牛病毒性腹泻病毒(BVDV)E0蛋白原核表达及间接ELISA方法的建立

BVDV-E0 gene prokaryotic expression and establishment of indirect ELISA method

牛病毒性腹泻病毒(BVDV)能够引起牛羊的持续感染,临床上很难对其根除,建立该病有效检测方法对净化畜群具有重要意义。本研究构建BVDV E0蛋白基因重组表达载体在大肠杆菌中诱导表达,用重组的E0蛋白为包被抗原,确定抗原最佳包被浓度为5μg/mL,最佳血清稀释比为1∶10,最佳封闭液为5%脱脂奶粉,最佳封闭条件为37℃封闭2 h,酶标二抗稀释度为1∶5000,作用时间为30 min,样品临界值X+2S为0.278。该方法与IDEXX的BVDV间接ELISA试剂盒比较总符合率为86%,与口蹄疫病毒、牛支原体、牛传染性鼻气管炎病毒、牛副结核、牛布鲁氏菌病阳性血清无交叉反应。该检测方法能够用于BVDV抗体水平的临床检测,可为感染病畜的合理淘汰提供实验依据。

Bovine viral diarrhea virus caused persistent infection in cattle and sheep and was hard to eradicate, establishingaeffective method to detect the antibody of bovine viral diarrhea virus （BVDV） was significant for cleaning out infected animals. Theenvelope protein E0 of BVDV was successfully expressed in expression system. The recombinant and purified E2 protein wascoated in ELISA plates as antigen. The optimal working parameters were determined as follows, the antigen concentration was5μg/mL, the serum dilution was1∶10袁the optimal coating buffer was 5% skimmed milk, the block time was 2h at 37 ℃袁thesecond-antibody concentration was1∶5000 for 30 min袁the cut-off value was 0.278. Compared with foreign commercial kits, thetotal coincidence rate was 86% . There was no cross-reaction with the positive sera of , FMDV, BPIV-3, IBRV,and . The indirect ELISA method can be applied in BVDV antibody detection forepidemiological investigation and offer experimental evidence for eliminating infected animals.