犬干扰素α4在CHO细胞中的稳定表达
发布时间：2019-06-03 16:59
【摘要】：为制备高活性的犬干扰素α4(CaIFN-α4),将犬IFN-α4和绿色荧光蛋白(GFP)基因用2个GGGGS五肽柔性肽连接,获得犬IFN-α4和GFP的融合基因(CaIFN-α4-GFP)。将该融合基因与真核表达载体pLeGFP连接,构建重组表达质粒pL-CaIFN-α4-GFP,并与其他3种慢病毒质粒(pC-Gp,pR-Rev和pC-VsvG)共同转染293FT细胞,包装获得重组慢病毒(vCaIFN-α4-GFP)。用获得的重组慢病毒感染中国仓鼠卵巢(CHO)细胞,经杀稻瘟菌素筛选、流式细胞仪分选和有限稀释法克隆转染细胞,获得表达CaIFN-α4-GFP的CHO细胞。用荧光显微镜观察构建的CHO细胞呈现绿色荧光,用MDCK与MDB K细胞检测CHO细胞培养上清液具有抗水疱性口炎病毒活性,活性分别为1.39×105和2.19×104U/m L。本研究成功地利用CHO细胞表达了具有活性的犬干扰素α4,为进一步研制高活性的犬IFN-α4生产系统奠定了试验基础。
[Abstract]:In order to prepare highly active canine interferon 伪 4 (CaIFN- 伪 4), canine IFN- 伪 4 and green fluorescent protein (GFP) gene were ligated with two GGGGS pentapeptide flexible peptides to obtain the fusion gene of dog IFN- 伪 4 and GFP (CaIFN- 伪 4 GFP). The fusion gene was ligated with eukaryotic expression vector pLeGFP, and the recombinant expression plasmid pL-CaIFN- 伪 4-GFP, was constructed and co-transfected into 293FT cells with three other lentivirus plasmids (pC-Gp,pR-Rev and pC-VsvG). Recombinant lentivirus (vCaIFN- 伪 4-GFP).) was obtained by packaging Chinese hamster ovarian (CHO) cells were infected with recombinant lentivirus. CHO cells expressing CaIFN- 伪 4-GFP were cloned by flow cytometry and limited dilution. The green fluorescence of CHO cells was observed by fluorescence microscope. MDCK and MDB K cells were used to detect the antivesicular stomatitis virus activity of CHO cells, which were 1.39 脳 10 ~ 5 and 2.19 脳 104U/m L, respectively. In this study, the active canine interferon 伪 4 was successfully expressed by CHO cells, which laid an experimental foundation for the further development of a highly active canine interferon 伪 4 production system.
【作者单位】： 南京农业大学动物医学院;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国家兽用生物制品工程技术研究中心;
【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项目[CX(15)1065]
【分类号】：S858.292
[Abstract]:In order to prepare highly active canine interferon 伪 4 (CaIFN- 伪 4), canine IFN- 伪 4 and green fluorescent protein (GFP) gene were ligated with two GGGGS pentapeptide flexible peptides to obtain the fusion gene of dog IFN- 伪 4 and GFP (CaIFN- 伪 4 GFP). The fusion gene was ligated with eukaryotic expression vector pLeGFP, and the recombinant expression plasmid pL-CaIFN- 伪 4-GFP, was constructed and co-transfected into 293FT cells with three other lentivirus plasmids (pC-Gp,pR-Rev and pC-VsvG). Recombinant lentivirus (vCaIFN- 伪 4-GFP).) was obtained by packaging Chinese hamster ovarian (CHO) cells were infected with recombinant lentivirus. CHO cells expressing CaIFN- 伪 4-GFP were cloned by flow cytometry and limited dilution. The green fluorescence of CHO cells was observed by fluorescence microscope. MDCK and MDB K cells were used to detect the antivesicular stomatitis virus activity of CHO cells, which were 1.39 脳 10 ~ 5 and 2.19 脳 104U/m L, respectively. In this study, the active canine interferon 伪 4 was successfully expressed by CHO cells, which laid an experimental foundation for the further development of a highly active canine interferon 伪 4 production system.
【作者单位】： 南京农业大学动物医学院;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国家兽用生物制品工程技术研究中心;
【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项目[CX(15)1065]
【分类号】：S858.292
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