猪传染性胃肠炎病毒NSP8蛋白单克隆抗体的制备及抗原表位的初步鉴定

doi:10.16431/j.cnki.1671-7236.2015.02.019

Abstract

Abstract: To identify the epitope of porcine transmissible gastroenteritis virus (TGEV),the NSP8 gene was cloned into the vector pGEX-6P-1 for expression in E.coli.BALB/c mice of 6 week-old were immunized with the recombinant NSP8 protein.Then the monoclonal antibody against NSP8 protein was prepared by lymphocyte hybridoma technique.The monoclonal antibody belonged to IgG1 subtype with κ chain.The titers in cell culture medium of the hybidomas was 1:3 200.Western blotting assay showed that the monoclonal antibody was able to recognize the recombinant NSP8 protein.Epitope on NSP8 protein was identified by truncated expression and the linear epitope of ³¹SPQILKQLTKAFNIAKSDFEREASV⁵⁵ was identified by Western blotting with the monoclonal antibody.This monoclonal antibody and its epitope mapping provided a basis for further study of the function of the TGEV NSP8.

Key words: porcine transmissible gastroenteritis virus; NSP8 gene; monoclonal antibody; antigenic epitope

CLC Number:

S858.28

DONG Hui, ZHANG Xin, CHEN Jian-fei, SHI Hong-yan, SHI Da, CHANG Tie-cheng, GU Feng-li, XU Tian, WANG Zhi-qin, LIU Ming-chun, FENG Li. Preparation of Monoclonal Antibody and Preliminary Identification of Antigenic Epitope of NSP8 Protein in Porcine Transmissible Gastroenteritis Virus[J]. , 2015, 42(2): 358-364.

References

[1] 殷相平,任晓峰,柳纪省.猪传染性胃肠炎病毒致病机制的研究进展[J].世界华人消化杂志,2013,21(1):39-43.
[2] 常志顺,汤德元,曾志勇,等.猪传染性胃肠炎病毒基因及其疫苗的研究进展[J].猪业科学,2008,12:26-32.
[3] te Velthuis A J,van den Worm S H,Snijder E J.The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension[J].Nucleic Acids Res,2012,40(4):1737-1747.
[4] Zola H著.周宗安,方元,房德兴,等译.单克隆抗体技术手册[M].南京:南京大学出版社,1991.
[5] 胡小华,李刚,史利军,等.猪圆环病毒2型间接ELISA抗体检测试剂盒的研制及初步应用[J].中国畜牧兽医,2011,38(3):199-203.
[6] 王永志,苏颖,米丽娟,等.山羊痘病毒p32基因的克隆、表达及单克隆抗体制备[J].中国畜牧兽医,2011,38(9):101-105.
[7] 迟延彬,陈建飞,时洪艳,等.A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定[J].中国畜牧兽医,2014,41(2):70-74.
[8] 赵鑫,张鑫,时洪艳,等.抗猪传染性胃肠炎病毒N蛋白单克隆抗体的制备及抗原表位的鉴定[J].中国预防兽医学报,2014,36(4):314-317.
[9] Leman A D,Straw B,Glock R D,et al.Disease of swine[M].7^thed.Ames:Iowa State University Press,1992.
[10] 田美娜,李永亮,付元芳,等.口蹄疫病毒非结构蛋白2C主要表位区的原核表达及其单克隆抗体的制备[J].中国预防兽医学报,2009,31(1):69-73.
[11] Imbert I,Guilenmot J C,Bourhis J M,et al.A second,non-canonical RNA-dependent RNA polymerase in SARS coronavirus[J]. EMBO J,2006,25(20):4933-4942.
[12] te Velthuis A J,Arnold J J,Cameron C E,et al.The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent[J].Nucleic Acids Res,2010,38(1):203-214.
[13] te Velthuis A J,van den Worm S H,Sims A C,et al.Zn²⁺ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc lonophores block the replication of these viruses in cell culture[J].PLoS Pathog,2010,6(11):e1001176.
[14] 赵琪.冠状病毒复制酶系结构与功能研究[D].北京:清华大学,2010.

