重组犬瘟热病毒抗原表位T'TB和犬细小病毒vp2基因的真核表达

Eukaryotic expression of recombined with T'TB antigen epitope of canine distemper virus and vp2 gene of canine parvovirus

将人工合成的犬瘟热病毒抗原表位T'TB基因克隆至Bac-to-Bac杆状病毒表达系统中的转移载体pFastBacHTc上,命名为pFastBacHTc-T'TB。应用PCR方法扩增犬细小病毒vp2基因,将其克隆至pMD18-T载体,测序后将vp2基因插入T'TB基因下游,命名为pFastBacHTc-T'TB-VP2,进而转化含穿梭载体Bacmid的感受态细胞DH10Bac中,获得携带犬瘟热病毒T'TB细胞表位和犬细小病毒vp2基因的重组转染质粒Bacmid-BacHT-T'TB-VP2,转染昆虫细胞Sf-9后获得融合重组T'TB-VP2蛋白,大小约为70ku。经Westernblot分析,表达的蛋白可与犬细小病毒阳性血清反应,具有良好的免疫原性。

Canine distemper virus T^ITB antigen epitope gene was cloned into the transfer vector pFastBacHTc of Bac-to-Bac baculovirus expression system and named pFastBacHTc-T^ITB, vp2 gene of canine parvovirus was amplified by PCR and cloned into the pMD18-T vector. After sequencing, vp2 gene was cloned into pFastBacHTc-T^ITB and named pFastBacHTc-T^ITB-VP2. pFastBacHTc-T^ITB-VP2 was transfered into E.coli DH10Bac, which contains baculovirus shuttle vector bacmid. Recombinant bacmid （Bacmid-BacHT-T^ITB-VP2） was constructed, and verified by PCR. The reconibinant bacmid was transfected into Sf-9 insect cells for T^ITB-VP2 fusion protein expression, The recombinant protein had MW of 70 ku and could react with CPV antiserum in Western blot.