牛病毒性腹泻病毒E0基因重组慢病毒载体构建和鉴定

Construction and identification of lentiviral vector for bovine viral diarrhea virus E0 gene

为构建携带牛病毒性腹泻病毒(BVDV)E0基因的重组慢病毒质粒,并检测其在HEK-293T细胞中的表达,本研究通过合成BVDV E0基因序列(AJ133739.1),构建包含Ig K信号肽的p ZJ-CMV-e GFP-Ig K-E0重组质粒和不含信号肽的p ZJ-CMV-e GFP-E0重组质粒,将其采用PEI方法分别转染HEK-293T细胞包装慢病毒,并浓缩后测定滴度,收集转染后上清液进行western blot检测蛋白表达。通过酶切、PCR及测序鉴定表明E0基因正确整合至慢病毒载体中,western blot结果表明其能够在HEK-293T细胞中表达。本研究构建获得携带BVDV E0基因的重组慢病毒,为BVDV E0蛋白哺乳动物细胞中的表达及其免疫原性研究奠定基础。

To construct the recombinant lentivirus expressing E0 gene of bovine viral diarrhea virus （BVDV）, the E0 gene according to the sequence of noncytopathie BVDV strain NADL （AJ133739.1） was synthesized and inserted into lentiviral vector of pZJ-CMV-eGFP to construct the recombinant plasmids of pZJ-CMV-eGFP-IgK-E0 and pZJ-CMV-eGFP-E0 with or without IgK signal peptide, respectively. In addition, the recombinated plasmids were transfected into HEK-293T cell with two other helper plasmids to pack the lentivirus, respectively. Moreover, the recombinant lentivirus expressing E0 gene of BVDVs were generated, and the E0 protein was expressed with a molecular weight of 45 ku from the recombinant lentivirus detected by western blot. The constructions of recombinant lentivirus facilitate further studies on the immunogenicity of BVDV E0 protein and development of the vaccine againt BVDV.