Bacteroicin against lysteria

FIELD: biotechnology, in particular provision storage.

SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.

EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.

13 cl, 2 dwg, 1 tbl, 3 ex

 

The technical field to which the invention relates.

The present invention relates to the field of biotechnology and, in particular, for bacteriocin from Lactobacillus sakei, namely, Lactobacillus sakei 2512, of a nucleotide sequence encoding the bacteriocins, and industrial applications of this bacteriocin as an active anti-pathogenic or undesirable flora in the manufacture of food products.

The level of technology

Lactic acid bacteria are widely used in food fermentation not only to improve the taste and texture of food, but also to increase the shelf life of the resulting product. In fact, a variety of lactic acid bacteria have the ability to suppress the growth of some gram-positive bacteria, including such sources of pathogens as Listeria monocytogenes, due to the selection of molecules antagonists, including peptide compounds. Such peptide compounds called bacteriocins, are of considerable interest to preserve the quality and sanitary protection of food products obtained by fermentation.

As an example of such bacteriocins can produce bacteriocins belonging to the subclass of proteins called bacteriocins against Listeria, bacteriocins of class IIA (S. Ennahar et al., 2000, FEMS Environ. Rev., 24: 85-106) and systemetically (Jack R. et al., 1995, Environ. Rev.,59(2): 171-200). It was recently reported on the possible use of one of these bacteriocins of class IIA, divertida V41, to suppress growth of Listeria monocytogenes in smoked salmon (Duffes F. et al., 1999, J. Food Prot, 62(12): 1394-1403).

The sequence of these polypeptides have a great similarity in their N-terminal parts, particularly the presence of a disulfide bridge. Hydrophobic C-terminal part of a much more variable, although for some of these bacteriocins related to the type of pediocin (pediocin RA-1, enterocin and diverting V41), characterized by the presence of cut larger than 40 residues and a second disulfide bridge at the end.

The authors of the present invention have discovered a new bacteriocins of class IIA derived from one specific strain of Lactobacillus sakei, which turned out to be particularly effective in suppressing the growth of Listeria, in particular Listeria monocytogenes.

According to Tagg J.R. et al., Bacteriol. Rev., 40; 722-756 (1976), the term "bacteriocins" in the present invention means a polypeptide produced by ribosomal synthesis by microorganisms that are able to specifically inhibit the growth of other bacteria.

The first object of the present invention is a polypeptide derived from Lactobacillus sakei 2512, possessing activity of bacteriocin.

Lactobacillus sakei 2512 was deposited on may 25, 2000, in the National Collection of Cultures of Microorganisms, where it is registered under depot is item number I-2479.

The bacteriocins of the present invention were named as Skatin G (Sakacine G). This polypeptide with a molecular mass of about the level of 3700-3900, preferably 3834 Yes, when defining a method of mass spectrometry. It has a range of inhibition of bacteria, a very close range of bacteriocins of class IIA. That is why he was particularly effective against strains of Lactobacillus sakei that is different from Lactobacillus sakei 2512, against Pediococcus cerevisiae, all strains of Listeria, against Enterococcus faecalis and durans. Conversely, it is ineffective against other species of Lactobacillus, for example, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei and one strain of Enterococcus faecium.

Like bacteriocins against Listeria type pediocin, Skatin G is a peptide structure two disulfide bridge.

Analysis of the genetic determinants of many bacteriocins of class IIA showed that the genes responsible for their production, transport and immunity, organized into one or more structures of type operons. These operons are often localized in the plasmid and typically include at least two genes encoding proteins, homologs ABC vector and the helper protein, which, apparently, is involved in the selection of bacteriocin.

Cloning of the nucleotide fragment containing the gene sacarina G, showed the existence of three complete open reading frames skgA1 (SEQ ID No. 1), skgA (SEQ ID No. 3) and skgDc (SEQ ID No. 13), including shortened frame reading skgD (SEQ ID No. 7), and shortened frame skgI (SEQ ID No. 5), which is schematically presented in figure 1. Nucleotide fragment consists of two circuits, one of which is the thread 5’-3’ shown in sequence ID No. 15.

