中国荷斯坦奶牛MD-2基因在HEK293细胞中的表达及稳定细胞系的构建  

Expression of the China Holstein dairy cow MD-2 gene in HEK293 cells and construction of the stable cell lines

作　　者：焦寒伟 赵宇[1] 帅学宏 伍莉[1] 陈吉轩 王红均 黄庆洲[1] JIAO Han-wei;ZHAO Yu;SHUAI Xue-hong;WU Li;CHEN Ji-xuan;WANG Hong-jun;HUANG Qing-zhou(Veterinary Scientific Engineering Research Center,College of Animal Sciences,Southwestern University,Chongqing 402460,China)

机构地区：[1]西南大学动物科学学院兽医科学工程研究中心,重庆荣昌402460

出　　处：《中国兽医科学》2019年第4期493-498,共6页Chinese Veterinary Science

基　　金：国家自然科学基金青年基金项目(31802215);重庆市自然科学基金项目(cstc2018jcyjA0807);中央高校基本科研业务费项目(XDJK2019C024;XDJK2019D013);西南大学博士启动基金项目(5360300101;20700505)

摘　　要：将中国荷斯坦奶牛髓样分化蛋白-2 (myeloid differentiation protein-2,MD-2) cDNA全长克隆至pEGFP-N1真核表达载体,利用双酶切与测序进行鉴定。经脂质体2000,将pMD-2-EGFP重组质粒转染至HEK293细胞中,应用倒置荧光显微镜、流式细胞术和Western-blot分别检测融合蛋白的表达,利用极限稀释的方法获取单细胞,并利用G418抗性筛选,获取稳定细胞系。结果发现,目的基因含有一个483 bp的开放阅读框,编码160个氨基酸。双酶切鉴定和测序结果均表明,MD-2基因编码区正确插入pEGFP-N1真核表达载体,读码框架正确,MD-2-EGFP融合蛋白在HEK293细胞中,能够成功表达;极限稀释获取单细胞,用250?g/mL的G418筛选得到的细胞系,经流式细胞术和Western-blot检测,在pMD-2-EGFP稳定细胞系组,分子质量约为46 ku处,发现了特异性条带,而pEGFP-N1空载稳定细胞组,以及空白的HEK293细胞对照组,均未发现条带。综上所述,本研究成功构建了真核表达载体pMD-2-EGFP,并在HEK293细胞中能够成功表达;经极限稀释,并利用G418抗性筛选,成功获得了pMD-2-EGFP和pEGFP-N1稳定细胞系,这为进一步研究中国荷斯坦奶牛奶牛MD-2基因的生物学功能奠定基础。The full length of myeloid differentiation protein-2(MD-2)cDNA of the China Holstein dairy cow was cloned to the eukaryotic expression vector of pEGFP-N 1,and identified by double enzyme digestion and sequeneed.The recombiriant plasmid pMD-2-EGFP was transfected into HEK293 cells by liposome 2000.The expression of fusion protein was detected by fluorescence microscope,flow cytometry and Wes tern-bl ot.The single cell was obtained by the method of lim iting dilution,and the stable cell line was obta ined by usi ng G418 resista nee screening.In result,the t ar get gene contains an open readi ng frame with 483 bp,encoding 160 amino acids.The double enzyme identification and sequencing results showed that the encoding region of the MD-2 gene was correctly inserted into the eukaryotic vector pEGFP-N 1,the reading frame was correct,and the MD-2-EGFP fusion protein can be expressed successfully in HEK293 cells.The single cell was obtained by limit dilution,and the signle cell was screened with 250 μg/mL G418.The flow cytometry and Western-blot detection showed that in the pMD-2-EGFP stable cell line group,the specific bands were found at the molecular weight of 46 ku,the pEGFP-Nl empty stable cell group and the blank HEK293 cell control group were not found.In conclusion,this experiment successfully constructed the eukaryotic expression vector pMD-2-EGFP,and it can be successfully expressed in HEK293 cells.Through the limiting dilution,and using G418 resistance screening,the stable cell lines of pMD-2-EGFP and pEGFPN1 were successfully obtained,which lay the foundation for further study of the biological function of the China Holstein dairy cow MD-2 gene.

关 键 词：中国荷斯坦奶牛 髓样分化蛋白-2 EGFP 真核表达 稳定细胞系 

分 类 号：S852.42[农业科学—基础兽医学]