CN101028522A - Function and use of MNSF-beta gene during process of embryo nidation - Google Patents
Function and use of MNSF-beta gene during process of embryo nidation Download PDFInfo
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Abstract
An application of MNSF beta gene or its coding protein in regulatin the nidation of embryo, and the application of the antagon of MNSF beta (such as the specific antibody or antisense nucleic acid of MNSF) in specifically suppressing the nidation of embryo are disclosed.
Description
Technical field
The present invention relates to biotechnology and medical domain, specifically, the present invention relates to function and the application thereof of MNSF β gene in the embryo nidation process.
Background technology
MNSF (the non-specific inhibitive factor of monoclonal, monoclonal nonspecific suppressor factor) is found to be a kind of lymphocyte factor (the Nakamura M etc. that generate by the mouse T cell hybridoma at first with nonspecific immunity depression effect, (1986), Isolation and characterization of amonoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma.Journal of Immunology, 136:2904-2909).And MNSF β is the subunit of MNSF, also be main composition (the Nakamura M etc. that MNSF brings into play its biological function, (1996), Ubiquitin-likemoiety of the monoclonal nonspecific suppressor factor beta is responsible for itsactivity.Journal of Immunology, 156:532-538), the synthesis secretion that can suppress IgG, and propagation (the Kondoh T etc. of T cell and B cell, (1999), Ubiquitin-like polypeptide inhibits theproliferative response of T cells in vivo.Immunobiology, 200:140-149).
For the parent uterine cancer cell, embryo and subsequent fetus all belong to half allohisto compatibility, it is exogenous immune material, therefore, in whole pregnant process, must between mother's immune system and embryo/fetal cell, set up a kind of special compatibility, to guarantee fetal survival and normal development.Though the molecular mechanism that the immune tolerance state between this female tire interface forms is unclear fully as yet, but the local immunity inhibit feature of some cytokines, and the change of Th1 cell/Th2 cell balance, between mother-tire, brought into play very important effect in the forming process of immune tolerance state.
Because MNSF β is a kind of inhibiting cytokine of nonspecific immunity that has, therefore will expects that very naturally this expression of gene level should improve during embryo nidation, thereby help suppressing the immunoreation between mother-tire.But according to bibliographical information (Nie GY, Deng, (2000), Identification of monoclonalnonspecific suppressor factor beta (MNSF β) as one of the genes differentiallyexpressed at implantation sites compared to interimplantation sits in the mouseuterus.Molecular Reproductive and Development, 55:351～363), MNSF β gene all has high-caliber expression in the Mouse Uterus tissue before unpregnancy and embryo nidation, and expression does not have significant change; But between the mice embryonic implantation period, MNSF β gene is tangible down regulation trend at the expression in implantation site with respect to non-implantation site, and therefore infer that this gene is not directly to bring into play its immunosuppressive action in the embryo nidation process, and may be decline by its expression, promote the propagation of Th2 cell in the endometrial tissue of implantation position and synthesizing of IL4, make Th1 cell/Th2 cell balance be partial to the Th2 cell, thereby help the formation of immune tolerance state.
To sum up, people are very few to the influence understanding of embryo nidation and phenolics to MNSF β gene and expression product thereof at present, and think that it is not directly to bring into play immunosuppressive action in the embryo nidation process.Yet above-mentioned result of study is the supposition on surface just, also is necessary to further investigate the mechanism of action of MNSF β gene at embryo nidation and phenolics.
Summary of the invention
The purpose of this invention is to provide the application of a kind of MNSF β gene in regulating embryo nidation.
Another object of the present invention provides the purposes of antagonist or the agonist of MNSF β, is used to regulate embryo's implantation.
In a first aspect of the present invention, the purposes of a kind of MNSF β gene or its encoding proteins is provided, described MNSF β gene or its encoding proteins are used as the promoter that promotes embryo nidation.
In a second aspect of the present invention, the purposes of a kind of MNSF β gene or its encoding proteins is provided, described MNSF β gene or its encoding proteins are used to prepare the medicine that promotes embryo nidation.
