JP2009031307A - Extravillous trophoblast-specific protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for isolating and tagging extravillous trophoblasts. <P>SOLUTION: Extravillous trophoblasts are isolated and tagged by using an antibody binding to a protein described in the following (a) or (b): (a) a protein including an amino acid sequence represented by sequence number 2, or (b) a protein including an amino acid sequence given by subjecting one or more amino acids in the amino acid sequence represented by sequence number 2 to deletion, substitution, or addition, and has properties or functions equal to those of the protein described in (a). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel protein, and more particularly, to a novel protein specifically expressed in human extravillous trophoblasts and a DNA encoding the same.

In the early placenta formation, human embryonic trophoblast (trophoblast) stem cells are divided into two main cell lineages: chorionic trophoblast cells and extravillous trophoblasts (hereinafter abbreviated as “EVT”). Differentiate. The former covers the placental chorionic villi and its function is to transport oxygen and nutrients from the mother to the fetus. On the other hand, the latter (EVT) is a cell that infiltrates the maternal endometrium (decidual membrane), is immunologically tolerant, and is not attacked by maternal immune cells. Infiltrate into the myometrium. Only when these blood vessels are replaced by EVT can the placental blood flow be maintained in the fetus in sufficient quantity, invasion of EVT into the uterus is an essential phenomenon for the maintenance of pregnancy. It is thought to be deeply involved in the onset of perinatal diseases such as addiction (Non-patent Document 1).
In particular, the number of EVT cells reached a peak from the 12th to the 19th week of pregnancy, and the EVT activity during this period is considered to influence the adaptability of the placental function as the pregnancy progresses.

  In addition, EVT infiltrates into normal tissues and destroys the tissues, just like cancer cells. However, EVT infiltrates in tissues according to certain rules and is different from cancer cells. The invasion stops without fail until the inner third of the myometrium. The regulatory mechanism involved in this arrest has not yet been elucidated, and the maternal uterus immunologically accepts EVT, the cell of another person, and allows its invasion into the uterine artery, but how to infiltrate it. It is extremely useful for the development of reproductive and perinatal medical care to clarify the problems such as how it is controlled and how EVT functions and differentiates in the process . In addition, elucidation of the mechanism of EVT invasion cessation can be achieved by applying the mechanism of EVT cessation used in nature to research for the suppression of cancer invasion. Expected to contribute to development. In addition, EVT can infiltrate the myometrium without being attacked by maternal immune cells, making it immunologically tolerant. The elucidation of the mechanism of EVT immune tolerance is expected to contribute to the treatment of immune diseases such as autoimmune diseases and rejection at the time of transplantation.

  As described above, in normal pregnancy, EVT invasion stops in the decidua of the endometrium in the early stages of pregnancy, but by about 15 weeks of gestation, it reaches 1/3 of the myometrium and reappears in the uterine spiral artery. Build. On the other hand, in cases of pregnancy toxemia, the invasion of EVT stops in the decidua and does not reach the myometrium, so that the reconstruction of the uterine spiral artery and the accompanying vasodilation do not occur sufficiently. The placenta then produces inflammatory cytokines in response to placental ischemia due to dysfunction of the uterine spiral artery, which causes leukocyte activation and vascular endothelial cell damage in maternal blood, hypertension, proteinuria, edema It is thought that pregnancy toxemia with 3 main features will develop (Non-patent Document 2).

  In this way, EVT functions are deeply involved in the mechanism of pregnancy toxemia, and the cause is said to have already been completed by the 15th week of pregnancy, and it develops as early as possible before the onset of pregnancy toxemia. It is important in perinatal management to be able to predict the possibility of this. However, no clinically satisfactory method has been established so far. In addition, the detection of EVT infiltration failure is expected to be realized because the specific marker for EVT, which is an indicator of its function and disorder, is not known and its functional control mechanism itself has not yet been elucidated. It is a situation that cannot stand.

  EVT is also known to exist in the egg membrane. The egg membrane has a structure consisting of three layers: fetal amnion and chorion laeve (smooth chorion) and maternal decidua from the fetal side to the maternal uterus side. However, it is the main constituent cell of the outer layer that forms the surface in contact with the maternal tissue (decidual membrane), and controls the fetal fluid layer environment that develops in amniotic fluid through the interaction with the maternal cell. It is speculated that. The smooth chorion is known to play an important role in the production of prostaglandins, which are important for the onset of labor, and the central mechanism of the regulation of onset of labor during natural labor in humans is the egg membrane It is speculated that chorion laeve's EVT is also deeply involved in this mechanism, but little is known about its details.

  In addition, although EVT has lost its proliferative ability, it has not been affected by immunological attack from the mother during pregnancy but has not fallen into apoptosis, but it disappears from the maternal tissue by some mechanism after childbirth. The EVT is a very special immunological cell that remains in the maternal tissues until the end of pregnancy as a cell of another person.

In this way, human EVT has various characteristics not found in other cells and contributes deeply to the maintenance mechanism of pregnancy, so to speak, it is a group of cells directly involved in the preservation of species as humans. However, there are problems such as difficulty in separation culture, and analysis has hardly progressed.
One solution to the above problem is to identify and clone molecules (proteins) that are specifically expressed in EVT. Since such a molecule can serve as a marker for predicting the function of EVT, the marker measurement system in blood can be created with extremely high specificity and sensitivity using the molecule or an antibody thereto. By monitoring the placental function from the early stage of pregnancy to the middle and later stages with this measurement system, it is possible to detect early cases of embryo implantation abnormalities, cases of intrauterine growth retardation, and cases of pregnancy poisoning.
EVT-specific molecules are thought to enable analysis of EVT function and differentiation, but in light of the extremely specific functions and characteristics of EVT in the living body, the results of the analysis are not only from industrial science but also from immunology and oncology It is expected to contribute greatly to the development of research in a wide range of medical fields including

Training Note No.64 Pregnancy Toxicosis (March 2001) p19-22 Published by Japan Maternal Protection Obstetrics and Gynecologists Association Journal of Japan Society of Obstetrics and Gynecology 55 vol.9 (September 2003) N265-N268

The present invention is useful for elucidating the physiological function of EVT, and for identifying, cloning, and cloning a molecule specifically expressed in EVT, for the diagnosis, prevention and treatment of pregnancy toxemia using it as a marker The purpose is to establish the means.
Another object of the present invention is to provide means for elucidating the function of EVT and the mechanism of differentiation, and to contribute to the development of not only obstetrics but also immunology and oncology.
Other objects will be apparent throughout the specification.

