|本期目录/Table of Contents|

[1]徐子豪,史小雨,王倩.重组质粒pcDNA4-FLAG-GLUT1构建及蛋白表达[J].天津医科大学学报,2020,26(06):521-525.
　XU Zi-hao,SHI Xiao-yu,WANG Qian.Construction of recombinant plasmid pcDNA4-FLAG-GLUT1 and protein expression[J].Journal of Tianjin Medical University,2020,26(06):521-525.
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重组质粒pcDNA4-FLAG-GLUT1构建及蛋白表达(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
26卷
期数:
2020年06期
页码:
521-525
栏目:
基础医学
出版日期:
2020-11-20

文章信息/Info

Title:
Construction of recombinant plasmid pcDNA4-FLAG-GLUT1 and protein expression
作者:
徐子豪; 史小雨; 王倩
天津医科大学基础医学院免疫学系，天津300070
Author(s):
XU Zi-hao;  SHI Xiao-yu;  WANG Qian
Department of Immunology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China
关键词:
葡萄糖转运蛋白1; 蛋白磷酸化; 恶性疟原虫
Keywords:
glucose transporter 1; protein phosphorylation; Plasmodium falciparum
分类号:
R382.31
DOI:
-
文献标志码:
A
摘要:
目的：为探究葡萄糖转运蛋白1（GLUT1）磷酸化水平对红细胞期疟原虫生长发育的影响，构建必要的分子工 具。方法：以pcDNATM4/TO/myc-His B为载体，构建在GLUT1第一个胞外区插入2×FLAG标签的重组质粒pcDNA4- 2×FLAG-GLUT1。将重组质粒转染到NIH/3T3细胞中，利用Western印迹和流式细胞术分别检测细胞FLAG-GLUT1蛋白总量 和细胞表面FLAG-GLUT1的表达水平。结果：酶切鉴定及DNA测序表明正确构建重组质粒pcDNA4-2×FLAG-GLUT1。通过识 别FLAG标签，利用Western印迹能够检测到FLAG-GLUT1在NIH/3T3细胞中的表达，流式细胞术结果显示，转染重组质粒 的细胞表面可检测到FLAG-GLUT1的表达，阳性群比例为（21.46 ± 2.375）%，明显高于对照组（0.01±0.00）%，差 异有统计学意义（t=9.035，P<0.01）。结论：成功构建重组质粒pcDNA4-2×FLAG-GLUT1，为研究GLUT1蛋白磷酸化在 疟原虫感染红细胞中的作用提供了必要的工具。
Abstract:
Objective: To study the effect of glucose transporter 1（GLUT1） phosphorylation on the growth of blood-stage Plasmodium, and construct a necessary molecular tool. Methods:Using pcDNATM 4/TO/myc-His B as a vector，the recombinant plasmid pcDNA4-2×FLAG-GLUT1 was constructed by inserting 2 ×FLAG tag into the first extracellular domain of GLUT1.The recombinant plasmid was transfected into NIH/3T3 cells.The total amount of FLAG-GLUT1 protein and the level of FLAG-GLUT1 on the cell surface were detected by Western blotting and flow cytometry，respectively. Results: The results of restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid pcDNA4-2×FLAG-GLUT1 was constructed correctly. By recognizing the FLAG tag，the expression of FLAG-GLUT1 in NIH/3T3 cells could be detected by Western blotting. The results of flow cytometry showed that the expression of FLAG-GLUT1 was detected on the cell surface transfected with recombinant plasmid，and the proportion of positive group was（21.46 ± 2.375）%，which was significantly higher than that of control group（0.01 ± 0.00） %（t = 9.035，P < 0.01）. Conclusion: The recombinant plasmid pcDNA4-2×FLAG-GLUT1 is successfully constructes which provides a necessary tool for studying the role of GLUT1 phosphorylation in Plasmodium-infected red blood cells.

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备注/Memo

备注/Memo:
文章编号 1006-8147（2020）06-0521-05
基金项目 天津市教委科研计划项目（2018KJ085）
作者简介 徐子豪（1996-），男，硕士在读，研究方向：免疫学；通信作者：王倩，E-mail:wangq@tmu.edu.cn。
更新日期/Last Update: 2020-11-20