Producción de una proteína recombinante de interés farmacéutico derivada del virus del síndrome reproductivo y respiratorio porcino (prrsv)

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a disease that causes mortality and severe respiratory problems, such as interstitial pneumonia, in pigs. It is characterized by prolonged viremia, and its replication occurs primarily in pulmonary alveolar macrophages. PRRSV is highly variable and antigenically complex, making it difficult to control and prevent. It is composed of proteins such as the nucleocapsid and the glycoproteins GP2, GP3, GP4, and GP5. The latter is crucial for the interaction of the virus with the host cell and contains immunodominant epitopes that generate an immune response against PRRSV. In Colombia, it is an endemic disease present in more than 11 departments, where 11 strains have been identified. The purpose of this study is the production of a recombinant protein based on the fusion of the GP5 glycoprotein with the heat shock protein Hsp90.3 from Nicotiana benthamiana, used as a carrier, to improve the folding of GP5 in bacterial systems. To this end, the GP5 gene was amplified with PstI and XhoI restriction sites using conventional PCR. The amplified DNA was purified and ligated into the pGEMT (cloning) and pRSET-A (expression) vectors. Subsequently, BL21 competent cells were transformed, and expression parameters of the recombinant protein were standardized. The identity and antigenic capacity of this protein were evaluated by Western blot using monoclonal antibodies and sera from PRRSV-seropositive sows, respectively. The results confirmed the successful amplification and cloning of the GP5 gene (615 bp), yielding 97.84% identity by sequencing, both in its single version and fused with Hsp90.3. Furthermore, the recombinant GP5 protein, weighing 22 kDa, was successfully expressed and identified using anti-His monoclonal antibodies, reacting with sera from seropositive sows circulating in the Atlantic. Additionally, the p35S:GATFH-GP5 vector was constructed to transiently express the protein in plants. These results represent a significant step forward in evaluating the antigenic functionality of the recombinant protein, as well as its potential application in PRRSV diagnosis. This study will allow for future research to validate the expression conditions of GP5 alone and fused to Hsp90.3 from Nicotiana benthamiana in plant systems for pharmaceutical applications against PRRSV.