13 research outputs found
Π£ΡΠΎΠ²Π΅ΡΡΠ΅Π½ΡΡΠ²ΠΎΠ²Π°Π½ΠΈΠ΅ ΡΠΊΠ·ΠΎΠ½ΠΎΠ²ΠΎΠ³ΠΎ ΠΌΠ΅ΡΠΎΠ΄Π° Π΄Π»Ρ ΡΡΠΊΠΎΡΠ΅Π½Π½ΠΎΠ³ΠΎ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΊΠΠΠ Π³Π΅Π½Π° ΡΠ΅Π½Π°Π»Π°Π·Ρ ΠΊΡΡΡΡ
We have improved our previously developed method of exon cloning of cDNA of eukaryotic genes to obtain the rat renalase gene cDNA. In contrast to the previously used step-by-step pairwise assembly of exons, in this work the procedure of full-length cDNA preparation was shortened due to simultaneous assembly of four neighboring exons at once (exons 1-4 and exons 6-9 of the rat renalase gene). The two obtained sequences (exons 1-4 and 6-9) were combined into a full-length cDNA of the rat renalase gene. The cDNA synthesized in this way was cloned into the prokaryotic vector pET-28a(+), which was then expressed in E. coli cells. The correctness of this approach was confirmed by sequencing resultant cDNA sequencing, which showed full (100%) identity with the nucleotide sequence available in the GenBank database (accession code: GenBankNM_001014167).Π£ΡΠΎΠ²Π΅ΡΡΠ΅Π½ΡΡΠ²ΠΎΠ²Π°Π½ ΡΠ°Π½Π΅Π΅ ΡΠ°Π·ΡΠ°Π±ΠΎΡΠ°Π½Π½ΡΠΉ Π½Π°ΠΌΠΈ ΠΌΠ΅ΡΠΎΠ΄ ΡΠΊΠ·ΠΎΠ½ΠΎΠ²ΠΎΠ³ΠΎ ΠΊΠ»ΠΎΠ½ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΊΠΠΠ ΡΡΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ
Π³Π΅Π½ΠΎΠ² Π΄Π»Ρ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΊΠΠΠ Π³Π΅Π½Π° ΡΠ΅Π½Π°Π»Π°Π·Ρ ΠΊΡΡΡΡ. Π ΠΎΡΠ»ΠΈΡΠΈΠ΅ ΠΎΡ ΡΠ°Π½Π΅Π΅ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½Π½ΠΎΠ³ΠΎ ΠΏΠΎΡΡΠ°Π΄ΠΈΠΉΠ½ΠΎΠ³ΠΎ ΠΏΠ°ΡΠ½ΠΎΠ³ΠΎ ΠΎΠ±ΡΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΡ ΡΠΊΠ·ΠΎΠ½ΠΎΠ², Π² Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅ ΠΏΡΠΎΡΠ΅Π΄ΡΡΠ° ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΉ ΠΊΠΠΠ Π±ΡΠ»Π° ΡΠΎΠΊΡΠ°ΡΠ΅Π½Π° Π·Π° ΡΡΠ΅Ρ ΡΠΎΠ³ΠΎ, ΡΡΠΎ ΠΌΡ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΎΠ±ΡΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠ΅ ΡΡΠ°Π·Ρ ΡΠ΅ΡΡΡΠ΅Ρ
ΡΠΎΡΠ΅Π΄Π½ΠΈΡ
ΡΠΊΠ·ΠΎΠ½ΠΎΠ² (1-4 ΠΈ 6-9 Π³Π΅Π½Π° ΠΊΡΡΡΡ). ΠΠ²Π΅ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΡΠ΅ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ (ΡΠΊΠ·ΠΎΠ½ΠΎΠ² 1-4 ΠΈ 6-9) ΠΎΠ±ΡΠ΅Π΄ΠΈΠ½ΡΠ»ΠΈ Π² ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΡΡ ΠΊΠΠΠ Π³Π΅Π½Π° ΡΠ΅Π½Π°Π»Π°Π·Ρ ΠΊΡΡΡΡ. Π‘ΠΈΠ½ΡΠ΅Π·ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ ΡΠ°ΠΊΠΈΠΌ ΠΎΠ±ΡΠ°Π·ΠΎΠΌ ΠΊΠΠΠ ΠΊΠ»ΠΎΠ½ΠΈΡΠΎΠ²Π°Π»ΠΈ Π² ΠΏΡΠΎΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΠΉ Π²Π΅ΠΊΡΠΎΡ pET-28a(+) Ρ ΠΏΠΎΡΠ»Π΅Π΄ΡΡΡΠ΅ΠΉ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠ΅ΠΉ Π² ΠΊΠ»Π΅ΡΠΊΠ°Ρ
E. coli. ΠΠΎΡΡΠ΅ΠΊΡΠ½ΠΎΡΡΡ ΡΠ°ΠΊΠΎΠ³ΠΎ ΠΏΠΎΠ΄Ρ
ΠΎΠ΄Π° ΠΏΠΎΠ΄ΡΠ²Π΅ΡΠΆΠ΄Π΅Π½Π° ΠΏΡΡΠ΅ΠΌ ΡΠ΅ΠΊΠ²Π΅Π½ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΠΎΠΉ ΠΊΠΠΠ, ΠΊΠΎΡΠΎΡΠ°Ρ ΠΏΠΎΠΊΠ°Π·Π°Π»Π° ΠΏΠΎΠ»Π½ΠΎΠ΅ (100%) ΡΠΎΠ²ΠΏΠ°Π΄Π΅Π½ΠΈΠ΅ Ρ Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡΡ Π±Π°Π·Ρ Π΄Π°Π½Π½ΡΡ
(ΠΊΠΎΠ΄ Π΄ΠΎΡΡΡΠΏΠ° GenBankNM_001014167)
ΠΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠΈ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΠΈ Π²ΡΠ΄Π΅Π»Π΅Π½ΠΈΡ ΡΠΊΠΎΡΠΎΡΠ΅Π½Π½ΠΎΠΉ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠΉ ΡΠ΅Π½Π°Π»Π°Π·Ρ Π² ΠΏΡΠΎΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ ΠΊΠ»Π΅ΡΠΊΠ°Ρ
Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for Β«accommodationΒ» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.Π Π΅Π½Π°Π»Π°Π·Π° (RNLS) β ΡΠ»Π°Π²ΠΎΠΏΡΠΎΡΠ΅ΠΈΠ½, N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠΉ ΠΏΠ΅ΠΏΡΠΈΠ΄ ΠΊΠΎΡΠΎΡΠΎΠ³ΠΎ (1-17 Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΡΡ
ΠΎΡΡΠ°ΡΠΊΠ° (Π°.ΠΎ.)) Π²ΡΠΏΠΎΠ»Π½ΡΠ΅Ρ Π½Π΅ΡΠΊΠΎΠ»ΡΠΊΠΎ Π²Π°ΠΆΠ½ΡΡ
ΡΡΠ½ΠΊΡΠΈΠΉ. Π ΠΊΠ»Π΅ΡΠΊΠ°Ρ
ΠΎΠ½ ΡΡΠ°ΡΡΠ²ΡΠ΅Ρ Π² ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ ΡΠ°ΠΊ Π³Π°Π·ΡΠ²Π°Π΅ΠΌΠΎΠΉ ΡΠΊΠ»Π°Π΄ΠΊΠΈ Π ΠΎΡΡΠΌΠ°Π½Π° (2-35 Π°.ΠΎ.), Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΠΎΠΉ Π΄Π»Ρ Β«ΡΠ°Π·ΠΌΠ΅ΡΠ΅Π½ΠΈΡΒ» ΠΊΠΎΡΠ°ΠΊΡΠΎΡΠ° FAD ΠΈ Π²ΡΠΏΠΎΠ»Π½Π΅Π½ΠΈΡ ΠΊΠ°ΡΠ°Π»ΠΈΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΡΠ½ΠΊΡΠΈΠΉ RNLS Π² ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ FAD-Π·Π°Π²ΠΈΡΠΈΠΌΠΎΠΉ ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Ρ (ΠΠ€ 1.6.3.5). ΠΡΠΈ ΡΠ΅ΠΊΡΠ΅ΡΠΈΠΈ RNLS Π²ΠΎ Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠ΅ ΠΏΡΠΎΡΡΡΠ°Π½ΡΡΠ²ΠΎ ΡΡΠΎΡ ΠΏΠ΅ΠΏΡΠΈΠ΄ ΠΎΡΡΠ΅ΠΏΠ»ΡΠ΅ΡΡΡ, Π° ΠΎΠ±ΡΠ°Π·ΡΡΡΠ°ΡΡΡ ΡΠΊΠΎΡΠΎΡΠ΅Π½Π½Π°Ρ Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS Π½Π΅ ΠΌΠΎΠΆΠ΅Ρ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡ FAD ΠΈ ΠΏΠΎΡΡΠΎΠΌΡ Π²ΡΠΏΠΎΠ»Π½ΡΠ΅Ρ ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ Π½Π΅ΠΊΠ°ΡΠ°Π»ΠΈΡΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΡΡΠ½ΠΊΡΠΈΠΈ. Π Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅ ΠΌΡ ΠΎΡΡΡΠ΅ΡΡΠ²ΠΈΠ»ΠΈ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡ Π³Π΅Π½Π΅ΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΠΊΠΎΠ½ΡΡΡΡΠΊΡΠΈΠΈ, ΠΊΠΎΠ΄ΠΈΡΡΡΡΠ΅ΠΉ Π»ΠΈΡΠ΅Π½Π½ΡΡ N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠ³ΠΎ ΡΠΈΠ³Π½Π°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΏΠ΅ΠΏΡΠΈΠ΄Π° RNLS (tRNLS), Π² ΠΊΠ»Π΅ΡΠΊΠ°Ρ
E. coli Rosetta (DE3). ΠΠ°ΠΊ ΠΈ Π² ΡΠ»ΡΡΠ°Π΅ ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΉ RNLS, ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½Π°Ρ tRNLS Π½Π°ΠΊΠ°ΠΏΠ»ΠΈΠ²Π°Π΅ΡΡΡ Π² ΡΠ΅Π»ΡΡΠ°Ρ
Π²ΠΊΠ»ΡΡΠ΅Π½ΠΈΡ Π² Π½Π΅ΡΠ°ΡΡΠ²ΠΎΡΠΈΠΌΠΎΠΉ ΡΠΎΡΠΌΠ΅, ΠΊΠΎΡΠΎΡΠ°Ρ ΠΌΠΎΠΆΠ΅Ρ Π±ΡΡΡ ΠΏΠ΅ΡΠ΅Π²Π΅Π΄Π΅Π½Π° Π² ΡΠ°ΡΡΠ²ΠΎΡΠΈΠΌΡΡ ΡΠΎΡΠΌΡ Π² ΠΏΡΠΈΡΡΡΡΡΠ²ΠΈΠΈ Π²ΡΡΠΎΠΊΠΎΠΉ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΈ ΠΌΠΎΡΠ΅Π²ΠΈΠ½Ρ ΠΈΠ»ΠΈ Π³ΡΠ°Π½ΠΈΠ΄ΠΈΠ½Ρ
Π»ΠΎΡΠΈΠ΄Π°. ΠΡΠΈ ΡΡΠΎΠΌ, Π² ΠΎΡΠ»ΠΈΡΠΈΠ΅ ΠΎΡ ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΉ RNLS, ΠΊΠΎΡΠΎΡΠ°Ρ ΡΠΎΠ»ΡΠ±ΠΈΠ»ΠΈΠ·ΠΈΡΠΎΠ²Π°Π»Π°ΡΡ Π² ΠΏΡΠΈΡΡΡΡΡΠ²ΠΈΠΈ 8 Π ΠΌΠΎΡΠ΅Π²ΠΈΠ½Ρ, Π±ΠΎΠ»Π΅Π΅ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½Π°Ρ ΡΠΎΠ»ΡΠ±ΠΈΠ»ΠΈΠ·Π°ΡΠΈΡ tRNLS Π±ΡΠ»Π° Π΄ΠΎΡΡΠΈΠ³Π½ΡΡΠ° Π² ΠΏΡΠΈΡΡΡΡΡΠ²ΠΈΠΈ 6 Π Π³ΡΠ°Π½ΠΈΠ΄ΠΈΠ½Ρ
Π»ΠΎΡΠΈΠ΄Π°
Π£ΠΏΡΠΎΡΠ΅Π½Π½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΊΠΠΠ Π½ΠΈΠ·ΠΊΠΎΠΊΠΎΠΏΠΈΠΉΠ½ΡΡ ΠΈ ΠΌΠΎΠ»ΡΠ°ΡΠΈΡ ΡΡΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ Π³Π΅Π½ΠΎΠ² Π½Π° ΠΏΡΠΈΠΌΠ΅ΡΠ΅ ΡΠ΅Π½Π°Π»Π°Π·Ρ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°
A simplified Β«exonΒ» method was developed for producing cDNA of low-copy and silent eukaryotic genes. It is based on assembly of the target gene from genomic DNA by direct synthesis of its exons, followed by their PCR-based joining without further purification of the amplicons. During the synthesis of exons, direct primers were used; these included about ~ 20 nucleotides of the 3`-terminal sequence previous (from the amplified) exon and ~ 20 nucleotides of the 5`-initial sequence of the amplified exon. Reverse primers included ~ 20 nucleotides complementary to the terminal sequence of the amplified exon. Forward and reverse primers flanking the gene to be assembled included the restriction sites necessary for insertion into the expression vector. Using this approach it is possible to assemble almost any eukaryotic gene with a known nucleotide sequence of genomic DNA available in the database.