Prokaryotic expression and polyclonal antibody preparation of N protein of PD-CoV
10.16303/j.cnki.1005-4545.2025.08.01
- VernacularTitle:PDCoV N蛋白原核表达与多克隆抗体的制备
- Author:
Mingyu LI
1
;
Yuhang JIANG
;
Guoqing ZHANG
;
Lingcong DENG
;
Letian LI
;
Jiayi HAO
;
Xue ZHANG
;
Jiani LI
;
Chang LI
Author Information
1. 西北农林科技大学动物医学院,陕西杨凌 712100;中国农业科学院长春兽医研究所,吉林长春 130122
- Publication Type:Journal Article
- Keywords:
PDCoV;
N protein;
prokaryotic expression;
polyclonal antibody
- From:
Chinese Journal of Veterinary Science
2025;45(8):1587-1592,1608
- CountryChina
- Language:Chinese
-
Abstract:
The synthetic PDCoV N protein gene was optimized and cloned into the pET-30a vector to obtain the pET-30a-N plasmid.Thenthe recombinant plasmid was transformed into three strains of BL21 E.coli using heat-shock to explore protein expression conditions.The expressed proteins was purified using Ni Focurose 6FF(IMAC)and used as antigen to immunize the New Zealand White rabbit to prepare the polyclonal antibody against the PDCoV N protein.The antibody titer was measured by indirect ELISA method.The specificity for the antibody was identified by West-ern blot and indirect immunofluorescence(IFA).The results showed that the pET-30a-N plasmid showed high expression level in BL21 StarTM(DE 3).The optimal expression condition was 37 ℃ 4 h.The purity of the target protein could reach 90.3％after purification.Indirect ELISA showed that the antibody titers was up to 1∶204 800.Western blot and IFA showed that the produced rabbit polyclonal antibody exhibited good specificity.In conclusion,the polyclonal antibody was prepared which specifically recognized the PDCoV N proteins.The results provided some references for the subsequent exploration of PDCoV N protein function and laid a foundation for establishing a diag-nostic method for PDCoV.
