Identification and Analysis of MHCⅡ Genes in Wuzhishan Pigs

doi:10.12300/j.issn.1674-5817.2024.135

Abstract

Abstract:

Objective To obtain the gene sequences of major histocompatibility complex (MHC ) Ⅱgenes of Wuzhishan pigs, analyze their genetic information, and explore the biological functions of their MHC system. Methods Spleen samples were collected from 3 adult male Wuzhishan pigs. Primers were designed according to MHCⅡ gene sequences, and the coding sequences of Wuzhishan pig MHCⅡ genes were amplified by RT-PCR. Sanger sequencing was performed to determine the full-length sequences. Bioinformatics tools were employed to analyze the physicochemical properties, phylogenetic relationships, conserved motifs, structural domains, chromosomal localization, and syntenic relationships of these genes. Results Eight MHCⅡ genes were identified in Wuzhishan pigs, designated as SLA-DRA, SLA-DQA, SLA-DQB, SLA-DRB, SLA-DOB, SLA-DMB, SLA-DMA and SLA-DOA. The full-length sequences of these genes were determined by Sanger sequencing and subsequently deposited in GenBank under accession numbers PQ182796, PQ182797, PQ182798, PQ182799, PQ182800, PQ182801, PQ182802, and PQ164779. Phylogenetic analysis showed that the six MHCⅡ genes of Wuzhishan pigs clustered separately from their counterparts in Duroc, Meishan, Large White, and Bama pigs, indicating distinct evolutionary trajectories. Bioinformatics analysis demonstrated that most MHC Ⅱ proteins were hydrophobic, with molecular weights ranging from 27 700 to 30 000 Da. Genes within the same subregion shared conserved motifs. Specifically, four MHCⅡ proteins encoded by SLA-DQB, SLA-DRB, SLA-DOB, and SLA-DMB contained the MHCⅡβ conserved domain, while those encoded by the genes SLA-DRA, SLA-DQA, SLA-DMA, and SLA-DOA contained the MHCⅡα conserved domain. The eight MHCⅡ genes were scattered along the long arm of chromosome 7 in the Wuzhishan pigs, exhibiting syntenic relationships with three human genes and five Duroc pig genes. Conclusion The MHCⅡ genes of Wuzhishan pigs may possess a unique evolutionary origin.

Key words: Wuzhishan pigs, Major histocompatibility complex, Bioinformatics, Gene sequence

CLC Number:

Q95-33

LIU Yuanyuan,XIN Wenshui,CHAO Zhe,et al. Identification and Analysis of MHCⅡ Genes in Wuzhishan Pigs[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 340-348. DOI: 10.12300/j.issn.1674-5817.2024.135.

Figures/Tables 6

Table 1 Information on PCR primers for amplification of major histocompatibility complex (MHC) genes

引物名称 Primer name	引物序列（5' to 3'） Primer sequence（5' to 3'）	扩增片段长度/bp Amplicon size /bp	退火温度/℃ Annealing temperature/℃
SLA-DRA-F	GCTTGTATTGCTGTCCATCC	851	55
SLA-DRA-R	CAAAGTCCATTCCCTGCAAG
SLA-DQA-F	ATGGTCCCAGGCCGAGTT	768	55
SLA-DQA-R	TCACAAGGACCCTTGGTGTC
SLA-DQB-F	CCATTACTTCTTCGTTTGCCC	995	55
SLA-DQB-R	GAAGAAGCTTCACAGCCAGAG
SLA-DRB-F	CACACTGTCCTCTCCTGTTC	886	58
SLA-DRB-R	CTCATGCTGTGAAGACGCTG
SLA-DOB-F	CCTCATTTTCTTTTTCCCCCTCC	926	55
SLA-DOB-R	GGAATCATCCAGAACATCGACC
SLA-DMB-F	CCGCTCAGTGTTTGGAGAT	967	55
SLA-DMB-R	CCATACGAGACCAAATTGCC
SLA-DMA-F	GGACCTGGGTTAGCTAGTTAG	1 028	55
SLA-DMA-R	ACATGGCAGTGATGTCGTAGG
SLA-DOA-F	GGTTAAAACACCAGAGGGCC	807	55
SLA-DOA-R	GTCTCTCTCACATCCCAGCC

Figure 1 PCR results of MHCⅡ genes in spleen tissues of Wuzhishan pigsNote： SLA refers to the swine leukocyte antigen ［also named as major histocompatibility complex（MHC）］. M denotes the DNA molecular weight marker （2000 bp ladder）.

Figure 2 Phylogenetic tree analysis of MHCⅡ genes in Wuzhishan pigsNote： SLA refers to the swine leukocyte antigen ［also named as major histocompatibility complex （MHC）］.

