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

Abstract

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

CLC Number:

S859.797

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.

Figures/Tables 10

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References 28

1	崔然. 猪α、β、γ干扰素的表达及抗病毒活性的检测[D]. 泰安: 山东农业大学, 2012.
	CUI R. Expression and antiviral activity of pig α, β and γ[D]. Taian: Shandong Agricultural University, 2012. (in Chinese)
2	孙波涛, 张涛清, 孙冰洁, 等. 重组猪干扰素α的真核表达纯化及其单克隆抗体制备[J]. 中国兽医科学, 2023, 53 (6): 684- 690.
	SUN B T , ZHANG T Q , SUN B J , et al. Eukaryotic expression and purification of recombinant porcine interferon-α and preparation of its monoclonal antibodies[J]. Chinese Veterinary Science, 2023, 53 (6): 684- 690.
3	DIAZ-SAN SEGUNDO F , MORAES MP , DE LOS SANTOS T , et al. Interferon-induced protection against foot-and -mouth disease virus infection correlates with enhanced tissue-specific innate immune cell infiltration and interferon-stimulated gene expression[J]. J Virol, 2010, 84 (4): 2063- 2077. doi: 10.1128/JVI.01874-09
4	朱江巍. 重组猪α干扰素治疗高致病性猪蓝耳病疗效观察[J]. 中国畜禽种业, 2018, 14 (3): 126. doi: 10.3969/j.issn.1673-4556.2018.03.108
	ZHU J W . Efficacy of recombinant porcine α interferon in the treatment of highly pathogenic porcine PRRSV[J]. The Chinese Livestock and Poultry Breeding, 2018, 14 (3): 126. doi: 10.3969/j.issn.1673-4556.2018.03.108
5	SONG B W , WEI W K , LIU X Y , et al. Recombinant porcine interferon-α decreases pseudorabies virus infection[J]. Vaccines (Basel), 2023, 11 (10): 1587. doi: 10.3390/vaccines11101587
6	CHEN J , XU W , LI P H , et al. Antiviral effect of pIFNLs against PEDV and VSV infection in different cells[J]. Int J Mol Sci, 2022, 23 (17): 9661. doi: 10.3390/ijms23179661
7	BARBÉ F , SAELENS X , BRAECKMANS D , et al. Role of IFN-l during the acute stage of a swine influenza virus infection[J]. Res Vet Sci, 2010, 88 (1): 172- 178. doi: 10.1016/j.rvsc.2009.07.001
8	曹瑞兵, 周国栋, 陈溥言. 猪α干扰素对猪圆环病毒2型亚单位疫苗免疫效果的影响[J]. 畜牧兽医学报, 2009, 40 (6): 867- 872. doi: 10.3321/j.issn:0366-6964.2009.06.014
	CAO R B , ZHOU G D , CHEN P Y . Effect of porcine interferon alpha on the immunogenicity of porcine circovirus type 2 subunit vaccine[J]. Acta Veterinaria et Zootechnica Sinica, 2009, 40 (6): 867- 872. doi: 10.3321/j.issn:0366-6964.2009.06.014
9	YU H Y , QU M S , ZHANG J L , et al. Recombinant porcine interferon alpha enhances immune responses to killed porcine reproductive and respiratory syndrome virus vaccine in pigs[J]. Viral Immunol, 2019, 32 (9): 383- 392. doi: 10.1089/vim.2019.0092
10	赵娜, 陈国华, 房永祥, 等. 猪IFN-γ和CpG基序双因子重组质粒增强亚洲Ⅰ型口蹄疫灭活病毒抗原免疫水平的研究[J]. 畜牧兽医学报, 2010, 41 (10): 1281- 1289.
	ZHAO N , CHEN G H , FANG Y X , et al. Enhanced immunity to killed FMDVA antigen by dual factors recombinant plasmid of porcine interfereon-gamma and CpG motifs[J]. Acta Veterinaria et Zootechnica Sinica, 2010, 41 (10): 1281- 1289.
11	LIU L R , FAN W H , ZHANG H , et al. Interferon as a mucosal adjuvant for an influenza vaccine in pigs[J]. Virol Sin, 2019, 34 (3): 324- 333. doi: 10.1007/s12250-019-00102-7
12	荣柳, 沈国顺. 猪α干扰素的研究进展[J]. 黑龙江畜牧兽医, 2015 (17): 84- 86.
	RONG L , SHEN G S . Research progress of porcine interferon alpha[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015 (17): 84- 86.
13	张杏, 李爽, 崔栩, 等. 猪α-干扰素表达系统及其应用的研究进展[J]. 中国兽药杂志, 2014, 48 (10): 60- 62.
	ZHANG X , LI S , CUI X , et al. Porcine interferon-α: advances in expression system and application[J]. Chinese Journal of Veterinary Drug, 2014, 48 (10): 60- 62.
14	董世娟, 于瑞嵩, 李震, 等. 重组猪α干扰素的纯化及其对猪繁殖与呼吸综合征病毒的抑制作用[J]. 中国兽医科学, 2018, 48 (5): 559- 565.
	DONG S J , YU R S , LI Z , et al. Purification of recombinant porcine interferon-α and its inhibitory effect on porcine reproductive and respiratory syndrome virus[J]. Chinese Veterinary Science, 2018, 48 (5): 559- 565.