The products of the genes skgA1 and skgA2 called prebaccalaureate, undergo a process of maturation process, in which their respective leader peptides hatshepsuts between residues 18 and 19, while relieving active Skatin G (residues 19-55).

5’-3’ chain nucleotide fragment containing skgAl, skgA2, skgD and skgI represented in SEQ ID No. 9.

The object of the present invention is also isolated (dedicated) a polypeptide corresponding to bacteriocins, including the sequence of SEQ ID No. 2 and/or SEQ ID No. 4. The sequence of the Mature bacteriocin corresponds to SEQ ID No. 12 and it comprises the sequence SEQ ID No. 2 and No. 4.

The open reading frame skgI encodes a protein of 52 residues. Comparison of this sequence with databases shows great similarity SkgI with proteins of the immune system. Probably it encodes a protein of the immune system, which protects the bacterium producing Skatin G.

The present invention also extends to an isolated polypeptide comprising the sequence SEQ ID No. 6, the corresponding frame reading skgI.

What comes after is it gene skgDc, it encodes a protein with homology to proteins of the family of ABC-carriers, in particular, the vector of pediocin RA-1. Gene skgDc probably encodes an ABC-carrier specific to Sabatino G.

The present invention also extends to an isolated polypeptide comprising the sequence SEQ ID No. 8, the corresponding gene skgDc, and an isolated polypeptide containing the sequence SEQ ID No. 14, the corresponding gene skgDc.

It is also envisaged that covered and homologous sequences, defined as:

i) sequence having a similarity of at least 70% with the sequence SEQ ID№ 2, № 4, № 6, № 8, № 12 or # 14,

ii) a sequence encoded by nucleic acids that are homologous to those identified below, i.e. nucleic acids, hybridizations sequences of SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 or # 15 or them with complementary sequences, in simple terms.

The term "similarity" means not only a complete similarity or identity (identity) amino acid comparison of homologous sequences, but incomplete similarity, defined as the similarity. When determining similarity between polypeptide sequences are considered conservative replacement is a replacement of one amino acid and the CSOs same class: replacement of amino acids with uncharged side chains (aspartic acid, glutamine, serine, threonine, tyrosine), amino acids with basic side chains (lysine, arginine, histidine), amino acids with kislotnimi side chains (aspartic acid, glutamic acid), amino acids with nonpolar side chains (glycine, alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan, cysteine).

In particular, under "homologous amino acid sequence" refers to all amino acid sequence differing from the sequence of SEQ ID№ 2, № 4, № 6, № 8, № 12 or # 14 replacement, deletion and/or insertion of one amino acid or a limited number of amino acids, especially the replacement of natural amino acids, synthetic amino acids or pseudo-amino acids in those positions, the modification of which do not cause significant damage to the biological activity of the selected polypeptide, preferably sacarina G.

Preferably, such a homologous amino acid sequence similarities at the level of at least 85% with the sequence SEQ ID№ 2, № 4, № 6, № 8, № 12 or # 14, preferably not less than 95%.

Homology is usually defined using sequence analysis, for example, a software package for sequence analysis of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Similar aminokislot the e sequence impose (match) each other so that to obtain the maximum degree of homology (identity or similarity, as defined above). This may require the introduction of artificial gaps in the sequence. Once the optimal alignment determine the degree of homology by calculating the sum of all rules by which the amino acids of the two compared sequences are identical, then dividing by the total number of provisions.

The biological activity of the isolated polypeptide, in particular, sacarina G, means its ability to suppress the growth of unwanted strains of bacteria and/or pathogens, preferably, bacteria Listeria, more preferably bacteria Listeria monocytogenes.

The object of the present invention is an isolated nucleic acid encoding the above-defined polypeptide.

More precisely, the subject of the present invention is an isolated nucleic acid comprising the sequence SEQ ID No. 1 and/or SEQ ID No. 3.

Was determined the complete nucleotide sequence region that is involved in the expression sacarina G (3055 base pairs, BP). This double-stranded DNA 5’-3’ chain which is presented in SEQ ID No. 15. Chain 3’-5’ are presented in figure 2. The object of the present invention is also a nucleic acid comprising the sequence.