Table 2 Physicochemical property analysis of MHC Ⅱ molecules in Wuzhishan pigs

基因ID Gene ID	染色体定位 Chromosomal localization	氨基酸长度/aa Amino acid length/aa	相对分子质量，×10³ Relative molecular mass, ×10³	等电点 Isoelectric point	亲水性平均系数 Grade average of hydropathicity
SLA-DRA	Chr07	252	28.4	5.03	0.029
SLA-DQA	Chr07	255	28.2	5.53	0.007
SLA-DQB	Chr07	261	29.5	6.97	-0.222
SLA-DRB	Chr07	266	30.0	8.59	-0.135
SLA-DOB	Chr07	264	29.8	5.32	0.024
SLA-DMB	Chr07	272	29.8	5.69	0.027
SLA-DMA	Chr07	260	28.9	4.69	0.152
SLA-DOA	Chr07	250	27.7	6.18	0.016

Figure 3 Analysis of conserved motifs （A） and structural domains （B） of MHCⅡ molecules in Wuzhishan pigsNote： Colored boxes represent different conserved motifs or conserved domains. The x-axis denotes the amino acid length of MHC Ⅱ molecules. SLA refers to the swine leukocyte antigen ［also named as major histocompatibility complex （MHC）］.

Figure 4 Chromosomal localization of MHCⅡ genes on chromosome 7 in Wuzhishan pigs（A）and syntenic relationship analysis of MHCⅡ genes between Wuzhishan pigs， Homo sapiens and Sus scrofa（B）Note： The human genome assembly used is GRCh38.p12； the Duroc pig genome assembly used is Sscrofa11.1. Genes exhibiting syntenic relationships with MHC class II molecules are depicted as blue lines， while other syntenic genes are represented by gray lines. SLA refers to the swine leukocyte antigen ［also named as major histocompatibility complex （MHC）］.

