FKBP12真核表达载体的构建及稳定转染A549细胞株的建立

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。

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

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 ...