JP5783557B2 - Mutant natriuretic peptide B receptor, gene encoding the receptor and use thereof - Google Patents

Description

  The present invention relates to a mutant natriuretic peptide B receptor that produces cyclic guanosine 3 ', 5'-monophosphate without ligand stimulation, a gene encoding the receptor, and use thereof.

  Cyclic guanosine 3 ', 5'-monophosphate (hereinafter also referred to as cGMP) is an important second messenger having various biological activities. cGMP is involved in vasodilation, cartilage and bone elongation, neurotransmission, and the like. cGMP is synthesized from GTP in cells by the natriuretic peptide B receptor (hereinafter also referred to as NPR-B). NPR-B is a transmembrane protein, has a ligand-binding domain that binds to C-type natriuretic peptide (hereinafter also referred to as CNP) outside the cell, and synthesizes cGMP from GTP in the cell. It has an active enzyme region. Wild-type NPR-B is activated by binding of CNP as a ligand, and synthesizes cGMP from GTP. Also, cGMP is hydrolyzed to 5'-GMP by the action of cyclic nucleotide phosphodiesterase.

  It is known that the decrease of intracellular cGMP is a primary or secondary cause of various diseases. Conventionally, as a method for suppressing the decrease in cGMP, a method of increasing intracellular cGMP concentration by using a phosphodiesterase inhibitor that degrades cGMP has been generally used (Non-patent Document 1). Inhibitors of cGMP-degrading enzymes have been developed as therapeutic agents for erectile dysfunction, pulmonary hypertension, heart failure and the like. However, currently developed cGMP-degrading enzyme inhibitors have problems such as specificity as an inhibitor and short-lasting effects, problems such as systemic side effects (such as hypotension), and are safer and more effective. In particular, development of a drug that increases intracellular cGMP levels (cGMP levels) is desired.

  As a substance that increases intracellular cGMP, riociguat BAY63-2521, a soluble guanylate cyclase stimulant, is under development by Bayer HealthCare (Non-patent Document 2). However, development of a drug that increases intracellular cGMP more safely and more effectively is desired. Non-Patent Document 3 reports that an active mutation of guanylate cyclase A (GC-A) was produced, but NPR-B, which is guanylate cyclase B (GC-B), is described. Absent.

Circulation Heart Failure, 2008; 1; 272-280 European Respiratory Journal; 2008, vol. 32, no. 4, 881-891 (http://erj.ersjournals.com/content/32/4/881.long) Proc. Natl. Acad. Sci. USA, 1997, vol.94, 459-462

  In view of the above problems, the present invention provides a mutant natriuretic peptide B receptor that produces cGMP without CNP stimulation, a gene that encodes the receptor, and an intracellular cGMP that effectively uses the receptor or gene. It is an object of the present invention to provide a non-human mammal or the like in which a drug capable of increasing the amount of the gene and a gene encoding a mutant natriuretic peptide B receptor are introduced into the genome.

  As a result of repeated studies to solve the above problems, the present inventors have found that wild-type NPR-B produces cGMP only when the ligand CNP is present (when CNP is bound). A mutant natriuretic peptide B receptor (mutant NPR-B) that always produces cGMP was found even in the absence of glycine. So far, several receptors with amino acid substitutions in the amino acid sequence of wild-type NPR-B have been reported, but all of the mutant receptors found so far have wild-type cGMP-producing ability. It was lower than type NPR-B. The mutant NPR-B consisting of the amino acid sequence of SEQ ID NO: 2 found by the present inventors is a polypeptide consisting of an amino acid sequence in which the 883rd valine is substituted with methionine in the amino acid sequence of wild-type human NPR-B. This is a novel one different from the mutant NPR-B found so far. The nucleotide sequence of wild type human NPR-B is registered in GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide) with an accession number of NM — 003995.3.

  The mutant NPR-B consisting of the amino acid sequence of SEQ ID NO: 2 found by the present inventors always produces cGMP even in the absence of CNP. Further, when CNP is added to the mutant NPR-B, the CNP concentration Since the amount of cGMP production increased dependently, it was found that activation of the catalyst region also occurred by a normal cycle.

  The cGMP-degrading enzyme inhibitors developed so far have problems such as specificity as an inhibitor, short-lasting effects, systemic side effects (such as hypotension), and the like. The mutant NPR-B can effectively increase intracellular cGMP concentration, and is useful for the prevention or treatment of diseases associated with low cGMP levels (low cGMP levels). Moreover, if such mutant NPR-B is administered using gene transfer technology, the drug duration per administration can be prolonged. That is, a single administration can improve low cGMP levels for a long time. In addition, for example, a novel pulmonary hypertension treatment can be achieved by introducing a vector that expresses the mutant NPR-B at the time of catheter examination.

Furthermore, by analyzing the three-dimensional structure of the mutant NPR-B, it is possible to elucidate the structural changes necessary for exhibiting sustained enzyme activity. If a low molecular weight compound or the like that brings about this structural change can be found by drug screening using cells incorporating a wild type receptor, it will lead to the development of a new drug via cGMP.
In addition, since the mutant NPR-B increases the amount of cGMP produced in a CNP concentration-dependent manner, screening for compounds that increase cGMP production is also possible by drug screening using cells incorporating the mutant NPR-B. can do.

In addition, if a substance that inhibits or regulates the enzyme activity of mutant NPR-B is found by drug screening using cells incorporating the mutant NPR-B, development of a preventive or therapeutic agent for diseases associated with high cGMP levels Leads to.
Therefore, by using the mutant NPR-B and a gene encoding the same, a new preventive or therapeutic agent for diseases associated with cGMP can be developed.

  The present inventors further injected a mouse fertilized egg with an expression vector containing the gene encoding the mutant NPR-B, and introduced the gene encoding the mutant NPR-B into the genome. Mammals were produced. In the transgenic non-human mammal in which the gene encoding this mutant NPR-B was introduced into the genome, bone elongation was observed compared to the wild-type non-human mammal. Therefore, by analyzing the transgenic non-human mammal histologically, etc., the bone elongation effect of the CNP / NPR-B / cGMP signal system, the bone elongation effect observed in patients with high stature, the cause of osteoporosis, etc. Etc. can be elucidated. Thereby, the therapeutic target with respect to a growth disorder, osteoporosis, etc. can be clarified. Further, by administering a candidate substance to this transgenic non-human mammal, a therapeutic agent for growth disorder, osteoporosis and the like can be screened.

The present invention has been completed based on the above findings, and provides the following inventions.
Item 1. The mutant natriuretic peptide B receptor which is a polypeptide of any one of the following (a) to (c), or a salt thereof.
(A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 (b) having an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2, and Polypeptide having the activity of producing cGMP without CNP stimulation (c) Hybridizing under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 1 or DNA consisting of a base sequence complementary to SEQ ID NO: 1 A polypeptide encoded by DNA that has the activity of producing cGMP without CNP stimulation; A gene encoding a polypeptide of any one of (a) to (c) below.
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2, and CNP (C) a polypeptide having the activity of producing cGMP without stimulation of DNA (c) hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 1 or DNA consisting of a base sequence complementary to SEQ ID NO: 1 2. A polypeptide encoded by DNA and having an activity of producing cGMP without stimulation of CNP. An expression vector comprising the DNA of any one of the following (a) to (c):
(A) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2
(B) a polypeptide comprising an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 are deleted, added, or substituted, and having an activity of producing cGMP without stimulation of CNP DNA to do
(C) DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 1 or DNA complementary to SEQ ID NO: 1, and without cNP stimulation DNA encoding polypeptide having activity to produce
Item 4. An agent for preventing or treating a disease associated with a low cGMP level, comprising the mutant natriuretic peptide B receptor according to Item 1 or a pharmacologically acceptable salt thereof.
Item 5. A prophylactic or therapeutic agent for a disease associated with a low cGMP level, comprising the gene according to Item 2 or the expression vector according to Item 3.
Item 6. Item 6. The prophylactic or therapeutic agent according to Item 4 or 5, wherein the disease associated with a low cGMP level is pulmonary hypertension, short stature, erectile dysfunction, heart failure, dementia, or cancer.
Item 7. (I) A method for screening a natriuretic peptide B receptor activator comprising the step of analyzing the structure of the mutant natriuretic peptide B receptor according to item 1.
Item 8. (II) a step of selecting a candidate substance that activates natriuretic peptide B receptor from the analysis result of step (I), (III) a wild-type natriuretic peptide B receptor in vitro or in a non-human mammal The step of contacting the candidate substance, (IV) the step of monitoring the amount of cGMP in the cell, and (V) the increase in the amount of cGMP in the cell detected in step (IV) as compared to the case of not contacting the candidate substance. The method for screening a natriuretic peptide B receptor activator according to item 7, comprising a step of selecting the candidate substance obtained as a natriuretic peptide B receptor activator.

Item 9. A transgenic non-human mammal, wherein the gene encoding the mutant natriuretic peptide B receptor according to Item 1 is introduced into the genome.
Item 10. Item 10. The transgenic non-human mammal according to Item 9, wherein the DNA encoding the mutant natriuretic peptide B receptor is under the control of an expression control sequence that enables cartilage-specific expression.
Item 11. Item 11. The transgenic nonhuman mammal according to Item 9 or 10, wherein the nonhuman mammal is a mouse or a rat.

  According to the present invention, a mutant natriuretic peptide B receptor that produces cGMP without CNP stimulation and a gene encoding the receptor can be provided. By using such a mutant receptor or a gene encoding the receptor, the intracellular cGMP concentration can be effectively increased, so that prevention or improvement of a disease or symptom caused by a decrease in cGMP or Drugs and the like effective for treatment can be developed. By using the mutant receptor, it is possible to efficiently screen a drug effective for the prevention, amelioration, or treatment of a disease or symptom caused by a decrease in cGMP. Moreover, the transgenic non-human mammal of the present invention is useful in the development of therapeutic agents for growth disorders, osteoporosis and the like.