In a third aspect of the present invention, a kind of purposes of MNSF beta antagonists is provided, described MNSF beta antagonists is used as the inhibitor that suppresses embryo nidation.
In a fourth aspect of the present invention, a kind of purposes of MNSF beta antagonists is provided, described MNSF beta antagonists is used to prepare the medicine that suppresses embryo nidation.
In a preference of the present invention, described medicine is a contraceptive.
In another one preference of the present invention, described antagonist is the antisensenucleic acids of MNSF β, the part of MNSF β, or the antibody of anti-MNSF beta polypeptides.
In a fifth aspect of the present invention, a kind of purposes of MNSF beta-agonists is provided, it is used as the promoter that promotes embryo nidation.
In a sixth aspect of the present invention, a kind of purposes of MNSF beta-agonists is provided, it is used to prepare the medicine that promotes embryo nidation.
In a preference of the present invention, described medicine is the assisted reproduction medicine.
In a seventh aspect of the present invention, provide whether a kind of definite test compounds is the method for antagonist or the agonist of MNSF β, it comprises step:
(a) embryo with test compounds and MNSF beta polypeptides adding In vitro culture organizes as test, and the embryo of MNSF beta polypeptides adding In vitro culture is organized in contrast;
(b) observation test group and matched group embryo nidation situation are if the embryo nidation quantity of test group, represents then that test compounds is the agonist of MNSF β greater than matched group; If the embryo nidation quantity of test group, represents then that test compounds is the antagonist of MNSF β less than matched group.
In a eighth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains MNSF beta polypeptides, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the expression of MNSF β albumen in mice ovum and body early embryo, and wherein 1 is ovum; 2 is 2-cell stage embryo; 3 is blastaea.(scale=10 μ m)
Fig. 2 has shown that the quantitative PCR in real time of Sprr2f gene expression dose detects data in the Mouse Uterus tissue, and wherein, A figure is that fluorescent quantitation detects data, and its vertical coordinate is a fluorescent value, and abscissa is a period, the corresponding illustrated in table 4 of each curve; B figure is a standard curve, and its vertical coordinate is for reaching threshold value institute through period, and abscissa is a concentration.
Fig. 3 has shown that the quantitative PCR in real time of Ovpg1 gene expression dose detects data in the Mouse Uterus tissue, and wherein, A figure is that fluorescent quantitation detects data, and its vertical coordinate is a fluorescent value, and abscissa is a period, the corresponding illustrated in table 5 of each curve; B figure is a standard curve, and its vertical coordinate is for reaching threshold value institute through period, and abscissa is a concentration.
The specific embodiment
The inventor finds first that through extensive and deep research the non-specific inhibitive factor β of monoclonal (MNSF β) albumen is that embryo nidation is necessary; And confirm that first the antagonist (as antibody or the antisensenucleic acids of MNSF β) of MNSF β can suppress embryo's implantation specifically.Finished the present invention based on this.
Existing research thinks that MNSF β directly brings into play immunosuppressive action.Yet the inventor is well thrashed out and proves, and finds that MNSF β is that embryo nidation is necessary, and MNSF β has multi-biological and learns function (as suppressing the immunoreation at female tire interface) in mice early pregnancy process.It can also regulate the expression of other several genes.And anti-MNSF β antibody can pass through to strengthen the immunoreation at female tire interface, thereby suppresses embryo's implantation.
In the present invention, term " MNSF β albumen ", " MNSF beta polypeptides ", " the MNSF β factor " or " the non-specific inhibitive factor β of monoclonal " etc. are used interchangeably, and all refer to have albumen or the polypeptide of the non-specific inhibitive factor beta amino acids of monoclonal sequence (SEQ ID NO:2).They can contain or not contain initial methionine.In addition, these terms also are included in homologue with function of the same race or the homologous protein in other mammals (as people, Canis familiaris L., cattle, sheep, monkey).
Mice MNSF β gene cDNA sequence (the GenBank accession number: D26610) as SEQ ID NO:1, its amino acid sequence coded such as SEQ ID NO:2.
Contain 2 functional areas in the mice MNSF β protein molecular, the ubiquitin sequence that first functional domain is made up of 70 aa residues (ubiquitin, UBQ) (1-70aa); Second ribosome S30 protein sequence (Ribosomal protein S30) (74-133aa) that functional domain is made up of 60 aa residues.
Existing research report (Xavier R, Deng, (1995), Human nonspecific suppressor factor (hNSF): cell source and effects on T and B lymphocytes.Immunology, 192:262-271), from a SLE patient's ascites separation and purification to people hNSF β (the non-special inhibitive factor β of people of mice MNSF β albumen homology, human nonspecific suppressor factor-beta) albumen, and confirm after deliberation, people hNSF β albumen also has the similar biological function with mice MNSF β, can suppress the synthetic of IgG, and the propagation of B cell and T cell.
Should be understood that because proteic nucleotide sequence of mammiferous MNSF β such as Mus and aminoacid sequence all are known, can obtain its albumen with the DNA recombinant technique of this area routine.
The particularly preferred albumen of one class is MNSF β analog, i.e. the homologous protein of MNSF β in other mammals (as cattle, sheep, rabbit, Canis familiaris L., monkey, people etc.).The coded sequence of the homologous protein of these other species can obtain by the method for hybridizing or increase, and then obtain these homologous proteins by conventional recombination method according to the sequence of MNSF β.
As used herein, " isolated M NSF β albumen or polypeptide " is meant that the MNSF beta polypeptides is substantially free of natural relative other albumen, lipid, saccharide or other material.Those skilled in the art can use the purified technology of protein purification MNSF beta polypeptides of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purification, or the product of chemosynthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (for example, antibacterial, yeast, higher plant, insecticide and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivant and the analog of MNSF beta polypeptides.As used herein, term " fragment ", " derivant " are meant with " analog " and keep identical biological function or the active polypeptide of the natural MNSF β factor of the present invention basically.Polypeptide fragment of the present invention, derivant or analog can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another chemical compound (such as the chemical compound that prolongs the polypeptide half-life, Polyethylene Glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as targeting sequencing or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purification, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivant and analog belong to the known scope of those skilled in the art.
In the present invention, term " MNSF beta polypeptides " refers to have the active SEQ ID of direct or indirect adjusting embryo nidation NO:2 polypeptide of sequence.This term also comprises having and variant form MNSF β factor identical function, SEQ IDNO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or one or more (being generally in 20 of N-terminal interpolation, preferably being in 10, more preferably is in 5) aminoacid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar aminoacid of performance.Again such as, add one or several aminoacid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragment and the reactive derivative of the MNSF β factor.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of MNSF β DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-MNSF beta polypeptides to obtain.The present invention also provides other polypeptide, as comprises MNSF beta polypeptides or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of MNSF beta polypeptides.Usually, this fragment have MNSF beta polypeptides sequence at least about 50 continuous amino acids, usually at least about 100 continuous amino acids, preferably at least about 150 continuous amino acids, more preferably at least about 200 continuous amino acids, best at least about 300 continuous amino acids.
The invention still further relates to the analog of MNSF beta polypeptides.The difference of these analog and natural MNSF beta polypeptides can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic agent and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars.Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(as D-aminoacid), and has non-natural analog that exist or synthetic aminoacid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylation or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as phosphotyrosine, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-Proteolytic enzyme performance or optimized solubility property by modifying.
MNSF beta nucleoside acid full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of conventional method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence than long time, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies be stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by conventional method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small fragments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the DNA sequence of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This DNA sequence can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The MNSF beta polypeptides is of use in many ways.These purposes include, but is not limited to: directly as the disease (infertile as what lowly cause because of the embryo nidation ability) due to Drug therapy MNSF β factor hypofunction or the forfeiture be used to screen antibody, polypeptide or other part that promotes or resist MNSF β factor function.The peptide molecule that can suppress or stimulate MNSF β factor function that can be used for seeking therapeutic value with the reorganization MNSF β factor screening peptide library of expressing.