[1]	ZHANG Guoqing, JIANG Yuhang, ZHANG Shuang, LI Letian, HAO Jiayi, CHEN Jing, ZOU Wancheng, LI Chang, LI Taiyuan, GAO Xu, JIN Ningyi. Preparation and Identification of PDCoV RBD Protein Peptide Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1575-1583.
[2]	ZHANG Jingxu, XU Yu, LIANG Haiying, ZENG Zhiyong, TANG Deyuan, WANG Bin, XU Songping, WAN Juan, ZHU Yang. Phylogenetic Evolution and Epitope Analysis of Pseudorabies Virus Guizhou Isolates and Vaccine Strains [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1093-1106.
[3]	HOU Haoyu, ZHENG Nannan, WU Hongju, CHEN Yinlong, LI Chao, ZHANG Angke, JI Pengchao, WAN Bo, DU Yongkun, ZHANG Gaiping. Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1241-1249.
[4]	WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.
[5]	MO Hongfang, JIE Kai, CHEN Xin, WEN Wei, LIU Wenqiang, CHEN Huanchun, QIAN Ping, LI Xiangmin. Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 307-317.
[6]	HU Huanyi, WANG Jianzhong, LIU Changjin, LIN Min, LIU Xiaolan, WEI Huangsiwu, DENG Shunzhou. Eukaryotic Expression of Green Fluorescent Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 328-337.
[7]	ZENG Jianyu, DONG Zhenyuan, JIA Wenfeng, ZHANG Guozhong, XUE Jia. Preparation and Identification of Monoclonal Antibody Against Newcastle Disease Virus M Protein [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3423-3431.
[8]	PAN Chenhao, ZHANG Xin, ZHAO Ruili, JIANG Xuan, JIN Tianming, YU Enyuan, LI Liuan, ZENG Jun, YU Xiaoxue, SONG Qiqi, HU Ye. Construction and Cytotoxicity Detection of Recombinant Epitope Protein DCpep-GSE of Streptococcus suis [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2990-3001.
[9]	LI Si, JIANG Wei, CHENG Tingting, LU Chunhui, XIN Sipei, CAO Jingzheng, WANG Quan, SUN Weidong. Establishment of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Sarafloxacin [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1772-1783.
[10]	YANG Songxin, FENG Jianyuan, GUO Xuan, WEI Liuyuan, LIANG Yiying, YAN Hao, ZHANG Ziyi, CHEN Hailan. Preparation of Monoclonal Antibodies for Small Molecular Compounds and Their Applications in the Field of Veterinary Medicine [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 1121-1131.
[11]	LI Jiesen, SUN Ronghang, KUANG Yanqi, CHEN Luman, LIU Qing, GUO Xiaofeng. Optimal Expression of Porcine Epidemic Diarrhea Virus S1D Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4641-4651.
[12]	WANG Zhiping, ZHANG Yunjing, SUN Yujie, XU Xin, WANG Zhiyan, HUANG Baicheng, TIAN Kegong. Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2248-2255.
[13]	SHI Jianzhou, LI Qingmei, WANG Yanhong, LIU Xiao, LI Ge, GUO Junqing, DENG Ruiguang, ZHANG Gaiping. Establishment of a Method of IPMA for Detection of the Monoclonal Antibody Against H1N1 Swine Influenza Virus [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2691-2698.
[14]	XIANG Zilai, WANG Ling, XIA Sugan, ZOU Hui, GU Jianhong, YUAN Yan, LIU Xuezhong, LIU Zongping, BIAN Jianchun. Preparation of Monoclonal Antibodies Against Phenylethanolamine A and Establishment of ELISA Method [J]. , 2019, 46(3): 849-857.
[15]	ZHOU Manli, ZHOU Yucheng, ZHANG Haiwei, QIAO Lianjiang, YANG Yanling. Preparation and Detection of Monoclonal Antibodies Against Mycobacterium bovis MPB70 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 3075-3083.

Preparation of Monoclonal Antibody and Preliminary Identification of Antigenic Epitope of NSP8 Protein in Porcine Transmissible Gastroenteritis Virus

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments