Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Definitions

• the subject of the invention is the method of obtaining synthetic genes encoding low molecular weight myelin proteins, derivatives of protein fragments of mammalian spinal cord, and the method of their microbial production using lactic acid bacteria, single and combined preparations of these peptides and their application in medicine for specific induction of oral tolerance.
• Multiple sclerosis is a destructive disease of the central nervous system that affects mainly young people, between 20 and 40 years of age. Incidence data indicate that around 60 000 people suffer from this disease in Poland. It is an autoimmune disease, for which, as yet, there is no effective treatment. Immunomodulatory drugs, such as interferon and Copaxone, only change the course of the disease, reducing the number of attacks, but after 6 years of treatment, the disability of people with multiple sclerosis is the same as without treatment. Moreover, treatment of this disease is quite expensive, and reimbursement of medical expenses – infrequent. Hence the need for intensive research on new effective and economically profitable methods of treating this chronic immune disease.
• Autoimmune diseases are based on a pathological recognition by the immune system of the body's own proteins as foreign antigens, which, in consequence, leads to induction of mechanims eliminating these antigens.
• fragments of myelin proteins of the central nervous system constitute antigens pathologically recognized by antibodies.
• myelin proteins The recognized targets in the attack on the immune system in multiple sclerosis and in the experimental allergic encephalomyelitis (EAE) animal model are myelin proteins. Main proteins components of myelin are three proteins:
• MBP myelin basic protein
• PGP proteolipid protein
• the present invention is based on own preliminary research.
• Application of a spinal cord hydrolysate preparation in a arbitrarily selected dose in rats with EAE led to reduction of inflammatory symptoms both when applied before and after EAE induction (6).
• Lactic acid bacteria constitute a large and taxonomically diverse group of Gram-positive bacteria. An indisputable advantage of these bacteria is their granted GRAS status (Generally Regarded As Safe), i.e. non-pathogenic and safe bacteria for humans and animals. Lactic acid bacteria are a significant object of biotechnological uses as well as basic and applicational research. Biotechnological use of lactic acid bacteria finds application in various industrial branches (7) – food (e.g. production of fermented milk products, food and feeds as well as feed additives), chemical (e.g. production of polymers and enzymes) and pharmaceutical (e.g. probiotic preparation, such as Lakcid or Lactovaginal).
• food e.g. production of fermented milk products, food and feeds as well as feed additives
• chemical e.g. production of polymers and enzymes
• pharmaceutical e.g. probiotic preparation, such as Lakcid or Lactovaginal
• the subject of the invention is development of a biological system, based on lactic acid bacteria, for production of peptide fragments of natural myelin proteins, to create a preparation for induction of oral tolerance.
• Peptide fragments of natural myelin proteins for use in the invention were selected based on own studies, which indicated that patients with multiple sclerosis are identified with the HLA-DR2 antigen that recognizes fragment 84-103 of the MBP protein (13). Antibodies against other fragments of the three main proteins are also identified (14, 15) and during progression of the disease the number of recognized antigens deriving from the three basic myelin proteins increases. The above observations are most probably connected with the availability of these proteins as well as their fragments in patients. Immunogenicity of the selected peptide fragments of natural myelin proteins was confirmed also on EAE animal models (16, 17, 18).
• Synthetic genes encoding peptide fragments of natural myelin proteins for induction of oral tolerance are characteristic by the fact that they contain at least one nucleotide sequence encoding a myelin protein fragment, selected from a group comprising: nucleotide sequence for MBP21-40 fragment, represented by formula 1 , encoding a peptide of amino acid sequence represented by formula 1a , and/or nucleotide sequence for MOG35-55 fragment, represented by formula 2 , encoding a peptide of amino acid sequence represented by formula 2a, and/or nucleotide sequence for MBP85-97 fragment, represented by formula 3 , encoding a peptide of amino acid sequence represented by formula 3a, and/or nucleotide sequence for PLP139-151 fragment, represented by formula 4 , encoding a peptide of amino acid sequence represented by formula 4a, and/or nucleotide sequence for PLP178-191 fragment, represented by formula 5 , encoding at least one nucleo
• the method of generating genes encoding peptide fragments of natural myelin proteins is based on synthesizing by PCR method of synthetic genes encoding peptide fragments of nautral myelin proteins using 2 complementary primers staurLONG”) presented in Table 1 specific for each peptide, which nucleotide sequences were designed in such a way so they would correspond to the codons preferably occurring in Lactococcus lactis bacteria.