FIG. 1 shows the results of examining the expression of wild-type NPR-B and mutant NPR-B by Western blotting. FIG. 2 is a diagram showing the results of analyzing the cGMP production activity of mutant NPR-B (*: P <0.05 with respect to the amount of cGMP produced by wild-type NPR-B expressing cells). FIG. 3 shows the structure of the Col11a2 promoter-HA tagged human NPR-B V883M transgene construct used for introduction into mice. FIG. 4 shows the expression level of mutant NPR-B gene (mRNA) in transgenic mice (Tg (+)) and wild-type mice (Tg (−)) in real time PCR (upper figure) and RT-PCR ( It is a figure which shows the result investigated by the following figure. FIG. 5 is a whole body X-ray photograph of a transgenic mouse (Tg) and a wild type mouse (Wt). FIG. 6 is an X-ray photograph of the back of a transgenic mouse (Tg) and a wild type mouse (Wt). FIG. 7 is a radiograph of tails of transgenic mice (Tg) and wild-type mice (Wt). FIG. 8 is a radiograph of the hind paw fingers of transgenic mice (Tg) and wild type mice (Wt).

Hereinafter, the present invention will be described in detail.
The mutant NPR-B of the present invention is a polypeptide having an activity of producing cGMP without stimulation of CNP.
The mutant natriuretic peptide B receptor (NPR-B) of the present invention is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. Preferably, it is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
The amino acid sequence shown in SEQ ID NO: 2 is a sequence in which the 883rd valine is substituted with methionine in the amino acid sequence of wild-type human NPR-B.

The mutant NPR-B of the present invention has an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 have been deleted, added, or substituted as long as the effects of the present invention are exhibited. It may be a thing. A polypeptide having an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 are deleted, added, or substituted and having an activity of producing cGMP without stimulation of CNP is also provided by the present invention. Of the mutant NPR-B. The number is usually about 10, preferably about 5, more preferably about 3, and still more preferably about 2. “1 to plural” is usually about 1 to 10, preferably about 1 to 5, more preferably about 1 to 3, further preferably about 1 to 2, and most preferably 1.
However, the amino acid substituted, deleted, or added at any one of the following sites or two or more sites in the amino acid sequence of wild-type human NPR-B (amino acid sequence in which the 883rd methionine is valine in the amino acid sequence shown in SEQ ID NO: 2) The sequence is usually not included in the amino acid sequence in which one to a plurality of amino acids are deleted, added, or substituted among the amino acid sequence shown in SEQ ID NO: 2 in the present invention;
P32T, W115G, D176E, Y338C, A409T, G413E, Y708C, R776W, L885R, R957C, G959A, Q282X, T297M, R371X + P438fsX441, A409T, D176E + R388X, P432fsX476, Q500X, G630fsX, Y708C, W769, K480X, R1020fsX1025, L658F, and I364fsX376 (Am. J. Hum. Genet. 75: 27-34, 2004, Human Molecular Genetics, 2009, Vol. 18, No. 2, 267-277, J. Clin. Endocrinol. Metab 2006 91: 1229-1232, J. Clin. Endocrinol. Metab. 2007 92: 4009-4014). “Fs” means a frameshift mutation, and “X” means a termination codon (termination). For example, “P32T” means that the 32nd proline in the amino acid sequence of wild-type human NPR-B has been replaced with threonine. “Q282X” means that the 282nd glutamine has been terminated (termination codon) by base substitution. In “R371X + P438fsX441”, the 371st arginine became a termination (termination codon) by base substitution, and the 438th proline caused a frameshift due to base deletion, and the 441st amino acid became termination (termination codon). Means.

  As a mutant NPR-B of the present invention, a DNA encoded by a DNA comprising a base sequence represented by SEQ ID NO: 1 or a DNA hybridizing under stringent conditions with a DNA comprising a base sequence complementary to SEQ ID NO: 1 Also suitable are polypeptides that are peptides and have the activity of producing cGMP without stimulation of CNP.

  It can be confirmed that the mutant NPR-B has cGMP production activity using a commercially available cGMP measurement kit. As a commercially available cGMP measurement kit, for example, cyclic GMP EIA kit (trade name, manufactured by Cayman Chemical Company, www.caymanchem.com (http://www.caymanchem.com/)) and the like can be used. Using such a cGMP measurement kit, the cGMP production activity can be measured by performing an operation according to the method described in the instruction manual attached to the kit. When cGMP production activity is detected without adding CNP in the method using the commercially available cGMP measurement kit, it is determined to have activity to produce cGMP without CNP stimulation.

  The mutant NPR-B of the present invention may form a salt. Examples of the salt of mutant NPR-B include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like. The salt of mutant NPR-B is prepared from mutant NPR-B by a method known per se.

  A gene encoding the mutant NPR-B of the present invention is also encompassed by the present invention. For example, a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 (preferably a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2) is one of the preferred embodiments of the present invention. The amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which one or more amino acids are deleted, added, or substituted (however, usually P32T, W115G in the amino acid sequence of wild type human NPR-B described above) , D176E, Y338C, A409T, G413E, Y708C, R776W, L885R, R957C, G959A, Q282X, T297M, R371X + P438fsX441, A409T, D176E + R388X, P432fsX476, Q500X, G630fsX, Y708C, W769X, R7710X, W769X, R7710W , L658F, and I364fsX376), and a gene encoding a polypeptide having an activity of producing cGMP without CNP stimulation is also included in the present invention. Is done.

The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is encoded by DNA consisting of the base sequence shown in SEQ ID NO: 1, for example. The nucleotide sequence of SEQ ID NO: 1 is a sequence in which the 2647th guanine (G) is substituted with adenine (A) in the DNA sequence encoding wild-type human NPR-B.
The gene encoding the mutant natriuretic peptide B receptor comprising DNA consisting of the base sequence shown in SEQ ID NO: 1 is also encompassed in the present invention.

  A DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and that encodes a polypeptide having an activity of producing cGMP without CNP stimulation is also included in the present invention. It is suitable as a gene encoding mutant NPR-B. A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and encodes a polypeptide that has the activity of producing cGMP without CNP stimulation DNA is also suitable as a gene encoding the mutant NPR-B of the present invention.

The “stringent conditions” refer to hybridization only when at least about 90% identity, preferably at least about 95% identity, most preferably at least about 98% identity exists between sequences. Means to occur, eg, binding at 60 ° C. under 2 × SSC wash conditions. The hybridization can be performed by a conventionally known method such as the method described in “Molecular Cloning (Third Edition)” (J. Sambrook & DW Russell, Cold Spring Harbor Laboratory Press, 2001). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize) and the more homologous DNA can be obtained.
Specifically, “hybridization under stringent conditions” means, for example, a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate ( pH 7.6) in 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA), followed by overnight incubation at 42 ° C. followed by 0.1 × SSC at about 65 ° C. This shows that a positive hybridization signal is still observed even under the condition of washing with a filter.

  Specifically, the DNA that hybridizes with the DNA having the base sequence shown in SEQ ID NO: 1 under stringent conditions is preferably about 90% or more, more preferably about 95% with the base sequence shown in SEQ ID NO: 1. As mentioned above, DNA having a base sequence having a homology of about 98% or more is particularly preferable.

  The mutant NPR-B of the present invention such as mutant NPR-B having the amino acid sequence shown in SEQ ID NO: 2 can be isolated from mammals such as humans. It can also be produced by recombinant DNA techniques and / or chemical synthesis. For example, in a production method using recombinant DNA technology, transformation is performed with an expression vector containing a gene encoding the amino acid sequence shown in SEQ ID NO: 2, specifically, for example, a DNA consisting of the base sequence shown in SEQ ID NO: 1. The mutant NPR-B of the present invention can be obtained by culturing the prepared host cells and collecting the target peptide from the culture. Extraction and purification of mutant NPR-B from transformed cells or the like, or production of a synthetic polypeptide may be performed according to a method known per se.

  Examples of the vector into which the gene encoding the mutant NPR-B of the present invention is incorporated include Escherichia coli vectors (pBR322, pUC18, pUC19, etc.), Bacillus subtilis vectors (pUB110, pTP5, pC194, etc.), and yeast vectors (YEp type). , YRp type, YIp type), or animal cell vectors (retrovirus, vaccinia virus, lentivirus, etc.), etc., but those that can stably hold the target gene in the host cell Any of them can be used. The vector is introduced into a suitable host cell. For example, the method described in Molecular Cloninng (Sambrook et al., 1989) can be used as a method for incorporating a target gene into a vector (plasmid) or a method for introducing it into a host cell. A vector in which a promoter is previously incorporated may be used.

  In order to express a target peptide gene in the vector (plasmid), a promoter is usually connected upstream of the gene so as to function.

  The promoter used in the present invention may be any promoter as long as it is appropriate for the host cell used for expression of the target gene. For example, if the host cell to be transformed is Escherichia, lac promoter, trp promoter, lpp promoter, λPL promoter, recA promoter, etc. can be used, and if it is Bacillus, SPO1 promoter, SPO2 promoter, etc. can be used. In the case of yeast, GAP promoter, PHO5 promoter, ADH promoter and the like can be used, and in the case of animal cells, SV40-derived promoter, retrovirus-derived promoter, cytomegalovirus-derived promoter and the like can be used.

  Using the expression vector (expression plasmid) containing the target gene obtained as described above, a host cell is transformed by a known technique to obtain a transformed cell. As the host cell, bacteria (eg, Escherichia genus, Bacillus genus, etc.), yeast (Saccharomyces genus, Pichia genus, Candida genus, etc.), animal cells (CHO cells, COS cells etc.) and the like can be used.