As stated above, this is the sequence has three complete open reading frames skgA1, skgA2, skgDc and one shorter - skgI. Putative genes skgA1 (SEQ ID No. 1), skgA2 (SEQ ID No. 3) and skgI (SEQ ID No. 5) is oriented in the opposite direction relative to skgDc (SEQ ID No. 13).

The present invention also encompasses nucleic acid comprising the sequence SEQ ID No. 5, a nucleic acid comprising the sequence SEQ ID No. 13, and a nucleic acid comprising the sequence SEQ ID No. 7.

It is also envisaged that covered and homologous sequences, defined as:

i) sequence having a similarity of at least 70% with the sequence SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 or # 15,

ii) sequence hybridizers sequences of SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 or # 15 or them with complementary sequences, in simple terms,

iii) a sequence encoding a polypeptide called sacarina G, which is defined above.

Preferably homologous to the nucleotide sequence according to the invention, has similarity at the level of at least 75% with the sequence SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 or # 15, more preferably at least 85% or at least 90%.

Preferably, such a homologous nucleotide sequence's hybrid specifically with sequences complementary to the sequence of SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 the sludge is No. 15, in severe conditions. The parameters defining the stringent conditions of hybridization depend on the temperature at which separation occurs at the 50% double chains (Tm).

For sequences with more than 30 bases, Tmis determined by the formula (Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory):

Tm= 81,5+of 0.41(%G+C)+16,6 log (concentration of cations) to 0.63 (% formamide)

- (600/number of bases).

For sequences with less than 30 bases, Tmis determined by the formula: Tm= 4(G+C) + 2(A+T).

In appropriate circumstances, stringent hybridization in which nonspecific sequences do not hybridize, hybridization temperature is preferably 5-10°below Tmand as a buffer, preferably, applied solutions of high ionic strength, for example, a solution of 6×SSC.

The term "similar sequences", as used above, means not only a complete similarity or identity (identity) compared nucleotides, but incomplete similarity, defined as the similarity. When determining similarity between polypeptide sequences are taken into account, for example, purines and pyrimidines.

Homologous nucleotide sequences with open reading frames shown in SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 or # 15, also include all sequences of nucleotides, about the tives such as the sequence SEQ ID No. 2, № 4, № 6, № 8, № 12 or # 14 by mutation, insertion, deletion and/or substitution of one or more bases, or degeneracy of the genetic code, if only they encode a polypeptide having biological activity sacarina G, as defined below.

Such homologous sequences are sequences of genes from other bacteria than Lactobacillus encoding Skatin G.

The polypeptides of the present invention can be synthesized by any methods well known in this area. The polypeptides according to the invention, for example, can be synthesized by chemical synthesis, for example, by using the Merrifield synthesis, which is preferred by reason of purity, antigenic specificity, absence of undesirable secondary products, as well as the ease of obtaining the target product.

The object of the present invention is also a method of obtaining a recombinant polypeptide, in which a vector comprising the nucleic acid of the present invention, is introduced into the host cells, which are cultured in conditions that ensure the expression of the polypeptide of the present invention or of the polypeptide encoded by the nucleic acid of the present invention.

Recombinant bacteriocins can also be obtained by a method in which a vector carrying a nucleic acid comprising sequence is alnost nucleotides, according to the invention, preferably, the sequence SEQ ID No. 1 and/or # 3, or a homologous sequence, is introduced into the host cells, which are cultured in conditions that ensure the expression of the corresponding polypeptide. The resulting protein can then select and clear. Apply the cleaning methods known in this field. The obtained recombinant polypeptide can be purified from lysates and extracts cells from the supernatant culture fluid using such methods or their combinations, as fractionation, chromatographic methods, immunoaffinity methods using specific mono - or polyclonal antibodies, etc.

The sequence of a given nucleic acid that encodes Skatin G can be embedded in expressing the vector in which it is embedded functional way, with elements that regulate its expression, such as promoters, activators and/or transcription terminators. The elements controlling the expression of nucleotide sequences (promoters, activators, sequence termination...), is selected depending on the host cells. For this sequence of nucleotides according to the invention can be embedded into a vector that can replicate autonomously in the body of the selected host, or vector built into the genome hostinga vectors get methods currently used in this field of technology, and the resulting clones enter the appropriate host by standard methods, for example, by electroporation or deposition of calcium phosphate.