On the other hand, the present invention also comprises MNSF β DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into MNSF β gene outcome or fragment.Preferably, refer to that those can combine with MNSF β gene outcome or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the molecule of the MNSF β factor, comprise that also those do not influence the antibody of MNSF β factor function.The present invention also comprise those can with modify or without the bonded antibody of MNSF β gene outcome of modified forms.
The present invention not only comprises complete monoclonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the MNSF β gene outcome of purification or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing the MNSF β factor or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block MNSF β function and the antibody that does not influence MNSF β function.Each antibody-like of the present invention can utilize the fragment or the functional areas of MNSF β gene outcome, obtains by the routine immunization technology.These fragments or functional areas can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene outcome of producing in the prokaryotic cell (for example E.Coli) with the bonded antibody of unmodified form of MNSF β gene outcome; With the bonded antibody of post translational modification form (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene outcome that produces in the eukaryotic cell (for example yeast or insect cell).
The antibody of anti-MNSF β can be used in the immunohistochemistry technology, detects the MNSF β in the biopsy specimen.
Antibody among the present invention also can be used for suppressing embryo nidation as the antagonist of MNSF β, thereby is used for contraception.The antibody that gives suitable dosage can be blocked generation or the activity of MNSF β.
Utilize albumen of the present invention,, can filter out with MNSF β interactional material takes place, as receptor, inhibitor, agonist or antagonist etc. by various conventional screening techniques.For example, can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various aminoacid that may make up by screening with the bonded peptide molecule of MNSF β obtains.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or receptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, Intradermal, intravaginal or topical.
The present invention also provides a kind of pharmaceutical composition, and it contains MNSF beta polypeptides of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the excipient of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the MNSF β albumen of safe and effective amount or its antagonist, agonist are applied to mammal, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The polynucleotide of MNSF β also can be used for multiple therapeutic purposes.Gene therapy technology can be used for treating because the embryo nidation level due to the expression of the MNSF β of the nothing expression of MNSF β or unusual/non-activity is low.Antisense nucleotide can suppress embryo nidation, thereby is used for contraception.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization MNSF β level.These tests are known in the art, comprise methods such as radioimmunoassay.The MNSF β level that is detected in the test, can with lay down a definition the MNSF β factor in disease such as infertile importance and be used for diagnosis.
A kind of method that whether has the MNSF β factor in the test sample that detects is to utilize the specific antibody of the MNSF β factor to detect, and it comprises: sample is contacted with MNSF β factor-specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample MNSF β factor.
Major advantage of the present invention is:
(1) finding that first MNSF β albumen is that embryo nidation is necessary, has negated that existing research thinks that MNSF β is not an argument of directly bringing into play immunosuppressive action.
(2) confirm that first the antagonist (as antibody or the antisensenucleic acids of MNSF β) of MNSF β can suppress embryo's implantation specifically.
(3) find that MNSF β has multi-biological and learns function in mice early pregnancy process, it can also regulate other multiple pregnant Expression of Related Genes.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The proteic expression of MNSF β is arranged in mice ovum and the prenidatory body early embryo
The inventor has carried out immunofluorescence analysis to ovum, 2-cell stage embryo and blastaea.The results are shown in Figure 1, find in ovum (1), 2-cell stage embryo (2) and blastaea (3), all to detect the proteic positive signal of MNSF β.
Because factor fixes,, infer according to fluorescence intensity therefore that the MNSF β albumen expression among the embryo in early days may improve with the pregnancy period so the power of signal can reflect the expression of testing protein to a certain extent.
Embodiment 2
The antibody enclosed experiment confirms that anti-MNSF β antibody capable specificity suppresses the implantation of mice embryonic
In the present embodiment, the coded sequence of ORF completely with conventional PCR method acquisition MNSF β inserts the multiple clone site (available from Amersham Pharmacia Biotech company) of pGEX-4T-2 then, thereby obtains expression plasmid pGEX-4T-2/MNSF β.With this plasmid at expression in escherichia coli GST-MNSF beta fusion proteins; Handle the GST-MNSF beta fusion proteins of purification then with thrombin of beef, isolate reorganization MNSF β albumen, and with it as immunogen, be immunostimulant with the freund adjuvant, immunizing rabbit according to a conventional method prepares the anti-MNSF β of the rabbit serum of high titre; By Protein A Sepharose CL-4B column chromatography, from antiserum, isolate the reorganization MNSF β (rMNSF β) of biologically active again, and in can be effectively with the anti-MNSF β-IgG of the bioactive rabbit of MNSF β.
