WO2015131254A1 - Inhibin alpha antigen, gene coding for inhibin alpha, gene coding for fusion protein, method for obtaining inhibin alpha antigen, antigenic composition, use of the inhibin alpha antigen and use of the antigenic composition - Google Patents
Inhibin alpha antigen, gene coding for inhibin alpha, gene coding for fusion protein, method for obtaining inhibin alpha antigen, antigenic composition, use of the inhibin alpha antigen and use of the antigenic composition Download PDFInfo
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- 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
- C07K14/575—Hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- ALPHA INHIBINE ANTIGEN ALPHA INHIBINE CODING GENE, MERGER PROTEIN CODING GENE, ALPHA INHIBINE ANTIGEN PROCESS, ANTIGENIC COMPOSITION, ALPHA INIBINE ANTIGEN USE, AND ANGENIC COMPOSITION USE
- the present invention relates to a modified alpha inhibin protein, antigenic fragments thereof and fusion protein comprising it, as well as an antigenic composition comprising modified alpha inhibin and / or an antigenic fragment thereof and / or a protein.
- fusion protein comprising modified alpha inhibin, the process of producing the modified alpha inhibin protein, antigenic fragments thereof and fusion protein comprising same, and the use of modified alpha inhibin antigenic fragments thereof or fusion protein comprising modified alpha inhibin in the increased ovulation in animals, especially mammals and birds.
- the present invention is based on modification of the mature sequence of the Bos taurus inhibin alpha subunit, specifically in the C-terminal end segment which, in addition to the modification of some amino acids, now has two immunogenic regions in that segment, the latter being most immunogenic region is that which is located at the C-terminal end.
- Inhibin is a protein (glycoprotein) secreted by granulosa (female) and Sertoli (male) cells in response to FSH (follicle stimulating hormone) and its main action is to control feedback. negative in FSH secretion from the pituitary gland. It is found in large quantities in seminal plasma and follicular fluid. It is a highly complex dimeric protein, the "mature" form of inhibin having a molecular weight of 32 kDa and consisting of an alpha chain (approximately 18 kDa) and a beta chain (14 kDa), these two chains being linked by disulfide bridges. The subunits alone have no known biological activity.
- Each subunit originates from a separate gene and is produced as a large precursor protein. It is also known that each subunit has multiple cleavage sites, so subunits of different sizes are routinely found in follicular fluid. It is estimated that various combinations are possible which give rise to numerous different dimeric forms of inhibin. Those often found in bovine follicular fluid are approximately 29, 34, 48, 58, 68, 77, 122 and> 160 kDa. The inhibin family is further complicated by the existence of two separate beta subunit genes. They are called beta-A subunit and beta-B subunit. While inhibins are dimers formed by alpha and beta subunits, dimers composed of beta-beta subunits form another hormone, activin.
- inhibin acts systematically to inhibit FSH release, it is appropriate to say that a reduction in inhibin secretion may increase FSH concentrations and thus have potential use in increasing fertility.
- inhibins and activins are multifunctional molecules that inhibit and activate, respectively, the secretion of FSH produced by the pituitary gland.
- Inhibins and activins have already been found to be involved in the regulation of numerous functions such as hypothalamus hormone secretion and pituitary gland, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, axial embryogenic development or bone growth, depending on its subunit composition. Inhibins are believed to oppose activin functions (see information available at www.uniprot.org).
- inhibins are currently recognized as ovarian and testicular paracrine regulators and have multiple paracrine effects on the utero-placenta unit, representing a promising marker for male and female infertility and gynecological diseases. and gestation.
- WO8600078 describes a method of purification and characterization of inhibin and its use as an antigen for antibody production. Also proposed is a method of constructing oligonucleotide probes for subsequent cloning and expression of inhibin genes, using the recombinant DNA technique, from protein subunit amino acid sequences. This development was carried out at a time when knowledge of inhibin and the techniques of sequencing, characterization and modification of genetic material lacked the resources available today. For this reason, there is no information in this document about the amino acid sequences of the protein and the nucleotide sequences encoding the whole protein or its subunits.
- US4864019 describes a method of treating a mammal for fertility control.
- an antibody is generated in response to the use of a synthetic peptide which includes the six N-terminal end residues of a mammalian alpha inhibin, wherein the antibody reacts with inhibin and acts on the activity of suppression of Gonadotropin release.
- the resulting antibody may be administered to a mammal in sufficient dosage to neutralize a significant portion of the inhibin content in peripheral blood serum, thereby blocking the hormonal activity of endogenous inhibin.
- the 32 kDa protein isolated from porcine follicular fluid is composed of two disulfide bonded 18 kDa and 14 kDa chains, which chains are rich in Cys residues.
- CA21581 16 describes a method for inducing superovulation in cattle by inhibin vaccination, followed by exogenously injecting FSH.
- Inhibin vaccination is by the use of an antigen obtained by binding albumin to a polypeptide synthesized from the amino acid sequence of positions 1-26 (from the N-terminus) of the porcine or bovine inhibin alpha subunit, said antigen and adjuvant mixture being injected subcutaneously into cattle several times.
- WO9522980 (PI9507426) describes a method for accelerating the onset of puberty in birds, particularly ratites and parrots, by administering a heterologous protein comprising inhibin, or a fragment thereof, and a carrier protein, wherein the inhibin or fragment thereof comprises the sequences identified herein as SEQ ID NO: 2 or SEQ ID NO: 4 and the carrier protein may be selected from maltose binding protein, bovine serum albumin, ovoalbumin, flagellin, serum albumin. , etc.
- WO9640219 (PI9609067) describes the compositions employed in the method described and claimed in WO9522980.
- PI0316084 describes multiple antigenic peptide compositions comprising immunogenic peptides (mature inhibin alpha subunit, or conservative substitutions thereof) linked to an amino acid backbone together with an acceptable carrier such as serum albumin, protein of binding to maltose, keyhole limpet hemocyanin etc. These compositions are employed in improving the production performance of animals, especially birds.
- PI0506043 describes a process for genetic evaluation of productive performance of animals from molecular markers associated with inhibin, leptin, LH, FSH, among others genes, and their receptors.
- SCHANBACHER SCHANBACHER B. Radioimmunoassay of inhibin: serum responses to bilateral unilateral and orchidectomy. Endocrinology 1988; 123 (5): 2323-30) developed a radioimmunoassay for the determination of inhibin concentration in serum and other biological fluids using rabbit antiserum to porcine inhibin alpha chain. This test was accurate, sensitive and specific for inhibin.
- TANNETTA D.S. and collaborators found that immunization of sheep with a synthetic peptide corresponding to the amino terminal portion (amino acids 1-29) of the mature alpha subunit of tuberculin-conjugated bovine inhibin was responsible for a three-fold increase in total number of mature follicles per sheep compared to a control group.
- PRASAD More recently, PRASAD, S. and collaborators (PRASAD, S. RASTOGI, SK GANGULY, B. Computational design of a polypitope fecundity vaccine for multiple species of livestock. Vaccine. 2013: Artcle in Press) described a computational method for the prediction of epitopes in a vaccine antigen candidate peptide.
- the proposed vaccine contains a new polyepitope that has been designed using different computational tools and targets inhibin and folistatin from different animals of veterinary interest.
- inhibin immunogenicity particularly inhibin alpha subunit, and fragments or fusion proteins containing inhibin alpha immunogenic sequences.
- This increase in immunogenicity has important application both in increasing the production of animals, including mammals and birds, as well as in the treatment, prophylaxis and diagnosis of diseases and medical conditions in humans.
- the present invention discloses novel inhibin alpha sequences containing more immunogenic regions by modification of the mature inhibin alpha subunit sequence of Bos taurus (obtained from the GenBank collection under accession No. AAI09838.1, Inhibin alpha Bos Taurus).
- an antigen comprising a modified alpha inhibin amino acid sequence, herein called INIBINA OF, comprising SEQ ID NO: 1, which antigen has higher immunogenicity as compared to that of Bos taurus inhibin, GenBank Accession No. AAI09838.1: SEQ ID NO: 1:
- amino acid sequences are provided having conservative substitutions in the sequence of INIBINA OF, as well as immunogenic fragments thereof comprising conservative substitutions.
- heterologous fusion proteins comprising INIBINA OF, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1, and a pharmacologically acceptable carrier / carrier protein are provided. preferably the carrier protein Rrp42.
- nucleotide sequence - SEQ ID NO: 2 - comprising the INIBINA OF amino acid sequence coding gene of the present invention comprising SEQ ID NO: 1.
- the present invention relates to an antigen comprising a modified alpha inhibin amino acid sequence, herein called INIBINA OF, and encoded by the synthetic gene, said antigen comprising SEQ ID NO: 3 having the same antigenicity. of the antigen comprising SEQ ID NO: 1.
- the present invention relates to an antigenic composition
- an antigenic composition comprising (i) the INIBINA OF antigen, comprising SEQ ID NO: 1 or SEQ ID NO: 3, analogs, derivatives or immunogenic fragments thereof. including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, (ii) optionally additives or substances enhancing the immunogenic activity of INIBINA OF and (iii) a pharmacologically acceptable carrier.
- the present invention relates to an antigenic composition
- an antigenic composition comprising (i) a heterologous fusion protein comprising the INIBINA OF antigen, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, and a pharmacologically acceptable carrier / carrier protein, preferably the carrier protein Rrp42; (ii) optionally additives or substances enhancing the antigenic activity of INIBINA OF and (iii) a pharmacologically acceptable carrier.
- the invention relates to the use (a) of INIBINA OF antigen, analogs, derivatives or antigenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3 (b) a heterologous fusion protein comprising the INIBINA OF amino acid sequence, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, and a carrier protein / pharmacologically acceptable carrier, preferably the carrier protein Rrp42 Pyrococcus abyssi and (c) an antigenic composition comprising said INIBINA OF, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, or said heterologous fusion protein of INIBINA OF , in the treatment or prophylaxis of medical conditions related to infertility in mammals and birds and in the increase in the production of mammals or birds.
- Figure 1 shows the amino acid sequence of the mature Bos taurus inhibin alpha subunit (Gen Bank Accession No. AAI09838.1, Inhibin alpha Bos taurus).
- Figure 2 illustrates the antigenicity analysis of Bos taurus inhibin alfa using the PROTEAN (Lasergene) program.
- Figure 3 shows the amino acid sequence as analyzed in Figure 2, highlighting the two sequences with good antigenic index (according to algorithms present in the PROTEAN program).
- Figure 4 represents the alpha inhibin structural model showing antigenic regions 1 (green) and 2 (orange), and the structural modeling of Bos taurus inhibin was performed with the SwissModel PDB Viewer program and the SwissModel server ( http://swissmodel.expasv.org) using the TGF-beta framework (PDB 2QCQ) as a template.
- PDB 2QCQ TGF-beta framework
- Figure 5 shows the structure formed at the C-terminal end of Bos taurus inhibin alpha.
- Figure 6 shows the original amino acid sequence containing the N-terminal end region (underlined) to be changed, the orange antigenic region shown in Figure 4 and the amino acids (underlined) after the antigenic region to be changed. changed.
- Figure 7 shows the amino acid sequence resulting from a first modification at the N-terminal end.
- Figure 8 shows the structural model conformation of the modified amino acid sequence at the C-terminal end of Bos inhibin alpha taurus according to a first modification according to the invention as shown in Figure 7.
- Figure 9 shows the INIBINA OF amino acid sequence of the invention resulting from the modifications shown in Figure 8 and including, at the C-terminal end, the antigenic region represented in green in Figure 3.
- Figure 10 shows the nucleotide sequence of the synthetic gene of the present invention encoding the inhibitor of the present invention.
- Figure 11 shows the amino acid sequence encoded by the synthetic gene of the invention comprising the INIBINA OF sequence of the invention.
- Figure 12 shows the sequence alignment of INIBINA OF with inhibins of selected organisms.
- Figure 13 shows the cloning scheme of the "INIBINA OF" antigen gene in the expression vector.
- Figure 14 shows analysis of purified protein by affinity and molecular exclusion chromatography.
- the present invention is based on obtaining a novel protein, herein called INIBINA OF, produced from the amino acid sequence of the mature protein corresponding to the Bos taurus inhibin alpha subunit (obtained from Gen Bank under No. access AAI09838.1), which contains two important antigenic regions that make INIBINA OF useful in increasing mammalian and poultry production and treating infertility in humans, among other uses.
- INIBINA OF novel protein, herein called INIBINA OF, produced from the amino acid sequence of the mature protein corresponding to the Bos taurus inhibin alpha subunit (obtained from Gen Bank under No. access AAI09838.1), which contains two important antigenic regions that make INIBINA OF useful in increasing mammalian and poultry production and treating infertility in humans, among other uses.
- the said mature sequence of the Bos taurus inhibin alpha subunit (Gen. Accession No. AAI09838.1) (see Figure 1) is as follows: Ser Thr Pro Pro Leu Pro Trp Pro Trp
- the new protein was designed to contain the most immunogenic regions of the Inhibin alpha subunit.
- Inhibin sequence antigenicity was analyzed using Lasergene's PROTEAN program. The result of this analysis is shown in Figure 2.
- the two sequences with good antigen index correspond to the fragments shown in green and orange of Figure 3. .
- Arg Pro Pro Glu Glu Pro Wing Ala His It is located in the first 29 amino acids of the N-terminal end of the Inhibin alpha subunit, and its use as an antigen, conjugate or fusion to carrier / carrier proteins is well known.
- the second sequence with good antigenicity (Gly Tyr ser Phe Lys Tyr Glu Met) is closer to the C-terminal end. alpha subunit and is not known to be used alone as an antigen.
- the Bos taurus Inhibin alpha structural model obtained using the SwissModel server, presents the first antigenic sequence located in a flexible framework region at the N-terminal end.
- the second sequence is located in a beta-leaf present in a structure formed at the C-terminal end of Inhibin alfa.
- cysteines As shown in Figure 5, at the base of the chosen structure, there are four cysteines (Cys) and a "fold" in the structure that is maintained by the hydrophobic interaction of the amino acids valine (Val) and leucine (Leu). These amino acids are located in the middle of a hydrophobic region of the Inhibin alfa molecule, more precisely in the phase that interacts with the receptor.
- Figure 8 shows the result of analysis of this new sequence by molecular dynamics using the NOC program (http://noch.sourceforqe.net) which showed the stability and maintenance of the desired structure during dynamics, despite all modifications / replacements performed.
- antigenic sequence 1 (Leu Leu Gin Arg Pro Giu Giu Pro Ala Ala H ⁇ S, amino acids 16-27 of the Bos taurus inhibin alpha subunit sequence, GenBank Accession No. AAI09838.1), which is naturally flexible, was added at the C-terminal end of the molecule depicted in Figure 8, which already contains antigenic sequence 2 (Giy Tyr Phe Lys Tyr Giu Met).
- Figure 9 shows the amino acid sequence of the antigen of the present invention, referred to herein as INIBINA OF.
- Alpha-inhibin belongs to the family of proteins "TGF-beta", and in Bos taurus there are other inhibin homologous proteins such as TGF-beta 1, TGF-beta 2, Morfogen, among others.
- the strategy followed in the present invention was to use only a subsequence comprised of the first 29 amino acids of alpha-inhibin, a region not present in any other member of the "TGF-beta” family.
- the immune response generated is specific against inhibin.
- the "INIBINA OF" antigen of the present invention brings together the two most immunogenic regions of inhibin alpha, which regions are now arranged in a molecule that retains only one structural region of the parent protein. Indeed, the antigen of the present invention is different from all inhibin-related antigens tested so far.
- the present invention also includes conservatively modified INIBINA OF variants of the invention and having at least 85% identity to any of the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3, and having substantially the same. immunogenic activity of the same.
- amino acid substitutions may be made in protein sequences without affecting its function or activity.
- conservative amino acid substitutions or similar amino acid substitutions may occur naturally or intentionally without modification of biological activity, e.g. protein immunogenicity.
- Similar amino acids may be those with similar size and / or charge properties.
- aspartate and glutamate and isoleucine and valine are similar amino acid pairs. Similarity between amino acid pairs can be assessed in a number of ways known to the skilled artisan. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, p.
- 345-352 which is incorporated herein by reference, provides frequency tables for amino acid substitutions that may be employed as a measure of similarity.
- the frequency tables of Dayhoff et al. are based on comparisons of amino acid sequences for proteins having the same function from a variety of different sources of evolution.
- the "percent sequence identity" for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, with the portion of the polynucleotide or polypeptide sequence in the comparison window, may comprise additions or deletions when compared to a reference sequence (which does not comprise deletions or additions) for optimal alignment of the two sequences.
- the percentage is calculated by determining the number of positions of nucleic acid base or identical amino acid residue that occurs in both sequences to produce the number of matched / matched positions by the total number of positions in the comparison window and multiplying the result by 100 to give the percentage.
- Optimal alignment of sequences for comparison can be conducted by computerized implementation of known algorithms (eg, GAP, BESTFIT, FASTA, and TFASTA present in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis. , or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. Sequences are typically compared using BlastN or BlastX with default parameters.
- Substantial identity of polynucleotide sequences means that a polynucleotide or gene comprises a sequence that has at least 75% sequence identity, preferably at least 80% and more preferably at least 90% sequence identity when compared to a sequence. of reference.
- two polypeptides or proteins are considered substantially identical if at least 40%, preferably at least 60%, more preferably 90% and more preferably 95% are identical sequences or have conservative substitutions.
- the sequences are preferably compared using GAP with default parameters.
- the INIBINA OF protein of the invention may be obtained by methods known in the art, for example by polymerase chain reaction (PCR). More particularly, a primer based on the amino acid sequence of the INIBINA OF protein of the invention, SEQ ID NO: 1 or SEQ ID NO: 3, was constructed.
- INIBINA OF antigen of the present invention may contain conservative substitutions, which permit the production of INIBINA OF protein analogs at sites other than the antigenic sequences shown in Figure 9, to preserve its antigenicity and therefore being suitable for the same uses.
- Table 1 shows examples of said substitutions:
- INIBINA OF protein fragments of the present invention that maintain the antigenicity of the integral protein as depicted in SEQ ID NO: 1 or SEQ ID NO: 3.
- the synthetic gene encoding said sequence was designed.
- the design of this synthetic gene is preferably performed using codons optimized for expression in an appropriate host, for example, a prokaryote expression system, preferably Escherichia coli. This technique is well known to those skilled in the art and requires no further description.
- a prokaryote expression system preferably Escherichia coli.
- This technique is well known to those skilled in the art and requires no further description.
- There are also several programs / software available on the market to design a synthetic gene For example, software such as GeneDesign 2.0, Vector may be employed and preferably GeneDesign (DNA2.0) software, which allows reverse translation from a sequence of polypeptides, all known in the art.
- the present invention also includes a heterologous fusion protein comprising the INIBINA OF protein of the invention and an appropriate carrier / carrier protein.
- the term "fused heterologous protein" as used herein is intended to define the set of two different proteins constituting a single molecule.
- the fused heterologous protein is expressed from an expression system comprising a fused gene containing a gene coding for the expression of the inhibin OF protein of the invention and a gene coding for the expression of the carrier protein.
- Carrier / carrier protein is not a critical aspect of the invention.
- the carrier protein coding gene may be selected from, but not limited to, coding genes for expression of the following proteins: Glutathione S-Transferase (GST), ubiquitin-related small modifier family (SUMO) proteins, Rrp42 protein Pyrococcus abyssi archaebacteria, maltose binding protein, bovine serum albumin, keyhole lympet hemocyanin, ovoalbumin, flagellin, serum albumin of any species, gamma globulin of any species, D- and / or L- amino acid polymers.