Vectors for cloning and/or expression as those described above, comprising the nucleotide sequence of the present invention, are also part of the present invention.

The subject of the invention are also of a host cell, temporarily or stably transformed expressing these vectors. These cells can be obtained by introducing into the host cell, preferably a prokaryotic sequence of nucleotides that is built into the vector described above, and culturing these cells under conditions which enable the replication and/or expression of the introduced nucleotide sequences.

Examples of host cells include bacteria such as Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia, and yeast.

Of a nucleotide sequence according to the invention can be both artificial and not artificial. This sequence of DNA or RNA obtained when skanirovaniya libraries using probes designed based on the sequences of SEQ ID№ 1, № 3, № 5, № 7, № 9, № 13 and/or No. 15. Such libraries can be obtained by classical methods mo is collaroy biology, known in this field.

Of a nucleotide sequence according to the invention can also be obtained by chemical synthesis or using mixed methods including the chemical or enzymatic modification of sequences obtained by skanirovaniya libraries.

The present invention also provides a method of suppressing the growth of Listeria, especially Listeria monocytogenes, in conditions associated or not associated with food that may be contaminated with Listeria monocytogenes.

Listeria monocytogenes is a pathogen that causes severe disease in humans and animals, which can easily spread through contaminated food, in particular, through meat, meat products, marine products, milk and dairy products. The present invention provides a method of suppressing the growth of Listeria monocytogenes in foods that may contain Listeria monocytogenes as pollution, the method includes adding a polypeptide according to the invention in a given product in a quantity sufficient to inhibit the growth of Listeria monocytogenes.

Bacteriocins, according to the invention, are used in all food systems, preferably in an amount of from 1 to 100000 conventional units (UE) bacteriocin per 1 g of food product.

A standard unit of bacteriocin is defined as 5 ál of the pain is on the cultivation supernatant of the culture fluid, calling education a particular zone of inhibition of growth in relation to the control of one strain of gram-positive bacteria on agar medium.

Although the food and most susceptible to infection with Listeria monocytogenes, however, veterinary and medical drugs may be susceptible to contamination with bacteria such as cosmetic and related products.

The bacteriocins of the present invention, in particular, Skatin G, is also useful for suppressing the growth of such pathogens in these products.

Thus, the object of the present invention is the use of bacteriocins according to the present invention as an active anti-pathogenic or undesirable flora, especially in the manufacture of food products, namely, to inhibit the growth and reproduction of Listeria, especially Listeria monocytogenes in food products.

The polypeptide can either be entered himself in foods or produced in them by Lactobacillus sakei 2512.

Thus, the object of the present invention is the use of Lactobacillus sakei 2512 in food to develop in them polypeptide of bacteriocin, according to the invention.

The invention also relates to compositions of bacteriocin comprising at least one polypeptide of the present invention, that is derived from Lactobacillus saei 2512 or comprising the sequence SEQ ID No. 2, or # 4, or # 12, or # 14 or Lactobacillus sakei 2512.

The invention also covers the use of Lactobacillus sakei 2512 for production of the polypeptide, as defined above, to inhibit the growth and reproduction of Listeria, especially Listeria monocytogenes, in food products, as well as compositions containing this strain.

The following examples and figures are provided to illustrate and not to limit the subject matter of this invention.

List of figures

Fig. 1. Schematic representation of the genetic locus involved in the production sacarina G.

Fig. 2. Complementary strand 3’-5’corresponding to the complete nucleotide sequence of the region that is involved in the expression sacarina G, in which the thread 5’-3’ represented in SEQ ID No. 15.

Information confirming the possibility of carrying out the invention

Materials and methods

Bacterial strains and culture medium. Lactobacillus sakei 2512 cultivated at 30°environment MRS (DIFCO Laboratories), sterilized 12 min at 110°C. the Indicator strains were cultured in medium BHI (brain-heart infusion", DIFCO Laboratories) at 37°C.

- Measurement of activity. In agar (10 g/l) environment BHI was inoculable 1% stationary culture of the indicator strain, and then poured into Petri dishes. In holes made in solidifying the agar with a punch, made of 50 μl of a solution of sakati the and G. Activity bacteriocin was expressed in the emergence of zones of inhibition of growth around the wells after incubation over night at 37°C.

