牛病毒性腹泻病毒E^rns84-170aa蛋白的可溶性表达及抗体间接ELISA检测方法的建立

Soluble expression of E protein of BVDV and establishment of indirect ELISA to detect bovine viral diarrhea virus antibodies

为建立一种检测牛病毒性腹泻病毒(BVDV)抗体的ELISA方法,对E^rns蛋白一个包含2个预测抗原表位的96氨基酸蛋白编码序列,经密码子优化后进行基因合成,构建重组表达载体pET-E84-170aarns,转化表达菌株BL21(DE3),在28℃用0.1 mmol/L IPTG诱导重组蛋白表达。将重组E84-170aarns蛋白通过Ni^2+-NTA树脂亲和层析方法纯化后,用Western-blot分析其抗原性。结果显示,重组E84-170aarns蛋白以完全可溶形式在大肠杆菌中高效表达,经Ni2+-NTA树脂纯化获得重组E84-170aarns蛋白的浓度和纯度分别为1.5 mg/mL和94%。Western-blot显示,该重组蛋白对BVDV阳性血清具有很强的抗原反应性。用纯化的重组E84-170aarns蛋白建立间接ELISA方法(E84-170aarns-iELISA)的结果显示,所建立方法的最佳抗原包被浓度为1.0μg/mL,血清稀释度为1∶100,酶标二抗的稀释度为1∶5000,血清D450的阴阳性临界值为0.283;该方法仅对BVDV阳性血清呈特异性反应,对1∶1280倍稀释的阳性血清仍可检出,批内试验和批间试验的变异系数均小于10%。用该方法对487份牛血清进行BVDV抗体检测,检出128份阳性血清。本研究建立的E84-170aarns-iELISA方法为BVDV抗体检测及流行病学调查提供了一种有效手段。

In order to establish an indirect ELISA for detection of BVDV antibodies,a genetic sequence,which encoded protein that composed of 96 amino acids(84-170 aa)of E^rnsprotein and conteined 2 predicted antigen eptopes,was synthesized after codon optimization,and recombinant expression vector of p ET-E84-170aarnswas constructed and then transformed into E.coli BL21(DE3)strain.At 28℃,the recombinant vector was induced and expressed by 0.1 mmol/L IPTG.After purification by affinity chromatography of Ni^2+-NTA resin,the antigenicity of recombinant protein was analyzed by Western-blot.The results of SDS-PAGE showed that ecombinant E84-170aarnsprotein was efficiently expressed in E.coli as a fully soluble form.After purified by Ni2+-NTA affinity chromatography,the concentration and purity of the purified the recombinant E84-170aarnsprotein was 1.5 mg/m L and 94%,respectively.The results of Western-blot showed that the recombinant E84-170aarnsprotein was specifically bounded to the anti-BVDV positive serum.Futhermore,an indirect enzyme-linked immunosorbent assay(E84-170aarns-i ELISA)based on the recombinant E84-170aarnsprotein was established,and the optimal dilutions of antigen was 1∶1500(1.0μg/m L),the optimal dilutions of serum was 1∶100,and the optimal dilutions of goat anti-bovine Ig G conjugate was 1∶5000.The cut-off value for the method was 0.283,and the analytical sensitivity of the method was 1∶1280.The results of specific evaluation showed that anti-BVDV sera did not cross-react with any other common bovine anti-virus serum.The coefficients of variation of the intra-assay and inter-assay were lower than 10%.E84-170aarns-i ELISA was applied to detect antibodies against BVDV in 487 clinical serum samples from cow,and 128 were detected to be positive.In conclusion,an effective method was provided for detection of antibodies against BVDV in serum samples and the epidemiological monitoring of BVD by the E84-170aarns-i ELISA established in this study.