Identification and Analysis of MHC II Genes in Wuzhishan Pig

doi:10.12300/j.issn.1674-5817.2024.135

Abstract

Abstract:

Objective To obtain the sequences of Wuzhishan pig MHC II genes and analyze their genetic information, the biological function of MHC complex of Wuzhishan pig was investigated. Methods Spleen samples were collected from 3 adult male Wuzhishan pigs, Primers were designed according to MHC II genes sequence, the CDS sequences of Wuzhishan pig MHC II genes was amplified by RT-PCR and sequenced by Sanger sequencing. The physical and chemical properties, phylogenetic relationships, conserved domains, motifs and chromosome locations of MHC II genes in Wuzhishan pigs were analyzed with bioinformatics tools. Results A total of 8 MHC II genes were identified in Wuzhishan pigs and GenBank entry numbers were obtained, namely SLA-DRA, SLA-DQA, SLA-DQB, SLA-DRB, SLA-DOB, SLA-DMB, SLA-DMA and SLA-DOA. The gene sequences were determined by Sanger sequencing and uploaded to GenBank to obtain accession numbers, namely PQ182796, PQ182797, PQ182798, PQ182799, PQ182800, PQ182801, PQ182802, and PQ164779. The 8 MHC II proteins are unevenly distributed on chromosome 7 of the Wuzhishan pig. Bioinformatics analysis shows that most of the MHC II proteins are hydrophobic proteins, with a relative molecular weight range of 27 700 to 30 000. Their genes belong to the same class contain similar conserved motifs, among which 4 MHC II proteins contain the MHC_II_beta conserved domain, and 4 MHC II proteins contain the MHC_II_alpha conserved domain. Phylogenetic analysis showed that the 6 MHC II genes of Wuzhishan pig were not in the same branch as the MHC II genes of Duroc pig, Meishan pig, Large White pig, Bama pig and other different breeds, so the MHC II genes of Wuzhishan pig may have a certain diversity. Conclusion The Wuzhishan pig MHC II gene sequence information is successfully obtained and the physicochemical properties of MHC II genes were analyzed comprehensively, which may provide basic data for immune research of Wuzhishan pig, new ideas for vaccine development and disease prevention, and basic theoretical basis for constructing disease models and exploring the application of gene editing technology in improving the health and production performance of Wuzhishan pig. basic data for immune research of Wuzhishan pig, new ideas for vaccine development and disease prevention, and basic theoretical basis for constructing disease models and exploring the application of gene editing technology in improving the health and production performance of Wuzhishan pig.

Key words: Wuzhishan pig, Major histocompatibility complex, Bioinformatics

LIU Yuanyuan,XIN Wenshui,CHAO Zhe,et al. Identification and Analysis of MHC II Genes in Wuzhishan Pig[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2024.135.

Figures/Tables 9

References 24

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引物名称 Primer name	引物序列（5'to3'） Primer sequence（5'to3'）	扩增片段 Amplified fragment	退火温度（℃） Annealing temperature（℃）
SLA-DRA-F	GCTTGTATTGCTGTCCATCC	851 bp	55
SLA-DRA-R	CAAAGTCCATTCCCTGCAAG	851 bp
SLA-DQA-F	ATGGTCCCAGGCCGAGTT	768 bp	55
SLA-DQA-R	TCACAAGGACCCTTGGTGTC	768 bp
SLA-DQB-F	CCATTACTTCTTCGTTTGCCC	995 bp	55
SLA-DQB-R	GAAGAAGCTTCACAGCCAGAG	995 bp
SLA-DRB-F	CACACTGTCCTCTCCTGTTC	886 bp	58
SLA-DRB-R	CTCATGCTGTGAAGACGCTG	886 bp
SLA-DOB-F	CCTCATTTTCTTTTTCCCCCTCC	926 bp	55
SLA-DOB-R	GGAATCATCCAGAACATCGACC	926 bp
SLA-DMB-F	CCGCTCAGTGTTTGGAGAT	967 bp	55
SLA-DMB-R	CCATACGAGACCAAATTGCC	967 bp
SLA-DMA-F	GGACCTGGGTTAGCTAGTTAG	1 028 bp	55
SLA-DMA-R	ACATGGCAGTGATGTCGTAGG	1 028 bp
SLA-DOA-F	GGTTAAAACACCAGAGGGCC	807 bp	55
SLA-DOA-R	GTCTCTCTCACATCCCAGCC	807 bp

引物名称 Primer name	引物序列（5'to3'） Primer sequence（5'to3'）	扩增片段 Amplified fragment	退火温度（℃） Annealing temperature（℃）
SLA-DRA-F	GCTTGTATTGCTGTCCATCC	851 bp	55
SLA-DRA-R	CAAAGTCCATTCCCTGCAAG	851 bp
SLA-DQA-F	ATGGTCCCAGGCCGAGTT	768 bp	55
SLA-DQA-R	TCACAAGGACCCTTGGTGTC	768 bp
SLA-DQB-F	CCATTACTTCTTCGTTTGCCC	995 bp	55
SLA-DQB-R	GAAGAAGCTTCACAGCCAGAG	995 bp
SLA-DRB-F	CACACTGTCCTCTCCTGTTC	886 bp	58
SLA-DRB-R	CTCATGCTGTGAAGACGCTG	886 bp
SLA-DOB-F	CCTCATTTTCTTTTTCCCCCTCC	926 bp	55
SLA-DOB-R	GGAATCATCCAGAACATCGACC	926 bp
SLA-DMB-F	CCGCTCAGTGTTTGGAGAT	967 bp	55
SLA-DMB-R	CCATACGAGACCAAATTGCC	967 bp
SLA-DMA-F	GGACCTGGGTTAGCTAGTTAG	1 028 bp	55
SLA-DMA-R	ACATGGCAGTGATGTCGTAGG	1 028 bp
SLA-DOA-F	GGTTAAAACACCAGAGGGCC	807 bp	55
SLA-DOA-R	GTCTCTCTCACATCCCAGCC	807 bp

组成成分 Constituent	体积 Volume
PrimeSTAR HS DNA Polymerase	0.5 μL
5×PrimeSTAR Buffer	10 μL
dNTP Mixture	4 μL
上游引物（10 μM）	1.0 μL
下游引物（10 μM）	1.0 μL
cDNA模板	1.0 μL
RNase-free H2O	32.5 μL

组成成分 Constituent	体积 Volume
PrimeSTAR HS DNA Polymerase	0.5 μL
5×PrimeSTAR Buffer	10 μL
dNTP Mixture	4 μL
上游引物（10 μM）	1.0 μL
下游引物（10 μM）	1.0 μL
cDNA模板	1.0 μL
RNase-free H2O	32.5 μL

基因ID	染色体定位	氨基酸长度	相对分子量（ⅹ10³）	等电点PI	GRAVY
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