猪肾上皮细胞SLA-1基因的克隆及分子结构特征分析

doi:10.16431/j.cnki.1671-7236.2022.05.008

Abstract

Abstract: 【Objective】 The purpose of this study was to establish a systematic porcine leukocyte antigen-1 (SLA-1) epitope screening system using porcine renal epithelial cells 15(PK15).【Method】 Total RNA was extracted from PK15 cells,specific primers were designed,and SLA-1 gene (SLA-1*PK15) was amplified by RT-PCR method.The SLA-1 gene was further cloned into pMD18-T vector and identified by double enzyme digestion and sequencing.Bioinformatics softwares DNAMAN 5.2.2,Mega 5.0,Multalin and homology modeling were used to analyze the phylogenetic tree,secondary structure and tertiary structure.【Result】 The results showed that about 1 400 bp band was obtained by RT-PCR amplification,the results of plasmid extraction and enzyme digestion showed that SLA-1 was successfully inserted into pMD18-T vector.Sequencing results showed that the gene had a total of 1 419 bp,of which 2－1 087 bp was the coding region,encoding 361 amino acids,and the signal peptide contained 21 amino acids,which was accorded with the characteristics of SLA-1 gene.The results of evolutionary tree analysis showed that SLA-1*PK15 had the closest evolutionary relationship with SLA-1*wxd (Chinese Meishan pig) and SLA-1*0401 (Chinese Bama miniature pig),but far from SLA-1*lr02 (Danish Landrace) and SLA-1*0509 (Chinese Tibetan wild boar).The comparative analysis of extracellular amino acids showed that the main variation sites in the extracellular region of SLA-1 gene in PK15 cells and other SLA-1 genes existed in α1 and α2 regions,there were few variation sites in α3 region,and there were no characteristic amino acid variation sites.The secondary structure of SLA-1 protein of PK15 cells was mainly based on α-helix and β-fold.Homology modeling showed that SLA-1 protein of PK15 cells had a typical three-level structure of SLA class Ⅰ,α1 and α2 regions constituted the antigen polypeptide binding region.【Conclusion】 SLA-1 gene existed stably in PK15 cells.PK15 cells had potential application value as SLA-1 antigen epitope screening system.

Key words: porcine renal epithelial cells 15 (PK15); swine leukocyte antigen(SLA-1); cloning; bioinformatic analysis; homology modeling

CLC Number:

S852.4

WANG Baobao, JIN Hang, XIAN Yuhan, FENG Hongsheng, GAO Fengshan. Cloning SLA-1 Gene from Porcine Kidney Epithelial Cells and Analyzing its Molecular Structure Characteristics[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1671-1678.

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Cloning SLA-1 Gene from Porcine Kidney Epithelial Cells and Analyzing its Molecular Structure Characteristics

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