Expression of severe fever with thrombocytopenia syndrome virus Gn-D Ⅲ-Ⅲ and development of indirect ELISA for antibody detection
10.16303/j.cnki.1005-4545.2024.08.17
- VernacularTitle:发热伴血小板减少综合征病毒Gn蛋白D Ⅲ-Ⅲ的表达与抗体间接ELISA检测方法的建立
- Author:
Mengyao ZHANG
1
;
Tianlai LIANG
;
Feihu YAN
;
Tao CHEN
;
Cuicui JIAO
;
Hongli JIN
;
Jiaoyan LUAN
;
Xiao WU
;
Pei HUANG
;
Haili ZHANG
;
Qin NING
;
Hualei WANG
;
Yuanyuan LI
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
- Keywords:
severe fever with thrombocytopenia syndrome virus(SFTSV);
Gn protein domain Ⅲ(Gn-D Ⅲ);
prokaryotic expression;
indirect ELISA
- From:
Chinese Journal of Veterinary Science
2024;44(8):1704-1712
- CountryChina
- Language:Chinese
-
Abstract:
The PCR-amplified severe fever with thrombocytopenia syndrome virus(SFTSV)Gn-DⅢ-Ⅲ gene was inserted into the pET-30a(+)prokaryotic expression vector to generate the re-combinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ.The plasmid was transformed into E.coli BL21(DE3)for Gn-DⅢ-m protein expression and the expression conditions were optimized.The Gn-DⅢ-Ⅲ protein purified with Ni-NTA column affinity chromatography was applied as the captured antigen to establish an indirect ELISA method for the detection of SFTSV antibody.The results demonstrated that the recombinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ was successfully constructed as identified by PCR and sequencing.The recombinant protein SFTSV Gn-D m-Ⅲ was soluble ex-pression in E.coli under the optimal induction conditions of 0.4 mmol/L IPTG at 25 ℃ for 4 h,and the protein purity was 91.77％after purification by Ni-NTA column.The optimal reaction con-ditions for the indirect ELISA of SFTSV antibody were as follows:coating antigen concentration(5 μg/mL),primary antibody(incubation at 37 ℃ for 1.5 h),and secondary antibody(diluted 1:10 000 and incubated at 37 ℃ for 1 h).The established method had no cross-reactivity with Rift Valley fever virus(RVFV),Ebola virus(EBOV),and tick-borne encephalitis virus(TBEV)posi-tive sera.The method had a high sensitivity,with P/N＞2.1 for SFTSV-positive sera diluted to 81920.Coefficients of variation for intra-and inter-batch reactions were less than 10％.Detection of four SFTSV-infected human clinical serum samples showed the serum samples from patients in re-mission were tested as positive(P/N＞2.1),while serum samples from patients with multiple or-gan failure were detected as negative(P/N＜2.1).The results indicated that the SFTSV Gn-D Ⅲ-Ⅲ protein was successfully expressed and purified,and it was used as the coating protein to estab-lish an indirect ELISA assay for SFTSV antibody,which possesses good specificity,sensitivity and reproducibility.This method might be applied to detect human SFTSV clinical serum samples.