Analysis of the protein. Skatin G were analyzed by the method of mass spectrometry instrument Sciex API 165 firm Perkin-Elmer, equipped with ionizer type lonspray. After lyophilization of the active fraction was dissolved in a mixture of acetonitrile/water (1:1)containing 0.1% formic acid and then introduced into the device through infusion with a speed of 5 μl/min

The concentration of protein was determined by the method of bicinchoninic acid using a set of ICA (Sigma), according to manufacturer's instructions.

Comparison of protein sequences was performed using the BLAST program (1)obtained from ExPASY, Swiss Institute of Bioinformatics.

Molecular cloning and transformation. Plasmids were extracted and isolated from strains of Escherichia coli and Lactobacillus sakei 2512 in accordance with the methods previously described Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory, and Muriana et Klaenhammer, 1987, Appl. Environ. Environ, 53: 553-560, respectively.

Restriction enzymes and enzymes for the modification of DNA was used according to the supplier's instructions (Gibco-BRL). Analytical and preparative gel electrophoresis agarose was performed in buffer Tris/borate/EDTA (pH 8.3) according to the methods described in Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory. Cleaved DNA fragments were isolated from agarose gels using the of abortion practices "Prep-a-Gene" (Bio-Rad). Cloning in plasmid pGEM-T (Promega) and pZERO2 (Invitrogen) was carried out in accordance with the recommendations of the suppliers. Southern-hybridization was performed by transfer to nylon membrane (Hybond-N+, Amersham)according to Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory. After the transfer was performed hybridization with radioactive probe labeled with32P using the kit for labeling DNA in a system of random primers" (Gibco-BRL). Competent cells of bacteria E. coli were obtained and their transformation was performed by the method of Hanahan, 1983, J. Mol. Biol., 166, 557-80.

Taq polymerase (Gibco-BRL) was used according to the recommendations of the provider.

Amplification of the DNA fragment encoding Skatin G, carried out with the help of the device Geneamp 9700®(Perkin-Elmer) under the following conditions: 35 cycles consisting of denaturation at 94°C for 30 s, hybridization at 45°C for 30 s, and chain elongation at 72°C for 1 min, after which another cycle of chain elongation at 72°C for 5 minutes

The DNA fragment carrying the locus sacarina G, sequenced using automated sequencing machine (ABI Prism 310®(Perkin-Elmer) with the use of the kit for sequencing "Big-dye terminator®" (Perkin-Elmer) and the appropriate primers.

Example 1

Isolation and purification of sacarina G

16-hour culture of Lactobacillus sakei 2512 (100 ml) were centrifuged for 15 min at 6000g. Selected supernatant and heated at 70°C for 20 minutes is supernatant was cooled and diluted with 1 volume of water (after dilution, the pH value should be less than 6, if necessary, add 1 M Hcl), and then was passed through a column (2,5×18 cm)filled with cation-exchange resins (carboxymethylcellulose: Cellufine C-200, Amicon), balanced water. After washing first with water (100 ml), and then 0.1 M NaCl solution (150 ml), Skatin G was suirable 0.5 M NaCI solution (200 ml). The pH of all solutions should be less than 6. After this active fraction was applied to a cartridge for solid phase extraction (Sep-pak plus C18, Waters), balanced water. After sequential washing of 20 mm solution of ammonium acetate (5 ml)containing 0, 10, 20 and 30% acetonitrile, Skatin G was suirable 10 ml of a 20 mm solution of ammonium acetate containing 80% acetonitrile. After drying the extract was dissolved in 1 ml of 40% aqueous solution of acetonitrile, and then through the injector was applied to the analytical column C8 HPLC reverse phase (Kromasil, 5 μm, 100 Å, 4,6×250 mm, A.I.T.). HPLC was performed on a system consisting of a pump Perkin-Elmer series 200 LC connected to the detector Perkin-Elmer 785A. Register absorption at 220 nm. Separation was carried out with a speed of 0.8 ml/min using a gradient of the following solvents: A = water/0.1% of triperoxonane acid; B = acetonitrile/water/0,07% triperoxonane acid. After washing for 5 min, 20%solution In elution was performed with a gradient of 20-40% solution for 10 min and 40-55% solution In 20 minutes