• nucleotide sequences of donFLONG” primers for peptides MBP85-97, PLP139-151 and PLP178-191 were modified by introducing a sequence corresponding to the translation START codon (ATG), just before the sequence encoding the first amino acid of the original peptide sequence.
• ATG translation START codon
• the 5’ ends of primers playedFLONG_peptide contain an additional, typical for Lactococcus lactis bacteria, RBS (ribosome-binding site) sequence recognized by the translation machinery and a ‘spacer’ sequence (linking sequence) localized between the RBS region and the translation START codon.
• Primers tendLONG are used as templates for synthesizing by PCR technique the synthetic genes using short primers CSSHORT”) homologous to the 5’ ends of primers favorFLONG_peptide” and expediRLONG_peptide”, where the 5’ ends of primers casualSHORT” carry additional sequences recognized by specific restriction enzymes, preferably SalI (for forward primer SHORT) and PstI (for reverse primer SHORT), while the 5’ ends of prmiers mayberevSHORT_peptide” (reverse SHORT) have additionally a twice repeated sequence corresponding to the translation STOP codon (TAA) (Table 2).
• TAA translation STOP codon
• the subject of the invention are also DNA fragments obtained through the above-mentioned PCR reaction using primers originally designed and ordered SHORT”, which nucleotide sequences, according to the invention, comprise the sequence of synthetic genes of selected peptides of natural myelin proteins (underlined sequences), according to the invention, represented by formula 6 , formula 7 , formula 8 , formula 9 and formula 10.
• the method of cloning the generated DNA fragments comprising synthetic genes is based on subjecting them to digestion by restriction enzymes, favorably PstI and SalI, and then ligating them with a plasmid vector that replicates in lactic acid bacteria cells, favorably in Lactococcus, in particular with the pIL253 vector, digested beforehand with the same restriction enzymes, to obtain recombinant plasmids, in which the orientation of the cloned PCR products (inserts) is in accordance with the direction of transcription directed from the internal promoter present on the vector, in order to introduce the recombinant DNA by electroporation method into cells of bacterial strains, which are cultured in a known manner.
• restriction enzymes favorably PstI and SalI
• Cells containing the new gene are isolated from the culture population, favorably by analyzing the obtained transformants by PCR technique for the presence of inserts which length corresponds to the length of DNA fragments comprising the expected synthetic genes. Nucleotide sequences of the cloned genes are examined for conformity with the nucleotide sequences of the synthetic genes by DNA sequencig technique.
• Lactococcus strains e.g. Lactococcus lactis IL1403, Lactococcus lactis IBB477 and Lactococcus lactis IBB360, differing in their viability in the mammalian gut as well as other genera of lactic acid bacteria, preferably Lactobacillus and Bifidobacterium.
• the method of producing selected peptides in a bacterial system is based on expression of synthetic genes of selected peptides in cells of lactic acid bacteria strains, in particular Lactococcus, by culturing them in a known medium specific for lactic acid bacteria, preferably on M17 medium or defined CDM medium ( chemically defined medium ), supplemented with sugar, favorably glucose or cellobiose, at a concentration no less than 0.5% or on milk, whey or other suitable medium, at a temperature ranging between 20 C-30 C, preferably at 30 C.
• a known medium specific for lactic acid bacteria preferably on M17 medium or defined CDM medium ( chemically defined medium )
• sugar favorably glucose or cellobiose
• the method of cloning the synthetic genes of selected myelin peptides in Gram-negative bacteria, favorably Escherichia coli species, according to the invention is based on ligation of the synthetic gene amplified using specific primers ‘SHORT’ (Table 2) with the vector, preferably with a commercially available pGEMT-Easy plasmid or other vector replicating in Gram-negative bacterial cells, favorably from Escherichia coli species , to obtain recombinant plasmids.