  As a medium for culturing transformed cells, a liquid medium is usually suitable, and it is particularly preferable that the medium contains a carbon source, a nitrogen source, and the like necessary for the growth of the transformed cells to be cultured. If desired, vitamins, growth promoting factors, serum, drugs and the like can be added.

  After culturing, the mutant NPR-B according to the present invention can be separated and purified from the culture by a conventional method. For example, in order to extract a target substance from cultured cells or cells, the cells or cells are collected after culturing, suspended in a buffer solution containing a protein denaturant (eg, guanidine hydrochloride), and then the cells by ultrasound. Alternatively, the cells are disrupted and then centrifuged. Next, in order to purify the target substance from the supernatant, gel filtration, ultrafiltration, dialysis, SDS-PAGE, various chromatographies are performed in consideration of the molecular weight, solubility, charge (isoelectric point), affinity, etc. of the target substance. Separation and purification methods such as chromatography can be appropriately combined.

  The mutant NPR-B according to the present invention can be chemically synthesized by a conventional method. For example, an amino acid having a protecting group is condensed by a liquid phase method and / or a solid phase method, the peptide chain is extended, all protecting groups are removed with an acid, and the resulting crude product is purified by the above purification method. Can be obtained. In addition, various methods for producing a peptide have already been known, and the production of mutant NPR-B according to the present invention can also be easily produced according to a known method. For example, a classical peptide synthesis method may be used. It can be easily produced according to the solid phase method. Moreover, you may use the manufacturing method which used recombinant DNA technique and chemical synthesis together.

  The gene encoding mutant NPR-B such as DNA containing the base sequence represented by SEQ ID NO: 1 in the present invention may be any of genomic DNA, cDNA derived from cells or tissues, and synthetic DNA. The gene encoding mutant NPR-B used in the present invention can be obtained by a known method.

  For example, the cloning of the gene encoding the mutant NPR-B of the present invention is performed by, for example, first cloning a gene encoding wild-type human NPR-B and then publicly known it based on the gene encoding the wild-type NPR-B. It can be carried out by introducing mutations by the method described above. Cloning of the gene encoding wild-type human NPR-B is performed by, for example, collecting mRNA from human cartilage or vascular tissue expressing wild-type NPR-B mRNA by a known technique and performing RT-PCR on the wild-type NPR-B. This can be done by preparing cDNA encoding -B. A gene encoding the mutant NPR-B of the present invention can be obtained by performing substitution with a mutant gene by a known technique using the prepared cDNA encoding wild-type human NPR-B. Specifically, for example, by substituting guanine (G) at position 2647 with adenine (A) in the DNA sequence encoding wild type human NPR-B, the gene encoding mutant NPR-B of the present invention One DNA having the base sequence represented by SEQ ID NO: 1 can be obtained.

When the gene encoding the mutant NPR-B of the present invention is directly cloned, for example, human or other cartilage or vascular tissue in which overgrowth of both mothers is observed is collected, and is known from the collected tissue. MRNA can be collected by the method described above, and a portion encoding NPR-B can be amplified by the PCR method and incorporated into an expression vector. By sequencing the obtained sequence, it can be confirmed that the cDNA has been recombined into the mutant type.
Moreover, it can also amplify by direct RT-PCR method using what prepared the mRNA fraction from the cell and the tissue. Furthermore, you may perform a well-known process with respect to the obtained gene. It can also be digested with restriction enzymes or added with a linker. Furthermore, a translation initiation codon (ATG) or a translation termination codon (TAA, TGA or TAG) can be added using an appropriate synthetic DNA adapter.

  The gene encoding the mutant NPR-B of the present invention may be modified in order to increase its stability in cells. A known technique can be used for such modification.

  The gene encoding the mutant NPR-B of the present invention may be used as it is, but for example, when formulated, it is preferably used in a form inserted into a vector. An expression vector containing a gene encoding the mutant NPR-B of the present invention is also one aspect of the present invention.

As the gene encoding mutant NPR-B in the expression vector of the present invention, the following DNAs (a) to (c) are preferred.
(A) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2
(B) a polypeptide comprising an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 are deleted, added, or substituted, and having an activity of producing cGMP without stimulation of CNP DNA to do
(C) DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 1 or DNA complementary to SEQ ID NO: 1, and without cNP stimulation DNA encoding polypeptide having activity to produce
Among these, an expression vector containing the DNA (a) is preferable. For example, an expression vector containing DNA consisting of the base sequence shown in SEQ ID NO: 1 is one of the preferred embodiments of the present invention.

  The expression vector only needs to be able to express the gene encoding the mutant NPR-B of the present invention, and is preferably capable of expressing the gene encoding the mutant NPR-B in mammalian cells. Examples thereof include an expression vector in which a DNA fragment encoding the mutant NPR-B of the present invention is linked downstream of an appropriate promoter.

  As the vector used for the expression vector, an existing vector can be used. For example, plasmids derived from E. coli (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), Bacteriophages such as lambda phage, retrovirus, adeno-associated virus (AAV), adenovirus, lentivirus, vaccinia virus, baculovirus, poxvirus, herpesvirus, herpes simplex virus, lentivirus (HIV), Sendai virus (HVJ- E vectors), Epstein-Barr virus (EBV), vaccinia virus, poliovirus, symbis virus, vectors derived from viruses such as SV40, pA1-11, pXT1 PRc / CMV, pRc / RSV, pcDNAI / Neo, etc. are used. Among these, vectors derived from viruses are preferable, and adeno-associated virus (AAV), adenovirus, retrovirus, poxvirus, herpes virus, herpes simplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV), vaccinia It is preferable to use a virus, a poliovirus, a synbis virus, a vector derived from SV40, or the like.

  The promoter may be any promoter as long as it is suitable for expression of a gene in a host, ie, a target cell or target tissue. For example, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, retrovirus-derived promoter, cytomegalovirus-derived promoter, adenovirus-derived promoter, phosphoglycerate kinase-1 (PGK-1) promoter, Col11a2 gene (XI type collagen α2 chain gene) promoter, CAG promoter, elongation factor 2 (EF-2) promoter, MC-1 promoter and the like. Of these, it is preferable to use, for example, CMV promoter, SRα promoter, and the like.

The expression vector may have an enhancer, a splicing signal, a poly A addition signal, a selection marker, or an SV40 replication origin, if desired, in addition to the gene encoding the mutant NPR-B of the present invention and a promoter. Examples of the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), an ampicillin resistance gene, a neomycin resistance gene (G418 resistance), and the like.
An expression vector containing a gene encoding the mutant NPR-B of the present invention can be prepared according to a method known per se using the above existing vector. For example, it can be produced by ligating a DNA fragment having a base sequence encoding the mutant NPR-B by a known method downstream of a promoter in an appropriate vector.

  In the present invention, a gene encoding the mutant NPR-B of the present invention or an expression vector containing the gene is formulated into a liposome, microcapsule, microsphere, cytofectin, DNA-protein complex, or bio It may be used in the form of being encapsulated in an artificial vector such as a polymer. Among these, it is preferable to encapsulate in liposomes or microcapsules.

  Here, a liposome is a closed endoplasmic reticulum made of a lipid bilayer membrane having an aqueous layer inside, and its lipid bilayer structure is known to be very close to that of a biological membrane. Examples of phospholipids used in the production of liposomes include phosphatidylcholines such as lecithin and lysolecithin, acidic phospholipids such as phosphatidylserine and phosphatidylglycerol, and sphingophospholipids such as phosphatidylethanolamine and sphingomyelin. Moreover, cholesterol etc. can also be added. Liposomes can be produced according to a method known per se. Liposomes include membrane fusion liposomes, HVJ-membrane fusion liposomes (Kaneda. Y et al., Biol. Chem, 264, p12126-12129 (1989), Kato. K et al., Biol. Chem, 266, p3361-3364. (1991), Tomita. N et al., Biochem. Biophys. Res., 186, p129-134 (1992), Tomota. N et al., Cric. Res., 73, p898-905 (1993)), positive Ionic liposomes (Japanese Patent Publication No. 2000-510151, Japanese Patent Publication No. 2000-516630) and the like are known. It is preferable in the present invention to use HVJ-membrane fusion liposomes fused with sender virus (HVJ). When polyethyleneglycol or the like is added by incorporating or covalently binding HVJ glycoprotein to the surface of the liposome, the efficiency of gene introduction into cells increases. Microcapsules are film-coated particles composed of film-coated polymer derivatives, hydrophobic plasticizers, surfactants and / or particles coated with a coating material consisting of a mixture of a lubricant nitrogen-containing polymer, etc. Can be mentioned.

  Since the mutant NPR-B of the present invention has an activity to produce cGMP without CNP stimulation, it can increase intracellular cGMP. Since the gene encoding the mutant NPR-B and the expression vector containing the gene can express the mutant NPR-B when introduced into the cell, cGMP in the cell can be increased. Therefore, the mutant NPR-B of the present invention, the gene encoding the same and the expression vector containing the gene can increase the cGMP level in the cell and efficiently improve the low cGMP level (low cGMP level). , Which are preferably used for the prevention or treatment of diseases associated with cGMP levels.

