Inhibition of myostatin signal by myostatin splice variant derived proteins and uses thereof

文档序号：74018 发布日期：2021-10-01 浏览：47次中文

阅读说明：本技术 肌肉生长抑制素剪接变体衍生蛋白对肌肉生长抑制素信号的抑制及其利用 (Inhibition of myostatin signal by myostatin splice variant derived proteins and uses thereof ) 是由松尾雅文冈崎宏亮前田和宏于 2020-02-26 设计创作，主要内容包括：本发明提供抑制肌肉生长抑制素信号的方法。以下的(a)或(b)的蛋白：(a)蛋白,该蛋白由下述的氨基酸序列构成；(b)蛋白,该蛋白由与下述的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成,并且可抑制肌肉生长抑制素信号。(The present invention provides methods of inhibiting myostatin signaling. A protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence set forth below; (b) a protein which comprises an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence described below and which inhibits myostatin signal.)

1. A protein of the following (a) or (b):

(a) a protein consisting of the amino acid sequence of SEQ ID NO 1;

(b) a protein consisting of an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and which inhibits myostatin signal.

2. A polynucleotide, the polynucleotide comprising: a nucleotide sequence encoding the protein of claim 1 or a sequence complementary thereto.

3. A vector comprising the polynucleotide of claim 2.

4. A cell comprising the vector of claim 3.

5. A method for producing the protein of (a) or (b) below, the method comprising: culturing the cell according to claim 4, wherein the cell is cultured,

(a) a protein consisting of the amino acid sequence of SEQ ID NO 1;

(b) a protein consisting of an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and which inhibits myostatin signal.

6. A composition for inhibiting myostatin signal, the composition comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

7. A composition for promoting muscle formation, the composition comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

8. A composition for preventing and/or treating a myostatin related disease, the composition comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

9. A medicament, the medicament comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

10. A food product, comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

11. A feed, comprising: at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4.

12. A method for preventing and/or treating a myostatin related disorder, the method comprising: administering to a subject at least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4 in a pharmaceutically effective amount.

13. At least one selected from the group consisting of the protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, and the cell of claim 4, for use in a method of preventing and/or treating a myostatin related disease.

Technical Field

The present invention relates to the inhibition of myostatin signal by a myostatin (myostatin) splice variant derivative protein and uses thereof.

Background

Myostatin is produced intracellularly as a precursor, which is cleaved by proteases to convert to mature myostatin. When the mature myostatin binds to a receptor on the cell surface layer, Smad2/3 is phosphorylated, and phosphorylated Smad2/3 is transferred into the nucleus. Phosphorylated Smad2/3 binds to Smad binding elements present within the promoter of the target gene, thereby inducing expression of the target gene (myostatin signal). Activation of myostatin signal induces gene expression, and the induced factor acts to inhibit muscle formation. In contrast, inhibition of myostatin signal promotes muscle formation. Since promotion of muscle formation is useful for treating muscular dystrophy and other muscular dystrophy diseases, inhibition of myostatin signal is considered to be a therapeutic method for muscular dystrophy (non-patent document 1). In addition, the growth of cancer cells is inhibited by reducing the expression level of myostatin (non-patent document 2). This implies that: reduction of myostatin levels and inhibition of myostatin signal is effective in the treatment of cancer. Furthermore, since myostatin expression is increased in type 2 diabetic patients, it is considered that: there is a certain relationship between myostatin and diabetes (non-patent document 3). From these cases, it is considered that: inhibition of myostatin signal is effective in inhibiting diabetes or inhibiting its progression.

As a method for inhibiting myostatin signal, use of a peptide that binds to myostatin is being examined. The peptide is derived from the sequence of myostatin itself and is capable of substantially inhibiting myostatin signaling. However, there is a problem of low stability when administered to a living body (non-patent document 4 and patent document 1).

Further, a method of inhibiting myostatin signal using a myostatin splice variant of sheep (sheep) is disclosed (non-patent document 5 and patent document 2). However, the same substance as the myostatin splice variant found in this sheep has not been found in humans.

Documents of the prior art

Non-patent document

Non-patent document 1: bogdannovich et al, Nature, 2002, 420; 418-;

non-patent document 2: han et al, Redox biol. 2018, 19; 412-4128;

non-patent document 3: palsgaard et al 2009, 4; e 6575;

non-patent document 4: ohsawa et al, Ploss one, 2015, 10, e 0133713;

non-patent document 5: jeannlong et al, Ploss, 2013, 8, e 81713;

patent document

Patent document 1: WO2014/119753A 1;

patent document 2: WO2006/036074A 1.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to: methods of inhibiting myostatin signaling are provided.

Means for solving the problems

As a result of diligent efforts, the present inventors have found that a protein translated from a myostatin splice variant (a diversified variant due to a change in splicing pattern) inhibits myostatin signaling. The protein translated from the mRNA of the myostatin splice variant, while not having the active region required for mature myostatin, retained more than 90% of the pro domain (fig. 3). Myostatin signal was inhibited by overexpression of myostatin variants in cultured cells (fig. 6). Since the protein translated from the myostatin splice variant is a protein naturally produced in an organism, it is considered to have better stability than an exogenous protein. By inhibiting myostatin signaling, muscle wasting diseases can be treated by promoting muscle formation. In addition, inhibition of cancer cell growth can be inhibited by inhibiting myostatin signaling. It has also been suggested that activation of myostatin signaling accompanied by an increase in myostatin may be associated with diabetes, and thus: inhibition of myostatin signal is effective in preventing diabetes, or inhibiting its progression. Therefore, the protein and the protein expression system can be used for a method for treating a myostatin related disease.

The gist of the present invention is as follows.

(1) A protein of the following (a) or (b):

(a) a protein consisting of the amino acid sequence of SEQ ID NO 1;

(b) a protein consisting of an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and which inhibits myostatin signal.

