猪繁殖与呼吸综合征病毒非结构蛋白7真核表达及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2023.03.030

摘要/Abstract

摘要： 【目的】对猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)非结构蛋白7(nonstructural protein 7,NSP7)进行真核表达及生物信息学分析,从而推测其在PRRSV复制过程中的功能。【方法】按照GenBank数据库中NSP7基因序列合成目的基因并构建真核表达载体P3×FLAG-CMV-NSP7,瞬时转染Marc-145细胞后通过Western blotting验证蛋白表达,通过间接免疫荧光试验(indirect immunofluorescence assay,IFA)对NSP7蛋白进行亚细胞定位,利用生物信息学分析软件预测NSP7蛋白的理化性质、结构及功能。【结果】成功构建P3×FLAG-CMV-NSP7真核表达载体;Western blotting结果显示,获得大小约为2.7 ku的目的条带,证实重组载体在Marc-145细胞中高效表达。IFA结果证实在PRRSV感染初期NSP7蛋白大部分分布于细胞质中,随着感染时间延长NSP7蛋白由细胞质进入细胞核。生物信息学分析结果显示,NSP7蛋白由259个氨基酸编码,分子式为C₁₂₈₄H₂₀₂₀N₃₄₈O₃₇₉S₈,分子质量为28652.75 u,理论等电点为5.88,为不稳定、亲水蛋白。结构预测发现,NSP7蛋白二级结构包括α-螺旋和β-转角,占比分别为35.91%和5.41%。核定位序列分析发现,NSP7蛋白具有一段跨膜序列RLNKKKRRRMEAVGIF。【结论】本研究成功构建高效表达的PRRSV NSP7真核表达载体,在PRRSV感染初期NSP7蛋白分布于细胞质,感染后期分布于细胞核,NSP7蛋白存在核定位序列。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); 非结构蛋白7(NSP7); 亚细胞定位

Abstract: 【Objective】 The purpose of this experiment was to express nonstructural protein 7 (NSP7) of Porcine reproductive and respiratory syndrome virus (PRRSV) in eukaryotic cells and analyze its bioinformatics,so as to speculate its function in the process of PRRSV replication.【Method】 The target gene was synthesized according to the sequence of NSP7 gene in GenBank database,and the P3×FLAG-CMV-NSP7 eukaryotic expression vector was constructed.The protein expression was verified by Western blotting after transient transfection of Marc-145 cells.The subcellular localization of NSP7 was performed by indirect immunofluorescence assay (IFA),and the physicochemical properties,structure and function of NSP7 were predicted by bioinformatics analysis software.【Result】 The eukaryotic expression vector P3×FLAG-CMV-NSP7 was successfully constructed.Western blotting results showed that the target band with a size of 2.7 ku was obtained,which confirmed that the recombinant vector was highly expressed in Marc-145 cells.IFA results confirmed that NSP7 protein was mostly distributed in the cytoplasm at the initial stage of PRRSV infection,and entered the nucleus from the cytoplasm with the extension of infection time.Bioinformatics analysis results showed NSP7 protein was encoded by 259 amino acids,the molecular formula was C₁₂₈₄H₂₀₂₀N₃₄₈O₃₇₉S₈,the molecular weight was 28 652.75 u,the theoretical isoelectric point was 5.88,and it was an unstable and hydrophilic protein.Structural prediction found that the secondary structure of NSP7 protein included alpha helix and beta turn accounted for 35.91% and 5.41%, respectively.Nuclear localization sequence analysis showed that NSP7 protein had a transmembrane sequence RLNKKKRRRMEAVGIF.【Conclusion】 The eukaryotic expression vector of PRRSV NSP7 was successfully constructed.NSP7 was distributed in cytoplasm at the initial stage of PRRSV infection and in nucleus at the later stage of PRRSV infection.It was found that NSP7 had nuclear localization sequence.

Key words: Porcine reproductive and respiratory syndrome virus (PRRSV); nonstructural protein 7 (NSP7); subcellular localization

中图分类号:

S852.65^＋1

李华玮, 郭科威, 王旭英, 张小雨, 路紫微, 李新锋, 马辉, 侯文静. 猪繁殖与呼吸综合征病毒非结构蛋白7真核表达及亚细胞定位[J]. 中国畜牧兽医, 2023, 50(3): 1160-1168.

LI Huawei, GUO Kewei, WANG Xuying, ZHANG Xiaoyu, LU Ziwei, LI Xinfeng, MA Hui, HOU Wenjing. Eukaryotic Expression and Subcellular Localization of Nonstructural Protein 7 of Porcine Reproductive and Respiratory Syndrome Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1160-1168.

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