过表达非洲猪瘟病毒D1133L蛋白的MA-104细胞系建立及其初步应用

doi:10.11843/j.issn.0366-6964.2022.06.021

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (6): 1877-1885.doi: 10.11843/j.issn.0366-6964.2022.06.021

过表达非洲猪瘟病毒D1133L蛋白的MA-104细胞系建立及其初步应用

张婷, 杨博, 崔卉梅, 袁兴国, 赵登率, 杨金柯, 郝雨, 陈学辉, 闫文倩, 申超超, 史喜绢, 张大俊, 杨行, 刘湘涛, 郑海学*, 张克山*

中国农业科学院兰州兽医研究所，兰州大学动物医学与生物安全学院家畜疫病病原生物学国家重点实验室，兰州 730000

收稿日期:2021-08-26 出版日期:2022-06-23 发布日期:2022-06-25
通讯作者: 郑海学，主要从事动物传染病与流行病学研究，E-mail: zhenghaixue@caas.cn;张克山，主要从事兽医微生物及其分子生物学研究，E-mail: zks009@126.com
作者简介:张婷(1996-)，女，甘肃武威人，硕士生，主要从事兽医微生物及其分子生物学，E-mail 1835631389@qq.com
基金资助:
国家重点研发计划(2021YF1D1801300)；甘肃省重大专项(20ZD7NA006；21ZD3NA001)；中国农业科学院基本科研业务费(Y2020XK09)；国家自然科学基金联合项目(31941002)

Establishment and Preliminary Application of MA-104 Cell Line Overexpressing African Swine Fever Virus D1133L Protein

State Key Laboratory of Veterinary Etiological Biology, College of Veterinary Medicine, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China

Received:2021-08-26 Online:2022-06-23 Published:2022-06-25

摘要/Abstract

摘要： 旨在研究非洲猪瘟病毒(African swine fever, ASFV)编码的D1133L基因功能，本文利用慢病毒表达系统构建了过表达D1133L的MA-104/D1133L细胞系，分析ASFV在MA-104/D1133L细胞系与MA-104细胞中的复制差异。利用RT-qPCR和Western blot技术分别检测了ASFV感染MA-104/D1133L细胞后p30和p72基因的转录和蛋白表达水平，同时测定病毒滴度和病毒拷贝数评价ASFV的复制能力。结果表明：ASFV p30和p72基因在MA-104/D1133L细胞系的转录和蛋白表达水平高于野生型细胞系，ASFV在MA-104/D1133L细胞系的病毒增殖能力高于野生型细胞。本研究成功构建了过表达ASFV D1133L基因的MA-104/D1133L细胞系，与野生型细胞相比，D1133L能促进ASFV增殖，MA-104/D1133L细胞系更利于ASFV的复制，本结果为进一步研究ASFV D1133L基因功能积累了生物材料。

关键词: 非洲猪瘟病毒, D1133L基因, MA-104细胞, 基因功能, 过表达细胞系

Abstract: To study the function of D1133L gene encoded by African swine fever virus (ASFV), we constructed MA-104/D1133L cell line for overexpression of D1133L using lentivirus expression vector, and compared ASFV replication ability in MA-104/D1133L and MA-104 cells. The transcription and protein expression levels of p30 and p72 in ASFV infected MA-104/D1133L cells were evaluated by real-time qPCR and Western blot respectively, and the proliferation of ASFV was evaluated according to virus titer and virus copies number. The transcriptional and protein levels of ASFV p30 and p72 proteins in MA-104/D1133L cell line were higher than those in wild MA-104 cell, and the viral proliferation capacity of ASFV in MA-104/D1133L cell line was higher than that in wild MA-104 cell line. The results showed that overexpressing ASFV D1133L gene of MA-104 cell line was successfully constructed in this study, that compared with wild type cells, MA-104/D1133L cell line was more conducive to ASFV replication, and D1133L promoted ASFV proliferation. These results provide biomaterials for further study of the function of ASFV D1133L gene.

Key words: African swine fever virus, D1133L genes, MA-104 cells, gene function, overexpressed cell lines

中图分类号:

S852.659.6

张婷, 杨博, 崔卉梅, 袁兴国, 赵登率, 杨金柯, 郝雨, 陈学辉, 闫文倩, 申超超, 史喜绢, 张大俊, 杨行, 刘湘涛, 郑海学, 张克山. 过表达非洲猪瘟病毒D1133L蛋白的MA-104细胞系建立及其初步应用[J]. 畜牧兽医学报, 2022, 53(6): 1877-1885.