- GST Glutathione S-Transferase
- SUMO ubiquitin-related small modifier family
- Rrp42 protein Pyrococcus abyssi archaebacteria maltose binding protein
- bovine serum albumin keyhole lympet hemocyanin
- ovoalbumin flagellin
- serum albumin of any species gamma globulin of any species
- the most preferred carrier protein for obtaining the protein fused to the INIBINA OF antigen sequence of the invention is Pyrococcus abyssi archeobacterium Rrp42 (GenBank Accession No.: NP_126302.1) It is part of the exosomal complex that participates in the processing and maturation of cellular RNAs.
- the amino acid sequence of the Pyrococcus abyssi archeobacterium Rrp2 is as follows (see Cohen et al., 2003, "An integrated analysis of the genome of the hyperthermophilicarchaeon Pyrococcus abyssi". Mol Microbiol. 47 (6): 1495-512):
- the method for producing a fused heterologous protein of the present invention comprises the steps of inserting a fusion gene into a coding region of a plasmid, transforming the host cell with the plasmid, and expressing the fused heterologous protein at a time. from the host cell by methods well known in the art. More particularly, the method of producing the fused heterologous protein of the present invention comprises inserting the cDNA that is encoded for expression of the INIBINA OF of the invention, or a fragment thereof, or a conservatively modified variant thereof, into a vector containing the coding information for the production of a carrier protein, preferably the Pyrococcus abyssi archaebacterium protein Rrp42. After insertion of the vector into the expression system, the fused heterologous protein is expressed by the system.
- the pET-SUMO vector is a commercial pET vector derivative and contains multiple cloning sites just after the SUMO-protease cleavage site ( Figure 13B).
- the expression system for producing the antigenic proteins of the invention may be any commercially available system.
- An example is the Escherichia coli, BL21 (DE3) and Rosetta (DE3) expression system, available from Novagen (see Novagen Catalog 2009/2010, pages 121 and 123, available on the Web at www.merckmillichina.com/ promart / library / Novagen-pET-E-coli-expression-system.pdf).
- the heterologous protein of fusion of the invention simply, with high purity and without the need for further purification methods such as chromatographic methods.
- composition of the present invention may comprise, in addition to the antigen of the invention, adjuvants, preservatives, diluents, emulsifiers, stabilizers and other pharmacologically acceptable ingredients which are employed in immunogenic preparations for human and animal use.
- adjuvants include incomplete Freund's adjuvant, Freund's complete adjuvant, ⁇ - (1,4) linked polydispersed acetylated mannan, polyoxyethylene-polyoxypropylene copolymer adjuvants, modified lipid adjuvants, saponin-derived adjuvants, dextran sulfate , aluminum hydroxide and aluminum phosphate.
- composition of the present invention may be administered to an animal, including man, by means known in the art.
- the composition may be administered parenterally, for example, subcutaneously, intraperitoneally, or intramuscularly, orally or intranasally, and may be administered in one or more daily doses.
- the amount of the composition of the invention administered to an animal varies according to the animal species.
- the amount of antigen per dose reported in the literature ranges from 50 to 125 micrograms for whole inhibin (Medan MS et al (2004). Effects of reimmunization of heifers against hormone profiles and ovulation rate. Reproduction. 128 (4): 475-82), and from 1 mg to 5 mg for peptides based on the first 29 loader-fused inhibin amino acids (Takedomi T et al. (2005) Active immunization against inhibitor improves superovulatory response to exogenous FSH in cattle. J Reprod Dev. 51 (3): 341-6).
- the dose of the composition of the invention to be administered to the subject contains the INIBINA OF antigen of the invention, or a conservatively modified variant or immunogenic fragments thereof or heterologous fusion protein comprising the even, in an amount of less than 1.0 mg, preferably at most 0.5 mg, more preferably in the range 0.2 to 0.4 mg.
- One skilled in the art based on the teachings of the present invention may be able to determine by routine testing the amount of INIBINA OF protein, or a conservatively modified variant or immunogenic fragments thereof or a heterologous fusion protein comprising the same. , which will be required to develop an adequate immune response to said protein by the animal.
- amino acid sequence expressed by the synthetic gene of SEQ ID NO: 2 has the amino acid sequence shown in Figure 11 and corresponds to SEQ ID NO: 3.
- the INIBINA OF molecule of the present invention contains the two most immunogenic segments of inhibin alpha: one segment that corresponds to the most immunogenic sequence of the N-terminal 29-amino acid peptide, and another more localized immunogenic segment. near the C-terminal end, which end has been modified by amino acid substitutions to facilitate purification of the resulting protein / antigen, make it more soluble, and avoid interference of the carrier protein in the fusion protein embodiment of the invention.
- the antigen sequence of the invention is relatively short compared to the full inhibin sequence and amino acid substitutions minimize or even eliminate the possibility of cross-reacting with other TGF-beta family proteins.
- Figure 12 shows the low identity of the INIBINA OF sequence of the invention with the other TFG-beta family members present in Bos taurus.
- the antigenic sequence 1 (Leu Leu Gln Arg Pro Pro Glu Glu Pro
- Ala corresponds to amino acids 16-27 of mature alpha inhibin. Peptides containing the first 29 amino acids have already been used as antigen, conjugated or fused to carrier / carrier proteins.
- Antigenic sequence 2 (Gly Tyr Ser Phe Lys Tyr Glu Met) corresponds to amino acids 1 15-122 of inhibin alfa. This sequence has not been used alone as an antigen.
- Bos taurus alpha-inhibin As there are no experimentally obtained inhibin structures, a modeling of the structure of Bos taurus alpha-inhibin was performed. automatically through the Swiss Model server (http://swissmodel.expasy.org), using as a template the structure of human bone morphogenetic protein (BMP-3), a member of the TGF- ⁇ protein superfamily (PDB 2QCQ chain B).
- BMP-3 human bone morphogenetic protein
- PDB 2QCQ chain B a member of the TGF- ⁇ protein superfamily
- Figure 4 shows the model obtained.
- the N-terminal antigenic sequence 1 shows a free coif "helix structure, while the antigenic sequence 2 is located on a beta-sheet formed at the C-terminal inhibin.
- the first 29 amino acids of inhibin alfa containing antigenic sequence 1 are not present in other members of the TGF- ⁇ protein superfamily.
- the amino acid sequences of these proteins align with inhibin from amino acid 30.
- antigenic sequence 2 For antigenic sequence 2, a sequence comparison, as illustrated in Figure 7, was performed with non-redundant proteins deposited in several gene banks using the Basic Local Alignment Search Tool "protein BLAST" on the server. http://blast.ncbi.nlm.nih.gov/Blast.cgi. This antigenic sequence 2 aligned only with inhibins, with an "e-value” (an index that indicates the likelihood of alignment being random) less than 1 e-06 (7e-12 for Bos taurus inhibin). Alignments with other non-inhibited proteins showed an e-value greater than 5. Therefore, cross-reactivity with non-inhibin proteins is unlikely.
- protein BLAST Basic Local Alignment Search Tool
- Figure 12 shows the alignment of the modified sequence with the C-terminal end of some selected inhibins.
- antigenic sequence 2 Giy Tyr and Phe Lys Tyr Giu Met
- Antigenic sequence 1 Leu Leu Gin Arg Pro Giu Giu Pro Ala Wing H ⁇ S
- the naturally flexible antigenic sequence 1 was added at the C-terminal end of the modified sequence containing the antigenic sequence 2, thereby obtaining the sequence corresponding to the "inhibin OF" antigen [SEQ ID NO: 1 ]:
- expression of the recombinant protein was performed by fusion with the SUMO ("small ubiquitin-like modifier") protein, which belongs to the family of those used to increase expression of the protein inhibitor OF. invention and improve its solubility.
- SUMO small ubiquitin-like modifier
- the tertiary structure of the SUMO protein is recognized by SUMO protease which cleaves exactly between SUMO and the target protein, resulting in the native fused protein.
- Figure 13 shows the cloning strategy.
- the synthetic gene was cut from plasmid pBSK-lniOF ( Figure 13A) using the enzymes BamHI and Hind III and cloned into the corresponding sites of the pET-SUMO vector ( Figure 13B).
- the resulting plasmid was named pET-SUMO-lniOF ( Figure 13C).
- Successful cloning was verified by sequencing using the T7 reverse primer universal primer (5 'GCTAGTTATTGCTCAGCGG 3').
- Cells were harvested by centrifugation at 6000 rpm for 30 minutes and resuspended in lysis buffer (5 mM imidazole, 0.5 M NaCl, 50 mM Tris- HCI, pH 8.0; 0.1% Triton X-100; 5 mM 2-mercaptoethanol) and lysed in the high pressure homogenizer.
- lysis buffer 5 mM imidazole, 0.5 M NaCl, 50 mM Tris- HCI, pH 8.0; 0.1% Triton X-100; 5 mM 2-mercaptoethanol
- the lysed and homogenized cells were subjected to 0.2 ⁇ membrane filtration (millipore end-filtration system) at room temperature.
- the lysate was clarified on a column containing Ni 2+ activated Chelating Sepharose (GE health care) resin. After absorption, the resin was washed with 0.5M NaCl, 50mM Tris-HCl, pH 8.0 buffer containing 5mM imidazole; and then with the same buffer containing 40 mM imidazole.
- the recombinant protein was eluted with elution buffer (0.5 M imidazole, 0.1 M NaCl, 50 mM Tris-HCl, pH 8.0) by collecting fractions corresponding to the elution peak.
- the protein eluted from the affinity chromatographic column was subjected to molecular exclusion chromatography on the column loaded with Sephacryl S-100 HR resin (GE Healthcare) using 0.1 M NaCI buffer, 20 mM Tris-HCI, pH 8.0.
- the SDS-PAGE of Figure 14 shows only an approximately 20 kDa band expected for the fusion of 6xHis-SUMO protein (13.5 kDa) and OF inhibin (6.5 kDa). Soluble protein expression, as well as the stability of the purified protein during storage at 4 ° C at a concentration of 1 mg / ml, indicate success in antigen design with reference to solubility and stability.
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Abstract
The present invention relates to a modified inhibin alpha protein, to antigenic fragments of same and to a fusion protein comprising same, as well as to an antigenic composition comprising a modified inhibin alpha and/or an antigenic fragment of same and/or a fusion protein comprising modified inhibin alpha, to the method for producing the modified inhibin alpha protein, antigenic fragments of same and fusion protein comprising same, to the use of modified inhibin alpha, antigenic fragments of same or fusion protein comprising inhibin alpha, and to the use of the antigenic composition to increase ovulation in animals, in particular mammals and birds. The present invention is based on the modification of the mature sequence of the alpha subunit of inhibin from Bos taurus, in particular in the segment at the C-terminus which, besides having some amino acids modified, becomes the segment having the two strongest immunogenic regions.
Description
ANTIGENO DE INIBINA ALFA, GENE CODIFICANTE DE INIBINA ALFA, GENE CODIFICANTE DE PROTEÍNA DE FUSÃO, PROCESSO DE OBTENÇÃO DO ANTIGENO DE INIBINA ALFA, COMPOSIÇÃO ANTIGENICA, USO DO ANTIGENO DE INIBINA ALFA E USO DA COMPOSIÇÃO ANTIGENICA" ALPHA INHIBINE ANTIGEN, ALPHA INHIBINE CODING GENE, MERGER PROTEIN CODING GENE, ALPHA INHIBINE ANTIGEN PROCESS, ANTIGENIC COMPOSITION, ALPHA INIBINE ANTIGEN USE, AND ANGENIC COMPOSITION USE
Campo Técnico Technical Field
[01 ] A presente invenção se refere a uma proteína inibina alfa modificada, fragmentos antigênicos da mesma e proteína de fusão compreendendo a mesma, assim como a uma composição antigênica compreendendo inibina alfa modificada e/ou um fragmento antigênico da mesma e/ou uma proteína de fusão compreendendo inibina alfa modificada, ao processo de produção da proteína inibina alfa modificada, fragmentos antigênicos da mesma e proteína de fusão compreendendo a mesma, e ao uso da inibina alfa modificada, fragmentos antigênicos da mesma ou proteína de fusão compreendendo inibina alfa modificada no aumento da ovulação em animais, especialmente mamíferos e aves. [01] The present invention relates to a modified alpha inhibin protein, antigenic fragments thereof and fusion protein comprising it, as well as an antigenic composition comprising modified alpha inhibin and / or an antigenic fragment thereof and / or a protein. fusion protein comprising modified alpha inhibin, the process of producing the modified alpha inhibin protein, antigenic fragments thereof and fusion protein comprising same, and the use of modified alpha inhibin antigenic fragments thereof or fusion protein comprising modified alpha inhibin in the increased ovulation in animals, especially mammals and birds.
[02] A presente invenção é baseada na modificação da sequência madura da subunidade alfa da inibina de Bos taurus, especificamente no segmento da extremidade C-terminal que, além da modificação de alguns aminoácidos, passa a ter duas regiões imunogênicas nesse segmento, sendo a região mais imunogênica aquela que está localizada no extremo C-terminal. [02] The present invention is based on modification of the mature sequence of the Bos taurus inhibin alpha subunit, specifically in the C-terminal end segment which, in addition to the modification of some amino acids, now has two immunogenic regions in that segment, the latter being most immunogenic region is that which is located at the C-terminal end.
Referência à Listagem de Sequencia Sequence Listing Reference
[03] A Listagem de Sequência submetida juntamente com este documento é ora incorporada para referência complementar. A cópia eletrônica da Listagem Sequencial está gravada em disco compacto não regravável (CD) nos termos da Resolução N- 228/2009 do Instituto Nacional da Propriedade Industrial - INPI, em uma forma legível por computador no formato de texto (.txt). [03] The Sequence Listing submitted together with this document is hereby incorporated for further reference. The electronic copy of the Sequential Listing is recorded on a non-rewritable compact disc (CD) pursuant to Resolution N-228/2009 of the National Institute of Industrial Property - INPI, in a computer readable text (.txt) format.
Histórico da Invenção Invention History
[04] A inibina é uma proteína (glicoproteína) secretada pelas células granulosa (feminina) e Sertoli (masculina) em resposta ao FSH (hormônio estimulante de folículo) e sua principal ação é o controle da realimentação
negativa na secreção de FSH da glândula pituitária. Ela é encontrada em grandes quantidades no plasma seminal e fluido folicular. É uma proteína dimérica de grande complexidade, sendo que a forma "madura" da inibina tem um peso molecular de 32 kDa e consiste de uma cadeia alfa (aproximadamente 18 kDa) e uma cadeia beta (14 kDa), essas duas cadeias sendo ligadas por pontes dissulfeto. As subunidades sozinhas não possuem atividade biológica conhecida. Cada subunidade é originada a partir de um gene separado e é produzida como uma grande proteína precursora. Também é conhecido o fato de que cada subunidade tem múltiplos sítios de clivagem, de modo que subunidades de diferentes tamanhos são rotineiramente encontradas no fluido folicular. Estima-se que são possíveis várias combinações que dão origem a inúmeras formas diméricas diferentes de inibina. As que são encontradas frequentemente no fluido folicular bovino são as de aproximadamente 29, 34, 48, 58, 68, 77, 122 e >160 kDa. A família da inibina é adicionalmente complicada pela existência de dois genes separados da subunidade beta. Eles são denominados subunidade beta-A e subunidade beta-B. Enquanto as inibinas são dímeros formados pelas subunidades alfa e beta, os dímeros compostos pelas subunidades beta-beta formam outro hormônio, a activina. [04] Inhibin is a protein (glycoprotein) secreted by granulosa (female) and Sertoli (male) cells in response to FSH (follicle stimulating hormone) and its main action is to control feedback. negative in FSH secretion from the pituitary gland. It is found in large quantities in seminal plasma and follicular fluid. It is a highly complex dimeric protein, the "mature" form of inhibin having a molecular weight of 32 kDa and consisting of an alpha chain (approximately 18 kDa) and a beta chain (14 kDa), these two chains being linked by disulfide bridges. The subunits alone have no known biological activity. Each subunit originates from a separate gene and is produced as a large precursor protein. It is also known that each subunit has multiple cleavage sites, so subunits of different sizes are routinely found in follicular fluid. It is estimated that various combinations are possible which give rise to numerous different dimeric forms of inhibin. Those often found in bovine follicular fluid are approximately 29, 34, 48, 58, 68, 77, 122 and> 160 kDa. The inhibin family is further complicated by the existence of two separate beta subunit genes. They are called beta-A subunit and beta-B subunit. While inhibins are dimers formed by alpha and beta subunits, dimers composed of beta-beta subunits form another hormone, activin.
[05] Como a inibina atua sistematicamente para inibir a liberação de FSH, é apropriado dizer que uma redução na secreção de inibina pode aumentar as concentrações de FSH e, consequentemente, ter um potencial uso no aumento da fertilidade. [05] As inhibin acts systematically to inhibit FSH release, it is appropriate to say that a reduction in inhibin secretion may increase FSH concentrations and thus have potential use in increasing fertility.
[06] Portanto, inibinas e activinas são moléculas multifuncionais que inibem e ativam, respectivamente, a secreção do FSH produzido pela glândula pituitária. [06] Therefore, inhibins and activins are multifunctional molecules that inhibit and activate, respectively, the secretion of FSH produced by the pituitary gland.
[07] Já foi verificado que as inibinas e activinas estão envolvidas na regulação de inúmeras funções, tais como secreção de hormônio do hipotálamo e glândula pituitária, secreção de hormônio gonadais, desenvolvimento e maturação de células germinativas, diferenciação do eritróide, secreção de insulina, sobrevivência de células nervosas, desenvolvimento embriogênico axial ou crescimento de ossos, dependendo de
sua composição de subunidade. Acredita-se que as inibinas se opõem às funções da activina (ver informação disponível em www.uniprot.org). [07] Inhibins and activins have already been found to be involved in the regulation of numerous functions such as hypothalamus hormone secretion and pituitary gland, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, axial embryogenic development or bone growth, depending on its subunit composition. Inhibins are believed to oppose activin functions (see information available at www.uniprot.org).
[08] Além de seu papel essencial no controle seletivo de secreção de FSH, as inibinas são atualmente reconhecidas como reguladores parácrinos ovariano e testicular e têm múltiplos efeitos parácrinos na unidade útero-placenta, representando um marcador promissor para infertilidade masculina e feminina e doenças ginecológicas e de gestação. [08] In addition to their essential role in the selective control of FSH secretion, inhibins are currently recognized as ovarian and testicular paracrine regulators and have multiple paracrine effects on the utero-placenta unit, representing a promising marker for male and female infertility and gynecological diseases. and gestation.
[09] Em virtude de sua importância no desenvolvimento de compostos e composições para a profilaxia, tratamento e diagnóstico de doenças e condições médicas em seres humanos, assim como no aumento da fertilidade de animais mamíferos, incluindo o homem, e aves, muitas pesquisas vêm sendo realizadas sobre as inibinas e sua obtenção, não só por purificação a partir de extratos naturais, como através de métodos empregando técnicas de DNA recombinante. Because of their importance in the development of compounds and compositions for the prophylaxis, treatment and diagnosis of diseases and medical conditions in humans, as well as in the increase in fertility of mammalian animals, including humans, and birds, much research has come. being carried out on inhibins and their obtaining, not only by purification from natural extracts, but by methods employing recombinant DNA techniques.
