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

doi:10.11843/j.issn.0366-6964.2023.05.025

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (5): 2042-2049.doi: 10.11843/j.issn.0366-6964.2023.05.025

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

王国超^1,2, 赵亚茹¹, 张忠辉¹, 张玉龙¹, 白鸽², 耿抒贤¹, 樊洁¹, 杨吉飞¹, 关贵全¹, 殷宏^1,3, 罗建勋^1*, 牛庆丽^1*

1. 中国农业科学院兰州兽医研究所, 国家非洲猪瘟区域实验室 (兰州), 家畜疫病病原生物学国家重点实验室, 兰州 730046;
2. 渭南市动物疫病预防控制中心, 渭南 715400;
3. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2022-08-01 出版日期:2023-05-23 发布日期:2023-05-20
通讯作者: 牛庆丽,主要从事预防兽医学研究,E-mail:niuqingli@caas.cn;罗建勋,主要从事预防兽医学研究,E-mail:luojianxun@caas.cn
作者简介:王国超(1985-),男,陕西韩城人,兽医师,主要从事动物疫病防控工作,E-mail:646929054@qq.com
基金资助:
国家自然科学基金面上资助项目(32072830);国家重点研发计划(2021YFD1800101);甘肃省科技重大专项(20ZD7NA006;22ZD6NA001);农业科技创新工程 (ASTIP,CAAS-ASTIP-2016-LVRI);甘肃省基础研究创新群体项目(22JR5RA024);甘肃省自然科学基金(21JR7RA018);渭南市高层次人才"特支计划"项目(20210086)

Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation

WANG Guochao^1,2, ZHAO Yaru¹, ZHANG Zhonghui¹, ZHANG Yulong¹, BAI Ge², GENG Shuxian¹, FAN Jie¹, YANG Jifei¹, GUAN Guiquan¹, YIN Hong^1,3, LUO Jianxun^1*, NIU Qingli^1*

1. African Swine Fever Regional Laboratory of China (Lanzhou)/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Weinan Animal Disease Prevent and Control Center, Weinan 715400, China;
3. Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis, Yangzhou University, Yangzhou 225009, China

Received:2022-08-01 Online:2023-05-23 Published:2023-05-20

摘要/Abstract

摘要： 非洲猪瘟病毒(African swine fever virus, ASFV)pD205R蛋白参与病毒基因的转录,属于ASFV RNA聚合酶,且与真核生物RNA聚合酶II RPB5亚基相似,为深入了解pD205R结构,功能及病毒与宿主之间的作用机制。利用DNAStar、MEGA7.0对GenBank中公布的不同毒株的ASFVD205R基因序列进行分析,利用ExPASy、GOR4、AlphaFold软件对该蛋白的理化性质及结构预测进行分析,并对其原核表达和制备抗pD205R蛋白多克隆抗体。结果显示:在不同基因型间D205R基因高度保守;利用ExPASy在线软件预测pD205R蛋白理化性质其分子式为C₁₀₈₈H₁₇₁₂N₂₇₈O₂₉₅S₈,属亲水性蛋白,稳定性差;二级结构及三级结构预测显示,α螺旋是其主要结构形式,占比为35.12%;同时以ASFV-CN/SC/2019毒株基因组为模板,构建原核表达质粒pET-28a-D205R并转入Rosetta (DE3)感受态细胞,经IPTG诱导重组蛋白成功表达,大小为23.3 ku。获得的蛋白经镍柱亲和层析纯化,Western blot鉴定可与ASFV阳性血清发生特异性反应。免疫新西兰大白兔制备的多克隆抗体效价达到1∶51 200,Western blot和IFA结果显示制备的多克隆抗体够特异性的识别ASFV感染的猪肺泡巨噬细胞中表达的天然pD205R蛋白,为进一步研究D205R基因的功能及其在宿主与病毒互作中的作用奠定了基础。

关键词: 非洲猪瘟病毒, pD205R蛋白, 原核表达, 多克隆抗体

Abstract: African swine fever virus (ASFV) pD205R protein is involved in virus gene transcription, which belongs to ASFV RNA polymerase, similar with RPB5 of eukaryotic RNA polymerase II. In this study, in order to further understand the structure, function of pD205R and interaction mechanism between virus and host, the sequence of D205R gene from different ASFV strains deposed in GenBank were analyzed by using DNAStar and Mega7.0 software. Physicochemical Properties and Structure of Protein were predicted by using ExPASy, GOR4 and AlphaFold software, and its prokaryotic expression and preparation of anti-pD205R protein polyclonal antibody were performed. Furthermore, the D205R gene was amplified and cloned into pET-28a vector, named as pET-28a-D205R. The results showed that the D205R gene was highly conserved among different genotypes of ASFV isolates. The physicochemical properties analysis showed that pD205R protein was hydrophilic with poor stability, its molecular formula was predicted as C₁₀₈₈H₁₇₁₂N₂₇₈O₂₉₅S₈. The secondary structure and tertiary structure indicated that α helix was the main structural form, proportion for 35.12%. Furthermore, the pET-28a-D205R recombinant plasmids were constructed successfully using ASFV CN/SC/2019 strain genome as template and transformed into E. coli Rosetta (DE3) competent cells, the recombinant proteins were successfully induced and expressed by IPTG, with the size of 23.3 ku. Then the obtained protein was purified by nickel column affinity chromatography, which could react specifically with ASFV positive serum by Western blot identification. The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits could reach to 1:51 200, and could specially recognize the native pD205R protein expressed in ASFV-infected porcine alveolar macrophages (PAMs) by Western blot and IFA detection. It laid a foundation for further study the function of D205R gene and its role in host-pathogen interaction.