With above-mentioned similar conventional method, the inventor has also made up recombinant expression plasmid pVL 1392/MNSF β (insect baculovirus transfer vector pVL1392 is available from Invitrogen company), behind linearisation nuclear polyhedrosis virus (AcNPV) DNA (available from Invitrogen company) cotransfection Sf9 insect cell, identify and collect Sf9 insect cell (available from Invitrogen company) condition training liquid (the rMNSF β that contains reorganization MNSF β albumen (rMNSF β)
+Sf9 condition training liquid).The inventor utilizes LPS energy inducing mouse splenocyte synthesis secretion IgG, and MNSF β has this feature of biological activity that suppresses this effect, (see Nakamura M etc. according to the method that Nakamura describes, J.Immunol., 156:532,1996), by bioassay, confirm that the rMNSF β albumen of insect cell expression has the biological activity of natural MNSF β, and the anti-MNSF β-IgG of rabbit can be effectively in and the biological activity of rMNSF β, illustrate that prepared rMNSF β and the anti-MNSF β of rabbit antibody are applicable to the antibody enclosed experiment.
In the experiment, will be divided into 10 groups at random with a collection of pregnant the 1st day ICR mice, the injection solution of each cornua uteri is respectively:
I group: 30 μ L normal saline;
II group: the 30 μ L normal saline that contain the positive rabbit anteserum-IgG of 300 μ g;
III group: the 30 μ L normal saline that contain the anti-GST-IgG of 300 μ g rabbits;
IV group: the 30 μ L normal saline that contain the anti-MNSF β-IgG of 500 μ g rabbits;
V group: the 30 μ L normal saline that contain the anti-MNSF β-IgG of 300 μ g rabbits;
VI group: the 30 μ L normal saline that contain the anti-MNSF β-IgG of 100 μ g rabbits;
VII group: contain 500 μ L rMNSF β
+30 μ L normal saline of Sf9 condition training liquid freeze dried powder;
VIII group: the 30 μ L normal saline that contain 500 μ L Sf9 conditions training liquid freeze dried powder;
IX group: contain anti-MNSF β-IgG of 100 μ g rabbits and 500 μ L rMNSF β
+30 μ L normal saline of Sf9 condition training liquid freeze dried powder;
X group: the 30 μ L normal saline that contain anti-MNSF β-IgG of 100 μ g rabbits and 500 μ L Sf9 conditions training liquid freeze dried powder.
All animal subjects all are in injection in pregnant the 3rd day morning, and put to death pregnant the 8th day morning, and embryo's quantity of each cornua uteri is counted and statistical analysis.Wherein, the laboratory animal of IX group and X group places injection solution earlier and hatched under 35 ℃ 1 hour before injection, so that generation neutralization reaction earlier between the antigen, antibody.
The results are shown in Table 1, show: positive rabbit anteserum-IgG does not have influence to the mice embryonic implantation; Compare with positive rabbit anteserum-IgG, the anti-GST-IgG of rabbit is to the also not influence of implantation of mice embryonic; The anti-MNSF β-IgG of rabbit then demonstrates the inhibitory action to the mice embryonic implantation, and this inhibitory action strengthens along with the increase of the anti-MNSF β of rabbit-IgG dosage, has tangible dosage effect; Though reorganization MNSF β albumen does not influence embryo's implantation, but behind anti-MNSF β-IgG of rabbit and reorganization MNSF β incubation, then the anti-MNSF β-IgG of rabbit no longer demonstrates the inhibitory action to embryo nidation, being the anti-MNSF β-IgG of rabbit can be neutralized by rMNSF β to embryo's inhibitory action, illustrates that this inhibitory action is that anti-MNSF β antibody is special.This result shows that anti-MNSF β antibody has specific inhibitory action to embryo's implantation, and the rMNSF β of biologically active does not have inhibitory action to embryo's implantation.
