鸡TRAF6和TIFA蛋白的序列分析与相互作用验证  

Sequence Analysis and Interaction Verification of Chicken TRAF6 and TIFA Proteins

作　　者：赵采芹 王燕碧 朱杰 唐宏 董运韬 段志强[1,2] ZHAO Caiqin;WANG Yanbi;ZHU Jie;TANG Hong;DONG Yuntao;DUAN Zhiqiang(Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction of Ministry of Education,Guizhou University,Guiyang 550025,China;Guizhou Province Key Laboratory of Animal Genetics,Breeding and Reproduction,Guizhou University,Guiyang 550025,China;Shandong Binzhou Wohua Biological Engineering Co.LTD.,Binzhou 256600,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [2]贵州大学贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [3]山东滨州沃华生物工程有限公司,滨州256600

出　　处：《畜牧兽医学报》2021年第6期1511-1522,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31960698,31760732);贵州省科学技术基金(黔科合基础[2020]1Y134号);贵州省地方家禽产业联合攻关项目(黔财农[2020]175号);贵州大学大学生“SRT计划”项目(贵大SRT字[2019]052号)。

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 TRAF 6和 TIFA 基因的CDS区,分别构建重组真核表达载体pCMV-HA- TRAF 6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 TRAF 6和 TIFA 基因,并构建其重组真核表达载体pCMV-HA- TRAF 6和pEGFP-C1- TIFA 。序列分析结果显示,鸡 TRAF 6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 TRAF 6 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- TRAF 6 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 TRAF 6 and TIFA genes were amplified, and the recombinant plasmids pCMV-HA- TRAF 6 and pEGFP-C1- TIFA were successfully constructed. Sequence analysis results showed that the CDS regions of chicken TRAF 6 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

关 键 词：鸡 TRAF6 TIFA 序列分析 蛋白质相互作用 

分 类 号：S831.2[农业科学—畜牧学]