In order to isolate human EVT, which has been conventionally considered difficult to immunize because of difficulty in separating from the implantation site, chorion laeve-derived cells were used in mice in order to separate it from chorion laeve (chorion) of the egg membrane. An antibody that recognizes molecules that are specifically expressed in EVT after immunization was prepared, and the molecules that were specifically expressed in EVT were identified and cloned.
That is, the present invention
(1) The protein according to the following (a) or (b):
(A) a protein containing the amino acid sequence represented by SEQ ID NO: 2, or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, And a protein having the same properties or functions as the protein described in (a) above,
(2) DNA encoding the protein according to (1) or (2) above,
(3) DNA described in the following (a) or (b):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1, or (b) hybridizing under stringent conditions with the DNA comprising the base sequence represented by SEQ ID NO: 1, and the above (a) DNA encoding a protein having properties equivalent to those of protein and h-function,
(4) a recombinant vector containing the DNA according to (3) above,
(5) A transformant comprising the recombinant vector according to (4) above,
(6) The transformant described in (5) above is cultured, and a protein having properties or functions equivalent to the protein represented by the amino acid sequence described in SEQ ID NO: 2 is recovered from the obtained culture. A method for producing the protein,
(7) an antibody against the protein according to (1) above,
(8) The antibody according to (7) above, wherein the antibody is a monoclonal antibody or a polyclonal antibody, or a fragment thereof,
(9) DNA having a sequence complementary to the DNA coding sequence consisting of the nucleotide sequence of SEQ ID NO: 1 or the fragment DNA in the 5 ′ non-coding sequence, RNA corresponding to the DNA, or a chemically modified product thereof,
(10) The protein according to (1) above, the DNA according to any of (1) to (3) above, the antibody according to any of (7) to (8) above, or the above (9) A pharmaceutical composition comprising an antisense polynucleotide of
(11) A method for measuring extravillous trophoblast cell function, characterized by quantifying the protein according to (1) above,
(12) A method for diagnosing pregnancy toxemia characterized by quantifying the protein according to (1) above, and (13) a pharmaceutical comprising as an active ingredient a compound that controls the expression of the protein according to (1) above It relates to a composition and the like.

The EVT-specific antigenic protein of the present invention makes it possible to establish a means for diagnosis, prevention and treatment of pregnancy toxemia using the same as a marker, and further means for elucidating the function and differentiation mechanism of EVT Can contribute to the development of not only obstetrics but also immunology and oncology.
Specifically, it is possible to develop and establish a diagnostic method capable of early detection of embryo implantation abnormal cases, intrauterine fetal growth delay cases and pregnancy poisoning cases.
The antigenic protein of the present invention and the antibody against it are considered to be effective as an essential component of a therapeutic agent for pregnancy toxemia. It is also useful for screening for compounds that regulate EVT activity and compounds that regulate expression by elucidating the function of EVT in placental formation and maintenance.
Furthermore, since the laeverin protein is thought to be involved in the migration and invasion of various cells in vivo, the recombinant laeverin protein can be used, for example, to regulate wound healing, nerve regeneration, and infarct site healing. It is useful as an active ingredient of a pharmaceutical composition used for regulation of placenta formation, regulation of organ regeneration, regulation of angiogenesis, regulation of malignant tumor infiltration, regulation of tissue degeneration and inflammatory response due to autoimmune disease, and the like.

The present invention relates to a novel protein specifically expressed in EVT, a DNA encoding the same, an antibody against the protein, an antisense, and use thereof. The present invention is not only a novel protein specifically expressed in the extravillous vegetative cell represented by SEQ ID NO: 2 (hereinafter referred to as “laeverin”), but also a mutant having functions and properties peculiar to laeverin protein. Also includes proteins.
In the present specification, “a protein having functions and properties peculiar to laeverin protein” refers to a protein having a regulatory function of EVT invasion, proliferation, immune response, cell adhesion, cell division, etc. Point to.

Identification of EVT-specific protein Chorion laeve containing human egg membrane was isolated and minced, and a mouse was used to immunize mice to produce antibodies, from which human implantation tissue was frozen. Hybridomas producing antibodies that react specifically with EVT were selected by immunohistochemical staining using sections. This antibody was named CHL-2 and produced and purified in large quantities. Based on this antibody, protein molecules that react with the CHL-2 antibody were purified from placenta and egg membrane tissue. This substance was a glycoprotein having a molecular weight of about 160 kDa, and was present at the cell surface site of EVT. The amino acid sequence of the N-terminal 11 residues was GPPSSSGFYVS (SEQ ID NO: 3). This sequence was not registered in the data bank, and analysis of this protein molecule was continued.

  A large amount of antigen protein was purified by immunoaffinity chromatography from chorion laeve using CHL-2 antibody, and part of the protein was immunized to produce antibody CHL-2J that reacts with antigen protein present in EVT as well. did. The remaining purified antigen protein was again subjected to amino acid sequence analysis, and 10 internal amino acid sequences at 4 positions were identified. These amino acid sequences were also novel sequences not registered in the data bank. Three of the identified internal amino acid sequences were completely identical to the amino acid sequence deduced from the estimated sequence tag (EST, 1672 bp, AK075131) registered in January 2003.

  Subsequently, using the 5'RACE (5 'rapid amplification of cDNA ends) method, a cDNA encoding the target antigen was identified from mRNA prepared from human EVT (2970 bp). The first amino acid sequence downstream of the start codon in the determined DNA sequence is exactly the same as that of the previously determined N-terminus of the antigen protein and exactly the other internal amino acid sequence Since it had a DNA sequence, it was concluded that it was a cDNA encoding the antigen protein of interest. This cDNA was not registered in the BLAST program on the NCBI website, and was judged to be a new protein that had not yet been identified, and was named “laeverin”. The nucleotide sequence of the obtained laeverin cDNA is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.

Analysis of laeverin The amino acid sequence encoded by laeverin DNA described in SEQ ID NO: 1 (SEQ ID NO: 2) contains a motif (amino acids 98-506) belonging to the M1 peptidase family, and further includes enzyme activity There is a Zn binding site motif (His-Glu-Xaa-Xaa-His-18 amino acids-Glu, 415-438), which is the central part of. A transmembrane site consisting of a 23 amino acid hydrophobic group residue (14-36) was also found near the N-terminal. A homology search revealed 36% identity with aminopeptidase N (EC 3.4.11.2), which belongs to the M1 peptidase family. Similarly, homology was observed with the M1 peptidase family of glutamylaminopeptidase (aminopeptidase A, EC 3.4.11.7) and oxytocinase / insulin-regulated aminopeptidase (EC 3.4.11.3). The number of bases is almost the same as these cell membrane-bound M1 peptidases. The protein molecular weight calculated from the base sequence of laeverin of the present invention is 113 kDa, which is smaller than the actual molecular weight, but is expected to be modified with a sugar chain as in the case of the peptidase.

