JP2006238844A - Cytostatic factor derived from retinal pericyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an amino acid sequence and a base sequence by isolating a cytostatic factor protein derived from retinal pericytes having cytostatic action related to angiogenesis. <P>SOLUTION: The target protein, its amino acid sequence and base sequence are obtained by the following procedures of (1) a protein group is narrowed down by using an endothelial cytostatic effect from a concentrated medium of a retinal capillary pericytes as an index, (2) a protein contained in the protein group is examined by a mass spectrometry system, etc., to identify its amino acid sequence, (3) a plurality of base sequences encoding a polypeptide containing the amino acid sequence is determined on the basis of the amino acid sequence, (4) the whole length of the base sequence encoding a protein having the corresponding endothelial cytostatic effect is determined by PCR etc., on the basis of base sequence information and (5) an isolated gene is transferred to Escherichia coli to prepare the protein. The protein has endothelial cytostatic action and is useful for a therapeutic agent for diabetic retinopathy accompanying angiogenesis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a protein having an inhibitory action on endothelial cell proliferation derived from retinal pericytes and a gene encoding the protein.

In diabetic retinopathy retinal capillaries, pericytes are selectively dropped and endothelial cells proliferate, resulting in angiogenesis. Non-Patent Document 1 discloses that pericytes suppress the proliferation of endothelial cells by co-culturing endothelial cells and pericytes. This finding leads to the presence of substances that inhibit endothelial cell proliferation in pericytes. Identification of an endothelial cell growth inhibitory factor in pericytes leads to suppression of angiogenesis in the retina and is considered to be applicable as a therapeutic agent for diabetic retinopathy.

As an endothelial cell growth inhibitory factor, transforming growth factor-β1 (transforming growth factor-β1 (TGF-β1)) is known, and TGF-β1 has no activity when present in pericytes. It is known that plasmin in cells is converted to an active form (Non-patent Document 2). However, although this phenomenon is observed in vitro, the inhibitory effect on endothelial cell proliferation in vivo cannot be explained. The culture concentrate of pericytes shows an inhibitory effect on endothelial cell proliferation, but when the action of TGF-β1 is neutralized with anti-TGF-β1 antibody, the effect of TGF-β1 is the inhibitory effect on endothelial cell proliferation of pericytes (Non-patent Document 3). In addition, as a factor that suppresses retinal neovascularization, there is a pigmented epithelium-derived factor (PEDF) that is a protein isolated from the culture supernatant of retinal pigment epithelial cells (Patent Document 1). Furthermore, endostatin, which is a C-terminal fragment of collagen XVIII, is also known as a cell growth inhibitory factor (Patent Document 2).

J. Cell Biol., 105, 1455-1462 (1987). J. Cell Biol., 109, 309-315 (1989). J. Cell Physiol., 203, 378-386 (2005). Kondo T, Hosoya K et al., Cell Struct. Funct., 28, 145-153 (2003) Special table 2003-522160 Special Table 2002-501362

As described above, a plurality of endothelial cell growth inhibitory factors have hitherto been known, but no factor that is specifically derived from retinal pericytes and specifically acts on endothelial cell growth inhibition has been identified. This is because the retina is a very small tissue and therefore the protein secreted from the retinal pericytes cannot be analyzed. That is, establishment of a technique for analyzing a protein having a specific activity secreted from retinal pericytes has been an issue. If a factor that is derived from retinal pericytes and specifically acts on the inhibition of endothelial cell proliferation can be identified, it is very useful as an anti-angiogenic agent, particularly a therapeutic agent for diabetic retinopathy. There are many unknowns regarding the abnormal growth mechanism of retinal capillary endothelial cells during diabetic retinopathy, and identification of new endothelial cell growth inhibitory factors prevents new angiogenesis inhibitors and diabetic retinopathy from becoming seriously injured Greatly help. For such a purpose, it is an object of the present invention to isolate a cell growth inhibitory factor protein derived from retinal pericytes having a cell growth inhibitory action related to a novel angiogenesis and to obtain its amino acid sequence and base sequence. .