• Recombinant DNA is introduced by a known method, e.g. electroporation, to cells of bacterial strains, which are then cultured in a known manner.
• Cells containing the new gene are isolated from the cultured population, favorably by analyzing the obtained transformants by PCR technique for the presence of inserts which length corresponds to the length of DNA fragments comprising the expected genes. Nucleotide sequences of the cloned DNA fragments are examined for conformity with the nucleotide sequences of the synthetic genes by DNA sequencing technique.
• the method of optimizing the expression of synthetic genes of selected peptide fragments of natural myelin proteins favorably is based on replacing the internal, constitutive promoter of the plasmid vector, replicating in L. lactis cells, with a regulated promoter, favorably with the promoter region of a gene deriving from the genome of L. lactis bacteria or other species or from the genome of a bacteriophage, preferably with the promoter region of ptcB gene engaged in sugar catabolism in lactic acid bacteria from Lactococcus genus, which is regulated by the presence in the medium of various sugars, such as: cellobiose, galactosa, salicin, esculin, glucose.
• the method of optimizing the production of peptides in a bacterial system favorably is based on culturing cells carrying the recombinant plasmid vector (with the cloned synthetic gene and a suitable promoter region, favorably the promoter of ptcB gene from the genome of Lactococcus bacteria) in a known medium specific for lactic acid bacteria, preferably on defined CDM medium ( chemically defined medium ), supplemented with sugar, favorably glucose or cellobiose, at a concentration of ⁇ 0.5%, or on milk, whey or other suitable medium, at a temperature ranging between 20 -30 C, preferably at 30 C.
• the subject of the invention is also the nucleotide sequence of the promoter region of gene ptcB, comprising the -10 and -35 region, represented by formula 11, for optimizing the production of synthetic genes encoding peptide fragments of natural myelin proteins, according to the invention.
• the method of producing the nucleotide sequence of the promoter region of L. lactis ptcB gene as well as the method of replacing the internal, constitutive promoter of a plasmid vector that replicates in L. lactis with the promoter region of L. lactis ptcB gene, according to the invention, is based on amplifying by PCR reaction the promoter region of ptcB gene using chromosomal DNA of L.
• lactis IL1403 strain as template and pair of primers (ptcBfor and ptcBrev), which sequences are complementary to the DNA sequences flanking the promoter region of the chromosomal ptcB gene and modified in such a way that their 5’ ends contain sequences recognized by specific restriction endonucleases, preferably VspI (for primer ptcBfor) and NciI (for primer ptcBrev) (Table 3).
• VspI for primer ptcBfor
• NciI for primer ptcBrev
• the product obtained in this manner is subjected to digestion by restriction enzymes, preferably VspI and NciI, and then ligated with the recombinant vectors carrying synthetic genes encoding the selected peptide fragments of natural myelin proteins, from which the original promoter region was removed (e.g. by digestion using the same restrictases).
• the recombinant DNA is introduced by electroporation method into cells of bacterial strains which are then cultured by known means. Cells containing the new promoter region are isolated from the cultured population, favorably by analyzing the obtained transformants by PCR technique for the presence of DNA fragments which expected length corresponds to the length of the selected promoter region of ptcB gene. Nucleotide sequences of the cloned DNA fragment are examined for conforminty with the nucleotide sequence of the promotor region of ptcB gene, according to the invention, by DNA sequencing technique.
• the subject of the invention is also the application of bacteria producing peptide fragments of natural myelin proteins according to the invention, for induction of oral tolerance.
• nucleotide sequence of the promoter region of ptcB gene which comprises the -10 and -35 region, represented by formula 11 , according to the invention, for optimizing the production of synthetic genes encoding peptide fragments of natural myelin proteins according to the invention, for induction of oral tolerance.