When we investigated the expression of laeverin in human normal tissues by immunohistochemical staining and RT-PCR, laeverin expression was not seen in reproductive organs such as ovary, endometrium and fallopian tube in adult women. Among the placental tissues, expression was confirmed only in EVT. Moreover, expression of laeverin gene was detected only in placental tissues and could not be confirmed in other organs even in the examination by Northern blot method using tissue blot membrane of mRNA from whole body organs. This finding indicates that laeverin is a specific cell differentiation marker for EVT.

laeverin features
When laeverin was expressed in COS7 cells, cell adhesion and proliferation were suppressed, and cell death was induced. This strongly suggests that laeverin plays an important role in the function of EVT, a special cell. (Data not shown)
In addition, a cell line expressing laeverin protein established by introducing laeverin gene into CHO (Chinese hamster ovary) cells is a Matrigel invasion assay method [Sato Y et al. “Involvement of dipeptidyl peptidase IV in extravillous trophoblast invasion and differentiation”, J Clin Endocrinol Metab 87: 4287-4296 (2002) and Sato Y et al. “Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype”, Development 130: 5519-5532 (2003)] Increase (Experimental Example 1, FIG. 6). In particular, strong laeverin expression was observed in many cells infiltrating the lower surface of the membrane. Furthermore, in the presence of the anti-laeverin antibody (CHL-2 antibody), a tendency for the infiltration of laeverin gene-introduced cells to increase was observed. This suggests that the laeverin protein may be involved in cell invasion control.

Quantification of laeverin
Since laeverin is specifically expressed in EVT, its function can be monitored by quantifying it. EVT plays an important role in the supply of placental blood flow to the fetus, and it is recognized in the art that it is involved in maintaining pregnancy and the development of pregnancy toxemia. A marker specific for EVT As a result of identification, changes associated with the progress of pregnancy can be monitored. Examples of the quantitative method include the following method using a blood concentration measurement system.
The antibody A (polyclonal or monoclonal antibody) against laeverin is immobilized on the bottom of a 96-well enzyme immunoassay (ELISA) plate, and patient serum adjusted to an appropriate concentration is reacted for a certain period of time. After washing, antibody B (monoclonal antibody) against enzyme-labeled laeverin is reacted for a certain period of time. After washing this, a color reaction solution containing a substrate for the enzyme is added, and colorimetric determination is performed. Next, using a standard curve created using purified laeverin protein, the amount of serum laeverin is calculated from the measured value.

laeverin mutant The present invention also includes a protein having a function equivalent to that of the laeverin protein represented by SEQ ID NO: 2 cloned by the above method. Examples of a protein having a function equivalent to that of laeverin protein include a protein having a variant in an amino acid sequence and a function and properties peculiar to laeverin protein. Such a variant has, for example, a deletion of at least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids in the amino acid sequence shown in SEQ ID NO: 2, or at least one. Or preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids may be added, or at least one of the amino acid sequence represented by SEQ ID NO: 2, preferably about 1 to 10, More preferably, 1 to 5 amino acids may be substituted with other amino acids. The protein having a function equivalent to that of laeverin protein includes about 50% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, most preferably the amino acid sequence shown in SEQ ID NO: 2. Preferably, a protein containing a sequence having a homology of about 95% or more and having substantially the same function and properties as the laeverin protein described in SEQ ID NO: 2 is also included. Production of mutants is known in the art, and can be performed, for example, by the method described in the literature (Molecular Cloning: A Laboratory Manual, 3rd edition, volume 1-3, Sambrook, J. et al., Published by Cold Spring Harber Labolatory Press). .
A protein or variant having a function equivalent to the laeverin protein of the present invention is recognized by, for example, a CHL-2 monoclonal antibody, exhibits high cell invasive ability in a cell invasion assay (see Experimental Example 1 described later), etc. Can be identified.
A method for introducing a mutation into an amino acid sequence is known to those skilled in the art and is usually performed by introducing a mutation into the base sequence of a gene encoding it. For example, the mutation is introduced by site-directed mutagenesis using a mutant oligonucleotide as a primer, PCR, or the like, but the mutation can also be introduced using a commercially available mutation introduction kit.

  Furthermore, the present invention relates to a DNA that hybridizes with a DNA encoding laeverin represented by SEQ ID NO: 2 under stringent conditions and encodes a protein having functions and properties peculiar to the laeverin protein, and is encoded by the DNA. It also includes the proteins that are present. Stringent conditions refer to conditions where the sodium concentration is 0.1 × SSC and the temperature is 50 ° C. More specifically, it can be obtained by the method described in Molecular Cloning: A Laboratory Manual 3rd edition, volumes 1-3, Sambrook, J. et al., Cold Spring Harber Labolatory Press publication.

  Since the nucleotide sequence of laeverin DNA has been determined according to the present invention, those skilled in the art can obtain the DNA of the present invention by chemical synthesis or by PCR using primers synthesized from the determined nucleotide sequence according to conventional methods. It can be used to obtain laeverin protein.

By inserting Laeverin DNA recombinant vectors present invention into a suitable vector to obtain a recombinant vector. Such a vector is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA.
Examples of plasmid DNA include Escherichia coli-derived plasmids (eg, pBR322), Bacillus subtilis-derived plasmids (eg, pUB110), and yeast-derived plasmids (eg, YEp13). Phage DNA includes λ phage (eg, λgt10). ). Moreover, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, and insect virus vectors such as baculoviruses can also be used.
In order to insert laeverin DNA into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, and if necessary, a promoter sequence for controlling transcription is added upstream thereof, and an appropriate restriction enzyme site or multiple of the vector DNA is added. Insert into the cloning site and link to vector. The expression vector preferably contains a promoter, an enhancer, a selection marker, etc. in order to express the target DNA when introduced into a suitable host. Methods for constructing such expression vectors are known in the art.

Transformant A transformant can be obtained by introducing the above recombinant vector into a host suitable for expression of laeverin DNA. The host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia genus such as Escherichia coli, Bacillus bacterium such as Bacillus subtilis, yeast such as Saccharomyces cerevisiae, animal cells such as COS cells, CHO cells, and the like Examples include insect cells such as Sf9 and Sf21.
Suitable promoters for each host (for example, trp promoter, lac promoter (bacteria), gal1 promoter (yeast), SV40 promoter (animal cell), etc.) and the like are known and can be appropriately selected from these.
Methods for introducing a recombinant vector into a host are also known in the art, and examples include a method using calcium ions, an electroporation method, a calcium phosphate method, and a lipofection method.

Production of Recombinant Protein The laeverin protein of the present invention can be produced by culturing the transformant prepared above in vivo or in vitro and collecting it from the culture.
The transformant of the present invention is cultured according to a normal method depending on the selected host, and a medium and culture conditions suitable for each host are also known.
Usually, as a medium when a microorganism is a host, any medium containing a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and capable of efficiently culturing a transformant is used. Either a medium or a synthetic medium may be used. The culture is usually carried out at 37 ° C. for 8 to 12 hours under aerobic conditions such as shaking culture or aeration and agitation culture.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary.

