吉林大学学报(医学版)

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FGFR3真核表达载体的构建及其在人白血病细胞系K562中的表达

许会静1,2，杜彤华3，孙艳3，李校堃1，肖业臣1   

  1. (1.吉林大学基础医学院生物化学教研室，吉林 长春 130021；2.吉林医药学院检验学院临床生物化学检验教研室，吉林 吉林 132013；3.吉林大学第二医院肿瘤血液内科，吉林 长春 130041）
  • 收稿日期:2013-09-04 出版日期:2014-05-28 发布日期:2014-05-28
  • 通讯作者: 肖业臣 E-mail:（Tel:0431-85619474，E-mail:yechenxiao2400@jlu.edu.cn）
  • 作者简介:许会静（1985-），女，河北省保定市人，在读理学硕士，主要从事白血病的基础研究。
  • 基金资助:

    国家自然科学基金资助课题（81370640）；吉林省科技厅重点科技攻关项目资助课题（20130206003YY）；吉林省科技厅医药产业发展专项资金资助课题（20130727041YY）

Construction of eukaryotic expression vectors of  FGFR3 geneand their expressions in human leukemia K562 cell line

XU Hui-jing1,2,DU Tong-hua3,SUN Yan3,LI Xiao-kun1,XIAO Ye-chen1   

  1. (1.Department of Biochemistry,School of Basic Medical Sciences,JilinUniversity, Chagnchun130021,China;2.Department of Biochemistry Laboratory,Jilin Medical College,Jilin 132013,China;3.Department of Tumor and Hematology,Second Hospital,Jilin University,Changchun 130041,China)
  • Received:2013-09-04 Online:2014-05-28 Published:2014-05-28

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摘要：

目的：构建成纤维细胞生长因子受体3（FGFR3）真核表达载体MSCV/puro-fgfr3-WT和MSCV/puro-fgfr3-DN，并检测FGFR3蛋白在人白血病细胞系K562中的表达。方法:通过PCR法获得FGFR3全长基因（fgfr3-WT）和截短失活型的FGFR3（fgfr3-DN），经双酶切后与真核表达载体MSCV/puro连接，构建MSCV/puro-fgfr3-WT和MSCV/puro-fgfr3-DN重组表达质粒。经PCR、双酶切及测序鉴定正确后，脂质体介导转染至人白血病细胞系K562中，经puromycin抗性筛选后，Western blotting法和流式细胞术检测细胞中FGFR3蛋白的表达。结果：PCR法鉴定MSCV/puro-fgfr3-WT和MSCV/puro-fgfr3-DN重组质粒，2个质粒分别扩增出2　400 bp的fgfr3-WT全长基因片段和1　200 bp的fgfr3-DN截短型片段,表明成功扩增fgfr3-WT全长基因和1　200 bp的fgfr3-DN基因；双酶切MS
CV/puro-fgfr3-WT重组质粒，获得2　400 bp的目的基因片段；测序显示，fgfr3-WT片段大小为2　400 bp,Blast对比分析表明测序结果与GenBank中的FGFR3序列完全一致。fgfr3-DN片段大小与预先设计的序列完全一致,表明成功构建野生型FGFR3和突变失活型FGFR3真核表达载体；Western blotting 检测，与对照组（K562-MSCV）比较，MSCV/puro-fgfr3-WT转染组（K562-WT）FGFR3蛋白表达水平增加，表达水平是对照组的10倍以上；MSCV/puro-fgfr3-DN转染组（K562-DN）的截短型的FGFR3（K562-DN）高表达，而对照组和K562-WT转染组则无此片段；流式细胞术检测，K562-WT组有57.5%的细胞高表达FGFR3，K562-DN组有41.5%的细胞高表达FGFR3-DN。结论：成功构建高表达野生型和突变型FGFR3的人白血病细胞系K562。

关键词: 受体, 成纤维细胞生长因子, 3型, 真核表达载体, K562细胞系, 基因转染, 白血病, 粒-单核细胞, 慢性

Abstract:

To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3)  MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN,and to detect their expressions in human chronic myeloid leukemia(CML) K562 cell line.Methods The full-length FGFR3(fgfr3-WT) and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR).The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN  which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry.
Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method,and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared　 with control  (K562 MSCV) group,the expression level  of FGFR3-WT  in MSCV/puro-fgfr3-WT transfection (K562-WT) group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN  transfection  (K562-DN) group,but there was no expressions in  control(K562 MSCV) group and  K562-WT  group.The flow cytometry results showed that the  high expressions of FGFR3-WT  were     in  57.5%   cells in K562-DN group and  the  high expressions of FGFR3-DN  were  in  41.5%  cells in K562-DN  group.Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Key words: receptor,fibroblast growth factor,type 3;eukaryotic expression vector, K562 cell line, gene transfection;leukemia,myelomonocytic,chronic

中图分类号: 

  • R733.72
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