In order to achieve such a problem, the present inventors established a retinal capillary pericyte cell line (Non-patent Document 4), concentrated the culture solution 5 times, and observed a growth inhibitory effect in the retinal capillary endothelial cell line. As a result, an inhibitory effect of about 65% was shown. Furthermore, administration of a concentrated medium to an ischemia-induced retinopathy model mouse showed an inhibitory effect on retinal neovascularization. That is, by establishing retinal capillary pericytes and using the culture supernatant, it becomes possible to purify the protein for the first time and to solve the above problems. The outline of the procedure is as follows.
(1) Proteins are separated from the concentrated medium of retinal capillary pericytes by molecular weight and charge fractions, and the proteins are narrowed down using the endothelial cell proliferation inhibitory effect as an index.
(2) The amino acid sequences of the proteins included in the narrowed protein group are identified by a mass spectrometry system or the like.
(3) Based on the identified amino acid sequence, a plurality of base sequences encoding a polypeptide containing the corresponding amino acid sequence are determined.
(4) Using the PCR method or hybridization method based on the determined nucleotide sequence information, the full length of the nucleotide sequence encoding the corresponding protein having an inhibitory effect on endothelial cell proliferation is determined.
(5) Using the isolated gene, the protein encoded by the gene of interest is prepared using Escherichia coli, mammalian cultured cells, and insect cells, and expressed in cultured retinal capillary endothelial cells or in vivo to express endothelial cell proliferation inhibitory activity Let
Hereinafter, the present invention will be described in detail.

The present invention includes a protein functionally equivalent to a cell growth inhibitory factor protein (SEQ ID NO: 1) derived from retinal pericytes. In the present invention, functionally equivalent proteins are referred to as homologs. Specifically, the homolog is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1, and a protein consisting of the amino acid sequence of SEQ ID NO: 1. Contains functionally equivalent proteins. The number and site of mutations in the amino acid sequence are not limited as long as the function of the protein can be maintained. The number of mutations is usually within 40% of all amino acids, preferably within 30% of all amino acids, and more preferably within 20% of all amino acids.

The homologue of the present invention can be obtained by introducing a mutation into DNA comprising a base sequence encoding the amino acid sequence. In order to introduce a mutation into DNA, for example, a site-directed mutagenesis method or PCR described in “Molecular Cloning: A Laboratory Manual 2nd edition, volumes 1 to 3” (Cold Spring Harber Laboratory Press published New York 1989) A method such as a method can be used. The DNA into which the mutation has been introduced may be expressed by genetic engineering using an appropriate vector and host system, for example, by the method described in “Molecular Cloning: A Laboratory Manual 2nd edition, volumes 1-3”.

The homolog in the present invention includes a protein functionally equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 1, encoded by DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence shown in SEQ ID NO: 2. included. The DNA consisting of the base sequence described in SEQ ID NO: 2 contains a base sequence encoding the amino acid sequence of the cell growth inhibitory factor protein derived from the retinal pericytes of the present invention. A DNA that can hybridize under stringent conditions is likely to encode a protein having high structural homology. Accordingly, such proteins include many functionally equivalent proteins. In the present invention, high homology means sequence identity of at least 60% or more, preferably 70% or more, more preferably 80% or more. Amino acid sequence homology can be determined using the BLAST search algorithm.

In the present invention, the stringent conditions can indicate the following conditions. For example, in the case of performing hybridization with a long probe (50 bases or more), a standard for the temperature (Tm) at which 50% dissociation occurs can be obtained from the following formula, and the hybridization temperature can be set as follows.
Tm = 81.5 + 16.6 (log10 [Na +]) + 0.41 (% G + C) -0.63 (% formamide)-(600 / n) -1.5 (% mismatch)
[Na +] is the Na ion concentration; [M], and n is the number of bases of the probe.
Hybridization temperature = Tm-25 ° C
If a probe with 100% homology and 100 bases or more is used, the temperature condition can be set as follows.
(1) 65-75 ° C. (no formamide added)
(2) 35 to 45 ° C. (in the presence of formamide)
When a short oligonucleotide is used as a probe, the following calculation formula can be set as a guide.
Hybridization temperature = Tm−5 ° C. = 2 ° C. × (number of A + T) + 4 ° C. × (number of C + G) −5 ° C.
The stringent condition in the present invention is that a positive signal is still observed even under the condition of washing at 65 ° C. in a 0.2 × SSC, 0.5% SDS solution after hybridization under the above conditions. Represents that. The combination of these conditions is merely an example. Those skilled in the art will readily adjust the various factors that determine the stringency of hybridization and empirically set the desired stringency. Factors that determine stringency include, for example, probe concentration and hybridization reaction time in addition to the above conditions.

