猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2024.10.046

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4773-4778.doi: 10.11843/j.issn.0366-6964.2024.10.046

猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

刘福康¹^,²(), 袁莉刚¹^,*(), 张达², 汤傲星², 刘光清², 朱杰²^,*()

1. 甘肃农业大学动物医学院, 兰州 730070
2. 中国农业科学院上海兽医研究所, 上海 200241

收稿日期:2023-10-07 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 袁莉刚,朱杰 E-mail:liufukang1015@163.com;yuan2918@126.com;zj121@shvri.ac.cn
作者简介:刘福康(1998-), 男, 甘肃庆阳人, 硕士生, 主要从事动物解剖与组织胚胎学研究, E-mail: liufukang1015@163.com
基金资助:
上海市市级科技重大专项(ZD2021CY001);上海市自然科学基金项目(22ZR1476300)

Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain

Fukang LIU¹^,²(), Ligang YUAN¹^,*(), Da ZHANG², Aoxing TANG², Guangqing LIU², Jie ZHU²^,*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2023-10-07 Online:2024-10-23 Published:2024-11-04
Contact: Ligang YUAN, Jie ZHU E-mail:liufukang1015@163.com;yuan2918@126.com;zj121@shvri.ac.cn

摘要/Abstract

摘要：

本研究旨在体外原核表达猫传染性腹膜炎病毒(feline infectious peritonitis virus, FIPV)SH2021株核衣壳蛋白(nucleocapsid, N)，制备兔源多克隆抗体。根据FIPV SH2021株N基因序列设计引物，构建pCold-I-N重组表达质粒。随后，将重组质粒转化至表达感受态细胞BL21(DE3)，在终浓度为1.0 mmol·L^-1的IPTG和16 ℃的诱导条件下进行表达。最后，利用His层析柱进行纯化，纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体。结果表明，纯化获得重组N蛋白大小约为45 ku，制备的N蛋白兔多克隆抗体效价可达1∶512 000，该抗体对N蛋白具有良好的反应原性。本研究成功所制备了FIPV N蛋白兔多克隆抗体，该抗体具有良好的反应原性和特异性，为FIPV抗原抗体检测以及研究N蛋白生物学功能研究提供重要工具。

关键词: 猫传染性腹膜炎病毒, 猫冠状病毒, N蛋白, 基因克隆, 原核表达, 多克隆抗体

Abstract:

This study aimed to express the nucleocapsid protein (N) of feline infectious peritonitis virus (FIPV) strain SH2021 in vitro and prepare rabbit polyclonal antibodies. Primers were designed based on the N gene sequence of FIPV strain SH2021 to construct the recombinant expression plasmid pCold-I-N. Subsequently, the recombinant plasmid was transformed into expression-competent cells BL21 (DE3), and expression was induced under conditions of 1.0 mmol·L^-1 IPTG and 16 ℃. Finally, purification was performed using a His column, and the purified recombinant protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies. The experimental results showed that the purified recombinant N protein had a size of approximately 45 ku, and the titer of the prepared rabbit polyclonal antibodies against the N protein reached 1∶512 000. The antibodies exhibited good reactivity and specificity towards the N protein. This study successfully prepared rabbit polyclonal antibodies against the FIPV N protein, which showed good reactivity and specificity, providing important tools for FIPV antigen-antibody detection and research on the biological functions of the N protein.

Key words: feline infectious peritonitis virus, feline coronavirus, N protein, gene cloning, prokaryotic expression, polyclonal antibody

中图分类号:

S852.659.6

刘福康, 袁莉刚, 张达, 汤傲星, 刘光清, 朱杰. 猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备[J]. 畜牧兽医学报, 2024, 55(10): 4773-4778.

Fukang LIU, Ligang YUAN, Da ZHANG, Aoxing TANG, Guangqing LIU, Jie ZHU. Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4773-4778.

