D896N突变型EGFR稳转细胞株的构建及其对吉非替尼敏感性的研究

The study of constructing the cell carrying EGFR D896N mutation and its sensitivity to gefitinib

目的构建携带D896N突变型EGFR基因的重组质粒,转染Ba/F3细胞后对其的表达及对吉非替尼的敏感性进行研究。方法 PCR构建重组质粒MSCV-Tel-EGFR D896N,与2种辅助质粒MLV和VSVG共转染293T细胞包装产生慢病毒,对Ba/F3细胞进行转染。Puromycin筛选后,Western blot检测EGFR及p-EGFR的表达;撤除IL-3培养观察细胞是否正常增殖;Western blot检测并比较Ba/F3-Tel-EGFR L858R、Ba/F3-Tel-EGFR D896N对于吉非替尼的敏感性。结果酶切及测序验证重组质粒构建无误,Western blot检测转染后的Ba/F3细胞中EGFR及p-EGFR有表达,撤除IL-3后细胞无法正常增殖,吉非替尼作用后p-EGFR明显下降。结论成功构建了表达D896N突变型EGFR的Ba/F3细胞株,初步判断该位点并非驱动基因,但是对吉非替尼有一定敏感性。

Objective To construct a lentiviral vector carrying D896 N mutant EGFR gene and to study its expression in BA/F3 cells. Methods PCR was used to construct the recombinant plasmid MSCV-Tel-EGFR D896 N.Recombinant plasmid MSCV-Tel-EGFR D896 N and two helper plasmids MLV and VSVG were co-transfected into293 T cells to produce lentivirus,which was used to transfect BA/F3 cells. After antibiotic screening,the expression of EGFR and p-EGFR were detected by Western blot,and observed whether cell proliferation was normal after removing IL-3. Western blot was used to compare the sensitivity of BA/F3-Tel-EGFR L858 R and BA/F3-Tel-EGFR D896 N to gefitinib. Results Enzyme digestion and sequencing confirmed that lentiviral vector was constructed correctly. Western blot showed that EGFR and p-EGFR were expressed in the transfected BA/F3 cells. After IL-3 was removed,the cells could not proliferate normally. The expression of p-EGFR decreased after treated with gefitinib. Conclusion We successfully construct the D896 N mutant EGFR gene lentiviral expression vector and the BA/F3 cell line with D896 N mutant EGFR. It is initially determined that this site is not a driver gene,but has some sensitivity to gefitinib.