[010] O documento WO8600078 descreve um método de purificação e caracterização da inibina e o uso da mesma como antígeno para a produção de anticorpos. Também é proposto um método de construção de sondas de oligonucleotídeo para subsequente clonagem e expressão dos genes de inibina, usando a técnica de DNA recombinante, a partir das sequências de aminoácidos das subunidades da proteína. Este desenvolvimento foi realizado em uma época em que o conhecimento da inibina e das técnicas de sequenciamento, caracterização e modificação de material genético não contava com os recursos que hoje estão disponíveis. Por essa razão, nesse documento não há nenhuma informação sobre as sequências de aminoácidos da proteína e nem das sequências de nucleotídeos codificantes da proteína integral ou de suas subunidades. WO8600078 describes a method of purification and characterization of inhibin and its use as an antigen for antibody production. Also proposed is a method of constructing oligonucleotide probes for subsequent cloning and expression of inhibin genes, using the recombinant DNA technique, from protein subunit amino acid sequences. This development was carried out at a time when knowledge of inhibin and the techniques of sequencing, characterization and modification of genetic material lacked the resources available today. For this reason, there is no information in this document about the amino acid sequences of the protein and the nucleotide sequences encoding the whole protein or its subunits.
[01 1 ] O documento US4864019 descreve um método para tratar um mamífero visando o controle da fertilidade. De acordo com esse método, um anticorpo é gerado em resposta ao uso de um peptídeo sintético, o qual inclui os seis resíduos da extremidade N-terminal de uma inibina alfa de mamífero, sendo que o anticorpo reage com a inibina e atua na atividade de supressão da
liberação de gonadotropina. O anticorpo resultante pode ser administrado a um mamífero em dosagem suficiente para neutralizar uma porção significativa do teor de inibina no soro sanguíneo periférico, bloqueando, assim, a atividade hormonal da inibina endógena. Nesse documento é descrito que a proteína de 32 kDa, isolada de fluido folicular suíno, é composta por duas cadeias, de 18 kDa e de 14 kDa, ligadas por pontes dissulfeto, cadeias essas que são ricas em resíduos Cys. [01 1] US4864019 describes a method of treating a mammal for fertility control. According to this method, an antibody is generated in response to the use of a synthetic peptide which includes the six N-terminal end residues of a mammalian alpha inhibin, wherein the antibody reacts with inhibin and acts on the activity of suppression of Gonadotropin release. The resulting antibody may be administered to a mammal in sufficient dosage to neutralize a significant portion of the inhibin content in peripheral blood serum, thereby blocking the hormonal activity of endogenous inhibin. It is disclosed herein that the 32 kDa protein isolated from porcine follicular fluid is composed of two disulfide bonded 18 kDa and 14 kDa chains, which chains are rich in Cys residues.
[012] O documento CA21581 16 descreve um método para induzir a superovulação em gado por meio de vacinação contra inibina, seguida de injeção de FSH exogenamente. A vacinação contra inibina é feita pelo uso de um antígeno obtido pela ligação de albumina a um polipeptídio sintetizado a partir da sequência de aminoácidos das posições 1 -26 (a partir da extremidade N-terminal) da subunidade alfa de inibina suína ou bovina, dita mistura de antígeno e adjuvante sendo injetada subcutaneamente no gado diversas vezes. CA21581 16 describes a method for inducing superovulation in cattle by inhibin vaccination, followed by exogenously injecting FSH. Inhibin vaccination is by the use of an antigen obtained by binding albumin to a polypeptide synthesized from the amino acid sequence of positions 1-26 (from the N-terminus) of the porcine or bovine inhibin alpha subunit, said antigen and adjuvant mixture being injected subcutaneously into cattle several times.
[013] O documento WO9522980 (PI9507426) descreve um método para acelerar o início da puberdade em aves, mais particularmente em ratitas e psitaciformes, pela administração de uma proteína heteróloga compreendendo inibina, ou um fragmento da mesma, e uma proteína portadora, sendo que a inibina ou fragmento da mesma compreende as sequências identificadas nesse documento como SEQ ID NO: 2 ou SEQ ID NO: 4 e a proteína portadora pode ser selecionada de proteína de ligação a maltose, albumina de soro bovino, ovoalbumina, flagelina, albumina de soro, etc. No documento WO9640219 (PI9609067) são descritas as composições empregadas no método descrito e reivindicado no WO9522980. WO9522980 (PI9507426) describes a method for accelerating the onset of puberty in birds, particularly ratites and parrots, by administering a heterologous protein comprising inhibin, or a fragment thereof, and a carrier protein, wherein the inhibin or fragment thereof comprises the sequences identified herein as SEQ ID NO: 2 or SEQ ID NO: 4 and the carrier protein may be selected from maltose binding protein, bovine serum albumin, ovoalbumin, flagellin, serum albumin. , etc. WO9640219 (PI9609067) describes the compositions employed in the method described and claimed in WO9522980.
[014] O documento PI0316084 descreve composições de peptídeo antigênico múltiplo compreendendo peptídeos imunogênicos (subunidade alfa de inibina madura, ou substituições conservativas das mesmas) ligados a uma cadeia principal de aminoácidos em conjunto com um veículo aceitável, tal como albumina de soro, proteína de ligação a maltose, hemocianina de keyhole limpet etc. Essas composições são empregadas na melhoria do desempenho de produção de animais, especialmente de aves.
[015] O documento PI0506043 descreve um processo para avaliação genética do desempenho produtivo de animais a partir de marcadores moleculares associados a genes da inibina, leptina, LH, FSH, entre outros, e seus receptores. PI0316084 describes multiple antigenic peptide compositions comprising immunogenic peptides (mature inhibin alpha subunit, or conservative substitutions thereof) linked to an amino acid backbone together with an acceptable carrier such as serum albumin, protein of binding to maltose, keyhole limpet hemocyanin etc. These compositions are employed in improving the production performance of animals, especially birds. [015] PI0506043 describes a process for genetic evaluation of productive performance of animals from molecular markers associated with inhibin, leptin, LH, FSH, among others genes, and their receptors.
[016] SCHANBACHER (SCHANBACHER B. Radioimmunoassay of inhibin: serum responses to unilateraland bilateral orchidectomy. Endocrinology 1988;123(5):2323-30) desenvolveu um radioimunoensaio para a determinação da concentração da inibina no soro e em outros fluidos biológicos, utilizando o antisoro do coelho para a cadeia alfa da inibina porcina. Este teste mostrou-se preciso, sensível e específico para a inibina. [016] SCHANBACHER (SCHANBACHER B. Radioimmunoassay of inhibin: serum responses to bilateral unilateral and orchidectomy. Endocrinology 1988; 123 (5): 2323-30) developed a radioimmunoassay for the determination of inhibin concentration in serum and other biological fluids using rabbit antiserum to porcine inhibin alpha chain. This test was accurate, sensitive and specific for inhibin.
[017] O'SHEA T, e colaboradores (O'SHEA T, BINDON BM, HILLARD MA, PIPER LR, FINDLAY JK, MIYAMOTO K. Increasein ovulation rate in Merino ewes after active immunization with inhibinpreparations obtained by immunoaffinity chromatography. Reprod Fértil Dev 1989;1 (4):347-55) verificaram um aumento de duas a quatro vezes na taxa de ovulação de ovelhas após a imunização com peptídeo sintético contendo a porção N- terminal da subunidade alfa da inibina porcina ovina ou bovina conjugada a uma proteína carreadora. [017] O'SHEA T, and collaborators (O'SHEA T, BINDON BM, HILLARD MA, PIPER LR, FINDLAY JK, MIYAMOTO K. Increasein ovulation rate in Merino ewes after active immunization with inhibitions prepared by immunoaffinity chromatography. Reprod Fertile Dev 1989; 1 (4): 347-55) found a two to four-fold increase in sheep ovulation rate following immunization with synthetic peptide containing the N-terminal portion of the protein-conjugated sheep or bovine porcine inhibin alpha subunit. carrier.
[018] Adicionalmente, TANNETTA D.S. e colaboradores (TANNETTA DS, FEIST SA, BLEACH EC, GROOME NP, EVANS LW, KNIGHT PG. Effects ofactive immunization of sheep against an amino terminal peptide of the inhibinalpha C subunit on intrafollicular leveis of activin A, inhibin A and follistatin. J Endocrinol 1998;157(1 ):157-68) verificaram que a imunização de ovelhas com um peptídeo sintético correspondente à porção amino terminal (aminoácidos 1 -29) da subunidade alfa madura da inibina bovina conjugada a tuberculina foi responsável pelo aumento de três vezes no número total de folículos maduros por ovelha comparada a um grupo controle. Additionally, TANNETTA D.S. and collaborators (TANNETTA DS, FEIST SA, BLEACH EC, GROOME NP, EVANS LW, KNIGHT PG. Effects of active immunization of sheep against an amino terminal peptide of the inhibinalpha C subunit on activin A, inhibin A and follistatin. J Endocrinol 1998; 157 (1): 157-68) found that immunization of sheep with a synthetic peptide corresponding to the amino terminal portion (amino acids 1-29) of the mature alpha subunit of tuberculin-conjugated bovine inhibin was responsible for a three-fold increase in total number of mature follicles per sheep compared to a control group.
[019] Recentemente, LI HAN, D.G. e colaboradores (LI HAN, D.G. MAO, D.K. ZHANG, A.X. LIANG, M. FANG, MUHAMMAD MOAEEN-UD-DIN, L.G. YANG, Development and evaluation of a novel DNA vaccine expressing inhibin α (1 - 32) fragment for improving the fertility in rats and sheep, Animal Reproduction
Science, 2008, 109, 1 -4, 251 -265) avaliaram a possibilidade de utilização de uma vacina de DNA contendo os aminoácidos 1 -32 da cadeia alfa da inibina como ferramenta para o aumento do desenvolvimento de folículo ovariano e tamanho da prole. [019] Recently, LI HAN, DG and collaborators (LI HAN, DG MAO, DK ZHANG, AX LIANG, M. FANG, MUHAMMAD MOAEEN-UD-DIN, LG YANG, Development and Evaluation of a novel DNA vaccine expressing inhibin α ( 1 - 32) fragment for improving fertility in rats and sheep, Animal Reproduction Science, 2008, 109, 1-4, 251-265) evaluated the possibility of using a DNA vaccine containing inhibin alpha chain amino acids 1-32 as a tool for increasing ovarian follicle development and offspring size.
[020] Mais recentemente ainda, PRASAD, S. e colaboradores (PRASAD, S. RASTOGI, S.K. GANGULY, B. Computational designing of a poly-epitope fecundity vaccine for multiple species of livestock. Vaccine. 2013: Artcle in Press) descreveram um método computacional para a predição de epítopos em um peptídeo candidato a antígeno vacinai. A vacina proposta contém um novo poli-epítopo que foi desenhado utilizando diferentes ferramentas computacionais e tem como alvo a inibina e a folistatina de diferentes animais de interesse veterinário. [020] More recently, PRASAD, S. and collaborators (PRASAD, S. RASTOGI, SK GANGULY, B. Computational design of a polypitope fecundity vaccine for multiple species of livestock. Vaccine. 2013: Artcle in Press) described a computational method for the prediction of epitopes in a vaccine antigen candidate peptide. The proposed vaccine contains a new polyepitope that has been designed using different computational tools and targets inhibin and folistatin from different animals of veterinary interest.
[021 ] O estado da técnica citado acima mostra a importância do aumento da imunogenicidade da inibina, particularmente da subunidade alfa da inibina, e de fragmentos ou proteínas de fusão contendo sequências imunogênicas da inibina alfa. Esse aumento de imunogenicidade tem aplicação importante tanto no aumento de produção de animais, incluindo mamíferos e aves, como no tratamento, profilaxia e diagnóstico de doenças e condições médicas em seres humanos. The above state of the art shows the importance of increased inhibin immunogenicity, particularly inhibin alpha subunit, and fragments or fusion proteins containing inhibin alpha immunogenic sequences. This increase in immunogenicity has important application both in increasing the production of animals, including mammals and birds, as well as in the treatment, prophylaxis and diagnosis of diseases and medical conditions in humans.
Sumário da Invenção Summary of the Invention
[022] A presente invenção revela novas sequências de inibina alfa contendo regiões mais imunogênicas por modificação da sequência madura da subunidade alfa da inibina de Bos taurus (obtida a partir da coleção do GenBank sob o No. de acesso AAI09838.1 , Inhibin alpha Bos taurus). [022] The present invention discloses novel inhibin alpha sequences containing more immunogenic regions by modification of the mature inhibin alpha subunit sequence of Bos taurus (obtained from the GenBank collection under accession No. AAI09838.1, Inhibin alpha Bos Taurus).
[023] De acordo com um primeiro aspecto da presente invenção, é provido um antígeno compreendendo uma sequência de aminoácidos de inibina alfa modificada, aqui denominada INIBINA OF, compreendendo a SEQ ID NO: 1 , antígeno esse que tem maior imunogenicidade quando comparada à da inibina de Bos taurus, No. de acesso AAI09838.1 no GenBank:
SEQ ID NO: 1 : According to a first aspect of the present invention, there is provided an antigen comprising a modified alpha inhibin amino acid sequence, herein called INIBINA OF, comprising SEQ ID NO: 1, which antigen has higher immunogenicity as compared to that of Bos taurus inhibin, GenBank Accession No. AAI09838.1: SEQ ID NO: 1:
Gly Ser Ala Pro Gly Thr Met Arg Pro Leu His Val Arg Thr Thr 1 5 10 15 Gly Ser Wing Pro Gly Thr Met Arg Pro Read His Val Arg Thr Thr 1 5 10 15
Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Met Asp Pro Asn Arg Be Asp Gly Gly Tyr Be Phe Lys Tyr Glu Met Asp Pro Asn Arg
20 25 30 20 25 30
Leu Thr Gln His Ser Ala Gly Leu Leu Gln Arg Pro Pro Glu Glu Leu Thr Gln His Ser Gly Wing Read Leu Gln Arg Pro Pro Glu Glu
35 40 45 35 40 45
Pro Ala Ala His Pro Wing His Wing
49 49
[024] De acordo com um segundo aspecto da invenção, são providas sequências de aminoácidos tendo substituições conservativas na sequência da INIBINA OF, assim como fragmentos imunogênicos da mesma compreendendo substituições conservativas. According to a second aspect of the invention, amino acid sequences are provided having conservative substitutions in the sequence of INIBINA OF, as well as immunogenic fragments thereof comprising conservative substitutions.
[025] De acordo com um terceiro aspecto da invenção, são providas proteínas heterólogas de fusão compreendendo a INIBINA OF, análogos, derivados ou fragmentos imunogênicos da mesma, incluindo substituições conservativas na SEQ ID NO:1 , e uma proteína portadora/carregadora farmacologicamente aceitável, preferencialmente a proteína portadora Rrp42. According to a third aspect of the invention, heterologous fusion proteins comprising INIBINA OF, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1, and a pharmacologically acceptable carrier / carrier protein are provided. preferably the carrier protein Rrp42.
[026] De acordo com um quarto aspecto da invenção, é provida uma sequência de nucleotídeos - SEQ ID NO:2 - compreendendo o gene codificante da sequência de aminoácidos da INIBINA OF da presente invenção que compreende a SEQ ID NO:1 . According to a fourth aspect of the invention, there is provided a nucleotide sequence - SEQ ID NO: 2 - comprising the INIBINA OF amino acid sequence coding gene of the present invention comprising SEQ ID NO: 1.
SEQ ID NO:2: SEQ ID NO: 2:
GGATCCCTCG AGGGTTCTGC TCCGGGCACC ATGCGTCCGC TGCACGTGCG 50 TACCACCTCC GATGGTGGCT ACTCTTTCAA ATACGAAATG GACCCGAACC 100 GCCTGACTCA GCATTCTGCT GGCCTGCTGC AGCGCCCGCC GGAAGAACCG 150 GCTGCGCACT AAGCTT 166 GGATCCCTCG AGGGTTCTGC TCCGGGCACC ATGCGTCCGC TGCACGTGCG 50 TACCACCTCC GATGGTGGCT ACTCTTTCAA ATACGAAATG GACCCGAACC 100 GCCTGACTCA GCATTCTGCT GGCCTGCTGC GACCGCGC
[027] Em um quinto aspecto, a presente invenção diz respeito a um antígeno compreendendo uma sequência de aminoácidos de inibina alfa modificada, aqui denominada INIBINA OF, e codificada pelo gene sintético, dito antígeno compreendendo a SEQ ID NO:3 tendo a mesma antigenicidade do antígeno que compreende a SEQ ID NO:1 .
SEQ ID NO:3: In a fifth aspect, the present invention relates to an antigen comprising a modified alpha inhibin amino acid sequence, herein called INIBINA OF, and encoded by the synthetic gene, said antigen comprising SEQ ID NO: 3 having the same antigenicity. of the antigen comprising SEQ ID NO: 1. SEQ ID NO: 3:
Gly Ser Leu Glu Gly Ser Ala Pro Gly Thr Met Arg Pro Leu His 1 5 10 15 Gly Seru Read Glu Gly Seru Pro Wing Gly Thr Met Arg Pro Leu His 1 5 10 15
Val Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Met Val Arg Thr Thr Be Asp Gly Gly Tyr Be Phe Lys Tyr Glu Met
20 25 30 20 25 30
Asp Pro Asn Arg Leu Thr Gln His Ser Ala Gly Leu Leu Gln Arg Asp Pro Asn Arg Read Thr Gln His Ser Wing Gly Read Leu Read Gln Arg
35 40 45 35 40 45
Pro Pro Glu Glu Pro Ala Ala His Pro Pro Glu Glu Pro Wing His Wing
50 50
[028] Em um sexto aspecto, a presente invenção diz respeito a uma composição antigênica compreendendo (i) o antígeno INIBINA OF, que compreende a SEQ ID NO:1 ou SEQ ID NO:3, seus análogos, derivados ou fragmentos imunogênicos da mesma, incluindo substituições conservativas na SEQ ID NO:1 ou SEQ ID NO:3, (ii) opcionalmente, aditivos ou substâncias potencializadoras da atividade imunogênica da INIBINA OF e (iii) um veículo farmacologicamente aceitável. In a sixth aspect, the present invention relates to an antigenic composition comprising (i) the INIBINA OF antigen, comprising SEQ ID NO: 1 or SEQ ID NO: 3, analogs, derivatives or immunogenic fragments thereof. including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, (ii) optionally additives or substances enhancing the immunogenic activity of INIBINA OF and (iii) a pharmacologically acceptable carrier.
[029] Em um sétimo aspecto, a presente invenção diz respeito a uma composição antigênica compreendendo (i) uma proteína heteróloga de fusão que compreende o antígeno INIBINA OF, análogos, derivados ou fragmentos imunogênicos da mesma, incluindo substituições conservativas na SEQ ID NO: 1 ou SEQ ID NO:3, e uma proteína portadora/carregadora farmacologicamente aceitável, preferencialmente a proteína portadora Rrp42; (ii) opcionalmente, aditivos ou substâncias potencializadoras da atividade antigênica da INIBINA OF e (iii) um veículo farmacologicamente aceitável. In a seventh aspect, the present invention relates to an antigenic composition comprising (i) a heterologous fusion protein comprising the INIBINA OF antigen, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, and a pharmacologically acceptable carrier / carrier protein, preferably the carrier protein Rrp42; (ii) optionally additives or substances enhancing the antigenic activity of INIBINA OF and (iii) a pharmacologically acceptable carrier.