Coat the Oia, the corresponding peak on 23 minutes, which proved to be active against Listeria ivanovii BUG 496, analyzed by the method of mass spectrometry with ionization type "ionspray". The isolated product had a purity of at least 95% and a molecular weight 3834,32±0,31 D. Quantity of clean sacarina G was 120 μg per 100 ml of culture. Exit when cleaning accounted for 55% of the total activity. Part of the primary sequence sacarina G established method mikroekonomia, and based on this sequence were designed two degenerate oligonucleotide.

Example 2

Cloning of the genetic locus involved in the production sacarina G

On the basis of the genetic code were synthesized two degenerate oligonucleotide: SakG01 (5’-AARTTATTATGGNAAYGGNGT-3’) (SEQ ID No. 10) and SakG02S (5’-ACATGATGNCCNCCRTTNGC-3’ (SEQ ID No. 11) in order to amplify the DNA fragment corresponding to the structural gene of the Mature sacarina G (SEQ ID No. 15) by the method of polymerization chain reaction (PCR). The amplification product size of about 100 BP cloned into plasmid pGEM-T, obtaining the plasmid pJMBYC01. Restriction PvuII fragment length 560 BP of pJMBYC01 containing the inserted fragment served as a probe when performing Southern-hybridization for localization of the structural gene in the genome of Lactobacillus sakei 2512. In plasmid extract of Lb. sakei 2512, treated with restriction enzymes HindIII and EcoRI, the probe obnarujivaet size of about 2.1 and 9 KBP, respectively. The HindIII fragment of 2.1 KBP was purified and incorporated into the vector pZER02 obtained plasmid was named pJMBYC02. The presence of the structural gene sacarina G pJMBYC02 was confirmed by PCR amplification with primers SakG01 and SakG02S and sequencing of DNA fragment embedded in pJMBYC02. The same strategy was used to identify the complete gene sequence skgD. Plasmid extract Lb. sakei 2512 uncoupled Xbal. The cleavage product was built into the plasmid pBluescript SK+. Clones containing the correct sequence was determined using a radioactive probe derived from plasmid pJMBYC02 by PCR using oligonucleotides SakG03 (5’-CCTTGGTCAGGCTATCG-3’) (SEQ ID No. 16) and SakG04 (5’-ATCACCTTTTTGAATTACCC-3’) (SEQ ID No. 17).

Analysis of the complete nucleotide sequence of this region (3051 p. O.) showed the presence of three complete open reading frames skgA1, skgA2, skgDc and one shorter, skgI. Putative genes skgA1, skgA2 and skgI oriented in the opposite direction relative to skgD.

Before each of the open reading frames is a potential binding site with ribosomes. Both genes skgA1 and skgA2 encode proteins of 55 amino acid residues, of which the remains 19-55 absolutely identical. Cut 19-52 corresponds to the sequence sacarina G, obtained by microsectioning. It should be noted the presence of 4 cysteine residues at positions 9, 14, 24 and on To the end. To the ome, the calculated molecular mass of this peptide is equal 3838,2 D and differs from the measured molecular weight (3834,32 D) for 4 D, which indicates the presence of two disulfide bonds in skazine G, as already shown for other bacteriocins against Listeria.

Fragment 1-18 proteins SkgA1 and SkgA2 differ only by 3 residues and have a strong homology with the "leader" of the peptides of class II bacteriocins, which are involved in the transport of these peptides specific ABC-carriers. In particular, for these "leaders" characteristic leaf motifs GG included in the site of maturation of these bacteriocins. Comparison of the nucleotide sequences of genes skgA1 and skgA2 also shows a sequence identity greater than 95% in the part of the gene, which encodes the Mature bacteriocins.

Incomplete open reading frame skgI encodes a protein of 52 residues. Comparison of this sequence with different databases shows strong homology SkgI protein immunity Lcc and Mesl. It was shown part MesI protection from mesenterica Y105. It can be assumed that skgI encodes a protein of immunity to Sabatino G.