Π Π°Π·ΡΠ°Π±ΠΎΡΠ°Π½ ΡΠΏΡΠΎΡΠ΅Π½Π½ΡΠΉ Β«ΡΠΊΠ·ΠΎΠ½ΠΎΠ²ΡΠΉΒ» ΠΌΠ΅ΡΠΎΠ΄ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΊΠΠΠ Π½ΠΈΠ·ΠΊΠΎΠΊΠΎΠΏΠΈΠΉΠ½ΡΡ
ΠΈ ΠΌΠΎΠ»ΡΠ°ΡΠΈΡ
ΡΡΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ
Π³Π΅Π½ΠΎΠ². ΠΠ½ ΠΎΡΠ½ΠΎΠ²Π°Π½ Π½Π° ΡΠ±ΠΎΡΠ΅ ΡΠ΅Π»Π΅Π²ΠΎΠ³ΠΎ Π³Π΅Π½Π° Ρ Π³Π΅Π½ΠΎΠΌΠ½ΠΎΠΉ ΠΠΠ ΠΏΡΡΠ΅ΠΌ ΠΏΡΡΠΌΠΎΠ³ΠΎ ΡΠΈΠ½ΡΠ΅Π·Π° Π΅Π³ΠΎ ΡΠΊΠ·ΠΎΠ½ΠΎΠ² Ρ ΠΏΠΎΡΠ»Π΅Π΄ΡΡΡΠΈΠΌ ΠΈΡ
ΠΎΠ±ΡΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠ΅ΠΌ ΠΠ¦Π Π±Π΅Π· Π΄ΠΎΠΏΠΎΠ»Π½ΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ ΠΎΡΠΈΡΡΠΊΠΈ Π°ΠΌΠΏΠ»ΠΈΠΊΠΎΠ½ΠΎΠ². ΠΡΠΈ ΡΠΈΠ½ΡΠ΅Π·Π΅ ΡΠΊΠ·ΠΎΠ½ΠΎΠ² ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ ΠΏΡΡΠΌΡΠ΅ ΠΏΡΠ°ΠΉΠΌΠ΅ΡΡ, ΠΊΠΎΡΠΎΡΡΠ΅ Π²ΠΊΠ»ΡΡΠ°ΡΡ ΠΏΡΠΈΠΌΠ΅ΡΠ½ΠΎ ~20 Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄ΠΎΠ² 3β-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΏΡΠ΅Π΄ΡΠ΄ΡΡΠ΅Π³ΠΎ ΠΎΡ Π°ΠΌΠΏΠ»ΠΈΡΠΈΡΠΈΡΡΠ΅ΠΌΠΎΠ³ΠΎ ΡΠΊΠ·ΠΎΠ½Π° ΠΈ ~20 Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄ΠΎΠ² 5β-Π½Π°ΡΠ°Π»ΡΠ½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π°ΠΌΠΏΠ»ΠΈΡΠΈΡΠΈΡΡΠ΅ΠΌΠΎΠ³ΠΎ ΡΠΊΠ·ΠΎΠ½Π°. ΠΠ±ΡΠ°ΡΠ½ΡΠ΅ ΠΏΡΠ°ΠΉΠΌΠ΅ΡΡ Π²ΠΊΠ»ΡΡΠ°Π»ΠΈ ~20 Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄ΠΎΠ² ΠΊΠΎΠΌΠΏΠ»Π΅ΠΌΠ΅Π½ΡΠ°ΡΠ½ΠΎΠΉ ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π°ΠΌΠΏΠ»ΠΈΡΠΈΡΠΈΡΡΠ΅ΠΌΠΎΠ³ΠΎ ΡΠΊΠ·ΠΎΠ½Π°. ΠΡΡΠΌΠΎΠΉ ΠΈ ΠΎΠ±ΡΠ°ΡΠ½ΡΠΉ ΠΏΡΠ°ΠΉΠΌΠ΅ΡΡ, ΡΠ»Π°Π½ΠΊΠΈΡΡΡΡΠΈΠ΅ ΡΠΎΠ±ΠΈΡΠ°Π΅ΠΌΡΠΉ Π³Π΅Π½, Π²ΠΊΠ»ΡΡΠ°Π»ΠΈ ΡΠ°ΠΉΡΡ ΡΠ΅ΡΡΡΠΈΠΊΡΠΈΠΈ, Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΡΠ΅ Π΄Π»Ρ Π²ΡΡΡΠ°ΠΈΠ²Π°Π½ΠΈΡ Π² ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡΡΡΡΠΈΠΉ Π²Π΅ΠΊΡΠΎΡ. Π’Π°ΠΊΠΎΠΉ ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ ΠΏΠΎΠ·Π²ΠΎΠ»ΡΠ΅Ρ Π±ΡΡΡΡΠΎ ΡΠΎΠ±ΡΠ°ΡΡ ΠΏΡΠ°ΠΊΡΠΈΡΠ΅ΡΠΊΠΈ Π»ΡΠ±ΠΎΠΉ ΡΡΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΠΉ Π³Π΅Π½ ΠΏΠΎ ΠΈΠ·Π²Π΅ΡΡΠ½ΠΎΠΉ Π½ΡΠΊΠ»Π΅ΠΎΡΠΈΠ΄Π½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ Π³Π΅Π½ΠΎΠΌΠ½ΠΎΠΉ ΠΠΠ, ΠΈΠΌΠ΅ΡΡΠ΅ΠΉΡΡ Π² Π±Π°Π·Π΅ Π΄Π°Π½Π½ΡΡ
ΠΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ ΠΏΡΠΎΡΠ΅ΠΊΡΠΈΠ²Π½ΡΡ ΡΠ²ΠΎΠΉΡΡΠ² ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ° Π±Π΅Π»ΠΊΠ° F Π½Π°ΡΡΠΆΠ½ΠΎΠΉ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ ΠΈ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π° Pseudomonas aeruginosa
Pseudomonas aeruginosa induces the complications after burns, injuries, surgical interventions and appears to be one of the main causative agents of nosocomial infections. This pathogen has the high resistance to the antibacterial preparations, therefore the immunoprophylaxis is considered as one of the major approaches to reduce Pseudomonas infection. Objective: The aim of our investigation is to study the protective properties of the recombinant complex of the outer membrane protein F (OprF) and a non-toxic variant of the exotoxin A (toxoid) against Pseudomonas infection. Methods: The recombinant proteins which contained