Therefore, the MNSF β albumen of uterine cancer cell synthesis secretion is that embryo nidation is necessary, the decline of implantation site MNSF beta gene expression level is to cause owing to stromal cell is divided into decidual cell, but not the MNSF β albumen of implantation site uterine cancer cell synthesis secretion, and the MNSF β albumen of embryonal tissue's synthesis secretion is directly to bring into play its immune suppression function, thereby plays a significant role in the forming process of immune tolerance state between mother-tire.
The anti-MNSF β of table 1 antibody is to the inhibitory action of mice embryonic implantation
| Group | Injection solution (each cornua uteri) | The cornua uteri quantity (n) that is used to detect | The embryo number of each cornua uteri (mean+SD) | Pb |
| I | 30 μ l normal saline | 16 | 5.1±1.4 | ＞0.05 |
| II | Positive rabbit anteserum-the IgG of 300 μ g/30 μ l normal saline | 10 | 4.8±1.8 | - |
| III | 300 μ g rabbits resist-GST IgG/30 μ l normal saline | 10 | 4.9±1.9 | ＞0.05 |
| IV | The anti-MNSF β of 500 μ g rabbits IgG/30 μ l normal saline | 12 | 1.2±0.9 | ＜0.001 |
| V | The anti-MNSF β of 300 μ g rabbits IgG/30 μ l normal saline | 10 | 1.9±1.5 | ＜0.001 |
| VI | The anti-MNSF β of 100 μ g rabbits IgG/30 μ l normal saline | 20 | 3.1±1.7 | ＜0.05 |
| VII | 500μl rMNSFβ +Sf9 condition training liquid powder/30 μ l normal saline | 20 | 4.9±1.7 | ＞0.05 |
| VIII | 500 μ l Sf9 conditions training liquid powder/30 μ l normal saline | 10 | 4.8±1.5 | ＞0.05 |
| IXa | The anti-MNSF β of 100 μ g rabbits IgG+ 500 μ l rMNSF β +Sf9 condition training liquid powder/30 μ l normal saline | 20 | 4.7±2.5 | ＞0.05 |
| Xa | The anti-MNSF β of 100 μ g rabbits IgG+ 500 μ l Sf9 conditions training liquid powder/30 μ l normal saline | 10 | 2.8±1.6 | ＜0.05 |
A: injection is prepended to 35 ℃ of following incubation 1hr.
B: when calculating the P value, compare with II group data with the t-method of inspection.
Embodiment 3 cDNA chip technologies are analyzed the molecular mechanism that anti-MNSF β antibody suppresses embryo nidation
In order further to understand the biological function of MNSF β gene in pregnant process in depth, the inventor uses the cDNA chip technology, use Affymetrix MOUSE 430A chip gene expression profile, to between a pair of like this uterine cancer cell anti-MNSF β antibody treatment and without anti-MNSF β antibody treatment, difference in gene expression is analyzed, the result adopts the Affymetrix gene chip scanning instrument to scan, and read and deal with data with little array analysis software Microarray Suite5.0 (Affymetrix company), the differential gene screening criteria is: " Change " is I or MI, Ratio＞or=1, " experimental group " Detection is the up-regulated gene that is decided to be of " P "; " Change " for D or MD, Ratio＜or=-1, " matched group " Detection is decided to be down-regulated gene table 2 for " P's "; And use quantitative real time pcr its partial analysis result is verified.Picked at random SPRR2f (little proline rich protein 2F, small proline-rich protein2F), DCK (deoxycytidine kinase, deoxycytidine kinase), INHBB (inhibin β B, inhibin betaB), ISP1 (implantation serine protease 1, implantation serine proteinase 1), OVPG1 (oviductal glycoprotein 1, oviductal glycoprotein 1) and the differential gene that screens of 6 cDNA chip technologies such as NPR2 be testing gene, with the beta-actin is house-keeping gene, carries out the real-time fluorescence quantitative PCR reaction respectively.According to the gradient dilution DNA standard curve of drawing, the concentration results of each testing gene and house-keeping gene is directly generated by instrument.Each testing gene concentration is divided by the concentration of its house-keeping gene, promptly for this reason relative amount after the correction of testing gene (table 3, Fig. 2, Fig. 3).