Examples of the medium when animal cells are hosts include generally used RPMI-1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. The culture is usually performed at 37 ° C. for 2 to 3 days in the presence of 5% CO ₂ . After culturing, when the expressed protein is accumulated in the microbial cells or cells, it is extracted by disrupting the microbial cells or cells. When secreted outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, a general method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. can be used alone or in appropriate combination to obtain the target from the culture. Proteins can be isolated and purified.

  The present invention includes not only a protein obtained by a recombinant method but also a protein isolated and purified from a tissue or a product produced by peptide chemical synthesis. However, it is preferably produced by recombinant methods.

To obtain polyclonal antibodies against the antibody (1) laeverin protein polyclonal antibody present invention against Laeverin, immunizing an animal with Laeverin protein or fragment thereof prepared as described above. The dose of antigen per animal is 50 μg in the case of mice. Immunization is carried out by administering intravenously, subcutaneously or intraperitoneally to mammals (eg, rats, mice, rabbits, humans, etc.). Further, the immunization interval is not particularly limited, and is 1 to 10 times, preferably 4 to 5 times, every few days to several weeks, preferably 2 to 3 weeks. The antibody titer is measured 7 to 10 days after the final immunization day, and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. The antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunohistochemical staining, and the like.
When purification of antibody from antiserum is required, it should be purified by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof. Can do.

(2) Monoclonal antibody 1) Immunization In order to obtain a monoclonal antibody against the laeverin protein of the present invention, an animal is immunized using the laeverin protein prepared as described above or a fragment thereof. Immunization is performed by administering intravenously, subcutaneously or intraperitoneally to a mammal (for example, rat, mouse, etc.). One dose of antigen is 30 μg per mouse in the case of mice. Further, the immunization interval is not particularly limited, and is performed at least 4 to 5 times at intervals of several days to several weeks, preferably 2-3 weeks. Then, antibody-producing cells are collected after the final immunization. Spleen cells are preferred as antibody producing cells.

2) Cell fusion Cell fusion between antibody-producing cells such as spleen cells and myeloma cells is performed. As myeloma cells, cell lines derived from animals such as mice and generally available can be used. As a cell line to be used, it has drug selectivity and cannot survive in an unfused state in a HAT selection medium (including hypoxanthine, aminopterin and thymidine), but can only survive in a state fused with antibody-producing cells. What has is preferable. For example, specific examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag and X63Ag8.653.
Antibody-producing cells and myeloma cells are mixed at a predetermined ratio (eg 3: 1) in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and cell fusion promoters such as polyethylene glycol are present Or by electrical pulse treatment (eg electroporation).

3) Selection of hybridoma For example, cells grown in a medium containing hypoxanthine (100 μm), aminopterin (0.4 μm) and thymidine (16 μM) can be obtained as hybridomas.
Next, it is screened whether the target antibody exists in the culture supernatant of the grown hybridoma. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by immunostaining, enzyme immunoassay (ELISA), RIA or the like.

4) Cloning Cloning of fused cells is performed by limiting dilution or the like, and finally, a hybridoma that is a monoclonal antibody-producing cell is established. In order to collect a monoclonal antibody from the established hybridoma, a normal cell culture method or the like can be employed.
In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum or MEM medium under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration) for 14 days, for example. Then, an antibody is obtained from the culture supernatant.

5) Purification When antibody purification is required, select a known method such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, gel chromatography, or a combination of these methods. Can be purified.
The monoclonal antibody of the present invention is selected based on an immunohistochemical staining method using an implantation site and an egg membrane tissue, an immunocytostaining method using gene-expressing cells, or an ELISA method using a generated recombinant protein. It is preferable to do.

laeverin antisense In the present specification, “DNA having a nucleotide sequence of SEQ ID NO: 1 or DNA having a complementary sequence to a fragment DNA in a 5 ′ non-coding sequence, or RNA corresponding to the DNA, or The “chemically modified product” is the DNA of the antisense strand of double-stranded DNA or RNA corresponding to the DNA of the antisense strand (hereinafter referred to as antisense polynucleotide), and binds to DNA or RNA, It means something that regulates the expression of laeverin. The antisense polynucleotide can be produced, for example, as DNA based on the base sequence of the gene encoding the laeverin protein, or can be produced as RNA by incorporating this DNA into an expression plasmid in the antisense direction. This antisense polynucleotide may be a sequence complementary to the DNA coding sequence of the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or the DNA fragment of any part of the 5 ′ non-coding sequence, but preferably a transcription initiation site, translation It is desirable that the sequence is complementary to the start site, 5 ′ untranslated region, border region between exon and intron, or 5′CAP region.

  The “chemically modified form” of the above “DNA or RNA corresponding to the DNA” is a derivative modified to enhance the transferability or stability of DNA or RNA into cells, for example, , Phosphorothioates, phosphorodithioates, alkylphosphotriesters, alkylphosphonates, alkylphosphoamidates and the like ("Antisense RNA and DNA" published by WILEY-LISS, 1992 P.1-50). This chemically modified product can be produced according to the method described in the literature. By using this antisense oligonucleotide or a chemical modification thereof to control the expression of the gene encoding laeverin protein, survival and metastasis of laeverin positive cells can be inhibited. When an antisense polynucleotide or a chemical modification thereof is administered as it is, a preferable length is, for example, 5 to 200 bases, more preferably 8 to 50 bases, particularly preferably. The thing of 10-30 bases is mentioned.

  When the antisense polynucleotide is incorporated into an expression plasmid, the preferred length is 1000 bases or less, preferably 500 bases or less, more preferably 150 bases or less. After being incorporated into an expression plasmid, it is introduced into a target cell according to a conventional method. The introduction can be performed by a method using a liposome or a recombinant virus. The expression plasmid of the antisense polynucleotide can be prepared by ligation using a normal expression vector in the downstream of the promoter and in the reverse direction, that is, so that the laeverin gene is transcribed in the 3 'to 5' direction.

Uses of laeverin protein, DNA, antibody, antisense polynucleotide, etc. DNA encoding the laeverin protein of the present invention is useful for screening for compounds that regulate the expression of the protein. For example, by using an in vivo or in vitro expression system containing the DNA, a target compound can be identified by a method usually used in the art for screening a substance that inhibits the expression of a specific protein.
In addition, laeverin protein, its mutants or antibodies can promote or suppress physiological activity related to laeverin to diagnose, treat, prevent or control general wound healing, regulate nerve regeneration, infarct It is useful for the regulation of site healing, the regulation of organ regeneration, the regulation of angiogenesis, the regulation of malignant tumor invasion, the regulation of tissue degeneration and inflammatory response due to autoimmune diseases, and the like.

