猪源牛病毒性腹泻病毒SD0803株E2蛋白的表达及抗体制备

Expression of SD0803 Strain from Pig and Preparation of Antibodies E2 Protein of Bovine Viral Diarrhea Virus SD0803 Strain

表达去除SD0803E2基因跨膜疏水区的sE2蛋白并制备其抗体,为猪源BVDV诊断奠定基础。RT-PCR扩增SD0803株sE2基因,构建重组原核表达载体pGEX-4T-1-sE2,转化BL21(DE3),IPTG诱导蛋白表达,以纯化的重组蛋白为抗原免疫家兔制备多克隆抗体。采用间接ELISA、Western blot、间接免疫荧光等方法鉴定获得的抗体。结果显示,抗体效价最高可达1∶256 000;制备的抗体可与rsE2蛋白特异性结合;该抗体可特异性识别BVDV感染MDBK细胞表达的E2蛋白,显示抗体高度的特异性。成功构建pGEX-4T-1-sE2原核表达质粒,诱导其表达的蛋白免疫家兔,成功制备的猪源BVDV SD0803株E2抗体。

To provide the method for detecting the bovine viral diarrhea virus from pig,sE2 gene,which removed C-terminal transmernbrane of E2 gene of BVDV SD0803 isolate,sE2 protein and anti-sE2 antibody were prepared.sE2 gene,which was amplified from SD0803 strain by PCR,was cloned into pGEX-4T-1 and transformed into BL21（DE3）.After induction with IPTG,the recombinant protein was obtained.Rabbits were immunized with the purified protein as antigen,and antiserum was acquired.The polyclonal antibodies were analyzed by indirect ELISA,Western blot and indirect immunofluorescence assay.The antiserum titer was determined by indirect ELISA,and was 1∶256 000.The results of Western blot and indirect immunofluorescence assay confirmed the antibodies reacted specifically with the protein expressed.A recombinant SD0803 sE2 protein and the specific antibodies were obtained.