[030] Também, de acordo com um oitavo aspecto, a invenção se refere ao uso (a) do antígeno INIBINA OF, análogos, derivados ou fragmentos antigênicos do mesmo, incluindo substituições conservativas na SEQ ID NO:1 ou SEQ ID NO:3, (b) de uma proteína heteróloga de fusão que compreende a sequência de aminoácidos da INIBINA OF, análogos, derivados ou fragmentos imunogênicos da mesma, incluindo substituições conservativas na SEQ ID NO:1 ou SEQ ID NO:3, e uma proteína portadora/carregadora farmacologicamente aceitável, preferencialmente a proteína portadora Rrp42
de Pyrococcus abyssi e (c) de uma composição antigênica compreendendo dita INIBINA OF, análogos, derivados ou fragmentos imunogênicos da mesma, incluindo substituições conservativas na SEQ ID NO:1 ou SEQ ID NO:3, ou dita proteína heteróloga de fusão de INIBINA OF, no tratamento ou profilaxia de condições médicas relacionadas com a infertilidade de animais mamíferos e aves e no aumento da produção de animais mamíferos ou aves. Also according to an eighth aspect, the invention relates to the use (a) of INIBINA OF antigen, analogs, derivatives or antigenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3 (b) a heterologous fusion protein comprising the INIBINA OF amino acid sequence, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, and a carrier protein / pharmacologically acceptable carrier, preferably the carrier protein Rrp42 Pyrococcus abyssi and (c) an antigenic composition comprising said INIBINA OF, analogs, derivatives or immunogenic fragments thereof, including conservative substitutions in SEQ ID NO: 1 or SEQ ID NO: 3, or said heterologous fusion protein of INIBINA OF , in the treatment or prophylaxis of medical conditions related to infertility in mammals and birds and in the increase in the production of mammals or birds.
Breve Descrição das Figuras Brief Description of the Figures
[031 ] A Figura 1 mostra a sequência de aminoácidos da subunidade alfa da inibina madura de Bos taurus (No. de acesso no Gen Bank AAI09838.1 , Inhibin alpha Bos taurus). [031] Figure 1 shows the amino acid sequence of the mature Bos taurus inhibin alpha subunit (Gen Bank Accession No. AAI09838.1, Inhibin alpha Bos taurus).
[032] A Figura 2 ilustra a análise de antigenicidade da inibina alfa de Bos taurus empregando o programa PROTEAN (Lasergene). [032] Figure 2 illustrates the antigenicity analysis of Bos taurus inhibin alfa using the PROTEAN (Lasergene) program.
[033] A Figura 3 apresenta a sequência de aminoácidos como analisada na Figura 2, destacando as duas sequências com bom índice antigênico (segundo algoritmos presentes no programa PROTEAN). [033] Figure 3 shows the amino acid sequence as analyzed in Figure 2, highlighting the two sequences with good antigenic index (according to algorithms present in the PROTEAN program).
[034] A Figura 4 representa o modelo estrutural da inibina alfa mostrando as regiões antigênicas 1 (verde) e 2 (laranja), sendo que a modelagem estrutural da inibina de Bos taurus foi realizada com o programa SwissModel PDB Viewer e o servidor SwissModel (http://swissmodel.expasv.org) usando como molde a estrutura do TGF-beta (PDB 2QCQ). [034] Figure 4 represents the alpha inhibin structural model showing antigenic regions 1 (green) and 2 (orange), and the structural modeling of Bos taurus inhibin was performed with the SwissModel PDB Viewer program and the SwissModel server ( http://swissmodel.expasv.org) using the TGF-beta framework (PDB 2QCQ) as a template.
[035] A Figura 5 apresenta a estrutura formada na extremidade C-terminal da inibina alfa de Bos taurus. Figure 5 shows the structure formed at the C-terminal end of Bos taurus inhibin alpha.
[036] A Figura 6 apresenta a sequência de aminoácidos original contendo a região da extremidade N-terminal (sublinhada) a ser mudada, a região antigênica em laranja mostrada na Figura 4 e os aminoácidos (sublinhados), após a região antigênica, a serem mudados. [036] Figure 6 shows the original amino acid sequence containing the N-terminal end region (underlined) to be changed, the orange antigenic region shown in Figure 4 and the amino acids (underlined) after the antigenic region to be changed. changed.
[037] A Figura 7 mostra a sequência de aminoácidos resultante de uma primeira modificação na extremidade N-terminal [037] Figure 7 shows the amino acid sequence resulting from a first modification at the N-terminal end.
[038] A Figura 8 apresenta a conformação do modelo estrutural da sequência de aminoácidos modificada na extremidade C-terminal da inibina alfa de Bos
taurus, de acordo com uma primeira modificação de acordo com a invenção, como mostrada na Figura 7. [038] Figure 8 shows the structural model conformation of the modified amino acid sequence at the C-terminal end of Bos inhibin alpha taurus according to a first modification according to the invention as shown in Figure 7.
[039] A Figura 9 mostra a sequência de aminoácidos da INIBINA OF da invenção resultante das modificações representadas na Figura 8 e incluindo, na extremidade C-terminal, a região antigênica representada em verde na Figura 3. [039] Figure 9 shows the INIBINA OF amino acid sequence of the invention resulting from the modifications shown in Figure 8 and including, at the C-terminal end, the antigenic region represented in green in Figure 3.
[040] A Figura 10 mostra a sequência de nucleotídeos do gene sintético da presente invenção, codificante da INIBINA OF da presente invenção. [040] Figure 10 shows the nucleotide sequence of the synthetic gene of the present invention encoding the inhibitor of the present invention.
[041 ] A Figura 1 1 mostra a sequência de aminoácidos, codificada pelo gene sintético da invenção, que compreende a sequência da INIBINA OF da invenção. [041] Figure 11 shows the amino acid sequence encoded by the synthetic gene of the invention comprising the INIBINA OF sequence of the invention.
[042] A Figura 12 apresenta o alinhamento da sequência da INIBINA OF com inibinas de organismos selecionados. Figure 12 shows the sequence alignment of INIBINA OF with inhibins of selected organisms.
[043] A Figura 13 apresenta o Esquema da clonagem do gene do antígeno "INIBINA OF" no vetor de expressão. [043] Figure 13 shows the cloning scheme of the "INIBINA OF" antigen gene in the expression vector.
[044] A Figura 14 apresenta a análise da proteína purificada por cromatrografia de afinidade e exclusão molecular. M.- Marcador de massa molecular. Figure 14 shows analysis of purified protein by affinity and molecular exclusion chromatography. M.- Molecular mass marker.
Descrição Detalhada da Invenção Detailed Description of the Invention
[045] A presente invenção baseia-se na obtenção de uma nova proteína, aqui denominada de INIBINA OF, produzida a partir da sequência de aminoácidos da proteína madura correspondente à subunidade alfa da inibina de Bos taurus (obtida do Gen Bank sob No. de acesso AAI09838.1 ), proteína essa contendo duas importantes regiões antigênicas que tornam a INIBINA OF útil no aumento da produção de animais mamíferos e aves e no tratamento de infertilidade em seres humanos, dentre outros empregos. [045] The present invention is based on obtaining a novel protein, herein called INIBINA OF, produced from the amino acid sequence of the mature protein corresponding to the Bos taurus inhibin alpha subunit (obtained from Gen Bank under No. access AAI09838.1), which contains two important antigenic regions that make INIBINA OF useful in increasing mammalian and poultry production and treating infertility in humans, among other uses.
[046] A dita sequência madura da subunidade alfa da inibina de Bos taurus (No. de acesso AAI09838.1 no Gen Bank) (ver Figura 1 ) é a seguinte:
Ser Thr Pro Pro Leu Pro Trp Pro Trp Ser Pro Ala Ala Leu ArgThe said mature sequence of the Bos taurus inhibin alpha subunit (Gen. Accession No. AAI09838.1) (see Figure 1) is as follows: Ser Thr Pro Pro Leu Pro Trp Pro Trp
1 5 10 151 5 10 15
Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asp Cys Leu Leu Gln Arg Pro Pro Glu Glu Pro Wing His Wing Asp Cys
20 25 30 20 25 30
His Arg Ala Ala Leu Asn lie Ser Phe Gln Glu Leu Gly Trp Asp His Arg Wing Wing Leu Asn lie Ser Phe Gln Glu Leu Gly Trp Asp
35 40 45 35 40 45
Arg Trp lie Val His Pro Pro Ser Phe lie Phe Tyr Tyr Cys His Arg Trp lie Val His Pro Be Phe lie Phe Tyr Tyr Cys His
50 55 60 50 55 60
Gly Gly Cys Gly Leu Ser Pro Pro Gln Asp Leu Pro Leu Pro Val Gly Gly Cys Gly Leu Ser Pro Pro Gln Asp Leu Pro Leu Pro Val
65 70 75 65 70 75
Pro Gly Val Pro Pro Thr Pro Val Gln Pro Leu Ser Leu Val Pro Pro Gly Val Pro Pro Thr Pro Val Gln Pro Read Be Read Valu Pro
80 85 90 80 85 90
Gly Ala Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Gly Wing Gln Pro Cys Cys Wing Wing Read Pro Gly Thr Met Arg Pro
95 100 105 95 100 105
Leu His Val Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Read His Val Arg Thr Thr Be Asp Gly Gly Tyr Be Phe Lys Tyr
110 115 120 110 115 120
Glu Met Val Pro Asn Leu Leu Thr Gln His Cys Ala Cys lie Glu Met Val Pro Asn Read Leu Read Gln His Cys Ala Cys lie
125 130 134 125 130 134
[047] A nova proteína foi desenhada para conter as regiões mais imunogênicas da subunidade alfa da Inibina. Para alcançar essa meta, a antigenicidade da sequência da Inibina foi analisada usando o programa PROTEAN da Lasergene. O resultado dessa análise está mostrado na Figura 2. As duas sequências com bom índice antigênico (segundo algoritmos presentes no programa PROTEAB), que estão ressaltados nos quadros 1 e 2 da Figura 2, correspondem aos fragmentos mostrados nas cores verde e laranja da Figura 3. [047] The new protein was designed to contain the most immunogenic regions of the Inhibin alpha subunit. To achieve this goal, Inhibin sequence antigenicity was analyzed using Lasergene's PROTEAN program. The result of this analysis is shown in Figure 2. The two sequences with good antigen index (according to the PROTEAB algorithms), which are highlighted in tables 1 and 2 of Figure 2, correspond to the fragments shown in green and orange of Figure 3. .
[048] A primeira sequência com bom índice de antigenicidade (Leu Leu Gln [048] The first sequence with good antigenicity index (Leu Leu Gln
Arg Pro Pro Glu Glu Pro Ala Ala His) Se localiza nos primeiros 29 aminoácidos da extremidade N-terminal da subunidade alfa da Inibina, sendo já conhecido seu emprego como antígeno, conjugado ou fusionado a proteínas portadoras/carregadoras. A segunda sequência com boa antigenicidade (Gly Tyr ser Phe Lys Tyr Glu Met) está mais perto da extremidade C-terminal
da subunidade alfa da Inibina e não é conhecido o seu emprego, isoladamente, como antígeno. Arg Pro Pro Glu Glu Pro Wing Ala His) It is located in the first 29 amino acids of the N-terminal end of the Inhibin alpha subunit, and its use as an antigen, conjugate or fusion to carrier / carrier proteins is well known. The second sequence with good antigenicity (Gly Tyr ser Phe Lys Tyr Glu Met) is closer to the C-terminal end. alpha subunit and is not known to be used alone as an antigen.
[049] Como mostrado na Figura 4, o modelo estrutural da Inibina alfa de Bos taurus, obtida usando o servidor SwissModel, apresenta a primeira sequência antigênica localizada numa região com estrutura flexível na extremidade N- terminal. Já a segunda sequência fica localizada numa folha-beta presente em uma estrutura formada na extremidade C-terminal da Inibina alfa. As shown in Figure 4, the Bos taurus Inhibin alpha structural model, obtained using the SwissModel server, presents the first antigenic sequence located in a flexible framework region at the N-terminal end. The second sequence is located in a beta-leaf present in a structure formed at the C-terminal end of Inhibin alfa.
[050] De acordo com a estratégia empregada na presente invenção, a estrutura formada na extremidade C-terminal da inibina alfa (ver Figuras 5 e 6), os aminoácidos 1 15-122 contendo a sequência 2 (Giy Tyr ser Phe Lys Tyr Giu Met), foi utilizada como o novo antígeno para reproduzir epítopos estruturais que possam estar presentes na sequência final escolhida. In accordance with the strategy employed in the present invention, the structure formed at the C-terminal end of inhibin alfa (see Figures 5 and 6), amino acids 15-122 containing sequence 2 (Giy Tyr ser Phe Lys Tyr Giu Met), was used as the new antigen to reproduce structural epitopes that may be present in the chosen final sequence.
[051 ] Como evidenciado na Figura 5, na base da estrutura escolhida, há quatro cisteínas (Cys) e uma "dobra" na estrutura que é mantida pela interação hidrofobica dos aminoácidos valina (Val) e Leucina (Leu). Estes aminoácidos estão localizados no meio de uma região hidrofobica da molécula da Inibina alfa, mais precisamente na fase que interage com o receptor. As shown in Figure 5, at the base of the chosen structure, there are four cysteines (Cys) and a "fold" in the structure that is maintained by the hydrophobic interaction of the amino acids valine (Val) and leucine (Leu). These amino acids are located in the middle of a hydrophobic region of the Inhibin alfa molecule, more precisely in the phase that interacts with the receptor.
[052] Na Figura 6 estão indicadas (aminoácidos sublinhados) as posições que foram modificadas para a obtenção da nova proteína da presente invenção, INIBINA OF. As modificações efetuadas na sequência representada na Figura 6 tiveram como objetivo: [052] In Figure 6 are indicated (underlined amino acids) the positions that have been modified to obtain the novel protein of the present invention, inhibit OF. The modifications made in the sequence represented in Figure 6 had as objective:
a. Facilitar a purificação da proteína recombinante: As duas cisteínas da extremidade C-terminal da sequência original (Figura 6) foram substituídas por Serina (S, homólogo estrutural da cisteína) e Glicina (G, para dar flexibilidade à sequência, no caso de se adicionar outra sequência nessa extremidade). As cisteínas podem formar pontes dissulfeto inespecíficos com outras moléculas e dificultar a purificação da proteína recombinante, por isso é conveniente evitar sua presença. b. Evitar a interferência da proteína portadora/carregadora na estrutura da INIBINA OF: Na extremidade N-terminal da sequência original (Figura The. Facilitate purification of recombinant protein: The two C-terminal cysteines of the original sequence (Figure 6) have been replaced by Serine (S, cysteine structural counterpart) and Glycine (G) to give sequence flexibility if added another sequence at that end). Cysteines can form nonspecific disulfide bridges with other molecules and hinder purification of the recombinant protein, so it is convenient to avoid its presence. B. Avoid carrier / carrier protein interference with INIBINA OF structure: At the N-terminal end of the original sequence (Figure
6), foi removida a Sequência "Pro Cys Cys Ala Ala Leu"
(aminoácidos 94-99) que contém duas cisteínas consecutivas. Assim esses aminoácidos foram substituídos pela sequência "Giy Ser Ai a" para dar flexibilidade à ligação, na concretização da invenção em que é empregada uma proteína/molécula carregadora. Os aminoácidos "Giy Ser Al a", na nova proteína fusionada, evitam a interferência da proteína carregadora na formação da estrutura tridimensional desejada, como representada na Figura 5. 6), the sequence "Pro Cys Cys Ala Ala Leu" has been removed (amino acids 94-99) containing two consecutive cysteines. Thus these amino acids were replaced by the sequence "Giy Ser Ai a" to give binding flexibility in the embodiment of the invention in which a protein / carrier molecule is employed. The amino acids "Giy Ser Al a" in the new fused protein prevent interference of the carrier protein in the formation of the desired three-dimensional structure, as shown in Figure 5.
c. Aumento da solubilidade da nova proteína INIBINA OF: Os aminoácidos valina (Val) e Leucina (Leu), mostrados na estrutura da Figura 5, foram substituídos pelo ácido Aspártico (D, Asp) e Arginina (R, Arg), respectivamente, que têm cargas opostas, podendo interagir para estabilizar a "dobra" na estrutura mostrada da Figura 5. Estas substituições possibilitam o aumento da solubilidade da proteína INIBINA OF da presente invenção e impedem uma possível interação com o receptor. Adicionalmente e vantajosamente obtém-se uma "quebra" da região hidrofóbica presente no segmento escolhido, melhorando, assim a solubilidade da proteína da invenção. ç. Increased solubility of the new protein INIBINA OF: The amino acids valine (Val) and leucine (Leu), shown in the structure of Figure 5, have been replaced by Aspartic acid (D, Asp) and Arginine (R, Arg), respectively. opposite charges, which may interact to stabilize the "fold" in the structure shown in Figure 5. These substitutions allow for increased solubility of the INIBINA OF protein of the present invention and prevent possible interaction with the receptor. Additionally and advantageously a "breakdown" of the hydrophobic region present in the chosen segment is obtained, thereby improving the solubility of the protein of the invention.
[053] A sequência de aminoácidos resultante dessas substituições e modificações está representada na Figura 7. [053] The amino acid sequence resulting from these substitutions and modifications is shown in Figure 7.
[054] A Figura 8 mostra o resultado da análise dessa nova sequência por dinâmica molecular, usando o programa NOC (http://noch.sourceforqe.net) que mostrou a estabilidade e manutenção da estrutura desejada durante a dinâmica, apesar de todas as modificações/substituições operadas. [054] Figure 8 shows the result of analysis of this new sequence by molecular dynamics using the NOC program (http://noch.sourceforqe.net) which showed the stability and maintenance of the desired structure during dynamics, despite all modifications / replacements performed.
[055] Finalmente, a sequência antigênica 1 (Leu Leu Gin Arg Pro Pro Giu Giu Pro Ala Ala HÍS, aminoácidos 16-27 da sequência da subunidade alfa da inibina de Bos taurus, No. de acesso AAI09838.1 no GenBank), que é naturalmente flexível, foi acrescentada na extremidade C-terminal da molécula representada na Figura 8, que já contém a sequência antigênica 2 (Giy Tyr ser Phe Lys Tyr Giu Met). A Figura 9 mostra a sequência de aminoácidos do antígeno da presente invenção, aqui denominado de INIBINA OF. A alfa- inibina pertence à família das proteínas "TGF-beta", e em Bos taurus há outras
proteínas homólogas à inibina como, por exemplo, TGF-beta 1 , TGF-beta 2, Morfogen, entre outras. Por esse motivo, a estratégia seguida na presente invenção foi a de usar apenas uma subsequência compreendida nos primeiros 29 aminoácidos da alfa-inibina, região essa que não está presente em nenhum outro membro da família das "TGF-beta". Assim a resposta imunológica gerada é específica contra a inibina. Finally, antigenic sequence 1 (Leu Leu Gin Arg Pro Giu Giu Pro Ala Ala HÍS, amino acids 16-27 of the Bos taurus inhibin alpha subunit sequence, GenBank Accession No. AAI09838.1), which is naturally flexible, was added at the C-terminal end of the molecule depicted in Figure 8, which already contains antigenic sequence 2 (Giy Tyr Phe Lys Tyr Giu Met). Figure 9 shows the amino acid sequence of the antigen of the present invention, referred to herein as INIBINA OF. Alpha-inhibin belongs to the family of proteins "TGF-beta", and in Bos taurus there are other inhibin homologous proteins such as TGF-beta 1, TGF-beta 2, Morfogen, among others. Therefore, the strategy followed in the present invention was to use only a subsequence comprised of the first 29 amino acids of alpha-inhibin, a region not present in any other member of the "TGF-beta" family. Thus the immune response generated is specific against inhibin.
[056] Em resumo, o antígeno "INIBINA OF" da presente invenção reúne as duas regiões mais imunogênicas da inibina alfa, regiões essas que agora se encontram dispostas numa molécula que conserva apenas uma região estrutural da proteína original. Efetivamente, o antígeno da presente invenção é diferente de todos os antígenos relacionados à inibina testados até o momento. In summary, the "INIBINA OF" antigen of the present invention brings together the two most immunogenic regions of inhibin alpha, which regions are now arranged in a molecule that retains only one structural region of the parent protein. Indeed, the antigen of the present invention is different from all inhibin-related antigens tested so far.