The last gene skgDc encodes a protein of 727 amino acids. According to databases, SkgDc has a high homology with proteins of the family of ABC-carriers, in particular, the vector of pediocin RA-1 PedD and PapD (Marugg et al., 1992, Appl. Environ. Environ. 58, 2360-7; Motlagh et al., 194, Lett. Appl. Environ. 18, 305-12), sacarina R-SppT (Huhne et al., 1996, Microbiology 142, 1437-48), sacarina And-SapT (Axelsson and Holck, 1995, J. Bacteriol. 177, 2125-37) and mesenterica Y105-MesD (Fremaux et al., 1995, Microbiology 141,1637-45).

Example 3

The range of inhibition of growth

Tested sensitivity to Sabatino G 17 strains of bacteria by the method of test holes (see Materials and Methods). The results are presented below in table 1.

Table 1
 The radius of the zone of inhibition of growth (mm)
Lc. lactis ATTC 114540
Ln. paramesenteroides DSM 202880
Ln. mesenteroides DSM 204840
Ln. mesenteroides DSM 202400
Lb. delbrueckii DSM 200810
Lb. plantarum DSM 201740
Lb. brevis DSM 200540
Lb. casei DSM 200110
Lb. sakei 25151
P. acidilactici ENSAIA 5830
P. cerevisiae IP 54921
E. faecium ENSAIA 6310
E. faecalis IP54302
E. faecalis ENSAIA 6361
E. durans ENSAIA 6302
L. inocua 88113
L. ivanovi BUG 4966

Range is davleniya growth this bacteriocin lactic acid bacteria seems to be quite narrow and limited to strains of Lactobacillus sakei and Pediococcus cerevisiae. This peptide, as well as other bacteriocins of class IIA, it is active against all tested strains Listeria, as well as against Enterococcus faecalis and durans, but not against Enterococcus faecium.

1. An isolated polypeptide representing the bacteriocins Skatin G originating from Lactobacillus sakei 2512 and having an amino acid sequence selected from the group consisting of SEQ ID No 2, SEQ ID No 4 and SEQ ID No 12.

2. The polypeptide according to claim 1, characterized in that it is used as an active anti-pathogenic or undesirable microflora in the manufacture of food products.

3. The polypeptide according to claim 2, characterized in that it is used to inhibit the growth and reproduction of Listeria, preferably Listeria monocytogenes in food products.

4. The polypeptide according to claim 2 or 3, characterized in that it is produced by a strain Lactobacills sakei 2512 directly in the food product.

5. The nucleotide sequence encoding the polypeptide according to claim 1.

6. The nucleotide sequence according to claim 5, characterized in that it comprises the sequence of SEQ ID No.1 and/or SEQ ID No.3.

7. The vector for cloning and/or expression of the polypeptide under item 1, comprising the nucleotide sequence according to claim 5 or 6.

8. The vector according to claim 7, characterized in that it is used to transform microorganisms selected from Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia or yeast.

9. A method of obtaining a recombinant polypeptide, comprising introducing a vector comprising the nucleotide sequence according to claim 5 or 6, in the host cell, culturing these cells under conditions enabling expression of the polypeptide according to claim 1 or the polypeptide encoded by the nucleotide sequence according to claim 5 or 6.

10. Lactobacillus sakei 2512, deposited in the National collection of cell cultures under the number I-2479, producer of the polypeptide according to claim 1, represents the bacteriocins Skatin G.

11. The strain of claim 10, characterized in that is used in food to develop in them the polypeptide according to claim 1, represents the bacteriocins Skatin G.

12. Bacteriocin the composition having the ability to inhibit the growth of gram-positive pathogenic bacteria, comprising the polypeptide according to claim 1, which is bacterial Skatin G, or Lactobacillus sakei 2512 of claim 10 in therapeutically effective amounts in combination with a carrier or diluent.

13. Bacteriocin the composition according to item 12, characterized in that it inhibits the growth of pathogenic microorganisms Listeria monocytogenes.



 

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2 tbl, 10 ex

FIELD: biotechnology, in particular epithelial cell growth factors useful in production of new keratinocyte growth factor (KGF).

