[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD]
Objective:
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.
Methods:
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.
Results:
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.
Conclusion:
The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.
Insights
Researchers successfully constructed lentiviral vectors for human protein kinase B (AKT2), phosphoinositide-dependent kinase 1 (PDK1), and bcl-2-associated death protein (BAD). These vectors were expressed in 293T cells, confirming successful gene delivery and protein production.
Area of Science:
- Molecular Biology
- Gene Expression
- Lentiviral Vector Technology
Background:
- Protein kinase B (AKT2), phosphoinositide-dependent kinase 1 (PDK1), and bcl-2-associated death protein (BAD) are key regulators in cellular signaling pathways.
- Understanding their expression is crucial for research in various biological processes, including cancer.
Purpose of the Study:
- To construct lentiviral expression vectors for human AKT2, PDK1, and BAD.
- To confirm the successful expression of these proteins in 293T cells.
Main Methods:
- Human AKT2, PDK1, and BAD cDNA were amplified from lung cancer tissue RNA.
- RT-PCR and subcloning were used to insert cDNA into the pCDF1-MCS2-EF1-copGFP lentiviral vector.
- 293T cells were transfected, and protein expression was verified using Western blot.
Main Results:
- Lentiviral vectors containing AKT2, PDK1, and BAD were successfully constructed.
- High transfection efficiencies (100% for AKT2, 95% for PDK1, 90% for BAD) were achieved.
- Western blot confirmed the successful expression of AKT2, PDK1, and BAD proteins in 293T cells.
Conclusions:
- The study successfully developed lentiviral vectors for AKT2, PDK1, and BAD.
- These vectors enable the expression of AKT2, PDK1, and BAD in 293T cells.
- This provides a valuable tool for further research into the functions of these proteins.
