Abstract：Duck (Anas platyrhynchos) interferon α (IFNα) has a broad spectrum of antiviral activity, and A. platyrhynchos avian β-defensin 2 (AvBD2) has a broad spectrum of antibacterial activity. In this study, Pichia pastoris was used to secrete and express A. platyrhynchos IFNα -AvBD2 fusion protein, and the biological activity of IFNα -AvBD2 was tested, which would be of great significance for the prevention and control of duck epidemic diseases under existing unresistant breeding conditions. The previous work has successfully constructed the prokaryotic expression plasmid pET32a-AplIFNα -AvBD2, and successfully expressed it in Escherichia coli. In this study, the gene fragment of mature protein AplIFNα -AvBD2 was amplified by PCR using laboratory-preserved pET32a-AplIFNα-AvBD2 as a template to construct recombinant plasmid pPICZαA- AplIFNα -AvBD2; the linearized recombinant plasmid was electroporated into Pichia pastoris KM71H competent cells, and the positive recombinant bacteria were induced by methanol to express, and the biological activity of the expression product in vitro was studied. The results of SDS-PAGE and Western blot showed that the molecular weight of AplIFNα -AvBD2 recombinant protein was about 24 kD; agar diffusion method and microdilution method showed that the recombinant protein had strong antibacterial activity against Staphylococcus aureus and E. coli; cytopathic effect inhibition method detected that the antiviral activity of the recombinant protein was 1.2×105 U/mL, and the specific activity was 5.2×105 U/mg; MTT cytotoxicity test showed that the recombinant protein was non-toxic to African green monkey kidney cells (Vero). The above results indicate that AplIFNα -AvBD2 achieves secretory expression in P. pastoris, with both antibacterial activity and antiviral activity. This study lays the foundation for further application of AplIFNα- AvBD2.
[1] 柴强强 .2017. 猪 β 干扰素在毕赤酵母中的分泌型表达及条件优化[D]. 硕士学位论文, 湖南农业大学, 导师: 黎满香, pp. 29.
(Chai Q Q.2017. Expression and production optimization of porcine beta interferon in Pichia pastoris [D]. Thesis for M.S., Hunan Agricultural University, Supervisor: Li M X, pp. 29.)
[2] 范逸文 .2019. 鸡 INF-γ-C3d 在毕赤酵母中的表达及抗病毒活性测定 [D]. 硕士学位论文 , 河南科技大学 , 导师 : 牛明福 , pp. 40.
(Fan Y W.2019. Expression of chicken IFN-γ-C3d in Pichia pastoris and determination of its antiviral activity[D]. Thesis for M.S., Henan Uni‐versity of Science and Technology, Supervisor: Niu M F, pp. 40.)
[3] 高佩, 黎玉莲, 李亚玲, 等 . 2016. 鸭干扰素研究进展与应用[J]. 养禽与禽病防治, (02): 6-9.
(Gao P, Li Y L, Li Y L, et al. 2016. Progress and application of duck interferon [J]. Poultry Husbandry and Disease Control, (02): 6-9.)
[4] 韩清林 .2010. 天府肉鸭 α 干扰素的原核表达及抗病毒活性研究[D]. 硕士学位论文, 四川农业大学, 导师: 刘菲, pp. 33-45.
(Han Q L.2010. Studies on prokaryotic expression and biological activity of Tianfu Rou duck interferon α[D]. Thesis for M. S., Sichuan Agricultural University, Supervisor: Liu F, pp. 33-45.)
[5] 蔺艳君, 董彬 . 2019. β-防御素 130 在大肠杆菌中的串联表达 、纯化及生物活性分析[J]. 生物工程学报, 35(06): 1088-1096.
(Lin Y J, Dong B.2019. Tandem expression, purification and biological activity of recombinant multimeric β-defensin130 in Escherichia coli[J]. Chinese Journal of Biotechnology, 35(06): 1088-1096.)
[6] 彭统全, 邵建伟, 张润祥, 等 . 2015. 牛 α0 干扰素在毕赤酵母中的分泌表达及抗病毒活性鉴定[J]. 畜牧兽医学报, 46(01): 124-129.
(Peng T Q, Shao J W, Zhang R X, et al.2015. Expression of the bovine interferon alpha0 with antiviral in Pichia pastoris[J]. Acta Veterinaria et Zootechnica Sinica, 46(01): 124-129.)
[7] 石水琴, 魏海婷, 祁克宗, 等 . 2016. 鸡 β-防御素 2 在大肠杆菌中的串联表达及抗菌活性分析[J]. 中国兽医科学, 46(04): 516-523.
