猪A3Z2基因生物信息学分析及其对PEDV复制的抑制作用

doi:10.16431/j.cnki.1671-7236.2023.09.026

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (9): 3695-3706.doi: 10.16431/j.cnki.1671-7236.2023.09.026

猪A3Z2基因生物信息学分析及其对PEDV复制的抑制作用

沈海燕^1,2, 王松祺³, 张斌⁴, 刘志成^1,2, 张建峰^1,2, 廖明^1,2, 张春红^1,2

1. 广东省农业科学院动物卫生研究所, 广东省畜禽疫病防治研究重点实验室, 农业农村部兽用药物与诊断技术广东科学观测实验站, 广州 510640;
2. 岭南现代农业科学与技术广东省实验室茂名分中心, 茂名 525032;
3. 华南农业大学兽医学院, 广州 510642;
4. 内蒙古农业大学兽医学院, 呼和浩特 010010

收稿日期:2023-01-05 出版日期:2023-09-05 发布日期:2023-08-24
通讯作者: 张春红 E-mail:13660450420@139.com
作者简介:沈海燕,E-mail:haiyan_0001@163.com;王松祺,E-mail:Wangsqi9797@163.com。沈海燕和王松祺对本文具有同等贡献,并列为第一作者。
基金资助:
十四五国家重点研发技术项目(2022YFD1800801-02);"十四五"广东省农业科技创新十大主攻方向"揭榜挂帅"项目(2022SDZG02);广东省基础与应用基础研究基金(2020A1515010950、2021A1515011125);茂名实验室自主科研项目(2022KF010);广州市基础研究计划(202002030456);广东省农业科学院"杰出人才"项目(R2020PY-JC001);国家自然科学基金(31302101)

Bioinformatics Analysis of Porcine A3Z2 Gene and Its Antiviral Activity of PEDV Replication

SHEN Haiyan^1,2, WANG Songqi³, ZHANG Bin⁴, LIU Zhicheng^1,2, ZHANG Jianfeng^1,2, LIAO Ming^1,2, ZHANG Chunhong^1,2

1. Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
2. Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525032, China;
3. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
4. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010010, China

Received:2023-01-05 Online:2023-09-05 Published:2023-08-24

摘要/Abstract

摘要： 【目的】对猪源载脂蛋白B mRNA编辑酶催化多肽蛋白3(apolipoprotein B mRNA-editing enzyme catalytic polypeptide protein 3,APOBEC3,简称A3)家族的A3Z2基因进行克隆、生物信息学分析,筛选稳定表达A3Z2的IPEC-J2细胞,研究A3Z2对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)增殖的影响,以期为研究A3Z2的抗病毒功能奠定基础。【方法】以IPEC-J2细胞总RNA为模板,采用RT-PCR法扩增猪A3Z2基因CDS区,并对其进行遗传进化和生物信息学分析。构建pcDNA3.1-3×Flag-A3Z2载体,将其转染IPEC-J2细胞后经G418筛选、有限稀释法和单克隆细胞培养,获得稳定表达A3Z2的细胞株。分别采用间接免疫荧光试验和Western blotting方法鉴定A3Z2的表达,采用实时荧光定量PCR和Western blotting方法分析PEDV N基因的mRNA和蛋白表达水平。【结果】猪A3Z2基因CDS区长843 bp,编码280个氨基酸,分子质量约为32.7 ku,不存在跨膜区及信号肽切割位点,主要存在于细胞核中。A3Z2蛋白二级结构由α-螺旋(27.14%)、延伸链(17.86%)、β-转角(6.43%)和无规则卷曲(48.75%)构成。通过细胞筛选获得稳定表达A3Z2的细胞株IPEC-J2-A3Z2;间接免疫荧光方法和Western blotting结果显示,A3Z2能在IPEC-J2细胞中表达。实时荧光定量PCR和Western blotting结果显示,细胞株IPEC-J2-A3Z2中PEDV N基因的mRNA和蛋白表达均被A3Z2抑制。【结论】本研究成功克隆了猪A3Z2基因,筛选获得了IPEC-J2-A3Z2细胞株,证实了A3Z2抑制PEDV复制的作用,为研究PEDV与A3Z2相互作用的分子机制和以此为靶点筛选抗PEDV新药提供了思路。

