新型鸭呼肠孤病毒DH13株σC蛋白的原核表达及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2020.09.030

Abstract

Abstract: The aims of the experiment was to optimize the prokaryotic expression system of σC protein,prepare polyclonal antibody against σC protein of novel duck reovirus (NDRV),and evaluate the titer of the antibody.The σC gene of NDRV-DH13 strain was amplified by RT-PCR,ligated into pET-30a(+) and pET-32a(+) expression vector,constructed prokaryotic expression plasmid,which were transformed into E.coli BL21(DE3) and the expression of the σC protein were induced by IPTG.The proteins expression were analyzed by SDS-PAGE.The recombinant protein without His tag was purified by digestion,and the recombinant protein with His-tagged was purified by Ni-NTA column.Then the polyclonal antibody was obtained from rabbits which had been immunized by the purified protein without His tag.Anti-His-labeled mAb and NDRV-σC positive serum were used as primary antibodies to evaluate antibodies specificity,the antibodies titer was detected by indirect ELISA (iELISA).SDS-PAGE results showed that the molecular weight of the expression on recombinant proteins were 34 and 37 ku respectively,the proteins were highly expression.Western blotting showed that they had the specific reaction and the prepared antibodies had higher affinity with σC protein,the titer were about 1:25 600 by iELISA detection.This study successfully constructed and optimized the prokaryotic expression system of the polyclonal antibody against σC protein,laid a foundation for the further study of σC protein and the research of genetically engineered vaccine.

Key words: novel duck reovirus; σC protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.4

LI Wenjun, HUANG Huilan, YANG Huihu, XIE Zimin, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Prokaryotic Expression and Polyclonal Antibody Preparation of σC Protein of Novel Duck Reovirus DH13 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2953-2959.

