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 inter-feron-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);sr-PoIFN-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 × 108IU/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.
Abstract
The objective of this research was to effectively produce soluble recombinant porcine in-terferon alpha(srPoIFN-α)with antiviral properties utilizing prokaryotic expression system.The mature peptide sequence of porcine IFN-α derived 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.Subse-quently,an assessment of srPoIFN-α expression levels was conducted on various factors including expression vectors,codon optimization,types of culture media,and purification methods.This eval-uation involved quantitative analysis of Western blot bands using ImageJ software or BCA assay,aiming to ascertain the optimal conditions for the expression of srPoIFNα in E coli.Furthermore,the antiviral activity of srPoIFN-α was examined by investigating its effect on VSV-GFP or PEDV infection in vivo.The results revealed that the implementation of codon optimiza-tion,utilization of HB-PET autoinduction medium,and purification through Ni-NTA MagBeads significantly enhanced the production efficiency of srPoIFN-α protein within a prokaryotic expres-sion system of pET30a(P＜0.001).Additionally,at a dilution of 1∶2-8.1,srPoIFN-α with a con-centration of 200 mg/L effectively suppressed VSV-GFP replication,and the relative activity of sr-PoIFN-α(concentration relative to recombinant human interferon α2b standard)was determined to be 1.71 ×108 IU/g.Similarly,at a dilution of 1∶2-10.29,srPoIFN-α with a concentration of 200 mg/L efficiently inhibited PEDV replication,with an anti-PEDV activity of 2.72 IU,which exhibi-ted notable anti-PEDV activity.The collective findings of this study provided valuable reference da-ta for the future development soluble recombinant interferon-α pharmaceutical formulations.
基金项目
西藏自治区科技重大专项(XZ202101ZD0005N)
江苏农牧科技职业学院校级科研项目(NSF2022CB15)
江苏农牧科技职业学院科技创新团队项目(NSF2023TC01)
江苏省高等学校优秀科技创新团队项目(2019)()
NETL
NSTL
万方数据