稳定表达狂犬病病毒CTN-1V株糖蛋白细胞系的建立

目的构建能稳定表达狂犬病病毒糖蛋白的CHO细胞系。方法用RT—PCR法扩增和分离CTN-1V株狂犬病毒糖蛋白基因，测序后克隆入pCDNA5．0FRT载体，构建重组质粒pCDNA5．0FRT—G，之后与POG44质粒共转染CHO细胞，采用潮霉素B抗性筛选，挑选阳性克隆，间接免疫荧光和WesternBlot进行稳定表达细胞系的鉴定。结果经酶切和测序鉴定，获得重组表达质粒pCDNA5．0FRT—G，共转染CHO细胞后，获得肉眼可见阳性细胞克隆，刮取阳性克隆进行传代扩大培养并定义为第2代，经过10代后免疫荧光和WesternBlot结果依然阳性。结论成功建立稳定表达狂犬病病毒糖蛋白的CHO细胞系，为深入研究糖蛋白的功能奠定基础。

Study on the mammalian cell lines of stable expression glycoprotein of rabies virus CTN-1V strain

Objective To build the mammalian cell line of stable expression of rabies virus glycoprotein. Methods A amplified rabies virus glycoprotein gene of CTN-1V strain by RT-PCR method, and cloned into pCDNAS. 0 FRT plasmid. Transfected the recombinant pCDNAS. 0 FRT-G plasmid into CHO cells with POG44 together. Selection screens the positive cell with hygromycin B resistance. Identify the cell which stable express rabies virus glycoprotein with indirect immunofluorescence and Western Blot. Results Recombinant expression plasmid pCDNAS. 0 FRT-G was successfully reconstruct by identification with enzyme digestion and sequencing. Get the visible positive cell clones, scraping into 96 well, it＇s the first passage. Subculture with 10 passages, the results of immunofluorescence and Western Blot were still positive. Conclusion Successfully established the stable expression of rabies virus glycoprotein CHO cell lines, and it＇s helpful to further study on the function of glycoprotein.