The inventor is by the cDNA chip technology, filter out 144 knowns that between two kinds of tissues, have differential expression altogether, some gene is relevant with reproduction activity, relevant such as SPRR2F genes such as (little proline rich protein 2F, small proline-rich protein 2F) with the generation and the oestrous cycle of gametid; SGK genes such as (kinases that serine/glucocorticoid is regulated, serum/glucocorticoid regulated kinase) is relevant with fetal development; Genes such as ISP1 are then directly relevant with the embryo nidation process.According to bibliographical information, these genes that expression raises after anti-MNSF β antibody treatment such as SGK, SPRR2F, the expression in normally enclosing implantation period Mouse Uterus tissue should be reduced; And the gene of some expression downward modulations after anti-MNSF β antibody treatment such as ISP1, the expression in normally enclosing implantation period Mouse Uterus tissue should raise; This is consistent with the phenomenon that anti-MNSF β antibody capable suppresses embryo nidation.But the biological function of these genes in reproductive process is different, and prompting MNSF β gene has multi-biological and learns function in mice early pregnancy process.
Except above-mentioned these genes relevant with reproductive process, also having series of genes is to participate in the regulation and control organism immune response, comprise: PSMA6 (the α 6 type proteasome subunits relevant with the conduction of TXi Baoshouti signal with antigen presentation, proteasome subunit, alpha type 6) gene such as, the genes such as DCK relevant with activated immune cell, with relevant VAV3 (the vav3 oncogene of lymphopoiesis differentiation, vav 3 oncogene) gene such as, and the CFI genes such as (complement Composition Factor 1, complementcomponent factor Is) relevant with complement system activation.Equally, report according to existing literature, the reduction of gene expression doses such as the raising of gene expression doses such as DCK and VAV3 will cause immunoreactive enhancing, and therefore, the immunoreation that strengthens female tire interface is the action pathway that anti-MNSF β antibody suppresses embryo nidation.
Except strengthening this approach of female tire interface immunoreation, anti-MNSF β antibody also other approach such as the wettability by the intercellular adhesion of influence, cell, cellular metabolism/fetal development suppresses embryo nidation.For example: the rise of TFPI gene expression doses such as (the tissue factor bypass suppress son, tissue factor pathway inhibitor) can reduce the wettability of cell, and the raising of gene expression doses such as INHBB then can suppress embryo's growth; And the downward modulation of MELA gene expression doses such as (melanoma-associated antigen, melanoma antigen) can weaken intercellular adhesion, and SLC14A2 (solute transfer body family 14 (carbamide transhipment), the member 2; Solute carrierfamily 14 (urea transporter), member 2) etc. the reduction of gene expression dose can cause cellular metabolism unusual.Therefore, the inventor can determine, MNSF β gene not only plays an important role in the forming process of female tire interface immune tolerance state by its immune suppression function of direct performance, and brings into play multiple action in the potential function of regulating and control trophoblastic cell wettability and embryonal tissue's development in the pregnant in early days process by this gene.
Also find in 144 knowns that screen, have nearly 30 genes that expression is also arranged by analyzing the inventor in different tumor cells.The characteristic of embryonal tissue's cell height propagation and differentiation and all similar to the wettability of normal structure to tumor tissues as half allosome tissue, therefore, further analyse in depth effect and the molecular mechanism of MNSF β gene in embryo nidation and fetal development, also help to understand the molecular mechanism that tumor tissues takes place, develops.