(2) A polynucleotide comprising a nucleotide sequence encoding the protein of (1) or a sequence complementary thereto.

(3) A vector comprising the polynucleotide of (2).

(4) A cell comprising the vector of (3).

(5) A method for producing the protein of (a) or (b) below, the method comprising: culturing the cells according to (4) above,

(a) a protein consisting of the amino acid sequence of SEQ ID NO 1;

(b) a protein consisting of an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and capable of inhibiting myostatin signal;

(6) a composition for inhibiting myostatin signal, the composition comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(7) A composition for promoting muscle formation, the composition comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(8) A composition for preventing and/or treating a myostatin related disease, the composition comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(9) A medicament, the medicament comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(10) A food product, comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(11) A feed, comprising: at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3) and the cell of (4).

(12) A method for preventing and/or treating a myostatin related disorder, the method comprising: administering to a subject at least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3), and the cell of (4) in a pharmaceutically effective amount.

(13) At least one selected from the group consisting of the protein of (1), the polynucleotide of (2), the vector of (3), and the cell of (4), for use in a method for preventing and/or treating a myostatin-related disease.

Effects of the invention

Myostatin signal can be inhibited by a protein translated from a myostatin splice variant.

The present specification includes contents described in japanese patent application, specification of japanese patent application 2019-37915 and/or drawings, which are the basis of priority of the present application.

Drawings

FIG. 1 shows an example of PCR amplification of myostatin V.

The results of PCR amplification of the Myostatin (MSTN) gene product of human rhabdomyosarcoma cells are shown. 2 amplification products (MSTN, MSTN-V) were obtained by amplification (left). The nucleotide sequence of each product was analyzed, and the resulting exon structure was shown schematically (right). In MSTN-V, the bases of MSTN range from 881 to 1843 are deleted. The sequence of the junction between exon 2 and exon 3 and a part of the nucleotide sequence of 1844 base (bottom right) of each product are shown.

FIG. 2 splicing of the myostatin gene.

MSTN is a gene in which introns 1 and 2 are removed from pre-mRNA of myostatin by splicing, and consists of exon 1, exon 2, and exon 3 (solid line). Intron 2 is the most general intron of sequences having GT and AG at both 5 'and 3' ends. On the other hand, in MSTN-V, intron 2 is spliced differently, and TG within exon 3, which is a potential splice acceptor site, is activated to form the GT-TG intron (dotted line). As a result, in myostatin V, 963 nucleotides 881 to 1843 of exon 3 were deleted.

[ FIG. 3] myostatin V protein.

The protein structures of myostatin and myostatin V are shown schematically. Myostatin is composed of a signal peptide (1 st to 18 th positions), a prodomain (19 th to 266 th positions), and mature myostatin (267 th to 375 th positions) from the N-terminus. In the nucleic acid sequences of myostatin and myostatin V, exon 1 and exon 2 are identical and are common up to amino acid 249. In myostatin V, since the nucleotide sequence of exon 3 is different, the amino acid at position 250 from the N-terminus is asparagine (N), valine (V) at position 251, and a stop codon (. + -.) (SEQ ID NO: 1) at position 252. Thus, myostatin V does not have the domain of mature myostatin.

FIG. 4 myostatin signal.

Myostatin activates downstream signaling by binding precursor-produced mature myostatin to receptors on cell surface layers. If mature myostatin binds to the receptor, Smad2/3 is phosphorylated and phosphorylated Smad2/3 is translocated into the nucleus. Phosphorylated Smad2/3 binds to Smad binding elements (binding elements) present in the promoter of the target gene, thereby inducing expression of the target gene.

[ FIG. 5] expression vectors and expression proteins for myostatin V.

The synthetic nucleic acid was inserted into pcDNA by adding a Nhe I recognition sequence (GCTTGC) to the 5 'side and a BamH I recognition sequence (GGATCC) to the 3' side of a sequence (SEQ ID NO: 3) obtained by optimizing the codon usage frequency of SEQ ID NO: 2^TM3.1(+) recognition sites for Nhe I and BamH I of vector, MSTN-V expression vector (left) was prepared. The prepared vector was introduced into muscle cells, and western blot analysis was performed on the expressed protein. In the sample extracted from the cells into which the MSTN-V expression vector was introduced, a specific band (arrow) was detected at a molecular weight of about 35kDa, and expression of myostatin V was confirmed (right). The molecular weight marker, Mock (Mock) (sample from cell into which empty vector was introduced), and sample from cell into which MSTN-V expression vector was introduced are shown in this order from the left.

[ FIG. 6] inhibition of myostatin signal by myostatin V.

Schematic (left) showing the effect of myostatin V and myostatin on an in vitro myostatin transcriptional activity assay system. In the myostatin signal analysis, the empty vector, the MSTN-N expression vector, and the MSTN-V expression vector were introduced into muscle cells, respectively, and evaluated based on the activity of luciferase expressed by Smad2/3 induction.

The measurement result of the extract solution from the empty vector-introduced cells was 1, and luciferase activity (center, right) was expressed as a relative value. In both human rhabdomyosarcoma cells and human skeletal myoblasts, increased luciferase activity was observed when myostatin was expressed. On the other hand, in the case of expressing myostatin V, a decrease in luciferase activity was observed, suggesting that myostatin V inhibits myostatin signaling.

Detailed Description

Hereinafter, embodiments of the present invention will be described in more detail.

The present invention provides a protein of the following (a) or (b):

(a) a protein consisting of the amino acid sequence of SEQ ID NO 1;

(b) a protein consisting of an amino acid sequence having at least 70% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and which inhibits myostatin signal.