  A prophylactic or therapeutic agent for a disease associated with a low cGMP level comprising the mutant natriuretic peptide B receptor or a pharmaceutically acceptable salt thereof is also encompassed in the present invention. The mutant natriuretic peptide B receptor is preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 2. More preferably, it is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. In addition, the amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which one to a plurality of amino acids are deleted, added, or substituted (however, usually the amino acid of wild-type human NPR-B described above) P32T, W115G, D176E, Y338C, A409T, G413E, Y708C, R776W, L885R, R957C, G959A, Q282X, T297M, R371X + P438fsX441, A409T, D176E + R388X, P432fsX476, Q500X, G630fsX, Y708C G959A + K480X, R1020fsX1025, L658F, and I364fsX376 or a polypeptide having an activity of producing cGMP without CNP stimulation may be used. it can. A polypeptide encoded by DNA consisting of the base sequence shown in SEQ ID NO: 1 or DNA hybridizing under stringent conditions with DNA consisting of a base sequence complementary to SEQ ID NO: 1, and without CNP stimulation Polypeptides having the activity of producing cGMP are also preferably used as mutant NPR-B. Examples of the pharmacologically acceptable salt of mutant NPR-B include the salts described above.

A preventive or therapeutic agent for a disease associated with a low cGMP level comprising the gene encoding the mutant NPR-B or an expression vector containing the gene is also encompassed in the present invention.
For example, a prophylactic or therapeutic agent for a disease associated with a low cGMP level comprising a gene comprising DNA comprising the nucleotide sequence of SEQ ID NO: 1 or an expression vector comprising the gene is one of the preferred embodiments of the present invention.
Preferably, an expression vector containing DNA consisting of the base sequence shown in SEQ ID NO: 1 is used as an active ingredient.

  The disease associated with a low cGMP level (cGMP level) may be a disease whose primary or secondary cause is a reduction in cGMP production and / or release. Examples of such diseases include pulmonary hypertension, short stature, erectile dysfunction, heart failure, dementia, or cancer. The preventive or therapeutic agent of the present invention is useful for the prevention or treatment of such diseases.

  In the present invention, “prevention” includes suppressing or delaying the onset. “Treatment” shall mean alleviating or curing and alleviating a symptom or disease and / or its attendant symptoms.

  Mammals, including humans, can be given as subjects for administration of the prophylactic or therapeutic agent of the present invention, but patients in need of treatment for increasing cGMP levels are suitable, for example, patients with diseases associated with low cGMP levels. Among them, patients with pulmonary hypertension, short stature, erectile dysfunction, heart failure, dementia, cancer and the like are preferable.

  The prophylactic or therapeutic agent of the present invention comprising the mutant NPR-B of the present invention or a pharmacologically acceptable salt thereof, a gene encoding the mutant NPR-B, or an expression vector containing the gene as an active ingredient. Individuals (for example, humans, dogs, cats, mice, rats, rabbits, cows, horses, pigs, monkeys, etc.) by mixing pharmacologically acceptable carriers, excipients, bulking agents and the like with active ingredients ).

  The dosage form of the preventive or therapeutic agent of the present invention is usually a parenteral administration agent, but can also be an oral administration agent. Since the active ingredient of the medicament of the present invention is a protein or gene, the dosage form is preferably a parenteral administration agent that is easily absorbed. For parenteral administration, injections, inhalants, catheters and the like are suitable. In the case of an injection, a predetermined amount can be administered to an individual to be administered, for example, intravenously, subcutaneously, intramuscularly or intraperitoneally in a single dose or divided into multiple doses. In addition, for example, when used for the treatment of pulmonary hypertension, an expression vector containing a gene encoding a mutant NPR-B such as DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 is pulmonary artery by the same technique as in cardiac catheterization. Alternatively, it can be inserted into the right ventricle, where the expression vector is injected and introduced into pulmonary vascular cells.

  In the present invention, the dose of the prophylactic or therapeutic agent is not particularly limited and can be appropriately selected depending on the purpose of use and the age, weight, type of individual, symptom, condition, concomitant drug, etc. of the individual to be administered. When administered to a mature individual twice to several times, it is preferable to administer the range of about 0.001 to 100 mg per time as mutant NPR-B, which is an active ingredient, and about 0.01 mg to 10 mg per time. More preferable. When the DNA encoding the mutant NPR-B is administered, it is preferably administered in the range of about 0.1 μg to 100 mg as the DNA, more preferably about 1 μg to 10 mg per time, and about 10 μg. 10 mg is more preferable.

  As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, lubricants, binders, disintegrants in solid preparations; solvents, dissolution aids in liquid preparations , Suspending agent, isotonic agent, buffering agent, soothing agent and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.

Preferable examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.

Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.
Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

  Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; Examples thereof include hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.
Preferable examples of the buffer include buffer solutions such as phosphate, acetate, carbonate, citrate and the like.
Preferable examples of the soothing agent include benzyl alcohol.

Preferable examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbic acid.

  For example, a medicine in the form of an injection or the like is injected with one or more formulation additives such as an appropriate buffer solution, sugar solution, isotonic agent, pH adjuster, soothing agent, preservative, etc. together with the active ingredient. It can be prepared and provided by dissolving in distilled water for use and sterilizing (filter) filtration and then filling an ampoule or vial, or lyophilizing the sterilized filtered solution into a lyophilized preparation. Such a preparation can be used as an injection or an instillation by adding and dissolving distilled water for injection or physiological saline at the time of use. An injection solution or an infusion solution can be administered through a catheter, for example, in the case of a lung. For transmucosal administration, intranasal administration agents (nasal administration agents) such as nasal sprays and intranasal sprays are suitable, and inhalation agents are also suitable for transpulmonary administration. Examples of the inhalant include aerosols, inhalation powders, inhalation liquids (for example, inhalation solutions, inhalation suspensions, etc.), capsule inhalants and the like. The liquid for inhalation can be produced by a known production method. The inhalation solution may be used in the form of being dissolved or suspended in water or other suitable medium at the time of use.

  The content of the active ingredient in one preparation is not particularly limited. For example, when the active ingredient is protein, it is preferably about 0.001 mg to 100 mg, more preferably about 0.01 mg to 10 mg. When the active ingredient is a gene, the content of the active ingredient is preferably about 0.1 μg to 100 mg, more preferably about 1 μg to 10 mg, more preferably as DNA encoding mutant NPR-B in one preparation. Is about 10 μg to 10 mg. The preparation can be administered once to several times a day.

The present invention also includes a screening method for a natriuretic peptide B receptor activator comprising the step of (I) analyzing the structure of the mutant natriuretic peptide B receptor.
Analysis of the structure of the mutant NPR-B reveals structural changes necessary for exerting sustained enzyme activity. As a structure analysis method, a known protein structure analysis method can be used. Mutant NPR-B may form a salt as described above.

  The screening method of the present invention comprises (II) a step of selecting a candidate substance that activates the natriuretic peptide B receptor from the analysis result of the step (I), (III) a wild-type in vitro or in a non-human mammal. Compared to the step of contacting the candidate substance with type NPR-B or the mutant NPR-B of the present invention, (IV) the step of monitoring the amount of cGMP in the cell, and (V) the case where the candidate substance is not contacted, It is preferable to include a step of selecting, as a natriuretic peptide B receptor activator, a candidate substance in which an increase in the amount of cGMP in cells is detected in step (IV). In step (III), the candidate substance is preferably contacted with wild-type NPR-B in vitro or in a non-human mammal.

In the step (II), based on the result obtained in the step (I), for example, a candidate substance that may change the enzyme region of NPR-B is preferably selected.
Examples of candidate substances in the present invention include nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammals (eg, mice, rats, Pig, cow, sheep, monkey, human etc.) tissue extract, plasma and the like. The candidate substance may be a novel substance or a known substance. These candidate substances may form salts, and as salts of candidate substances, salts with physiologically acceptable acids and bases are used.

  For example, when a candidate substance is brought into contact with wild-type or mutant NPR-B (preferably wild-type NPR-B), the amount of cGMP in the cell (cGMP level) compared to the case where the candidate substance is not contacted ) Is detected, the candidate substance used can be selected as an NPR-B activator. The amount of cGMP in the cell is the above-mentioned commercially available cGMP measurement kit such as cyclic GMP EIA kit (trade name, manufactured by Cayman Chemical Company, www.caymanchem.com (http://www.caymanchem.com/)), etc. Can be measured according to the method described in the attached instruction manual.

  The NPR-B activator selected by the above method increases NPR-B's cGMP production activity. Such an NPR-B activator is useful for the prevention, amelioration or treatment of the diseases associated with the low level of cGMP described above.

  As a method of bringing a candidate substance into contact with wild-type NPR-B, a method of administering a candidate substance to a non-human mammal expressing wild-type NPR-B, a cell expressing wild-type NPR-B in vitro And a method of adding a candidate substance to the above. Expression of wild-type NPR-B means that tissues (cartilage, blood vessels, etc.) that express a large amount of wild-type NPR-B are collected from mammals by a known technique, and the known technique is used from the tissues. This can be confirmed by collecting and analyzing the gene (mRNA).

  As a method of bringing the candidate substance into contact with the mutant NPR-B, a method of administering the candidate substance to the non-human mammal expressing the mutant NPR-B, expressing the mutant NPR-B in vitro Examples include a method of adding a candidate substance to cells. The non-human mammal expressing mutant NPR-B is preferably a transgenic non-human mammal described later. The transgenic non-human mammal expressing the mutant NPR-B is, for example, a transgene construct (for example, the base shown in SEQ ID NO: 1) containing the cDNA encoding the mutant NPR-B. And an expression vector containing a gene encoding the mutant NPR-B such as DNA comprising a sequence. As an example, a transgene incorporating a mutant NPR-B cDNA having a promoter specifically expressed in any organ or tissue (for example, downstream of the promoter specifically expressed in any organ, An expression vector into which a DNA comprising the indicated base sequence is inserted) into a fertilized egg of a non-human mammal, and the fertilized egg is transplanted into a foster parent and then a mutant NPR-B is incorporated. (Transgenic animals) are obtained. The expression of mutant NPR-B means that tissue expressing NPR-B specifically from a non-human mammal (transgenic animal) into which the construct has been introduced by a known technique (for example, the promoter is cartilage If it is a specific expression promoter, it can be collected from the cartilage), and mRNA can be collected from the tissue by a known technique and analyzed, for example, by RT-PCR.

