| The structure and function of PTEN, Akt and Trx were summarized in this paper, and the relationship between these genes and Insulin secretion was discussed at the same time. The eukaryotic expression vectors of PTEN, Akt and Trx were constructed, and the method of detecting the efficiency of amplification of Trx gene in cell was established by Real-time Flurescent Quantitive Polymerase Chain Reaction (FQ-PCR). All of these were important for the project that research on molecular mechanisms of insulin secretion in INS-1 cell stimulated with reactive oxygen species.(1) Total mRNA was extracted from rat's liver. The DNA of Trx obtained by RT-PCR was cloned into pcDNA3.0 vector, then the recombinant plasmid was selected by Amp, digested, detected by PCR and sequence, and the pcDNA3.0-Trx was coustructed successfully.(2) The NIH 3T3 cell was transfected with pcDNA3.0-Trx, pcDNA3.0 plasmid or nothing by lipofectamine, and half of cell was stimulated with 50μΜH2O2, then the six kinds total mRNA were extracted from the cell. The cDNA obtained by RT-PCR was diluted from 10-1 to 10-5, and standard curve of 18s was formed from FQ-PCR.(3) The CT of Trx and 18s from FQ-PCR was studied with the 2 -△△CT method. The results showed that not only the plasmids had been transfected into cell, but also the expression of Trx with 50μΜH2O2 was higher than not stimulated.(4) The pTRE-WT-PTEN, pTRE-G129R-PTEN, pTRE-G129E-PTEN, pECE-Akt plasmid were took as plates to clone PTEN-WT, PTEN-G129R, PTEN-G129E and anti-sense PTEN genes by PCR that were subcloned into eukaryotic expression vector pcDNA3.0 correctly.(5) The pcDNA3.0-WT-PTEN was mutanted to pcDNA3.0-C124S-PTEN by PCR site-directed mutagenesis.All the sequences of Trx, PTEN and Akt related recombinant plasmid were vertified correctly in GenBank. Construction of these expression vectors are to investigate the function of the genes, and would finally contribute to study the molecular mechanisms of insulin secretion.
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