China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (8): 2843-2854.doi: 10.16431/j.cnki.1671-7236.2022.08.001

• Biotechnology • Previous Articles     Next Articles

Cloning,Bioinformatics Analysis and Subcellular Localization of Interferon-stimulated Gene 15 in Goats

TANG Jingyu1, DU Hanyu1,2, MENG Chunchun1, LIU Guangqing1   

  1. 1. Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China;
    2. Xinjiang Agricultural University, Xinjiang 830052, China
  • Received:2022-03-04 Online:2022-08-05 Published:2022-07-21

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

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