Interacción insecto-baculovirus. Aplicaciones en la mejora de baculovirus como vector para la expresión heteróloga de proteínas

Resumen

Baculoviruses are a large group of pathogenic insect viruses that are characterized by their high specificity. The main application is their use as a bioinsecticidal against different crop pests, although some of their characteristics have allowed the development of technologies to use the baculoviruses as vectors for the heterologous protein expression, as well as in gene therapy. The expression system based on baculoviruses, known as BEVS (Baculovirus Expression Vector System), was developed in the 80’s and since then it has allowed the expression of a great diversity of recombinant proteins in insect cell cultures, in a faster and more economical way than using other expression systems. However, this production system has some limitations affecting mostly to the large-scale productions. These limitations are usually related with low yields of expression, decreasing the production efficiency. Moreover, the continuous multiplication of the baculoviruses in cell cultures results in the emergence of defective viruses which are characterized by the loss of parts of the genome, affecting to the recombinant proteins production due to the loss of the corresponding recombinant gene. Therefore, for the BEVS to remain competitive as a production system, it is necessary the introduction of improvements to increase the production levels with respect to the conventional system. Taking advantage of the knowledge of previous studies about the interaction of the baculoviruses with their host, in this thesis has been addressed the introduction of improvements in the BEVS from different perspectives and developing different strategies, which are reflected in each of the following chapters. In the first chapter, a sequence derived from the baculovirus genome of S. exigua (SeMNPV) with promoter activity is described. This sequence is derived from a viral gene highly expressed in insects infected with SeMNPV, and has been named as pSeL. This new promoter shows an expression levels of the GFP protein about twice higher compared to those obtained using the promoter of the polyhedrin gene (polh) in different insect cell lines. Moreover, the combination of both promoters pSeL and polh shows an additive effect in comparison with the expression obtained with both promoters used separately. In the second chapter, the transcriptional study of the infective process of the baculoviruses AcMNPV and SeMNPV both in cell culture and S. exigua larvae has revealed a gene, lef5, highly overexpressed in larvae (with respect to its expression in cell culture). This, gives us the opportunity to hypothesize about the possible role of this protein in the genomic stability of the baculoviruses during their replication in cell culture. The study of the overexpression of the lef5 gene from SeMNPV (Se-lef5) in recombinant baculoviruses, that also express GFP, confirms the influence of this gene in the viral stability. Through several successive infective passages, it is observed that the stability of the gfp transgene and the ability to express GFP, is higher in those viruses expressing Se-lef5 compared with the control viruses. The third chapter focuses on the search of non-viral regulatory or enhancer sequences (coming from the host), which can influence in the expression of viral genes and increase the production of recombinant proteins of interest. For that, it was generated a viral library containing genes expressed in the gut of S. exigua larvae, and it was selected by flow cytometry, using a cell sorter, for those viruses showing high expression levels of GFP. The subsequent selection and characterization of those individual baculoviruses showing the best expression levels of GFP, allowed us to identify some of the transgenes present in those baculoviruses. Among the identified sequences we found, multiple times, genes similarly to P450 cytochrome, heat-shock proteins and REPAT proteins. The isolated viruses that carry these sequences show expression levels of GFP between 3-4 times higher to the expression levels before the library selection. However, the generation of equivalent recombinant baculoviruses expressing these sequences, show expression levels of GFP similar to the control baculovirus. This suggests that the increased GFP expression observed can be due to other factors, as the emergence of point mutations, and not only to the presence of the transgene in the selected baculoviruses. The results presented in this thesis represent new improvements in the production of recombinant proteins in cell culture, through the use of promoters other than conventional and the increase of the genomic stability of the recombinant baculoviruses. The introduction of regulatory or enhancer sequences of the expression has also been explored to improve the production levels. Although in this case it could not be validated that the observed increase of expression was associated to the presence of these sequences (it exists the possibility that this improvement was due to mutations on the viral genome), our results validate the methodology employed for the selection of viruses with higher expression levels of recombinant protein. Thus, the strategies developed in this work are able to increase the expression levels of a recombinant protein. Therefore, they show a great potential to be used in the vectors usually employed in the baculovirus expression system, increasing its profitability in the large-scale production of recombinant proteins.