鸡TRAF6和TIFA蛋白的序列分析与相互作用验证

doi:10.11843/j.issn.0366-6964.2021.06.007

摘要/Abstract

摘要： 旨在对鸡肿瘤坏死因子受体相关因子6（tumor necrosis factor（TNF）receptor-associated factor 6，TRAF6）和肿瘤坏死因子受体相关因子相互作用的具有叉形头相关结构域蛋白（TNF receptor associated factor（TRAF）-interacting protein with a forkhead-associated（FHA）domain，TIFA）进行序列分析，并进行蛋白质相互作用验证。本研究首先以鸡胚成纤维细胞提取的总RNA反转录产物为模板扩增鸡TRAF6和TIFA基因的CDS区，分别构建重组真核表达载体pCMV-HA-TRAF6和pEGFP-C1-TIFA；运用生物信息学软件ProtParam、SOPMA、SWISS-MODEL、MegAlign和PSORTⅡ分别对鸡TRAF6和TIFA蛋白理化性质、二级和三级结构、氨基酸同源性、功能结构域保守性和亚细胞定位进行分析。将重组真核表达载体共转染细胞，通过荧光共定位和免疫共沉淀（Co-IP）试验验证鸡TRAF6和TIFA蛋白之间的相互作用。结果，成功扩增鸡TRAF6和TIFA基因，并构建其重组真核表达载体pCMV-HA-TRAF6和pEGFP-C1-TIFA。序列分析结果显示，鸡TRAF6和TIFA基因CDS区分别为1 638和564 bp，共编码545和187个氨基酸，分子量约为62和22 ku。蛋白质二级和三级结构分析结果表明，鸡TRAF6和TIFA蛋白均以无规则卷曲和α螺旋为主。氨基酸同源性分析结果发现，鸡与人和其他哺乳动物TRAF6和TIFA蛋白的同源性分别为76.4%~80.3%和49.7%~53.3%，而与非洲爪蟾的同源性分别为69.4%和49.7%，并且鸡TRAF6和TIFA蛋白的功能结构域存在多个氨基酸位点变异。亚细胞定位预测结果表明，鸡TRAF6和TIFA蛋白主要定位在细胞核。荧光共定位和Co-IP试验结果显示，鸡TRAF6和TIFA蛋白在细胞核中具有共定位，并且存在相互作用。本研究结果显示，鸡与人和其他哺乳动物以及非洲爪蟾TRAF6和TIFA蛋白的同源性及功能结构域保守性均较低，但鸡TRAF6和TIFA蛋白能在细胞核中发生相互作用，这可为进一步研究鸡TRAF6和TIFA蛋白相互作用调控鸡相关病毒复制的作用机制奠定基础。

关键词: 鸡, TRAF6, TIFA, 序列分析, 蛋白质相互作用

Abstract: The purpose of this study was to analyze the sequence of chicken tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF receptor associated factor(TRAF)-interacting proteins with a forkhead-associated(FHA) domain (TIFA), and verify their interaction. In this study, the reverse transcription products of total RNA extracted from chicken embryo fibroblasts were used as templates to amplify the CDS region of chicken TRAF6 and TIFA genes. The obtained fragments were then inserted into the plasmids pCMV-HA and pEGFP-C1 to construct the recombinant eukaryotic expression vectors pCMV-HA-TRAF6 and pEGFP-C1-TIFA, respectively. The bioinformatics software ProtParam, SOPMA, SWISS-MODEL, MegAlign and PSORTⅡ were employed to analyze the physicochemical properties, secondary and tertiary structures, amino acid homology, functional domain conservation and subcellular localization of chicken TRAF6 and TIFA proteins. The fluorescence co-localization and co-immunoprecipitation (Co-IP) assays were performed to verify the interaction between chicken TRAF6 and TIFA. The chicken TRAF6 and TIFA genes were amplified, and the recombinant plasmids pCMV-HA-TRAF6 and pEGFP-C1-TIFA were successfully constructed. Sequence analysis results showed that the CDS regions of chicken TRAF6 and TIFA genes were 1 638 and 564 bp in length, respectively, which encoded 545 and 187 amino acids with molecular weights of about 62 and 22 ku. The results of secondary and tertiary structure analysis revealed that both chicken TRAF6 and TIFA proteins were mainly composed of irregular coil and alpha helix. The amino acid homology analysis showed that the homologies of TRAF6 and TIFA proteins between chicken and human, other mammals were 76.4%-80.3% and 49.7%-53.3%, respectively, while those with Xenopus laevis were 69.4% and 49.7%, respectively. In addition, multiple amino acid site variations existed in the functional structural domains of chicken TRAF6 and TIFA proteins. Subcellular localization analysis indicated that both chicken TRAF6 and TIFA proteins were localized in the nucleus. Further fluorescence co-localization and Co-IP assays demonstrated that chicken TRAF6 protein interacted with TIFA protein in the nucleus. This study confirmed that although the low homology and conservation were presented in the TRAF6 and TIFA proteins of chicken, human, other mammals and Xenopus laevis, chicken TRAF6 protein could interact with TIFA protein in the nucleus, which lay a foundation for further studying the role of chicken TRAF6 and TIFA proteins interaction in regulating the replication of chicken related viruses.

Key words: chicken, TRAF6, TIFA, sequence analysis, protein-protein interaction

中图分类号:

S831.2

赵采芹, 王燕碧, 朱杰, 唐宏, 董运韬, 段志强. 鸡TRAF6和TIFA蛋白的序列分析与相互作用验证[J]. 畜牧兽医学报, 2021, 52(6): 1511-1522.

ZHAO Caiqin, WANG Yanbi, ZHU Jie, TANG Hong, DONG Yuntao, DUAN Zhiqiang. Sequence Analysis and Interaction Verification of Chicken TRAF6 and TIFA Proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1511-1522.

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