Key words: African swine fever virus, pD205R protein, prokaryotic expression, polyclonal antibodies

中图分类号:

S852.659.1

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.

WANG Guochao, ZHAO Yaru, ZHANG Zhonghui, ZHANG Yulong, BAI Ge, GENG Shuxian, FAN Jie, YANG Jifei, GUAN Guiquan, YIN Hong, LUO Jianxun, NIU Qingli. Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2042-2049.

参考文献 21

[1]	ARABYAN E, KOTSYNYAN A, HAKOBYAN A, et al. Antiviral agents against African swine fever virus[J]. Virus Res, 2019, 270:197669.
[2]	周彦铸, 朱羽婕, 张彤彤, 等. 抗非洲猪瘟病毒药物靶点及抗病毒活性物质研究进展[J]. 病毒学报, 2022, 38(5):1225-1236.ZHOU Y Z, ZHU Y J, ZHANG T T, et al. Progress in research on potential drug targets and antiviral constituents against African swine fever virus infection[J]. Chinese Journal of Virology, 2022, 38(5):1225-1236. (in Chinese)中华人民共和国农业农村部. 中华人民共和国农业农村部公告第573号[EB/OL]. (2022-08-30)[2023-03-21]. http://www.moa.gov.cn/nybgb/2022/202208/202208/t20220830_6408131.htm. Ministry of Agriculture and Rural Affairs, PRC. Announcement No.573 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China[EB/OL]. (2022-08-30)[2023-03-21]. http://www.moa.gov.cn/nybgb/2022/202208/202208/t20220830_6408131.htm. (in Chinese)
[3]	欧云文, 马小元, 王俊, 等. 非洲猪瘟分子病原学及分子流行病学研究进展[J]. 中国兽医学报, 2018, 38(2):416-420.OU Y W, MA X Y, WANG J, et al. Advances in studies of molecular etiology and molecular epidemiology for African swine fever[J]. Chinese Journal of Veterinary Science, 2018, 38(2):416-420. (in Chinese)
[4]	王清华, 任炜杰, 包静月, 等. 我国首例非洲猪瘟的确诊[J]. 中国动物检疫, 2018, 35(9):1-4.WANG Q H, REN W J, BAO J Y, et al. The first outbreak of African swine fever was confirmed in China[J]. China Animal Health Inspection, 2018, 35(9):1-4. (in Chinese)
[5]	CHAPMAN D A G, TCHEREPANOV V, UPTON C, et al. Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates[J]. J Gen Virol, 2008, 89(2):397-408.
[6]	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.
[7]	朱利敏, 邹兴启, 赵启祖. 非洲猪瘟病毒多样性[J]. 病毒学报, 2021, 37(3):719-725.ZHU L M, ZOU X Q, ZHAO Q Z. Diversity of African swine fever virus[J]. Chinese Journal of Virology, 2021, 37(3):719-725. (in Chinese)
[8]	SUN E C, HUANG L Y, ZHANG X F, et al. Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection[J]. Emerg Microbes Infect, 2021, 10(1):2183-2193.
[9]	王晓丽, 孙蕾, 刘文军, 等. 非洲猪瘟病毒编码蛋白功能研究进展[J]. 微生物学通报, 2019, 46(7):1827-1836.WANG X L, SUN L, LIU W J, et al. Advances in the functions of African swine fever virus-encoded proteins[J]. Microbiology China, 2019, 46(7):1827-1836. (in Chinese)
[10]	张锦, 陈艳, 邹剑文, 等. 非洲猪瘟病毒p30蛋白的原核表达及间接ELISA抗体检测方法的建立与应用[J]. 畜牧与兽医, 2022, 54(3):83-90.ZHANG J, CHEN Y, ZOU J W, et al. Prokaryotic expression of p30 protein and establishment and application of an indirect ELISA antibody detection method for ASFV[J]. Animal Husbandry & Veterinary Medicine, 2022, 54(3):83-90. (in Chinese)
[11]	戴建华, 王豪伟, 张紫菡. 基于p54的非洲猪瘟抗体胶体金检测方法建立[J]. 中国动物传染病学报, 2021, 29(6):50-54.DAI J H, WANG H W, ZHANG Z H. Development of p54 based colloidal gold detection method for detection of African swine fever antibodies[J]. Chinese Journal of Animal Infectious Diseases, 2021, 29(6):50-54. (in Chinese)
[12]	刘靖. 非洲猪瘟病毒CD2v、p30和pK205R蛋白的表达纯化与p30、pK205R蛋白单克隆抗体的制备[D]. 北京:中国农业科学院, 2021.LIU J. Expression and purification of CD2v, p30 and pK205R proteins of African swine fever virus and preparation of monoclonal antibodies against p30 and pK205R proteins[D]. Beijing:Chinese Academy of Agricultural Sciences, 2021. (in Chinese)
[13]	杨文兵, 邹亚文, 蒋一凡, 等. 非洲猪瘟血清学诊断靶点的研究进展[J]. 畜牧兽医学报, 2021, 52(5):1208-1217.YANG W B, ZOU Y W, JIANG Y F, et al. Advances research on African swine fever serological diagnostic targets[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5):1208-1217. (in Chinese)
[14]	欧云文, 刘俐君, 贾宁, 等. 非结构蛋白在非洲猪瘟病毒感染中作用[J]. 病毒学报, 2021, 37(4):910-921.OU Y W, LIU L J, JIA N, et al. Roles of African swine fever virus nonstructural proteins in viral infection[J]. Chinese Journal of Virology, 2021, 37(4):910-921. (in Chinese)
[15]	RODRÍGUEZ J M, SALAS M L. African swine fever virus transcription[J]. Virus Res, 2013, 173(1):15-28.
[16]	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.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)
[17]	刘弘知, 熊丹, 潘志明, 等. 非洲猪瘟病毒逃逸天然免疫的研究进展[J]. 中国兽医科学, 2022, 52(10):1323-1327.LIU H Z, XIONG D, PAN Z M, et al. Research progresson in innate immune evasion of African swine fever virus[J]. Chinese Veterinary Science, 2022, 52(10):1323-1327. (in Chinese)
[18]	赵旭阳, 靳家鑫, 路闻龙, 等. 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.ZHAO X Y, JIN J X, LU W L, et al. Advances in the molecular mechanism of immune escape of african swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7):2074-2082. (in Chinese)
[19]	ZHANG D W, RODRÍGUEZ-MOLINA J B, TIETJEN J R, et al. Emerging views on the CTD code[J]. Genet Res Int, 2012, 2012:347214.
[20]	IYER L M, BALAJI S, KOONIN E V, et al. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses[J]. Virus Res, 2006, 117(1):156-184.
[21]	钱虹萍, 陈博, 林金星, 等. RNA聚合酶II动态调控及其成像技术的研究进展[J]. 生物技术通报, 2021, 37(4):293-302.QIAN H P, CHEN B, LIN J X, et al. Recent advances on dynamic regulation and imaging techniques of RNA polymerase II[J]. Biotechnology Bulletin, 2021, 37(4):293-302. (in Chinese)