the additional histidine residues were synthesized into Escherichia coli with isopropyl-Ξ²D-thyogalactopyranoside (IPTG). The recombinant proteins were purified by affinity chromatography on Ni-Sepharose. The preparations of recombinant proteins were injected intraperitoneally into the mice. Aluminum hydroxide was used as an adjuvant. For an experimental infection in mice, animals were challenged intraperitoneally by a live virulent culture of P. aeruginosa (PA-103 strain). Results: The best protective effect for the complex containing 25 ΞΌg OprF and 50 ΞΌg toxoid was identified when we used the double immunization of mice (Index of efficiency of the protective properties in this case was 4.0). Indexes of efficiency of separated recombinant proteins which were injected twice in the same doses were 2.0 for OprF ΠΈ 2.3 for toxoid. The triple immunization of animals was inefficient for separated recombinant proteins in the same doses. The injection of doses which were lowered twice (12.5 ΞΌg for OprF and 25 ΞΌg for toxoid) resulted in increased survival of mice immunized by individual proteins (indexes of efficiency: 3 for OprF and ΠΈ 3,5 for toxoid). However when we administered to the complex of proteins with the same doses Index of efficiency was 2.8. Conclusion: It was shown that the maximum protective effect in a short time is achieved by the combination of double immunization and the mixture of the recombinant proteins OprF and the 25 and 50 ΞΌg doses of recombinant toxoid .Β Pseudomonas aeruginosa Π²ΡΠ·ΡΠ²Π°Π΅Ρ ΠΎΡΠ»ΠΎΠΆΠ½Π΅Π½ΠΈΡ ΠΏΠΎΡΠ»Π΅ ΠΎΠΆΠΎΠ³ΠΎΠ², ΡΡΠ°Π²ΠΌ ΠΈ Ρ
ΠΈΡΡΡΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
Π²ΠΌΠ΅ΡΠ°ΡΠ΅Π»ΡΡΡΠ², ΡΠ²Π»ΡΡΡΡ ΠΎΠ΄Π½ΠΎΠΉ ΠΈΠ· ΠΎΡΠ½ΠΎΠ²Π½ΡΡ
ΠΏΡΠΈΡΠΈΠ½ Π½ΠΎΠ·ΠΎΠΊΠΎΠΌΠΈΠ°Π»ΡΠ½ΡΡ
ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΉ. ΠΡΠΎΡ ΠΏΠ°ΡΠΎΠ³Π΅Π½ ΠΎΠ±Π»Π°Π΄Π°Π΅Ρ Π²ΡΡΠΎΠΊΠΎΠΉ ΡΠ΅Π·ΠΈΡΡΠ΅Π½ΡΠ½ΠΎΡΡΡΡ ΠΊ Π±ΠΎΠ»ΡΡΠΈΠ½ΡΡΠ²Ρ Π°Π½ΡΠΈΠ±Π°ΠΊΡΠ΅ΡΠΈΠ°Π»ΡΠ½ΡΡ
ΡΡΠ΅Π΄ΡΡΠ², ΠΏΠΎΡΡΠΎΠΌΡ ΠΈΠΌΠΌΡΠ½ΠΎΠΏΡΠΎΡΠΈΠ»Π°ΠΊΡΠΈΠΊΠ° ΡΠ°ΡΡΠΌΠ°ΡΡΠΈΠ²Π°ΡΡΡΡ ΠΊΠ°ΠΊ ΠΎΠ΄ΠΈΠ½ ΠΈΠ· ΠΏΡΠΈΠΎΡΠΈΡΠ΅ΡΠ½ΡΡ
ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΎΠ² Π΄Π»Ρ Π±ΠΎΡΡΠ±Ρ Ρ ΡΠΈΠ½Π΅Π³Π½ΠΎΠΉΠ½ΠΎΠΉ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠ΅ΠΉ. Π¦Π΅Π»Ρ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ: ΠΠ·ΡΡΠΈΡΡ Π·Π°ΡΠΈΡΠ½ΡΠ΅ ΡΠ²ΠΎΠΉΡΡΠ²Π° ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ° Π±Π΅Π»ΠΊΠ° F Π½Π°ΡΡΠΆΠ½ΠΎΠΉ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ (OprF) ΠΈ Π°ΡΠΎΠΊΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΡΠΎΡΠΌΡ ΡΠΊΠ·ΠΎΡΠΎΠΊΡΠΈΠ½Π°Β Π (Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π°) ΠΎΡ ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΠΎΠΉ ΡΠΈΠ½Π΅Π³Π½ΠΎΠΉΠ½ΠΎΠΉ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠΈ. ΠΠ΅ΡΠΎΠ΄Ρ: Π Π΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠ΅ Π±Π΅Π»ΠΊΠΈ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΠ΅ Π΄ΠΎΠΏΠΎΠ»Π½ΠΈΡΠ΅Π»ΡΠ½ΡΡ ΡΠ΅ΡΡΠΈ Π³ΠΈΡΡΠΈΠ΄ΠΈΠ½ΠΎΠ²ΡΡ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ, ΡΠΈΠ½ΡΠ΅Π·ΠΈΡΠΎΠ²Π°Π»ΠΈ Π² ΠΊΠ»Π΅ΡΠΊΠ°Ρ
Escherichia coli c ΠΏΠΎΠΌΠΎΡΡΡ ΠΈΠ·ΠΎΠΏΡΠΎΠΏΠΈΠ»-b-d-ΡΠΈΠΎΠ³Π°Π»Π°ΠΊΡΠΎΠΏΠΈΡΠ°Π½ΠΎΠ·ΠΈΠ΄Π° (ΠΠΠ’Π) ΠΈ ΠΎΡΠΈΡΠ°Π»ΠΈ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ Π°ΡΡΠΈΠ½Π½ΠΎΠΉ Ρ
