猪传染性胃肠炎病毒核衣壳蛋白的表达及在ELISA中的初步应用

重组质粒PET-N转化大肠杆菌（Escherichia coli） BL21菌株，在1.0 mmol/L IPTG和37℃条件下诱导，外源基因获得高效表达。Western blot检测证明，表达产物具有良好的反应原性。以纯化的蛋白为抗原建立了检测猪传染性胃肠炎抗体的间接ELISA方法，确定抗原最佳包被浓度为15 μg/mL，抗体稀释度1∶40，最适封闭液为5% FBS，血清反应时间为90 min，酶标二抗HRP SPA的工作浓度为1∶5 000，反应时间为60 min，底物在室温显色时间为10 min，待检血清阳性标准定为光密度OD450≥0.35。与 Svanova TGEV/PRCV 抗体诊断试剂盒比较，此方法的特异性、敏感性和符合率分别为93.8%、93.5%和93.5%。

Prokaryotic Expression of Nucleoprotein Gene of Transmissible gastroenteritis virus and Development of ELISA Based on the Expressed Protein

The recombinant PET-N plasmid, which includes the N gene of Transmissible gastroenteritis virus(TGEV), was transformed into Escherichia coli BL21(DE3) and expressed by 1.0 mmol/L IPTG at 37 ℃. The indirect ELISA for detecting TGEV nucleocapsid protein antibody was established after the reactionogenicity of the recombinant protein was proved by Western blot. The optional working circumstances for the ELISA are as follows: antigen concentration 15 μg/mL, serum dilution 1∶40, blocking solution 0.5% FBS, serum sample incubated for 90 min, concentration of HRP-spa 1∶5 000 incubated for 60 min, the substrate incubated at room temperature for 10 min, and the threshold value of ELISA OD450≥0.35 tried out with chess titration. Sensitivity , specificity and concordance of this method were 93.5%, 93.8% and 93.5%, respectively compared to Svanova TGEV/PRCV antibody diagnosis kit．