犬干扰素α2与犬白蛋白融合基因在昆虫细胞中的表达与活性分析  

Expression and Antiviral Activity Analysis of Canine Interferon Alpha 2 and Canine Albumin Fusion Gene in Insect Cells Expression System

作　　者：王晶宇[1] 欧阳伟[1] 钱晶[1] 王晓丽[1] 夏兴霞[1] 诸玉梅[1] 毕振威[1] 王永山[1] WANG Jingyu;OUYANG Wei;QIAN Jing;WANG Xiaoli;XIA Xingxia;ZHU Yumei;BI Zhenwei;WANG Yongshan(Key Laboratory of Veterinary Biological Engineering and Technology,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室

出　　处：《畜牧兽医学报》2019年第10期2113-2118,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2016YFD0501000);公益性行业(农业)科研专项(201303042);江苏省农业科技自主创新资金(CX(15)1065)

摘　　要：本研究旨在利用Bac-to-Bac杆状病毒表达系统表达具有生物活性的犬白蛋白-犬干扰素α2(albumin-CaIFN-α2)。将蜂素信号肽(HBM)、犬血清白蛋白(Alb)与犬干扰素α2的融合基因经密码子优化后,连接至转移载体pFastBac1中,获得了重组pFastBac1-HBM-Alb-CaIFN-α2,将重组质粒转化DH10Bac感受态细胞,经过三次蓝白斑筛选纯化,得到的重组穿梭质粒rBac-HBM-Alb-CaIFN-α2转染Sf9细胞,72 h后可观察到明显的细胞病变。用第三代重组杆状病毒rBac-HBM-Alb-CaIFN-α2感染Sf9昆虫细胞,感染3 d后,收获昆虫细胞和细胞培养物,间接免疫荧光(IFA)结果显示,重组杆状病毒感染的细胞呈现绿色荧光;Western blot检测结果显示,在约90 ku左右可观察到特异性条带,证实重组Alb-CaIFN-α2能够在昆虫细胞中表达。利用MDCK/VSV微量细胞病变抑制法检测重组Alb-CaIFN-α2的抗病毒活性,结果显示重组Alb-CaIFN-α2的活性为1.70×10 6 U·mL^-1,为进一步研究新型犬用干扰素制品奠定了试验基础。In this study,a recombinant canine serum albumin-canine interferon alpha 2(albumin-CaIFN-α2)with antiviral activity were expressed by a Bac-to-Bac baculovirus expression system.The gene HBM-Alb-CaIFN-α2,encoding canine serum albumin and the mature protein gene of canine interferon alpha 2 and linked with a honeybee signal peptide(HBM),was inserted into the pFastBac1 vector,and then transformed into DH10Bac competent cells.The shuttle plasmid rBac-HBM-Alb-CaIFN-α2 was obtained after three times of blue and white spots screening and purification,and transfected into Sf9 cell.Cytopathic changes could be observed 72 h later.Under infection with the third generation recombinant baculovirus rBac-HBM-Alb-CaIFN-α2,cells and supernatant were harvested at 3 days after infection.Indirect immunofluorescence(IFA)results showed that infected cells could show green fluorescence.Western blot analysis showed that there were about 90 kDa expression products in culture supernatant,indicating that secretory expression was consistent with the expected results.The MDCK/VSV system was used to detect the antiviral activity of recombinant Alb-CaIFN-α2,the antiviral activity was 1.70×10 6 U·mL^-1.This study laid a foundation for further study on novel interferon products for dogs.

关 键 词：犬干扰素α2 犬血清白蛋白 昆虫细胞表达 抗病毒活性 

分 类 号：S852.42[农业科学—基础兽医学]