ZHANG Ting, YANG Bo, CUI Huimei, YUAN Xingguo, ZHAO Dengshuai, YANG Jinke, HAO Yu, CHEN Xuehui, YAN Wenqian, SHEN Chaochao, SHI Xijuan, ZHANG Dajun, YANG Xing, LIU Xiangtao, ZHENG Haixue, ZHANG Keshan. Establishment and Preliminary Application of MA-104 Cell Line Overexpressing African Swine Fever Virus D1133L Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1877-1885.

参考文献 31

[1]	TULMAN E R, DELHON G A, KU B K, et al. African swine fever virus[M]//VAN ETTEN J L. Lesser Known Large dsDNA Viruses. Berlin: Springer, 2009: 43-87.
[2]	TEKLUE T, SUN Y, ABID M, et al. Current status and evolving approaches to African swine fever vaccine development[J]. Transbound Emerg Dis, 2020, 67(2): 529-542.
[3]	WU K K, LIU J M, WANG L X, et al. Current State of Global African swine fever vaccine development under the prevalence and transmission of ASF in China[J]. Vaccines, 2020, 8(3): 531.
[4]	SÁNCHEZ E G, RIERA E, NOGAL M, et al. Phenotyping and susceptibility of established porcine cells lines to African Swine Fever Virus infection and viral production[J]. Sci Rep, 2017, 7(1): 10369.
[5]	GÓMEZ-VILLAMANDOS J C, BAUTISTA M J, SÁNCHEZ-CORDÓN P J, et al. Pathology of African swine fever: the role of monocyte-macrophage[J]. Virus Res, 2013, 173(1): 140-149.
[6]	RAI A, PRUITT S, RAMIREZ-MEDINA E, et al. Identification of a continuously stable and commercially available cell line for the identification of infectious African swine fever virus in clinical samples[J]. Viruses, 2020, 12(8): 820.
[7]	IYER L M, ARAVIND L, KOONIN E V. Common origin of four diverse families of large eukaryotic DNA viruses[J]. J Virol, 2001, 75(23): 11720-11734.
[8]	DIXON L K, ISLAM M, NASH R, et al. African swine fever virus evasion of host defences[J]. Virus Res, 2019, 266: 25-33.
[9]	YÁÑEZ R J, RODRÍGUEZ J M, BOURSNELL M, et al. Two putative African swine fever virus helicases similar to yeast 'DEAH' pre-mRNA processing proteins and vaccinia virus ATPases D11L and D6R[J]. Gene, 1993, 134(2): 161-174.
[10]	CACKETT G, SYKORA M, WERNER F. Transcriptome view of a killer: African swine fever virus[J]. Biochem Soc Trans, 2020, 48(4): 1569-1581.
[11]	ALEJO A, MATAMOROS T, GUERRA M, et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92(23): e01293-18.
[12]	DIXON L K, CHAPMAN D A G, NETHERTON C L, et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173(1): 3-14.
[13]	JIA X X, LIU Y, MENG D M, et al. Screening and identification of Vero cell line to stably express capsid protein of peste des petits ruminants virus[J/OL]. Chinese Journal of Animal Infectious Diseases. (2020-02-12). S.20200211.1624.044.html." target="_blank">http://kns.cnki.net/kcms/detail/31.2031.S.20200211.1624.044.html. (in Chinese)贾雪霞, 刘莹, 孟德梅, 等. 稳定表达小反刍兽疫病毒衣壳蛋白的Vero细胞系的筛选和鉴定[J/OL]. 中国动物传染病学报. (2020-02-12). S.20200211.1624.044.html" target="_blank">http://kns.cnki.net/kcms/detail/31.2031.S.20200211.1624.044.html.
[14]	LI Z H, SONG Z A, YANG Z X, et al. Construction of BHK-21 cell line stably expressing VP6 protein of Bluetongue virus[J]. Journal of Agricultural Biotechnology, 2021, 29(4): 780-788. (in Chinese)李占鸿, 宋子昂, 杨振兴, 等. 稳定表达蓝舌病病毒VP6蛋白的BHK-21细胞系构建[J]. 农业生物技术学报, 2021, 29(4): 780-788.
[15]	WUDONG G W, WEN Y S, GUO H, et al. Construction of a Marc-145 cell line stably expressing PRRSV GP2 protein[J/OL]. Chinese Journal of Animal Infectious Diseases. (2021-04-27). S.20210427.1447.055.html" target="_blank">http://kns.cnki.net/kcms/detail/31.2031.S.20210427.1447.055.html. (in Chinese)乌东高娃, 温永胜, 郭昊, 等. 稳定表达PRRSV GP2蛋白的Marc-145细胞系的构建[J/OL]. 中国动物传染病学报. (2021-04-27). S.20210427.1447.055.html" target="_blank">http://kns.cnki.net/kcms/detail/31.2031.S.20210427.1447.055.html.