[057] A presente invenção também inclui as variantes da INIBINA OF da invenção conservativamente modificadas e tendo pelo menos 85% de identidade com qualquer uma das sequências de aminoácidos de SEQ ID NO:1 ou SEQ ID NO:3, e tendo substancialmente a mesma atividade imunogênica das mesmas. The present invention also includes conservatively modified INIBINA OF variants of the invention and having at least 85% identity to any of the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3, and having substantially the same. immunogenic activity of the same.
[058] É amplamente conhecido da técnica de bioquímica que certas substituições de aminoácidos podem ser feitas em sequências de proteínas sem afetar a função ou atividade da mesma. Em geral, substituições conservativas de aminoácidos ou substituições de aminoácidos similares podem ocorrer naturalmente ou intencionalmente sem que haja modificação da atividade biológica, por exemplo, imunogenicidade da proteína. Os aminoácidos similares podem ser aqueles com tamanho e/ou propriedades de carga similares. Por exemplo, aspartato e glutamato e isoleucina e valina são pares de aminoácidos similares. A similaridade entre pares de aminoácidos pode ser avaliada por inúmeras maneiras conhecidas do técnico no assunto. Por exemplo, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Suplemento 3, Capítulo 22, pp. 345-352, que é aqui incorporado a título de referência, fornece tabelas de frequência para substituições de aminoácidos que podem ser empregadas como uma medida de similaridade. As tabelas de frequência de Dayhoff et al. são baseadas em comparações de
sequências de aminoácidos para proteínas tendo a mesma função, a partir de uma variedade de diferentes fontes de evolução. [058] It is widely known in the biochemistry art that certain amino acid substitutions may be made in protein sequences without affecting its function or activity. In general, conservative amino acid substitutions or similar amino acid substitutions may occur naturally or intentionally without modification of biological activity, e.g. protein immunogenicity. Similar amino acids may be those with similar size and / or charge properties. For example, aspartate and glutamate and isoleucine and valine are similar amino acid pairs. Similarity between amino acid pairs can be assessed in a number of ways known to the skilled artisan. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, p. 345-352, which is incorporated herein by reference, provides frequency tables for amino acid substitutions that may be employed as a measure of similarity. The frequency tables of Dayhoff et al. are based on comparisons of amino acid sequences for proteins having the same function from a variety of different sources of evolution.
[059] A "percentagem de identidade de sequência" para polinucleotídeos e polipeptídios é determinada por comparação de duas sequências alinhadas de modo ótimo ao longo de uma janela de comparação, sendo que a porção da sequência de polinucleotídeos ou polipeptídios, na janela de comparação, pode compreender adições ou deleções quando comparadas a uma sequência de referência (que não compreende deleções ou adições) para o alinhamento ótimo das duas sequências. A percentagem é calculada pela determinação do número de posições da base de ácido nucléico ou resíduo aminoácido idêntico que ocorre em ambas as sequências para produzir o número de posições igualadas/coincidentes pelo número total de posições na janela de comparação e multiplicando o resultado por 100 para dar a percentagem. O alinhamento ótimo de sequências para comparação pode ser conduzido por implementação computadorizada de algoritmos conhecidos (por exemplo, GAP, BESTFIT, FASTA e TFASTA presentes no Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., ou BlastN e BlastX disponíveis a partir do National Center for Biotechnology Information), ou por inspeção. Sequências são tipicamente comparadas usando BlastN ou BlastX com parâmetros default. The "percent sequence identity" for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, with the portion of the polynucleotide or polypeptide sequence in the comparison window, may comprise additions or deletions when compared to a reference sequence (which does not comprise deletions or additions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions of nucleic acid base or identical amino acid residue that occurs in both sequences to produce the number of matched / matched positions by the total number of positions in the comparison window and multiplying the result by 100 to give the percentage. Optimal alignment of sequences for comparison can be conducted by computerized implementation of known algorithms (eg, GAP, BESTFIT, FASTA, and TFASTA present in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis. , or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. Sequences are typically compared using BlastN or BlastX with default parameters.
[060] Identidade substancial de sequências de polinucleotídeos significa que um polinucleotídeo ou gene compreende uma sequência que possui pelo menos 75% de identidade de sequência, preferivelmente pelo menos 80% e mais preferivelmente pelo menos 90% de identidade de sequência quando comparada a uma sequência de referência. Tipicamente, dois polipeptídios ou proteínas são considerados substancialmente idênticos se pelo menos 40%, preferivelmente pelo menos 60%, mais preferivelmente 90% e mais preferivelmente 95% são sequências idênticas ou têm substituições conservativas. As sequências são preferivelmente comparadas usando GAP com parâmetros default.
[061 ] A proteína INIBINA OF da invenção pode ser obtida por métodos conhecidos na técnica, por exemplo, por reação em cadeia da polimerase (PCR). Mais particularmente, foi construído um primer com base na sequência de aminoácidos da proteína INIBINA OF da invenção, SEQ ID NO:1 ou SEQ ID NO:3. Substantial identity of polynucleotide sequences means that a polynucleotide or gene comprises a sequence that has at least 75% sequence identity, preferably at least 80% and more preferably at least 90% sequence identity when compared to a sequence. of reference. Typically, two polypeptides or proteins are considered substantially identical if at least 40%, preferably at least 60%, more preferably 90% and more preferably 95% are identical sequences or have conservative substitutions. The sequences are preferably compared using GAP with default parameters. The INIBINA OF protein of the invention may be obtained by methods known in the art, for example by polymerase chain reaction (PCR). More particularly, a primer based on the amino acid sequence of the INIBINA OF protein of the invention, SEQ ID NO: 1 or SEQ ID NO: 3, was constructed.
[062] É importante observar que o antígeno INIBINA OF da presente invenção pode conter substituições conservativas, as quais permitem a obtenção de análogos da proteína INIBINA OF em sítios diferentes das sequências antigênicas mostradas na Figura 9, para preservar a sua antigenicidade e, portanto, sendo apropriados para os mesmos usos. A Tabela 1 mostra os exemplos de ditas substituições: [062] It is important to note that the INIBINA OF antigen of the present invention may contain conservative substitutions, which permit the production of INIBINA OF protein analogs at sites other than the antigenic sequences shown in Figure 9, to preserve its antigenicity and therefore being suitable for the same uses. Table 1 shows examples of said substitutions:
Tabela 1 : Exemplos de substituições conservativas Table 1: Examples of Conservative Substitutions
[063] Também estão incluídos na presente invenção os fragmentos da proteína INIBINA OF da presente invenção que mantêm a antigenicidade da proteína integral como representada na SEQ ID NO:1 ou SEQ ID NO:3. Also included in the present invention are the INIBINA OF protein fragments of the present invention that maintain the antigenicity of the integral protein as depicted in SEQ ID NO: 1 or SEQ ID NO: 3.
[064] Após ser obtida a sequência de aminoácidos desejada (SEQ ID NO:1 ou SEQ ID NO:3) foi realizado o desenho do gene sintético codificante de dita sequência. O desenho desse gene sintético preferencialmente é realizado empregando códons otimizados para expressão em um hospedeiro apropriado,
por exemplo, um sistema de expressão procarioto, preferencialmente Escherichia coli. Essa técnica é bem conhecida dos técnicos no assunto e não requer descrição mais detalhada. Também há vários programas/softwares disponíveis no mercado para realizar o desenho de um gene sintético. Por exemplo, podem ser empregados softwares tais como o GeneDesign 2.0, Vector sendo preferencialmente empregado o software GeneDesign (DNA2.0), que permite a tradução inversa a partir de uma sequência de polipeptídios, todos conhecidos no estado da técnica. After obtaining the desired amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 3) the synthetic gene encoding said sequence was designed. The design of this synthetic gene is preferably performed using codons optimized for expression in an appropriate host, for example, a prokaryote expression system, preferably Escherichia coli. This technique is well known to those skilled in the art and requires no further description. There are also several programs / software available on the market to design a synthetic gene. For example, software such as GeneDesign 2.0, Vector may be employed and preferably GeneDesign (DNA2.0) software, which allows reverse translation from a sequence of polypeptides, all known in the art.
[065] Para alcançar a otimização em Escherichia coli, na sequência de nucleotídeos projetada foram adicionados os sítios de corte para as enzimas de restrição BamHI (GGATCC), Xhol (CTCGAG) e Hindlll (AAGCTT) para realizar a clonagem do gene sintético em vetores de expressão. Também foi adicionando o códon "stop" (TAA) para terminar a tradução após o último aminoácido (H, His) da sequência "INIBINA OF". A sequência de nucleotídeos resultante corresponde à SEQ ID NO:2 e está representada na Figura 10. Deve ser observado que, em razão da degenerescência do código genético é possível haver modificação de nucleotídeos que codifiquem os mesmos aminoácidos da SEQ ID NO:1 ou SEQ ID NO:3, desde que seja preservada a otimização de expressão no sistema de expressão utilizado. [065] In order to achieve optimization in Escherichia coli, in the engineered nucleotide sequence the BamHI (GGATCC), Xhol (CTCGAG) and Hindlll (AAGCTT) restriction enzymes have been added to clone the synthetic gene into vectors. of expression. The stop codon (TAA) was also added to terminate translation after the last amino acid (H, His) of the sequence "INIBINA OF". The resulting nucleotide sequence corresponds to SEQ ID NO: 2 and is shown in Figure 10. It should be noted that, due to the degeneracy of the genetic code, nucleotides encoding the same amino acids as SEQ ID NO: 1 or SEQ may be modified. ID NO: 3, provided that expression optimization is preserved in the expression system used.
[066] A presente invenção também inclui uma proteína heteróloga de fusão compreendendo a proteína INIBINA OF da invenção e uma proteína portadora/carregadora apropriada. O termo "proteína heteróloga fusionada" como aqui empregado tem a intenção de definir o conjunto de duas proteínas diferentes constituindo uma só molécula. Por exemplo, uma proteína compreendendo a proteína INIBINA OF da invenção, ou um fragmento da mesma, ou uma variante conservativamente modificada da mesma, fundida com uma proteína carregadora. A proteína heteróloga fusionada é expressada a partir de um sistema de expressão compreendendo um gene fusionado que contém um gene codificante para a expressão da proteína INIBINA OF da invenção e um gene codificante para expressão da proteína carregadora.
[067] A proteína portadora/carregadora não é um aspecto crítico da invenção. Por exemplo, o gene codificante da proteína carregadora pode ser selecionado de, mas não limitado a, genes codificantes para expressão das seguintes proteínas: Glutationa S-Transferase (GST), proteínas da família de pequeno modificador relacionado à ubiquitina (SUMO), proteína Rrp42 da arqueobactéria Pyrococcus abyssi, proteína de ligação a maltose, albumina de soro bovino, hemocianina de keyhole lympet, ovoalbumina, flagelina, albumina de soro de qualquer espécie, gama globulina de qualquer espécie, polímeros de aminoácidos D- e/ou L-. O gene de proteína carregadora preferido é o gene de Rrp42 da arqueobactéria Pyrococcus abyssi. [066] The present invention also includes a heterologous fusion protein comprising the INIBINA OF protein of the invention and an appropriate carrier / carrier protein. The term "fused heterologous protein" as used herein is intended to define the set of two different proteins constituting a single molecule. For example, a protein comprising the INIBINA OF protein of the invention, or a fragment thereof, or a conservatively modified variant thereof, fused to a carrier protein. The fused heterologous protein is expressed from an expression system comprising a fused gene containing a gene coding for the expression of the inhibin OF protein of the invention and a gene coding for the expression of the carrier protein. [067] Carrier / carrier protein is not a critical aspect of the invention. For example, the carrier protein coding gene may be selected from, but not limited to, coding genes for expression of the following proteins: Glutathione S-Transferase (GST), ubiquitin-related small modifier family (SUMO) proteins, Rrp42 protein Pyrococcus abyssi archaebacteria, maltose binding protein, bovine serum albumin, keyhole lympet hemocyanin, ovoalbumin, flagellin, serum albumin of any species, gamma globulin of any species, D- and / or L- amino acid polymers. The preferred carrier protein gene is the Pyrococcus abyssi archaebacterium Rrp42 gene.
[068] Como mencionado acima, a proteína carregadora mais preferida para a obtenção da proteína fusionada com a sequência do antígeno INIBINA OF da invenção é a Rrp42 da arqueobactéria Pyrococcus abyssi (No. de acesso no GenBank: NP_126302.1 ), proteína esta que faz parte do complexo exossômico que participa no processamento e maturação dos RNAs celulares. A sequência de aminoácidos da Rrp2 da arqueobactéria Pyrococcus abyssi é a seguinte (ver Cohen et al., 2003, "An integrated analysis of the genome of the hyperthermophilicarchaeon Pyrococcus abyssi". Mol Microbiol. 47(6):1495-512): As mentioned above, the most preferred carrier protein for obtaining the protein fused to the INIBINA OF antigen sequence of the invention is Pyrococcus abyssi archeobacterium Rrp42 (GenBank Accession No.: NP_126302.1) It is part of the exosomal complex that participates in the processing and maturation of cellular RNAs. The amino acid sequence of the Pyrococcus abyssi archeobacterium Rrp2 is as follows (see Cohen et al., 2003, "An integrated analysis of the genome of the hyperthermophilicarchaeon Pyrococcus abyssi". Mol Microbiol. 47 (6): 1495-512):
MSDNE IVAGIMRDHI INLLKEGKRIDDRGFEDYRPIE IEVGVIEKAEGSALVKLGSTQVLVGIKTSLGEP FPDTPNMGVMTTNVELVPLASPTFEPGPPDERAIELARVIDRGIRESKALNLEKMVIVPGKIVRVVFIDV HVLDHDGNLMDAIGIAAIAALLNARVPKVRYNEETGEVETLDETEPLPVEKIPVPVTFAKIGNILVVDPS LDEELVMDGKITITTDETGHISAVQKSEGGAFKLEEVMYAVETAFKKAEEIRKLILEAVEKAKQ MSDNE IVAGIMRDHI INLLKEGKRIDDRGFEDYRPIE IEVGVIEKAEGSALVKLGSTQVLVGIKTSLGEP FPDTPNMGVMTTNVELVPLASPTFEPGPPDERAIELARVIDRGIRESKALNLEKMVIVPGKIVRVVFIDV HVLDHDGNLMDAIGIAAIAALLNARVPKVRYNEETGEVETLDETEPLPVEKIPVPVTFAKIGNILVVDPS LDEELVMDGKITITTDETGHISAVQKSEGGAFKLEEVMYAVETAFKKAEEIRKLILEAVEKAKQ
[069] Com base nesta sequência, foi desenhado um gene sintético codificante dessa proteína, mas empregando códons otimizados para expressão melhorada em Escherichia coli, utilizando o software GeneDesign/DNA2.0. Nesta nova sequência de nucleotídeos foram adicionados os sítios de corte para as enzimas de restrição Xhol (CTCGAG) visando realizar a clonagem para fusionar outras proteínas no extremo C-terminal da Rrp42. O novo gene sintético codificante da proteína Rrp2 tem a seguinte sequência de nucleotídeos:
SEQ ID NO:4: Based on this sequence, a synthetic gene encoding this protein was designed, but employing codons optimized for enhanced expression in Escherichia coli using the GeneDesign / DNA2.0 software. In this new nucleotide sequence were added the cutoff sites for restriction enzymes XhoI (CTCGAG) to clone to fuse other proteins at the C-terminal end of Rrp42. The new synthetic gene encoding the Rrp2 protein has the following nucleotide sequence: SEQ ID NO: 4:
ATGTCCGACA ACGAAATCGT TGCTGGTATC ATGCGTGACC ATATCATCAA 50 ATGTCCGACA ACGAAATCGT TGCTGGTATC ATGCGTGACC ATATCATCAA 50
CCTGCTGAAA GAAGGTAAAC GTATTGACGA CCGCGGCTTT GAAGACTACC 120CCTGCTGAAA GAAGGTAAAC GTATTGACGA CCGCGGCTTT GAAGACTACC 120
GTCCGATTGA AATTGAAGTG GGTGTTATTG AAAAAGCTGA AGGCTCTGCG 100GTCCGATTGA AATTGAAGTG GGTGTTATTG AAAAAGCTGA AGGCTCTGCG 100
CTGGTGAAAC TGGGTTCTAC TCAGGTTCTG GTTGGCATCA AAACTTCTCT 150CTGGTGAAAC TGGGTTCTAC TCAGGTTCTG GTTGGCATCA AAACTTCTCT 150
GGGCGAACCG TTCCCGGACA CCCCGAACAT GGGTGTTATG ACCACCAACG 250GGGCGAACCG TTCCCGGACA TRANSGAACAT GGGTGTTATG ACCACCAACG 250
TTGAACTGGT TCCGCTGGCT TCTCCGACCT TCGAACCGGG TCCGCCGGAT 300TTGAACTGGT TCCGCTGGCT TCTCCGACCT TCGAACCGGG TCCGCCGGAT 300
GAACGCGCTA TCGAACTGGC GCGTGTGATC GATCGTGGTA TTCGCGAATC 350GAACGCGCTA TCGAACTGGC GCGTGTGATC GATCGTGGTA TTCGCGAATC 350
TAAAGCGCTG AACCTGGAAA AAATGGTTAT CGTTCCGGGT AAAATCGTTC 400TAAAGCGCTG AACCTGGAAA AAATGGTTAT CGTTCCGGGT AAAATCGTTC 400
GTGTTGTTTT CATCGACGTT CATGTTCTGG ACCACGACGG CAACCTGATG 450GTGTTGTTTT CATCGACGTT CATGTTCTGG ACCACGACGG CAACCTGATG 450
GACGCTATCG GCATCGCTGC TATTGCGGCT CTGCTGAACG CTCGTGTTCC 500GACGCTATCG GCATCGCTGC TATTGCGGCT CTGCTGAACG CTCGTGTTCC 500
GAAAGTTCGT TACAACGAAG AAACCGGCGA AGTTGAAACC CTGGACGAAA 550GAAAGTTCGT TACAACGAAG AAACCGGCGA AGTTGAAACC CTGGACGAAA 550
CTGAACCGCT GCCGGTTGAA AAAATTCCGG TTCCGGTTAC CTTCGCGAAA 600CTGAACCGCT GCCGGTTGAA AAAATTCCGG TTCCGGTTAC CTTCGCGAAA 600
ATTGGCAACA TTCTGGTTGT GGACCCGTCC CTGGACGAAG AACTGGTTAT 650ATTGGCAACA TTCTGGTTGT GGACCCGTCC CTGGACGAAG AACTGGTTAT 650
GGACGGCAAA ATTACTATTA CTACCGACGA AACCGGTCAC ATTTCCGCTG 700GGACGGCAAA ATTACTATTA CTACCGACGA AACCGGTCAC ATTTCCGCTG 700
TTCAGAAATC CGAAGGCGGT GCTTTCAAAC TGGAAGAAGT TATGTACGCG 750TTCAGAAATC CGAAGGCGGT GCTTTCAAAC TGGAAGAAGT TATGTACGCG 750
GTTGAAACCG CTTTTAAAAA AGCGGAAGAA ATCCGTAAAC TGATTCTGGA 800GTTGAAACCG CTTTTAAAAA AGCGGAAGAA ATCCGTAAAC TGATTCTGGA 800
AGCGGTTGAA AAAGCGAAAC AGCTCGAG 828 AGCGGTTGAA AAAGCGAAAC AGCTCGAG 828
[070] Muitos métodos de isolamento de genes e de produção de produtos de genes de fusão são conhecidos do estado da técnica. Pode ser citado, por exemplo, o método descrito em Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 2- Ed., Cold Spring Harbor Laboratory Press, 1989, Vols I, II e III. Também muitos kits de vetor comercialmente disponíveis, tal como o kit pMAL™-c podem ser usados para preparar o produto de gene de fusão da presente invenção. [070] Many methods of gene isolation and production of fusion gene products are known from the state of the art. For example, the method described in Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 2- Ed., Cold Spring Harbor Laboratory Press, 1989, Vol. I, II and III, may be cited. Also many commercially available vector kits, such as the pMAL ™ -c kit can be used to prepare the fusion gene product of the present invention.
