Zhu Jing, Chen Bo-Jiang, Huang Na, Li Wei-Min
Sichuan Da Xue Xue Bao Yi Xue Ban. 2014 Mar;45(2):299-303.
To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells.
Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot.
AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot.
The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.
构建人蛋白激酶B（ATK2）、磷酸肌醇依赖性激酶1（PDK1）和bcl-2相关死亡蛋白（BAD）慢病毒表达载体，并检测它们在293T细胞中的表达。
从肺癌组织中提取总RNA。使用特异性引物通过RT-PCR扩增人ATK2、BAD和PDK1 cDNA的全长编码区，亚克隆至PGEM-Teasy载体，然后进行测序确认。用特异性限制性内切酶消化切出全长编码序列，亚克隆至pCDF1-MCS2-EF1-copGFP载体。采用磷酸钙法将质粒转染至293T细胞。通过蛋白质免疫印迹法检测AKT2、BAD和PDK1的过表达情况。
AKT2、PDK1和BAD亚克隆至pCDF1-MCS2-EF1-copGFP载体，转染效率分别为100%、95%和90%。上清液中病毒滴度为6.7×10(6) PFU/mL。感染后，通过蛋白质免疫印迹法检测到AKT2、PDK1和BAD蛋白。
成功构建了含有AKT2、BAD和PDK1的慢病毒载体pCDF1-MCS2-EF1-copGFP，并在293T细胞中表达。
