重组犬α6干扰素的酵母表达及其抗病毒活性评价

试验旨在构建犬α6干扰素毕赤酵母表达系统，并对其进行优化和筛选，以期获得高活性的重组犬α6干扰素（CaIFN-α6）。根据CaIFN-α6基因序列，按毕赤酵母菌密码子偏好性对CaIFN-α6全基因序列进行优化与合成，用Xho Ⅰ和Not Ⅰ双酶切将其连接至载体pPICZαA中，构建pPICZαA-CaIFN-α6重组表达质粒，转化大肠杆菌DH5α感受态细胞。提取质粒pPICZαA-CaIFN-α6并线性化，电转入酵母感受态细胞X33中制备重组菌。采用甲醇进行诱导表达，收集上清，超滤浓缩，最终获得纯化的重组CaIFN-α6。利用BCA法测得纯化后的CaIFN-α6蛋白浓度为1.5 mg/mL，Western blotting分析表明CaIFN-α6蛋白具有良好的反应原性，SDS-PAGE显示其纯度约在95%以上，MDCK/VSV法检测其效价为2.37×10⁷ IU/mL，比活性为1.58×10⁷ IU/mg。结果表明犬α6干扰素在毕赤酵母pPICZαA表达载体系统中成功表达，且具有较高的生物活性，为后期的犬病毒病的临床预防与治疗提供了良好的支撑。

Yeast Expression of Recombinant Canine Alpha Interferon 6 and Evaluation of Their Antiviral Activities

The aim of the experiment was to construct the expression system of canine interferon alpha 6 (CaIFN-α6) in Pichia pastoris,optimize and screen the expression system in order to obtain recombinant canine interferon alpha 6 with high activity.According to the sequence of CaIFN-α6 gene and the codon preference of Pichia pastoris,the whole gene sequence of CaIFN-α6 was optimized and synthesized.The recombinant expression plasmid of pPICZαA-CaIFN-α6 was constructed using Xho Ⅰ and Not Ⅰ and linked to the vector pPICZαA.The recombinant expression plasmid was transformed into E.coli DH5α competent cells.The plasmid pPICZαA-CaIFN-α6 was extracted and linearized.The recombinant strain was prepared by electroporation into yeast competent cell X33.The recombinant CaIFN-α6 was purified by methanol induction,supernatant collection and ultrafiltration concentration.The concentration of purified CaIFN-α6 protein was 1.5 mg/mL by BCA.Western blotting analysis showed that CaIFN-α6 protein had good reactivity.SDS-PAGE showed that the purity of CaIFN-α6 protein was above 95%.The titer of CaIFN-α6 protein was 2.37×10⁷ IU/mL by MDCK/VSV detection and the specific activity was 1.58×10⁷ IU/mg.The results showed that CaIFN-α6 was successfully expressed in Pichia pastoris pPICZαA expression vector system with high biological activity,which provided a good support for the clinical prevention and treatment of canine viral disease in the later stage.