15	王彦彬, 崔保安, 陈红英, 等. 猪干扰素α在昆虫细胞中分泌表达及其抗病毒活性检测[J]. 中国农业科学, 2009, 42 (4): 1435- 1441. doi: 10.3864/j.issn.0578-1752.2009.04.038
	WANG Y B , CUI B A , CHEN H Y , et al. Secreted expression of porcine interferon-alpha in insect cells and its antiviral activity detection[J]. Scientia Agricultura Sinica, 2009, 42 (4): 1435- 1441. doi: 10.3864/j.issn.0578-1752.2009.04.038
16	ZHAO J , YU H Y , GAN L , et al. Soluble expression, protein purification and quality control of recombinant porcine interferon-α[J]. Kafkas Univ Vet Fak Derg, 2017, 23 (5): 825- 829.
17	刘延亭, 宫瑞雪, 张美美, 等. 重组猪α干扰素的安全性及体外抗病毒效果评价[J]. 今日畜牧兽医, 2021, 37 (4): 11- 12. doi: 10.3969/j.issn.1673-4092.2021.04.007
	LIU Y T , GONG R X , ZHANG M M , et al. Safety and evaluation of antiviral effect of recombinant porcine α interferon in porcine[J]. Today Animal Husbandry and Veterinary Medicine, 2021, 37 (4): 11- 12. doi: 10.3969/j.issn.1673-4092.2021.04.007
18	WANG P T , XIA L , LIANG X L , et al. Expression of porcine interferon-α and its bioactivity analysis in vitro and in vivo[J]. Bioprocess Biosyst Eng, 2021, 44 (3): 473- 482. doi: 10.1007/s00449-020-02457-8
19	LEE G , KIM A , KANG H R , et al. Porcine interferon-α linked to the porcine IgG-Fc induces prolonged and broad-spectrum antiviral effects against foot-and-mouth disease virus[J]. Antiviral Res, 2024, 223, 105836. doi: 10.1016/j.antiviral.2024.105836
20	朱亚召, 王明丽, 俞海洋, 等. 表达重组猪α干扰素的大肠埃希工程菌分批发酵动力学模型的建立[J]. 中国兽药杂志, 2018, 52 (9): 9- 15.
	ZHU Y Z , WANG M L , YU H Y , et al. Building a fermentation kinetic model for the expression of recombinant porcine interferon-α in bioengineered Escherichia coli[J]. Chinese Journal of Veterinary Drug, 2018, 52 (9): 9- 15.
21	何新柳. 猪α干扰素在大肠杆菌中的表达及生产工艺研究[D]. 广州: 华南理工大学, 2017.
	HE X L. Expression and production process of porcine interferon alpha in Escherichia coli [D]. Guangzhou: South China University of Technology, 2017. (in Chinese)
22	FANG J Y , ZHANG Q X , XI Y Y , et al. Analysis of the differential expression and antiviral activity of porcine interferon-α in vitro[J]. Int J Pept Res Ther, 2023, 29 (3): 42- 42. doi: 10.1007/s10989-023-10508-3
23	HUANG L , CAO R B , WANG N , et al. The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α[J]. Cytokine, 2012, 57 (1): 37- 45. doi: 10.1016/j.cyto.2011.10.011
24	王朋涛, 梁秀丽, 徐盟龙, 等. 新型猪α干扰素YNS突变体的构建及其抗病毒活性检测[J]. 畜牧与兽医, 2020, 52 (12): 116- 120.
	WANG P T , LIANG X L , XU M L , et al. Construction of a novel mutant of porcine interferon-α-YNS and detection of its antiviral activity[J]. Animal Husbandry & Veterinary Medicine, 2020, 52 (12): 116- 120.
25	李爽, 张杏, 崔栩, 等. 猪α干扰素基因突变、表达及其高效抗病毒活性筛选研究[J]. 中国兽药杂志, 2015, 49 (4): 12- 16.
	LI S , ZHANG X , CUI X , et al. The mutations, expression and antiviral activity screening of porcine interferon-α[J]. Chinese Journal of Veterinary Drug, 2015, 49 (04): 12- 16.
26	WU H X , QI H S , WANG B , et al. The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant[J]. J Biol Chem, 2023, 299 (11): 105347. doi: 10.1016/j.jbc.2023.105347
27	龙琴琴, 魏敏, 王雨婷, 等. 羊口疮病毒与宿主的博弈: 免疫应答与病毒免疫逃逸机制[J]. 畜牧兽医学报, 2023, 54 (5): 1845- 1853.
	LONG Q Q , WEI M , WANG Y T , et al. The battle between Orf virus and host: immune responseand viralImmune evasion mechanisms[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (5): 1845- 1853.
28	ZHANG R , TANG J . Evasion of I interferon-mediated innate immunity by pseudorabies virus[J]. Front Microbiol, 2021, 12801257

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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Prokaryotic Expression of PoIFN-α 8s and Identification of Its Activity in vitro and in vivo

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