犬瘟热病毒抗原表位T’TB和犬细小病毒VP2蛋白的共表达及免疫小鼠特异性抗体的测定

Co-expression of T'TB antigen epitope of canine distemper virus and VP2 protein of canine parvovirus and determination of specific antibodies in immunized mice

应用PCR方法扩增犬细小病毒VP2基因,将其克隆至Bac-to-Bac杆状病毒表达系统中的转移载体pFastBacHTc上,命名为pFastBacHTc-VP2,将人工合成的犬瘟热病毒抗原表位基因T’TB克隆至VP2基因的上游,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,将其转染昆虫细胞Sf-9后获得融合重组T’TB-VP2蛋白,大小约为70ku。经Westernblot分析,结果显示:表达的蛋白具有良好的免疫原性。表达的重组蛋白在无佐剂参与的情况下,按确定的免疫程序免疫6～8周龄的BALB/c小鼠,检测小鼠的体液免疫学指标。结果表明:表达蛋白能诱导小鼠产生抗CDV和CPV的特异性中和抗体。本实验为重组犬瘟热与犬细小病毒新型亚单位疫苗的研制奠定了重要的物质基础。

The VP2 gene of canine parvovirus （CPV） was amplified by applying the PCR approach. The amplicon was ligated into the transfer vector pFastBacHTc of Bac-to-Bac baculovirus expression system. The vector carrying VP2 gene was named pFastBacHTc-VP2. The Artificially synthesized T＇ TB antigen epitope gene of canine distemper virus （CDV） was inserted into the vector pFastBacHTc-VP2 at the upstream position of VP2 to create a vector carrying both VP2 gene and T＇ TB gene named pFastBacHTc-T＇ TB-VP2. pFastBacHTc-T＇ TB-VP2 was transferred into E. coil DH10Bac that contains baculovirus shuttle vector bacmid to obtain recombinant transfecting plasmid Bacmid-BacHT-T＇ TB-VP2. Bacmid-BacHT- T＇ TB-VP2 was then used to transfect Sf-9 insect cells to express recombinant protein T＇ TB-VP2. Western blot analysis in- dicated that the molecular weight of T＇ TB-VP2 was 70 ku and could react with CPV and CDV antiserum. Purified T＇ TB- VP2 protein was used to challenge BALB/c mice at the age of 6 -8 weeks without adjuvant. ELISA testing indicated that T＇TB-VP2 protein was able to induce high level of antibodies specifically neutralizing CDV and CPV. This study laid a foundation for developing the new sub-unit vaccine of canine distemper and canine parvovirus.