鸭IFNα-AvBD2融合蛋白在毕赤酵母中的分泌表达及生物学活性检测  

Secretory Expression and Bioactivity Detection of Duck(Anas platyrhynchos)IFNα-AvBD2 Fusion Protein in Pichia pastoris

作　　者：豆子航 李秀丽[1] 庞洪泽 韩颖[1] 雷白时[1,2] 赵款 庞敬红 袁万哲 DOU Zi-Hang;LI Xiu-Li;PANG Hong-Ze;HAN Ying;LEI Bai-Shi;ZHAO Kuan;PANG Jing-Hong;YUAN Wan-Zhe(College of Animal Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Engineering Technology Innovation Center,Baoding 071001,China;Hebei Borui Animal Pharmaceutical Co.,Ltd.,Shijiazhuang 050800,China)

机构地区：[1]河北农业大学动物医学院,保定071001 [2]河北省兽医生物技术创新中心,保定071001 [3]河北伯瑞动物药业有限公司,石家庄050800

出　　处：《农业生物技术学报》2022年第5期957-965,共9页Journal of Agricultural Biotechnology

基　　金：2020年度石家庄市重点研发项目(201208)。

摘　　要：鸭(Anas platyrhynchos)α干扰素(interferonα,IFNα)具有广谱的抗病毒活性,禽β防御素2(avianβ-defensin 2,AvBD2)具有广谱的抗菌活性。本研究利用毕赤酵母(Pichia pastoris)分泌表达鸭IFNα-AvBD2融合蛋白并明确IFNα-AvBD2的生物学活性,对于现有无抗养殖条件下的鸭疫病防控具有重要意义。本课题组在前期工作中已成功构建了原核表达质粒pET32a-AplIFNα-AvBD2,并在大肠杆菌(Escherichia coli)中成功表达。本研究以实验室保存的pET32a-AplIFNα-AvBD2为模板,通过PCR方法扩增得到AplIFNα-AvBD2成熟蛋白的基因片段,构建重组质粒pPICZαA-AplIFNα-AvBD2;将线性化的重组质粒电转化至毕赤酵母KM71H感受态细胞,阳性重组菌经甲醇诱导表达,对表达产物进行体外生物学活性研究。SDS-PAGE和Western blot结果显示,AplIFNα-AvBD2重组蛋白分子量约为24 kD;琼脂扩散法和微量稀释法表明重组蛋白对金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌有较强的抗菌活性;细胞病变抑制法检测重组蛋白抗病毒活性为1.2×10^(5) U/mL,比活性为5.2×10^(5) U/mg;MTT细胞毒性试验显示重组蛋白对非洲绿猴肾细胞(Vero)无毒。上述结果说明,AplIFNα-AvBD2可在毕赤酵母中分泌表达,同时具有抗菌活性和抗病毒活性。本研究为AplIFNα-AvBD2的进一步应用提供了基础资料。Duck(Anas platyrhynchos)interferonα(IFNα)has a broad spectrum of antiviral activity,and A.platyrhynchos avianβ-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 1.2×10^(5) U/mL,and the specific activity was 5.2×10^(5) 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.

关 键 词：α干扰素(IFNα) β防御素2(AvBD2) 毕赤酵母 融合蛋白 抗菌活性 抗病毒活性 

分 类 号：S852.4[农业科学—基础兽医学]