Optimizing Сonditions for Сultivation and Selecting a Producer Strain for Enhanced Expression of a Recombinant Fragment of Circovirus Capsid Protein

Porcine Circovirus Type 2 (PCV2) is an infectious viral disease that causes significant damage to the livestock industry. The virus affects the immune system of pigs, making them more susceptible to other diseases. Implementing veterinary control programs and vaccination are effective methods for preventing the spread of this infection. The main component of commercial vaccines is the recombinant capsid protein of the circovirus, encoded by the orf2 gene. The main capsid protein (ORF2) is essential for assembling viral particles and is a key target for the immune system. This thesis focuses on the development and optimization of methods for obtaining recombinant capsid protein, specifically full-length ORF2 with a nuclear localization signal, in the Escherichia coli expression system. The main goal of this work is to develop efficient methods for producing the ORF2 protein by creating a highly productive producer strain of the recombinant protein and selecting and optimizing cultivation conditions. During the research, optimal cultivation conditions were selected for the E. coli Rosetta (DE3) strain to predominantly obtain the target product in inclusion bodies - cultivation on LB medium at 40°C, induction with 1 mM IPTG at OD590=1.0. Protein production yields reached 120 mg/L. For further analysis, the Clear coli BL21(DE3) strain with modified lipopolysaccharides (LPS) was chosen, allowing safe use of the producer for obtaining viral vaccines. An optimal plasmid with the orf2 gene adapted for expression in E. coli was selected. Induction conditions were chosen based on high production of the target protein in inclusion bodies. As a result, optimal cultivation parameters were determined for the developed vaccine against porcine circovirus, achieving yields of approximately 120 mg/L with an 18-hour cultivation time, 0.2 mM IPTG concentration, OD590 of 0.7, and a cultivation temperature of 40°C. During the research, two auxiliary constructs were developed: pArg2 with arginine aminoacyl-tRNA genes and pArgS with the argS gene encoding arginyl-tRNA synthetase. The aim of these constructs was to increase the yield of the target protein ORF2. The presence of the pArgS plasmid did not affect the protein production yield, while the pArg2 plasmid contributed to increased production of the capsid protein ORF2. However, it was found that the presence of both constructs slowed down bacterial cell growth. Optimal cultivation conditions without using auxiliary constructs led to increased protein yield. Nevertheless, the use of these auxiliary constructs may be beneficial for obtaining proteins with a high content of the amino acid arginine. The most effective method for obtaining the target product was found to be autoinduction in TB medium without lactose supplementation. This method allowed for maximum yields of the investigated full-length ORF2 and truncated variant (without nuclear localization signal) of ORF2 - 240 mg/L and 500 mg/L, respectively. Autoinduction is a convenient and practical method that can be efficiently used for scaling up production and fermentation processes.