Expression of porcine deltacoronavirus S protein in insect cells and preparation and identification of monoclonal antibody
10.16303/j.cnki.1005-4545.2024.11.01
- VernacularTitle:猪丁型冠状病毒S蛋白在昆虫细胞中的表达及单克隆抗体的制备与鉴定
- Author:
Dantong LI
1
;
Qing HE
;
Dongliang WANG
;
Beilei YU
;
Qian YUAN
;
Weijiao LIU
;
Yi YANG
;
Naidong WANG
Author Information
1. 湖南农业大学动物医学院兽用蛋白质工程疫苗湖南省重点实验室/兽用疫苗逆向创制湖南省工程研究中心,湖南长沙 410128
- Keywords:
porcine deltacoronavirus;
S protein;
baculovirus expression vector system;
monoclonal antibody;
neutralizing antibody
- From:
Chinese Journal of Veterinary Science
2024;44(11):2309-2315
- CountryChina
- Language:Chinese
-
Abstract:
Porcine deltacoronavirus(PDCoV),a newly discovered virus in recent years,can cause severe diarrhea,vomiting,dehydration and even death in piglets.S protein is an important structur-al protein of PDCoV,which determines the host or tissue tropism of the virus,and is an important target for the development of PDCoV vaccines and detection methods.In order to prepare mono-clonal antibody(MAb)against the S protein of PDCoV,the recombinant plasmid of S protein was constructed based on the extracellular domain sequence of S protein of PDCoV epidemic strain in China and transformed into DH10Bac competent cells.The recombinant bacmid was identified by blue-white spot screening and PCR.BALB/c mice were immunized with S protein,and the spleen cells of immunized mice were fused with myeloma cells.The positive hybridoma cells that secreted stable antibodies were screened by indirect ELISA and subcloning.Five hybridoma cell superna-tants(MAb)specifically recognizing S protein(2E5,4D5,5D10,2F7 and 5A9)were identified by Western blot and immunofluorescence assay(IFA).Subsequently,the neutralization test showed that three of the monoclonal antibodies(2E5,4D5 and 5D10)could neutralize the virus to varying degrees.The S protein was successfully expressed and 5 monoclonal antibodies that can stably se-crete and specifically bind to PDCoV and S protein were prepared,which laid an important founda-tion for further research on the structure and function of S protein,the development of detection methods for PDCoV infection,and the prevention or treatment of PDCoV infection.
