摘要
目的从乳腺癌文库中钓取葡萄糖转运体基因12(glucose transporter 12 gene,GLUT12)全长编码序列,克隆至pCMV-Myc中,并在293T细胞中实现其真核表达,检测其对乳腺癌细胞葡萄糖摄取功能的影响。方法利用PCR法从乳腺文库中获得GLUT12编码区全长序列,将其以正确相位与pCMV-Myc载体中的Myc编码序列融合,将重组质粒转化E.coli DH5α后,通过限制性内切酶酶切和DNA测序等方法对重组质粒进行鉴定。将该重组质粒转染293T细胞,利用Western印迹法检测pCMV-Myc-GLUT12重组质粒的表达。在此基础上,检测GLUT12对乳腺癌细胞葡萄糖摄取功能的影响。结果构建了GLUT12真核表达载体,经过酶切电泳和DNA测序,鉴定出插入序列正确的GLUT12表达载体,在真核细胞内实现其真核表达,表达产物能促进乳腺癌细胞对葡萄糖的摄取。结论成功构建了GLUT12真核表达载体,在真核细胞中成功表达,表达产物具有生物学活性。
Objective To construct the expression vector of GLUT12(glucose transporter 12)gene by using pCMV-Myc plasmids and detect the effect of GLUT12 overexpression on the capability of glucose uptake in breast cancer cell lines.Methods The full-length coding sequence of human GLUT12 was amplified from the mammary library by PCR.It was then fused in frame with the coding region of Myc in the pCMV-Myc vector to generate the pCMV-Myc-GLUT12 recom-binant plasmid that was transfected into 293 T cells and then characterized by Western blotting.The effects of GLUT12 on the capability of glucose uptake in breast cancer cells were examined.Results TheGLUT12 eukaryotic expression vector was constructed and the pCMV-Myc-GLUT12 recombinant plasmid was verified by restriction endonulease digestion and DNA sequencing.The upregulation of GLUT12 promoted the glucose uptake level in breast cancer cells.Conclusion The GLUT12 eukaryotic expression vector is successfully constructed and expressed in 293 T cells,the product of which has the expected biological function.
作者
施亚娇
冉芳
罗沙柳
张亚楠
刘婕
丁丽华
叶棋浓
SHI Ya jiao;RAN Fang;LUO Sha liu;ZHANG Ya nan;LIU Jie;DING Li hua;YE Qi nong(Medical School of Guizhou University,Guiyang 550025,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《军事医学》
CAS
北大核心
2019年第12期935-938,共4页
Military Medical Sciences
基金
国家重点研发计划(2017YFA0505602).