[16]	REN T W, QIN N, QUAN D Q, et al. Construction of a Marc-145 cell line stably expressing PRRSV N protein[J]. Chinese Journal of Veterinary Parasitology, 2022, 30(1): 15-19. (in Chinese)任同伟, 覃念, 全东群, 等. 稳定表达PRRSV N蛋白的Marc-145细胞系的构建[J]. 中国动物传染病学报, 2022, 30(1): 15-19.
[17]	JIA Y H, LIU W, XU Z K, et al. Construction of BHK-21 cell line stably expressing TIGAR gene and evaluation of its proliferation effect on Newcastle disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 440-449. (in Chinese)栗永华, 刘伟, 徐智凯, 等. 稳定表达TIGAR基因的BHK-21细胞系的构建及其对新城疫病毒增殖效果的评价[J]. 畜牧兽医学报, 2021, 52(2): 440-449.
[18]	HOU J, SHEN C C, ZHANG D J, et al. Gene sequence analysis, protein structure prediction and subcellular localization of African swine fever virus helicase D1133L[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1953-1962. (in Chinese)侯景, 申超超, 张大俊, 等. 非洲猪瘟病毒解旋酶D1133L基因序列分析、蛋白结构预测及亚细胞定位[J]. 畜牧兽医学报, 2021, 52(7): 1953-1962.
[19]	BORCA M V, RAMIREZ-MEDINA E, SILVA E, et al. Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic Eurasia strain[J]. J Virol, 2020, 94(7): e02017-19.
[20]	YANG B, ZHANG D J, SHI X J, et al. Construction, identification and analysis of the interaction network of African swine fever virus MGF360-9L with host proteins[J]. Viruses, 2021, 13(9): 1804.
[21]	KING D P, REID S M, HUTCHINGS G H, et al. Development of a TaqMan® PCR assay with internal amplification control for the detection of African swine fever virus[J]. J Virol Methods, 2003, 107(1): 53-61.
[22]	TEKLUE T, WANG T, LUO Y Z, et al. Generation and evaluation of an African swine fever virus mutant with deletion of the CD2v and UK genes[J]. Vaccines, 2020, 8(4): 763.
[23]	PORTUGAL R, GOATLEY L C, HUSMANN R, et al. A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus[J]. Emerg Microbes Infect, 2020, 9(1): 1245-1253.
[24]	RAI A, PRUITT S, RAMIREZ-MEDINA E, et al. Detection and quantification of African swine fever virus in MA-104 cells[J]. Bio-protocol, 2021, 11(6): e3955.
[25]	WANG T, WANG L, HAN Y, et al. Adaptation of African swine fever virus to HEK293T cells[J]. Transbound Emerg Dis, 2021, 68(5): 2853-2866.
[26]	RODRIGUEZ J M, SALAS M L, SANTARÉN J F. African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis[J]. Proteomics, 2001, 1(11): 1447-1456.
[27]	ZHAO D M, LIU R Q, ZHANG X F, et al. Replication and virulence in pigs of the first African swine fever virus isolated in China[J]. Emerg Microbes Infect, 2019, 8(1): 438-447.
[28]	TABARÉS E, OLIVARES I, SANTURDE G, et al. African swine fever virus DNA: deletions and additions during adaptation to growth in monkey kidney cells[J]. Arch Virol, 1987, 97(3-4): 333-346.
[29]	MONTEAGUDO P L, LACASTA A, LÓPEZ E, et al. BA71ΔCD2: a new recombinant live attenuated African swine fever virus with cross-protective capabilities[J]. J Virol, 2017, 91(21): e01058-17.
[30]	KRUG P W, HOLINKA L G, O'DONNELL V, et al. The progressive adaptation of a Georgian isolate of African swine fever virus to Vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome[J]. J Virol, 2015, 89(4): 2324-2332.
[31]	BORCA M V, RAI A, RAMIREZ-MEDINA E, et al. A cell culture-adapted vaccine virus against the current African swine fever virus pandemic strain[J]. J Virol, 2021, 95(14): e0012321.

