猪SENP1基因克隆、生物信息学分析与表达研究

Cloning,Bioinformatics Analysis and Expression of Porcine SENP1 Gene

试验旨在对猪SUMO特异性蛋白酶1(sentrin-specific protease 1,SENP1)基因CDS区进行克隆和生物信息学分析,并探讨SENP1基因在乙脑病毒(Japanese encephalitis virus,JEV)感染PK15细胞后的表达情况。根据GenBank中公布的猪SENP1基因预测序列(登录号:XM_013997974.2)设计特异性引物,通过RT-PCR和T/A克隆技术对猪SENP1基因CDS区序列进行克隆测序,并进行生物信息学分析,利用实时荧光定量PCR分析猪SENP1基因在JEV感染PK15细胞不同时间点(0、12、24、36、48 h)的表达情况。结果显示,猪SENP1基因CDS序列全长2034 bp,共编码677个氨基酸。序列比对和系统进化树分析结果表明,猪SENP1蛋白序列和山羊、犬、人、牛的相似性分别为94.8%、94.2%、93.9%和93.8%,说明在这些物种中SENP1的保守性较强;猪与犬的SENP1分子亲缘关系较近。生物信息学分析结果显示,猪SENP1蛋白相对分子质量为76825.76,等电点为8.53,体外半衰期为30 h,不稳定系数为53.59,总平均亲水指数为-0.622。SENP1蛋白不含信号肽序列,无跨膜结构域。二级结构分析结果显示,SENP1蛋白中α-螺旋、无规则卷曲、延伸链和β-转角分别占31.31%、46.68%、16.25%和5.76%。三级结构分析结果显示,猪SENP1蛋白中存在8个α-螺旋区和7个β-转角区。实时荧光定量PCR结果显示,猪SENP1基因在JEV感染的PK15细胞中呈现上调表达趋势,其中在感染后48 h SENP1基因表达量显著高于对照组(P<0.05)。本试验结果为后续深入研究SENP1基因功能提供了参考。

The objective of this study was to clone and bioinformatics analyze the CDS of porcine sentrin-specific protease 1(SENP1)gene,and investigate the expression pattern of SENP1 gene in PK15 cells infected with Japanese encephalitis virus(JEV).The specific primers were designed according to the predicted sequence of porcine SENP1 gene(accession No.:XM_013997974.2)published in GenBank.The sequence of SENP1 gene CDS was cloned and sequenced by RT-PCR and T/A cloning technology,and bioinformatics analysis was carried out.Real-time quantitative PCR was used to analyze the expression pattern of SENP1 gene in JEV-infected PK15 cells at different time points(0,12,24,36 and 48 h).The results showed that the full length of SENP1 gene CDS was 2034 bp,encoding a total of 677 amino acids.The sequence alignment and phylogenetic tree analysis results showed that the similarity of amino acid sequence of porcine SENP1 with Capra hircus,Canis lupus,Homo sapiens and Bos taurus was 94.8%,94.2%,93.9%and 93.8%,respectively.The results indicated that porcine SENP1 was highly conserved among these species.The genetic relationship of SENP1 between Sus scrofa and Canis lupus was close.The results of bioinformatics analysis showed that the relative molecular weight of SENP1 protein was 76825.76,the isoelectric point was 8.53,the half-life in vitro was 30 h,the instability coefficient was 53.59,and the total average hydrophilic index was-0.622.The porcine SENP1 protein contained no signal peptide sequence and transmembrane domain.The ratio of alpha helix,random coil,extended chain and beta turn in secondary structure was 31.31%,46.68%,16.25%and 5.76%,respectively.The tertiary structure analysis showed that there were 8 alpha helix and 7 beta turn regions in porcine SENP1 protein.Real-time quantitative PCR results showed that the expression of SENP1 gene was up-regulated in JEV infected PK15 cells,and the expression of SENP1 gene at 48 h after infection was significantly higher than control group(P<0.05).This study provided reference materials for further study of porcine SENP1 gene function.