Construction Of Mutant P PVenus-cdc25b Expression Vector And Preliminary Study Of Its Function

Cdc25b(cell division cycle 25b),a member of the cdc25 phosphatase family.It was a Thr/Tyr bispecific protein phosphatase,which played a reverse inhibitory role in the regulation of mammalian cell cycle.During meiotic recovery of germ cells,cdc25 b can dephosphorylate 14/Thr and 15/Tyr on CDK1(cyclin dependent kinase1).This results in the recovery of CDK1 activity and the binding of cyclin B to play a role in initiating cell division.There were two kinds of short sequence specific cdc25 b protein phosphatase,which was nuclear export sequences and nuclear localization sequences.During the dynamic shuttle of cytoplasm and nucleus,the protein phosphatase enters the nucleus from the cytoplasm before the initiation of meiosis,and the cell meiosis resumes from the nucleus to the cytoplasm.At the same time,the activity of protein phosphatase of cdc25 b was regulated by phosphorylation or dephosphorylation.There were also 149 and 321 sites of phosphorylation modification on cdc25 b.Protein kinase PKA modifies these two sites on cdc25 b by phosphorylation or dephosphorylation to regulate the activity of protein phosphatase.Inhibiting or eliminating the blockade of cell meiosis can further regulate the process of cell cycle.In this study,RNA was retrieved from ovarian tissues of female mice by molecular biological method,and the mouse cdc25 b gene and pVenus-cdc25 b eukaryotic expression vector were constructed.The specific phosphorylation sites of PKA binding on cdc25 b were analyzed by proteomics to achieve gene site-specific modification and construct mutant vector to overexpress cdc25 b gene.The wild type pVenus-cdc25b-WT and mutant pVenus-cdc25b-Mut eukaryotic expression vectors were transfected into 293 T cells and Hela cells in vitro for functional verification.The results were as follow: 1.The mouse cdc25 b gene was amplified by specific amplification primers,and the eukaryotic expression vector of pVenus-cdc25-WT was constructed.The wild type plasmid of pVenus-cdc25-WT was identified by Hind III and BamH I digestion and sequencing analysis,which indicated that the mouse cdc25 b gene was successfully amplified.2.Mutations were made at the 30 th,149th,321 th and 351 th serine sites of cdc25 b protein phosphatase.The construction of pVenus-cdc25b-S321 A,pVenus-cdc25b-S149 A,pVenuscdc25b-S321A-S149 A,pVenus-cdc25b-S351 A,pVenus-cdc25b-S30 A mutant vectors was completed.The results of Hind III and BamH I digestion and sequencing analysis showed that: The site 30 th,149th,321 th and 351 th of cdc25 b have been mutated from serine to alanine,and the mutation of specific site of cdc25 b phosphatase has been successfully realized.3.The recombinant plasmids pVenus-cdc25b-WT and pVenus-cdc25b-Mut were used to transfect 293 T cells and Hela cells respectively.The results showed that after pVenus and pVenus-cdc25b-WT transfection,cdc25 b phosphatase was mainly localized in the cytoplasm of the two kinds of cells,and only a small amount was localized in the nucleus.However,after pVenus-cdc25b-Mutant transfection,the expression of cdc25 b phosphatase was localized only in the nucleus of both cells,but not in the cytoplasm.This indicated that cdc25 b phosphatase maintained the dynamic shuttle process in the cell nucleus,and there was obvious nuclear retention after mutation of specific site.CONCLUSION: The specific phosphorylation site of mouse cdc25 b gene was successfully mutated in this experiment.Various mutation vectors of pVenus-cdc25 b were constructed.The results showed that 293 T cells and Hela cells were transfected with mutant vectors.Cdc25 b phosphatase was a dynamic process of nuclear shuttle during cell division.After mutation of specific phosphorylation sites on cdc25 b phosphatase,nuclear localization and functional changes of nuclear output sequence showed obvious nuclear retention.