(a) The protein of (1) is a protein consisting of the amino acid sequence of SEQ ID NO. 1, which is translated from a human myostatin splice variant. As shown in FIG. 3, myostatin (375aa, 43kDa) is composed of the signal peptide (1-18), the prodomain (19-266) and the region of mature myostatin (267-375), while the myostatin variant is composed of the signal peptide (1-18) and a portion of the prodomain (19-251), and the C-terminal amino acid (251) is substituted with proline to valine. Thus, mature myostatin is not formed in the myostatin variants.

(a) The protein of (a) can inhibit myostatin signal (fig. 4).

(a) The protein of (3) can be produced as follows: the protein of (a) can be produced by extracting RNA from human rhabdomyosarcoma cells (CRL-2061, ATCC), synthesizing cDNA using reverse transcriptase and random primers, amplifying the cDNA by PCR, determining the sequence by sequence analysis, adding restriction enzyme recognition sequences to the 5 'side and 3' side of the sequence obtained by optimizing the codon usage frequency of the open reading frame, inserting the sequence into an appropriate vector, introducing the vector into an appropriate host cell, and producing the protein as a recombinant protein.

(b) The protein of (1) is a protein which is composed of an amino acid sequence having at least 70% or more sequence identity with the amino acid sequence of SEQ ID NO. 1 and which can inhibit myostatin signal. As described in examples described later, when myostatin activates signal transduction, transcription factor (Smad protein) binds to the Smad binding sequence to induce transcription, and therefore, this phenomenon is utilized, and a reporter gene in which a luciferase gene is arranged downstream of the Smad binding sequence and a vector expressing the protein of (b) are introduced into cells together, and luminescence of luciferase is measured, whereby whether myostatin signal is inhibited or not can be evaluated.

(b) The sequence identity of the protein of (a) with the amino acid sequence of the protein of (a) is at least 70% or more, and more preferably 80% or more, 90% or more, 95% or more, and 98% or more in this order. (b) The protein of (1) may be a protein having an amino acid sequence in which 1 or more (2 to 76, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 75, 76.) amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO. 1, and which can inhibit myostatin signal.

(b) The protein of (4) can be prepared by substituting an arbitrary amino acid in the protein of (a) with another amino acid by a site-specific mutagenesis method.

The present invention provides a polynucleotide comprising: a nucleotide sequence encoding the protein of (a) or (b) or a sequence complementary thereto.

The polynucleotide of the present invention may be single-stranded or double-stranded. In the case of double strands, it is composed of a polynucleotide comprising a nucleotide sequence encoding the protein of (a) or (b) and the complementary strand thereof.

The polynucleotide may be any of DNA, RNA, and a chimeric of DNA and RNA, and the nucleotides constituting the polynucleotide may be modified. As the modified nucleotide, there can be exemplified: sugar-modified nucleotides (e.g., nucleotides in which D-ribofuranose is 2 ' -O-alkylated, nucleotides in which D-ribofuranose is 2 ' -O, 4 ' -C-alkyleneated), nucleotides in which phosphodiester bond is modified (e.g., thioated), nucleotides in which base is modified, and nucleotides in which these are combined.

Examples of the nucleotide sequence encoding the protein of (a) include: 2, SEQ ID NO. The nucleotide sequence of SEQ ID NO 2 is the sequence of the mRNA of a myostatin variant extracted from human rhabdomyosarcoma cells (CRL-2061, ATCC). The nucleotide sequence of SEQ ID NO: 2 comprises a 5 'untranslated region, an open reading frame (sequence between the start codon (atg) and the stop codon (tga)), a 3' untranslated region, and poly (A). The nucleotide sequence encoding the protein of (a) may be a sequence between the start codon (atg) and the stop codon (tga) in the nucleotide sequence of SEQ ID NO. 2 or a sequence comprising the same. The nucleotide sequence of the open reading frame can be optimized for codon usage frequency, and as an example, the nucleotide sequence obtained by optimizing the codon usage frequency of the open reading frame of SEQ ID NO. 2 is shown in SEQ ID NO. 3.

The polynucleotide comprising the nucleotide sequence encoding the protein of (a) can be prepared, for example, by the method described in example 1, which will be described later.

A polynucleotide comprising a sequence complementary to a nucleotide sequence encoding the protein of (a) can be synthesized from mRNA of a myostatin variant comprising a nucleotide sequence encoding the protein of (a) having a poly (a) chain at the 3' end using reverse transcriptase and an oligo dT primer. The mRNA can be degraded by alkali treatment, and then double-stranded with reverse transcriptase or DNA polymerase using the resulting single-stranded DNA as a template.

The nucleotide sequence encoding the protein of (b) and the sequence complementary thereto can be obtained by, for example, introducing base substitution mutations into the nucleotide sequence encoding the protein of (a) and the sequence complementary thereto by site-specific mutagenesis.

The protein of (a) or (b) can be produced by inserting a DNA encoding the protein of (a) or (b) into a vector to produce a recombinant vector, introducing the recombinant vector into a host cell, transforming the host cell, and culturing the transformed cell to produce the protein of (a) or (b). Accordingly, the present invention provides a method of making a protein of (a) or (b), the method comprising: culturing a cell comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding the protein of (a) or (b) or a sequence complementary thereto. The present invention also provides a vector (recombinant vector) comprising a polynucleotide comprising a nucleotide sequence encoding the protein of (a) or (b) or a sequence complementary thereto. In addition, the present invention provides a cell comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding the protein of (a) or (b) or a sequence complementary thereto.

The recombinant vector of the present invention can be obtained by inserting a polynucleotide comprising a nucleotide sequence encoding the protein of (a) or (b) and a sequence complementary thereto into an appropriate vector.