SUBSTANCE: KGF protein is obtained by cultivation of recombinant host cell, transformed with vector containing DNA which encodes amini acid sequence of KGF protein. Obtained KGF protein in pharmaceutical composition is used for forcing of epithelial cell proliferation. Method of present invention makes it possible to produce KGF protein with specific mitogenic activity of 3.4 x 104 U/mg of protein in relation to keratinocyte cells.

EFFECT: new keratinocyte growth factor.

52 cl, 14 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: medicine, microbiology, immunology.

SUBSTANCE: method involves culturing cells of Yersinia pestis EV in solid nutrient medium up to the concentration 100 x 109 cells/ml with physiological solution and treated with equal volume of 0.125% cetavlon solution for 48 h. After control for the specific sterility suspension is centrifuged at 6 000-8 000 rev/min at 4oC for 20-30 min and supernatant is centrifuged at 16 000 rev/min at 4oC for 20-30 min. Precipitate is washed out twice with physiological solution, distilled water and centrifuged again at 16 000 rev/min for 20-30 min after each washing out. Precipitate is resuspended in 0.1% sodium deoxycholate and shaken at room temperature for 10-20 min followed by treatment by ultrasonic oscillation at frequency 20 kHz by 3-4 cycles by 5-7 s in ice bath and centrifuged again at 16 000 rev/min at 4oC for 30-40 min. Obtained precipitate is resuspended in distilled water, centrifuged at 16 000 rev/min for 30-40 min and precipitate is resuspended in minimal volume of distilled water and dried by lyophilization. Method is simple in performing and provides preparing the preparation with high immunogenicity. Invention can be used in manufacturing the chemical vaccine.

EFFECT: improved preparing method.

2 ex

FIELD: biotechnology, food industry, agriculture, microbiology.

SUBSTANCE: the strain Propionibacterium freudenreichii subsp. shermanii Ac-103/12 is obtained by multiple re-inoculations of the parent strain Propionibacterium freudenreichii subsp. shermanii Ac-103 cells and deposited in All-Russian State collection of microorganism strains used in veterinary science and animal husbandry at number VGNKI - 03. 04. 12. - DEP. This strain is a producer of fodder protein that allows excluding the environment pollution in manufacturing the protein fodder, to enhance the specific yield of protein, to reduce energy consumptions in manufacturing protein fodder, to simplify and to accelerate the process for its preparing, to simplify the equipment, to utilize wastes in processes using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: immunotherapeutic agents.

SUBSTANCE: antigenic preparations are obtained from keratinophilic fungi Trichophiton or Microsporum species or yeast species Candida by alkali hydrolysis techniques. Thus obtained preparations can be, in particular used, as vaccines and for treating allergy and modulating immune response.

EFFECT: expanded immunotherapeutic possibilities.

17 cl, 5 dwg, 12 tbl, 20 ex

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

Anti-plague vaccine // 2197988
The invention relates to a vaccine used to protect against infection by the bacterium Yersinia pestis, as well as to a method for protection of human or animal against infection Y

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention represents group of Neisseria proteins eliciting antigen properties. Protein eliciting antigen properties comprises fragment of protein ORF 40 (amino acid sequence of ORF 40 is given in the invention claim) that involves 7 or more conservative amino acids arranging in succession. Proteins as components of proteins in the claimed group are used as a medicinal agent or for it manufacturing for treatment and prophylaxis of infection caused by Neisseria, and for manufacturing the diagnostic preparation. Invention relates also nucleic acid encoding the Neisseria protein. Nucleic acid is used as the protein. Applying the invention provides the maximal recognition and reactivity between strains of Neisseria. Invention can be used in manufacturing curative-prophylactic preparations with respect to Neisseria meningitides.

EFFECT: valuable biological and medicinal properties of antigen.

27 cl, 51 dwg, 10 ex

The invention relates to biotechnology and can be used for the treatment and prevention of disease associated with infection by Streptococcus or G+ bacteria
The invention relates to biotechnology, namely, construction of Escherichia coli - producer cholera toxin, and can be used to create a diagnostic test systems, allowing to detect toxigenic clones of Vibrio cholerae and Escherichia coli, as well as for the design of live vaccine strains against diarrhoeal diseases caused by pathogenic strains of V. cholerae and E.
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