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参考文献 14

1	张丽, 李月勤, 宋予震, 等. 猫传染性腹膜炎的研究进展[J]. 现代牧业, 2017, 1 (4): 51- 53.
	ZHANG L , LI Y Q , SONG Y Z , et al. Research progress in feline infectious peritonitis[J]. Modern Animal Husbandry, 2017, 1 (4): 51- 53.
2	袁健. 基因组重配与重组的分子进化研究——以H6N1、MERS-CoV及鸟类mtDNA为例[D]. 芜湖: 安徽师范大学, 2014.
	YUAN J. Molecular evolution of genomic recombination and reassortment: a case study of H6N1, MERS-CoV and avian mtDNA[D]. Wuhu: Anhui Normal University, 2014. (in Chinese)
3	李少晗, 张广智, 崔尚金, 等. 犬猫冠状病毒研究进展[J]. 中国畜牧兽医, 2020, 47 (12): 4059- 4068.
	LI S H , ZHANG G Z , CUI S J , et al. Research progress of canine coronavirus and feline coronavirus[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47 (12): 4059- 4068.
4	何奕豪, 何杰珩, 岑晓锋, 等. 猫冠状病毒病研究进展[J]. 今日畜牧兽医, 2021, 37 (1): 8- 10.
	HE Y H , HE J H , CEN X F , et al. Research progress on feline coronavirus disease[J]. Today's Animal Husbandry and Veterinary Medicine, 2021, 37 (1): 8- 10.
5	尹伊娅, 刘晓雅, 王朝好, 等. 猫传染性腹膜炎研究进展[J]. 畜牧兽医学报, 2020, 51 (5): 933- 942.
	YIN Y Y , LIU X Y , WANG C H , et al. Advances in research on feline infectious peritonitis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51 (5): 933- 942.
6	RIEMER F , KUEHNER K A , RITZ S , et al. Clinical and laboratory features of cats with feline infectious peritonitis—a retrospective study of 231 confirmed cases (2000-2010)[J]. J Feline Med Surg, 2016, 18 (4): 348- 356. doi: 10.1177/1098612X15586209
7	OLYSLAEGERS D A J , DEDEURWAERDER A , DESMARETS L M B , et al. Altered expression of adhesion molecules on peripheral blood leukocytes in feline infectious peritonitis[J]. Vet Microbiol, 2013, 166 (3/4): 438- 449.
8	段聪, 汪霞. 猪δ冠状病毒蛋白功能研究进展[J]. 中国兽医杂志, 2023, 59 (5): 95- 99.
	DUAN C , WANG X . Research advances of porcine deltacoronavirus protein and its function[J]. Chinese Journal of Veterinary Medicine, 2023, 59 (5): 95- 99.
9	CHE X Y , HAO W , WANG Y D , et al. Nucleocapsid protein as early diagnostic marker for SARS[J]. Emerg Infect Dis, 2004, 10 (11): 1947- 1949. doi: 10.3201/eid1011.040516
10	CHANG C K , LO S C , WANG Y S , et al. Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein[J]. Drug Discov Today, 2016, 21 (4): 562- 572. doi: 10.1016/j.drudis.2015.11.015
11	LIN C N , CHAN K R , OOI E E , et al. Animal coronavirus diseases: parallels with COVID-19 in humans[J]. Viruses, 2021, 13 (8): 1507. doi: 10.3390/v13081507
12	JUNG K , HU H , SAIF L J . Porcine deltacoronavirus infection: Etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis[J]. Virus Res, 2016, 226, 50- 59. doi: 10.1016/j.virusres.2016.04.009
13	龙云凤, 王毅谦, 姜珊, 等. 猪δ冠状病毒检测方法研究进展[J]. 畜牧与兽医, 2017, 49 (3): 116- 121.
	LONG Y F , WANG Y Q , JIANG S , et al. Research progress on diagonosis of porcine deltacoronavirus[J]. Animal Husbandry & Veterinary Medicine, 2017, 49 (3): 116- 121.
14	MCBRIDE R , VAN ZYL M , FIELDING B C . The coronavirus nucleocapsid is a multifunctional protein[J]. Viruses, 2014, 6 (8): 2991- 3018.

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猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain

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