For use in therapeutic applications, these are aseptically mixed with appropriate pharmaceutically acceptable carriers and excipients, for example, injectable preparations (solutions, suspensions, emulsions), implantable preparations. Prepare as The composition of the present invention is usually administered parenterally, preferably into a body cavity such as intravenous, intramuscular, subcutaneous tissue or abdominal cavity, pleural cavity, joint cavity. The dose of laeverin protein, antibody, etc. is determined by a clinician based on various factors such as therapeutic purpose, patient weight, age, symptoms, etc., but is generally more than about 0.01 mg / kg body weight Is about 0.1 mg or more, preferably about 1 mg or more. The upper limit of the dose varies from case to case, but is generally 1,000 mg or less, preferably 100 mg or less, more preferably 10 mg or less, per kg of body weight as a total for one week.
Diagnosis of pregnancy toxemia by quantifying laeverin protein can be performed using the quantification method described in the quantification method of laeverin protein.

Using laeverin DNA or atticence DNA, cell invasion can be inhibited or regulated by suppressing laeverin expression in EVT, for example, regulation of wound healing, regulation of nerve regeneration, regulation of infarct site healing, placental formation It is useful for the treatment and prevention of regulation, regulation of organ regeneration, regulation of angiogenesis, regulation of malignant tumor invasion, regulation of tissue degeneration and inflammatory response due to autoimmune diseases.
Polynucleotides such as laeverin DNA and antisense polynucleotides or their derivatives can be formulated after mixing with stabilizers, buffers, solvents, etc., and then administered simultaneously with antibiotics, anti-inflammatory agents, anesthetics, etc. . This formulation can be administered every day or every few days to every few weeks. In order to avoid frequent administration, it is also possible to prepare a sustained-release mini-pellet preparation and implant it near the affected area. Or it can also administer to an affected part gradually gradually using an osmotic pump etc. Usually, the dosage is adjusted so that the concentration at the site of action is 0.1 nM-10 μM.
EXAMPLES The present invention will be described in detail below with reference to examples, but the examples are not intended to limit the present invention.

Example 1 Cloning of EVT-specific protein (1) Reagent Mouse anti-human cytokeratin 8, 18 monoclonal antibody (clone 5D3, IgG1) was purchased from Ylem SRL (Rome, Italy). Mouse IgG1 (clone DAK-GO1, DAKO, Glostrup, Denmark) was used as a negative control stain. FITC-conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) was used as a secondary antibody for immunostaining.

(2) Tissue specimens The placenta (n = 15) and egg membranes were obtained from normal delivery. For RNA extraction, tissues were quickly frozen in liquid nitrogen and stored at -80 ° C. For immunostaining, each tissue was embedded with OCT compound (Tissue-Tec, Miles Inc. Diagnostic Division, Elkhart, IN, USA), frozen in liquid nitrogen and stored at -80 ° C.

(3) Monoclonal antibody for EVT-specific protein screening Preparation and selection of a monoclonal antibody were performed by the following method.
Chorion laeve was manually peeled from the egg membrane and minced with scissors. 1 mg of this small piece (in PBS) was injected into the abdominal cavity of 8-week-old BALB / c mice 4 times every 4 weeks. On the fifth day after the final immunization, the spleen cells of these mice were collected and mixed with X63Ag8.653 myeloma cells at a ratio of 3: 1, and cell fusion was performed using 50% polyethylene glycol 3000. After allowing to stand, the fused cells were cultured on a 96-well microplate suspended in HAT medium containing 15% fetal bovine serum and thymocytes previously collected and isolated from 3-week-old mice as feeder cells. The culture supernatant of the proliferated hybridoma was appropriately collected and subjected to indirect fluorescent immunostaining using a frozen section prepared from egg membrane tissue to screen for a hybridoma containing an antibody that specifically reacts with EVT. For hybridomas producing important antibodies, the culture medium is appropriately shifted to HT medium and then RPMI1640 culture medium containing 10% fetal bovine serum, and cloning by limiting dilution is performed three times to stably produce antibody cells. A stock was selected. This was further cultured and proliferated, and 2 × 10 ⁷ was intraperitoneally injected into a BALB / c female mouse treated with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Aldrich, Milwaukee, Wis., USA) (administered intraperitoneally). The cells were administered at the rate of cells. About 10 days later, ascites containing a large amount of antibody produced by the hybridoma grown in the peritoneal cavity was collected, and immunoglobulin fraction was collected from this using Affi-gel protein A (Bio-Rad Laboratories, Hercules, CA, USA). Separation and purification of the desired monoclonal antibody.
Thus, one hybridoma was selected by immunostaining. The antibody CHL-2 produced by the hybridoma belonged to IgG1 isotype.

(4) Immunohistochemical staining Indirect fluorescent immunostaining was performed by the following method.
A 7-μm-thick section of frozen tissue was prepared with a cryostat microtome (Cryocut 1800, Reichert-Jung, Heiderberg, Germany) and immediately placed on a slide glass coated with Neoplane (Nisshin EM, Tokyo, Japan). Air-dried and fixed with acetone at −20 ° C. for 5 minutes.

CHL-2 monoclonal antibody (5μg / ml), anti-human cytokeratin 8, 18 monoclonal antibody (5μg / ml) or negative control antibody (mouse IgG1 negative control (clone DAK-GO1, DAKO, Glostrup, Denmark) ), 5 μg / ml) for 60 minutes at room temperature. The antibody was diluted with RPMI 1640 culture medium containing 10% fetal bovine serum and 0.1% NaN ₃ . After washing, it was reacted with FITC-conjugated rabbit anti-mouse immunoglobulin (dilution ratio 1:40) for 30 minutes in the dark. The specimen was washed with PBS, covered with an anti-fade agent (Perma Fluor Aqueous Mounting Medium; Immunon, Pittsburgh, PA, USA), and observed with a fluorescence microscope (Olympus, Tokyo, Japan). Some serial specimen sections were stained with hematoxylin-eosin. The results are shown in FIG.
AC of FIG. 1 shows the result in the placenta of term pregnancy. A is the result of hematoxylin-eosin staining, B is the FITC-staining using anti-human cytokeratin 8, 18 mAb, and C is the result of FITC-staining using CHL-2 mAb. In the figure, AE represents amniotic epithelial tissue, and CL represents chorion laeve. Further, the chorion laeve (CL) layer is indicated by a dotted line. Cytokeratin 8, 18 was highly detected in EVT in amniotic epithelium and chorion laeve (arrow). Immunoreactive laeverin was clearly detected in the cell surface region of EVT outside of chorion laeve (arrow head, C). The bar is 100 μm.
Thus, CHL-2 antigen was detected in EVT in the chorion laeve. However, its expression was not observed in fetal amniotic epithelium or maternal decidual cells. CHL-2 antigen was also expressed in EVT, which is invading maternal decidual tissue (data not shown). The main expression site was the cell surface region of EVT.