As the vector, there can be used a plasmid derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13, pUC19, pET-44, pBluescriptII), a plasmid derived from Bacillus subtilis (e.g., YEp13, pYES2, YRp7, YIp5, pYAC2, pUB110, pTP5, pC194), a plasmid derived from yeast (e.g., pSH19, pSH15), a plasmid derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13, pUC19, and a plasmid derived from Bacillus subtilis (e.g., YEp13, pYES2, YRp 7), a plasmid derived from yeast (e.g., pSH19, pSH15), a plasmid derived from Bacillus subtilis, a plasmid derived from a strain, such as a plasmid derived from a strain, such as a plasmid derived from a strain, such as a strain, a,λBacteriophage (A)λphage (bacteriophages), such as phage), animal viruses such as retrovirus, adenovirus, lentivirus, adeno-associated virus, vaccinia virus, and the like, and entomopathogenic viruses such as baculovirus.

Promoters, enhancers, terminators, splicing signals, Poly a-added signals, selection markers, SV40 replication origins, and the like may be added to the expression vector.

In addition, the expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercially available, and can be exemplified by: pGEX series (GE Healthcare), Novagen's (registered trademark) pET Systems (Merck), Clontech fluorescent protein vector series (Takara), expression vectors for adding His6 and HaloTag (Promega), FLAG tag fusion protein expression system (Sigma-Aldrich), pCruz^TMExpression vector series for mammalian cells (Santa Cruz Biotechnology Co., Ltd.), and the like.

Transformed cells can be obtained by introducing the recombinant vector of the present invention into host cells. The present invention also provides a cell (host cell) into which the recombinant vector has been introduced.

As the host, there can be exemplified: bacterial cells (e.g., Escherichia bacteria, Bacillus subtilis, etc.), fungal cells (e.g., yeast, Aspergillus, etc.), insect cells (e.g., S2 cells, Sf cells, etc.), animal cells (e.g., CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells, and the like.

Introduction of the recombinant vector into the host can be carried out by a method described in Molecular Cloning, 2 nd edition, J.Sambrook et al, Cold Spring Harbor Lab. Press, 1989 (for example, calcium phosphate method, DEAE-dextran method, transfection method, microinjection method, lipofection method, electroporation method, transduction method, scrape-loading method, shotgun method, etc.) or infection.

The transformed cells may be cultured in a medium, and the protein of (a) or (b) may be collected from the culture. When the protein (a) or (b) is secreted into the medium, the medium may be recovered, and the protein (a) or (b) may be isolated from the medium and purified. In the case where the protein of (a) or (b) is produced in the transformed cell, the cell may be lysed, and the protein of (a) or (b) may be isolated from the lysate and purified.

In the case where the protein of (a) or (b) is expressed as a fusion protein with other proteins (functioning as tags), a factor Xa or enzyme (enterokinase) treatment may be performed after the fusion protein is isolated and purified, thereby cleaving the other proteins to obtain the protein of (a) or (b) of interest.

(a) The isolation and purification of the protein of (a) or (b) can be carried out by known methods. As known separation and purification methods, there can be used: a method utilizing solubility such as salting-out or solvent precipitation, a method utilizing a difference in molecular weight such as dialysis, ultrafiltration, gel filtration, or SDS-polyacrylamide gel electrophoresis, a method utilizing a difference in charge such as ion exchange chromatography, a method utilizing specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, and a method utilizing a difference in isoelectric point such as isoelectric point electrophoresis.

Muscle formation can be promoted by inhibiting myostatin signal using a protein of the invention, a polynucleotide comprising a nucleotide sequence encoding a protein of the invention or a sequence complementary thereto, a vector comprising a nucleotide of the invention and/or a cell of the invention. Accordingly, the present inventionProviding a composition for inhibiting myostatin signal, the composition comprising: at least one of a protein selected from (a) and/or (b), a polynucleotide comprising a nucleotide sequence encoding the protein or a sequence complementary thereto, a vector comprising the polynucleotide, and a cell comprising the vector. In addition, the present invention also provides a composition for promoting muscle formation, the composition comprising: at least one of a protein selected from (a) and/or (b), a polynucleotide comprising a nucleotide sequence encoding the protein or a sequence complementary thereto, a vector comprising the polynucleotide, and a cell comprising the vector. When the vector contains a polynucleotide comprising a nucleotide sequence encoding the protein of (a) and/or (b) or a sequence complementary thereto, the vector may be any vector capable of introducing a polynucleotide comprising a nucleotide sequence encoding the protein of (a) and/or (b) or a sequence complementary thereto into a cell, and examples thereof include: adenovirus, retrovirus, lentivirus, adeno-associated virus, Sendai virus, liposome, plasmid and other gene therapy vectors. In addition, the product obtained by introducing the polynucleotide or vector of the present invention into cells (self, homologous species) can be used for cell therapy. Methods for introducing a target gene into a vector, methods for introducing a recombinant vector into a cell, methods for administering a cell into which a recombinant vector or gene has been introduced to a human, and administration sites are known, and they can be applied to the present invention as they are or after modification. Genome editing techniques can be utilized in gene therapy or cell therapy. ZFN can be used in genome editing: (zinc-finger nuclase: zinc finger nucleases), TALEN: (transcription activator-like effector nuclase: transcription activator-like effector nuclease), CRISPR/Cas9 (CRISPR/Casclustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9: clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins9) And the like.

The composition of the present invention can be used for medicines, experimental reagents, foods, feeds, etc.

