Estratégias para produção de proteínas recombinantes em células eucariontes e suas aplicações para o diagnóstico de arboviroses

Abstract

There are two main types of diagnostic tests for arboviruses: serological tests, which detect the presence of antibodies against the virus or viral antigens, and molecular tests, which detect the presence of the virus's genetic material. The production of serological tests for these diseases presents limitations: recombinant proteins from these viruses are challenging to express in eukaryotic cells, while those produced in prokaryotic systems have shown limited specificity and/or sensitivity. When produced in eukaryotic cells, these proteins undergo post-translational modifications that can interfere with the antigen-antibody interaction and consequently, the sensitivity of the diagnostic test, which does not occur in prokaryotic cells. This study describes the production and purification of the E protein from serotypes 1 to 4 of DENV and the E2 protein from CHIKV in mammalian cells and bacteria. The protein sequences were modified by excluding the transmembrane domain and adding a signal peptide to the N-terminal portion to allow secretion. Additionally, the Kozak consensus sequence was incorporated into the expression vector to optimize mRNA translation in eukaryotic cells. The pcDNA 3.1 plasmid was initially used for transient expression. Subsequently, antibiotics were used to select a pool of cells capable of stably expressing the cloned gene in the plasmid. Alternatively, eukaryotic cells were transduced with lentiviral vectors to establish stable cell lines of expression. The CHIKV E2 protein was produced in EXPI cells using a plasmid vector, and after purification, it was used in ELISA assays with sera from infected and uninfected individuals. These assays resulted in high sensitivity and specificity rates for the detection of IgM and IgG antibodies. For comparison purposes, the CHIKV E2 protein was also produced in E. coli and tested in ELISA, demonstrating similar potential for IgG detection but poorer performance for IgM detection compared to proteins produced in eukaryotic cells. The study demonstrated that cell lines stably transfected with pcDNA 3.1 followed by selection or transduction by lentiviruses are capable of expressing the proteins of interest. ELISA assays suggest that proteins produced in eukaryotic cells have better capacity for IgM and IgG antibodies detection than those produced in prokaryotic cells. Through the evaluation of antibody recognition in the serum of a larger number of infected individuals, these antigens are being employed in the prototyping of diagnostic tests in ELISA and imunocromatografic rapid test formats.