The quantitative PCR in real time measurement result of each testing gene of table 3 expression in two kinds of tissues
1, real-time quantitative measured value
| Beta-actin | Sprr2f | Dck | Inhbb | ISP1 | Ovgp1 | Npr3 | |
| Matched group | 2.71×10 -2 | 5.03×10 -4 | 1.85×10 -5 | 1.51×10 -3 | 4.34×10 -2 | 1.75×10 -3 | 1.01×10 -4 |
| Experimental group | 3.57×10 -2 | 4.71×10 -3 | 8.65×10 -5 | 5.42×10 -3 | 9.49×10 -3 | 7.73×10 -5 | 4.67×10 -5 |
2, confidential reference items are proofreaied and correct the back tabulation
| Sprr2f/beta-actin | Dck/beta-actin | Inhbb/beta-actin | ISP1/beta-actin | Ovgp1/beta-actin | Npr3/beta-actin | |
| Matched group | 1.86×10 -2 | 6.83×10 -4 | 5.57×10 -2 | 1.60E+00 | 6.46×10 -2 | 3.73×10 -3 |
| Experimental group | 1.32×10 -1 | 2.42×10 -3 | 1.52×10 -1 | 2.66×10 -1 | 2.17×10 -3 | 1.31×10 -3 |
| Experimental group/matched group | 7.1/1 | 3.5/1 | 2.7/1 | 1/6.0 | 1/30 | 1/2.8 |
Table 4 Sprr2f gene quantification detects tables of data
Table 5 Ovgp1 gene quantification detects tables of data
The antagonist of embodiment 4 screening MNSF β
Press embodiment 2 described methods, following two groups of materials (replacing antisensenucleic acids and transfection agents) be applied to pregnant mouse, observe then inhibitory action in the body of embryo nidation:
(a) candidate substances+MNSF β factor;
(b) the MNSF β factor.
If the embryo nidation rate of group (a) significantly is lower than the embryo nidation rate of group (b) statistically, show that then this candidate substances is the antagonist of MNSF β.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Family Planning Science and Research Institute.
＜120〉function and the application thereof of MNSF β gene in the embryo nidation process
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Ala Arg Ala Gly Lys Val Arg Gly Gln Thr Pro Lys Val Ala Lys Gln
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Pro Asn Ala Asn Ser
130
Claims (10)
1. the purposes of a MNSF β gene or its encoding proteins is characterized in that, described MNSF β gene or its encoding proteins are used as the promoter that promotes embryo nidation.
2. the purposes of a MNSF β gene or its encoding proteins is characterized in that, described MNSF β gene or its encoding proteins are used to prepare the medicine that promotes embryo nidation.
3. the purposes of a MNSF beta antagonists is characterized in that, described MNSF beta antagonists is used as the inhibitor that suppresses embryo nidation.
4. the purposes of a MNSF beta antagonists is characterized in that, described MNSF beta antagonists is used to prepare the medicine that suppresses embryo nidation.
5. purposes as claimed in claim 4 is characterized in that, described medicine is a contraceptive.
6. purposes as claimed in claim 4 is characterized in that, described antagonist is the antisensenucleic acids of MNSF β, the part of MNSF β or the antibody of anti-MNSF beta polypeptides.
7. the purposes of a MNSF beta-agonists is characterized in that, it is used as the promoter that promotes embryo nidation.
8. the purposes of a MNSF beta-agonists is characterized in that, it is used to prepare the medicine that promotes embryo nidation.
9. whether a definite test compounds is the method for antagonist or the agonist of MNSF β, it is characterized in that it comprises step:
(a) embryo with test compounds and MNSF beta polypeptides adding In vitro culture organizes as test, and the embryo of MNSF beta polypeptides adding In vitro culture is organized in contrast;
(b) observation test group and matched group embryo nidation situation are if the embryo nidation quantity of test group, represents then that test compounds is the agonist of MNSF β greater than matched group; If the embryo nidation quantity of test group, represents then that test compounds is the antagonist of MNSF β less than matched group.
10. a pharmaceutical composition is characterized in that, it contains MNSF beta polypeptides, its agonist or its antagonist and the pharmaceutically acceptable carrier of safe and effective amount.
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| CN104805070A (en) * | 2014-01-24 | 2015-07-29 | 上海市计划生育科学研究所 | Establishment and application of immunosuppressive factor MNSFbeta gene conditional knockout mice model |
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