猪干扰素α的原核可溶性表达及抗PEDV活性  

Analysis of factors influencing soluble-expression of recombinant porcine interferon-αin prokaryotic expression system

作　　者：朱睿 张弘石 吴植[1] 谢军 张蕾[2] 覃光让 房崇琴 成大荣[3] 朱善元[1] ZHU Rui;ZHANG Hongshi;WU Zhi;XIE Jun;ZHANG Lei;QIN Guangrang;FANG Chongqin;CHENG Darong;ZHU Shanyuan(Engineering TechnologyResearch Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development,Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals,Jiangsu Agri-Animal Husbandry Vocational College,Taizhou,Jiangsu 225300,China;Jiangsu Agri-Animal Husbandry Vocational College,Taizhou,Jiangsu 225300,China;College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009,China;Gaoyou Com prehensive Center for Quality Inspection&Test of Product,Yangzhou,Jiangsu 225600,China)

机构地区：[1]江苏农牧科技职业学院、江苏省兽用生物制药高技术研究重点实验室、江苏现代畜牧与新兽药工程技术中心,江苏泰州225300 [2]江苏农牧科技职业学院,江苏泰州225300 [3]扬州大学兽医学院,江苏扬州225009 [4]高邮市产品质量综合检验检测中心,江苏扬州225600

出　　处：《中国兽医学报》2024年第3期550-557,共8页Chinese Journal of Veterinary Science

基　　金：西藏自治区科技重大专项资助项目(XZ202101ZD0005N);江苏农牧科技职业学院校级科研资助项目(NSF2022CB15);江苏农牧科技职业学院科技创新团队资助项目(NSF2023TC01);江苏省2019年度高校优秀科技创新团队资助项目(动物疫病防控技术研究)。

摘　　要：旨在利用原核表达系统高效表达具有抗病毒活性的可溶性重组猪干扰素α(soluble recombinant porcine interferon-α,srPoIFN-α)。通过PCR方法扩增姜曲海猪IFNα基因成熟肽序列,将其分别克隆至原核表达载体pCold-Ⅱ、pET22b、pET30a和pET30a-ELP中并在大肠杆菌中进行表达;通过Western blot结合Image J软件或BCA定量分析不同表达载体、密码子偏好优化、培养基类别、纯化方式等因素对猪干扰素α基因在大肠杆菌中可溶性表达的影响,鉴定提高srPoIFN-α表达的有利因素;利用VSV-GFP和PEDV检测srPoIFN-α的抗病毒活性。结果显示,原核表达载体pET30a、密码子优化、HB-PET自诱导培养基和磁珠纯化均能显著提高srPoIFN-α产量(P<0.001);srPoIFN-α有效抑制VSV-GFP复制的稀释倍数为2-8.1,与重组人干扰素α2b标准品的比活为1.71×10^(8)IU/g,有效抑制PEDV复制的稀释倍数为2^(-10.29),抗PEDV活性为2.72 IU,表明srPoIFN-α具有良好抗PEDV活性。这些结果为进一步开发可溶性IFNα药物制剂提供参考。The objective of this research was to effectively produce soluble recombinant porcine interferon alpha(srPoIFN-α)with antiviral properties utilizing prokaryotic expression system.The mature peptide sequence of porcine TFN-αderived from the Jiangquhai pig was amplified using the PCR technique.The amplified fragment was inserted into prokaryotic expression vectors,namely pCold-Ⅱ,pET22b,pET30a,and pET30a-ELP,and then the protein was expressed in E coli.Subsequently,an assessment of srPoIFN-αexpression levels was conducted on various factors including expression vectors,codon optimization,types of culture media,and purification methods.This evaluation involved quantitative analysis of Western blot bands using ImageJ software or BCA assay,aiming to ascertain the optimal conditions for the expression of srPoIFNαin E coli.Furthermore,the antiviral activity of srPoIFN-αwas examined by investigating its effect on VSVGFP or PEDV infection in vivo.The results revealed that the implementation of codon optimization,utilization of HB-PET autoinduction medium,and purification through Ni-NTA MagBeads significantly enhanced the production efficiency of srPoTFN-αprotein within a prokaryotic expression system of pET30a(P<0.001).Additionally,at a dilution of 1:2~(-8.1),srPoIFN-αwith a concentration of 200 mg/L effectively suppressed VSV-GFP replication,and the relative activity of srPoIFN-α(concentration relative to recombinant human interferonα2b standard)was determined to be 1.71×10^(8) IU/g.Similarly,at a dilution of 1:2~(-10.29),srPoIFN-αwith a concentration of 200mg/L efficiently inhibited PEDV replication,with an anti-PEDV activity of 2.72 IU,which exhibited notable anti-PEDV activity.The collective findings of this study provided valuable reference data for the future development soluble recombinant interferon-αpharmaceutical formulations.

关 键 词：原核表达 猪可溶性重组干扰素α 抗病毒活性 PEDV 

分 类 号：S852[农业科学—基础兽医学]