(Shi S Q, Wei H T, Qi K Z, et al.2016.Tandem expression of chicken β-defensin 2 and analysis of its antimicrobial activity[J]. Chinese Veterinary Science, 46(04): 516-523.)
[8] 宋天琪, 侯绍华, 郭晓宇, 等 . 2019. 重组犬 α6 干扰素的酵母表达及其抗病毒活性评价[J]. 中国畜牧兽医, 46(07): 2144-2150.
(Song T Q, Hou S H, Guo X Y, et al.2019.Yeast expression of recombinant canine alpha interferon 6 and evaluation of their antiviral activities[J]. China Animal Husbandry & Veterinary Medicine, 46(07): 2144-2150.
[9] 王瑞琴, 廖文艳, 马得莹, 等 . 2009. 鸭 β-防御素 2 基因的克隆、表达和表达产物的生物学特性分析[J]. 中国农业科学, 42(10): 3685-3692.
(Wang R Q, Liao W Y, Ma DY, et al.2009. Cloning, expression and bioactivity characterization of duck avian β-defensin 2[J]. Scientia Agricultura Sinica, 42(10): 3685-3692.)
[10] 张定勇, 孙蕾, 杨利敏, 等 . 2010. 猪 β 防御素 2 与猪 γ 干扰素在毕赤酵母中的融合表达及生物学活性比较[J]. 生物工程学报, 26(12): 1652-1659.
(Zhang D Y, Sun L, Yang L M, et al.2010. Fusion expression and bioactivity com‐
parison of porcine β -defensin-2 and porcine interferon-gamma in Pichia pastoris[J]. Chinese Journal of Biotechnology, 26(12): 1652-1659.)
[11] 张可心 .2012. 五个新的鸭 β-防御素的分离、鉴定及其抗病毒机制的初探[D]. 硕士学位论文, 东北农业大学, 导师: 马得莹, pp. 2-4.
(Zhang K X.2012. Isolation, characterization and mechanism of anti-viral activity of five novel duck avian beta-defensins[D]. Thesis for M. S., Northeast Agricultural University, Supervisor: Ma D Y, pp. 2-4.)
[12] Baghbeheshti S, Hadadian S, Eidi A, et al.2021. Effect of flexible and rigid linkers on biological activity of recombinant tetramer variants of S3 antimicrobial peptide[J]. International Journal of Peptide Research and Therapeutics, 27(1): 457-462.
[13] Cao Y P, Ma Q Q, Shan A S, et al.2012. Expression in Pichia pastoris and biological activity of avian β-defensin 6 and its mutant peptide without cysteines[J]. Protein and Peptide Letters, 19(10): 1064-1070.
[14] Chu H L, Yip B S, Chen K H, et al.2015. Novel antimicrobial peptides with high anticancer activity and selectivity[J]. PLOS ONE, 10(5): e0126390.
[15] Fan W H, Jiao P T, Zhang H, et al.2020. Inhibition of African swine fever virus replication by porcine type Ⅰ and type Ⅱ interferons[J]. Frontiers in Microbiology, 11(4):1203.
[16] Gao M C, Guo Y L, Luo X X, et al.2019. Design, biological activity and signaling pathway of bovine consensus omega interferon expressed in Pichia pastoris[J]. Molecular Immunology, 106(2): 46-52.
[17] Huang L, Cao R B, Wang N, et al.2012. The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α[J]. Cytokine, 57(1): 37-45.
[18] Li N N, Liu P, Chen S J, et al.2010. Construction and expression of a novel bioactive IFN-α2b/CM4 fusion protein in Escherichia coli[J]. Microbiological Research, 165(2):116-121.
[19] Lin Q, Xie K H, Chen D W, et al.2020. Expression and functional characterization of a novel antimicrobial peptide: Human beta-defensin118[J]. BioMed Research International, 2020(11): 1-10.
[20] Lynn D J, Higgs R, Lloyd A T, et al.2007. Avian beta-defensin nomenclature: A community proposed update[J]. Immunology Letters, 110(1): 86-89.
[21] Soman S S, Arathy D S, Sreekumar E.2009. Discovery of Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions[J]. Molecular Immunology, 46(10): 2029-2038.
[22] Zhang T, Liu Y C, Chen Y M, et al.2021. Antiviral activity of porcine interferon delta 8 against Pesudorabies virus in vitro[J]. International Journal of Biological Macromolecules, 177(4): 10-18.