  As the non-human mammal used in the screening method, a wild-type non-human mammal (normal non-human mammal) or the transgenic animal expressing a mutant NPR-B can be used. It is preferable to use a model animal. Preferably, it is a rat or a mouse, more preferably a mouse.

  Examples of methods for administering candidate substances to non-human mammals include oral administration, intravenous injection, application, subcutaneous administration, intradermal administration, intraperitoneal administration, administration by catheter, inhalation, etc. In addition, it can be appropriately selected. The dosage of the candidate substance can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.

  Cells expressing wild type or mutant NPR-B used in the in vitro method introduce a wild type or mutant NPR-B gene introduced into a vector into a host cell according to a known genetic engineering technique. Cells having wild-type or mutant NPR-B in the cell membrane can be obtained. Host cells and vectors used can be the same as those described above. The method for measuring the expression of wild-type NPR-B and the expression of mutant NPR-B is not particularly limited, and can be measured by a method such as Western blotting.

  The substance selected by the screening method can be mixed with other components to obtain a medicine or the like. Furthermore, the usefulness with respect to a target disease can be evaluated by administering the selected NPR-B activator to the model animal of a target disease.

  As a specific example of the screening method of the present invention, for example, first, a higher order structure of mutant NPR-B is predicted from the amino acid sequence by a computer, and it is clarified that the structure is important for producing cGMP. The difference in structure between mutant and wild-type NPR-B is depicted on a computer, and a chemical substance that can bring about the change is predicted. Further, for example, structural analysis can be performed using X-ray crystal analysis, NMR, CD, ultraviolet absorption, fluorescence analysis, and the like. Then, whether the chemical actually promotes cGMP production in cells incorporating wild-type NPR-B will be examined. Furthermore, this chemical substance is named guanylyl cyclase stimulator and administered to the above-mentioned disease model animal (mouse, rat, etc.) to be treated for clinical application, and its effect is judged. This method leads to the discovery of new therapeutic agents.

(I) contacting the candidate substance with the mutant NPR-B of the present invention in an in vitro or non-human mammal, (II) monitoring the amount of cGMP in the cell, and (III) contacting the candidate substance A method for screening an NPR-B inhibitor comprising a step of selecting a candidate substance in which a decrease in the amount of cGMP in cells in step (II) is detected as a natriuretic peptide B receptor inhibitor as compared with the case of not allowing Included in the present invention. The method for bringing the candidate substance into contact with mutant NPR-B, the method for measuring the amount of cGMP in the cell, and the like are the same as described above.
Substances selected by such screening methods are useful for the prevention, amelioration or treatment of diseases associated with high levels of cGMP.

The present invention also includes a transgenic non-human mammal in which the gene encoding the mutant NPR-B is introduced into the genome.
The gene encoding mutant NPR-B and preferred embodiments thereof are as described above. Among these, a gene containing DNA consisting of the base sequence shown in SEQ ID NO: 1 is preferable.

The transgenic non-human mammal of the present invention will be described in more detail.
The non-human mammal is not particularly limited, and examples thereof include rats, mice, rabbits, guinea pigs, dogs, pigs, goats, sheep, and the like, preferably rats or mice, and more preferably mice. For example, if it is a mouse | mouth, C57BL / 6 system | strain mouse | mouth etc. are used suitably.

As described above, the transgenic non-human mammal of the present invention can be prepared by introducing an expression vector containing cDNA encoding the mutant NPR-B into the non-human mammal. If it is a transgenic mouse, it can be produced by the method described in Proc. Natl. Acad. Sci. USA 77, 7380-7384 (1980), for example.
More specifically, an expression vector containing cDNA encoding the mutant NPR-B is introduced into a totipotent cell of a non-human mammal, and the cell is generated into an individual, whereby the mutant NPR-B. A transgenic non-human mammal in which a gene coding for is introduced into the genome can be obtained.
Introducing such an expression vector containing a cDNA encoding mutant NPR-B into a totipotent cell of a non-human mammal, and producing a transgenic non-human mammal derived from the totipotent cell A method for producing a transgenic non-human mammal is also encompassed by the present invention.

  The expression vector may be any expression vector as long as it can express mutant NPR-B in the introduced animal cell. For example, the above-described expression vector of the present invention can be appropriately selected and used. An expression control sequence such as a vector (vector skeleton) or a promoter used for the expression vector may be any expression sequence that can express the target gene in the introduced animal cell. Animals and tissues that express mutant NPR-B What is necessary is just to select suitably according to etc.

  In the expression vector, the gene encoding mutant NPR-B is controlled by an expression control sequence such as a promoter. Examples of the expression control sequence include a promoter that enables systemic (non-tissue specific) expression, or a promoter that is specifically expressed in a specific organ or tissue. For example, a CAG promoter etc. are mentioned as a promoter which enables whole body (tissue non-specific) expression. For example, when producing a transgenic mouse in which mutant NPR-B is expressed in a cartilage-specific manner, a cartilage-specific expression promoter or the like can be preferably used.

  In an expression vector used for production of a transgenic non-human mammal, for example, when an expression control sequence that enables systemic (non-tissue specific) expression is used as an expression control sequence, mutant NPR-B is expressed systemically Transgenic non-human mammals can be produced. In addition, when an expression control sequence that is specifically expressed in a specific organ or tissue is used, a transgenic non-human mammal that expresses mutant NPR-B specifically in the organ or tissue can be produced. In the present invention, for example, when targeting a growth disorder (such as a model animal for growth disorder), it is preferable to use an expression control sequence that enables cartilage-specific expression. Specific examples include promoters and / or enhancers that enable cartilage-specific expression. Examples of such a promoter include promoters such as Col11a2 gene (XI type collagen α2 chain gene), type II collagen gene and the like. A transgenic non-human mammal in which the gene encoding mutant NPR-B is under the control of an expression control sequence that allows for cartilage-specific expression is one preferred embodiment of the present invention. Such a transgenic non-human mammal is usually a transgenic non-human mammal that specifically expresses the mutant NPR-B in cartilage.

  Examples of totipotent cells include pluripotent cells such as ES cells (embryonic stem cells) in addition to fertilized eggs and embryonic eggs. When an early embryo is used, an embryo before the 8-cell stage is preferred. The method for introducing the expression vector into the cell is not particularly limited, and a known method can be adopted. For example, the microinjection method, particle gun method, calcium phosphate method, electric pulse method, lipofection method, DEAE-dextran method, retrovirus infection method, aggregation method and the like can be used. Among these, the microinjection method is preferable, and microinjection into a fertilized egg is more preferable. More preferably, it is a mouse fertilized egg, and microinjection into the male pronucleus of the mouse fertilized egg is more preferable.

  Subsequently, the cells into which the gene encoding the mutant NPR-B has been introduced are usually transplanted into the uterus or fallopian tube of a foster parent, and a baby is born. As the temporary parent, for example, a female that has been pseudopregnant by a known technique is suitable.

After transplantation, an individual in which an expression vector containing a gene encoding mutant NPR-B is integrated into the genome is selected from offspring born from a temporary parent. The selection can usually be performed by a method of confirming that the target expression vector is contained in the chromosomal DNA separated and extracted from the tail of the litter by a method such as Southern hybridization or PCR. The selected individual (founder (F ₀ )) is usually a heterozygote (heterotype transgenic non-human mammal) for gene transfer. An F ₁ non-human mammal can be produced by crossing the founder (F ₀ ) with a wild-type non-human mammal of the same species. A heterozygote having a gene encoding a mutant NPR-B introduced into the genome is selected from an F ₁ non-human mammal by the same method as described above. In the present invention, since the heterozygote is a phenotype that expresses mutant NPR-B, the heterozygote (heterozygote transgenic non-human mammal) is preferably used as the transgenic non-human mammal of the present invention. Can be used. The maintenance of the heterozygote can be performed by a known method.
Moreover, a homozygote (homo-type transgenic non-human mammal) can be obtained by crossing selected heterozygotes for one generation or more. Such homozygotes are also encompassed by the present invention.

  The transgenic non-human mammal into which the gene encoding the mutant NPR-B of the present invention has been introduced into the genome is preferably a transgenic non-human mammal that expresses the mutant NPR-B in the whole body or a target tissue or organ. It is a human mammal. The expression of mutant NPR-B can be confirmed by performing RT-PCR using primers specific to the incorporated vector.

  A transgenic non-human mammal expressing the mutant NPR-B in cartilage is one of the preferred embodiments of the present invention. More preferably, it is a transgenic non-human mammal expressing the mutant NPR-B specifically in cartilage. Such a transgenic non-human mammal can be usually produced by using an expression control sequence such as a promoter that enables cartilage-specific expression in the expression vector used in the production method described above.

  In the transgenic non-human mammal of the present invention, for example, when mutant NPR-B is expressed in cartilage, bone phenotypes such as high height, scoliosis, long fingers, and flexion deformation of fingertips appear. . The transgenic non-human mammal of the present invention can be used for elucidating the mechanism of bone elongation, elucidating the mechanism of osteoporosis, and the like. Moreover, it can utilize as a model animal, such as a disease in which abnormality of a NPR-B gene is concerned, for example, a model animal of a NPR-B gene function acquisition type mutant family line (family of Example 1), a growth disorder, osteoporosis, etc. Moreover, the non-human mammal of the present invention can be used for screening for therapeutic agents such as growth disorders and osteoporosis. Furthermore, it can be used for screening or development of target molecular drugs that target the NPR-B gene.