The homologue of the present invention can also be obtained using gene amplification techniques such as PCR. That is, it is possible to design a primer based on the DNA sequence consisting of the base sequence shown in SEQ ID NO: 2 and to isolate a DNA fragment encoding the protein of the present invention or a DNA fragment having high homology with a part thereof. It is.

In the present invention, “functionally equivalent to a retinal pericyte cell-derived cell growth inhibitory factor protein (SEQ ID NO: 1)” means that the target protein has endothelial cell growth inhibitory activity.

This activity can be confirmed by a known method. Specifically, if the protein solution of interest is added to a culture solution of a retinal capillary endothelial cell line and cultured for a certain period of time to suppress cell proliferation, the protein is a cell derived from retinal pericytes. It is determined to be functionally equivalent to the growth inhibitory protein.

The cell growth inhibitory factor protein derived from the retinal pericytes of the present invention is not only a biomaterial such as a cell or tissue in which the protein is expressed, but also a gene that encodes the gene is incorporated into an appropriate expression system for genetic recombination. It can also be obtained as a recombinant. In order to obtain a retinal pericyte-derived cell growth inhibitory factor protein by a genetic engineering technique, a gene encoding a retinal pericyte-derived cell growth inhibitory factor protein is incorporated into an appropriate expression system as described later. It can be expressed. On the other hand, a gene encoding a cell growth inhibitory factor protein derived from retinal pericytes can be obtained by amplification by PCR or the like using a cDNA library of retinal pericytes or the like as a template.

The present invention relates to a DNA encoding the protein of the present invention or a homologue thereof.
The origin of the DNA of the present invention is not limited as long as it encodes the amino acid sequence shown in SEQ ID NO: 1 or a homologue thereof, such as cDNA, genomic DNA, or synthetic DNA. For example, the DNA of the present invention can be obtained by chemically synthesizing DNA consisting of the coding region of the base sequence shown in SEQ ID NO: 2. In addition, the DNA of the present invention can be amplified by PCR using primers designed based on the nucleotide sequence, using a cDNA library of retinal pericytes or other cells / tissues as a template.

The homologue of DNA included in the present invention is DNA containing a base sequence having at least 80% or more, more preferably 90% or more homology to the coding region of the base sequence shown in SEQ ID NO: 2. The homology of the base sequence constituting the DNA can be determined by the gene analysis software GENETYX-SV / RC Ver4.01 (manufactured by Software Development Co.) or the gene analysis program BLAST. Furthermore, by using cDNA derived from other species as a template, DNA encoding a homolog based on the present invention can also be amplified.

The DNA of the present invention can also be isolated by screening a cDNA library prepared from cells expressing the gene of the present invention, such as retinal pericytes, using the DNA consisting of the base sequence as a probe. Similar to PCR, if cDNA derived from other species is used, DNA encoding a homolog of a retinal pericyte-derived cell growth inhibitor derived from another species can be isolated. The probe is hybridized under stringent conditions as described above. Examples of animals other than rats include mice, rabbits, dogs, and humans.

In addition, eukaryotic genes are generally considered to exhibit polymorphism as known by interferon genes, and one or more amino acids are replaced by this polymorphism. In some cases, the amino acid may not change at all even if the base sequence changes. DNA in which the nucleotide sequence is mutated based on these polymorphisms is also included in the DNA of the present invention.

The protein of the present invention can be obtained as a recombinant using the DNA of the present invention. The operations necessary to produce recombinants are described in detail in many documents such as “Molecular Cloning” described above. Specifically, a translation start codon is added upstream of the DNA to be expressed, and a translation stop codon is added downstream. Furthermore, a control gene such as a promoter sequence that controls transcription (eg, trp, lac, T7, SV40 early promoter) is added, and the gene is inserted into an appropriate vector and replicated in a host cell to produce a functional expression plasmid. Can do. Suitable expression vectors include, for example, pRSET, pET, pGEMEX, pKK233-2 for bacteria, pYES2 for yeast, pVL1393, pFastBac1 for insect cells, pEF-BOS, pSRα, pDR2, pCR3. 1 etc. are mentioned.

