重组犬α干扰素在昆虫细胞中的高效表达与抗病毒活性鉴定

High-efficiency expression and antiviral activity of recombinant canine interferon-αexpressed in insect cells

目的利用杆状病毒表达系统制备犬α干扰素(Canine interferon-α,CaIFN-α)。方法根据昆虫细胞密码子偏爱性优化CaIFN-α基因,构建含双拷贝CaIFN-α优化基因的载体pFastBacDual-CaIFNα-CaIFNα,转化DH10Bac感受态后获得重组基因组rBacmid-CaIFNα-CaIFNα,转染Sf9细胞获得重组杆状病毒rBac-CaIFNα-CaIFNα,接种Sf9细胞表达rCaIFN-α。采用PCR和透射电子显微镜鉴定重组杆状病毒的外源基因与病毒形态,采用间接免疫荧光染色法检测rCaIFN-α蛋白的表达,采用细胞病变抑制法测定rCaIFN-α抑制水疱性口炎病毒(VSV-GFP)在MDCK细胞的复制能力。结果重组杆状病毒rBac-CaIFNα-CaIFNα构建成功,rCaIFN-α在rBac-CaIFNα-CaIFNα感染的Sf9细胞中表达,其抗病毒活性为8.73×105 IU/ml。结论构建的杆状病毒表达系统能高效表达rCaIFN-α,该干扰素具有高效抗病毒活性,这为犬干扰素制剂的研究奠定了基础。

Objective To efficiently express canine interferon-αusing a baculovirus/insect cell expression system.Methods The canine interferon-αgene(CaIFN-α)was optimized based on preferred codons in insect cells.The vector pFastBacDual-CaIFNα-CaIFNαcontaining the 2 copies of the CaIFN-αgene was transformed into DH10 Bac host bacteria to obtain the recombinant plasmid rBacmid-CaIFNα-CaIFNα.The recombinant baculovirus rBac-CaIFNα-CaIFNαwas obtained by transfecting Sf9 cells,and rCaIFN-αwas expressed by inoculation of Sf9 cells.The recombinant baculovirus was identified using PCR and transmission electron microscopy.CaIFN-αprotein was identified using IFA.Cytopathic inhibition was used to determine the ability of CaIFN-αprotein to inhibit the replication of vesicular stomatitis virus(VSVGFP)in MDCK cells. Results The rBacmid-CaIFNα-CaIFNαplasmid was verified as correct using restriction endonuclease analysis and DNA sequencing.The rBac-CaIFNα-CaIFNαbacmid was verified as correct using PCR and transmission electron microscopy.In IFA,specific green fluorescence was observed in Sf9 cells infected with rBac-CaIFNα-CaIFNα,indicating the expression of interferon.An antiviral activity assay indicated that recombinant CaIFN-αefficiently inhibits the replication of VSV-GFP in MDCK cells.The level of biological activity of recombinant CaIFN-αwas as high as 8.73×105 IU/ml. Conclusion This study successfully developed Sf9 cells expressing CaIFN-αwith a high level of antiviral activity.This finding lays the foundation for the further development and utilization of canine interferon.