The pharmaceutical composition can be used for the prevention and/or treatment of myostatin related diseases (myostatin related may be direct or indirect), specifically, muscular dystrophy diseases (e.g., muscular dystrophy, spinal cord muscular atrophy, sarcopenia, disuse muscular atrophy), circulatory diseases (e.g., cardiac insufficiency, arteriosclerosis, etc.), renal diseases (e.g., chronic renal insufficiency, etc.), bone diseases (e.g., inflammatory arthritis, etc.), cancer, or diabetes. The myostatin related disease can be a disease in which a reduction in the amount of myostatin or inhibition of myostatin signal is effective. The present invention provides a method for preventing and/or treating a myostatin related disease, the method comprising: administering to a subject at least one of a protein selected from (a) and/or (b), a polynucleotide comprising a nucleotide sequence encoding the protein or a sequence complementary thereto, a vector comprising the polynucleotide, and a cell comprising the vector in a pharmaceutically effective amount. In addition, the present invention also provides: at least one of a protein selected from (a) and/or (b), a polynucleotide comprising a nucleotide sequence encoding the protein or a sequence complementary thereto, a vector comprising the polynucleotide, and a cell comprising the vector, for use in a method for preventing and/or treating a myostatin-associated disease.

Since inhibition of myostatin also results in an increase in skeletal muscle mass, it is useful in the treatment of all diseases that present muscle atrophy, regardless of the cause of muscle atrophy. The increase in skeletal muscle mass can increase exercise amount and contribute to improvement of systemic metabolism. In addition, it is expected to act on the myocardium to restore its function.

On the other hand, myostatin inhibition is also expected to: acting on osteoclast to inhibit bone destruction, activating the homeostatic maintenance ability of vascular endothelial cells, inducing apoptosis, and increasing insulin sensitivity.

At least one of the protein selected from (a) and/or (b), a polynucleotide comprising a nucleotide sequence encoding the protein or a sequence complementary thereto, a vector comprising the polynucleotide, and a cell comprising the vector (hereinafter, referred to as "active ingredient") may be used alone or in combinationOr orally or parenterally administered to mammals (e.g., humans, rabbits, dogs, cats, rats, mice) in the form of pharmaceutical compositions in appropriate dosage forms, together with pharmacologically acceptable carriers, diluents or excipients. The dose varies depending on the subject, the disease, the symptom, the administration route, etc., and for example, in the case of preventing/treating a muscle-wasting disease (for example, muscular dystrophy), the dose is usually 0.1 in the case of 1 dose of the active ingredient, and in the case of a protein as the active ingredientμAbout g to 100mg/kg body weight, preferably about 0.5mg to 100mg/kg body weight, usually about 0.1 to 50 mg/kg body weight, preferably about 0.5 mg/kg body weight in the case where the active ingredient is a polynucleotide, and usually about 1X 10 in the case where the active ingredient is a vector containing a polynucleotide¹⁴～9×10¹⁴Genome copy/kg body weight of about, preferably 1X 10¹²The genome copy/kg body weight may be administered (preferably continuously or every other day) by oral administration/intramuscular injection/subcutaneous injection/intravenous injection at a frequency of about 1 time per week to about 1 time per month or about 1 time per year, preferably about 1 time per year. In the case of cells containing a carrier as an active ingredient, the administration may be carried out by intramuscular injection, subcutaneous injection, intravenous injection, preferably intravenous injection, at a frequency of about 1 time per week to about 1 time per month or about 1 time per year, preferably about 1 time per year, in 100,000 cells in 1 dose of the active ingredient.

In the case of other parenteral and oral administrations, amounts based thereon may also be administered. In cases where symptoms are particularly severe, the amount may be increased according to the symptoms.

As compositions for oral administration, mention may be made of: solid or liquid dosage forms, specifically tablet (including sugar-coated tablet and film-coated tablet), pill, granule, powder, capsule (including soft capsule), syrup, emulsion, suspension, etc. Such compositions may be prepared by conventional methods and may contain carriers, diluents or excipients commonly used in the formulation art. Examples of the carrier and excipient for tablets include: lactose, starch, sucrose, magnesium stearate, and the like.

As compositions for parenteral administration, for example, there may be mentioned: the injection can be intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous drip injection, etc. Such injections are prepared by a conventional method, that is, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily liquid generally used for injections. Examples of the aqueous injection solution include: physiological saline, isotonic solution containing glucose or other adjuvants, etc., and suitable cosolvents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50mol) adduct of hydrogenated castor oil), etc.) may be used in combination. Examples of the oily liquid include: sesame oil, soybean oil, etc., and benzyl benzoate, benzyl alcohol, etc. can be used together as cosolvent. The prepared injection is usually filled in an appropriate ampoule. Suppositories for rectal administration can be prepared by mixing the active ingredient in a usual suppository base.

The pharmaceutical composition for oral administration or parenteral administration may be formulated into dosage forms of administration units (dosage units) suitable for the administration amount of the active ingredient. The dosage form of such administration unit includes: tablets, pills, capsules, injections (ampoules), suppositories and the like, each of which preferably contains 0.1 to 1.000mg of the active ingredient per administration unit dosage form.

Can be used as food and feed for promoting muscle formation of human or animal. The animal may be any animal expressing myostatin, and examples thereof include: mammals such as cats, dogs, sheep, pigs, cattle, chickens, turkeys, etc.; working and edible breeding animals such as salmon, trout, cod, tuna, yellow tail fish, etc.

The food or feed of the present invention may be added with general components such as protein, lipid, carbohydrate, and sodium, minerals such as potassium, calcium, magnesium, and phosphorus, trace elements such as iron, zinc, copper, selenium, and chromium, vitamin A, vitamin B, vitamin C, vitamin E, vitamin C, and vitamin D,βCarotene, vitamin B₁Vitamin B₂Vitamin B₆Vitamin B₁₂Vitamin C, nicotinic acid, folic acid and vitamin D₃Vitamins such as vitamin E, biotin and pantothenic acid, coenzyme Q10,αLipoic acid, galacto-oligosaccharides, dietary fibres, excipients (water, carboxymethyl cellulose, lactose, etc.), sweeteners, flavours (malic acid, citric acid, amino acids, etc.), flavours, etc. When the food or feed of the present invention is prepared as a liquid preparation, water, physiological saline, soup, milk, fruit juice, or the like can be used as a liquid for dispersing or dissolving the food or feed components. The food and feed of the present invention can be made into the form of powder, granule, tablet, liquid preparation, etc. In order to make it easy for patients or the elderly to take, it is preferable to prepare a gel product such as jelly.

