五指山猪MHCⅡ类分子基因鉴定与分析

doi:10.12300/j.issn.1674-5817.2024.135

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (3): 340-348.DOI: 10.12300/j.issn.1674-5817.2024.135

• 实验动物资源开发与利用 • 上一篇下一篇

五指山猪MHCⅡ类分子基因鉴定与分析

刘园园¹^,², 辛文水², 晁哲², 曹宗喜¹^,², 蔡艺菲³, 李强¹^,², 李凌伟¹^,², 刘光亮¹^,²^,³()()

^1.新疆农业大学动物医学学院, 乌鲁木齐 830052
^2.海南省农业科学院畜牧兽医研究所, 海南省热带动物繁育与疫病研究重点实验室, 海口 571100
^3.中国农业科学院兰州兽医研究所, 兰州 730046

收稿日期:2024-09-18 修回日期:2024-12-15 出版日期:2025-06-25 发布日期:2025-06-25
通讯作者: 刘光亮（1975—），男，博士，研究员，博士生导师，研究方向：猪消化道感染与黏膜免疫。E-mail：LiuGuangliang01@caas.cn。ORCID：0000-0001-8185-5749
作者简介:刘园园（1996—），女，硕士研究生，研究方向：预防兽医学。E-mail：3125177841@qq.com
基金资助:
海南省重大科技计划“五指山猪实验动物模式化研究”(ZDKJ2021030);科技部重点研发计划“发育和代谢疾病猪突变品系资源库的建立”(2021YFA0805905);国家重点研发计划课题“异种移植供体猪保障培育体系建立”(2023YFC3404302)

Identification and Analysis of MHCⅡ Genes in Wuzhishan Pigs

LIU Yuanyuan¹^,², XIN Wenshui², CHAO Zhe², CAO Zongxi¹^,², CAI Yifei³, LI Qiang¹^,², LI Lingwei¹^,², LIU Guangliang¹^,²^,³()()

^1.College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
^2.Hainan Provincial Key Laboratory of Tropical Animal Breeding and Disease Research, Institute of Animal Husbandry and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, China
^3.Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2024-09-18 Revised:2024-12-15 Published:2025-06-25 Online:2025-06-25
Correspondence to: LIU Guangliang (ORCID: 0000-0001-8185-5749), E-mail: LiuGuangliang01@caas.cn

摘要/Abstract

摘要：

目的获得五指山猪主要组织相容性复合体（major histocompatibility complex，MHC）Ⅱ类分子基因序列，并分析其遗传信息，探究五指山猪MHC的生物学功能。方法采集3头成年雄性五指山猪的脾脏样本，根据MHC Ⅱ类分子基因序列设计引物，采用RT-PCR法扩增五指山猪MHC Ⅱ类分子基因的编码序列，通过Sanger测序确定MHC Ⅱ类分子基因序列，利用生物信息学软件分析五指山猪MHC Ⅱ类分子基因的理化性质、系统进化关系、保守基序和结构域，以及染色体位置和共线性关系。结果共鉴定出8个五指山猪MHC Ⅱ类分子基因，分别为SLA-DRA、SLA-DQA、SLA-DQB、SLA-DRB、SLA-DOB、SLA-DMB、SLA-DMA和SLA-DOA。Sanger测序后确定MHC Ⅱ类分子基因序列，并将序列上传至GenBank获得登录号，分别为PQ182796、PQ182797、PQ182798、PQ182799、PQ182800、PQ182801、PQ182802和PQ164779。系统进化树分析表明，五指山猪6个MHCⅡ类分子基因与杜洛克猪、梅山猪、大白猪和巴马猪等不同品种猪的MHC Ⅱ类分子基因不在同一个分支上。生物信息学分析表明，MHC Ⅱ类分子大部分为疏水性蛋白，其相对分子质量为27 700 ~ 30 000，归于同一亚区的基因含有相似的保守基序，其中SLA-DQB、SLA-DRB、SLA-DOB和SLA-DMB基因编码的4个MHC Ⅱ类分子包含MHC Ⅱβ保守结构域，SLA-DRA、SLA-DQA、SLA-DMA和SLA-DOA基因编码的4个MHC Ⅱ类分子包含MHC Ⅱɑ保守结构域。8个MHC Ⅱ类分子基因散在分布于五指山猪的7号染色体长臂上，与人的3个基因之间具有共线性关系，而与杜洛克猪的5个基因之间呈现共线性关系。结论五指山猪的MHC Ⅱ类分子基因可能具有特殊的遗传学起源。

关键词: 五指山猪, 主要组织相容性复合体, 生物信息学, 基因序列

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

中图分类号:

Q95-33

刘园园,辛文水,晁哲,等. 五指山猪MHCⅡ类分子基因鉴定与分析[J]. 实验动物与比较医学, 2025, 45(3): 340-348. DOI: 10.12300/j.issn.1674-5817.2024.135.

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.

图/表 6

表 1 主要组织相容性复合体基因的PCR扩增引物信息

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

注：SLA，猪白细胞抗原，也称主要组织相容性复合体。M，DNA分子量标准2000。
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图 1 五指山猪脾脏组织中MHC Ⅱ类分子基因的PCR结果
注：SLA，猪白细胞抗原，也称主要组织相容性复合体。M，DNA分子量标准2000。

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

注：SLA，猪白细胞抗原，也称主要组织相容性复合体。
">

图 2 五指山猪MHC Ⅱ类分子基因的系统进化树分析
注：SLA，猪白细胞抗原，也称主要组织相容性复合体。

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

表 2 五指山猪MHC Ⅱ类分子的理化性质分析

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

注：不同颜色的方框代表了不同的保守基序或保守结构域；横坐标为MHCⅡ类分子的氨基酸长度。SLA，猪白细胞抗原，也称主要组织相容性复合体。
">

图 3 五指山猪MHC Ⅱ类分子的保守基序（A）和保守结构域（B）分析
注：不同颜色的方框代表了不同的保守基序或保守结构域；横坐标为MHCⅡ类分子的氨基酸长度。SLA，猪白细胞抗原，也称主要组织相容性复合体。

Figure 3 Analysis of conserved motifs （A） and structural domains （B） of MHCⅡ molecules in Wuzhishan pigs
Note： 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）］.

注：人类基因组组装GRCh38.p12；杜洛克猪基因组组装Sscrofa11.1；具有共线性的MHC Ⅱ类分子基因为蓝色线条，具有共线性的其他基因为灰色线条。SLA，猪白细胞抗原，也称主要组织相容性复合体。
">

图 4 MHC Ⅱ类分子基因在五指山猪7号染色体上的分布（A）和五指山猪与人及杜洛克猪的MHC Ⅱ类分子基因共线性分析（B）
注：人类基因组组装GRCh38.p12；杜洛克猪基因组组装Sscrofa11.1；具有共线性的MHC Ⅱ类分子基因为蓝色线条，具有共线性的其他基因为灰色线条。SLA，猪白细胞抗原，也称主要组织相容性复合体。

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）］.

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五指山猪MHCⅡ类分子基因鉴定与分析

Identification and Analysis of MHCⅡ Genes in Wuzhishan Pigs

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