关键词: 猪; A3Z2基因; 生物信息学; 猪流行性腹泻病毒(PEDV); IPEC-J2细胞

Abstract: 【Objective】 The aim of this study was to perform cloning and bioinformatics of A3Z2 gene of porcine apolipoprotein B mRNA-editing enzyme catalytic polypeptide protein 3B (APOBEC3,A3) family,screen IPEC-J2 cells that stably expressed A3Z2,and investigate the effects of A3Z2 on the replication of Porcine epidemic diarrhea virus(PEDV),in order to lay the foundation for studying the antiviral function of A3Z2.【Method】 Porcine A3Z2 gene was amplified from the total RNA of IPEC-J2 cells as a template,the CDS region of porcine A3Z2 gene was amplified by RT-PCR,and its genetic evolution and bioinformatics analysis were performed.The pcDNA3.1-3×Flag-A3Z2 vector was constructed and transfected into IPEC-J2 cells screened by G418,the stable A3Z2 expressing cell line was obtained using limited dilution method and monoclone cell culture.The expression of A3Z2 was identified using indirect immunofluorescence experiment and Western blotting,the mRNA and protein levels of PEDV N gene were analyzed using Real-time quantitative PCR and Western blotting,respectively.【Result】 The full-length of A3Z2 gene CDS region was 843 bp,encoded 280 amino acids,the molecular weight of A3Z2 protein was 32.7 ku. There was no transmembrane region and signal peptide catting site,mainly in the nucleus. The secondary structure of A3Z2 protein was mainly composed by 27.14% alpha helix,17.86% extended chain,6.43% beta turn and 48.57% random coil.The A3Z2 stable expression cell line IPEC-J2-A3Z2 was obtained through cell screening.The results of indirect immunofluorescence assay and Western blotting showed that A3Z2 expressed in IPEC-J2-A3Z2 cells.The results of Real-time quantitative PCR and Western blotting found that the mRNA and protein expression of PEDV N gene in IPEC-J2-A3Z2 cells were inhibited by A3Z2.【Conclusion】 Porcine A3Z2 gene was successfully cloned and IPEC-J2-A3Z2 cell line was obtained.Moreover,A3Z2 had the antiviral activity of PEDV replication.The results provided insight for studying the molecular mechanism of PEDV and A3Z2,and as a target to screen new anti-PEDV drugs.

Key words: pig; A3Z2 gene; bioinformatics; Porcine epidemic diarrhea virus (PEDV); IPEC-J2 cells

中图分类号:

S852.65⁺1

沈海燕, 王松祺, 张斌, 刘志成, 张建峰, 廖明, 张春红. 猪A3Z2基因生物信息学分析及其对PEDV复制的抑制作用[J]. 中国畜牧兽医, 2023, 50(9): 3695-3706.

SHEN Haiyan, WANG Songqi, ZHANG Bin, LIU Zhicheng, ZHANG Jianfeng, LIAO Ming, ZHANG Chunhong. Bioinformatics Analysis of Porcine A3Z2 Gene and Its Antiviral Activity of PEDV Replication[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3695-3706.