For example, in the screening, a candidate substance is administered to the transgenic non-human mammal of the present invention (preferably a transgenic non-human mammal expressing mutant NPR-B in cartilage) as described above, and the transgenic Substances that can be used as therapeutic agents for growth disorders, osteoporosis and the like can be selected based on whether or not at least one of the following effects is observed in a non-human mammal.
(I) Relieve bone elongation.
(Ii) Suppress the progression of osteoporosis.

  As described above, the intracellular cGMP concentration can be effectively increased by using the mutant NPR-B of the present invention or the gene encoding the mutant NPR-B, resulting in a decrease in cGMP. A drug or the like effective for preventing, improving or treating a disease or symptom to be developed can be developed. By using the mutant NPR-B, it is possible to efficiently screen a drug effective for prevention, amelioration, or treatment of a disease or symptom caused by a decrease or increase in cGMP. Moreover, the transgenic non-human mammal of the present invention is useful in elucidating the causes of growth disorders, osteoporosis, etc., and developing therapeutic agents for growth disorders, osteoporosis, and the like.

Example 1
In 1996, it was reported that CNP (C-type Na diuretic peptide) -NPRB (Na diuretic peptide receptor B) signaling system plays an important role in endochondral ossification. In 2004, this system was shown to suppress FGFR3 via cGMP and is expected as a therapeutic target for achondroplasia. This time, we report a family with high height due to abnormalities in the CNP-NPRB system.

Case
A 12-year-old, 10-month-old boy, progress: Received referral to our department because of two fractures and low bone density. Height 177.0cm (+ 2.7SD), weight 55.6kg (+ 0.8SD), arm span 176.5cm, normal blood pressure, no facial abnormalities. The fingers were spider-like and the overgrowth of both mothers was characteristic. Similar physical findings were found in the mother and maternal grandmother. The phenotype was similar to that of CNP overexpression due to chromosomal translocation (2007 Bocciardi R et al.), But no chromosomal abnormality was observed in this child.

the purpose
Functional analysis of CNP-NPRB gene abnormality search and identified mutations.
Methods and results
The NPPC and NPRB genes were analyzed by the direct sequence method, and point mutations with amino acid substitutions in the same site of the NPRB catalytic domain were identified in the infant, mother and maternal grandmother. Analysis of cGMP response to CNP after forced expression of mutant NPRB gene in HEK293A cells revealed that cGMP concentration had already increased in the mutant receptor-expressing cells in the absence of CNP. In particular, it was elevated from cells expressing the wild type receptor. In fact, blood and urine cGMP levels were high in the child, mother and maternal grandmother.

Summary
We found the world's first tall stature with a gain-of-function mutation in the NPRB gene. Although NPRB activation and cGMP increase were observed, symptoms were limited to bone and cartilage.

(Example 2)
The sequence of the human NPPB gene isolated from the 12-year-old 10-month-old boy in Example 1 was amplified by using the PCR (polymerase chain reaction) method, followed by fluorescent labeling and direct sequencing.
Specifically, it is as follows.

  In 1996, it was reported that CNP (C-type Na diuretic peptide) -NPRB (Na diuretic peptide receptor B) signaling system plays an important role in endochondral ossification. In 2004, this system was shown to suppress FGFR3 via cGMP and is expected as a therapeutic target for achondroplasia. This time, I experienced a family with high stature due to abnormalities in the CNP-NPRB system. 12-year-old, 10-month-old boy, progress: history of two fractures, low bone density, etc. admitted to our department (ie Osaka University Hospital Pediatrics (Yamadaoka 2-15, Suita, Osaka, Japan)) . Height 177.0cm (+ 2.7SD), weight 55.6kg (+ 0.8SD), arm span 176.5cm, normal blood pressure, no facial abnormalities. The fingers were spider-like and the overgrowth of both mothers was characteristic. Similar physical findings were found in the mother and maternal grandmother. The phenotype was similar to that of CNP overexpression due to chromosomal translocation (2007 Bocciardi R et al.), But no chromosomal abnormality was observed in this child. Therefore, the CNP ligand and the NPR-B gene receptor were suspected, and human CNP and human NPR-B genes were analyzed using DNA isolated from the peripheral blood of a child (12-year-old boy). DNA isolation was performed using QuickGene DNA whole blood kit (manufactured by FUJIFILM). The human NPP-B gene was sequenced by amplifying the gene using PCR (polymerase chain reaction), followed by fluorescent labeling and direct sequencing.

1. Sequencing
The sequence of Primer of genomicNPR-B gene and PCR conditions are shown below.
(Human NPR-B gene primer)
Ex.1 (1)
F: CTGTAGGCCAGAGCAGCC (SEQ ID NO: 3)
R: AACCAGAGGCCACAGCAC (SEQ ID NO: 4)

Ex.1 (2)
F: GACCCCGACCTGCTGTTAG (SEQ ID NO: 5)
R: AGCTTTAGGGACACAGCGAC (SEQ ID NO: 6)

Ex.2
F: GGGACATCCCAGTGATTCAG (SEQ ID NO: 7)
R: ACAATTCCTGCCTGGTTCC (SEQ ID NO: 8)

Ex.3
F: AAGTTTCCCTGTCCTGCATC (SEQ ID NO: 9)
R: AGAGAAGCCCTTCCCCAAAG (SEQ ID NO: 10)

Ex.4 + 5
F: ACCCCAGAGAGAGGGGAAG (SEQ ID NO: 11)
R: ACTGAACCCTTGAAAGCTGG (SEQ ID NO: 12)

Ex.6 + 7
F: GAAACAGCTGGGGTCTGG (SEQ ID NO: 13)
R: CTTCAGCCAGAGGTTGTGTG (SEQ ID NO: 14)

Ex.8 + 9
F: AACCCTGCTGTTGGCTTG (SEQ ID NO: 15)
R: GATGGGGATGAGACAGGATG (SEQ ID NO: 16)

Ex.10 + 11
F: CCACTGCTCTCTAGCATTTCC (SEQ ID NO: 17)
R: CAGTATAACTTTGGGTCTATCCTTCC (SEQ ID NO: 18)

Ex.12
F: ATGCTGGGTGATAGCTGGTG (SEQ ID NO: 19)
R: CCACAATCAAAGAACAGTGGAAG (SEQ ID NO: 20)

Ex.13 + 14
F: AGCTAGCCAGTGCCCATCTC (SEQ ID NO: 21)
R: TCTCTGAGGGTGATCAAAGG (SEQ ID NO: 22)

Ex.15 + 16
F: TACCCACAGCCTCTTCTTCC (SEQ ID NO: 23)
R: GATGAGGCAGAGCCCAAC (SEQ ID NO: 24)

Ex.17 + 18
F: TGCAGCCACATACACTTTCC (SEQ ID NO: 25)
R: TGAGTTTCAAAGCGGGTCTC (SEQ ID NO: 26)

Ex.19 + 20
F: GTGTCAAGCTTGTCTCCCTC (SEQ ID NO: 27)
R: GGGATTATGAAAAGAAGAAAAGGG (SEQ ID NO: 28)

Ex.21 + 22
F: TCGGGCACGGTGCTATAC (SEQ ID NO: 29)
R: GTGTGTGCAGACATGGTGG (SEQ ID NO: 30)

(Human NPR-B gene PCR conditions)
Ex.1-1, 1-2, 2, 3, 4 + 5, 6 + 7, 8 + 9, 12, 13 + 14, 15 + 16, 17 + 18, and 21 + 22 PCR was performed.
Enzyme: Hot Star Taq
Conditions: 95 ° C for 15 minutes, 35 cycles: 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 10 minutes.

For Ex.10 + 11 and 19 + 20, PCR was performed under the following conditions.
Enzyme: Ex Taq
Conditions: 94 ° C. for 3 minutes, 35 cycles: 94 ° C. for 30 seconds, 64 ° C. for 1 minute, 72 ° C. for 1 minute, and finally 72 ° C. for 7 minutes.

  The human NPR-B gene derived from the patient amplified by PCR under the above conditions was subjected to a sequencing reaction using Big Dye terminator cycle sequencing kit (version 3.1; Applied Biosystems, Foster City, Calif.), And then a sequencer (device name: ABI The sequence was analyzed by 3130x automated sequencer (Applied Biosystems). The sequence of the human NPR-B gene isolated from the patient is shown in SEQ ID NO: 1. The amino acid sequence of mutant NPR-B encoded by this gene is shown in SEQ ID NO: 2. The amino acid sequence of this mutant NPR-B (SEQ ID NO: 2) was a sequence in which the 883rd valine was substituted with methionine in the amino acid sequence of wild-type human NPR-B. In the patient, no mutation was found in the CNP gene (PCR conditions were omitted).

  This mutant NPR-B was not found in the healthy adult 112 allele and was considered to be a novel mutation. When cGMP in patient plasma and urine was measured (measured with a cGMP measurement kit (Yamasa)), both values were high, and NPR-B gain-of-function mutation was suspected.

2. Production of a vector that expresses mutant NPR-B (pcDNA3.1 (+) / HA hNPR-B (mutant)) Expression vector containing human wild-type NPR-B labeled with an HA tag (National University Tokyo Medical) An expression vector containing mutant NPR-B was constructed using the Department of Molecular Metabolic Medicine (assigned from 2-3-10 Kanda Surugadai, Chiyoda-ku, Tokyo, Japan), Institute of Intractable Diseases, Dental University.
This expression vector containing human wild-type NPR-B labeled with an HA tag can be prepared by the technique introduced in, for example, J Clin Endocrinol Metab, October 2007, 92 (10): 4009-4014. In this paper, a pcDNA3.1 (+) vector (Invitrogen) was used as an expression vector, and the expression vector containing human wild-type NPR-B labeled with the HA tag used this time was pcDNA3. It is named 1 (+) / HA hNPR-B (wild type). The gene sequence of this pcDNA3.1 (+) / HA hNPR-B is shown in SEQ ID NO: 31. The mutant base observed this time is 3594 in pcDNA3.1 (+) / HA hNPR-B (wild type) (the first G (guanine) in SEQ ID NO: 31 is the first base of this gene sequence). The 3rd guanine (G) and the 3500th BsrGI (BioLabs) and 4092th XhoI (BioLabs) were replaced with the mutant gene in such a way as to be sandwiched between the restriction enzyme sites.
The procedure for preparing the replacement gene by the PCR method is as follows.