The DNA of the present invention is useful for gene therapy. The protein encoded by the DNA of the present invention functions as a cell growth inhibitory factor protein. Therefore, by introducing and expressing the DNA of the present invention in a living body, it can be used, for example, for the treatment of diabetic retinopathy associated with angiogenesis.
The DNA of the present invention can be introduced into a living body by a known method. A technique for introducing a foreign gene into a living body for the purpose of treatment is known. For example, the DNA for introduction of the present invention can be obtained by incorporating the DNA of the present invention into a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or the like. The vector of the present invention can be introduced into a living body ex vivo by infecting this vector with, for example, isolated lymphocytes and then returning it to the living body again.

A transformant can be obtained by introducing the expression plasmid into an appropriate host cell. Examples of host cells include prokaryotes such as E. coli, unicellular eukaryotes such as yeast, and cells of multicellular organisms such as insects and mammals. Mammalian cells are preferred, and examples of mammalian cells include CHO cells, 293 cells, and COS7 cells. By culturing this transformant, a protein can be produced.

The obtained protein can be purified by one or a combination of ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography and hydrophobic chromatography well known to those skilled in the art. In addition, a purification method using affinity chromatography can be applied by expressing the protein of the present invention as a fusion protein with a protein having binding affinity such as His tag or HA tag.

The present invention relates to a DNA that hybridizes to the DNA of the present invention or a complementary strand thereof, and has a chain length of at least 14 nucleotides. Such DNA can be used as a primer for synthesizing the DNA of the present invention, or as a probe for detecting the DNA of the present invention or mRNA transcribed therefrom. Designing the base sequence of the primer based on the given base sequence is usually possible for those skilled in the art. It is also obvious to those skilled in the art that an appropriate probe is designed by comparing a base sequence to be detected with a known base sequence. When the DNA of the present invention is used as a probe, it can be labeled by a known method using an affinity substance such as digoxigenin or biotin, or a fluorescent substance such as FITC or rhodamine. DNA having a necessary base sequence can be obtained chemically or enzymatically synthesized.

The probe or primer based on the present invention can be used for the detection of the DNA of the present invention and the mRNA to which it has been transcribed. mRNA can be detected by Northern blotting or RT-PCR.

Furthermore, the present invention relates to an antibody against the protein of the present invention. The antibody of the present invention includes both a monoclonal antibody and a polyclonal antibody. These antibodies can be prepared based on a known method using the protein of the present invention or a fragment thereof as an immunogen.
The monoclonal antibody of the present invention can be obtained, for example, as follows. Specifically, mice, rats, hamsters or the like are immunized using the protein of the present invention as an antigen. Lymphocytes are removed from the spleen or lymph node of the immunized animal and fused with myeloma cells, and the method of Koher and Milstein (Nature, 256, 495-497, 1975), or an improved method of Ueda et al. (Proc. Natl. Acad. Sci. USA, 79, 4386-4390, 1982) and the like. Among the obtained hybridomas, a monoclonal antibody can be produced by cloning and producing an antibody that produces an antibody having the desired reaction characteristics. A monoclonal antibody can be made into a monoclonal antibody that specifically recognizes the protein by selecting one that recognizes a structure that is characteristically found in the protein of the immunogen. The characteristic structure of the protein of the present invention can be estimated by, for example, selecting an amino acid sequence specifically found in the amino acid sequence constituting the protein of the present invention.

The monoclonal antibody according to the present invention can be made into a chimeric antibody by recombining its constant region with a human immunoglobulin molecule. A chimeric antibody is one of the monoclonal antibodies produced by genetic engineering. For example, a mouse / human chimera whose variable region (V) is a mouse immunoglobulin-derived variable region (VH, VL) and whose constant region (C) is a human immunoglobulin-derived constant region (CH, CL) Monoclonal antibodies are known. The constant region derived from human immunoglobulin can be used as the constant region of human immunoglobulin belonging to any class of IgG, IgM, IgA and IgE. Preferably, the constant region of human IgG is used.

