[Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring]. / Primenenie DNK-aptamerov dlia obogashcheniia nizkopredstavlennykh belkov v kletochnykh ékstraktakh dlia kolichestvennoi detektsii metodom monitoringa vybrannykh reaktsii.

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

[Way to the peptide vaccine against hepatitis C].

In order to surpass the problem of genetic variability of hepatitis C virus envelope proteins during vaccine development, we used the so-called "reverse vaccinology"approach--"from genome to vaccine". Database of HCV protein sequences was designed, viral genome analysis was performed, and several highly conserved sites were revealed in HCV envelope proteins in the framework of this approach. These sites demonstrated low antigenic activity in full-size proteins and HCV virions: antibodies against these sites were not found in all hepatitis C patients. However, two sites, which contained a wide set of potential T-helper epitope motifs, were revealed among these highly conserved ones. We constructed and prepared by solid-phase peptide synthesis several artificial peptide constructs composed of two linker-connected highly conserved HCV envelope E2 protein sites; one of these sites contained a set of T-helper epitope motifs. Experiments on laboratory animals demonstrated that the developed peptide constructs manifested immunogenicity compared with one of protein molecules and were able to raise antibodies, which specifically bound HCV envelope proteins. We succeeded in obtaining antibodies reactive with HCV from hepatitis C patient plasma upon the immunization with some constructs. An original preparation of a peptide vaccine against hepatitis C is under development on the basis of these peptide constructs.