ΡΠΎΠΌΠ°ΡΠΎΠ³ΡΠ°ΡΠΈΠΈ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ Ni-ΡΠ΅ΡΠ°ΡΠΎΠ·Ρ. ΠΡΠ΅ΠΏΠ°ΡΠ°ΡΠ°ΠΌΠΈ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΡ
Π±Π΅Π»ΠΊΠΎΠ² Π²Π½ΡΡΡΠΈΠ±ΡΡΡΠΈΠ½Π½ΠΎ ΠΈΠΌΠΌΡΠ½ΠΈΠ·ΠΈΡΠΎΠ²Π°Π»ΠΈ ΠΌΡΡΠ΅ΠΉ. Π ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ Π°Π΄ΡΡΠ²Π°Π½ΡΠ° ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π»ΠΈ Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΠ΄ Π°Π»ΡΠΌΠΈΠ½ΠΈΡ. ΠΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΠΎΠ΅ Π·Π°ΡΠ°ΠΆΠ΅Π½ΠΈΠ΅ ΠΎΡΡΡΠ΅ΡΡΠ²Π»ΡΠ»ΠΈ Π²Π½ΡΡΡΠΈΠ±ΡΡΡΠΈΠ½Π½ΡΠΌ Π²Π²Π΅Π΄Π΅Π½ΠΈΠ΅ΠΌ ΠΆΠΈΠ²ΠΎΠΉ Π²ΠΈΡΡΠ»Π΅Π½ΡΠ½ΠΎΠΉ ΠΊΡΠ»ΡΡΡΡΡ P.Β aeruginosa ΡΡΠ°ΠΌΠΌΠ° Π Π103. Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ: ΠΡΠΈ Π΄Π²ΡΠΊΡΠ°ΡΠ½ΠΎΠΉ ΠΈΠΌΠΌΡΠ½ΠΈΠ·Π°ΡΠΈΠΈ ΠΌΡΡΠ΅ΠΉ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠΌ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΠΌ 25 ΠΌΠΊΠ³ OprF ΠΈ 50 ΠΌΠΊΠ³ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π°, Π²ΡΡΠ²Π»Π΅Π½ Π½Π°ΠΈΠ»ΡΡΡΠΈΠΉ ΠΏΡΠΎΡΠ΅ΠΊΡΠΈΠ²Π½ΡΠΉ ΡΡΡΠ΅ΠΊΡ (ΠΈΠ½Π΄Π΅ΠΊΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π·Π°ΡΠΈΡΠ½ΡΡ
ΡΠ²ΠΎΠΉΡΡΠ² Π² ΡΡΠΎΠΌ ΡΠ»ΡΡΠ°Π΅ ΡΠΎΡΡΠ°Π²ΠΈΠ» 4,0). ΠΠ½Π΄Π΅ΠΊΡΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π΄Π»Ρ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΡ
Π±Π΅Π»ΠΊΠΎΠ², Π²Π²ΠΎΠ΄ΠΈΠΌΡΡ
Π΄Π²ΡΠΊΡΠ°ΡΠ½ΠΎ Π² ΡΠ΅Ρ
ΠΆΠ΅ Π΄ΠΎΠ·Π°Ρ
ΠΏΠΎ ΠΎΡΠ΄Π΅Π»ΡΠ½ΠΎΡΡΠΈ, ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²ΠΎΠ²Π°Π»ΠΈ 2,0 Π΄Π»Ρ OprF ΠΈ 2,3 Π΄Π»Ρ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π° ΠΈ Π½Π΅ ΡΠ²Π΅Π»ΠΈΡΠΈΠ²Π°Π»ΠΈΡΡ ΠΏΠΎΡΠ»Π΅ ΡΡΠ΅Ρ
ΠΊΡΠ°ΡΠ½ΠΎΠΉ ΠΈΠΌΠΌΡΠ½ΠΈΠ·Π°ΡΠΈΠΈ ΠΆΠΈΠ²ΠΎΡΠ½ΡΡ
. Π£ΠΌΠ΅Π½ΡΡΠ΅Π½Π½ΡΠ΅ Π² Π΄Π²Π° ΡΠ°Π·Π° Π΄ΠΎΠ· (12,5 ΠΌΠΊΠ³ OprF ΠΈ 25 ΠΌΠΊΠ³ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π°) ΠΏΡΠΈ ΡΡΠ΅Ρ
ΠΊΡΠ°ΡΠ½ΠΎΠΌ Π²Π²Π΅Π΄Π΅Π½ΠΈΠΈ ΡΠΏΠΎΡΠΎΠ±ΡΡΠ²ΠΎΠ²Π°Π»ΠΎ ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ Π²ΡΠΆΠΈΠ²Π°Π΅ΠΌΠΎΡΡΠΈ ΠΌΡΡΠ΅ΠΉ ΠΈΠΌΠΌΡΠ½ΠΈΠ·ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΎΡΠ΄Π΅Π»ΡΠ½ΡΠΌΠΈ Π±Π΅Π»ΠΊΠ°ΠΌΠΈ (ΠΈΠ½Π΄Π΅ΠΊΡΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ: 3 Π΄Π»Ρ OprF ΠΈ 3,5 Π΄Π»Ρ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½Π°), ΠΎΠ΄Π½Π°ΠΊΠΎ ΠΏΡΠΈ Π²Π²Π΅Π΄Π΅Π½ΠΈΠΈ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ° Π±Π΅Π»ΠΊΠΎΠ² Π² ΡΠ΅Ρ
ΠΆΠ΅ Π΄ΠΎΠ·Π°Ρ
ΠΈΠ½Π΄Π΅ΠΊΡ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π½Π΅ ΡΠ²Π΅Π»ΠΈΡΠΈΠ²Π°Π»ΡΡ ΠΈ ΡΠΎΡΡΠ°Π²ΠΈΠ» 2,8. ΠΠ°ΠΊΠ»ΡΡΠ΅Π½ΠΈΠ΅: ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΠΌΠ°ΠΊΡΠΈΠΌΠ°Π»ΡΠ½ΡΠΉ ΠΏΡΠΎΡΠ΅ΠΊΡΠΈΠ²Π½ΡΠΉ ΡΡΡΠ΅ΠΊΡ ΡΠΎΡΠΌΠΈΡΡΠ΅ΡΡΡ Π² ΠΊΠΎΡΠΎΡΠΊΠΈΠ΅ ΡΡΠΎΠΊΠΈ ΠΏΡΠΈ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠΈ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ½ΠΎΠΉ ΠΈΠΌΠΌΡΠ½ΠΈΠ·Π°ΡΠΈΠΈ Π΄Π²ΡΠΌΡ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΌΠΈ Π±Π΅Π»ΠΊΠ°ΠΌΠΈ OprF ΠΈ Π°Π½Π°ΡΠΎΠΊΡΠΈΠ½ΠΎΠΌ Π² Π΄ΠΎΠ·Π°Ρ
25 ΠΈ 50 ΠΌΠΊΠ³
Low-energy dynamics of the reaction
We calculate the one-quark-loop amplitude for the low energy
collision in the context of the Nambu and Jona-Lasinio
model with scalar and pseudoscalar four quark couplings to all orders in the
external momenta. We show that the NJL predictions for the
reaction are not far from the Born amplitude, which
is close to the data, and is compatible with the chiral perturbation theory
estimations. We determine the corrections given by the NJL model in leading
order of to the chiral loop amplitude for .