Furthermore, humanized antibodies can be obtained by humanizing the framework in the variable region. A human-type antibody (CDR-grafted antibody) is also one of monoclonal antibodies produced by genetic engineering. For example, a humanized antibody in which the hypervariable region (HV) in the variable region (V) is derived from a mouse and the framework region of the variable region (V) is derived from a human immunoglobulin is known. HV is also called a complementarity determining region (CDR), and there are three locations in the variable region. They are named HV1 (CDR1), HV2 (CDR2), and HV3 (CDR3), respectively. These regions are important regions that determine antigen binding specificity in the variable region.
On the other hand, the framework region is a region where the diversity of amino acid sequences is relatively small in the V region. The V region occupies four regions with HV sandwiched between them. They are called FR1, FR2, FR3, and FR4, respectively. Humanized antibodies can also consist of variable regions only. Alternatively, it can be constructed as a complete immunoglobulin molecule by having a structure derived from human immunoglobulin as its constant region.

A method of introducing a human antibody gene into a mouse and immunizing the mouse by transgenic technology for manipulating animal embryos has also been established (Nikkei Science 1995, June, P40- 50). The immunoglobulin molecules that can be obtained by this method are fully human immunoglobulin molecules. Thus, the humanized part or all of the immunoglobulin is also included in the present invention as long as the antibody recognizes the cell growth inhibitory protein derived from the retinal pericytes of the present invention.

A chimeric antibody can be obtained, for example, as follows: First, from a hybridoma that produces the mouse monoclonal antibody of the present invention, an active VH gene (a reconstituted VDJ gene encoding a heavy chain variable region), An active VL gene (a rearranged VJ gene encoding the light chain variable region) is isolated. On the other hand, a CH gene (C gene encoding an H chain constant region) and a CL gene (C gene encoding an L chain constant region) are obtained from DNA encoding human immunoglobulin. Next, CH is arranged downstream of VH so that it can be expressed and incorporated into an expression vector (pSV2neo, etc.). Similarly, VL is also arranged downstream of the VL so that it can be expressed and incorporated into an expression vector. VH + CL and VL + CL can be incorporated on one vector or can be inserted on another vector. The expression vector thus prepared is introduced into an appropriate host cell. When VH + CL and VL + CL are inserted into different vectors, they are co-transfected with two types of vectors. In order to facilitate screening, the host cell is advantageously a cell line that does not produce antibodies by itself. Specifically, myeloma cells such as P3X63Ag8.653 cells (ATCC CRL1580) and SP2 / 0-Ag14 cells (ATCC CRL1581) can be used. The vector can be transformed by DEAE-dextran method, calcium phosphate method, electroporation method or the like. Transformed cells are first screened using the drug resistance gene inserted into the expression vector as an indicator. Furthermore, a transformant expressing the target antibody molecule can be screened to obtain a target chimeric monoclonal antibody-producing cell.

Furthermore, a humanized antibody can be obtained, for example, as follows.
First, the mouse H chain CDR gene and the corresponding mouse L chain CDR gene are isolated. On the other hand, from the human immunoglobulin gene, a human H chain gene encoding the entire region other than the human H chain CDR corresponding to the mouse H chain CDR and a human L chain gene corresponding to the mouse L chain CDR are isolated. The isolated mouse H chain CDR gene and human H chain gene are introduced into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene and the human L chain gene are introduced into another appropriate expression vector so that they can be expressed. Alternatively, the mouse H chain CDR / human H chain gene and the mouse L chain CDR / human L chain gene can be introduced so that they can be expressed in the same expression vector. By transforming an appropriate host cell with the expression vector thus prepared, a humanized antibody-producing transformed cell can be obtained. Transformation and screening of host cells can be performed in the same manner as for chimeric antibodies.

Polyclonal antibodies can be prepared according to the method described in, for example, “Antibodies; A Laboratory Manual, Lane, HD et al.” (Cold Spring Harber Laboratory Press published New York 1989).
That is, by using the purified protein of the present invention or a fragment thereof as an antigen and immunizing an animal by an appropriate method, an antibody that specifically recognizes the antigen protein can be easily produced and purified. . As an immunized animal for obtaining a polyclonal antibody, a mouse, rat, hamster, rabbit or the like is generally used. In any case, in order to use a protein fragment as an immunogen, immunogenicity can be enhanced by binding to a carrier protein such as KLH or albumin.

The antibody of the present invention can be used for detection of the protein of the present invention. A method for detecting a protein in a sample with an antibody is known as an immunoblot method, an immunoassay method, an immunostaining method, or the like. In addition, a method for directly or indirectly labeling an antibody for use in these detection methods is also known. In general, an enzyme, a radioisotope, or a fluorescent substance is used for the label. The antibody of the present invention can be used as a reagent kit for detecting a growth inhibitory factor protein derived from retinal pericytes of the present invention by appropriately combining a buffer, a stabilizer, a suspension, a solvent such as a salt, and a solute. .
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these at all.

