomic sequence and analysis of the swine major histocompatibility complex[J]. Genomics.2006. 88, 96-110. [14] Smith, D.M., Martens, G.W., Ho, C.S., et al., DNA sequence based typing of swine leukocyte antigens in Yucatan miniature pigs[J]. Xenotransplantation. 2005c.12, 481-488.[15] 莫斯科. 中国合作猪SLAⅠ类和Ⅱ类分子的克隆及基因结构与多态性分析[D].甘肃农业大学,2009.Mo Si-ke.Cloning and analysis of genomic structure and genetic polymorphism of swine leukocyte antigen class I and II molecules in Chinese Hezuo pig[D]. Gansu Agriculture University.2009[16] 陶璇. SLAⅠ类经典基因多态性及正选择检验分析[D].四川农业大学,2008.Tao Xuan. P olymorphic and evolutional analysis of SLA classical class I genes[D]. Sichuan Agricultural University.2008.[17] 权金强,苟潇,赵生国. 云南地方猪种mtDNA D-loop区遗传多样性研究[J]. 四川农业大学学报,2015,04:422-428.QUAN Jinqiang GOU xiao ZHAO Shengguo . The Genetic Diversity of Mitochondrial DNA D-LOOP in Yunnan Native Pigs[J]. Journal of Sichuan Agricultural University. 2015,04:422-428.[18] Tajima F. St atistical method for t esting the neutral mutation hypothesisby DNA polymorphism[ J] .Genetics,1989 .123 :185 -195 .[19] Gui-ShengWu, Yong-Gang Yao, Kai-Xing Qu, et al. Population phylogenomic analysis of mitochondrial DNA in wild boars and domestic pigs revealed multiple domestication events in East Asia[J].Genome Biology.2007.8,(11);245-246.[20] Ho, C.S., Rochelle, E.S., Martens, G.W., et al., Characterization of swine leukocyte antigen polymorphism by sequence-based and PCR-SSP methods in Meishan pigs[J]. Immunogenetics.2006. 58, 873-882.[21] Ho, C.S., Martens, G.W., Amoss, M.S., et al., Swine leukocyte antigen (SLA) diversity in Sinclair and Hanford swine[J]. Developmental & Comparative Immunology. 2010b.34, 250-257.[22] Smith, D., Lunney, J., Ho, C.S., et al., Nomenclature for factors of the swine leukocyte antigenclass II system[J],Tissue Antigens 2005a.66, 623-639.[23] Zhang, N., Qi, J., Feng, S., et al., Crystal structure of swine major histocompatibility complexclass I SLA-1* 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic tlymphocyte epitope peptides[J]. Journal of virology. 2011. 85, 11709-11724.[24] Gleimer, M., Parham, P., Stress management: MHC class I and class I-like molecules asreportersof cellular stress[J]. Immunity .2003.19, 469-477.[25] Donnelly CE, Yatko C, Johnson EW, et al. Human natural killercells account for MHC class I restricted cytolysis of porcineCells[J]. Cell Immunol, 1997, 175: 171-178．[26] Yamada K, Sachs DH, DerSimonian H. Human anti-porcine xenogeneic T cell response Evidence for allelic specificity of mixedleukocyte reaction and for both direct and indirect pathway of recognition[J]. J Immunol,1995,155: 5249-5256．[27] Bravery CA, Batten P, Yacoub MH, et al. Direct recognition of SLA-and HLA-like class II antigens on porcine endothelium by human T cells results in T cell activation and release of interleukin-2[J]. Transplatation,1995,60: 1024-1032．[28] Xie J, Chen FX, Li NL, et al. Novel SLA class I alleles of Chinese pig strains and their significance in xenotrans plantation[J].J Immunol, 2000, 20: 136-139．[29] Vaiman M, Renard C, Lafage P, et al. Evidence for a histocompatibility system in swine ( SLA-A)[J]. Transplatation, 1970, 10: 155-164．
