| Abstrakt: |
High-level expression vector pAZ was constructed for in vivo delivery of bioactive recombinant proteins, antigenic determinants, among other things. This vector meets the requirements to construction of recombinant bacteria as live oral carriers. It has a strong constitutive promoter, high stability in E.coli and vaccine strain Salmonella cells, and, moreover, encodes in addition for the marker protein (beta-galactosidase) which will later help follow up the fate of bacterial carriers and their interactions in the microorganism. Several recombinant plasmids encoding for beta-galactosidase variants with insertions of short fragments of HIV-1 gp41 and gp120 proteins, which were previously shown to be antigenic determinants, have been constructed on the basis of pAZ. E.coli and vaccine strain Salmonella cells were transformed by recombinant plasmids. To a considerable extent the level of hybrid protein synthesis depends on the structure of the antigenic determinant inserted. The maximal level of synthesis in E.coli is 16%. This hybrid protein could be isolated and purified (up to 90%) with the yield of 4 to 6 mg/g of wet cells. Almost all the hybrid proteins were immunologically reactive, as shown by ELISA with nonfractionated lysates and purified hybrids. In both strains in vitro stability of the vector and recombinant plasmids was at least 90% after 10 passages (about 140 generations) under random conditions. This paper sums up the first stage of construction of recombinant bacteria as live oral carriers. |
