Construcción de vectores de expresión para la obtención y caracterización de una proteína recombinante derivada de la cápside del circovirus porcino tipo 2 (pcv2) en sistemas bacterianos y vegetales

Abstract

Porcine circovirus type 2 (PCV2) is a viral agent with a wide global distribution and is associated with reproductive disorders, causing porcine circovirus-associated diseases (PCVAD). In Colombia, it has been widely reported in the main swine-producing regions, where it is an endemic virus with a high mutation rate that generates new emerging genotypes associated with vaccine failures and more severe outbreaks, making its control more difficult. PCV2 is a single-stranded DNA (ssDNA) virus, 17 nm in diameter and non-enveloped. Its genome contains opening reading frame 2 (ORF2), also called the Cap gene, which encodes the capsid protein, considered the main immunogenic protein. This capsid protein has 234 amino acids and an approximate size of 28 kDa. Based on the above, the objective of this study was to construct vectors for the expression of an antigenic protein derived from the PCV2 capsid in bacterial and plant expression systems for its application in the serological detection of this infectious agent. To this end, PCR amplification of the gene encoding the capsid protein was performed using PCV2 DNA copies isolated from infected sows. Subsequently, this gene was ligated into the pGEM-T cloning vector and the pRSET-A expression vector, both in isolation and fused with the Hsp90.3 gene from Nicotiana benthamiana, which possesses chaperone properties, improving the stability and accumulation of the recombinant protein in any expression system. The recombinant plasmids were transformed into competent Escherichia coli cells. Expression levels were then evaluated, and the recombinant protein was detected by Western blot with monoclonal antibodies. The results obtained showed an amplification of the PCV2 Cap gene of 719 bp including the restriction sites XhoI and PstI. This gene was successfully cloned, transformed, and expressed in bacterial systems, and the protein was identified using anti-PCV2 and anti-polyhistidine monoclonal antibodies. Additionally, the recombinant protein obtained was reactive against sera from seropositive sows. Complementarily, the 6xHis-Cáp gene was amplified and successfully cloned into the pCR™8/GW/TOPO™ entry vector. Then, using Gateway recombination technology, it was transferred to the p35S:GAT-FH target vector, yielding recombinant plasmids for expression in plant systems. This work represents one of the first reports in Colombia on the construction of recombinant plasmids and the production of recombinant protein from the PCV2d genotype currently circulating in the country, with potential for use as diagnostic tools, enabling the effective control of porcine circovirus, differentiating it from other diseases, and reducing economic losses for the sector. Additionally, the results obtained will allow for studies on expression in plant systems, which represents a more efficient alternative for the production of antigenic proteins of pharmaceutical interest