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

摘要/Abstract

摘要： 【目的】对羊口疮病毒（Orf virus，ORFV） ORFV114蛋白进行生物信息学分析、转录动力学、真核表达及亚细胞定位研究。【方法】使用DNAStar软件对ORFV-JS株ORFV114基因进行序列比对分析；分别使用在线网站ExPASy、TMHMM-2.0、SignalP-5.0、SOPMA、SWISS-MODEL对ORFV114蛋白进行理化性质分析，以及跨膜区、信号肽、结构预测；在阿糖胞苷（cytarabine，Arac）存在或不存在的情况下，于ORFV感染HeLa细胞后多个时间点收获细胞，RT-PCR扩增ORFV114基因，确定ORFV114蛋白的动态转录水平；PCR扩增ORFV114基因，将其亚克隆到真核表达载体pEGFP-N1中，构建pEGFP-ORFV114重组质粒，经酶切、测序鉴定正确后，经脂质体Lipofectamine 3000瞬时转染HEK293细胞，通过Western blotting鉴定ORFV114-EGFP融合蛋白的表达；将pEGFP-N1质粒和pEGFP-ORFV114重组质粒瞬时转染HeLa细胞，24 h后使用Hoechst 33342对细胞核染色，倒置荧光显微镜下观察ORFV114蛋白的亚细胞定位。【结果】 ORFV-JS株ORFV114基因大小为1 041 bp，在ORFV毒株中高度保守；ORFV114蛋白包含346个氨基酸，预测分子质量大小为38 ku；ORFV114为弱亲水性不稳定蛋白，无跨膜区域，无信号肽；其二级结构由无规则卷曲、α-螺旋、延伸链和β-转角组成，分别占45.09%、25.14%、21.68%和8.09%；在Arac存在或不存在的情况下，ORFV114的转录在ORFV感染后2 h即可被检测到，且随感染时间的延长转录水平不断提高，即Arac并未抑制ORFV114转录，ORFV114为ORFV早期基因；成功构建了pEGFP-ORFV114重组质粒，ORFV114-EGFP融合蛋白在HEK293细胞中成功表达，融合蛋白大小约65 ku；ORFV114蛋白主要定位在HeLa细胞的细胞质中且呈点状分布。【结论】本研究成功地对ORFV114蛋白进行了转录动力学分析、真核表达及亚细胞定位，为进一步探索ORFV114蛋白功能及筛选互作蛋白奠定基础。

关键词: 羊口疮病毒(ORFV); ORFV114蛋白; 生物信息学分析; 转录动力学; 真核表达; 亚细胞定位

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

中图分类号:

S852.65^＋4

庞峰, 龙琴琴, 梁绍波, 成虹松, 程振涛. 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[J]. 中国畜牧兽医, 2022, 49(11): 4150-4158.

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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