• bacteria producing the peptide fragments of natural myelin proteins will induce oral tolerance as described in literature referring to oral administration of peptide mix in the form of the animal spinal cord hydrolysate, published in result of own studies (1, 6).
• Bacterial strains, culture conditions and plasmids used Bacterial strains, culture conditions and plasmids used.
• Lactococcus lactis strains were cultured in M17 medium (Oxoid, Yale) supplemented with 0.5 % glucose or in defined CDM medium ( chemically defined medium ) supplemented with glucose or cellobiose (0.5% - 1%) at a temperature between 20 C - 30 C.
• Escherichia coli strains were culture on Luria-Bertani (LB) medium, at a temperature of 37 C. When needed, for selection purposes, the following antibiotic were used: erythromycin 5 ⁇ g ml -1 for L. lactis and ampicillin 100 ⁇ g ml -1 for E. coli.
• the two DNA molecules were recombined with each other and introduced by electroporation method into the cells of selected Lactococcus lactis strains, which were cultured on solid M17 agar medium supplemented with 0.5% glucose and erythromycin at a final concetration of 5 ⁇ g ml -1 , and cells containing the expected synthetic genes were isolated from the cultured population by PCR method using specific primers. Nucleotide sequences of the cloned DNA fragments were examined for their conformity with the nucleotide sequences of the synthetic genes by DNA sequencing technique.
• the resulting proper recombinant plasmids were isolated using a known method from Lactococcus bacterial cells and introduced by electroporation into cells of other lactic acid bacteria, e.g. Bifidobacterium and Lactobacillus.
• Production of selected peptides in lactic acid bacteria cells was based on expression of synthetic genes in bacterial cells by culturing them on defined CDM medium ( chemically defined medium ), supplemented with cellobiose at a concentration no less than 0.5%, at a temperature ranging between 20 -30 C, preferably at 30 C, until reaching optical density of OD 600 0.6.
• defined CDM medium chemically defined medium
• revSHORT_peptide reverse primers
• two DNA fragments were obtained which length corresponded to the lenght of each of the genes.
• the promoter region of ptcB gene was amplified by PCR method using genomic DNA of L. lactis IL1403 strain as template and specific primers (Table 3). In result of the PCR amplification, a product was obtained of expected length, which was subsequently digested with NciI and VspI enzymes and ligated with the pIL253 plasmid, replicating in Lactococcus bacteria cells, digested beforehand with the same enzymes. The recombinant DNA was introduced by electroporation into cells of L.
• lactis strain which were cultured on solid M17 agar medium supplemented with 0.5% glucose and erythromycin at a final concentration of 5 ⁇ g ml -1 , and from the cultivated population cells containing the promoter region of ptcB gene were isolated using the PCR method and specific primers.
• Synthetic peptide genes were amplified by PCR method using specific primers ‘SHORT’ (Table 2) and then ligated with a vector replicating in cells of Escherichia coli bacteria - pGEMT-Easy. Recombinant DNA was introdiced by electroporation method into cells of E . coli strain, which were cultivated on LB agar medium supplemented with ampicillin at a final concentration of 100 ⁇ g ml -1 , and from the cultivated population cells containing the expected synthetic genes were isolated using the PCR method and specific primers. Nucleotide sequences of the cloned DNA fragments were examined for conformity with nucleotide sequences of synthetic genes by DNA sequencing technique.
• E . coli cells were transformed using electroporation technique (22).
• nucleotide sequence of synthetic genes of selected myelin peptides as well as of the promoter region of ptcB gene , according to the invention was confirmed by sequencing of DNA fragments using the BigDye Terminator set (Promega, USA) and ABI377 sequencing apparatus (Applied Biosystem, USA), and recombinant plasmids as template as well as primers complementary to the extremities of each of the inserts. Obtained nucleotide sequences were analyzed using the BLAST program (23).

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• 2012
  • 2012-01-13 WO PCT/EP2012/050483 patent/WO2013104424A1/fr not_active Ceased

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