Construction of eukaryotic recombinant plasmid pCDNA3.1-Flag-PTEN and prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN

ObjectiveTo construct the eukaryotic recombinant plasmid pCDNA3.1-Flag-PTEN and prokaryotic recombinant plasmid pGEX-6P-1-GST-PTEN of phosphatase and tension homologue deleted on chromosome ten(PTEN), and study the biological effects of PTEN fusion plasmid.

MethodsThe primers of PTEN gene were designed according to the sequence in Gene Bank.The target fragment was inserted into pCDNA3.1-Flag and pGEX-6P-1-GST empty vector by genetic recombination technique, and the inserted gene sequence was identified by PCR and DNA sequencing.The pCDNA3.1-Flag-PTEN was transfected into HEK293 cells and pGEX-6P-1-GST-PTEN was transfected into Escherichia coli BL-21.The expression of PTEN protein was detected by Western blot and Coomassie brilliant blue staining.

ResultsThe PCR results showed that the target gene was successfully inserted.The results of DNA sequencing showed that the inserted sequence was completely consistent with the target gene sequence, and no site-mutation was found.The results of Western blot and Coomassie brilliant blue staining confirmed that the fusion plasmid could be correctly expressed in vivo.

ConclusionsThe successful construction and correct expression of PTEN in eukaryotic and prokaryotic expression vectors can provide a basis for further study of the mechanism of PTEN in Parkinson's disease.