羊口疮病毒ORFV114蛋白生物信息学分析、真核表达及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2022.11.005

Abstract

Abstract: 【Objective】 This study was aimed to perform bioinformatics analysis, transcription kinetics, eukaryotic expression and subcellular localization of Orf virus (ORFV) ORFV114 protein.【Method】 DNAStar software was used to perform alignment analysis of ORFV114 gene.The online websites ExPASy, TMHMM-2.0, SignalP-5.0, SOPMA and SWISS-MODEL were used to predict the physical and chemical properties, hydrophobicity, the presence of transmembrane helices and signal peptide, the structure of ORFV114 protein.In the presence or absence of cytarabine (Arac), HeLa cells were harvested at various time post infection.RT-PCR was used to amplify ORFV114 gene to determine the dynamic transcriptional level of ORFV114.The ORFV114 gene was amplified by PCR and subcloned into eukaryotic expression vector pEGFP-N1 to construct the recombinant plasmid pEGFP-ORFV114.After restriction enzyme digestion and Sanger sequencing identification, the pEGFP-ORFV114 plasmid was transiently transfected into HEK293 cells with Lipofectamine 3000.The expression of ORFV114-EGFP fusion protein in HEK293 cells was detected by Western blotting.HeLa cells were transiently transfected with pEGFP-N1 plasmid and pEGFP-ORFV114 recombinant plasmid.After 24 h, the nucleus was stained with Hoechst 33342, and the subcellular localization of ORFV114 protein was observed under an inverted fluorescence microscope.【Result】 The ORFV114 gene of ORFV-JS strain was 1 041 bp, which was highly conserved among ORFV strains.The ORFV114 protein was 346 amino acids in length, with predicted molecular masses 38 ku.It was a weakly hydrophilic unstable protein without transmembrane region nor signal peptide.Its secondary structure was composed of random coil, alpha helix, extended chain and beta turn, which were 45.09%, 25.14%, 21.68% and 8.09%, respectively.Transcription of ORFV114 was detected as early as 2 h post infection, and the levels of expression increased during the infection cycle in the presence or absence of Arac.The results indicated that Arac did not inhibit the transcription of ORFV114, that was, ORFV114 belonged to an early gene of ORFV.The pEGFP-ORFV114 recombinant plasmid was successfully constructed and ORFV114-EGFP fusion protein with a molecular mass of 65 ku was successfully expressed in HEK293 cells.The ORFV114 protein mainly localized to the cytoplasm of HeLa cells, exhibiting a punctate distribution pattern in this compartment.【Conclusion】 In this study, the transcription kinetics, eukaryotic expression and subcellular localization of ORFV114 protein of ORFV were successfully performed, laying a foundation for further research on ORFV114 protein function and its interaction proteins.

Key words: Orf virus (ORFV); ORFV114 protein; bioinformatics analysis; transcription kinetics; eukaryotic expression; subcellular localization

CLC Number:

S852.65^＋4

PANG Feng, LONG Qinqin, LIANG Shaobo, CHENG Hongsong, CHENG Zhentao. Bioinformatics Analysis,Eukaryotic Expression and Subcellular Localization of Orf Virus ORFV114 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4150-4158.

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Bioinformatics Analysis,Eukaryotic Expression and Subcellular Localization of Orf Virus ORFV114 Protein

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