Numerical results for the cross sections and
for pion polarizabilities are given.Comment: 20 pages in LaTex, 3 figures in 1 Postscript fil
Heavy baryon properties with NLO accuracy in perturbative QCD
We present an analysis of the static properties of heavy baryons at
next-to-leading order in the perurbative expansion of QCD. We obtain analytical
next-to-leading order three-loop results for the two-point correlators of
baryonic currents with one finite mass quark field for a variety of quantum
numbers of the baryonic currents. We consider both the massless limit and the
HQET limit of the correlator as special cases of the general finite mass
formula and find agreement with previous results. We present closed form
expressions for the moments of the spectral density. We determine the residues
of physical baryon states using sum rule techniques.Comment: 43 pages in LaTeX, including 3 figure
ΠΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ ΠΊ ΠΏΡΠΎΡΠ΅ΠΎΠ»ΠΈΠ·Ρ ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΉ ΠΈ ΡΠΊΠΎΡΠΎΡΠ΅Π½Π½ΠΎΠΉ ΡΠΎΡΠΌ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠΉ ΡΠ΅Π½Π°Π»Π°Π·Ρ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡΠΎΠ²Π°Π½Π½ΡΡ Π² ΠΏΡΠΎΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΠ΅
Renalase (RNLS) is a flavoprotein; its N-terminal peptide (amino acid residues 1-17) performs various important functions. Inside cells, it is involved in the Rossmann fold formation (residues 2-35), which is necessary for the binding of the FAD cofactor and the manifestation of the enzymatic activity of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). When RNLS is secreted into the extracellular space, this peptide is cleaved off, and the resulting truncated extracellular RNLS can no longer bind FAD and, therefore, numerous effects described in the literature are carried out by non-catalytic mechanisms. In this work, we have investigated the sensitivity to trypsinolysis of two recombinant forms of human RNLS expressed in prokaryotic cells: (a) full-length RNLS containing the FAD cofactor; (b) a truncated RNLS lacking the 1-17 N-terminal peptide (truncatedRNLS, tRNLS) unable to bind the FAD cofactor. Trypsin (1 unit/20 ΞΌL of medium) effectively cleaved both forms of renalase (RNLS and tRNLS). When exposed to a lower concentration of trypsin (0.1 U/20 ΞΌL of medium), full length RNLS was more trypsin resistant than tRNLS. We suggest that the different sensitivity of RNLS and tRNLS is apparently determined by the presence of the FAD cofactor in the full-length recombinant protein, which contributes to the formation of a spatial structure that is more resistant to the action of certain proteases.Π Π΅Π½Π°Π»Π°Π·Π° (RNLS) β ΡΠ»Π°Π²ΠΎΠΏΡΠΎΡΠ΅ΠΈΠ½, N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠΉ ΠΏΠ΅ΠΏΡΠΈΠ΄ ΠΊΠΎΡΠΎΡΠΎΠ³ΠΎ (Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΡΠ΅ ΠΎΡΡΠ°ΡΠΊΠΈ (Π°.ΠΎ.) 1-17) Π²ΡΠΏΠΎΠ»Π½ΡΠ΅Ρ ΡΡΠ΄ Π²Π°ΠΆΠ½ΡΡ
ΡΡΠ½ΠΊΡΠΈΠΉ. Π ΠΊΠ»Π΅ΡΠΊΠ°Ρ
ΠΎΠ½ ΡΡΠ°ΡΡΠ²ΡΠ΅Ρ Π² ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ ΡΠΊΠ»Π°Π΄ΠΊΠΈ Π ΠΎΡΡΠΌΠ°Π½Π° (2-35 Π°.ΠΎ.), Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΠΎΠΉ Π΄Π»Ρ ΡΠ²ΡΠ·ΡΠ²Π°Π½ΠΈΡ ΠΊΠΎΡΠ°ΠΊΡΠΎΡΠ° FAD ΠΈ ΠΏΡΠΎΡΠ²Π»Π΅Π½ΠΈΡ ΡΠ΅ΡΠΌΠ΅Π½ΡΠ°ΡΠΈΠ²Π½ΠΎΠΉ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ RNLS Π² ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ FAD-Π·Π°Π²ΠΈΡΠΈΠΌΠΎΠΉ ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Ρ (ΠΠ€ 1.6.3.5). ΠΡΠΈ ΡΠ΅ΠΊΡΠ΅ΡΠΈΠΈ RNLS Π²ΠΎ Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠ΅ ΠΏΡΠΎΡΡΡΠ°Π½ΡΡΠ²ΠΎ ΡΡΠΎΡ ΠΏΠ΅ΠΏΡΠΈΠ΄ ΠΎΡΡΠ΅ΠΏΠ»ΡΠ΅ΡΡΡ, Π° ΠΎΠ±ΡΠ°Π·ΡΡΡΠ°ΡΡΡ ΡΠΊΠΎΡΠΎΡΠ΅Π½Π½Π°Ρ Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS ΡΠΆΠ΅ Π½Π΅ ΠΌΠΎΠΆΠ΅Ρ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡ FAD, ΠΈ ΠΏΠΎΡΡΠΎΠΌΡ ΠΌΠ½ΠΎΠ³ΠΎΡΠΈΡΠ»Π΅Π½Π½ΡΠ΅ ΡΡΡΠ΅ΠΊΡΡ, ΠΎΠΏΠΈΡΠ°Π½Π½ΡΠ΅ Π² Π»ΠΈΡΠ΅ΡΠ°ΡΡΡΠ΅, ΠΎΡΡΡΠ΅ΡΡΠ²Π»ΡΡΡΡΡ Π½Π΅ΠΊΠ°ΡΠ°Π»ΠΈΡΠΈΡΠ΅ΡΠΊΠΈΠΌΠΈ ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΠ°ΠΌΠΈ. Π Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅ ΠΌΡ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π»ΠΈ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΊ ΡΡΠΈΠΏΡΠΈΠ½ΠΎΠ»ΠΈΠ·Ρ Π΄Π²ΡΡ
ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΡ
ΡΠΎΡΠΌ RNLS ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
Π² ΠΏΡΠΎΠΊΠ°ΡΠΈΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΊΠ»Π΅ΡΠΊΠ°Ρ
: (Π°) ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΉ RNLS, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠ΅ΠΉ ΠΊΠΎΡΠ°ΠΊΡΠΎΡ FAD; (Π±) ΡΠΊΠΎΡΠΎΡΠ΅Π½Π½ΠΎΠΉ RNLS, Π»ΠΈΡΠ΅Π½Π½ΠΎΠΉ N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠ³ΠΎ ΠΏΠ΅ΠΏΡΠΈΠ΄Π° 1-17 (truncated RNLS, tRNLS), Π½Π΅ΡΠΏΠΎΡΠΎΠ±Π½ΠΎΠΉ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡ ΠΊΠΎΡΠ°ΠΊΡΠΎΡ FAD. Π’ΡΠΈΠΏΡΠΈΠ½ (1 Π΅Π΄./20 ΠΌΠΊΠ» ΡΡΠ΅Π΄Ρ) ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎ ΡΠ°ΡΡΠ΅ΠΏΠ»ΡΠ» ΠΎΠ±Π΅ ΡΠΎΡΠΌΡ ΡΠ΅Π½Π°Π»Π°Π·Ρ (RNLS ΠΈ tRNLS). ΠΡΠΈ Π²ΠΎΠ·Π΄Π΅ΠΉΡΡΠ²ΠΈΠΈ Π±ΠΎΠ»Π΅Π΅ Π½ΠΈΠ·ΠΊΠΎΠΉ ΠΊΠΎΠ½ΡΠ΅Π½ΡΡΠ°ΡΠΈΠΈ ΡΡΠΈΠΏΡΠΈΠ½Π° (0.01 Π΅Π΄./20 ΠΌΠΊΠ» ΡΡΠ΅Π΄Ρ) ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½Π°Ρ RNLS Π±ΡΠ»Π° Π±ΠΎΠ»Π΅Π΅ ΡΡΡΠΎΠΉΡΠΈΠ²Π° ΠΊ ΡΡΠΈΠΏΡΠΈΠ½Ρ, ΡΠ΅ΠΌ tRNLS. ΠΡ ΠΏΡΠ΅Π΄ΠΏΠΎΠ»Π°Π³Π°Π΅ΠΌ, ΡΡΠΎ ΡΠ°Π·Π»ΠΈΡΠ½Π°Ρ ΡΡΠ²ΡΡΠ²ΠΈΡΠ΅Π»ΡΠ½ΠΎΡΡΡ RNLS ΠΈ tRNLS, ΠΏΠΎ-Π²ΠΈΠ΄ΠΈΠΌΠΎΠΌΡ, ΠΎΠΏΡΠ΅Π΄Π΅Π»ΡΠ΅ΡΡΡ ΠΏΡΠΈΡΡΡΡΡΠ²ΠΈΠ΅ΠΌ ΠΊΠΎΡΠ°ΠΊΡΠΎΡΠ° FAD Π² ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠΌ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠΌ Π±Π΅Π»ΠΊΠ΅, ΠΊΠΎΡΠΎΡΡΠΉ ΡΠΏΠΎΡΠΎΠ±ΡΡΠ²ΡΠ΅Ρ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΠΏΡΠΎΡΡΡΠ°Π½ΡΡΠ²Π΅Π½Π½ΠΎΠΉ ΡΡΡΡΠΊΡΡΡΡ, Π±ΠΎΠ»Π΅Π΅ ΡΡΡΠΎΠΉΡΠΈΠ²ΠΎΠΉ ΠΊ Π΄Π΅ΠΉΡΡΠ²ΠΈΡ Π½Π΅ΠΊΠΎΡΠΎΡΡΡ
ΠΏΡΠΎΡΠ΅Π°Π·
ΠΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΈ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡ Ρ ΠΈΠΌΠ΅ΡΠ½ΠΎΠ³ΠΎ Π³Π΅Π½Π° ΡΠ΅Π½Π°Π»Π°Π·Ρ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, ΠΊΠΎΠ΄ΠΈΡΡΡΡΠ΅Π³ΠΎ N-ΠΊΠΎΠ½ΡΠ΅Π²ΡΡ ΡΠΈΠ³Π½Π°Π»ΡΠ½ΡΡ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΡΠ΅ΠΊΡΠ΅ΡΠΎΡΠ½ΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ° ΠΏΡΠΎΠ»Π°ΠΊΡΠΈΠ½Π°
Renalase (RNLS) is a protein that performs various protective functions both inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5). Extracellular RNLS lacking an N-terminal peptide does not interact with FAD and exhibits various protective effects on the cell through interaction with receptor proteins. The mechanisms and factors responsible for RNLS transport out of the cell are not fully understood. It is well known that the signal sequence plays a key role in the classical mechanism of protein transport outside cells. One of the approaches to study the secretion of RNLS from the cell can be the creation of chimeric forms of the protein with a modified N-terminal amino acid signal sequence. Bioinformatics analysis showed that the signal sequence of the prolactin gene (PRL), connected to the template sequence of the RNLS gene, gave the classic signal characteristic of secretory proteins. On this basis, this paper describes: (i) a method for constructing the human RNLS gene in which the N-terminal sequence encoded by the RNLS gene was replaced by the N-terminal sequence encoded by the PRL gene; (ii) expression of this chimeric genetic construct.Π Π΅Π½Π°Π»Π°Π·Π° (RNLS) - Π±Π΅Π»ΠΎΠΊ, ΠΊΠΎΡΠΎΡΡΠΉ Π²ΡΠΏΠΎΠ»Π½ΡΠ΅Ρ ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ Π·Π°ΡΠΈΡΠ½ΡΠ΅ ΡΡΠ½ΠΊΡΠΈΠΈ ΠΊΠ°ΠΊ Π²Π½ΡΡΡΠΈ, ΡΠ°ΠΊ ΠΈ ΡΠ½Π°ΡΡΠΆΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΠ½ΡΡΡΠΈΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS ΠΏΡΠΎΡΠ²Π»ΡΠ΅Ρ ΡΠ²ΠΎΠΉΡΡΠ²Π° FAD-Π·Π°Π²ΠΈΡΠΈΠΌΠΎΠΉ ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Ρ (ΠΠ€ 1.6.3.5). ΠΠ½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS, Π»ΠΈΡΠ΅Π½Π½Π°Ρ N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠ³ΠΎ ΠΏΠ΅ΠΏΡΠΈΠ΄Π°, Π½Π΅ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΡΠ΅Ρ Ρ FAD, ΠΏΡΠΎΡΠ²Π»ΡΠ΅Ρ ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ Π·Π°ΡΠΈΡΠ½ΡΠ΅ ΡΡΡΠ΅ΠΊΡΡ Π½Π° ΠΊΠ»Π΅ΡΠΊΡ ΠΏΠΎΡΡΠ΅Π΄ΡΡΠ²ΠΎΠΌ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ Π½Π° ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠ½ΡΠ΅ Π±Π΅Π»ΠΊΠΈ. ΠΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΡ ΠΈ ΡΠ°ΠΊΡΠΎΡΡ, ΠΎΡΠ²Π΅ΡΡΡΠ²Π΅Π½Π½ΡΠ΅ Π·Π° ΡΡΠ°Π½ΡΠΏΠΎΡΡ RNLS ΠΈΠ· ΠΊΠ»Π΅ΡΠΊΠΈ, Π΄ΠΎ ΠΊΠΎΠ½ΡΠ° Π½Π΅ ΠΈΠ·ΡΡΠ΅Π½Ρ. Π₯ΠΎΡΠΎΡΠΎ ΠΈΠ·Π²Π΅ΡΡΠ½ΠΎ, ΡΡΠΎ ΡΠΈΠ³Π½Π°Π»ΡΠ½Π°Ρ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ ΠΈΠ³ΡΠ°Π΅Ρ ΠΊΠ»ΡΡΠ΅Π²ΡΡ ΡΠΎΠ»Ρ Π² ΠΊΠ»Π°ΡΡΠΈΡΠ΅ΡΠΊΠΎΠΌ ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΠ΅ ΡΡΠ°Π½ΡΠΏΠΎΡΡΠ° Π²Π½ΡΡΡΠΈΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