The food or feed can be ingested in an amount, frequency, or period of ingestion to confirm the desired effect.

Examples

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 1 cloning and characterization of myostatin variants

In RNaseOUT^TM500ng of RNA extracted from human rhabdomyosarcoma cells (CRL-2061, ATCC) using a high purity RNA isolation kit (#11828665001, Roche Life Science) was reverse transcribed into cDNA by M-MLV reverse transcriptase (#28025013, Thermo Fisher Scientific) using random primers (#48190011, Thermo Fisher Scientific) in the presence of recombinant ribonuclease inhibitor (#10777-019, Thermo Fisher Scientific). The resulting cDNA was subjected to PCR using primers MSTN Ex1_ F1, 5'-agattcactggtgtggcaag-3' (SEQ ID NO: 6), MSTN R2, 5'-tgcatgacatgtctttgtgc-3' (SEQ ID NO: 7), TaKaRa Ex Taq (registered trademark) DNA polymerase (# RR001A, Takara). The PCR product was subjected to agarose gel electrophoresis. Amplified bands were obtained as PCR fragments of 2.5kbp and 1.5kbp between 2kbp and 3kbp and between 1kbp and 2kbp of the DNA size marker by electrophoresis (electrophoresis) (FIG. 1, left). DNA was extracted from each fragment region using MinElute (registered trademark) gel extraction kit (#28604, Qiagen), and the extracted fragments were passed through DNALigation kit version 2.1 (#6022, Takara) was subcloned into pT7Blue (#69820, Novagen). The subcloned sequences were amplified by PCR using the primers MSTN Ex1_ F1, MSTN R2, TaKaRa Ex Taq (registered trademark) DNA polymerase (# RR001A, TaKaRa), purified by a MinElute (registered trademark) PCR purification kit (#28006, QIAGEN), and then subjected to sequencing analysis by Sanger method (Sanger method).

As a result of the sequencing, the PCR fragment of about 2.5kbp was a normal splicing product (MSTN) consisting of all of exon 1(Ex1), exon 2(Ex2) and exon 3(Ex3) of the Myostatin (MSTN) gene. The MSTN is shown schematically in the right part of FIG. 1, and the sequence of the junction of exons 2 and 3 and a part of the sequence of the 1844 th base region within an exon are shown in the lower right part of FIG. 1. On the other hand, the nucleotide sequence (SEQ ID NO: 2) of a fragment of about 1.5kbp of myostatin variant (MSTN-V) was identical to MSTN up to exon 1 and exon 2 of the myostatin gene. However, it is completely different from MSTN in the sequence corresponding to exon 3. The sequence starting with aat ccg ttt becomes the sequence starting with aat gtc tga. The sequence below aat gtc tga perfectly matched the sequence downstream of base 1844 within exon 3 of MSTN. This shows the deletion of the region of exon 3 corresponding to positions 881 to 1843 of the nucleic acid sequence.

Intron 2 of MSTN is the most common intron with sequences of GT and AG at both 5 'and 3' ends. On the other hand, intron 2 in MSTN-V splices differently, and TG within exon 3, which is a potential splice acceptor site, is activated to form the GT-TG intron. As a result, in myostatin V, 963 nucleotides 881 to 1843 of exon 3 were deleted (FIG. 2).

The identified MSTN-V mRNA has a start codon and a stop codon, suggesting a protein translated into 251 amino acids. The amino acids from the N-terminal side to position 250 of myostatin V are identical sequences to myostatin, but valine is at position 251 and a stop codon is at position 252. Since mature myostatin is composed of amino acids 267 to 375 of myostatin, mature myostatin is not produced from MSTN-V mRNA (SEQ ID NO: 1, FIG. 3).

Myostatin acts via myostatin signal. That is, when mature myostatin produced from a precursor binds to a receptor present in a cell membrane, Smad2/3 is phosphorylated. Phosphorylated Smad2/3 was transferred into the nucleus, and the gene expression was accelerated by binding to a Smad binding element located upstream of the target gene (fig. 4). The amino acid sequence of the myostatin precursor is shown in SEQ ID NO. 5, and the sequence of mRNA is shown in SEQ ID NO. 4.

EXAMPLE 2 expression of myostatin variants in cultured cells

In order to express myostatin V in cultured cells, an MSTN-V expression vector was prepared. A nucleic acid was artificially synthesized by Fasmac, Inc., in which the codon usage frequency of the open reading frame of SEQ ID NO. 2 was optimized (SEQ ID NO: 3), a Nhe I recognition sequence (GCTTGC) was added to the 5 'side, and a BamH I recognition sequence (GGATCC) was added to the 3' side, and then inserted into cDNA^TM3.1(+) vector Nhe I, BamH I site of (# V79020, Thermo Fisher Scientific) (FIG. 5 left).