[071 ] Resumidamente, o método para produzir uma proteína heteróloga fusionada da presente invenção compreende as etapas de inserir um gene de fusão em uma região codificante de um plasmídeo, transformação da célula hospedeira com o plasmídeo e expressão da proteína heteróloga fusionada a
partir da célula hospedeira por métodos bem conhecidos no estado da técnica. Mais particularmente, o método de produção da proteína heterologa fusionada da presente invenção compreende a inserção do cDNA que é codificado para expressão da INIBINA OF da invenção, ou um fragmento da mesma, ou uma variante conservativamente modificada da mesma, em um vetor que contém a informação codificante para a produção de uma proteína carregadora, preferencialmente a proteína Rrp42 da arqueobactéria Pyrococcus abyssi. Após a inserção do vetor no sistema de expressão, a proteína heterologa fusionada é expressada pelo sistema. Briefly, the method for producing a fused heterologous protein of the present invention comprises the steps of inserting a fusion gene into a coding region of a plasmid, transforming the host cell with the plasmid, and expressing the fused heterologous protein at a time. from the host cell by methods well known in the art. More particularly, the method of producing the fused heterologous protein of the present invention comprises inserting the cDNA that is encoded for expression of the INIBINA OF of the invention, or a fragment thereof, or a conservatively modified variant thereof, into a vector containing the coding information for the production of a carrier protein, preferably the Pyrococcus abyssi archaebacterium protein Rrp42. After insertion of the vector into the expression system, the fused heterologous protein is expressed by the system.
[072] A obtenção tanto da sequência de nucleotídeos do gene codificante da proteína INIBINA OF da invenção como da proteína carregadora permite construir o vetor de expressão para produção do antígeno e da proteína heterologa antigênica de fusão da invenção. Inúmeros vetores de expressão estão comercialmente disponíveis para a construção do gene codificante das proteínas antigênicas da invenção, tal como o vetor comercial pRSETA da Invitrogen (Life Technologies). O especialista no assunto será capaz de, a partir dos ensinamentos da presente invenção e dos sistemas de expressão comercialmente disponíveis, selecionar o vetor apropriado para emprego na produção da INIBINA OF e da proteína de fusão contendo a mesma. Em particular, foi empregado o vetor não comercial pET-SUMO. O vetor pET- SUMO é um derivado de vetor comercial pET e contém sítios múltiplos de clonagem logo após o sitio de clivagem da SUMO-protease (Figura 13B). Obtaining both the nucleotide sequence of the gene encoding the INIBINA OF protein of the invention and the carrier protein allows the expression vector for the production of the antigen and the fusion heterologous antigenic protein of the invention to be constructed. Numerous expression vectors are commercially available for constructing the gene coding for the antigenic proteins of the invention, such as Invitrogen's commercial vector pRSETA (Life Technologies). One skilled in the art will be able, from the teachings of the present invention and commercially available expression systems, to select the appropriate vector for use in the production of INIBINA OF and the fusion protein containing it. In particular, the non-commercial vector pET-SUMO was employed. The pET-SUMO vector is a commercial pET vector derivative and contains multiple cloning sites just after the SUMO-protease cleavage site (Figure 13B).
[073] O sistema de expressão para a produção das proteínas antigênicas da invenção pode ser qualquer sistema disponível comercialmente. Um exemplo é o sistema de expressão Escherichia coli, cepas BL21 (DE3) e Rosetta (DE3), disponível a partir da empresa Novagen (ver Novagen Catalog 2009/2010, páginas 121 e 123, disponível na Web em www.merckmillichina.com/promart/library/Novagen-pET-E-coli-expression- system.pdf). Com o emprego deste sistema de expressão preferido e do vetor de expressão pET-SUMO e com a proteína carregadora Rrp42 obtida a partir do gene modificado de SEQ ID NO:4 é possível obter a proteína heterologa de
fusão da invenção de modo simples, com elevado nível de pureza e sem a necessidade de métodos de purificação adicional, tal como os métodos cromatográficos. [073] The expression system for producing the antigenic proteins of the invention may be any commercially available system. An example is the Escherichia coli, BL21 (DE3) and Rosetta (DE3) expression system, available from Novagen (see Novagen Catalog 2009/2010, pages 121 and 123, available on the Web at www.merckmillichina.com/ promart / library / Novagen-pET-E-coli-expression-system.pdf). By employing this preferred expression system and the pET-SUMO expression vector and with the Rrp42 loading protein obtained from the modified SEQ ID NO: 4 gene, the heterologous protein of fusion of the invention simply, with high purity and without the need for further purification methods such as chromatographic methods.
[074] A composição da presente invenção pode compreender, além do antígeno da invenção, adjuvantes, preservativos, diluentes, emulsificantes, estabilizantes e outros ingredientes farmacologicamente aceitáveis e que são empregados em preparações imunogênicas para uso humano e animal. Exemplos não limitantes de adjuvantes incluem o adjuvante incompleto de Freund, o adjuvante completo de Freund, manana acetilada polidispersa β-(1 ,4) ligada, adjuvantes de copolímero de polioxietileno-polioxipropileno, adjuvantes lipídicos modificados, adjuvantes derivados de saponina, sulfato de dextrana, hidróxido de alumínio e fosfato de alumínio. [074] The composition of the present invention may comprise, in addition to the antigen of the invention, adjuvants, preservatives, diluents, emulsifiers, stabilizers and other pharmacologically acceptable ingredients which are employed in immunogenic preparations for human and animal use. Non-limiting examples of adjuvants include incomplete Freund's adjuvant, Freund's complete adjuvant, β- (1,4) linked polydispersed acetylated mannan, polyoxyethylene-polyoxypropylene copolymer adjuvants, modified lipid adjuvants, saponin-derived adjuvants, dextran sulfate , aluminum hydroxide and aluminum phosphate.
[075] A composição da presente invenção pode ser administrada a um animal, incluindo o homem, por meios conhecidos no estado da técnica. Por exemplo, a composição pode ser administrada por via parenteral, por exemplo, subcutânea, intraperitoneal, ou intramuscular, ou por via oral, ou intranasal e pode ser administrada em uma ou mais doses diárias. [075] The composition of the present invention may be administered to an animal, including man, by means known in the art. For example, the composition may be administered parenterally, for example, subcutaneously, intraperitoneally, or intramuscularly, orally or intranasally, and may be administered in one or more daily doses.
[076] A quantidade da composição da invenção administrada a um animal varia de acordo com a espécie do animal. Para bovinos, a quantidade de antígeno por dose relatada na literatura varia de 50 a 125 microgramas, para a inibina inteira (Medan MS et al (2004) Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate. Reproduction. 128(4):475-82), e de 1 mg até 5 mg para peptídeos baseados nos primeiros 29 aminoácidos da inibina fusionados a carregadores (Takedomi T et al. (2005) Active immunization against inhibin improves superovulatory response to exogenous FSH in cattle. J Reprod Dev. 51 (3):341 -6).Dado que a invenção contém duas regiões antigênicas é esperado que seja mais imunogênica que os peptídeos baseados nos primeiros 29 aminoácidos. A dose da composição da invenção a ser administrada ao indivíduo contém o antígeno INIBINA OF da invenção, ou uma variante conservativamente modificada ou fragmentos imunogênicos do mesmo ou proteína heteróloga de fusão compreendendo o
mesmo, em uma quantidade menor que 1 ,0 mg, preferivelmente no máximo 0,5 mg, mais preferivelmente ainda na faixa de 0,2 até 0,4 mg. O especialista na técnica, com base nos ensinamentos da presente invenção pode ser capaz de determinar, por meio de testes rotineiros, a quantidade de proteína INIBINA OF, ou uma variante conservativamente modificada ou fragmentos imunogênicos da mesma ou uma proteína heteróloga de fusão compreendendo a mesma, que será necessária para desenvolver uma resposta imunológica adequada à dita proteína pelo animal. [076] The amount of the composition of the invention administered to an animal varies according to the animal species. For cattle, the amount of antigen per dose reported in the literature ranges from 50 to 125 micrograms for whole inhibin (Medan MS et al (2004). Effects of reimmunization of heifers against hormone profiles and ovulation rate. Reproduction. 128 (4): 475-82), and from 1 mg to 5 mg for peptides based on the first 29 loader-fused inhibin amino acids (Takedomi T et al. (2005) Active immunization against inhibitor improves superovulatory response to exogenous FSH in cattle. J Reprod Dev. 51 (3): 341-6). Since the invention contains two antigenic regions it is expected to be more immunogenic than peptides based on the first 29 amino acids. The dose of the composition of the invention to be administered to the subject contains the INIBINA OF antigen of the invention, or a conservatively modified variant or immunogenic fragments thereof or heterologous fusion protein comprising the even, in an amount of less than 1.0 mg, preferably at most 0.5 mg, more preferably in the range 0.2 to 0.4 mg. One skilled in the art, based on the teachings of the present invention may be able to determine by routine testing the amount of INIBINA OF protein, or a conservatively modified variant or immunogenic fragments thereof or a heterologous fusion protein comprising the same. , which will be required to develop an adequate immune response to said protein by the animal.
[077] A sequência de aminoácidos expressada pelo gene sintético da SEQ ID NO:2 tem a sequência de aminoácidos representada na Figura 1 1 e corresponde à SEQ ID NO:3. The amino acid sequence expressed by the synthetic gene of SEQ ID NO: 2 has the amino acid sequence shown in Figure 11 and corresponds to SEQ ID NO: 3.
[078] É importante reafirmar que a molécula da INIBINA OF da presente invenção contém os dois segmentos mais imunogênicos da inibina alfa: um segmento que corresponde à sequência mais imunogênica do peptídeo de 29 aminoácidos da extremidade N-terminal, e outro segmento imunogênico localizado mais próximo da extremidade C-terminal, extremidade esta que foi modificada por substituições de aminoácidos para facilitar a purificação da proteína/antígeno resultante, torná-lo mais solúvel e evitar a interferência da proteína carregadora na concretização da invenção correspondente à proteína de fusão. A sequência do antígeno da invenção é relativamente curta, se comparada à sequência completa da inibina e as substituições de aminoácidos reduzem ao máximo ou até mesmo eliminam a possibilidade de reação cruzada com outras proteínas da família das TGF-beta. A Figura 12 mostra a baixa identidade da sequência da INIBINA OF da invenção com os outros membros da família TFG-beta presentes em Bos taurus. It is important to reaffirm that the INIBINA OF molecule of the present invention contains the two most immunogenic segments of inhibin alpha: one segment that corresponds to the most immunogenic sequence of the N-terminal 29-amino acid peptide, and another more localized immunogenic segment. near the C-terminal end, which end has been modified by amino acid substitutions to facilitate purification of the resulting protein / antigen, make it more soluble, and avoid interference of the carrier protein in the fusion protein embodiment of the invention. The antigen sequence of the invention is relatively short compared to the full inhibin sequence and amino acid substitutions minimize or even eliminate the possibility of cross-reacting with other TGF-beta family proteins. Figure 12 shows the low identity of the INIBINA OF sequence of the invention with the other TFG-beta family members present in Bos taurus.
EXEMPLOS EXAMPLES
Exemplo 1 : Desenho do antígeno Inibina OF Example 1: Inhibin OF Antigen Design
[079] A sequência de 134 aminoácidos, correspondente à subunidade alfa madura da inibina de Bos taurus, foi deduzida a partir da sequencia do Gen Bank No AAI09838.1 :
Ser Thr Pro Pro Leu Pro Trp Pro Trp Ser Pro Ala Ala Leu ArgThe 134 amino acid sequence, corresponding to the mature alpha subunit of Bos taurus inhibin, was deduced from Gen Bank sequence No AAI09838.1: Ser Thr Pro Pro Leu Pro Trp Pro Trp
1 5 10 151 5 10 15
Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asp Cys Leu Leu Gln Arg Pro Pro Glu Glu Pro Wing His Wing Asp Cys
20 25 30 20 25 30
His Arg Ala Ala Leu Asn lie Ser Phe Gln Glu Leu Gly Trp Asp His Arg Wing Wing Leu Asn lie Ser Phe Gln Glu Leu Gly Trp Asp
35 40 45 35 40 45
Arg Trp lie Val His Pro Pro Ser Phe lie Phe Tyr Tyr Cys His Arg Trp lie Val His Pro Be Phe lie Phe Tyr Tyr Cys His
50 55 60 50 55 60
Gly Gly Cys Gly Leu Ser Pro Pro Gln Asp Leu Pro Leu Pro Val Gly Gly Cys Gly Leu Ser Pro Pro Gln Asp Leu Pro Leu Pro Val
65 70 75 65 70 75
Pro Gly Val Pro Pro Thr Pro Val Gln Pro Leu Ser Leu Val Pro Pro Gly Val Pro Pro Thr Pro Val Gln Pro Read Be Read Valu Pro
80 85 90 80 85 90
Gly Ala Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Gly Wing Gln Pro Cys Cys Wing Wing Read Pro Gly Thr Met Arg Pro
95 100 105 95 100 105
Leu His Val Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Read His Val Arg Thr Thr Be Asp Gly Gly Tyr Be Phe Lys Tyr
110 115 120 110 115 120
Glu Met Val Pro Asn Leu Leu Thr Gln His Cys Ala Cys lie Glu Met Val Pro Asn Read Leu Read Gln His Cys Ala Cys lie
125 130 134 125 130 134
[080] Esta sequência foi analisada usando o software Lasergene Protean (DNAstar) para identificar peptídeos com alto índice de antigenicidade pelo algoritmo de Jameson & Wolf [ver Jameson B.A., Wolf H. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput AppI Biosci. 4(1 ):181 -6]. A Figura 2 mostra o resultado gráfico desta análise, indicando com os números 1 e 2 os picos com maior antigenicidade [080] This sequence was analyzed using Lasergene Protean (DNAstar) software to identify high antigenicity peptides by the Jameson & Wolf algorithm [see Jameson BA, Wolf H. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. . Comput AppI Biosci. 4 (1): 181-6]. Figure 2 shows the graphical result of this analysis, indicating with the numbers 1 and 2 the peaks with greater antigenicity.
[081 ] A sequência antigênica 1 (Leu Leu Gln Arg Pro Pro Glu Glu Pro [081] The antigenic sequence 1 (Leu Leu Gln Arg Pro Pro Glu Glu Pro
Ala Ala HÍS) corresponde aos aminoácidos 16-27 da alfa-inibina madura. Peptídeos contendo os primeiros 29 aminoácidos já foram usados como antígeno, conjugado ou fusionado a proteínas carregadoras/portadoras. Ala (H1A) corresponds to amino acids 16-27 of mature alpha inhibin. Peptides containing the first 29 amino acids have already been used as antigen, conjugated or fused to carrier / carrier proteins.
[082] A sequência antigênica 2 (Gly Tyr Ser Phe Lys Tyr Glu Met) corresponde aos aminoácidos 1 15-122 da inibina alfa. Esta sequência ainda não foi usada, isoladamente, como antígeno. [082] Antigenic sequence 2 (Gly Tyr Ser Phe Lys Tyr Glu Met) corresponds to amino acids 1 15-122 of inhibin alfa. This sequence has not been used alone as an antigen.
[083] Como não se tem estruturas de inibinas obtidas experimentalmente, foi realizada uma modelagem da estrutura da alfa-inibina de Bos taurus
automaticamente através do servidor Swiss Model (http://swissmodel.expasy.org), usando como molde a estrutura da proteína morfogênica óssea (BMP-3) humana, um membro da superfamília de proteínas TGF-β (PDB 2QCQ chain B). A Figura 4 mostra o modelo obtido. Neste modelo, a sequência antigênica 1 , no extremo N-terminal mostra uma estrutura em hélice "coif livre, enquanto que a sequência antigênica 2 se localiza numa folha-beta formada no extremo C-terminal da inibina. As there are no experimentally obtained inhibin structures, a modeling of the structure of Bos taurus alpha-inhibin was performed. automatically through the Swiss Model server (http://swissmodel.expasy.org), using as a template the structure of human bone morphogenetic protein (BMP-3), a member of the TGF-β protein superfamily (PDB 2QCQ chain B). Figure 4 shows the model obtained. In this model, the N-terminal antigenic sequence 1 shows a free coif "helix structure, while the antigenic sequence 2 is located on a beta-sheet formed at the C-terminal inhibin.
[084] Com intuito de usar a sequência antigênica 2 como novo antígeno, foi selecionada toda a estrutura de folha beta que a contém, de modo a possibilitar a reprodução estrutural deste epítopo. A Figura 5 mostra a folha beta escolhida. [084] In order to use antigenic sequence 2 as the new antigen, the entire beta-leaf structure containing it was selected to enable the structural reproduction of this epitope. Figure 5 shows the chosen beta sheet.
[085] Na base da estrutura escolhida há quatro Cisteínas (duas delas mostradas na Figura 6) e uma estrutura "dobra" que é mantida pela interação hidrofobica dos aminoácidos Valina e Leucina (Val e Leu na Figura 6). Estes aminoácidos se encontram no meio de uma região hidrofobica da molécula da inibina, na fase que interage com o receptor. At the base of the chosen structure are four Cysteines (two of them shown in Figure 6) and a "fold" structure that is maintained by the hydrophobic interaction of the amino acids Valine and Leucine (Val and Leu in Figure 6). These amino acids are in the middle of a hydrophobic region of the inhibin molecule in the phase that interacts with the receptor.
[086] Sobre esta sequência foram realizadas as seguintes mudanças: [086] The following changes were made to this sequence:
1 . - As duas Cisteínas do extremo C-terminal, da sequência original, foram substituídas por Serina (S, homólogo estrutural da Cisteína) e Glicina (G, para dar flexibilidade no caso de se adicionar outra sequência neste extremo). 1 . - The two C-terminus Cysteines of the original sequence have been replaced by Serine (S, Cysteine structural counterpart) and Glycine (G, to give flexibility if another sequence is added at this end).
2. - No extremo N-terminal, da sequência original, foi removida a sequência "Pro Cys Cys Aia Ala Leu L" que contém duas Cisteínas consecutivas. No seu lugar, foi adicionada a sequência "Giy Ser Ala" para dar flexibilidade à ligação com a molécula carregadora, de modo que a fusão não interfira com a formação da estrutura tridimensional desejada que é mostrada na Figura 5. 2. At the N-terminal end of the original sequence, the "Pro Cys Cys Aia Ala Leu L" sequence containing two consecutive cysteines has been removed. In its place, the sequence "Giy Ser Ala" has been added to give flexibility in binding to the carrier molecule so that fusion does not interfere with the formation of the desired three-dimensional structure shown in Figure 5.
3. - Para incrementar a solubilidade da molécula final e impedir uma possível interação do antígeno com o receptor, os aminoácidos Valina (Val) e Leucina (Leu), mostrados na Figura 6, foram substituídos por ácido Aspártico (Asp, D) e Arginina (Arg, R), respectivamente, que possuem cargas opostas e,
portanto, podem interagir para estabilizar a "dobra" na estrutura mostrada na Figura 5 e simultaneamente diminuir a hidrofobicidade do segmento escolhido. 3. To increase solubility of the final molecule and prevent possible interaction of the antigen with the receptor, the amino acids Valine (Val) and Leucine (Leu), shown in Figure 6, have been replaced by Aspartic Acid (Asp, D) and Arginine. (Arg, R), respectively, which have opposite charges and, therefore, they may interact to stabilize the "fold" in the structure shown in Figure 5 and simultaneously decrease the hydrophobicity of the chosen segment.
