摘要
利用PCR技术扩增发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)Gn-DⅢⅢ基因,将其插入pET-30a(+)原核表达载体,构建重组质粒pET-SFTSV-Gn-D Ⅲ-Ⅲ,将测序正确的pET-SFTSV-Gn-DⅢ-Ⅲ转化至大肠杆菌BL21(DE3)中,优化Gn-D Ⅲ-Ⅲ蛋白表达表达条件.利用镍柱亲和层析法纯化的Gn-D Ⅲ-Ⅲ蛋白作为包被抗原,建立检测SFTSV抗体的间接ELISA方法,并进行评价.结果显示,通过PCR和测序鉴定重组质粒pET-SFTSV-Gn-D Ⅲ-111构建成功;重组Gn-D Ⅲ-Ⅲ蛋白为可溶性表达,其最佳诱导条件为:0.4 mmol/L IPTG于25 ℃诱导4 h;经镍柱纯化后蛋白纯度高达91.77％;SFTSV抗体间接ELISA检测方法的的最佳反应条件为:5 mg/L的包被质量浓度,一抗在37 ℃孵育1.5 h,二抗1:10 000稀释后在37 ℃孵育1 h.特异性试验结果显示该方法与裂谷热病毒(Rift Valley fever virus;RVFV)、埃博拉病毒(Ebola virus,EBOV)和蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)阳性血清均无交叉反应;敏感性试验结果显示该方法有较高的敏感性,SFTSV阳性血清稀释至81 920倍时其P/N仍大于2.1;重复性结果表明批内和批间反应变异系数均小于10％.应用所建立方法检测了 4份人类感染SFTSV不同阶段的临床血清样本,结果缓解期患者血清P/N值大于2.1,为阳性,多器官衰竭期患者血清P/N值小于2.1,为阴性.结果表明,本研究成功表达并纯化了 SFTSV Gn-D Ⅲ-Ⅲ蛋白,并以此为包被蛋白建立SFTSV抗体间接ELISA检测方法,该方法具有良好的特异性、敏感性和重复性,可用于检测人类SFTSV临床血清样本.
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.
基金项目
"十四五"国家重点研发计划基金资助项目(2021YFC2600202)
万方数据