Protein expression from the MSTN-V expression vector was confirmed by western blotting. Human rhabdomyosarcoma cells (CRL-2061, ATCC) were introduced with MSTN-V expression vector and empty vector (pcDNA) to be compared using Lipofectamin (registered trademark) 2000 (11668019, Thermo Fisher Scientific)^TM3.1(+)). 24 hours after the introduction of the vector, the cells were disrupted with Cell lysis buffer (#9803, Cell Signaling) (1 mM PMSF (#8553, Cell Signaling) was added) to obtain a soluble fraction as a sample. Protein quantification of the obtained sample was performed using the Qubit (registered trademark) protein assay kit (# Q33211, Thermo Fisher Scientific). The SDS-PAGE sample was prepared by mixing the sample with 4X Laemmli sample buffer (#1610747, Bio-Rad) (2-mercaptoethanol (#1610710, Bio-Rad) was added), and then heat-treating the mixture. In SDS-PAGE, Mini-PROTEAN (registered trademark) TGX was used^TMPrecast Gels 4-20% Gel (# 456-. As a molecular weight marker, Precision Plus Protein was used^TMTwo-color standard (# 161)0374. BIO-RAD). Transcription onto membranes Using Trans-Blot Turbo^TMTranscription System (Bio-Rad). Membranes after protein transcription were incubated with 2% ECL at room temperature^TMAfter 1 hour of Prime blocking agent (# RPN418, Amersham) blocking, an antibody recognizing the N-terminal side of myostatin (anti-GDF 8/myostatin antibody, # ab71808, abcam) or an antibody recognizing actin was used as a primary antibody ((ii) (iii))βActin antibodies (C4), # sc-47778, Santa Cruz Biotechnology) were treated overnight at 4 ℃. The secondary antibody was treated with HRP-labeled anti-rabbit IgG antibody (# NA934, GE), HRP-labeled anti-mouse IgG antibody (# NA931, GE) at room temperature for 1 hour. Detection was performed using Amersham^TM ECL Select^TMWestern blot assay reagents (# RPN2235, GE) by ChemiDoc^TMXRS + system (Bio-Rad). As a result, a band of myostatin V was detected at around 35kDa (FIG. 5, right). Actin was also analyzed at the same time, resulting in bands.

EXAMPLE 3 inhibition of myostatin Signal by myostatin variants

The inhibitory activity of myostatin V on myostatin signals was evaluated using an in vitro myostatin transcriptional activity assay system. In this evaluation system, a reporter gene (SBE4-Luc plasmid, #16495, Addgene) in which a luciferase gene is arranged downstream of the Smad binding sequence was introduced into cells, and the luminescence of luciferase induced by expression was measured to evaluate myostatin signals (fig. 6 left). In addition to the MSTN-V expression vector, a myostatin (MSTN-N) expression vector was used for the study. The MSTN-N expression vector is a nucleic acid obtained by adding a Nhe I recognition sequence (GCTTGC) to the 5 'side of myostatin cDNA (SEQ ID NO: 4) and a BamH I recognition sequence (GGATCC) to the 3' side of myostatin cDNA (SEQ ID NO: 4) artificially synthesized by Fasmac, Ltd, and inserted into pcDNA^TM3.1(+) vector (V79020, Thermo Fisher Scientific) at Nhe I, BamH I site. Expression of myostatin (SEQ ID NO: 5) from the MSTN-N expression vector was confirmed by Western blotting in the same manner as for myostatin V.

Using Lipofectamin (registered trademark) 2000 (#11668019, Thermo Fisher Scientific) 2 vectors were simultaneously introduced into Human rhabdomyosarcoma cells (CRL-2061, ATCC) and Human Skeletal myoblasts (Human Skeletal Myoblast). One is SBE4-Luc plasmid, the other is MSTN-V expression vector or MSTN-N expression vector or empty vector (pcDNA)^TM3.1 (+)). Cells were disrupted using a reporter gene lysis buffer of a luciferase assay system (E4030, Promega) with a reporter gene lysis buffer 24 hours after vector introduction to obtain a soluble fraction as a sample. Protein quantification of the obtained sample was performed using the Qubit (registered trademark) protein assay kit (# Q33211, Thermo Fisher Scientific). Luciferase activity was measured by using a luciferase assay system of the luciferase assay system (# E4030, Promega) with a reporter gene lysis buffer as a substrate and using a multi-label plate reader (Multi-label plate reader) ARVO^TM3 (PerkinElmer) luciferase luminescence signal was measured for evaluation.

The luciferase activity was expressed as a relative value, with the measurement result of the extract solution from the empty vector-introduced cells being 1 (center, right in FIG. 6). In both human rhabdomyosarcoma cells and human skeletal muscle myoblasts, an increase in specific luciferase activity was observed when myostatin was expressed. On the other hand, in the case of expressing myostatin V, a decrease in specific luciferase activity was observed. The luciferase activity of the experimental system correlated with myostatin signal, an increase in luciferase activity indicated an increase in myostatin signal, and a decrease in luciferase activity indicated an inhibition of myostatin signal. Thus, it can be seen that: myostatin signal is inhibited by expression of myostatin V.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

Industrial applicability

The invention can be used to promote muscle formation in humans or animals.

Sequence Listing free text

＜SEQ ID NO: 1＞

The amino acid sequence of the myostatin variant protein is shown (all 251 amino acids).

＜SEQ ID NO: 2＞

The nucleotide sequence of the myostatin variant is shown (all 1860 bases. start codon (atg) and stop codon (tga) are shown in boxes).

＜SEQ ID NO: 3＞

Myostatin V (MSTN-V) nucleotide sequence inserted into an expression vector

An Nhe I site was added to the 5 'side, and a BamH I site was added to the 3' side, and inserted into the vector.

(all 1860 bases. Start codon (atg) and stop codon (tga) in boxes)

＜SEQ ID NO: 4＞

A myostatin (MSTN-N) nucleotide sequence inserted into the expression vector.

Add Nhe I site on the 5 'side, BamH I site on the 3' side, and insert into vector (all 2823 bases. start codon (atg) and stop codon (tga) in box)

＜SEQ ID NO: 5＞

Amino acid sequence information of myostatin.

Amino acid sequence (all 375 amino acids)

＜SEQ ID NO: 6＞

Sequence of primer MSTN Ex1_ F1. 5'-agattcactggtgtggcaag-3'

＜SEQ ID NO: 7＞

Sequence of primer MSTN R2. 5'-tgcatgacatgtctttgtgc-3' are provided.