(5) Purification of CHL-2 antigen from chorion laeve and placenta
Purification of CHL-2 antigen was performed as described above.
Tissue material (chorion laeve, 1 g; placenta, 5 g; wet weight) 10 to 50 ml, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 (Sigma, St. Louis, MO, USA), 0.2 mg / in 40 mM phosphate buffer, pH 7.3 containing ml phenylmethylsulfonyl fluoride hydrochloride (Wako Pure Chemicals), 10 μg / ml leupeptin (Peptide Institute Inc.) and 10 μg / ml peptidatin (Peptide Institute Inc.) Stir. After centrifugation (11,000 × g; 30 minutes), the Nonidet P-40 concentration was lowered by diluting to 0.3% with a tissue solution excluding Nonidet P-40. The supernatant was Affigel 10 (Bio-Rad Labs., Hercules, CA, 2 mg IgG / conjugated with 10 ml anti-trinitrophenyl (TNP, an irrelevant monoclonal antibody) to remove non-specifically bound components. ml column) was passed through the column at 4 ° C. The filtered fraction was reacted with 0.15 ml of CHL-2 conjugated Affigel 10 (2 mg IgG / ml gel) at 4 ° C. for 2 hours. After thoroughly washing the gel, the antigen was eluted with 0.5 M NH ₄ OH containing 0.1% Nonidet P-40. The eluate was dried in vacuo at room temperature. Samples were dissolved in 0.1 M dithiothreitol and separated by 8% sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were silver stained using a silver staining kit (Wako Pure Chemical Industries). The same experiment was repeated three times with different tissue materials. The results are shown in FIG.

In FIG. 2, lane 1 is a molecular weight marker (Bio-Rad Laboratories, Hercules, CA, USA, 200 kDa: Myosin, 116 kDa: β-galactosidase, 97.4 kDa: PhosphorylaseB, 66 kDa: Serum albumin), and lanes 2-5 are Laeverin purified independently from 4 placental tissues, lane 6 is a protein purified from placenta using an anti-TNP (control) mAb, an irrelevant monoclonal antibody. A major protein band (arrow) was observed around 160 kDa.
As apparent from FIG. 2, the CHL-2 antigen molecule was purified from chorion laeve and placenta. The CHL-2 antigen purified from these tissues by SDS-PAGE had the same protein band and a molecular weight of about 160 kDa.

(6) Analysis of partial amino acid sequence of CHL-2 antigen purified from placenta
300 g of human placental tissue was stirred in a lysis solution containing 800 ml of 0.2 mg / ml phenylmethylsulfonyl fluoride hydrochloride. After centrifugation (15,000 × g; 30 min), the supernatant was filtered through a 10 μm pore membrane and diluted to 0.3% with a tissue solution excluding Nonidet P-40 to reduce Nonidet P-40 concentration. After reacting with Affigel 10 conjugated with 20 ml of anti-TNP antibody for 1 hour at 4 ° C., the gel was removed. The lysate was reacted with 0.6 ml Affigel 10 to which CHL-2 had been bound for 2 hours at 4 ° C. Antigen was eluted as described above. This process was repeated 7 times using 3 samples. The purified antigen was dialyzed against 3 mM Tris HCl buffer, pH 6.8 (containing 0.1% SDS) and dried. The purified antigen was electrophoresed on 8% SDS-PAGE. Proteins in polyacrylamide gels were blotted onto PVDF membranes (Millipore Co., Bedford, MA, USA) in 50 mM Tris, 15 mM boric acid, 0.05% SDS, and 20% ethanol. The protein was stained with Coomassie Blue, the main protein band was excised and analyzed with the amino acid sequencer PSQ-1 (Shimazu Co., Kyoto, Japan). The purified CHL-2 antigen was further cleaved inside and separated into peptide fractions by high performance liquid chromatography. The amino acid sequence at the N-terminus of each peptide fraction was analyzed in the same manner as described above. Amino acid sequence homology was analyzed using BLAST from the NCBI website (http: www.ncbi.nlm.nih.gov/BLAST/). The results are shown in Table 1.

N末端領域の１１残基からなるアミノ酸配列は報告されたアミノ酸配列とは相同性がなかった。独立して決定された４個の内部の部分アミノ酸配列のうち、１つは報告されていない配列であった (内部配列1)。しかし、他の３個の部分アミノ酸配列(内部配列2-4)は３’側1672塩基対からなるEST（AK075131 発現遺伝子配列断片から翻訳される配列と完全に一致していた。 Table 1 Partial amino acid sequence of laeverin purified from human placenta

The amino acid sequence consisting of 11 residues in the N-terminal region was not homologous with the reported amino acid sequence. Of the four internal partial amino acid sequences determined independently, one was an unreported sequence (internal sequence 1). However, the other three partial amino acid sequences (internal sequence 2-4) completely matched the sequence translated from the EST (AK075131 expressed gene sequence fragment) consisting of 1672 base pairs on the 3 ′ side.

(7) Total RNA extraction and 5 'RACE (5'rapid amplification of cDNA ends)
Based on the information of EST (AK075131), the 5'-side sequence of cDNA encoding CHL-2 antigen was analyzed by 5'RACE method.
Total RNA was extracted from chorion laeve (1 g) using RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the supplier's instructions. cDNA with an adapter added to the 5 ′ end of mRNA was prepared using SMART RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, Calif., USA), and using this as a template, a sense primer having a partial base sequence of the adapter (5 A PCR reaction was performed using ' -AGAATTC CTAATACGACTCACTATAGGGCAAG-3', SEQ ID NO: 8) and an antisense primer (5'- AGAATTC TTTCCACCTTCATGGCCACAGCTC-3 ', SEQ ID NO: 9) (the underlined portion is a restriction enzyme EcoRI cleavage sequence). PCR reaction conditions are 94 ° C 5 seconds, 72 ° C 3 minutes 5 cycles, 94 ° C 5 seconds, 70 ° C 10 seconds, 72 ° C 3 minutes 5 cycles, 94 ° C 5 seconds, 68 ° C 10 seconds, 72 ° C 3 The minute was 25 cycles. The obtained cDNA was isolated and the DNA sequence was analyzed. The homology of the DNA sequence determined by the above 5′RACE method was analyzed by BLAST on the NCBI website.

The nucleotide sequence of the obtained cDNA is shown in FIG. 3 and SEQ ID NO: 1, and the deduced amino acid sequence is shown in FIG. 4 and SEQ ID NO: 2.
The sequence (FIG. 4 and SEQ ID NO: 2) encoded by the cDNA clone described in FIG. 3 (SEQ ID NO: 1) matched the partial amino acid sequence of the N-terminal region of the purified CHL-2 antigen identified previously. The full-length sequence (2970 base pairs) of cDNA encoding CHL-2 antigen has not been reported so far. The coding region was the region of bases 53-3022 in SEQ ID NO: 1, and the amino acid sequence to be translated was amino acid 990 (FIG. 4). This amino acid sequence contained the last partial amino acid sequence (268-277). This new antigenic protein was named “laeverin”.

