Construction Of Flag-Tagged CDK4 Eukaryotic Expression Vector And Its Effects On Laryngeal Carcinoma Proliferation

ObjectiveCell cycle regulation plays a key role in the process of cell growth,and the imbalance of cell cycle also leads to the occurrence and development of tumor.Cyclin D-CDK4/6-INK4-Rb is an important pathway in the process of cell cycle regulation.However,Cyclin-dependent kinase 4(CDK4),as one of the key factors in this pathway,the specific mechanism of biological behavior in squamous cell carcinoma of head and neck still needs further study.In this experiment,pcDNA3.0-Flag was constructed.The eukaryotic expression vector of CDK4 gene and the effects of the gene on the biological behavior of laryngeal carcinoma cell(Hep-2)were preliminarily verified,which lead a foundation for the further study of laryngeal cancer cells mechanism.MethodsUsing human mammary gland library as template,the CDS region of CDK4 was amplified by polymerase chain reaction(PCR),then the pcDNA3.0-Flag was inserted into the amplified CDK4 sequence by double enzyme technology,and the product was transformed into DH5α cells,finding the positive clone liquid and extracting the plasmid that transfect into 293 T cells,gene protein expression and gene sequencing were identified.Untill the results were correct,the pcDNA3.0-Flag and pcDNA3.0-Flag-CDK4 eukaryotic expression vector were transfected into laryngeal carcinoma cells(Hep-2),the transcription level of CDK4 gene in laryngeal carcinoma cells was detected by real-time fluorescence quantitative PCR(qRT-PCR).The expression of the target gene protein in Hep-2 cells was detected by Western bloting.The effect of the gene on the proliferation of Hep-2 cells was confirmed by CCK-8 assay and plate cloning assay.ResultsThe results of protein expression identification and gene sequencing prove that the eukaryotic expression vector of pcDNA3.0-Flag-CDK4 was successfully constructed.The extracted plasmid was transfected into Hep-2 cells.The transcription level of CDK4 gene in Hep-2 cells detected by qRT-PCR was significantly higher than that in the control group.The results of Western bloting showed that the target gene protein was successfully expressed in Hep-2 cells.These experiments proved that the target gene was successfully transferred to Hep-2 cells.CCK-8 assay and plate cloning assay showed that CDK4 gene could promote the proliferation of Hep-2 cells.ConclusionsIn this study,the eukaryotic expression vector of human CDK4 gene was successfully constructed and it was preliminarily verified that the gene could promote the proliferation of laryngeal cancer cells(Hep-2),which lead a foundation for the further study of laryngeal cancer cells mechanism.