【摘要】 为建立检测早期旋毛虫抗体的间接ELISA方法，本研究将本实验室前期构建的旋毛虫海藻糖-6-磷酸磷酸酶（TsTPP）的重组质粒p ET-30a(+)-TsTPP转化E. coli BL21(DE3)感受态细胞中，诱导表达重组TsTPP蛋白（rTsTPP），经Ni-NTA-琼脂糖亲和层析柱纯化该目的蛋白后，通过SDS-PAGE和Western blot鉴定其表达、纯化效果和反应原性。将纯化后的r TsTPP作为包被抗原，优化间接ELISA方法各反应参数。结果显示rTsTPP表达、纯化效果和反应原性均较好。该间接ELISA方法最佳反应条件为TPP抗原包被浓度为5.0μg/mL,4℃孵育12 h,5%BSA 37℃封闭2 h，兔rTsTPP阳性血清稀释度为1:1 000，羊抗兔IgG-HRP稀释度为1:6 000,TMB 37℃显色10 min。利用建立的间接ELISA方法检测鼠源旋毛虫、华支睾吸虫、血吸虫和猪弓形虫等不同寄生虫阳性血清，进行特异性试验；利用该方法检测2倍倍比稀释（1:10～1:5 120）的旋毛虫阳性血清，进行敏感性试验；利用同一批次或不同批次包被的酶标板检测5份鼠源旋毛虫阳性血清，进行重复性试验。结果显示，该方法除对旋毛虫阳性血清检测为阳性外，与其他寄生虫阳性血清均无交叉反应，特异性强；对鼠源旋毛虫阳性血清的检测限为1:2 560，敏感性高；批内和批间重复性试验的变异系数均小于10%，重复性好。利用该方法和商品化旋毛虫抗体快速检测卡分别检测感染不同数量和不同时间旋毛虫小鼠的阳性血清，结果显示本实验建立的方法于旋毛虫感染7 d检出阳性血清样品，早于快速检测卡。本研究基于rTsTPP建立的旋毛虫抗体间接ELISA检测方法具有良好的特异性、敏感性和重复性，为旋毛虫病的早期和血清学诊断提供了可行技术手段。更多还原

【Abstract】 To establish an indirect ELISA method for detecting early antibodies against Trichinella spiralis, this study transformed the recombinant plasmid p ET-30a(+)-TsTPP, which harbors the Trichinella spiralis trehalose-6-phosphate phosphatase(TsTPP) gene previously constructed in our laboratory, into E. coli BL21(DE3) competent cells. The recombinant TsTPP protein(rTsTPP) was induced for expression and subsequently purified using a Ni-NTA agarose affinity chromatography column. The expression, purification efficiency, and immunoreactivity of the target protein were assessed by SDS-PAGE and Western blot analysis.The purified rTsTPP was used as the coating antigen to optimize reaction parameters. The results showed that the expression,purification, and immunoreactivity of rTsTPP were satisfactory. The optimal conditions for the established indirect ELISA method were as follows: TPP antigen coating concentration of 5.0μg/mL, incubation at 4℃ for 12 hours, blocking with 5% BSA at 37℃ for 2 hours, rabbit anti-rTsTPP positive serum diluted at 1:1000, enzyme-labeled secondary antibody(goat anti-rabbit IgG/HRP) diluted at1:6000, and TMB substrate development at 37℃ for 10 min. The method showed no cross-reactivity with positive sera from Clonorchis sinensis, Schistosoma, or Toxoplasma gondii infections, indicating high specificity. The detection limit for mouse anti-T.spiralis-positive serum was 1:2560, indicating high sensitivity. The coefficients of variation for both intra-batch and inter-batch reproducibility were less than 10%, demonstrating good reproducibility. Compared to a commercial T. spiralis antibody rapid detection kit, the established method detected positive serum samples as early as 7 days post-infection in mice infected with varying doses and durations. These results demonstrate that the purified rTsTPP was successfully employed as an antigen to establish an indirect ELISA antibody detection method for T. spiralis. This method exhibits high sensitivity, specificity, and reproducibility,making it suitable for early and serological diagnosis of trichinellosis.更多还原