[087] Como resultado dessas modificações, foi obtida a sequência representada na Figura 7. [087] As a result of these modifications, the sequence shown in Figure 7 was obtained.
[088] O efeito dessas modificações na estrutura da folha beta selecionada foi avaliado por simulação de dinâmica molecular usando o programa NOC 3.0 (http://noch.sourceforge.net). A Figura 8 mostra a representação da estrutura após a simulação, indicando a manutenção da estrutura desejada após as modificações realizadas. [088] The effect of these modifications on the selected beta sheet structure was assessed by molecular dynamics simulation using the NOC 3.0 program (http://noch.sourceforge.net). Figure 8 shows the representation of the structure after the simulation, indicating the maintenance of the desired structure after the modifications made.
[089] Como mencionado anteriormente, os primeiros 29 aminoácidos da inibina alfa que contém a sequência antigênica 1 não estão presentes em outros membros da superfamília de proteínas TGF-β. As sequências de aminoácidos destas proteínas alinham com a inibina a partir do aminoácido 30. As mentioned previously, the first 29 amino acids of inhibin alfa containing antigenic sequence 1 are not present in other members of the TGF-β protein superfamily. The amino acid sequences of these proteins align with inhibin from amino acid 30.
[090] Para a sequência antigênica 2, foi realizada uma comparação da sequência, como ilustrada na Figura 7, com proteínas não-redundantes depositadas em diversos bancos de genes, usando a ferramenta "protein BLAST" (Basic Local Alignment Search Tool) no servidor http://blast.ncbi.nlm.nih.gov/Blast.cgi. Essa sequência antigênica 2 alinhou apenas com inibinas, com um "e-value" (um índice que indica a probabilidade do alinhamento ser ao acaso) menor que 1 e-06 (7e-12 para a inibina de Bos taurus). Os alinhamentos com outras proteínas não relacionadas à inibida mostraram um "e-value" maior que 5. Portanto, é pouco provável a ocorrência de reação cruzada com outras proteínas que não sejam inibina. For antigenic sequence 2, a sequence comparison, as illustrated in Figure 7, was performed with non-redundant proteins deposited in several gene banks using the Basic Local Alignment Search Tool "protein BLAST" on the server. http://blast.ncbi.nlm.nih.gov/Blast.cgi. This antigenic sequence 2 aligned only with inhibins, with an "e-value" (an index that indicates the likelihood of alignment being random) less than 1 e-06 (7e-12 for Bos taurus inhibin). Alignments with other non-inhibited proteins showed an e-value greater than 5. Therefore, cross-reactivity with non-inhibin proteins is unlikely.
[091 ] A Figura 12 mostra o alinhamento da sequência modificada com o extremo C-terminal de algumas inibinas selecionadas. Pela identidade na região correspondente à sequência antigênica 2 (Giy Tyr ser Phe Lys Tyr Giu Met) é possível afirmar que ela pode gerar anticorpos contra as inibinas listadas nesta Figura 12, a saber: Bovino, Ovino, Porco, Cabra, Cavalo, Gato, Cachorro, Camundongo e Humano. A sequência antigênica 1 (Leu Leu Gin Arg Pro Pro Giu Giu Pro Ala Ala HÍS), também está presente em todas as inibinas relatadas na base de dados do GenBank.
[092] Finalmente, a sequência antigênica 1 , que é naturalmente flexível, foi adicionada no extremo C-terminal da sequência modificada contendo a sequência antigênica 2, obtendo-se assim a sequência correspondente ao antígeno "inibina OF" [SEQ ID No: 1 ]: [091] Figure 12 shows the alignment of the modified sequence with the C-terminal end of some selected inhibins. By identity in the region corresponding to antigenic sequence 2 (Giy Tyr and Phe Lys Tyr Giu Met) it can be stated that it can generate antibodies against the inhibins listed in this Figure 12, namely: Cattle, Sheep, Pig, Goat, Horse, Cat, Dog, Mouse and Human. Antigenic sequence 1 (Leu Leu Gin Arg Pro Giu Giu Pro Ala Wing HÍS) is also present in all inhibins reported in the GenBank database. Finally, the naturally flexible antigenic sequence 1 was added at the C-terminal end of the modified sequence containing the antigenic sequence 2, thereby obtaining the sequence corresponding to the "inhibin OF" antigen [SEQ ID NO: 1 ]:
Gly Ser Ala Pro Gly Thr Met Arg Pro Leu His Val Arg Thr Thr 1 5 10 15Gly Ser Wing Pro Gly Thr Met Arg Pro Read His Val Arg Thr Thr 1 5 10 15
Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Met Asp Pro Asn Arg Be Asp Gly Gly Tyr Be Phe Lys Tyr Glu Met Asp Pro Asn Arg
20 25 30 20 25 30
Leu Thr Gln His Ser Ala Gly Leu Leu Gln Arg Pro Pro Glu Glu Leu Thr Gln His Ser Gly Wing Read Leu Gln Arg Pro Pro Glu Glu
35 40 45 35 40 45
Pro Ala Ala His Pro Wing His Wing
49 49
Exemplo 2: Desenho do gene sintético para o antígeno Inibina OF Example 2: Synthetic gene design for Inibina OF antigen
[093] Estabelecida a sequência de aminoácidos do antígeno OF, procedeu-se ao desenho de um gene sintético que codifique esta sequência, usando códons otimizados e harmonizados para sua melhor expressão em Escherichia coli. [093] Once the OF antigen amino acid sequence was established, a synthetic gene encoding this sequence was designed using optimized and harmonized codons for best expression in Escherichia coli.
[094] Para a harmonização de códons [ver Angov et al. (201 1 ) Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding. Methods Mol Biol. 705:1 -13], foram localizados, no gene original (de Bos taurus), os códons raros ou usados com menor frequência, empregando o programa ANACONDA[094] For codon harmonization [see Angov et al. (201 1) Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding. Methods Mol Biol. 705: 1-13], were found in the original gene (Bos taurus), the rare or less frequently used codons using the ANACONDA program
(http://bioinformatics.ua.pt/software/anaconda) alimentado com as tabelas de uso de códons de Bos taurus depositado no "Codon usage database" (http://www.kazusa.or.jp/codon). Na harmonização foram considerados os códons raros, presentes entre as duas estruturas secundárias e loops, visualizados no modelo estrutural usando o programa DeepView-Swiss- PdbViewer (http://spdbv.vital-it.ch). Os códons raros ou menos frequentes de E. coli foram introduzidos manualmente no gene sintético, empregando a interface do programa Gene Design v2.0 (firma DNA2.0). Também foram introduzidos manualmente: o sítio de corte para a enzima Xhol (CTCGAG) no extremo 5', o códon stop junto com o sítio de corte para Hinde III (TAAGCTT, em negrito o
códon stop e sublinhado o sítio de Hind III, na Figura 10). Além disso, foi adicionado, na base de dados de sequências a serem evitadas do programa Gene Design v2.0, o sítio "chi" de E. coli 5' GCTGGTGG 3'. Este sítio favorece a recombinação por homologia e pode causar instabilidade do plasmídeo de expressão. (http://bioinformatics.ua.pt/software/anaconda) fed with the Bos taurus codon usage tables deposited in the "Codon usage database" (http://www.kazusa.or.jp/codon). In the harmonization were considered the rare codons, present between the two secondary structures and loops, visualized in the structural model using the DeepView-Swiss-PdbViewer program (http://spdbv.vital-it.ch). Rare or less frequent E. coli codons were manually introduced into the synthetic gene using the Gene Design v2.0 program interface (DNA2.0 firm). Also manually introduced: the cut-off site for the enzyme Xhol (CTCGAG) at the 5 'end, the stop codon along with the cut-off site for Hinde III (TAAGCTT, in bold the stop codon and underlining the Hind III site in Figure 10). In addition, in the database of sequences to be avoided from the Gene Design v2.0 program, the E. coli 5 'GCTGGTGG 3' chi site was added. This site favors homology recombination and may cause expression plasmid instability.
[095] Finalmente, foi realizada a síntese do gene desenhado, correspondente à SEQ ID NO:2, codificante da SEQ ID NO: 3. O gene sintético foi clonado num vetor sem o sitio de clonagem múltipla derivado do pBlueScript II SK cortado com Smal denominado pBSK-lniOF. A sequência do gene sintético (SEQ ID NO:2) foi confirmada por sequenciamento. Finally, the design gene corresponding to SEQ ID NO: 2 encoding SEQ ID NO: 3 was synthesized. The synthetic gene was cloned into a vector without the Smal cut pBlueScript II SK derived multiple cloning site called pBSK-lniOF. The synthetic gene sequence (SEQ ID NO: 2) was confirmed by sequencing.
Exemplo 3: Clonagem do gene sintético da Inibina OF em fusão com a proteína SUMO Example 3: Cloning of synthetic Inibin OF gene in fusion with SUMO protein
[096] Em uma das concretizações da presente invenção, a expressão da proteína recombinante foi realizada por fusão com a proteína SUMO (de "small ubiquitin-like modifier"), que pertence à família daquelas usadas para aumentar a expressão da proteína INIBINA OF da invenção e melhorar sua solubilidade. Além disso, a estrutura terciária da proteína SUMO é reconhecida pela SUMO protease que cliva exatamente entre a SUMO e a proteína alvo, dando como resultado a proteína nativa sem fusão. [096] In one embodiment of the present invention, expression of the recombinant protein was performed by fusion with the SUMO ("small ubiquitin-like modifier") protein, which belongs to the family of those used to increase expression of the protein inhibitor OF. invention and improve its solubility. In addition, the tertiary structure of the SUMO protein is recognized by SUMO protease which cleaves exactly between SUMO and the target protein, resulting in the native fused protein.
[097] A Figura 13 mostra a estratégia de clonagem. O gene sintético foi cortado do plasmídeo pBSK-lniOF (Figura 13A) usando as enzimas BamHI e Hind III e clonado nos sítios correspondentes do vetor pET-SUMO (Figura 13B). O plasmídeo resultante foi denominado pET-SUMO-lniOF (Figura 13C). O sucesso da clonagem foi verificado por sequenciamento usando o primer universal T7 reverse primer (5' GCTAGTTATTGCTCAGCGG 3'). [097] Figure 13 shows the cloning strategy. The synthetic gene was cut from plasmid pBSK-lniOF (Figure 13A) using the enzymes BamHI and Hind III and cloned into the corresponding sites of the pET-SUMO vector (Figure 13B). The resulting plasmid was named pET-SUMO-lniOF (Figure 13C). Successful cloning was verified by sequencing using the T7 reverse primer universal primer (5 'GCTAGTTATTGCTCAGCGG 3').
Exemplo 4: Produção da Inibina OF em fusão com a proteína SUMO Example 4: Production of Inhibin OF in Fusion with SUMO Protein
[098] A cepa de Escheríchia coli BL21 (DE3) transformada com o plasmídeo pET-SUMO-lniOF foi crescida em 200 ml de meio LB Kanamicina (25 ug/ml) e incubada com o material de um crio tubo do bando de sementes. A cultura foi incubada a 37°C, a 200 rpm, por 16 horas.
[099] Os 200 ml de cultura foram inoculados em 5 Litros do meio MCHD (Meio Complexo de Alta Densidade) em fermentador de 15 litros. A fermentação em regime constante de batelada alimentada, a 37°C, 30% de oxigénio dissolvido, tendo glicerol como fonte de carbono. Na DO6oonm = 15, a alimentação por glicerol foi trocada por lactose (indução) e a fermentação continuou por mais 12 horas a 28°C. The Escheríchia coli BL21 strain (DE3) transformed with the pET-SUMO-lniOF plasmid was grown in 200 ml LB Kanamycin medium (25 µg / ml) and incubated with the material from a cryo tube of the seed flock. The culture was incubated at 37 ° C at 200 rpm for 16 hours. The 200 ml culture was inoculated into 5 Liters of MCHD Medium (High Density Complex Medium) in a 15 liter fermenter. Constant batch fermentation fed at 37 ° C 30% dissolved oxygen with glycerol as carbon source. At OD 6 oonm = 15, glycerol feeding was switched to lactose (induction) and fermentation continued for a further 12 hours at 28 ° C.
[0100] As células foram coletadas por centrifugação a 6000 rpm por 30 minutos e ressuspendidas no tampão de lise (5 mM imidazol, 0,5M NaCI, 50 mM Tris- HCI, pH 8.0; 0,1 % Triton X-100; 5mM 2-mercaptoetanol) e lisadas no homogeneizador de alta pressão. Cells were harvested by centrifugation at 6000 rpm for 30 minutes and resuspended in lysis buffer (5 mM imidazole, 0.5 M NaCl, 50 mM Tris- HCI, pH 8.0; 0.1% Triton X-100; 5 mM 2-mercaptoethanol) and lysed in the high pressure homogenizer.
[0101 ] As células lisadas e homogeneizadas foram submetidas a filtração através de membrana de 0,2 μιη (sistema "end-filtration" millipore) à temperatura ambiente. O lisado foi clarificado em coluna contendo a resina Chelating-Sepharose (GE health care) ativada com Ni2+. Após a absorção, a resina foi lavada com o tampão 0,5M NaCI, 50 mM Tris-HCI, pH 8.0 contendo 5 mM de imidazol; e em seguida com o mesmo tampão contendo 40 mM de imidazol. A proteína recombinante foi eluída com o tampão de eluição (0,5 M imidazol, 0,1 M NaCI, 50 mM Tris-HCI, pH 8.0) coletando-se frações correspondentes ao pico de eluição. The lysed and homogenized cells were subjected to 0.2 μιη membrane filtration (millipore end-filtration system) at room temperature. The lysate was clarified on a column containing Ni 2+ activated Chelating Sepharose (GE health care) resin. After absorption, the resin was washed with 0.5M NaCl, 50mM Tris-HCl, pH 8.0 buffer containing 5mM imidazole; and then with the same buffer containing 40 mM imidazole. The recombinant protein was eluted with elution buffer (0.5 M imidazole, 0.1 M NaCl, 50 mM Tris-HCl, pH 8.0) by collecting fractions corresponding to the elution peak.
[0102] A proteína eluída da coluna cromatográfica de afinidade foi submetida a uma cromatografia de exclusão molecular na coluna carregada com a resina Sephacryl S-100 HR (GE Healthcare) usando o tampão 0,1 M NaCI, 20 mM Tris-HCI, pH 8.0. O SDS-PAGE da Figura 14 mostra apenas uma banda de aproximadamente 20 kDa, esperada para a fusão da proteína 6xHis-SUMO (13,5 kDa) e a Inibina OF (6,5 kDa). A expressão de proteína solúvel, assim como a estabilidade da proteína purificada durante seu armazenamento a 4°C na concentração de 1 mg/ml, indicam o sucesso no desenho do antígeno com referência à solubilidade e à estabilidade.
The protein eluted from the affinity chromatographic column was subjected to molecular exclusion chromatography on the column loaded with Sephacryl S-100 HR resin (GE Healthcare) using 0.1 M NaCI buffer, 20 mM Tris-HCI, pH 8.0. The SDS-PAGE of Figure 14 shows only an approximately 20 kDa band expected for the fusion of 6xHis-SUMO protein (13.5 kDa) and OF inhibin (6.5 kDa). Soluble protein expression, as well as the stability of the purified protein during storage at 4 ° C at a concentration of 1 mg / ml, indicate success in antigen design with reference to solubility and stability.
Claims
1 . Antígeno de inibina alfa caracterizado por compreender uma sequência de aminoácidos selecionada de: 1 . Inhibin alpha antigen characterized by comprising an amino acid sequence selected from:
(a) SEQ ID NO:1 ; (a) SEQ ID NO: 1;
(b) SEQ ID NO:3; (b) SEQ ID NO: 3;
(c) variantes conservativamente modificadas tendo pelo menos 85% de identidade com qualquer uma das sequências de aminoácidos de SEQ ID NO:1 ou SEQ ID NO:3, e tendo substancialmente a mesma atividade imunogênica das mesmas; (c) conservatively modified variants having at least 85% identity to any of the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3, and having substantially the same immunogenic activity thereof;
(d) fragmentos de SEQ ID NO:1 ou SEQ ID NO:3, ditos fragmentos contendo as duas regiões imunogênicas da SEQ ID NO:1 ou da SEQ ID NO:3; e (d) fragments of SEQ ID NO: 1 or SEQ ID NO: 3, said fragments containing the two immunogenic regions of SEQ ID NO: 1 or SEQ ID NO: 3; and
(e) uma proteína heteróloga de fusão compreendendo a SEQ ID NO:1 ou SEQ ID NO:3, ou um fragmento ou variante conservativamente modificada das mesmas, e uma proteína carregadora farmacologicamente aceitável. (e) a heterologous fusion protein comprising SEQ ID NO: 1 or SEQ ID NO: 3, or a conservatively modified fragment or variant thereof, and a pharmacologically acceptable carrier protein.
2. Antígeno de acordo com a reivindicação 1 , caracterizado pelo fato de compreender uma sequência selecionada do grupo consistindo de: Antigen according to claim 1, characterized in that it comprises a sequence selected from the group consisting of:
(i) SEQ ID NO:1 ; (i) SEQ ID NO: 1;
(ii) uma variante da SEQ ID NO:1 compreendendo aminoácidos conservativamente modificados e tendo pelo menos 90% de identidade de sequência com a SEQ ID NO:1 ; e (ii) a variant of SEQ ID NO: 1 comprising conservatively modified amino acids and having at least 90% sequence identity with SEQ ID NO: 1; and
(iii) um fragmento da SEQ ID NO:1 compreendendo as regiões imunogênicas que se ligam especificamente a anticorpos produzidos em resposta à inibina. (iii) a fragment of SEQ ID NO: 1 comprising immunogenic regions that specifically bind antibodies raised in response to inhibin.
3. Antígeno de acordo com a reivindicação 1 , caracterizado pelo fato de compreender uma sequência selecionada do grupo consistindo de: Antigen according to claim 1, characterized in that it comprises a sequence selected from the group consisting of:
(i) SEQ ID NO:3; (i) SEQ ID NO: 3;
(ii) uma variante da SEQ ID NO:3 compreendendo aminoácidos conservativamente modificados e tendo pelo menos 90% de identidade de sequência com a SEQ ID NO:3; e
(iii) um fragmento da SEQ ID NO:3 compreendendo as regiões imunogênicas que se ligam especificamente a anticorpos produzidos em resposta à inibina. (ii) a variant of SEQ ID NO: 3 comprising conservatively modified amino acids and having at least 90% sequence identity to SEQ ID NO: 3; and (iii) a fragment of SEQ ID NO: 3 comprising immunogenic regions that specifically bind antibodies raised in response to inhibin.
4. Antígeno de acordo com a reivindicação 1 , caracterizado pelo fato de compreender a fusão de proteínas que compreendem: An antigen according to claim 1, characterized in that it comprises fusion of proteins comprising:
(a) uma sequência de aminoácidos tendo antigenicidade com relação à inibina, dita sequência sendo selecionada do grupo consistindo de: (a) an amino acid sequence having inhibin antigenicity, said sequence being selected from the group consisting of:
(i) SEQ ID NO:1 ; (i) SEQ ID NO: 1;
(ii) uma variante da SEQ ID NO:1 , compreendendo aminoácidos conservativamente modificados e tendo pelo menos 90% de identidade de sequência com a SEQ ID NO:1 ; (ii) a variant of SEQ ID NO: 1, comprising conservatively modified amino acids and having at least 90% sequence identity to SEQ ID NO: 1;
(iii) um fragmento da SEQ ID NO:1 compreendendo as regiões imunogênicas que se ligam especificamente a anticorpos produzidos em resposta à inibina; (iii) a fragment of SEQ ID NO: 1 comprising immunogenic regions that specifically bind antibodies raised in response to inhibin;
(iv) SEQ ID NO:3; (iv) SEQ ID NO: 3;
(v) uma variante da SEQ ID NO:3 compreendendo aminoácidos conservativamente modificados e tendo pelo menos 90% de identidade de sequência com a SEQ ID NO:3; e (v) a variant of SEQ ID NO: 3 comprising conservatively modified amino acids and having at least 90% sequence identity to SEQ ID NO: 3; and
(vi) um fragmento da SEQ ID NO:3 compreendendo as regiões imunogênicas que se ligam especificamente a anticorpos produzidos em resposta à inibina, e (vi) a fragment of SEQ ID NO: 3 comprising immunogenic regions that specifically bind antibodies raised in response to inhibin, and
(b) uma sequência de aminoácidos de proteína carregadora da proteína antigênica (a), farmacologicamente aceitável. (b) a pharmacologically acceptable antigenic protein-carrying protein amino acid sequence (a).