References 23

[1]	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. DOI:10.1177/0300985811402846 .
[2]	兰宗宝, 王修文, 许惠艳. 猪器官异种移植研究进展[J]. 广西农业科学, 2008, 39(3):380-384. DOI: 10.3969/j.issn.2095-1191.2008.03.030 .
	LAN Z B, WANG X W, XU H Y. Progress of porcine organs xenotransplantation[J]. Guangxi Agric Sci, 2008, 39(3):380-384. DOI: 10.3969/j.issn.2095-1191.2008.03.030 .
[3]	CARRIER A N, VERMA A, MOHIUDDIN M, et al. Xenotransplantation: a new era[J]. Front Immunol, 2022, 13: 900594. DOI:10.3389/fimmu.2022.900594 .
[4]	PROSSER A, HUANG W H, LIU L, et al. Dynamic changes to tissue-resident immunity after MHC-matched and MHC-mismatched solid organ transplantation[J]. Cell Rep, 2021, 35(7):109141. DOI:10.1016/j.celrep.2021.109141 .
[5]	冯书堂, 戴一凡, 章金刚, 等. 五指山小型猪近交系异种移植产业化研发进展[J]. 器官移植, 2018, 9(6):469-473. DOI: 10.3969/j.issn.1674-7445.2018.06.014 .
	FENG S T, DAI Y F, ZHANG J G, et al. The progress of xenograft industrialization on Wuzhishan miniature pig inbred line[J]. Organ Transplant, 2018, 9(6):469-473. DOI: 10.3969/j.issn.1674-7445.2018.06.014 .
[6]	晁哲, 李娇伦, 秦烨, 等. 五指山猪SLA-DQA和SLA-DQB基因SNP检测及生物信息学分析[J]. 养猪, 2017(6):57-60. DOI: 10.13257/j.cnki.21-1104/s.2017.06.018 .
	CHAO Z, LI J L, QIN Y, et al. Detection of single nucleotide polymorphism and bioinformatics analysis of SLA-DQA and SLA-DQB genes in Wuzhishan pig[J]. Swine Prod, 2017(6):57-60. DOI: 10.13257/j.cnki.21-1104/s.2017.06.018 .
[7]	NIU M M, LIU Y Q, XIANG L, et al. Long-term case study of a Wuzhishan miniature pig with diabetes[J]. Animal Model Exp Med, 2020, 3(1):22-31. DOI:10.1002/ame2.12098 .
[8]	ZHAO Y Q, XIANG L, LIU Y Q, et al. Atherosclerosis induced by a high-cholesterol and high-fat diet in the inbred strain of the Wuzhishan miniature pig[J]. Anim Biotechnol, 2018, 29(2):110-118. DOI:10.1080/10495398.2017.1322974 .
[9]	HUANG Q, XU H B, YU Z, et al. Inbred Chinese Wuzhishan (WZS) minipig model for soybean glycinin and beta-conglycinin allergy[J]. J Agric Food Chem, 2010, 58(8):5194-5198. DOI:10.1021/jf904536v .
[10]	LI X, HUANG Y, LIANG Q F, et al. Local immunosuppression in Wuzhishan pig to Rhesus monkey descemet's stripping automated endothelial keratoplasty: an innovative method to promote the survival of xenografts[J]. Ophthalmic Res, 2022, 65(2):196-209. DOI:10.1159/000521193 .
[11]	韦习会, 王林云, 徐银学, 等. 五指山猪白细胞抗原血清学研究[J]. 畜牧兽医学报, 1996(3): 199-206.
	WEI X H, WANG L Y, XU Y X, et al. Preliminary serological study on swine lymphocyte antigen (sla) of the Wuzhishan pig[J]. Acta Vet Zootechnica Sin, 1996(3): 199-206.
[12]	孙俊丽. 五指山猪近交系群体三个白细胞抗原基因序列和功能分析[D]. 咸阳: 西北农林科技大学, 2005.
	SUN J L. Analysis of sequence and its function on three swine leutocyte antigen gene in inbreeding line of wuzhishan pig population[D]. Xianyang: Northwest Agriculture and Forestry University, 2005.
[13]	RABIN M, FRIES R, SINGER D, et al. Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization[J]. Cytogenet Cell Genet, 1985, 39(3):206-209. DOI:10.1159/000132136 .
[14]	BARBOSA A, DEMEURE O, URIEN C, et al. A physical map of large segments of pig chromosome 7q11-q14: comparative analysis with human chromosome 6p21[J]. Mamm Genome, 2004, 15(12):982-995. DOI:10.1007/s00335-004-3008-6 .
[15]	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. DOI:10.1146/annurev-animal-020518-115014 .
[16]	VAIMAN M, CHARDON P, ROTHSCHILD M F. Porcine major histocompatibility complex[J]. Rev Sci Tech, 1998, 17(1):95-107. DOI:10.20506/rst.17.1.1093 .
[17]	MERKENSCHLAGER J, EKSMOND U, DANELLI L, et al. MHC class II cell-autonomously regulates self-renewal and differentiation of normal and malignant B cells[J]. Blood, 2019, 133(10):1108-1118. DOI:10.1182/blood-2018-11-885467 .
[18]	CHARDON P, RENARD C, VAIMAN M. The major histocompatibility complex in swine[J]. Immunol Rev, 1999, 167:179-192. DOI:10.1111/j.1600-065x.1999.tb01391.x .
[19]	刘丽霞. 三个猪种SLA-DQA和DRA基因分子遗传特征及其与仔猪腹泻的关联研究[D]. 兰州: 甘肃农业大学, 2015.
	LIU L X. Molecular genetic characteristics of SLA-DQA and DRA genes and their association with piglet diarrhea in three pig breeds[D]. Lanzhou: Gansu Agricultural University, 2015.
[20]	VIRET C, JANEWAY C A Jr. MHC and T cell development[J]. Rev Immunogenet, 1999, 1(1):91-104.
[21]	吴群, 熊平, 陈实, 等. 近交系海南五指山猪SLA经典I类和II类分子序列分析[J]. 现代免疫学, 2004(1):23-26. DOI: 10.3969/j.issn.1001-2478.2004.01.007 .
	WU Q, XIONG P, CHEN S, et al. Sequence analysis of classical swine leukocyte antigens (SLA) class I and class II molecules in inbred strain of Chinese Wuzhishan pigs[J]. Curr Immunol, 2004(1):23-26. DOI: 10.3969/j.issn.1001-2478.2004.01.007 .
[22]	常瑞刚, 舒丹, 刘丽霞. SLA-DRB基因多态性与仔猪腹泻的关联性研究进展[J]. 甘肃畜牧兽医, 2017, 47(6):20-21. DOI: 10.15979/j.cnki.cn62-1064/s.2017.06.002 .
	CHANG R G, SHU D, LIU L X. Research progress on the correlation between SLA-DRB gene polymorphism and piglet diarrhea[J]. Gansu Anim Husb Vet, 2017, 47(6):20-21. DOI: 10.15979/j.cnki.cn62-1064/s.2017.06.002 .
[23]	顾茂松. 五指山小型猪SLA-DQA基因的SNPs及猪人MHC Ⅰ类区自然杀伤细胞KIRs结合区氨基酸变异的比较分析[D]. 武汉: 华中农业大学, 2005.
	GU M S. Single nucleotide polymorphism of SLA-DQA gene in wuzhishan miniature pigs and comparision of amino acids variability of NK cell KIRs binding sites between swine and human MHC class Ⅰ region[D]. Wuhan: Huazhong Agricultural University, 2005.