猪β防御素2与α干扰素重组融合蛋白的表达及其活性评价  

Expression and activity evaluation of porcineβ-defensin-2and interferonαrecombinant fusion protein

作　　者：雷白时[1,2] 李秀丽 康佳梦[1] 张会文 李伯森 赵款 张武超 梁飞 袁万哲 LEI Baishi;LI Xiuli;KANG Jiameng;ZHANG Huiwen;LI Bosen;ZHAO Kuan;ZHANGWuchao;LIANG Fei;YUAN Wanzhe(Hebei Agricultural University,Baoding,Hebei 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China;Chengde Veterinary Medicine Management Station,Chengde,Hebei 067000,China;ShijiazhuangAnimal Genetic Engineering Drug Technology Innovation Center,Shijiazhuang 050800,China;Hebei Borui Animal Pharmaceutical Co.,Ltd.,Zhengding,Hebei 050800,China)

机构地区：[1]河北农业大学,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001 [3]承德市兽药管理站,河北承德067000 [4]石家庄市动物基因工程药物技术创新中心,河北石家庄050800 [5]河北伯瑞动物药业有限公司,河北正定050800

出　　处：《中国兽医学报》2024年第12期2585-2590,共6页Chinese Journal of Veterinary Science

基　　金：承德市科技计划资助项目(202205B037);2024年石家庄市政府与中国农科院战略合作资助项目(24008)。

摘　　要：为获得同时具有广谱抗细菌和抗病毒活性的猪β防御素2(PBD2)与猪α干扰素(PoIFN-α)重组融合蛋白,将2种蛋白的基因串联融合后插入pPICZαA载体,电转导入KM71H毕赤酵母细胞构建工程菌,经甲醇诱导表达后,浓缩分离重组蛋白,MTT法和猪红细胞溶血试验测定其细胞毒性,并对重组融合蛋白的体外抗细菌和抗病毒活性进行评价。结果显示,成功获得了pPICZαA-PBD2-IFNα-KM71H毕赤酵母工程菌株,经诱导、表达、浓缩纯化后的融合蛋白PBD2-IFN-α的质量浓度为1.116g/L,相对分子质量为25kDa,当蛋白质量浓度低于4^(-4) g/L时对PK-15细胞和猪红细胞无明显毒性,融合蛋白在大肠杆菌或金黄色葡萄球菌混菌平板上产生的抑菌圈直径为(15.0±0.9)mm,均具有比较明显的抑菌活性,Reed-Muench法计算融合蛋白在PK-15中对水泡性口炎病毒(VSV)的抗病毒活性为8.89×10^(5) U/mL。为进一步开发该重组融合蛋白作为抗细菌和抗病毒产品奠定了理论基础。In order to obtain a recombinant fusion protein of porcineβ-defensin-2and interferonαwith broad-spectrum antibacterial and antiviral activities at the same time,the genes encoding both proteins were fused in series and inserted into the pPICZαA vector.This construct was then electrotransferred into Pichia pastoris KM71H cells to construct an engineered strain.Following methanol-induced expression,the recombinant protein was concentrated and isolated.The cytotoxicity of the protein was assessed using the MTT assay and a porcine red blood cell hemolysis test.Subsequently,the in vitro antibacterial and antiviral activities of the recombinant fusion protein were evaluated.The results showed that the engineered strain pPICZαA-PBD2-IFNα-KM71Hof Pichia pastoris was successfully obtained,and the fusion protein PBD2-IFN-αwas obtained by concentrating and purifying the fermentation broth after fermentation induction and expression.Its concentration was 1.116g/L and molecular weight was 25kDa.When the concentration of fusion protein was lower than 4^(-4) g/L,it had no obvious toxicity to PK-15cells and porcine red blood cells.The diameter of the inhibition zone produced by the fusion protein on the mixed plate of Escherichia coli and Staphylococcus aureus was(15.0±0.9)mm,which had obvious antibacterial activity.The antiviral activity of the fusion protein against VSV in PK-15cells was 8.89×10^(5) U/mL measured by Reed-Muench method.This study laid a theoretical foundation for further developing the recombinant fusion protein as an antibacterial and antiviral product.

关 键 词：猪β防御素2 猪Α干扰素 重组融合蛋白 抗细菌 抗病毒 

分 类 号：S852.23[农业科学—基础兽医学]