牛病毒性腹泻病毒E2蛋白的截短表达与鉴定

Expression and characterization of truncate E2 protein of bovine viral diarrhea virus in Escherichia coli

利用牛病毒性腹泻病毒(BVDV)BA株接种MDBK细胞,提取病毒RNA。参照已发表的BVDV基因组序列,利用Oligo 6生物学软件设计扩增E2基因的1对引物,引入酶切位点并去掉E2蛋白的跨膜区及疏水区。通过RT-PCR扩增了长约1 000 bp的E2基因片段,克隆到pMD18-T载体上,酶切并测序鉴定。然后将目的片段进一步定向克隆到pET30a表达载体,转化BL21表达菌。取转化菌培养,并用IPTG诱导,获得了以包涵体形式表达的重组蛋白。将重组蛋白变性、纯化和复性后,用免疫印迹与间接ELISA检测表明纯化的重组蛋白具有良好的免疫原性,为牛病毒性腹泻病毒诊断试剂的研制奠定了基础。

An truncate E2 of bovine viral diarrhea virus （BVDV） gene without transmembrane domain and hydrophobic region was amplified by RT-PCR from RNA of BVDV BA strain. The target fragment of about 1 000 bp was sequenced and cloned into pET30a vector. The recombinant plasmid was transformed into E.coli BL21 and induced with IPTG for expression. Recombinant protein was mainly expressed in inclusion body forms and could be recognised by specific antibodies in Western blot and indirect enzyme-linked immtmosorbent assay.