5. Antígeno de acordo com a reivindicação 4, caracterizado pelo fato de que a dita proteína carregadora farmacologicamente aceitável é selecionada do grupo consistindo de proteínas da família de pequeno modificador relacionado à ubiquitina (SUMO), proteína Rrp42 da arqueobactéria Pyrococcus abyssi, proteína de ligação a maltose, albumina de soro bovino, hemocianina de keyhole lympet, ovoalbumina, flagelina, albumina de soro de qualquer espécie gama globulina de qualquer espécie, polímeros de aminoácidos D- e/ou L-.
An antigen according to claim 4, characterized in that said pharmacologically acceptable carrier protein is selected from the group consisting of proteins from the small ubiquitin-related modifier (SUMO) family, Pyrococcus abyssi archaebacterium protein Rrp42, binding protein. maltose, bovine serum albumin, keyhole lympet hemocyanin, ovoalbumin, flagellin, serum albumin of any gamma globulin species of any species, D- and / or L- amino acid polymers.
6. Antígeno de acordo com a reivindicação 5, caracterizado pelo fato de dita proteína carregadora farmacologicamente aceitável ser a proteína Rrp42 da arqueobactéria Pyrococcus abyssi codificada pela SEQ ID NO:4. An antigen according to claim 5, characterized in that said pharmacologically acceptable carrier protein is the Pyrococcus abyssi archeobacterium protein Rrp42 encoded by SEQ ID NO: 4.
7. Antígeno de inibina alfa caracterizado por compreender a SEQ ID NO:1 . Inhibin alpha antigen comprising SEQ ID NO: 1.
8. Antígeno de inibina alfa caracterizado por compreender a SEQ ID NO:3.8. Inhibin alpha antigen comprising SEQ ID NO: 3.
9. Antígeno de inibina alfa caracterizado por compreender a fusão da proteína antigênica SEQ ID NO:1 com a proteína carregadora Rrp42 da arqueobactéria Pyrococcus abyssi codificada pela SEQ ID NO:4. An inhibin alpha antigen comprising the fusion of the antigenic protein SEQ ID NO: 1 with the Pyrrococcus abyssi archeobacterial carrier protein Rrp42 encoded by SEQ ID NO: 4.
10. Antígeno de inibina alfa caracterizado por compreender a fusão da proteína antigênica SEQ ID NO:3 com a proteína carregadora Rrp42 da arqueobactéria Pyrococcus abyssi codificada pela SEQ ID NO:4. An inhibin alpha antigen comprising the fusion of the antigenic protein SEQ ID NO: 3 with the Pyrrococcus abyssi archeobacterial carrier protein Rrp42 encoded by SEQ ID NO: 4.
1 1 . Gene codificante de um antígeno de inibina alfa caracterizado pelo fato de compreender uma sequência de nucleotídeos selecionada do grupo consistindo de: 1 1. A gene encoding an inhibin alpha antigen characterized in that it comprises a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO:2; e (a) SEQ ID NO: 2; and
(b) uma sequência de nucleotídeos tendo pelo menos 75% de identidade com relação à SEQ ID NO:2. (b) a nucleotide sequence having at least 75% identity to SEQ ID NO: 2.
12. Gene codificante de uma proteína de fusão compreendendo o antígeno como definido na reivindicação 4, caracterizado pelo fato de compreender: A gene encoding a fusion protein comprising the antigen as defined in claim 4, comprising:
(a) uma sequência de nucleotídeos selecionada do grupo consistindo de: (a) a nucleotide sequence selected from the group consisting of:
(i) SEQ ID NO:2; (i) SEQ ID NO: 2;
(ii) uma sequência codificante do antígeno compreendendo a SEQ ID NO:1 ; (ii) an antigen coding sequence comprising SEQ ID NO: 1;
(iii) uma sequência codificante de variantes conservativamente modificadas tendo pelo menos 90% de identidade com a SEQ ID NO:1 e tendo substancialmente a mesma atividade imunogênica da SEQ ID NO:1 ; e (iii) a conservatively modified variant coding sequence having at least 90% identity to SEQ ID NO: 1 and having substantially the same immunogenic activity as SEQ ID NO: 1; and
(iv) uma sequência codificante de fragmentos da SEQ ID NO:1 ditos fragmentos contendo as regiões imunogênicas da SEQ ID NO:1 , e
(b) uma sequência codificante de uma proteína carregadora selecionada do grupo consistindo de proteínas da família de pequeno modificador relacionado à ubiquitina (SUMO), proteína Rrp42 da arqueobactéria Pyrococcus abyssi, proteína de ligação a maltose, albumina de soro bovino, hemocianina de keyhole lympet, ovoalbumina, flagelina, albumina de soro de qualquer espécie gama globulina de qualquer espécie, polímeros de aminoácidos D- e/ou L-. (iv) a sequence encoding fragments of SEQ ID NO: 1 said fragments containing the immunogenic regions of SEQ ID NO: 1, and (b) a coding sequence for a carrier protein selected from the group consisting of proteins from the small ubiquitin-related modifier (SUMO) family, Pyrococcus abyssi archaebacterium protein Rrp42, bovine serum albumin, keyhole lympet hemocyanin , ovoalbumin, flagellin, serum albumin of any gamma globulin species of any species, D- and / or L- amino acid polymers.
13. Gene codificante de uma proteína de fusão compreendendo o antígeno como definido na reivindicação 4, caracterizado pelo fato de compreender: A gene encoding a fusion protein comprising the antigen as defined in claim 4, comprising:
(a) uma sequência de nucleotídeos selecionada do grupo consistindo de: (a) a nucleotide sequence selected from the group consisting of:
(i) SEQ ID NO:2; (i) SEQ ID NO: 2;
(ii) uma sequência codificante do antígeno compreendendo a SEQ ID NO:3; (ii) an antigen coding sequence comprising SEQ ID NO: 3;
(iii) uma sequência codificante de variantes conservativamente modificadas tendo pelo menos 90% de identidade com a SEQ ID NO:3 e tendo substancialmente a mesma atividade imunogênica da SEQ ID NO:3; e (iii) a conservatively modified variant coding sequence having at least 90% identity to SEQ ID NO: 3 and having substantially the same immunogenic activity as SEQ ID NO: 3; and
(iv) uma sequência codificante de fragmentos da SEQ ID NO:3 ditos fragmentos contendo as regiões imunogênicas da SEQ ID NO:3, e (iv) a sequence encoding fragments of SEQ ID NO: 3 said fragments containing the immunogenic regions of SEQ ID NO: 3, and
(b) uma sequência codificante de uma proteína carregadora selecionada do grupo consistindo de proteínas da família de pequeno modificador relacionado à ubiquitina (SUMO), proteína Rrp42 da arqueobactéria Pyrococcus abyssi, proteína de ligação a maltose, albumina de soro bovino, hemocianina de keyhole lympet, ovoalbumina, flagelina, albumina de soro de qualquer espécie gama globulina de qualquer espécie, polímeros de aminoácidos D- e/ou L-. (b) a coding sequence for a carrier protein selected from the group consisting of proteins from the ubiquitin-related small modifier (SUMO) family, Pyrococcus abyssi archaebacterium protein Rrp42, bovine serum albumin, keyhole lympet hemocyanin , ovoalbumin, flagellin, serum albumin of any gamma globulin species of any species, D- and / or L- amino acid polymers.
14. Gene codificante de acordo com a reivindicação 12 ou reivindicação 13, caracterizado pelo fato de que a dita sequência codificante de uma proteína
carregadora é a SEQ ID NO:4 que codifica a proteína Rrp2 da arqueobactéria Pyrococcus abyssi. A coding gene according to claim 12 or claim 13, characterized in that said coding sequence for a protein The carrier is SEQ ID NO: 4 encoding the Pyrococcus abyssi archaebacterium protein Rrp2.
15. Processo de obtenção do antígeno de inibina alfa definido nas reivindicações 1 -3 e 7-8, caracterizado pelo fato de compreender as etapas de: A process for obtaining the inhibin alpha antigen as defined in claims 1-3 and 7-8, comprising the steps of:
(i) preparação de um sistema de expressão compreendendo: (i) preparing an expression system comprising:
(a) o gene codificante de um antígeno de inibina alfa definido na reivindicação 1 1 ; (a) the gene encoding an inhibin alpha antigen defined in claim 11;
(b) um sistema de expressão apropriado, preferencialmente uma cepa de Escherichia coli selecionada das cepas BL21 (DE3) e Rosetta (DE3), e (b) an appropriate expression system, preferably an Escherichia coli strain selected from the BL21 (DE3) and Rosetta (DE3) strains, and
(c) um promotor de expressão apropriado, selecionado de pRSETA e pET, preferencialmente o promotor pET-SUMO; (c) an appropriate expression promoter selected from pRSETA and pET, preferably the pET-SUMO promoter;
(ii) inoculação das células hospedeiras com o sistema de expressão preparado na etapa (i); (ii) inoculating the host cells with the expression system prepared in step (i);
(iii) lise das células para obtenção do antígeno de inibina alfa; e (iii) cell lysis for inhibin alpha antigen; and
(iv) purificação do dito antígeno de inibina alfa obtido. (iv) purifying said inhibin alpha antigen obtained.
16. Processo de obtenção do antígeno como definido nas reivindicações 4-5 e 9-10, caracterizado pelo fato de compreender as etapas de: Process for obtaining the antigen as defined in claims 4-5 and 9-10, characterized in that it comprises the steps of:
(i) preparação de um sistema de expressão compreendendo: (i) preparing an expression system comprising:
(a) o gene codificante de um antígeno de inibina alfa como definido na reivindicação 12, ou reivindicação 13 ou reivindicação 14; (a) the gene encoding an inhibin alpha antigen as defined in claim 12, or claim 13 or claim 14;
(b) um sistema de expressão apropriado, preferencialmente uma cepa de Escherichia coli selecionada das cepas BL21 (DE3) e Rosetta (DE3), e (b) an appropriate expression system, preferably an Escherichia coli strain selected from the BL21 (DE3) and Rosetta (DE3) strains, and
(c) um promotor de expressão apropriado, selecionado de pRSETA e pET, preferencialmente o promotor pET-SUMO; (c) an appropriate expression promoter selected from pRSETA and pET, preferably the pET-SUMO promoter;
(ii) inoculação das células hospedeiras com o sistema de expressão preparado na etapa (i); (ii) inoculating the host cells with the expression system prepared in step (i);
(iii) lise das células para obtenção do antígeno de inibina alfa; e (iii) cell lysis for inhibin alpha antigen; and
(iv) purificação do antígeno como definido nas reivindicações 4, 9 ou 10.
(iv) antigen purification as defined in claims 4, 9 or 10.
17. Composição antigênica, caracterizada por compreender: 17. Antigenic composition, characterized in that it comprises:
(a) o antígeno de inibina alfa como definido em qualquer uma das reivindicações 1 -3 e 7-8; (a) the inhibin alpha antigen as defined in any one of claims 1-3 and 7-8;
(b) opcionalmente, um adjuvante; e (b) optionally an adjuvant; and
(c) um veículo farmacologicamente aceitável. (c) a pharmacologically acceptable carrier.
18. Composição antigênica, caracterizada por compreender: 18. Antigenic composition, characterized in that it comprises:
(a) o antígeno de inibina alfa como definido em qualquer uma das reivindicações 4-6 e 9-10; (a) inhibin alpha antigen as defined in any one of claims 4-6 and 9-10;
(b) opcionalmente, um adjuvante; e (b) optionally an adjuvant; and
(c) um veículo farmacologicamente aceitável. (c) a pharmacologically acceptable carrier.
19. Composição antigênica de acordo com a reivindicação 17 ou reivindicação 18, caracterizada pelo fato de que dito adjuvante é selecionado do grupo consistindo de: adjuvante incompleto de Freund, o adjuvante completo de Freund, manana acetilada polidispersa β-(1 ,4) ligada, adjuvantes de copolímero de polioxietileno-polioxipropileno, adjuvantes lipídicos modificados, adjuvantes derivados de saponina, sulfato de dextrana, hidróxido de alumínio e fosfato de alumínio. An antigenic composition according to claim 17 or claim 18, characterized in that said adjuvant is selected from the group consisting of: incomplete Freund's adjuvant, Freund's complete adjuvant, β- (1,4) polydispersed acetylated mannan , polyoxyethylene-polyoxypropylene copolymer adjuvants, modified lipid adjuvants, saponin-derived adjuvants, dextran sulfate, aluminum hydroxide and aluminum phosphate.
20. Composição antigênica de acordo com a reivindicação 17 ou reivindicação 18, caracterizada pelo fato de que dito veículo farmacologicamente aceitável inclui preservativos, diluentes, emulsificantes, estabilizantes e outros ingredientes farmacologicamente aceitáveis. Antigenic composition according to claim 17 or claim 18, characterized in that said pharmacologically acceptable carrier includes preservatives, diluents, emulsifiers, stabilizers and other pharmacologically acceptable ingredients.
21 . Uso do antígeno de inibina alfa como definido nas reivindicações 1 -3 e 7-8, caracterizado por ser no aumento da produção de animais mamíferos ou aves. 21 Use of the inhibin alpha antigen as defined in claims 1-3 and 7-8, characterized in that it increases the production of mammalian or avian animals.
22. Uso do antígeno de inibina alfa como definido nas reivindicações 4-6 e 9-10, caracterizado por ser no tratamento de infertilidade de animais mamíferos, incluindo o homem Use of the inhibin alpha antigen as defined in claims 4-6 and 9-10, characterized in that it is in the treatment of infertility in mammalian animals, including humans.
23. Uso da composição antigênica como definida em qualquer uma das reivindicações 17-20, caracterizado por ser no aumento da produção de animais mamíferos ou aves.
Use of the antigenic composition as defined in any one of claims 17-20, characterized in that it is in increased production of mammalian or avian animals.
24. Uso da composição antigênica como definida em qualquer uma das reivindicações 17-20, caracterizado por ser no tratamento de infertilidade de animais mamíferos, incluindo o homem.
Use of the antigenic composition as defined in any one of claims 17-20, characterized in that it is in the treatment of infertility of mammalian animals, including man.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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BRBR1020140053760 | 2014-03-07 | ||
BR102014005376A BR102014005376A2 (en) | 2014-03-07 | 2014-03-07 | inhibin alpha antigen, inhibin alpha coding gene, fusion protein encoding gene, process of obtaining inhibin alpha antigen, antigenic composition, use of inhibin alpha antigen, and use of antigenic composition |
Publications (1)
Publication Number | Publication Date |
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WO2015131254A1 true WO2015131254A1 (en) | 2015-09-11 |
Family
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Application Number | Title | Priority Date | Filing Date |
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PCT/BR2015/050025 WO2015131254A1 (en) | 2014-03-07 | 2015-03-04 | Inhibin alpha antigen, gene coding for inhibin alpha, gene coding for fusion protein, method for obtaining inhibin alpha antigen, antigenic composition, use of the inhibin alpha antigen and use of the antigenic composition |
Country Status (4)
Country | Link |
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AR (1) | AR099671A1 (en) |
BR (1) | BR102014005376A2 (en) |
UY (1) | UY36020A (en) |
WO (1) | WO2015131254A1 (en) |
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US5215893A (en) * | 1985-10-03 | 1993-06-01 | Genentech, Inc. | Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid |
WO1995022980A1 (en) * | 1994-02-28 | 1995-08-31 | Agritech Technologies, Ltd. | Inhibin compositions and methods of use thereof |
JPH07238097A (en) * | 1994-09-07 | 1995-09-12 | Masao Igarashi | Igg antibody comprising inhibin as antigen |
CA2158116A1 (en) * | 1994-12-09 | 1996-06-10 | Toshiro Takedomi | Method for inducing superovulation in cattle |
US20040220098A1 (en) * | 1994-02-28 | 2004-11-04 | Satterlee Daniel G. | Inhibin compositions and methods of enhancing fertility and growth |
BR0316084A (en) * | 2002-11-07 | 2005-09-27 | Agritech Technology Ltd | Multiple inhibin-related antigenic peptide compositions that improve the performance of poultry production. |
BRPI0506043A (en) * | 2005-07-12 | 2007-03-13 | Univ Fed Do Rio Grande Do Sul | animal evaluation and selection process |
CN101407781A (en) * | 2008-12-01 | 2009-04-15 | 华中农业大学 | Inhibin DNA vaccine capable of improving animal fertility, and preparation and use thereof |
-
2014
- 2014-03-07 BR BR102014005376A patent/BR102014005376A2/en not_active Application Discontinuation
-
2015
- 2015-03-04 WO PCT/BR2015/050025 patent/WO2015131254A1/en active Application Filing
- 2015-03-05 AR ARP150100658A patent/AR099671A1/en unknown
- 2015-03-05 UY UY0001036020A patent/UY36020A/en not_active Application Discontinuation
Patent Citations (13)
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WO1986000078A1 (en) * | 1984-06-08 | 1986-01-03 | Monash University | Inhibin isolated from ovarian follicular fluid |
WO1986006076A1 (en) * | 1985-04-18 | 1986-10-23 | Biotechnology Australia Pty. Ltd. | Recombinant inhibin |
EP0222491A1 (en) * | 1985-10-03 | 1987-05-20 | Genentech, Inc. | Nucleic acid encoding the alpha or beta chains of inhibin and method for synthesizing polypeptides using such nucleic acid |
US5215893A (en) * | 1985-10-03 | 1993-06-01 | Genentech, Inc. | Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid |
US4864019A (en) * | 1985-11-08 | 1989-09-05 | The Salk Institute For Biological Studies | Antibodies to inhibin and conjugates produced therefrom |
WO1991010445A1 (en) * | 1990-01-08 | 1991-07-25 | Genentech, Inc. | Method for increasing fertility in females |
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US20040220098A1 (en) * | 1994-02-28 | 2004-11-04 | Satterlee Daniel G. | Inhibin compositions and methods of enhancing fertility and growth |
JPH07238097A (en) * | 1994-09-07 | 1995-09-12 | Masao Igarashi | Igg antibody comprising inhibin as antigen |
CA2158116A1 (en) * | 1994-12-09 | 1996-06-10 | Toshiro Takedomi | Method for inducing superovulation in cattle |
BR0316084A (en) * | 2002-11-07 | 2005-09-27 | Agritech Technology Ltd | Multiple inhibin-related antigenic peptide compositions that improve the performance of poultry production. |
BRPI0506043A (en) * | 2005-07-12 | 2007-03-13 | Univ Fed Do Rio Grande Do Sul | animal evaluation and selection process |
CN101407781A (en) * | 2008-12-01 | 2009-04-15 | 华中农业大学 | Inhibin DNA vaccine capable of improving animal fertility, and preparation and use thereof |
Also Published As
Publication number | Publication date |
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AR099671A1 (en) | 2016-08-10 |
UY36020A (en) | 2015-09-30 |
BR102014005376A2 (en) | 2016-02-10 |
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