Transfecção do gene GUCA2A codiûcante da guanilina em células embrionárias de rim humano

Abstract

The synthesis of recombinant proteins is one of the most important tools in modern biotechnology, widely used in biomedical research and drug development. This process involves introducing genetic material into host cells through transfection, using vectors such as plasmids. Among the available expression systems, mammalian cells4such as the HEK 293T cell line4stand out for their ability to perform post-translational modifications essential to the functionality of human proteins. This study aimed to establish an experimental system for the expression of the GUCA2A gene in HEK 293T cells, with the goal of producing the recombinant protein guanylin, which has therapeutic potential. The recombinant plasmid containing the GUCA2A gene was constructed using the restriction enzymes BamHI and XbaI, and its incorporation into Escherichia coli dH5alpha strains was analyzed by Polymerase Chain Reaction (PCR), showing early-cycle amplification. Plasmid DNA was extracted with suitable purity and concentration, as confirmed by spectrophotometric analysis. HEK 293T cells were cultured under controlled temperature and CO¢ conditions and transfected via lipofection. Transfection efficiency was assessed by fluorescence microscopy, observing the expression of the EGFP marker present in the vector. The presence of green fluorescence in multiple cells indicated plasmid DNA uptake. Subsequently, selection with puromycin enriched the population of transfected cells. Cell morphology remained preserved, indicating that transfection with the GUCA2A gene did not compromise cell integrity. The results confirmed the success of cloning, amplification, and transfection steps involving the GUCA2A gene in HEK 293T cells. The established system proved to be efficient and reproducible, providing a foundation for future analyses such as RT-qPCR, Western blot, and functional assays of the recombinant protein. This study contributes to advancing gene expression systems aimed at producing proteins with therapeutic potential