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当前位置: 首页 > 文章 > 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 中国兽医科学 2019 (4) 493-498
Position: Home > Articles > Expression of the China Holstein dairy cow MD-2 gene in HEK293 cells and construction of the stable cell lines Chinese Veterinary Science 2019 (4) 493-498

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

作  者：
焦寒伟;赵宇;帅学宏;伍莉;陈吉轩;王红均;黄庆洲
单  位：
西南大学动物科学学院兽医科学工程研究中心
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
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。
译  名：
Expression of the China Holstein dairy cow MD-2 gene in HEK293 cells and construction of the stable cell lines
作  者：
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;
关键词：
China Holstein dairy cow;;MD-2;;EGFP;;fusion protein;;eukaryotic expression;;the stable cell line
摘  要：
The full length of myeloid differentiation protein-2(MD-2)c DNA of the China Holstein dairy cow was cloned to the eukaryotic expression vector of p EGFP-N1,and identified by double enzyme digestion and sequenced.The recombinant plasmid p MD-2-EGFP was transfected into HEK293 cells by liposome 2000.The expression of fusion protein was detected by fluorescence microscope,flow cytometry and Western-blot.The single cell was obtained by the method of limiting dilution,and the stable cell line was obtained by using G418 resistance screening.In result,the target gene contains an open reading 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 p EGFP-N1,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 ?g/m L G418.The flow cytometry and Western-blot detection showed that in the p MD-2-EGFP stable cell line group,the specific bands were found at the molecular weight of 46 ku,the p EGFP-N1 empty stable cell group and the blank HEK293 cell control group were not found.In conclusion,this experiment successfully constructed the eukaryotic expression vector p MD-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 p MD-2-EGFP and p EGFPN1 were successfully obtained,which lay the foundation for further study of the biological function of the China Holstein dairy cow MD-2 gene.

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