<110> Shenhu Natural products chemical Co., Ltd

School courts and courts

<120> inhibition of myostatin signal by myostatin splice variant derived protein and use thereof

<130> FP-277PCT

<150> JP2019-37915

<151> 2019-03-01

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 251

<212> PRT

<213> Homo sapiens

<400> 1

Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile

1 5 10 15

Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn

20 25 30

Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr

35 40 45

Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu

50 55 60

Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu

65 70 75 80

Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val

85 90 95

Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His

100 105 110

Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu

115 120 125

Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser

130 135 140

Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu

145 150 155 160

Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu

165 170 175

Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu

180 185 190

Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val

195 200 205

Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly

210 215 220

Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr

225 230 235 240

Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Val

245 250

<210> 2

<211> 1860

<212> DNA

<213> Homo sapiens

<400> 2

agattcactg gtgtggcaag ttgtctctca gactgtacat gcattaaaat tttgcttggc 60

attactcaaa agcaaaagaa aagtaaaagg aagaaacaag aacaagaaaa aagattatat 120

tgattttaaa atcatgcaaa aactgcaact ctgtgtttat atttacctgt ttatgctgat 180

tgttgctggt ccagtggatc taaatgagaa cagtgagcaa aaagaaaatg tggaaaaaga 240

ggggctgtgt aatgcatgta cttggagaca aaacactaaa tcttcaagaa tagaagccat 300

taagatacaa atcctcagta aacttcgtct ggaaacagct cctaacatca gcaaagatgt 360

tataagacaa cttttaccca aagctcctcc actccgggaa ctgattgatc agtatgatgt 420

ccagagggat gacagcagcg atggctcttt ggaagatgac gattatcacg ctacaacgga 480

aacaatcatt accatgccta cagagtctga ttttctaatg caagtggatg gaaaacccaa 540

atgttgcttc tttaaattta gctctaaaat acaatacaat aaagtagtaa aggcccaact 600

atggatatat ttgagacccg tcgagactcc tacaacagtg tttgtgcaaa tcctgagact 660

catcaaacct atgaaagacg gtacaaggta tactggaatc cgatctctga aacttgacat 720

gaacccaggc actggtattt ggcagagcat tgatgtgaag acagtgttgc aaaattggct 780

caaacaacct gaatccaact taggcattga aataaaagct ttagatgaga atggtcatga 840

tcttgctgta accttcccag gaccaggaga agatgggctg aatgtctgag gctaccaggt 900

ttatcacata aaaaacattc agtaaaatag taagtttctc ttttcttcag gtgcattttc 960

ctacacctcc aaatgaggaa tggattttct ttaatgtaag aagaatcatt tttctagagg 1020

ttggctttca attctgtagc atacttggag aaactgcatt atcttaaaag gcagtcaaat 1080

ggtgtttgtt tttatcaaaa tgtcaaaata acatacttgg agaagtatgt aattttgtct 1140

ttggaaaatt acaacactgc ctttgcaaca ctgcagtttt tatggtaaaa taatagaaat 1200

gatcgactct atcaatattg tataaaaaga ctgaaacaat gcatttatat aatatgtata 1260

caatattgtt ttgtaaataa gtgtctcctt ttttatttac tttggtatat ttttacacta 1320

aggacatttc aaattaagta ctaaggcaca aagacatgtc atgcatcaca gaaaagcaac 1380

tacttatatt tcagagcaaa ttagcagatt aaatagtggt cttaaaactc catatgttaa 1440

tgattagatg gttatattac aatcatttta tattttttta catgattaac attcacttat 1500

ggattcatga tggctgtata aagtgaattt gaaatttcaa tggtttactg tcattgtgtt 1560

taaatctcaa cgttccatta ttttaatact tgcaaaaaca ttactaagta taccaaaata 1620

attgactcta ttatctgaaa tgaagaataa actgatgcta tctcaacaat aactgttact 1680

tttattttat aatttgataa tgaatatatt tctgcattta tttacttctg ttttgtaaat 1740

tgggattttg ttaatcaaat ttattgtact atgactaaat gaaattattt cttacatcta 1800

atttgtagaa acagtataag ttatattaaa gtgttttcac atttttttga aagacaaaaa 1860

<210> 3

<211> 1860

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized sequence

<400> 3

agattcactg gtgtggcaag ttgtctctca gactgtacat gcattaaaat tttgcttggc 60

attactcaaa agcaaaagaa aagtaaaagg aagaaacaag aacaagaaaa aagattatat 120

tgattttaaa atcatgcaga agctccagct ttgcgtgtac atctacctgt tcatgctgat 180

agttgcaggc ccagtggatc tgaatgagaa cagcgaacag aaggagaacg tagagaagga 240

aggcttgtgc aatgcctgta cttggcggca gaatacgaaa tcttcccgta ttgaggccat 300

caagatccag attctcagca aactgcgcct tgaaactgca cctaacatca gcaaggacgt 360

aatcagacag cttctgccca aagctcctcc actgagagag ctcattgacc agtacgacgt 420

ccaacgagat gacagttcag atggctcact tgaggatgac gactatcatg ccactaccga 480

aaccatcatt acaatgccga ccgaaagcga tttcctgatg caagtggatg ggaaaccaaa 540

gtgttgcttc ttcaagtttt cctccaagat ccagtacaac aaagtcgtca aggcgcaact 600

gtggatatat ctgaggcccg ttgagactcc aacaaccgtg tttgtgcaga ttttgaggct 660

gatcaagccc atgaaagacg gaacacgcta taccggaata cggagtctga aactggacat 720

gaatcccggt acagggattt ggcagtctat cgacgtcaaa acggttctcc agaactggct 780

gaaacaaccg gagtctaatc tcgggattga gatcaaggcc ttggacgaaa atggccacga 840

tctggctgtg acctttcctg gtcctggaga agatggcctg aacgtgtgag gctaccaggt 900