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217 The Investigation of Plasmid DNA Stability in Attenuated Strains of Salmonella
I. V. Dukhovlinov1, Y. N. Dukhovlinova1, B. V. Murashev1, M. N. Boychenko2, N. A. Klimov*1, A. A. Vorobyov2, and A. P. Kozlov1
1Biomedical Ctr., St. Petersburg, Russia and 2I. M. Sechenov Moscow Med. Academy, Russia
Objectives: To study the stability of recombinant plasmid DNA molecules in attenuated Salmonella strains used as carrier of DNA-vaccines against HIV-1.
Materials and Methods: Three attenuated strains of Salmonella were studied: Salmonella typhimurium SL 7207, Salmonella typhimurium T10, and Salmonella enteritidis Å-23. Instability of plasmid DNA in Salmonella strains was shown as percentage of cells loosing plasmid during one generation in logarithmic phase of growth. The time of generation was determined graphically in semilogarithmic scale. Cell retention rate in vivo was determined by plating of spleen homogenates of infected mice on brilliant green agar. The rate of plasmid-containing cells was shown by plating of spleen homogenates on LB-agar containing the antibiotic (ampicillin 100 mkg/ml) and without antibiotic. The following plasmids were studied: pBMC (3.2 kb) was constructed for the expression of genes in eucaryotic cells; pBMC-ENV (5.8 kb) expressing gene of gp120 glycoprotein of HIV1 subtype B in eucaryotic cells.
Results: It was shown that percentage of cells loosing plasmid DNA per generation varies in different strains of Salmonella (Table 1). The best stability was observed in S. typhimurium T-10.
Table 1.
Strain of Bacteria Percent of Cells Loosing
Plasmid DNA per Generation
pBMC pBMC-ENV
Salmonella
typhimurium SL7207 - 27%
Salmonella
typhimurium T-10 - 2.8%
Salmonella
enteritidis E-23 3.0% 75%
Instability was shown also in spleens of infected mice. Mice were infected per os by Salmonella typhimurium T10 strain, which carries pBMC-ENV plasmid. We have determined that after 3 weeks 20% of Salmonella cells contained plasmid DNA. This corresponds to 60 generations of Salmonella in mouse organism.
Conclusions: Plasmid DNA stability in Salmonella depends on host strain and on DNA sequences in plasmid DNA. After immunization of mice per os with recombinant Salmonella bearing plasmid DNA, Salmonella replicates in spleens of animals with the rate of 20 generations/week.
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