Upstream is the following primer from the 3405th to 3607th gene in the nucleotide sequence of SEQ ID NO: 31 (primer with mutations replaced in M1-reverse)
M1-forward: 5'ACACAGGCCTATCTGGAGGA3 '(SEQ ID NO: 32)
M1- reverse: 5'TTAAGAAGTGTCA T TACCTGCA3 '(SEQ ID NO: 33)
Using,
Enzyme: Hot Star taq
Conditions: 95 ° C for 15 minutes, 35 cycles: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 10 minutes.

In the downstream, the 3586th to 4203rd genes in the nucleotide sequence of SEQ ID NO: 31 are the following primers (primers with mutations replaced in M2-forward):
M2- forward: 5'TGCAGGTA A TGACACTTCTTAA3 '(SEQ ID NO: 34)
M2- reverse: 5 'CACCTTCCAGGGTCAAGGA3' (SEQ ID NO: 35)
Using,
Enzyme: Hot Star taq
Conditions: 95 ° C for 15 minutes, 35 cycles: 94 ° C for 30 seconds, 56 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 10 minutes.

After purifying these two PCR products with Gel Extraction kit (QIAGEN), Primer M1-forward (SEQ ID NO: 32) and M2-reverse (SEQ ID NO: 35) were used with these upstream and downstream PCR products as templates. And
Enzyme: Hot Star taq
Conditions: 95 ° C for 15 minutes, 35 cycles: 94 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° C for 1 minute, and finally at 72 ° C for 10 minutes, 3405 to 4203 with mutant gene PCR products up to were prepared.

  The PCR product and pcDNA3.1 (+) / HA hNPR-B (wild type) were digested at 37 ° C for 90 minutes at the 3500th BsrGI (BioLabs) and 4092th XhoI (BioLabs) restriction enzyme sites, respectively. After digestion (referred to as insertDNA BsrGI * XhoI and pcDNA3.1NPR-B (wild type) BsrGI * XhoI, respectively) and purification with Gel Extraction kit (QIAGEN) respectively, insertDNA BsrGI * XhoI and pcDNA3.1NPR-B (Wild type) BsrGI * XhoI was ligated using DNA ligation kit ver.2.1 (TaKaRa) according to the package insert, transformed into competent cells, and cultured in LB plate medium containing ampicillin. After confirming the formation of colonies after about 15 hours, each colony is cultured in ampicillin-containing LB liquid medium, and then the mutant NPR-B prepared using QIA prep Spin Miniprep kit (QIAGEN) is expressed. Purify the vector (hereinafter referred to as “pcDNA3.1 (+) / HA hNPR-B (mutant)”), and the pcDNA3.1 (+) / HA hNPRB specific primer (listed below) The total length of the NPR-B gene was confirmed by the direct sequencing method as described above.

pcDNA3.1 (+) / HA hNPRB specific primer
pcDNA3.1hNPRB 1F: TAATACGACTCACTATAGGG (SEQ ID NO: 36)
pcDNA3.1hNPRB 1R: ACCAGGGTACGATAATGGTC (SEQ ID NO: 37)
pcDNA3.1hNPRB 2F: ACCTCAAGCTGTACCATGAC (SEQ ID NO: 38)
pdDNA3.1hNPRB 2R: AGACTCTCCCCAAAGACATC (SEQ ID NO: 39)
pcDNA3.1hNPRB 3F: GGGCGCATTGTGTATATCT (SEQ ID NO: 40)
pcDNA3.1hNPRB 3R: TCTCGGTCATTGTTCTTGTC (SEQ ID NO: 41)
pcDNA3.1hNPRB 4F: TGATGGGATCCTGCTATATG (SEQ ID NO: 42)
pcDNA3.1hNPRB 4R: GTGATAACGCTCTGAGTTGC (SEQ ID NO: 43)
pcDNA3.1hNPRB 5F: TTCCAGCTTCCTAATTTTCC (SEQ ID NO: 44)
pcDNA3.1hNPRB 5R: CGACTATCCACCACACAGTT (SEQ ID NO: 45)
pcDNA3.1hNPRB 6F: AAATGACAGCATCAACTTGG (SEQ ID NO: 46)
pcDNA3.1hNPRB 6R: AGCCCTTAATCTGTCCAAAG (SEQ ID NO: 47)
pcDNA3.1hNPRB 7F: ATATTTCCGGCCAAGCAT (SEQ ID NO: 48)
pcDNA3.1hNPRB 7R: GCCAGATACCACCATGTAAG (SEQ ID NO: 49)
pcDNA3.1hNPRB 8F: AGTGACATTGTTGGCTTCAC (SEQ ID NO: 50)
pcDNA3.1hNPRB 8R: CAGTATGTTCGCATCTTTCC (SEQ ID NO: 51)
pcDNA3.1hNPRB 9F: CGTATGGCCCTAGCATTACT (SEQ ID NO: 52)
pcDNA3.1hNPRB 9R: AAGGCACAGTCGAGG (SEQ ID NO: 53)

  The newly prepared pcDNA3.1 (+) / HA hNPR-B (mutant) gene sequence is shown in SEQ ID NO: 54 (the first G (guanine) is the first base of this gene sequence). The gene sequence (SEQ ID NO: 54) of pcDNA3.1 (+) / HA hNPR-B (mutant type) is the gene sequence (SEQ ID NO: 31) of pcDNA3.1 (+) / HA hNPR-B (wild type). This is a sequence in which the 3594th guanine (G) is replaced with adenine (A).

3. Preparation of GFP-expressing vector (control) Further, as a control, except that a gene encoding GFP was used, 2. In the same manner, a vector expressing GFP was prepared (hereinafter, this vector is referred to as pcDNA4 GFP).

4). Confirmation of cGMP production ability of HEK293A cells into which DNA encoding wild-type NPR-B or mutant NPR-B has been introduced As a gene introduction experiment, an expression vector (pcDNA3.1) having the wild-type NPR-B gene prepared above is used. (+) HA hNPR-B (wild type)) and an expression vector (pcDNA3.1 (+) HA hNPR-B (mutant)) having a mutated NPR-B gene, respectively, by lipofection (FuGENE6 Roche) Introduced into HEK293A cells, wild type NPR-B or mutant NPR-B was forcibly expressed. Similarly, a vector expressing GFP (pcDNA4 GFP) was also introduced into HEK293A cells.

Specifically, (100,000) HEK293A cells were inoculated into each well of a 12-well plate (FALCON). After adding 1 mL of DMEM (manufactured by nacalai tesque) + 10% FBS, 0.1 mM non-essential amino acid, 2 mM L-glutamine + 1% penicillin / streptomycin (available from any manufacturer), culture for 24 hours, then FuGene6 ( Roche), expression vector having wild type NPR-B gene (pcDNA3.1 (+) HA hNPR-B (wild type)), expression having mutant NPR-B gene A vector (pcDNA3.1 (+) HA hNPR-B (mutant)) and a vector expressing GFP (pcDNA4 GFP) as a control were transfected.
48 hours after transfection of the expression vector, the culture supernatant was collected, the medium was replaced with 300 μL of 0.1M HCl, the cells were thawed and recovered, and cGMP was measured according to the instructions of the cGMP measurement kit (manufactured by Cayman Chemical).

It was confirmed by Western blotting that the introduced wild-type NPR-B or mutant NPR-B was expressed in HEK293A cells using an HA tag as a label.
In Western blot, anti-HA tag antibody (cellsignaling, 1: 1000) is used as the primary antibody, β-actin (Sigma, 1: 4000) is used as the primary antibody, and anti-mouse IgG, horseradish peroxidase is used as the secondary antibody. -Linked sheep antibody (Promega, 1: 1000) was used.
The Western blot procedure was according to the method described in J Clin Endocrinol Metab, October 2007, 92 (10): 4009-4014 except that the antibody was used as an antibody.
The results are shown in FIG.

  FIG. 1 is a diagram showing the results of examining the expression of wild type NPR-B (Wt) and mutant NPR-B (Mut) in HEK293A cells by Western blotting. As shown in FIG. 1, cells introduced with an expression vector having a wild type NPR-B gene (Wt in FIG. 1) and cells introduced with an expression vector having a mutant NPR-B gene (Mut in FIG. 1). Then, a band was observed at a position corresponding to the band of NPR-B, and it was confirmed that wild type NPR-B and mutant NPR-B were expressed, respectively. NPR-B expression was not observed in cells (GFP in FIG. 1) into which a GFP-expressing vector (control) was introduced. From FIG. 1, β-actin is expressed to the same extent in cells into which any of a vector expressing GFP, an expression vector having a wild type NPR-B gene, and an expression vector having a mutant NPR-B gene has been introduced. I understand that.

