FKBP12真核表达载体的构建及稳定转染A549细胞株的建立

Construction of Eukaryotic Expressing Vector of FKBP12 and Establishment of Stable Transfectant A549 Cell Line

目的:构建FK506结合蛋白12(FK506 binding proteins 12,FKBP12)真核表达载体并建立稳定转染A549细胞株。方法:RT-PCR扩增人平滑肌细胞FKBP12基因片断,构建pcDNA3.1/Hygro(+)-FKBP12真核表达载体,经琼脂糖电泳、特异性内切酶切割及测序验证其正确性。脂质体法转染真核细胞A549,HygromycinB筛选建立稳定转染的细胞株,免疫印迹法检测稳定转染的细胞株。结果:构建了FKBP12真核表达载体并建立了稳定转染的A549细胞株,成功表达FKBP12蛋白。结论:FKBP12真核表达载体成功构建及稳定转染A549细胞株的建立,为深入研究基于FKBP12靶点药物的机制奠定基础,进而为探索安全、高效的免疫抑制剂提供新的途径。

Objective： To construct the eukaryotic plasmid of FK506 Binding proteins 12 （FKBP12）and transfect A549 cell so as to establish stable cell line. Methods： The whole objective gene was cloned by RT-PCR from VSMCs. Eukaryotic victor pcDNA3.1/Hygro（＋）-FKBP12 was connected and by enzyme digestion and DNA sequencing . We transfected the recombinant vector into A549 cell by lipofectamine TM 2000.Stable transfected A549 cell line was established after screening culture by Hygromycin B and was identified by Western blot. Results： The eukaryotic expression vector pcDNA3.1/Hygro（＋）FKBP12 was constructed, stable transfected A549 cell line was established and FKBP12 protein was expressed successfully. Conclusion： The construction of the eukaryotic expression vector pcDNA3.1/Hygro （＋）FKBP12 and the establishment of stable transfected A549 cell line not only lay the foundation for the further research into the mechanism of drug targets based on FKBP12 but also provide a safe and efficient way for myocardial new immunosuppressive agents.