Π±Π΅Π»ΠΊΠΎΠ². ΠΠ΄Π½ΠΈΠΌ ΠΈΠ· ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΎΠ² Π΄Π»Ρ ΠΈΠ·ΡΡΠ΅Π½ΠΈΡ ΡΠ΅ΠΊΡΠ΅ΡΠΈΠΈ RNLS ΠΈΠ· ΠΊΠ»Π΅ΡΠΊΠΈ ΠΌΠΎΠΆΠ΅Ρ Π±ΡΡΡ ΡΠΎΠ·Π΄Π°Π½ΠΈΠ΅ Ρ
ΠΈΠΌΠ΅ΡΠ½ΡΡ
ΡΠΎΡΠΌ Π±Π΅Π»ΠΊΠ° Ρ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠΉ N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠΉ ΡΠΈΠ³Π½Π°Π»ΡΠ½ΠΎΠΉ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠΈ. ΠΠΈΠΎΠΈΠ½ΡΠΎΡΠΌΠ°ΡΠΈΡΠ΅ΡΠΊΠΈΠΉ Π°Π½Π°Π»ΠΈΠ· ΠΏΠΎΠΊΠ°Π·Π°Π», ΡΡΠΎ ΡΠΈΠ³Π½Π°Π»ΡΠ½Π°Ρ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ Π³Π΅Π½Π° ΠΏΡΠΎΠ»Π°ΠΊΡΠΈΠ½Π° (PRL), ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½Π½Π°Ρ Ρ ΠΌΠ°ΡΡΠΈΡΠ½ΠΎΠΉ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡΡ Π³Π΅Π½Π° RNLS, Π΄Π°Π²Π°Π»Π° ΠΊΠ»Π°ΡΡΠΈΡΠ΅ΡΠΊΠΈΠΉ ΡΠΈΠ³Π½Π°Π», ΡΠ²ΠΎΠΉΡΡΠ²Π΅Π½Π½ΡΠΉ ΡΠ΅ΠΊΡΠ΅ΡΠΎΡΠ½ΡΠΌ Π±Π΅Π»ΠΊΠ°ΠΌ. ΠΡΡ
ΠΎΠ΄Ρ ΠΈΠ· ΡΡΠΎΠ³ΠΎ, Π² Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅: (i) ΠΎΠΏΠΈΡΠ°Π½ ΠΌΠ΅ΡΠΎΠ΄ ΠΊΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΡ Π³Π΅Π½Π° RNLS ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, Π² ΠΊΠΎΡΠΎΡΠΎΠΌ N-ΠΊΠΎΠ½ΡΠ΅Π²Π°Ρ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ, ΠΊΠΎΠ΄ΠΈΡΡΠ΅ΠΌΠ°Ρ Π³Π΅Π½ΠΎΠΌ RNLS, Π±ΡΠ»Π° Π·Π°ΠΌΠ΅Π½Π΅Π½Π° Π½Π° N-ΠΊΠΎΠ½ΡΠ΅Π²ΡΡ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΡ, ΠΊΠΎΠ΄ΠΈΡΡΠ΅ΠΌΡΡ Π³Π΅Π½ΠΎΠΌ PRL; (ii) ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½Π° ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡ ΡΡΠΎΠΉ Ρ
ΠΈΠΌΠ΅ΡΠ½ΠΎΠΉ Π³Π΅Π½Π΅ΡΠΈΡΠ΅ΡΠΊΠΎΠΉ ΠΊΠΎΠ½ΡΡΡΡΠΊΡΠΈΠΈ
982 Induction of apoptosis in herpes semplex virus thymidine kinase/aciclovir transfected experimental breast cancer models
ΠΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ Ρ ΠΈΠΌΠ΅ΡΠ½ΠΎΠ³ΠΎ Π³Π΅Π½Π° ΡΠ΅Π½Π°Π»Π°Π·Ρ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° Ρ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΡΠΌ N-ΠΊΠΎΠ½ΡΠΎΠΌ
Renalase (RNLS) is a recently discovered protein that plays different roles inside and outside cells. Extracellular RNLS exhibits protective
effects on the cell, acting on its receptor proteins, while intracellular RNLS acts as FAD-dependent oxidoreductase (EC 1.6.3.5). The ratio of
the intracellular and extracellular forms of this protein, as well as the mechanisms and factors responsible for its transport from the cell, remain
unknown. One of the approaches to studying these issues can be the creation of chimeric forms of this protein with modified fragments of its amino
acid sequences. This work describes a method for constructing a chimeric human RNLS gene encoding RNLS without its N-terminal peptidΠ Π΅Π½Π°Π»Π°Π·Π° (RNLS) - Π½Π΅Π΄Π°Π²Π½ΠΎ ΠΎΡΠΊΡΡΡΡΠΉ Π±Π΅Π»ΠΎΠΊ, ΠΊΠΎΡΠΎΡΡΠΉ Π²ΡΠΏΠΎΠ»Π½ΡΠ΅Ρ ΡΠ°Π·Π»ΠΈΡΠ½ΡΠ΅ ΡΡΠ½ΠΊΡΠΈΠΈ Π²Π½ΡΡΡΠΈ ΠΈ ΡΠ½Π°ΡΡΠΆΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ. ΠΠ½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS
ΠΎΠΊΠ°Π·ΡΠ²Π°Π΅Ρ Π·Π°ΡΠΈΡΠ½ΡΠ΅ ΡΡΡΠ΅ΠΊΡΡ Π½Π° ΠΊΠ»Π΅ΡΠΊΡ, Π΄Π΅ΠΉΡΡΠ²ΡΡ, ΠΏΠΎ Π΄Π°Π½Π½ΡΠΌ ΡΡΠ΄Π° Π°Π²ΡΠΎΡΠΎΠ², Π½Π° ΡΠ²ΠΎΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠ½ΡΠ΅ Π±Π΅Π»ΠΊΠΈ, Π²Π½ΡΡΡΠΈΠΊΠ»Π΅ΡΠΎΡΠ½Π°Ρ RNLS ΠΏΡΠΎΡΠ²Π»ΡΠ΅Ρ
ΡΠ²ΠΎΠΉΡΡΠ²Π° FAD-Π·Π°Π²ΠΈΡΠΈΠΌΠΎΠΉ ΠΎΠΊΡΠΈΠ΄ΠΎΡΠ΅Π΄ΡΠΊΡΠ°Π·Ρ (ΠΠ€ 1.6.3.5). Π‘ΠΎΠΎΡΠ½ΠΎΡΠ΅Π½ΠΈΠ΅ Π²Π½ΡΡΡΠΈΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΠΈ Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
ΡΠΎΡΠΌ ΡΡΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ°, Π° ΡΠ°ΠΊΠΆΠ΅
ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΡ ΠΈ ΡΠ°ΠΊΡΠΎΡΡ, ΠΎΡΠ²Π΅ΡΡΡΠ²Π΅Π½Π½ΡΠ΅ Π·Π° Π΅Π³ΠΎ ΡΡΠ°Π½ΡΠΏΠΎΡΡ ΠΈΠ· ΠΊΠ»Π΅ΡΠΊΠΈ ΠΎΡΡΠ°ΡΡΡΡ Π½Π΅ΠΈΠ·Π²Π΅ΡΡΠ½ΡΠΌΠΈ. ΠΠ΄Π½ΠΈΠΌ ΠΈΠ· ΠΏΠΎΠ΄Ρ
ΠΎΠ΄ΠΎΠ² Π΄Π»Ρ ΠΈΠ·ΡΡΠ΅Π½ΠΈΡ ΡΡΠΈΡ
Π²ΠΎΠΏΡΠΎΡΠΎΠ²
ΠΌΠΎΠΆΠ΅Ρ Π±ΡΡΡ ΡΠΎΠ·Π΄Π°Π½ΠΈΠ΅ Ρ
ΠΈΠΌΠ΅ΡΠ½ΡΡ
ΡΠΎΡΠΌ Π±Π΅Π»ΠΊΠ° Ρ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΡΠΌΠΈ ΡΡΠ°Π³ΠΌΠ΅Π½ΡΠ°ΠΌΠΈ Π΅Π³ΠΎ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΡΡ
ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°ΡΠ΅Π»ΡΠ½ΠΎΡΡΠ΅ΠΉ. Π Π΄Π°Π½Π½ΠΎΠΉ
ΡΠ°Π±ΠΎΡΠ΅ ΠΎΠΏΠΈΡΠ°Π½ ΠΌΠ΅ΡΠΎΠ΄ ΠΊΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΈΡ Ρ
ΠΈΠΌΠ΅ΡΠ½ΠΎΠ³ΠΎ Π³Π΅Π½Π° RNLS ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°, ΠΊΠΎΠ΄ΠΈΡΡΡΡΠ΅Π³ΠΎ RNLS Π±Π΅Π· N-ΠΊΠΎΠ½ΡΠ΅Π²ΠΎΠ³ΠΎ ΠΏΠ΅ΠΏΡΠΈΠ΄Π°