猪α干扰素8s突变体原核表达及其体内外活性鉴定

doi:10.11843/j.issn.0366-6964.2025.05.038

摘要/Abstract

摘要：

猪α干扰素(PoIFN-α)可用于治疗和预防病毒感染，但天然猪α干扰素抗病毒活性低，寻找高生物活性的突变体序列对猪传染性疾病的防治措施研究具有重要意义。运用生物信息学方法分析和比对PoIFN-α的各亚型氨基酸序列，对天然PoIFN-α序列的八个位点进行定点突变构建PoIFN-α 8s突变体并进行原核表达纯化；获得重组蛋白后在PK-15细胞上针对水疱性口炎病毒(VSV)进行活性测定，同时在小鼠体内验证PoIFN-α 8s突变体对猪伪狂犬病病毒(PRV)的抑制能力。结果发现，重组PoIFN-α 8s测得抗病毒活性为1.13×10⁷ IU·mg^-1，对比PoIFN-α产品，PoIFN-α 8s可降低PK-15细胞上VSV的病毒滴度和基因拷贝数达10倍以上，具有更高的体外抑制VSV增殖能力；动物试验表明，PoIFN-α 8s可降低感染PRV小鼠体内各器官的病毒载量。综上，本研究成功构建高效的PoIFN-α 8s突变体，并验证了其良好的体内外抗病毒活性。

关键词: 猪α干扰素, 原核表达, 突变, 生物信息学分析, 抗病毒活性

Abstract:

Porcine α interferon (PoIFN-α) can be used to treat and prevent viral infection. However, the natural pig α interferon has low antiviral activity, so it is of great significance to find the mutant sequence of high biological activity for the prevention and control measures of swine infectious diseases. After using bioinformatics methods to analyze and compare the amino acid sequences of various subtypes of PoIFN-α, PoIFN-α 8s mutants were constructed by site-directed mutagenesis of eight sites in the native PoIFN-α sequence and purified by prokaruclear expression. After obtaining the recombinant protein, activity assays were conducted against the vesicular stomatitis virus (VSV) in PK-15 cells and validated the inhibitory effect of the PoIFN-α 8s mutant on the pseudorabies virus (PRV) in mice. The results showed that, antiviral activity of recombinant PoIFN-α 8s was 1.13×10⁷ IU·mg^-1, which reduced the viral load and gene copy number of VSV on PK-15 cells by more than 10 times compared to the PoIFN-α product. Animal experiments indicated that PoIFN-α 8s can reduce the viral load in various organs of mice infected with PRV. Thus, this study successfully constructed an efficient PoIFN-α 8s mutant and verified its good antiviral activity both in vivo and in vitro.

Key words: porcine α interferon, prokaryotic expression, mutations, bioinformatics analysis, antiviral activity

中图分类号:

S859.797

吴琼, 李凌丹, 袁辉, 宾晨, 邓可, 李伟, 叶十一, 李国攀, 沈青春, 熊涛. 猪α干扰素8s突变体原核表达及其体内外活性鉴定[J]. 畜牧兽医学报, 2025, 56(5): 2413-2423.

WU Qiong, LI Lingdan, YUAN Hui, BIN Chen, DENG Ke, LI Wei, YE Shiyi, LI Guopan, SHEN Qingchun, XIONG Tao. Prokaryotic Expression of PoIFN-α 8s and Identification of Its Activity in vitro and in vivo[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2413-2423.

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引物名称 Primer names	核苷酸序列(5′→3′) Nucleotide Sequences (5′→3′)	扩增片段长度/bp Amplification fragment length	退火温度/℃ Tm
VSV-G-qF	ACGCTCGGCTTCCTCTCC	181	60
VSV-G-qR	GGTAGTCGTCGCTCTCGTG
PRV-gH-qF	ACGCTCGGCTTCCTCTCC	215	60
PRV-gH-qR	GGTAGTCGTCGCTCTCGTG

名称Name	AB369102	PoIFN-α 8s
分子式Formula	C₈₃₀H₁₃₀₉N₂₄₃O₂₄₉S₁₀	C₈₃₃H₁₃₂₀N₂₄₂O₂₅₂S₉
分子质量Molecular weight	18 996.6	19 045.65
等电点Theoretical pI	6.4	5.97
溶解性值Solubility value	0.365	0.481
失稳指数Instability index	66.74	65.43
脂肪族指数Aliphatic index	80.60	82.95
亲水性平均值Grand average of hydropathicity	-0.396	-0.414
磷酸化位点Phosphorylation site	丝氨酸：8、25、28、38、69、137、153、155；苏氨酸：87、109、128	丝氨酸：8、25、28、69、116、137、153、155；苏氨酸：109、128

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