(8) Homology search for amino acid sequence of CHL-2 antigen
The amino acid sequence of the CHL-2 antigen had 36% (identities) and 54% (positives) homology with aminopeptidase N (EC 3.4.11.2), respectively. In addition, glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) (L. Li, et al., Genomics 17 (1993) 657-664) and oxytocinase / insulin-regulated aminopeptidase (EC 3.4. 11.3) (PG Laustsen, et al., Biochim. Biophys. Acta. 1352 (1997) 1-7 and SR Keller, et al., J. Biol. Chem. 270 (1995) 23612-23618) These all belong to the MA family gluzincin aminopeptidases M1 family zinc aminopeptidase (AJ Barrett, Methods Enzymol. 244 (1994) 1-15).

(9) Search for gene expression by Northern blotting Screening experiments show that the novel protein laeverin of the present invention having a molecular weight of about 160 kDa is applied to the placenta and ovary other than the egg membrane and uterus where EVT is present by immunohistochemical staining. Was not expressed, indicating that it was expressed specifically in EVT.
The results of gene expression search by Northern blot method are shown in FIG. Northern blot was performed according to the following procedure. First, the vector pCMV-script plasmid was cleaved with the restriction enzyme BstXI, and a PCR product encoding laeverin prepared with 5′RACE was inserted, and then E. coli was transformed. E. coli was grown and the plasmid was extracted. The linearized plasmid was labeled with ³² P as a probe. MRNA samples extracted from human multiple organs were purchased from Clontech as a Multiple Tissue membrane. The obtained membrane (Human 12-Lane Multiple Tissue Northern Blot) was incubated in a prehybridization buffer (Pharmacia) at 65 ° C. for 30 minutes, and then hybridized with a ³² P-labeled laeverin probe. Wash with 2 x standard saline citrate (SCC, 2 mM sodium acetate, 20 mM sodium chloride) solution containing 0.1% SDS for 15 minutes at room temperature, then wash with 0.2 x SSC solution containing 0.1% SDS for 30 minutes at 65 ° C. . Subsequently, autoradiography was performed at -80 ° C for 1 day, and a gene corresponding to laeverin was detected on the membrane. As a result, the tissues tested (Brain, Heart, Skeletal muscle, Colon (no mucosa) (Colon (no mucosa), Thymus, Spleen), Kidney (Kidney) ), Small intestine, placenta (Placenta), lung (Lung), and peripheral blood mononuclear cells (PBMC), laverin gene expression was confirmed at 4.0 kb and 3.0 kb positions only in the placenta. As a result of examination using placenta tissue and egg membrane tissue of various weeks, the 4.0 kb band was the main mRNA, and the 3.0 kb band was considered to be a splicing variant. Expression was not observed in organs and cells such as muscle, large intestine, thymus, spleen, kidney, small intestine, lung and peripheral blood immune cells.

Experimental Example 1 Invasion ability of cells introduced with Laverin gene
The laeverin gene was introduced into CHO (Chinese hamster ovary) cells, and four cell lines were established.The membrane invasion ability was examined by Matrigel invasion assay, and an empty vector was introduced as a control. laeverin gene negative cells were used, and the above-mentioned antibody for ETV-specific protein screening (CHL-2 antibody) was used as an antibody.

(1) Establishment of cell lines B2D1, B2D2, D1D1 and D1D2
The laeverin gene was introduced into CHO cells using lipofectamine, and the transfected cells were selectively cultured in a nutrient mixture F-12 / HAM medium containing antibiotic G418 and 10% FCS. Subsequently, the obtained cell group was cloned and the presence or absence of expression of laeverin protein was confirmed by fluorescent immunostaining using an anti-laeverin antibody. The positive cell group is further cultured, and cells expressing laeverin protein on the cell surface are selected by flow cytometry using an anti-laeverin antibody (FACScalibur, Becton Dickinson), cultured and cloned, and cell lines B2D1, B2D2 D1D1 and D1D2 were obtained.

(2) Assay of invasive ability of laeverin-expressing cells The assay was performed according to a method already reported (Sato Y et al., 2002 J Clin Endocrinol Metab 87: 4287-4296: Sato Y, et al., 2003 Development 130: 5519- 5532).
Each cell line was adjusted to a concentration of ⁵ × 10 ⁵ / ml in a nutrient mixture F-12 / HAM culture medium containing G418 and 10% FCS to prepare a cell suspension.
A chamber (cell culture insert 8.0 micron pore size PET membrane; Beckton Dickinson Labware) in which a Matrigel basement membrane matrix (MATRGEL (registered trademark) Basement Membrane; Beckton Dickinson Labware, Bedford, MA) was previously coated on the bottom membrane (pore size: 8 microns) , Bedford, MA), and this was placed in a 24-well culture plate to which 800 μl of culture medium (nutrient mixture F-12 / HAM culture medium name) was added. 200 μl of the above cell suspension was added to the chamber and cultured at 37 ° C. for 20 hours. Non-infiltrating cells cannot pass through the Matrigel basement membrane matrix, but infiltrating cells can invade from the Matrigel basement membrane matrix into the 8 micron membrane pore.
Next, non-infiltrated cells were removed, the membrane was fixed with methanol, stained with hematoxylin, infiltrated into the Matrigel basement membrane matrix, passed through the 8 μm diameter Millipore present in the bottom membrane, and reached the lower surface of the membrane The invasive ability of the cells was tested by counting the number.

(3) Effect of laeverin antibody on the invasion ability of laeverin-expressing cells A mouse anti-human laeverin monoclonal antibody (10 mg / ml) prepared by immunizing laeverin protein with 10% of G418 and 10% In addition to the cell suspension (5 × 10 ⁵ / ml) in the nutrient mixture F-12 / HAM culture medium containing FCS, the cell invasion ability was assayed in the same manner as in (2) above. In this experiment, no significant difference was observed in the rate of cell growth between each cell group and under culture conditions.

The results are shown in FIG. Among the four cell lines into which the laeverin gene was introduced, the number of infiltrating cells was significantly increased compared to the negative cell lines in which the laeverin gene was not introduced with B2D2, D1D1, and D1D2 except for B2D1. Among them, B2D2 and D1D2 enhanced the invasive cell ability in the cell group by 6 times.
The laeverin antibody did not act on the laeverin gene non-introduced negative cell line, but laeverin gene-introduced cells showed a tendency to increase infiltration.
Furthermore, it was confirmed by immune cell staining that cells with strong laeverin expression accounted for many of the cells infiltrating the lower surface of the membrane.

The higher invasion ability of B2D2, D1D1 and D1D2 than in the negative cell line indicates that laeverin protein may be involved in the control of cell invasion. The result that the anti-laeverin antibody affects the invasion ability of laeverin transgenic cells is thought to support this reasoning.
Considering that laeverin protein is an enzyme, experimental results show that the presence of laeverin protein can change the migration and invasion ability of various cells such as vascular endothelial cells and nerve cells in the living body. It strongly suggests the possibility of a reversible laeverin as a therapeutic agent.

