Preparation of polyclonal antibodies against VP8 protein of porcine rotavirus A and determination of neutralizing antibody titers
10.16303/j.cnki.1005-4545.2025.06.02
- VernacularTitle:猪A群轮状病毒VP8*蛋白多克隆抗体的制备及其中和抗体效价的测定
- Author:
Jiachao XU
1
;
Guangli HU
;
Qingqing WU
;
Xiaomei PAN
;
Sun HE
;
Yidi GUO
;
Changchun TU
;
Wenjie GONG
Author Information
1. 吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春 130062
- Publication Type:Journal Article
- Keywords:
porcine rotavirus A;
VP8* protein;
neutralization assay;
polyclonal antibody
- From:
Chinese Journal of Veterinary Science
2025;45(6):1109-1116,1131
- CountryChina
- Language:Chinese
-
Abstract:
This study investigates the feasibility of the VP8*protein as a subunit vaccine target for porcine rotavirus A(PoRVA),a major causative agent of diarrhea in piglets.The VP8* genes of PoRVA P[13]and P[23]genotype strains were amplified by RT-PCR.These genes were then liga-ted into the pET-28a(+)vector,yielding recombinant plasmids pET-28a-XJWF1-VP8*-P[23]and pET-28a-ShXYW13-VP8*-P[13].These plasmids were subsequently transformed into BL21(DE3)competent cells.The VP8*protein,induced by IPTG,was purified using affinity chroma-tography,and its expression and purification were verified by SDS-PAGE and Western blot.The purified VP8* protein was used to immunize mice,and serum samples were collected after three immunizations.Cross-neutralization assays were conducted to evaluate the ability of the VP8*protein immune serum to neutralize different genotype strains.The results demonstrated the ex-pression of soluble VP8*protein,with SDS-PAGE and Western blot analyses showing that the purified VP8*protein existed in both monomeric(27 kDa)and homodimeric(54 kDa)forms.ELISA results indicated that high levels of antibodies were produced in mice immunized with VP 8*-P[13]and VP8*-P[23]after three immunizations.Serum cross-neutralization assays revealed that the neutralizing titers of PoRVA VP8*-P[13]and VP8*-P[23]immune sera against homol-ogous genotype strains ranged from 1∶4 800 to 1∶19 200,significantly higher than those against heterologous genotype strains(1∶1 200).This suggests that the VP8*protein of different geno-type strains exhibits both antigenic conservation and distinct variability.The data obtained in this study provide a solid foundation for further exploration of the antigenic structure of the PoRVA VP8* protein and the development of novel subunit vaccines.
