山羊干扰素刺激基因15克隆、生物信息学分析及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2022.08.001

摘要/Abstract

摘要： 【目的】对山羊干扰素刺激基因15(interferon-stimulated gene 15,ISG15)的CDS区进行克隆、表达和生物信息学分析,并探讨小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)感染山羊子宫内膜上皮细胞(caprine endometrial epithelial cells,EEC)对ISG15的影响。【方法】根据GenBank中公布的山羊ISG15基因预测序列(登录号:XM_005690795)设计特异性引物,利用RT-PCR方法扩增山羊ISG15基因CDS区,连接至真核表达载体进行测序并表达,对不同物种ISG15核苷酸及氨基酸序列进行比对,并构建系统进化树,利用生物信息学方法对ISG15蛋白理化性质、跨膜结构、修饰位点、二级结构、三级结构、亚细胞定位等进行分析。通过构建真核表达载体转染及PPRV感染EEC细胞,利用间接免疫荧光的方法观察其外源性和内源性亚细胞定位,并探究PPRV感染对EEC细胞的影响。【结果】成功克隆出山羊ISG15基因CDS区并进行了真核表达。山羊ISG15核苷酸和氨基酸相似性及进化树分析都与盘羊和绵羊亲缘关系最近。生物信息学分析结果显示,山羊ISG15基因位于16号染色体上,全长474 bp,编码157个氨基酸,分子质量约为17.47 ku,为亲水性蛋白,无跨膜区和信号肽,有1个潜在的N-糖基化位点,16个潜在的O-糖基化位点和10个潜在的磷酸化位点。二级结构与三级结构预测显示,山羊ISG15蛋白由无规则卷曲、延伸链、α-螺旋和β-转角组成,比例分别为34.39%、31.21%、21.66%和12.74%。可以与干扰素和泛素化相关的蛋白相互作用,并参与机体的抗感染作用。亚细胞定位显示,外源性和内源性ISG15均定位于细胞质中。【结论】试验成功克隆出山羊ISG15基因CDS区序列,亚细胞定位发现内源性和外源性ISG15均定位于EEC细胞的细胞质中。结果为后续ISG15基因细胞内功能研究奠定基础。

关键词: 山羊; ISG15基因; 克隆; 表达; 生物信息学分析; 亚细胞定位

Abstract: 【Objective】 This study was aimed to clone,express and bioinformatics analyze the CDS region of goats interferon-stimulated gene 15 (ISG15) and investigate the effect of Peste des petits ruminants virus (PPRV) infection on ISG15 in caprine endometrial epithelial cells (EEC).【Method】 According to the prediction sequence of ISG15 gene of goats published in GenBank (accession No.:XM_005690795),specific primers were designed and RT-PCR was used to amplify the CDS region of ISG15 gene in goats which was connected to the eukaryotic expression vector for sequencing and expression.The nucleotide and amino acid sequences of ISG15 from different species were compared, and the phylogenetic tree was constructed. Physicochemical properties,transmembrane structure,modification sites,secondary structure,tertiary structure,and subcellular localization of ISG15 protein were analyzed by the bioinformatics method.EEC cells were transfected by constructing eukaryotic expression vectors and infected with PPRV.The exogenous and endogenous subcellular localization were observed by the indirect immunofluorescence method,and the effects of PPRV infection on EEC cells were investigated.【Result】 The CDS region of ISG15 gene in goats was successfully cloned and expressed in eukaryotes.The nucleotide and amino acid similarities and evolutionary tree analysis of ISG15 in goats were closest to Ovis aries and Ovis ammon.Bioinformatics analysis showed that the ISG15 gene in goats was located on chromosome 16 with a total length of 474 bp,encoding 157 amino acids,and a molecular weight of 17.47 ku. It was a hydrophilic protein without transmembrane region and signal peptide and had 1 potential N-glycosylation site,16 potential O-glycosylation sites,and 10 potential phosphorylation sites.The prediction of the secondary and tertiary structure showed that ISG15 protein was composed of the random coil,extended chain,alpha helix,and beta turn,accounting for 34.39%,31.21%,21.66%,and 12.74%,respectively.It could interact with interferon and ubiquitination-related proteins and participate in the body's anti-infection effects action.Subcellular localization showed that both exogenous and endogenous ISG15 were localized in the cytoplasm.【Conclusion】 The CDS region of ISG15 gene in goats was successfully cloned,and subcellular localization showed that both exogenous and endogenous ISG15 were localized in the cytoplasm,which laid a foundation for subsequent intracellular functional studies of ISG15 gene.

Key words: goats; ISG15 gene; cloning; expression; bioinformatics analysis; subcellular localization

中图分类号:

S827

唐井玉, 杜汉宇, 孟春春, 刘光清. 山羊干扰素刺激基因15克隆、生物信息学分析及亚细胞定位[J]. 中国畜牧兽医, 2022, 49(8): 2843-2854.

TANG Jingyu, DU Hanyu, MENG Chunchun, LIU Guangqing. Cloning,Bioinformatics Analysis and Subcellular Localization of Interferon-stimulated Gene 15 in Goats[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2843-2854.

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