Discussion on laeverin molecule The laeverin of the present invention has a long region called peptidase M1 motif (98-506). Within this region laeverin has a highly conserved motif, namely His-Glu-Xaa-Xaa-His- (18 amino acid residues) -Glu (415-438) (FIG. 4). This zinc-binding motif is common to many enzyme activities, and this structure is named gluzincin aminopeptidase (AJ Barrett, Methods Enzymol. 244 (1994) 1-15). There is a highly hydrophobic region (14-36, 23 residues) at the N-terminus of laeverin, which is thought to be a transmembrane domain. Therefore, laeverin is presumed to be a membrane-bound protein with a short intracellular part.

  The amino acid sequence of laeverin is aminopeptidase N (J. Olsen, et al., FEBS Lett. 238 (1988) 307-314). And have homology. Aminopeptidase N is a 160 kDa zinc-dependent membrane-bound peptidase (B.L. Vallee, et al., Biochemistry 29 (1990) 5647-5659). It has been reported that this peptidase is expressed in ovarian inner lining cells and endometrial stromal cells, and may be involved in the regulation of follicular development and decidualization of endometrial stromal cells. (H. Fujiwara et al., J. Clin. Endocrinol. Metab. 74 (1992) 91-95; K. Imai, et al., Biol. Reprod. 46 (1992) 328-334; T. Tachibana, et al ., Hum. Reprod. 11 (1996) 497-502; K. Nakamura, et al., Hum. Reprod. 11 (1996) 1952-1957; T. Inoue, et al., J. Clin. Endocrinol. Metab. 79 (1994) 171-175).

The molecular weight of laeverin calculated from the translated region of cDNA is 113 kDa, which is smaller than the molecular weight of Laevelin purified from placenta (160 kDa). As in the case of aminopeptidase N, this discrepancy is thought to be due to the modification of laeverin by glycosylation (sugar chain). From these findings, this new protein can be identified as a kind of membrane-bound metallopeptidase.
Human chorion laeve expresses membrane-bound peptidase dipeptidyl peptidase IV (dipeptidyl peptidase IV, DDPIV, EC3.4.14.5) and neutral endopeptidase (neutral endopeptidase, NEP, EC3.4.24.11) (K. Imai, et al., Am. J. Obstet. Gynecol. 170 (1994) 1163-1168). Recently, the present inventors have expressed DPPIV and another membrane-bound peptidase, carboxypeptidase M (carboxypeptidase M, CP-M, EC3.4.17.12), in EVT that has invaded maternal decidual tissue. (Y. Sato, et al., J. Clin. Endocrinol. Metab. 87 (2002) 4287-4296). These peptidases are expressed on the cell membrane surface and can degrade some biologically active peptides outside the cell. Kenny et al. Suggest that peptidases on the surface of the cell membrane regulate the growth and differentiation of many cells by regulating peptide factor activity and reaching the target cell (AJ Kenny, et al., Lancet 2 (1989) 785-787).

  It is thought that the chorion laeve of the egg membrane is wrapped in amniotic membrane containing fetus and amniotic fluid in the womb to form the boundary between the fetus and the mother and to control the environmental maintenance of the water phase around the fetus. On the other hand, EVT infiltrating the maternal decidual tissue further invades the maternal spiral artery and is replaced to dilate the blood vessels to supply sufficient blood flow to the intervillous space. Membrane-bound peptidases expressed in these trophoblasts seem to play some role in the regulation of trophoblast function. To reinforce this concept, several chemokines, including RANTES, have recently been shown to be substrates for DPPIV. It was also reported that CCR1, a RANTES receptor, was expressed in EVT in accordance with DPPIV expression. Furthermore, it is suggested that RANTES promotes invasion of EVT, and that DDPIV and CCR1 on EVT regulate EVT invasion, metabolism and responsiveness to chemokines (Y. Sato, et al., Development 130). (2003) 5519-5532). DPPIV and CP-M have also been proposed to be involved in luteinization of human granulosa cells in luteinization after ovulation (H. Fujiwara, et al., J. Clin. Endocrinol. Metab. 5 (1992) 1352-1357; H. Fujiwara, et al., J. Clin. Endocrinol. Metab. 79 (1994) 1007-1011; S. Yoshioka, et al ,, Mol. Hum. Reprod. 4 (1998): 709-717] These peptidases are also known to be membrane surface molecules of immune cells as cluster of differentiation (CD) markers, and membrane-bound peptidases are also known to function and differentiate immune cells. (U. Lendeckel, et al., Adv. Exp. Med. Biol. 477 (2000) 1-24). This suggests the existence of an event that supports the involvement of the membrane-bound peptidase containing laeverin of the invention in reproductive phenomena (H. Fujiwara, et al., Endocr. J. 46 (1999) 11-25). , This departure It is intended to support the utility of Laeverin.

  The novel EVT-derived protein laeverin of the present invention is a protein that can serve as a molecular marker for EVT having extremely specific functions and immunological properties in vivo, and the present invention provides a large amount of the protein and its gene. It provides a means to do. Analysis of EVT-specific proteins is useful in a wide range of medical fields including immunology, oncology, etc. Through this analysis, the present invention provides an understanding of the regulatory mechanism of pregnancy maintenance, and further development of methods for suppressing invasion of cancer cells, It can greatly contribute to the development of medicine in a wide range of fields, such as elucidating the mechanism of immune tolerance and developing autoimmune disease treatment.

It is a reproduction of the photograph which shows distribution of laeverin expressed on EVT of chorion laeve analyzed based on immunohistochemical staining. It is a reproduction of a photograph showing a profile on silver-stained 8% SDS-PAGE of laeverin produced from human placenta using CHL-2 monoclonal antibody. laeverin cDNA sequence. This is the deduced amino acid sequence of laeverin. It is a reproduction of a photograph showing the results of a gene expression search by Northern blot method. It is a graph which shows the invasive cell number of the CHO cell which introduce | transduced the laeverin gene in the Matrigel invasion assay, and the CHO cell which is not introduce | transduced.

Claims (7)

  A reagent for detecting extravillous trophoblast cells, comprising an antibody specific for a protein comprising the amino acid sequence represented by SEQ ID NO: 2.   A method for isolating extravillous trophoblast cells, which uses an antibody that binds to a protein comprising the amino acid sequence represented by SEQ ID NO: 2.   A method for isolating extravillous trophoblast cells, which uses an antibody that specifically binds to a protein comprising the amino acid sequence represented by SEQ ID NO: 2.   A method for labeling extravillous trophoblast cells using an antibody that binds to a protein comprising the amino acid sequence represented by SEQ ID NO: 2.   A method of labeling extravillous trophoblast cells using an antibody that specifically binds to a protein comprising the amino acid sequence represented by SEQ ID NO: 2.   The reagent according to claim 1, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a fragment thereof.   The method according to claim 2, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or a fragment thereof.

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