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

Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

[1]	谢青云, 邢蕙萱, 于岩飞, 袁厅, 熊祺琰, 熊富强, 冯志新. 猪肺炎支原体解螺旋酶RuvA的原核表达、多克隆抗体制备及活性鉴定[J]. 畜牧兽医学报, 2024, 55(1): 271-281.
[2]	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.
[3]	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.
[4]	王静瑜, 潘阳阳, 徐庚全, 张瑞, 张文兰, 王筱珊, 乌仁套迪, 照日格图, 崔燕, 余四九. 牦牛Fas相关因子1多克隆抗体制备及初步应用[J]. 畜牧兽医学报, 2023, 54(8): 3369-3382.
[5]	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.
[6]	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.
[7]	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.
[8]	荆婷婷, 尹号, 黄蓉, 兰仕梅, 李章程, 尤留超, 郝华芳, 付磊, 储岳峰. 牛支原体0580基因C端截短体的原核表达及生物学功能初步分析[J]. 畜牧兽医学报, 2023, 54(7): 2991-3001.
[9]	王辉, 许梦缘, 刘灵康, 郑喜邦, 李恭贺, 吴文德. PhiC31整合酶电穿孔鸡成纤维细胞诱发位点特异性基因盒交换[J]. 畜牧兽医学报, 2023, 54(6): 2330-2342.
[10]	焦正兴, 潘阳阳, 王萌, 王靖雷, 马文斌, 高翔, 张晖, 崔燕, 余四九, 王立斌. 牦牛LC3B蛋白多克隆抗体制备及在生殖器官表达检测中的应用[J]. 畜牧兽医学报, 2023, 54(6): 2436-2447.
[11]	高真贞, 蒙泽菁, 何小兵, 房永祥, 田慧慧, 陈国华, 景志忠. 鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定[J]. 畜牧兽医学报, 2023, 54(6): 2468-2477.
[12]	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.
[13]	程天印, 吴聪颖, 刘雨珂, 段德勇. 褐黄血蜱HSP70-b2及其类14-Mer肽的作用研究[J]. 畜牧兽医学报, 2023, 54(6): 2570-2580.
[14]	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.
[15]	翟云逸, 袁野, 李俊玫, 田路路, 刁梓洋, 李彬, 陈家露, 周栋, 靳亚平, 王爱华. 布鲁氏菌外膜蛋白16单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2083-2091.