过表达非洲猪瘟病毒D1133L蛋白的MA-104细胞系建立及其初步应用

Establishment and Preliminary Application of MA-104 Cell Line Overexpressing African Swine Fever Virus D1133L Protein

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 31

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘传霞, 王晓, 李雪雯, 鲍苗菲, 李婷婷, 陈欣, 翁长江, 郑君. 非洲猪瘟病毒pE120R蛋白单克隆抗体的制备[J]. 畜牧兽医学报, 2024, 55(1): 388-394.
[2]	王慧, 冯保亮, 吴丹, 向光明, 王楠, 牟玉莲, 李奎, 刘志国. CD163基因在猪繁殖与呼吸综合征抗病育种中的研究进展[J]. 畜牧兽医学报, 2023, 54(8): 3127-3138.
[3]	冯永智, 龚婷, 吴东东, 高琦, 郑晓宇, 张桂红, 孙彦阔. 影响非洲猪瘟病毒对培养细胞感染性的因素分析[J]. 畜牧兽医学报, 2023, 54(8): 3406-3414.
[4]	刘桃雪, 苏冰倩, 齐艳丽, 郭江涛, 刘忠虎, 褚贝贝, 王江, 曾磊. 非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3415-3423.
[5]	丁晓艳, 何久香, 周晓杨, 周伃欣, 李晋涛. 非洲猪瘟病毒感染相关调控基因以及毒力基因初步筛选[J]. 畜牧兽医学报, 2023, 54(7): 2964-2971.
[6]	王映, 朱家宏, 赵加凯, 纪品品, 陈旭, 张路, 刘宝元, 孙亚妮, 赵钦. 抗非洲猪瘟病毒NP419L蛋白纳米抗体的筛选鉴定及其在抗体检测中的初步应用[J]. 畜牧兽医学报, 2023, 54(6): 2509-2520.
[7]	刘文豪, 朱彦策, 张冬萱, 王智豪, 张超. 稳定表达非洲猪瘟病毒E165R蛋白PK 15细胞系的构建[J]. 畜牧兽医学报, 2023, 54(6): 2662-2666.
[8]	王国超, 赵亚茹, 张忠辉, 张玉龙, 白鸽, 耿抒贤, 樊洁, 杨吉飞, 关贵全, 殷宏, 罗建勋, 牛庆丽. 非洲猪瘟病毒RNA聚合酶亚基D205R基因生物信息学分析及多克隆抗体制备[J]. 畜牧兽医学报, 2023, 54(5): 2042-2049.
[9]	张婷, 冯涛, 杨金柯, 郝雨, 杨行, 张大俊, 史喜绢, 闫文倩, 陈玲玲, 刘湘涛, 郑海学, 张克山. 条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性[J]. 畜牧兽医学报, 2023, 54(2): 706-714.
[10]	张芳源, 杨大为, 仇德洋, 姜国骞, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白的表达及抗体液相芯片检测方法的建立[J]. 畜牧兽医学报, 2023, 54(10): 4300-4310.
[11]	齐艳丽, 刘桃雪, 于海深, 张超, 鲁维飞, 王江, 褚贝贝, 张改平. 非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(1): 281-292.
[12]	王洋, 崔帅, 鑫婷, 王西西, 于海男, 陈世钰, 蒋亚君, 高新桃, 庞忠宝, 姜一曈, 郭晓宇, 贾红, 朱鸿飞. 非洲猪瘟病毒MGF360-14L靶向MAVS抑制Ⅰ型干扰素的产生[J]. 畜牧兽医学报, 2022, 53(9): 3272-3278.
[13]	许李锋, 贾晨宇, 陈福再, 方梦园, 刘孝丹, 陈吉龙, 李训良. 1株鸡滑液囊支原体的分离鉴定与全基因组分析[J]. 畜牧兽医学报, 2022, 53(8): 2663-2676.
[14]	赵旭阳, 靳家鑫, 路闻龙, 张帅, 黄丽, 张改平, 孙爱军, 庄国庆. 非洲猪瘟病毒免疫逃逸分子机制研究进展[J]. 畜牧兽医学报, 2022, 53(7): 2074-2082.
[15]	陈孝军, 马俊, 陈熊男, 梁祎凡, 高琦, 黄钊, 许润达, 郑佳琛, 郑泽中, 张桂红, 王衡, 龚浪. 1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2239-2251.