[Example]
(Identification of cytostatic protein)
Protein was collected from the culture concentrate of the retinal capillary pericyte cell line by ammonium sulfate precipitation, and passed through an anion exchange column (Econo-Pac High Q 1 mL cartrige; Bio-rad) to retain the protein. Protein elution from the anion exchange column was fractionated using 0.01-1 M sodium chloride solution. A portion of the fraction sample was buffer-exchanged by ultrafiltration, dissolved in 10% FBS-DMEM, added to the retinal capillary endothelial cell line, and the amount of 5-bromo-2′-deoxyuridine (BrdU) incorporated was determined. By measuring, cell growth inhibitory activity was evaluated. Of the fractions divided into a total of 60, No. 21 to 35 showed significant inhibition of BrdU incorporation. Next, for further fractionation by the hydroxyapatite chromatography method, a fraction of proteins showing inhibition of BrdU incorporation was retained on a hydroxyapatite column (CHT-1, ceramic hydroxyapatite 2 mL colume; Bio-rad), and 0.01 to Fractionation was performed using 0.5M phosphate buffer. Part of the fraction sample was buffer-exchanged by ultrafiltration, dissolved in 10% FBS-DMEM, added to the retinal capillary endothelial cell line, and when BrdU incorporation was measured, strong BrdU incorporation was suppressed in the vicinity of fraction 25 It has been shown. Furthermore, fractionation was performed with a hydroxyapatite column, and fractions of proteins showing inhibition of BrdU incorporation were separated by two-dimensional electrophoresis. The first-dimension isoelectric focusing was performed using Imbibiline DryStrip (pH 3.0 to 10.0 NL, 7 cm; Amersham Biosciences) and the second dimension using SDS-PAGE (ExcelGel 2-D Homogeneous 12.5; Amersham Biosciences). . The gel after electrophoresis was stained with Coomassie brilliant blue and visualized. The obtained spots were cut out and fragmented by in-gel digestion using trypsin. As a protein analysis method, a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOFMS, AXIMA-CFR; Shimadzu) was used. Based on the calculated peak value of monoisotopic ions, the protein was identified by the PMF method by searching the database ProFound. As a result, it was suggested that it was tropomyosin [tropomyosin (TM)]. At this stage, TMα (MW = 28.33 kDa, pI = 4.8, NCBI number = 167850), TM3 (MW = 28.77 kDa, pI = 4.7, NCBI number = NP476556), TM6 (MW = 29.30 kDa, pI = 4.7, NCBI number) = Np775134). A primer specifically recognizing rat TM3 was prepared and an open reading frame (ORF) was cloned by RT-PCR. Furthermore, RT-PCR analysis showed expression in rat retina and retinal capillary pericyte cell lines. As a result of DNA sequencer analysis of the PCR product of the amplified retinal capillary pericyte cell line, the sequences shown in Sequence Table 2 were obtained. The obtained sequences were different from those of rat TM3 and rat TM6. Furthermore, when the base sequence was converted into an amino acid (Sequence Table 1), it was still different from the both.

(Cell growth inhibitory activity of the identified protein)
PCR was performed with a primer such that a restriction enzyme site was attached to the gene sequence shown in Sequence Listing 2, and the resulting amplified DNA fragment was introduced into a pGEX-6P-1 vector (Amersham Biosciences) in order to induce protein expression. Add 100 μL of BL21 (DE3) competent cell (STRATGENE) to 5 μL of pGE-6P-1 solution into which ORF of the target gene has been introduced, add heat shock, add 200 μL of SOC medium, and incubate at 37 ° C. for 1 hour. Colonies were formed by plating on Amplate and incubating at 37 ° C. for 12-16 hours. Colonies formed in 4 to 5 mL of LB Amp medium were added, and after mass cultivation by incubation at 37 ° C. for 11 hours, the cells were collected by centrifugation.

The collected BL21 (DE3) was suspended in a phosphate buffer (pH 7.5), disrupted with an ultrasonic cell disrupter (MISONIX; Farmingdale, NY), and centrifuged to collect the supernatant. The resulting protein was purified using Glutaion Sepharose 4B (Amersham Biosciences). The purified protein was confirmed by measuring the protein amount by MALDI-TOFMS and further analyzing the N-terminal amino acid sequence using a protein sequencer (PPSQ-21; Shimadzu).

