Construction Of Eukaryotic Recombinant Plasmid PCDNA3.1-FLAG-PTEN And Prokaryotic Recombinant Plasmid PGEX-6P-1-GST-PTEN And Their Respective Expression In HEK 293 Cells And Escherichia Coli BL-21

Background:The tumor suppressor gene PTEN(phosphatase and tension homologue deleted from chromosome 10)is a gene located in the human tenth autosome,and its expression product belongs to the protein tyrosine phosphatases(PTP)family.The PTEN genes of different species have high homology.PTEN gene is an important regulatory factor in a variety of organisms and can participate in the proliferation,differentiation and apoptosis of various cells.In addition,the latest scientific research evidence shows that PTEN gene plays an important regulatory role in the growth,differentiation,axon repair,and synaptic plasticity of central nervous system neurons,and participates in important links in the pathogenesis of various neurodegenerative diseases.Some studies have pointed out that PTEN is particularly related to the mechanisms of the dopamine neurotransmitter,?-synuclein toxicity,and mitochondrial dysfunction.It provides new ideas for the pathogenesis of Parkinson's disease,potential genetic diagnostic sites and therapeutic targets.This is why this study chose to construct multiple kinds of recombinant plasmids of PTEN gene and purify the fusion protein.It can be used for further research on the sequence characteristics of PTEN gene,the structural biological function of protein,and the research and future of PTEN in the pathogenesis of PD.Further development of relevant clinical experiments has created favorable conditions.Objective:1.Designing the corresponding primers based on the PTEN gene sequence found in the gene bank and the different cell types is the most basic step in subsequent experiments.2.Construction of PTEN eukaryotic recombinant plasmid pCDNA 3.1-FLAG-PTEN and prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN using genetic recombination technology,which provides basic substances of molecular studies for studying the biological effects of PTEN fusion plasmids and point mutation experiments.3.The eukaryotic recombinant plasmid pCDNA 3.1-FLAG-PTEN was transfected into HEK 293 cells,and then it was verified that the recombinant plasmid can be normally expressed in the cells.This is to further study the biological effect of PTEN gene on PD model cells and to be used for PD gene diagnosis and treatment Provide scientific experimental basis.4.The prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN was transformed into Escherichia coli BL-21 to induce its correct expression of the corresponding PTEN recombinant protein,and the fusion protein was extracted and purified.This was laid for the experimental basis study of various biological function,such as the phosphorylation site and degradation mode of PTEN protein and its relationship with PD-related signaling pathways.Methods:According to the PTEN gene sequence found in GeneBank,primers were designed and synthesized,and the target fragment was inserted into pCDNA 3.1-FLAG and pGEX-6P-1-GST empty plasmids using gene recombination technology.The insertion was successfully identified gene sequence by PCR and DNA sequencing.PCDNA 3.1-FLAG-PTEN was transfected into HEK 293 cells to detect the expression of PTEN protein by egg and white blot techniques.PGEX-6P-1-GST-PTEN was transferred to Escherichia coli BL-21 and the expression of PTEN protein was detected by Coomassie blue staining.Results:1.PCR showed that the target gene was successfully inserted.DNA sequencing showed that the inserted sequence was completely consistent with the target gene sequence,and there was no site mutation.2.Prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN was successfully constructed.3.The eukaryotic recombinant plasmid pCDNA3.1-FLAG-PTEN was successfully constructed.4.Western blotting and Coomassie blue staining were used to confirm that the prokaryotic and eukaryotic fusion plasmids can be correctly expressed in the corresponding types of organisms.Conclusion:The successful fusion and expression of PTEN-related eukaryotic and prokaryotic fusion plasmids have successfully laid the foundation for further research on the specific molecular mechanism and effect of PTEN in PD.