鸡DCAF1生物信息学分析及对J亚群禽白血病病毒复制的影响

doi:10.16431/j.cnki.1671-7236.2024.05.005

摘要/Abstract

摘要： 【目的】为分析鸡损伤特异性DNA结合蛋白和泛素连接酶复合物骨架蛋白库林4相关因子1(DDB1 and CUL4 associated factor 1,DCAF1)蛋白的生物学特性,构建DCAF1基因的真核表达载体,并初步探究其对J亚型禽白血病病毒(ALV-J)复制的影响。【方法】在NCBI中下载相关物种DCAF1蛋白氨基酸序列,采用Mega 11.0软件构建系统进化树;利用ProtParam、ProtScale、TMHMM等在线程序分析鸡DCAF1蛋白的氨基酸序列,预测其理化性质、保守结构域及二、三级结构和互作蛋白等生物信息;构建鸡DCAF1基因重组真核表达载体,通过实时荧光定量PCR和Western blotting技术检测ALV-J感染对DCAF1 mRNA和ALV-J Env蛋白表达水平的影响及过表达DCAF1对ALV-J复制的影响。【结果】鸡DCAF1蛋白由1 496个氨基酸组成,其分子质量约为170 ku;系统进化树结果表明,鸡与红鸭亲缘关系最近,与斑马鱼亲缘关系最远。生物信息学预测结果表明,鸡DCAF1蛋白无跨膜结构域,无信号肽表达,包含ARM折叠、LisH和WD40重复3个主要结构域与末尾1个酸性结构域,其互作蛋白为DDB1、CUL4A、SAMHD1等。鸡DCAF1蛋白二级结构主要由无规则卷曲和α-螺旋组成,分别占比44.85%和41.18%,延伸链和β-折叠占比次之,分别是9.76%和4.21%。三级结构与二级结构基本一致,建模一致性为93.79%。ALV-J感染24、48 h后,DCAF1基因和蛋白表达水平均极显著高于感染0 h时(P＜0.01);过表达DCAF1后,与感染对照组相比,ALV-J Env蛋白显著升高(P＜0.05)。【结论】鸡DCAF1蛋白由1 496个氨基酸组成,分子质量约170 ku,属于稳定的亲水蛋白,不含跨膜结构域。本研究成功构建了DCAF1基因真核表达载体。在ALV-J感染的细胞中,DCAF1蛋白和基因表达水平极显著升高,而过表达DCAF1则显著促进ALV-J复制。研究结果为进一步阐明鸡DCAF1的生物学功能及其与ALV-J互作的分子机制奠定了基础。

关键词: 鸡; DCAF1; ALV-J; 真核表达载体; 生物信息学

Abstract: 【Objective】 The purpose of this study was to analyze the protein characteristics of chicken DDB1 and CUL4 associated factor 1(DCAF1) and construct an eukaryotic expression vector,with investigating the effect of DCAF1 on ALV-J replication.【Method】 The amino acid sequence of DCAF1 protein of related species were downloaded from NCBI and a phylogenetic tree was constructed using Mega 11.0 software.Online programs such as ProtParam,ProtScale,and TMHMM were utilized to analyze the amino acid sequence of DCAF1 protein in chicken,predict its biological information such as physicochemical properties,conserved domains,secondary and tertiary structures,and interacting proteins.The recombinant eukaryotic expression vector of DCAF1 gene in chicken was constructed,and the effects of ALV-J infection on the expression of DCAF1 mRNA and ALV-J Env protein,as well as the effects of overexpression of DCAF1 on ALV-J replication were detected using Real-time quantitative PCR and Western blotting techniques.【Result】 The DCAF1 protein of chicken was about 1 496 amino acids,and its molecular weight was about 170 ku. The results of phylogenetic tree showed the chicken was closet related to Oxyura jamaicensis and farthest related to Danio rerio.The bioinformatics prediction results indicated that DCAF1 protein of chicken had no transmembrane domain,no signal peptide expression,and contained three main structural domains:ARM folding,LisH and WD40 duplication,and one acidic domain at the end,and interacted with proteins such as DDB1,CUL4A and SAMHD1.The secondary structure of DCAF1 protein of chicken was mainly consisted of random coil and alpha helix,which accounted for 44.85% and 41.18%;followed respectively by extended chain and beta turn,which accounted for 9.76% and 4.21%.The tertiary structure results were basically consistent with the secondary structure,with a modeling consisitency of 93.79%.After 24 and 48 h of ALV-J infection,the protein and gene expression of DCAF1 were extremely significantly higher than those at 0 h of infection (P＜0.01).After overexpression of DCAF1,compared with the infection control group,the ALV-J Env protein was significantly increased (P＜0.05).【Conclusion】 The DCAF1 protein of chicken consisted of 1 496 amino acids with a molecular weight of about 170 ku.It was a stable hydrophilic protein and did not contain a transmembrane domain.This study successfully constructed eukaryotic expression vector of DCAF1 gene.In ALV-J infected cells,the expression of DCAF1 protein and gene were extremely significantly increased,while overexpression of DCAF1 significantly promoted ALV-J replication.This results laid the foundation for further elucidating the biological function of chicken DCAF1 and its molecular mechanism of interaction with ALV-J.

Key words: chicken; DCAF1; ALV-J; eukaryotic expression vector; bioinformatics

中图分类号:

S831

潘诗雨, 尹娜, 王佳兴, 王强州, 陈世豪, 白皓, 常国斌. 鸡DCAF1生物信息学分析及对J亚群禽白血病病毒复制的影响[J]. 中国畜牧兽医, 2024, 51(5): 1837-1845.

PAN Shiyu, YIN Na, WANG Jiaxing, WANG Qiangzhou, CHEN Shihao, BAI Hao, CHANG Guobin. Bioinformatic Analysis of Chicken DCAF1 and Its Impact on the Replication of ALV-J[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1837-1845.

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