A retinal capillary endothelial cell line was seeded on a collagen-coated plate (Biocoat Collagen I Cellware 96-Well plate; BD Biosciences) at a rate of 750 cells / well and cultured for 24 hours. Dilute the concentrated recombinant protein solution to the set concentration, and add 10% FBS-DMEM containing each concentration of protein or culture concentrate of retinal capillary pericyte cell line (5x, 5x rPCT1-CM) For 48 hours. Cell measurement was performed using a cell proliferation assay kit (Cell Counting Kit-8) manufactured by Dojindo Laboratories. The cell growth inhibitory activity is shown in Table 1. The protein of the present invention 1 to 37 μM showed a remarkable endothelial cell proliferation inhibitory effect.
Table 2 shows the results using the same concentration of bovine serum albumin. Since bovine serum albumin has no effect on endothelial cell proliferation even when the same concentration is used, it was shown that the effect of the endothelial cell growth factor protein obtained in the present invention is specific.

Control:培地のみ
5x CM:培地に網膜毛細血管周皮細胞の培養濃縮液(５倍)を添加
0.1μM：培地に本発明タンパク質(TM3 sv)濃度0.1μMで添加
1μM：培地に本発明タンパク質濃度(TM3 sv)1μMで添加
10μM：培地に本発明タンパク質濃度(TM3 sv)10μMで添加

Control: Medium only
5x CM: culture concentrate (5 times) of retinal capillary pericytes added to medium
0.1 μM: The protein of the present invention (TM3 sv) is added to the medium at a concentration of 0.1 μM
1 μM: The protein concentration of the present invention (TM3 sv) added to the medium at 1 μM
10 μM: The protein concentration of the present invention (TM3 sv) added to the medium at 10 μM

Control:培地のみ
5x CM:培地に網膜毛細血管周皮細胞の培養濃縮液(５倍)を添加
0.1μM：培地にウシ血清アルブミン(BSA)濃度0.1μMで添加
1μM：培地にウシ血清アルブミン(BSA)濃度1μMで添加
10μM：培地にウシ血清アルブミン(BSA)濃度10μMで添加

Control: Medium only
5x CM: culture concentrate (5 times) of retinal capillary pericytes added to medium
0.1 μM: added to medium with bovine serum albumin (BSA) concentration of 0.1 μM
1 μM: added to the medium at a bovine serum albumin (BSA) concentration of 1 μM
10 μM: added to the medium at a bovine serum albumin (BSA) concentration of 10 μM

According to the present invention, a protein having a cell growth inhibitory action related to a novel angiogenesis and a gene encoding the protein were obtained. The protein encoded by the gene of the present invention has an endothelial cell growth inhibitory action and is useful as a therapeutic agent for diabetic retinopathy associated with angiogenesis. Furthermore, the protein having an inhibitory effect on cell growth related to angiogenesis according to the present invention is various as a cell growth inhibitor in diseases associated with angiogenesis in retinopathy of prematurity, angiogenesis in malignant tumors, angiogenesis in inflammatory diseases, and the like. Applicable to diseases.

It is an amino acid sequence of a cytostatic protein derived from rat retinal capillary pericytes. It is a DNA sequence of a cytostatic protein derived from rat retinal capillary pericytes.

Claims (14)

A protein comprising the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted, and is functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Protein. A protein that is encoded by a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 and is functionally equivalent to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1. DNA encoding any of the proteins according to claims 1-3. The DNA according to claim 4, comprising a coding region of the base sequence shown in SEQ ID NO: 2. A vector into which the DNA according to claim 4 or 5 is inserted. The vector according to claim 6, which is used for gene therapy. A transformant carrying the vector according to claim 6. A method for producing any one of the proteins according to claims 1 to 3, comprising a step of culturing the transformant according to claim 8 and recovering the expression product. An antibody against any of the proteins according to claims 1-3. The antibody according to claim 10, wherein the antibody is a monoclonal antibody. The antibody according to claim 10 or 11, wherein the antibody is humanized at least partially. The protein detection reagent according to claim 1, comprising the antibody according to claim 10. A hybridoma producing the monoclonal antibody according to claim 13.

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