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目的 干扰素具有广谱抗病毒活性，在临床上具有重要应用价值，开发犬长效干扰素是当前宠物抗病毒药物研究的重点。方法 本研究将犬IFN-α3成熟肽基因通过密码子优化后，构建到pPICZα A酵母表达载体上，上、下游分别引入Eco RⅠ和XhoⅠ限制性酶切位点，阳性质粒经SacⅠ限制酶线性化后，电转化X33酵母菌感受态细胞，在含有100 mg/mL Zecoin抗性YPD培养基上挑取规则菌落，经AXO1鉴定引物PCR鉴定为阳性，阳性菌株经甲醇诱导表达，SDS-PAGE表明犬IFN-α3成功在酵母中分泌表达，表达产物大小为20～25 kDa，表达量0.95 mg/mL。应用微量细胞病变抑制法测定发酵原液及纯化rCaIFN-α3在MDCK上对VSV-GFP的抗病毒活性，发酵原液活性为8×106 IU/mL，纯化产物活性为2×108 IU/mg。在F81上对CPV、RDPV、MEV复制均有抑制作用，抑制效果无显著差异(P>0.05)。结果 根据标准曲线计算血清中rCaIFN-α3血药浓度，在注射后40 h达到药物浓度峰值，为458.733 pg/mL，随后浓度缓慢下降。注射rCaIFN-α剂量4×105 IU/kg、4×106 IU/kg、4×107 IU/kg，对体温、白细胞、淋巴细胞、中性粒细胞数量没有影响，主要组织HE染色无明显病理变化。与辅助治疗组和RDPV对照组相比，4×105 IU/kg剂量注射rCaIFN-α3预防用药联合辅助治疗组和rCaIFN-α3联合辅助治疗组显著降低不同时间貉粪便中病毒载量，其中预防用药组效果最好，有效率80%，死亡率20%。结论综上所述，本研究建立了高效表达r CaIFN-α3的毕赤酵母表达系统，表达产物具有显著的抗病毒活性和良好的安全性，为开发长效重组犬IFN-α抗病毒制剂奠定了基础。
Abstract：Objective Interferon has broad-spectrum antiviral activity and has important clinical application value. The development of canine long-acting interferon is the focus of current pet antiviral drug research.Methods the mature peptide gene of canine IFN-α3 was codon-optimized and inserted into the Pichia pastoris expression vector pPICZα A, with Eco R Ⅰ and Xho Ⅰ restriction sites introduced at the 5′ and 3′ ends, respectively. The positive plasmid was linearized with SacI and transformed into competent P. pastoris X33 cells by electroporation. Transformants were selected on YPD plates containing 100 μg/mL Zeocin. Positive colonies were identified by PCR using AOX1-specific primers. SDS-PAGE analysis confirmed that canine IFN-α3 was successfully expressed and secreted in yeast, with a molecular mass of approximately 20 – 25 kDa and an expression yield of 0.95 g/L. The antiviral activity of recombinant canine IFN-α3(rCaIFN-α3) was evaluated using the micro-cytopathic effect inhibition assay. The antiviral titer against VSV-GFP in MDCK cells was 8 × 106 IU/mL in fermentation broth and 2 × 108 IU/mg in the purified product. rCaIFN-α3 also inhibited the replication of CPV, RDPV, and MEV in F81 cells, with no significant differences among these viruses(P > 0.05).Results Serum pharmacokinetics of rCaIFN-α3 were determined using a commercial ELISA kit, and concentrations were calculated from a standard curve. The serum drug concentration reached a peak of 458.733 pg/mL at 40 h post-injection and declined gradually thereafter. Administration of rCaIFN-α3 at doses of 4 × 105, 4 × 106, and 4 × 107 IU/kg caused no observable effects on body temperature, white blood cell count, lymphocyte count, or neutrophil count. Histopathological examination of major tissues revealed no significant pathological alterations. Compared with the adjuvant therapy group and the RDPV control group, both the prophylactic and adjuvant combination treatment groups receiving 4 × 105 IU/kg of rCaIFN-α3 exhibited significantly reduced the viral loads in the feces of raccoon dogs at different time points, with the prophylactic group showing the best efficacy(80 % protection, 20 % mortality). Conclusion Overall, this study established an efficient Pichia pastoris expression system for producing biologically active rCaIFN-α3 and demonstrated its potent antiviral activity and good safety profile, providing a foundation for the development of long-acting recombinant canine IFN-α antiviral formulations.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250111
中图分类号:S852.65
引用信息:
[1]张东亮,刘方圆,张海玲,等.重组犬干扰素-α3抗病毒活性分析及初步应用[J].病毒学报,2026,42(01):242-252.DOI:10.13242/j.cnki.bingduxuebao.250111.
基金信息:
中国农业科学院特产研究所科技创新项目（项目号：CAAS-ASTIP-ISAPS-2021-1）,题目：基于微纳米微球缓释的犬重组干扰素-α的制备及初步应用~~