0.5 mM IBMX (3-isobutyl-1-methylxanthine) (manufactured by Wako) after 48 hours have passed since the wild-type NPR-B or mutant NPR-B was forcibly expressed (after transfection of the vector) After incubating for 10 minutes at 37 ° C., various concentrations (0 mol / L, 10 ⁻⁸ mol / L, 10 ⁻⁷ mol / L and 10 ⁻⁶ mol / L) of CNP (purchased from Biochem) Was added and incubated for 10 minutes at 37 ° C. Thereafter, the cell culture medium was removed by aspiration, and then a cell recovery solution was obtained with 300 μL of 0.1M HCL according to the instructions of the cGMP measurement kit. Using the obtained cell recovery solution, the cGMP concentration was measured with a cGMP measurement kit (Cayman). The results are shown in FIG. The horizontal axis of FIG. 2 is the added CNP concentration, and the vertical axis is the amount of cGMP produced. In FIG. 2, the gray bar represents the amount of cGMP produced by wild type NPR-B expressing cells, and the black bar represents the amount of cGMP produced by mutant NPR-B expressing cells. The data in FIG. 2 shows the mean value ± standard error (SE) (n = 3). The significant difference in the cGMP production amount of the mutant NPR-B expressing cells relative to the cGMP production amount of the wild type NPR-B expressing cells was determined by Student t test (*: P <0.05).
Analysis of the cGMP reaction against CNP revealed that, as shown in FIG. 2, the cGMP concentration was already increased in the mutant NPR-B-expressing cells in the absence of CNP (CNP: 0 mol / L). It was confirmed that the concentration was higher than that of the wild type NPR-B expressing cells in a concentration-dependent manner.

From the above results, the substitution of the 883rd valine by methionine in the amino acid sequence of wild-type NPR-B resulted in a structural change of the enzyme region in NPR-B. Therefore, mutant NPR-B is in a ligand-free state. However, it was always considered to have activity as a guanylate cyclase.
In addition, the addition of CNP increases the cGMP concentration in a CNP concentration-dependent manner, thus confirming that activation of the catalyst region by a normal cycle also occurs. Mutant NPR-B having such properties has not been known so far, and is an interesting finding as protein structural chemistry.

(Example 3)
Preparation of cartilage-specific expression transgenic mouse In preparation of a transgenic mouse (Tg mouse) expressing cartilage-specific mutant NPR-B (p.Val883Met), p742-Int lacZ vector (Tsumaki N et al. Separable cis -regulatory elements that contribute to tissue-and site-specific alpha 2 (XI) collagen gene expression in the embryonic mouse cartilage. J Cell Biol 1996; 134: 1573-82). This vector contains the promoter and intron enhancer of the Col11a2 gene (XI type collagen α2 chain gene), cuts out the insert lacZ (3.5 kb) with the restriction enzyme NotI, discards it, and blunts the vector part with T4DNA polymerase. After that, CIAP treatment was performed using Alkaline Phosphatase (Calf intestine) (trade name, manufactured by Takara Bio Inc.).

  In order to prepare an insert (mutant NPR-B), a fragment excised from the pcDNA3.1 (+) / HA hNPR-B (mutant) prepared in Example 2 with restriction enzymes HindIII / XhoI was blunt-ended with Klenow. did. Ligation was performed using the resulting blunted fragment and the p742-Int lacZ vector treated above (the resulting plasmid was called p742NPR-B V883M Int). To produce Tg mice, this p742NPR-B V883M Int was treated with the restriction enzymes HindIII / PvuI to remove extra portions other than the promoter / HA-NPR-B / intron, and then the fragment of interest (about 7.3 kb) Was purified using QIAquick Gel Extraction Kit (manufactured by Quiagen). The obtained construct (Col11a2 promoter-HA tagged human NPR-B V883M transgene construct) was outsourced to Japan SLC Co., Ltd. (3371-8 Kotomachi, Nishi-ku, Hamamatsu City, Shizuoka Prefecture 431-1103) After inserting 250 fertilized eggs by the microinjection method, they were returned to the temporary parent mice. The baby was born naturally. FIG. 3 shows the structure of the Col11a2 promoter-HA tagged human NPR-B V883M transgene construct introduced into mice. In this transgene construct, the vector-derived sequences are Col11a2 promote (Col11a2 gene promoter), SV40 intron (SV40 intron), SV40 polyA, and Coi11a2 first intron (first intron of Col11a2 gene).

In order to confirm whether or not the mouse born was a Tg mouse, DNA was extracted from the tail of the mouse and PCR was performed under the following conditions using primers (SEQ ID NO: 55 and SEQ ID NO: 56) (genotyping). ).
Equipment used: Quiagen enzyme: Hot Star Taq
95 ° C 15 minutes, (94 ° C 30 seconds + 58 ° C 30 seconds + 72 ° C 1 minute) x 30 times, 72 ° C 10 minutes

As a result, 4 of the born mice were positive for transgene. That is, a construct containing mutant NPR-B was introduced. These transgene positive mice (Tg positive mice) were designated as Tg positive mice No. 19, 23, 28, and 4-1, respectively. In addition, RNA was extracted from the tail bones of mice using RNeasy Mini Kit (Quiagen) to determine whether mutant NPR-B (NPR-B V883M) mRNA was expressed in these 4 Tg-positive mice. This was confirmed by RT-PCR using the same primers (SEQ ID NO: 55 and SEQ ID NO: 56). As a result, it was confirmed that NPR-B V883M mRNA was positive in all four mice. For quantification of the expression level of NPR-B V883M mRNA in each mouse, the same primer (SEQ ID NO: 55 and SEQ ID NO: 55) was used as the housekeeping gene (denominator) as the mRNA expression level of the SOX9 gene expressed specifically in cartilage. Realtime-PCR was performed using SEQ ID NO: 56). Confirmation of mRNA expression of the SOX9 gene was performed using the primers of SEQ ID NO: 57 and SEQ ID NO: 58. RT-PCR was performed under the following conditions.
Equipment used: Quiagen enzyme: Hot Star Taq
95 ° C 15 minutes, (94 ° C 30 seconds + 58 ° C 30 seconds + 72 ° C 1 minute) x 30 times, 72 ° C 10 minutes

  The results are shown in FIG. The graph (upper figure) in FIG. 4 shows NPR-B V883M mRNA expression level (Realtime-PCR) in Tg mice. The lower diagram in FIG. 4 shows the results of examining the expression of mutant NPR-B (NPR-B V883M) mRNA in Tg mice by RT-PCR (upper in the lower diagram in FIG. 4: mutant NPR-B (NPR- B V883M) mRNA, bottom: SOX9 mRNA). In FIG. 4, Tg (−) 10 is a wild type mouse (C57BL / 6 strain mouse). Tg (+) 19, Tg (+) 23, Tg (+) 28, and Tg (+) 4-1 are Tg positive mice No. 19, 23, 28, and 4-1, respectively.

Tg positive mouse No.4-1 (Tg (+) 4-1) with the highest expression level of NPR-B V883M mRNA died at 6 weeks of age due to bone deformation including severe glans. NPR-B V883M Tg-positive mouse No.19 (Tg (+) 19) and Tg-positive mouse No.23 (Tg (+) 23) with low mRNA expression were deformed on the outer surface, including the glans (Tg-positive mouse No. 19 died due to trouble during delivery after mating). Tg-positive mouse No.28 (Tg (+) 28), which showed moderate expression, was tall on the outer surface and had a glans, long finger and tail, and a deformed bone with a bent tip. Admitted. Radiographs of this Tg positive mouse No. 28 are shown in FIGS. 4 to 8, Tg is Tg positive mouse No. 28 (Tg (+) 28), and Wt is a wild type mouse.
Currently, Tg-positive mice No. 19 and Tg-positive mice No. 28 are crossed with wild-type C57BL / 6 mice, and colonies are being prepared.

  Tg-positive mouse No.28 (Tg (+) 28) has a bone phenotype such as height, scoliosis, long fingers, and flexion deformation of the fingertips similar to the previously analyzed tall stature. Model mice could be produced. Using this model mouse, it is possible to analyze how the NPR-B gene is involved in signal transduction at the histochemical, cellular, or molecular level when the bone grows in endochondral ossification. In addition, osteoporosis is recognized in this family, but by using this model mouse, the relationship between the NPR-B gene and osteoporosis can be studied.

Claims (8)

A mutant natriuretic peptide B receptor which is a polypeptide of the following (a) or (b) , or a salt thereof:
(A) Polypeptide having the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one to a plurality of amino acids other than the 883th methionine were deleted, added, or substituted Polypeptide having an amino acid sequence and having an activity of producing cGMP without stimulation of CNP A gene encoding the following polypeptide (a) or (b) .
(A) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids other than the 883rd methionine were deleted, added, or substituted A polypeptide comprising an amino acid sequence and having an activity of producing cGMP without stimulation of CNP An expression vector comprising the following DNA (a) or (b) :
(A) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2
(B) Among the amino acid sequence shown in SEQ ID NO: 2, it comprises an amino acid sequence in which one or more amino acids other than the 883rd methionine are deleted, added or substituted, and produces cGMP without stimulation of CNP. DNA encoding a polypeptide having activity   (I) A method for screening a natriuretic peptide B receptor activator comprising the step of analyzing the structure of the mutant natriuretic peptide B receptor according to claim 1.   (II) a step of selecting a candidate substance that activates natriuretic peptide B receptor from the analysis result of step (I), (III) a wild-type natriuretic peptide B receptor in vitro or in a non-human mammal The step of contacting the candidate substance, (IV) the step of monitoring the amount of cGMP in the cell, and (V) the increase in the amount of cGMP in the cell detected in step (IV) as compared to the case of not contacting the candidate substance. 5. The method for screening a natriuretic peptide B receptor activator according to claim 4, further comprising the step of selecting the candidate substance as a natriuretic peptide B receptor activator.   A transgenic non-human mammal, wherein the gene encoding the mutant natriuretic peptide B receptor according to claim 1 is introduced into the genome.   The transgenic non-human mammal according to claim 6, wherein the gene encoding the mutant natriuretic peptide B receptor is under the control of an expression control sequence enabling cartilage-specific expression.   The transgenic non-human mammal according to claim 6 or 7, wherein the non-human mammal is a mouse or a rat.