References

[1] 胡奇林,陈少莺,林锋强,等.番鸭呼肠孤病毒的鉴定[J].病毒学报,2004,20(3):242-248. HU Q L,CHEN S Y,LIN F Q,et al.Identification of muscovy duck reovirus[J].Chinese Journal of Virology,2004,20(3):242-248.(in Chinese)
[2] 袁远华,王俊峰,吴志新,等.1株番鸭源新型鸭呼肠孤病毒(QY株)生物学鉴定[J].中国兽医学报,2013,33(8):1174-1178. YUAN Y H,WANG J F,WU Z X,et al.Biological identification of a new-type duck reovirus (QY strain) isolated from Muscovy duck[J].Chinese Journal of Veterinary Science,2013,33(8):1174-1178.(in Chinese)
[3] 陈少莺,陈仕龙,林锋强,等.一种新的鸭病(暂名鸭出血性坏死性肝炎)病原学研究初报[J].中国农学通报,2009,25(16):28-31. CHEN S Y,CHEN S L,LIN F Q,et al.The primary study of pathogen of duck hemorrhagic-necrotic hepatitis[J].Chinese Agricultural Science Bulletin,2009,25(16):28-31.(in Chinese)
[4] LI N,HONG T Q,WANG Y,et al.The pathogenicity of novel duck reovirus in Cherry Valley ducks[J].Veterinary Microbiology,2016,192(25):181-185.
[5] 范辉.一例番鸭新型鸭呼肠孤病毒感染的诊治[J].当代畜牧,2018,35(2):35-36. FAN H.Diagnosis and treatment of a new duck reovirus infection in Muscovy ducks[J].Contemporary Animal Husbandry,2018,35(2):35-36.(in Chinese)
[6] 武鸿,康亚男,唐荣宏,等.3株樱桃谷肉鸭源和2株番鸭源新型鸭呼肠孤病毒的分离与鉴定[J].南京农业大学学报,2019,42(2):328-335. WU H,KANG Y N,TANG R H,et al.The isolation and identification of novel duck reovirus from Cherry Valley ducks (3 strains) and Muscovy ducks (2 strains)[J].Journal of Nanjing Agricultural University,2019,42(2):328-335.(in Chinese)
[7] 高斌.新型鸭呼肠孤病毒的分离鉴定及致病性研究[D].泰安:山东农业大学,2019. GAO B.Isolation,identification and pathogenicity of a new-type duck reovirus[D].Tai'an:Shandong Agricultural University,2019.(in Chinese)
[8] ZHANG X L,SHAO J W,LI X W,et al.Molecular characterization of two novel reoviruses isolated from Muscovy ducklings in Guangdong,China[J].BMC Veterinary Research,2019,15(1):143.
[9] YUN T,YU B,NI Z,et al.Genomic characteristics of a novel reovirus from Muscovy duckling in China[J].Veterinary Microbiology,2014,168(2-4):261-271.
[10] ZHU Y Q,LI C F,BI Z L,et al.Molecular characterization of a novel reovirus isolated from Pekin ducklings in China[J].Archives of Virology,2015,160(1):365-369.
[11] BI Z L,ZHU Y Q,CHEN Z Y,et al.Induction of a robust immunity response against novel duck reovirus in ducklings using a subunit vaccine of sigma C protein[J].Scientific Reports,2016,6:39092.
[12] SHIH W L,HSU H W,LIAO M H,et al.Avian reovirus σC protein induces apoptosis in cultured cells[J].Virology,2004,321(1):65-74.
[13] 郑献进.两株北京鸭源呼肠孤病毒生物学特性比较[D].北京:中国农业大学,2016. ZHENG X J.Comparison of biological characteristics of two Pekin duck-origin reoviruses[D].Beijing:China Agricultural University,2016.(in Chinese)
[14] 于可响,路晓,刘存霞,等.山东地区新型鸭呼肠孤病毒致病特点与变异分析[J].中国动物传染病学报,2018,26(4):24-28. YU K X,LU X,LIU C X,et al.Pathogenic characteristics and variation analysis of novel duck reovirus isolation from Shandong province[J].Chinese Journal of Animal Infectious Diseases,2018,26(4):24-28.(in Chinese)
[15] 陆新浩,陈秋英,刘鸿,等.宁波地区鸭呼肠孤病毒病的流行病学及防控措施[J].浙江畜牧兽医,2015,40(5):34-35. LU X H,CHEN Q Y,LIU H,et al.Epidemiology and control measures of duck reovirus in Ningbo area[J].Zhejiang Journal Animal Science and Veterinary Medicine,2015,40(5):34-35.(in Chinese)
[16] 梅敏敏,叶颖,黄翠英,等.新致病型番鸭呼肠孤病毒对雏番鸭致病性的研究[J].黑龙江畜牧兽医,2019,12(6):95-97. MEI M M,YE Y,HUANG C Y,et al.Pathogenicity of a new-type pathogenic Muscovy duck reovirus to Muscovy ducklings[J].Heilongjiang Animal Science and Veterinary Medicine,2019,12(6):95-97.(in Chinese)
[17] KUNTZ-SIMON G,BLANCHARD P,CHERBONNEL M,et al.Baculovirus-expressed Muscovy duck reovirus sigmaC protein induces serum neutralizing antibodies and protection against challenge[J].Vaccine,2002,20(25-26):3113-3122.
[18] 刘红,郁宏伟,朱朝辉,等.鸭源呼肠孤病毒DRV-GZ株S2基因的序列分析及其表达[J].中国兽医科学,2008,38(3):224-228. LIU H,YU H W,ZHU C H,et al.Sequence analysis and expression of S2 gene of reovirus strain DRV-GZ originated from duck[J].China Veterinary Science,2008,38(3):224-228.(in Chinese)
[19] 邹光盛.番鸭呼肠孤病毒σC基因真核表达及其间接ELISA方法的建立[D].福州:福建农林大学,2007. ZOU G S.Eukaryotic expression and indirect ELISA development of MDRV σC gene[D].Fuzhou:Fujian Agriculture and Forestry University,2007.(in Chinese)
[20] 毕庄莉.新型鸭呼肠孤病毒σC基因在昆虫细胞中的表达及其免疫原性的初步研究[D].合肥:安徽农业大学,2014. BI Z L.Expression of novel duck reovirus σC gene in insect cells and immunogenicity preliminary study of expressed protein[D].Hefei:Anhui Agricultural University,2014.(in Chinese)
[21] ZHENG X J,WANG D,NING K,et al.A duck reovirus variant with a unique deletion in the sigma C gene exhibiting high pathogenicity in Pekin ducklings[J].Virus Research,2016,215(6):37-41.
[22] 王劭,陈少莺,林锋强,等.pMAL-p2X系统表达番鸭呼肠孤病毒MW9710株σC蛋白[J].福建农业学报,2009,24(5):396-399. WANG S,CHEN S Y,LIN F Q,et al.Expression of σC gene of Muscovy duck reovirus MW9710 strain by pMAL-p2X system[J].Fujian Journal of Agricultural Sciences,2009,24(5):396-399.(in Chinese)

Prokaryotic Expression and Polyclonal Antibody Preparation of σC Protein of Novel Duck Reovirus DH13 Strain

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[5]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[6]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[7]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[8]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[9]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[10]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[11]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[12]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[13]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[14]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[15]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.