[Different systems of delivery of HIV-1 DNA vaccine encoding the multiepitope CTL-immunogene].

We used, within the case study, virus-like particles (VLP) and attenuated strains of salmonella for the delivery of HIV-1 DNA vaccine encoding the multiepitope CTL-immunogene. The immunogenicity of the thus obtained vaccine constructions was comparatively analyzed. All constructions were shown to be able of inducing, in immunized animals, both the specific T-cell responses and the synthesis of virus-specific antibodies. The lowest level of immune response was registered in animals immunized by "naked" plasmid DNA. The delivery by plasmid DNA involving VLP or the attenuated strain of salmonella enhances the efficiency of the DNA-vaccine presentation to the immune system.

[Chemico-enzymatic synthesis, cloning and expression of a gene for an analog of human anaphylatoxin C5a]. / Khimiko-fermentativnyi sintez, klonirovanie i ékspressiia gena analoga chelovecheskogo anafilatoksina C5a.

Chemical-enzymatic synthesis and cloning of a gene for a human anaphylatoxin C5a analog were carried out. Recombinant plasmid pRC5a providing the expression of the synthetic gene in the Escherichia coli cells was obtained. The biological activity of the expression product was demonstrated by the chemotaxis activity test and by the release of myeloperoxidases from rat peritoneal cells that was induced by bacterial cell lysates containing the recombinant protein.

[Effective expression of genes for interleukin-2 and its mutant analogs in E. coli cells]. / Effektivnaia ékspressiia v kletkakh E. coli genov interleikina-2 cheloveka i ego mutantnykh analogov.

Recombinant plasmids were constructed for the efficient expression in E. coli cells of the human interleukin-2 (HIL-2) gene and two its mutant analogues obtained by of chemical-enzymic synthesis and polymerase chain reaction (deletion of 14 C-terminal amino acids and a change of the codon for Trp121 to Phe). The recombinant HIL-2 but not the mutant analogues were shown to be biologically active. Both analogues obtained were weak antagonists to HIL-2.

[Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]. / Effektivnyi sintez i klonirovanie gena interleikina-2 cheloveka i egoanaloga: ékspressiia v kletkakh E. coli gena interleikina-2.

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.

[Design of recombinant-stable plasmids of the pFH series]. / Konstruirovanie rekombinantno-ustoichivykh plazmid serii pFH.

By cloning synthetic oligonucleotides into pUC18 plasmid, pFH123--pFH127 plasmids have been constructed. Their polylinker area, along with sites of widely used restriction endonucleases, contains two pairs each of FokI and HgaI sites in the opposite orientation to provide subfragment with unique predetermined 5'-ends. Comparative stability of the new plasmids and their derivatives has been studied and compared with that of the earlier constructed pMB plasmids.

[Reconstruction of the synthetic gene for human interleukin-2]. / Rekonstruktsiia iskusstvennogo gena chelovecheskogo interleikina-2.

Chemical-enzymatic synthesis and cloning of the DNA fragment coding for the human interleukin-2 signal sequence was accomplished. A hybrid plasmid pSIL-2 containing the gene of the human interleukin-2 with this signal sequence was constructed for effective expression of the gene in eukaryotic systems. A variant permitting the removal of the interleukin-2 stop-codons was obtained, which is suitable for the construction of chimeric genes containing the interleukin-2 gene sequence at the 5'-end.

[Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends]. / Plazmidy pMB123 i pMB124--vektory dlia polucheniia subfragmentov DNK s prizvol'nymi "lipkimi" kontsami.

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.

[A simplified variant of the Maxam-Gilbert method for determining the primary structure of oligonucleotides and DNA fragments]. / Uproshchennyi variant metoda Maksama-Gilberta dlia opredeleniia pervichnoi struktury oligonukleotidov i fragmentov DNK.

A modification to the Maxam-Gilbert method is proposed that involves precipitation of the nucleotide material with the acetone solution of lithium perchlorate after the completion of chemical reactions to remove the reagents. Modification of cytosine residues is carried out in the presence of lithium chloride. The new mode of precipitation simplifies and speeds up the analysis of oligonucleotides and DNA fragments.

[Synthesis and cloning of a DNA fragment containing a probable site for eukaryotic mRNA binding to ribosome]. / Sintez i klonirovanie fragmenta DNK, soderzhashchego predpolagaemyi sait sviazyvaniia éukarioticheskoi mRNK s ribosomoU

A series of oligonucleotides, including two polynucleotides of 33 bases long, were synthesized by a solid-phase phosphotriester method. Potassium salt of 3-nitro-1,2,4-triazole in the presence of 18-crown-6 ether was used as nucleophilic catalyst. The partly complementary polynucleotides were elongated by DNA-polymerase I (Klenow fragment) to the full duplex, which was digested with SalGI and was inserted into a plasmid pUR222. Phe synthesized DNA fragment precedes the gene of human gamma-interferon in the chromosome and contains the site for mRNA binding to ribosome.

[Chemico-enzymatic synthesis of genetic elements for expression of synthetic genes in Bacillus subtilis cells]. / Khimiko-fermentativnyi sintez geneticheskikh élementov dlia ékspressii iskusstvennykh genov v kletkakh Bacillus subtilis.

In order to obtain the recombinant Bacillus subtilis strain, a transcriptional-translational control unit of the alpha-amylase gene of B. amyloliquefaciens was synthesized. The oligodeoxyribonucleotides were prepared by the modified triester method in solution and by the solid-phase approach. Then these oligonucleotides were joined by DNA ligase into two fragments which were cloned in the phage M13mp9 DNA and the plasmid pBR327. A plasmid harboring the site regulating the transcription of the alpha-amylase gene may be employed as vector for cloning the promoter-containing fragments in E. coli cells.

[Complexes of nonionic triether analogs of oligonucleotides with polynucleotides]. / Kompleksy neionnykh triéfirnykh analogov oligonukleotidov s polinukleotidami.

Alkyl triester analogues of oligodeoxynucleotides were synthesized: [Tp(CH3)]8T(Ac), [Tp(C2H5)]8T, [dGp(C2H5)]2G, [dAp(C2H5)]2A. Their binding to complementary polyribo-and polydeoxyribonucleotides was studied by UV-spectroscopy and gel-filtration. The DNA complexes of analogues were shown to be more thermostable than the RNA ones independent on the nucleic base nature and alkyl residue size of the analogue used. Mg-salt increased the thermostability of the oligonucleotide analogue--RNA complexes.