发热伴血小板减少综合征病毒Gn蛋白DⅢ-Ⅲ的表达与抗体间接ELISA检测方法的建立

Expression of severe fever with thrombocytopenia syndrome virus Gn-DⅢ-Ⅲand development of indirect ELISA for antibody detection

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-DⅢ-Ⅲ基因,将其插入pET-30a(+)原核表达载体,构建重组质粒pET-SFTSV-Gn-DⅢ-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-DⅢ-Ⅲ蛋白表达表达条件。利用镍柱亲和层析法纯化的Gn-DⅢ-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-DⅢ-Ⅲ构建成功;重组Gn-DⅢ-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∶10000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81920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-DⅢ-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。

The PCR-amplified severe fever with thrombocytopenia syndrome virus(SFTSV)Gn-DⅢ-Ⅲgene was inserted into the pET-30a(+)prokaryotic expression vector to generate the recombinant plasmid pET-SFTSV-Gn-DⅢ-Ⅲ.The plasmid was transformed into E.coli BL21(DE3)for Gn-DⅢ-Ⅲprotein expression and the expression conditions were optimized.The Gn-DⅢ-Ⅲprotein 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-DⅢ-Ⅲwas successfully constructed as identified by PCR and sequencing.The recombinant protein SFTSV Gn-DⅢ-Ⅲwas soluble expression in E.coli under the optimal induction conditions of 0.4mmol/L IPTG at 25℃for 4h,and the protein purity was 91.77%after purification by Ni-NTA column.The optimal reaction conditions for the indirect ELISA of SFTSV antibody were as follows:coating antigen concentration(5μg/mL),primary antibody(incubation at 37℃for 1.5h),and secondary antibody(diluted 1∶10000and incubated at 37℃for 1h).The established method had no cross-reactivity with Rift Valley fever virus(RVFV),Ebola virus(EBOV),and tick-borne encephalitis virus(TBEV)positive sera.The method had a high sensitivity,with P/N>2.1for 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 remission were tested as positive(P/N>2.1),while serum samples from patients with multiple organ failure were detected as negative(P/N<2.1).The results indicated that the SFTSV Gn-DⅢ-Ⅲprotein was successfully expressed and purified,and it was used as the coating protein to establish 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.