禽脑脊髓炎病毒VP1蛋白间接ELISA检测方法的建立

Establishment of indirect ELISA for detecting avian encephalomyelitis virus with VP1 as coating protein

对AEV陕西分离株VP1基因进行原核表达,采用SDS-PAGE和Western blotting检测表达产物;以纯化的蛋白为包被抗原建立ELISA方法,并对反应条件进行优化。结果表明,VP1蛋白在大肠杆菌中成功表达,产物约为50 000的融合蛋白,具有良好的反应原性;优化的ELISA最佳工作条件为:重组抗原包被质量浓度3.8mg/L,37℃2h后4℃过夜,1%BSA 37℃封闭2h,待检血清1∶50稀释37℃孵育0.5h,酶标抗体1∶4 000稀释,37℃作用30min,底物37℃显色5min,临界值为0.360;建立的ELISA方法特异性强、重复性好、敏感性高;临床检测180份样品,与美国IDEXX公司AEV抗体检测试剂盒检测结果符合率为91.9%。该方法可用于临床样品的大批量检测。

Prokaryotic expression of VP1 gene of AEV Shaanxi strain was done. SDS-PAGE and Western blotting were employed for analyzing the recombinant protein. The purified recombinant protein was used for establishment of an indirect ELISA for detecting of anti-AE antibody. The resuits showed that target fusion protein was successfully expressed in E. coli, it was about 50 000 in molecular weight and has a good reactogenicity;the optimal reaction condition were determined. coating antigen at 4℃ overnight after 37℃ for 2 h at a concentration of 3.8 mg/L, 1%BSA as blocking agent at 37℃ for 2 h,serum sample（1 ： 50） at 37℃ for 30 min,HRP labeled goat antichicken（1 ： 4 000） at 37℃ for 30 min,the substrate TMB for ELISA being incubated at 37℃ for 5 min;the threshold of positive and negative sera was 0. 360 by statistical analysis. T.he ELISA assay was confirmed to have a good repeatability, sensitivity and specificity. The coincidence rate of the two assays was up to 91.9% compared with the bird AE-Ab ELISA kit of IDEXX by detecting 180 clinical sera. The method can be used for the detection of large quantities of clinical samples.