Expression of Rift Valley fever virus Gn-D Ⅱ-Ⅲ and development of indirect ELISA for RVFV antibody detection
10.16303/j.cnki.1005-4545.2025.06.11
- VernacularTitle:裂谷热病毒Gn蛋白D Ⅱ-Ⅲ的表达与间接ELISA抗体检测方法的建立
- Author:
Jiaoyan LUAN
1
;
Mengyao ZHANG
1
;
Cuicui JIAO
1
;
Xiangyang ZHANG
1
;
Lisi AI
1
;
Pei HUANG
1
;
Yuanyuan LI
1
;
Haili ZHANG
1
;
Hualei WANG
1
Author Information
1. 吉林大学动物医学学院人畜共患传染病重症诊治全国重点实验室,吉林长春 130062
- Publication Type:Journal Article
- Keywords:
Rift Valley fever virus;
prokaryotic expression;
Gn-D Ⅱ-Ⅲ protein;
indirect ELISA
- From:
Chinese Journal of Veterinary Science
2025;45(6):1186-1193,1209
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to establish an indirect ELISA method for detecting RVFV antibodies u-sing recombinant proteins of Rift Valley fever virus(RVFV)Gn protein Ⅱ-Ⅲ structural domains as the encapsulated antigen which was expressed by the Escherichia coli(E.coli)expression sys-tem.The gene sequences encoding the Ⅱ and Ⅲ subdomains of RVFV Gn protein were inserted in-to pET-30a(+)to construct the recombinant plasmid pET-RVFV Gn-D Ⅱ-Ⅲ.After transforma-tion of the recombinant plasmid into DE3(BL21)competent cells,the recombinant Gn-D Ⅱ-Ⅲ protein was induced with IPTG and purified using affinity chromatography.An indirect ELISA method for the detection of RVFV antibodies was developed using purified recombinant protein as coating antigen and SPA-HRP as the enzyme-labelled secondary antibody.Western blot analysis confirmed that the RVFV Gn-D Ⅱ-Ⅲ protein was successfully expressed.The optimal expression conditions for RVFV Gn-D Ⅱ-Ⅲ protein were induced with 0.8 mmol/L IPTG at 37 ℃ for 5 h.The Gn-D Ⅱ-Ⅲ protein was purified using affinity chromatography with a purity of 91.9％,and the purified protein was used as the encapsulated antigen to develop an ELISA assay for RVFV anti-bodies.The specificity evaluation showed that the method specifically detected RVFV-positive sera and did not cross-react with sera positive for West Nile virus(WNV),Ebola virus(EBOV),Mar-burg virus(MARV)and tick-borne encephalitis virus(TBEV).When the RVFV Gn-D Ⅲ-Ⅲ posi-tive serum was diluted to 6 400 times,the test result still showed positive results,demonstrating the method had good sensitivity.The repeatability evaluation results indicated that the variation co-efficients for both intra-and inter-batch responses was less than 10％,indicating that the method had good repeatability.In conclusion,the RVFV Gn-D Ⅱ-Ⅲ protein was successfully expressed u-sing the E.coli expression system.The purified recombinant Gn-D Ⅱ-Ⅲ protein was used as the encapsulated antigen to develop an indirect ELISA assay for RVFV antibodies,which provides a preliminary basis for the diagnosis of RVF and the research and development of RVF vaccines.