参考文献

[1] SADEGHPOUR S,KHPDAEE S,M RAHNAMA,et al.Human APOBEC3 variations and viral infection [J].Viruses,2021,13(7):1366.
[2] URIU K,KOSUGI Y,ITO J,et al.The battle between retroviruses and APOBEC3 genes:Its past and present [J].Viruses,2021,13(1):124.
[3] OLSON M E,HARRIS R S,HARKI D A.APOBEC enzymes as targets for virus and cancer therapy [J].Cell Chemical Biology,2018,25(1):36-49.
[4] MIKL M C,WATT I N,LU M,et al.Mice deficient in APOBEC2 and APOBEC3[J].Molecular and Cellular Biology,2005,25(16):7270-7277.
[5] MUNK C,BECK T,ZIELONKA J,et al.Functions,structure,and read-through alternative splicing of feline APOBEC3 genes [J].Genome Biology,2008,9(3):R48.
[6] LARUE R S,JONSSON S R,SILVERSTEIN K A,et al.The artiodactyl APOBEC3 innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals [J].BMC Molecular Biology,2008,9:104.
[7] BOGERD H P,TALLMADGE R L,OAKS J L,et al.Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism [J].Journal of Virology,2008,82(23):11889-11901.
[8] EVA D,FISCHER N,BRAVO I G,et al.Restriction of porcine endogenous retrovirus by porcine APOBEC3 cytidine deaminases [J].Journal of Virology,2011,85(8):3842-3857.
[9] YUAN C,LI Y,ZHANG E,et al.The Mechanism of PEDV-Carrying CD3⁺ T cells migrate into the intestinal mucosa of neonatal piglets [J].Viruses,2021,13(3):469.
[10] 郭思杰,刘俊琦,李玉娟,等.猪流行性腹泻及新型疫苗研究进展[J].中国畜牧兽医,2020,47(9):2935-2944. GUO S J,LIU J Q,LI Y J,et al.Research advances on porcine epidemic diarrhea and new vaccine [J].China Animal Husbandry & Veterinary Medicine,2020,47(9):2935-2944.(in Chinese)
[11] 刘雨桐,姚妍婷,黎芮伶,等.中国西南部分地区2020―2021年猪流行性腹泻病毒遗传变异分析[J].中国畜牧兽医,2022,49(6):2279-2290. LIU Y T,YAO Y T,LI R L,et al.Analysis of genetic variation of Porcine epidemic diarrhea virus in parts of Southwest China from 2020 to 2021[J].China Animal Husbandry & Veterinary Medicine,2022,49(6):2279-2290.(in Chinese)
[12] JUNG K,SAIF L J,WANG Q.Porcine epidemic diarrhea virus (PEDV):An update on etiology,transmission,pathogenesis,and prevention and control [J].Virus Research,2020,286:198045.
[13] TIKHONOV A S,MINTAEV R R,GLAZKOVA D V,et al.HIV restriction factor APOBEC3G and prospects for its use in gene therapy for HIV [J]. Molecular Biology,2022,56(4):546-556.
[14] MILEWSKA A,KINDLER E,VKOVSKI P,et al.APOBEC3-mediated restriction of RNA virus replication [J].Scientific Reports,2018,8(1):5960.
[15] WANG H,ZHONG M,LI Y,et al.APOBEC3G is a restriction factor of EV71 and mediator of IMB-Z antiviral activity [J].Antiviral Research,2019,165:23-33.
[16] KIM K,CALABRESE P,WANG S,et al.The roles of APOBEC-mediated RNA editing in SARS-CoV-2 mutations,replication and fitness [J].Scientific Reports,2022,Preprint.Doi:10.21203/rs.3.rs-1524060/v1.
[17] POULAIN F,LEJEUNE N,WILLEMART K,et al.Footprint of the host restriction factors APOBEC3 on the genome of human viruses [J].PLoS Pathogens,2020,16(8):e1008718.
[18] SHARMA S,PATNAIK S K,TAGGART R T,et al.The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme [J].Scientific Reports,2016,6:39100.
[19] 朱红晓,王秋霞,李鹏,等.猪流行性腹泻病毒逃避宿主干扰素抗病毒作用机制的研究进展[J].中国畜牧兽医,2019,46(7):2096-2102. ZHU H X,WANG Q X,LI P,et al.Research progress on mechanism of Porcine epidemic diarrhea virus evading the antiviral effect of host interferon [J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2096-2102.(in Chinese)
[20] 吴小霞,许天委,李国寅.人类APOBEC3蛋白的结构与功能分析[J].中国免疫学杂志,2020,36(4):477-490. WU X X,XU T W,LI G Y.Structural and functional analysis of human APOBEC3 protein [J].Chinese Journal of Immunology,2020,36(4):477-490.(in Chinese)
[21] 程杰.PRRSV两种毒株GP5蛋白糖基化位点鉴定及其突变体核酸疫苗诱导免疫反应的研究[D].泰安:山东农业大学,2022. CHENG J.Identification of glycosylation sites in GP5 protein of two PRRSV strains and study on immune responses induced by mutants vaccine [D].Tai’an:Shandong Agricultural University,2022.(in Chinese)
[22] 向田,章晓联.病毒与宿主细胞的糖基化修饰及相关功能[J].生物化学与生物物理进展,2017,44(10):898-907. XIANG T,ZHANG X L.Glycosylation modification and related functions of viruses and host cells [J].Progress in Biochemistry and Biophysics,2017,44(10):898-907.(in Chinese)
[23] 胡汪来,吴缅.p53磷酸化修饰及其功能研究进展[J].中国科学:生命科学,2015,45(11):1093-1100. HU W L,WU M.Progress in p53 phosphorylation and function [J]. Scientia Sinica (Vitae),2015,45(11):1093-1100.(in Chinese)
[24] 梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):75-81. LIANG Q J,WANG P C,BAI Y R.Summarization on the progress in protein phosphorylation [J].Science & Technology Review,2012,30(31):75-81.(in Chinese)
[25] SALTER J D,BENNETT R P,SMITH H C.The APOBEC protein family:United by structure,divergent in function [J].Trends in Biochemical Sciences,2016,41(7):578-594.

