猪肾上皮细胞SLA-1基因的克隆及分子结构特征分析

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

【目的】利用猪肾上皮细胞15(PK15)建立系统的猪白细胞抗原1(SLA-1)抗原表位筛选系统。【方法】提取PK15细胞总RNA,设计特异性引物,应用RT-PCR方法扩增SLA-1基因(SLA-1*PK15),将该基因克隆到pMD18-T载体上,并进行双酶切及测序鉴定;利用DNAMAN 5.2.2、Mega 5.0、Multalin及同源建模进行系统进化树、二级结构、三级结构分析。【结果】RT-PCR扩增获得约1400 bp条带,质粒提取和酶切鉴定结果表明SLA-1成功插入pMD18-T载体;测序结果证实该基因共1419 bp,其中2-1087 bp为编码区,共编码361个氨基酸,信号肽为21个氨基酸,符合SLA-1基因特征。进化树分析结果显示,SLA-1*PK15与SLA-1*wxd(中国梅山猪)和SLA-1*0401(中国巴马小型猪)进化关系最近,而与SLA-1*lr02(丹麦长白猪)及SLA-1*0509(中国西藏野猪)进化关系较远。胞外区氨基酸比较分析表明,PK15细胞SLA-1基因与其他SLA-1基因胞外区主要变异位点存在于α1区和α2区,α3区的变异位点较少,无特征性氨基酸变异位点。PK15细胞SLA-1蛋白的二级结构主要以α-螺旋和β-折叠为主。同源建模结果显示,SLA-1蛋白具有SLA classⅠ典型的三级结构,α1区和α2区构成抗原多肽结合区。【结论】SLA-1基因稳定存在于PK15细胞,PK15细胞具有作为SLA-1抗原表位筛选系统的潜在应用价值。

【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 1400 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 1419 bp,of which 2-1087 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 inα1 andα2 regions,there were few variation sites inα3 region,and there were no characteristic amino acid variation sites.The secondary structure of SLA-1 protein of PK15 cells was mainly based onα-helix andβ-fold.Homology modeling showed that SLA-1 protein of PK15 cells had a typical three-level structure of SLA classⅠ,α1 andα2 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.