Avaliação da estabilidade físico-química e biológica de plasmídeos com potencial biotecnológico

Abstract

Studies involving plasmid stability have started for at least two decades and have been growing in recent years, since pDNAs have had enormous potential as vectors in gene therapy, however the therapeutic use of these vectors has been hampered by stability issues, especially in Refers to the process of production and purification, storage for long periods and being susceptible to degradation by nucleases. Thus, assays that allow the analysis of this degradation process can be important tools for its understanding, associated to other variables, such as temperature, storage time, pDNA size and nucleoside action. The competent E. coli DH5-α was produced, transformed with the pDNAs studied (pVAX1, pVAX1lacZ and MSPpVAX1), purified and stored at different temperatures for a predetermined time and to establish a relationship between the stability of the different pDNAs and their Biological function as vectors, the resistance of the supercoiled isoform to the action of serum nucleases at different concentrations and over time was studied. For this purpose, agarose gel electrophoresis and transformation in E. coli with calculation of cell transformation efficiency were performed. It was observed over time that the integrity of the supercoiled pDNA was lost as a function of time and storage temperature, in addition, pDNAs containing only prokaryotic sequences proved to be more resistant to these factors when compared to that having procaryotic sequence pDNA, showing that these factors influence the stability of pDNA. In relation to the nuclease action, the bigger plasmid was more affected by the activity of these enzymes. Regarding biological function, transformation efficiency assays in E. coli indicated that there was a higher percentage of transformed cells when plasmid was used in the supercoiled conformation. It was verified in all the tests that the supercoiled isoform was always more efficient than the other isoforms, possibly due to its greater cytoplasmic stability and the faster diffusion of this isoform towards the nucleus. Thus, this work showed the degradation kinetics, step by step, of the studied pDNAs, showing that the loss of supercoiled form compromises the stability of the pDNAs, thus affecting the biological function of the same, compromising their use in gene therapy and vaccines DNA.