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We describe here a novel approach for the construction of recombinant fusion proteins in a bacterial expression vector that facilitates the production of protein immunogens. This approach is based on the introduction of a copy of the cloned gene into a constitutively active plasmid via homologous recombination. Sequences necessary for the in-frame ligation with the cloned gene’s own 5′ sequences are encoded by the targeting vector. The presence of a small number of extra nucleotides (2 bp) after the desired gene allows the recombination to occur between the recombination sites and the target gene. In order to produce a subunit vaccine protein, the targeting vector is integrated into the chromosome of a recombinant E. coli clone. The insertion of the genetic material is confirmed by the identification of antibiotic markers, which can be eliminated by homologous recombination. The vector constructs used are designed to facilitate the expression of covalently linked glycoprotein subunits by directing them to the periplasmic space of the bacteria. Subsequently, they are processed via proteolytic enzymes and chemically linked to carrier proteins. The feasibility of this approach was demonstrated in the construction of a recombinant fusion protein of tetanus toxin. The recombinant protein could be highly purified and induce protection in mice. Since it can be produced in large quantities, this approach is feasible for the production of vaccine antigens.Q:

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