猪A3Z2基因生物信息学分析及其对PEDV复制的抑制作用

Bioinformatics Analysis of Porcine A3Z2 Gene and Its Antiviral Activity of PEDV Replication

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	朱凯晴, 朱悦, 刘迎迎, 崔亚男, 蒋秉妤, 李妍. ETEC感染猪小肠上皮细胞初期差异表达cricRNA的筛选[J]. 中国畜牧兽医, 2023, 50(9): 3490-3498.
[2]	袁冬冬, 宋国华, 陈孟娟, 王智通, 柏中峰, 蔡含芳, 李明, 许会芬. 新西兰白兔CTSB、CTSS基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(9): 3499-3509.
[3]	字嘉鑫, 连英杰, 陈敏仪, 李秋煦, 张伟泽, 陆梦可, 张阳洋, 区浩炫, 马骏, 白挨泉. 4株广东地区2型猪链球菌耐药性及致病性研究[J]. 中国畜牧兽医, 2023, 50(9): 3740-3751.
[4]	何娅, 王召贺, 陈波, 宋玉平, 周明光, 徐高原. 猪源A型多杀性巴氏杆菌灭活疫苗制备及免疫效果评估[J]. 中国畜牧兽医, 2023, 50(9): 3752-3761.
[5]	史超, 张伟, 刘素平, 张梦琦, 何欣, 赵明彦, 周霞, 王震, 张辉. 单增李斯特菌IspD蛋白生物信息学分析及表达纯化[J]. 中国畜牧兽医, 2023, 50(9): 3799-3810.
[6]	王首元, 贠红梅, 史明月, 秦云梦, 李熊, 陈军舟, 周琛帛, 曹果清. 猪RPL36A基因对PK15细胞增殖过程的影响[J]. 中国畜牧兽医, 2023, 50(8): 3035-3044.
[7]	张婷, 魏立民, 刘光亮, 王峰, 晁哲, 任钰为, 刘海隆, 郑心力, 黄丽丽, 孙瑞萍. 五指山猪和长白猪断奶仔猪肠道免疫性能及微生物多样性比较分析[J]. 中国畜牧兽医, 2023, 50(8): 3113-3122.
[8]	文伟, 胡友军, 程皇座, 何军. 有机酸对猪肠道菌群的调节作用及其抑菌机制研究进展[J]. 中国畜牧兽医, 2023, 50(8): 3133-3141.
[9]	李寒妹, 颜华, 刘欣, 王立贤. 母猪的母性行为及其影响因素研究进展[J]. 中国畜牧兽医, 2023, 50(8): 3171-3179.
[10]	梁小芬, 冉雪琴, 牛熙, 李升, 黄世会, 王嘉福. 香猪CYP3A29基因3'-UTR结构变异及其与肌肉雄激素水平的关联分析[J]. 中国畜牧兽医, 2023, 50(8): 3199-3209.
[11]	马静, 杨丹娇, 韦龙拼, 甘代冰, 龙芷欣, 何宇琪, 卿霆, 范燕迪, 周泷. 1株藏猪源Moraxella pluranimalium的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2023, 50(8): 3275-3285.
[12]	李倩倩, 蔺思函, 王丽媛, 杨兵兵, 张明达, 沈秀丽, 杜志强. 猪种布鲁氏菌TIR结构域蛋白的生物信息学分析、重组表达及蛋白互作研究[J]. 中国畜牧兽医, 2023, 50(8): 3303-3312.
[13]	王林青, 赵丽, 陈曦艋, 吴少峰, 韩莹莹, 刘芳, 金钺, 陈红英. 河南省猪伪狂犬病病毒野毒感染血清学调查及gE基因遗传变异分析[J]. 中国畜牧兽医, 2023, 50(8): 3364-3372.
[14]	倪黎纲, 周春宝, 徐盼, 张亚琴, 陶勇, 张君胜. 苏姜猪不同生长阶段背膘组织差异表达基因及其调控通路分析[J]. 中国畜牧兽医, 2023, 50(7): 2595-2605.
[15]	王慧, 冯保亮, 向光明, 黄雷, 刘志国, 李奎, 牟玉莲. CD163单等位基因表达的iPAMs细胞系的构建及其介导PRRSV感染的特征分析[J]. 中国畜牧兽医, 2023, 50(7): 2617-2628.