犬干扰素α4在CHO细胞中的稳定表达  

Stable expression of canine interferon alpha4 in CHO cells

作　　者：王晶宇[1,2] 欧阳伟[2] 王永山[1,2] 夏兴霞[2] 诸玉梅[2] 王晓丽[2] 潘群兴[2] 孟凯[2] 毕振威[2] 安欣[1,2] 楼晶瑶 姚火春[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]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,江苏南京210014

出　　处：《中国兽医科学》2017年第4期448-454,共7页Chinese Veterinary Science

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]

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。In order to obtain canine interferon alpha4（CaIFN-α4） with high activity,we prepared the fusion gene of canine interferon alpha4 and green fluorescent protein（GFP）,CaIFN-alpha4-GFP,containing the canine interferon alpha4 and GFP with two GGGGS-five peptide（2G4S）. The recombinant expression plasmidp L-CaIFN-α4-GFPwas constructed bycloningthefusiongene（CaIFN-α4-GFP）into p L-e GFPeukaryotic expression vector. Recombinant lentivirus,v CaIFN-α4-GFP,was harvested following transfection of293 FT cells with p L-CaIFN-α4-GFP eukaryotic expression plasmid and other three lentivirus plasmids.Finally,we successfully established Chinese hamster ovary（CHO） cell expressing CaIFN-α4-GFP through infecting the CHO cells with the harvested recombinant lentivirus,followed by screened with blast fungus element,flow cytometry sorting and limited dilution transfection cell cloning.Green fluorescence could be observed in the established CHO cells,demonstrating the expression of interferon.The CHO cell culture supernatant had the antiviral activity agianst vesicular stomatitis virus that was detected with MDCK and MDBK,and the antiviral activities were 1.39×105U/m L and 2.19×104U/m L,respectively.This study successfully developed CHO cells expressing the canine interferon alpha4 with activity, which laid the experimental foundations for the development of the production system of dogs interferon alpha4 with high activity.

关 键 词：犬干扰素α4(CaIFN-α4) CHO细胞 真核表达 慢病毒 绿色荧光蛋白 

分 类 号：R979.5[医药卫生—药品]