CN108828228A - Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia - Google Patents
Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia Download PDFInfo
- Publication number
- CN108828228A CN108828228A CN201810503358.4A CN201810503358A CN108828228A CN 108828228 A CN108828228 A CN 108828228A CN 201810503358 A CN201810503358 A CN 201810503358A CN 108828228 A CN108828228 A CN 108828228A
- Authority
- CN
- China
- Prior art keywords
- nanos2
- azoospermatism
- protein
- antibody
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of pair of azoospermatism or aspermia or oligospermia disease forecasting or the NANOS2 markers of diagnosis, wherein the NANOS2 marker can be DNA, mRNA or protein encoded by it of NANOS2.The present invention also provides a kind of NANOS2 polyclonal antibodies, and the external diagnosis reagent based on NANOS2 marker preparation, the prediction or diagnosis of external diagnosis reagent of the invention for azoospermatism or aspermia or oligospermia, have the advantages that easy, quick, accuracy and specificity with higher, it can be used as extensive screening to use, also can be used as detection means clinically, the diagnosis detection of the present invention in particular SCO type azoospermatism provides a kind of new way.
Description
Technical field
The present invention relates to biomedicine technical field, it is related to predicting and diagnosing the NANOS2 marker of azoospermatism or aspermia or oligospermia
And its application, specifically, protein and its antibody including NANOS2 gene coding are in SCO type azoospermatism diagnostic application.
Background technique
One of the main reason for male's azoospermatism and aspermia or oligospermia are male sterilities.Male's azoospermatism and the aspermia or oligospermia cause of disease are multiple
Miscellaneous, some are due to inadequate natural endowment, some cause due to lacking of proper care after one's birth, including chromosome number and textural anomaly, microorganism sense
Dye, drug influence etc..The method for treating this disease at present includes the medicinal treatment based on tonifying kidney and strengthening yang production of sperm drug, or passes through hand
Art treatment, but effect is poor, slow effect, therefore therapeutic effect is undesirable.Male spermatogenesis dysfunction is just paid more and more attention, but due to
The complexity of During spermatogenesis, spermatogenetic pathologic, physiologic is recognized for a long time it is insufficient, cause the prediction of patient, diagnosis,
Treatment and Index for diagnosis are more difficult.
The prior art mainly has chromosome karyotype analysis and AZF micro-deleted the clinical testing procedure of azoospermatism and aspermia or oligospermia
Detection etc., but time-consuming, low efficiency for the detection of these technologies.Therefore, it is necessary to develop a kind of kit or detection method, come fast
Speed prediction or diagnosis azoospermatism or aspermia or oligospermia.
Summary of the invention
The present invention is to solve the drawbacks described above of the prior art, is provided a kind of for predicting or diagnosing azoospermatism or aspermia or oligospermia
NANOS2 marker.Wherein, NANOS2 gene is located at No. 19 chromosome long arms of people, 1577 base (such as SEQ ID of overall length
Shown in NO.1), it is made of 1 exon and 0 introne.The ORF for the NANOS2 that the present invention is cloned into has 414 bases, compiles
The albumen of one 138 amino acid of code, predicted molecular weight 15kDa.Fig. 1 is the gene structure display of NANOS2, NANOS2
Gene ORF is 138 amino acid, and oblique line portion is EF-hand (62-116aa) structural domain；Lower section show the albumen of NANOS2
Sequence, or as shown in SEQ ID NO.2.By the detection to NANOS2 marker, improves and azoospermatism or aspermia or oligospermia are carried out in advance
The accuracy and specificity surveyed or diagnosed.
In the present invention, the disease for predicting or diagnosing is azoospermatism or aspermia or oligospermia, particularly for prediction or diagnosis cell
Type (sertoli cell only, SCO) azoospermatism.
In the present invention, the marker of prediction or diagnosis as azoospermatism or aspermia or oligospermia can be DNA, mRNA of NANOS2
Or protein encoded by it.When the DNA of NANOS2 in test individual has can pathogenic mutation and/or mRNA or encoded by it
When the level of protein is substantially less than Normal group, predictable test individual has the risk for suffering from azoospermatism or aspermia or oligospermia, or
It diagnoses test individual and suffers from azoospermatism or aspermia or oligospermia.
In the present invention, the homology with the NANOS2 DNA is 80%, 85%, 90%, 95%, 96%, 97%, 98%
And 99% sequence, also can be used as prediction or the diagnosis marker of azoospermatism or aspermia or oligospermia.
In the present invention, with the homology of the NANOS2 mRNA be 80%, 85%, 90%, 95%, 96%, 97%,
98% and 99% sequence also can be used as prediction or the diagnosis marker of azoospermatism or aspermia or oligospermia.
In the present invention, with the homology of the NANOS2 protein be 80%, 85%, 90%, 95%, 96%, 97%,
98% and 99% sequence also can be used as prediction or the diagnosis marker of azoospermatism or aspermia or oligospermia.
The present invention also provides DNA, mRNA of NANOS2 as described above or protein encoded by it as marker
Application in the product of preparation prediction or diagnosis azoospermatism or aspermia or oligospermia.
The present invention also provides the levels of DNA, mRNA or protein encoded by it for detecting NANOS2 as described above
Reagent preparing the application in in-vitro diagnosis product for predicting or diagnosing azoospermatism or aspermia or oligospermia.
The present invention also provides a kind of prediction or the in-vitro diagnosis product of diagnosis azoospermatism or aspermia or oligospermia, the in-vitro diagnosis
Reagent includes the examination of the reagent, and/or specific detection NANOS2 mRNA of specific detection NANOS2 DNA as described above
The reagent of agent and/or specific detection NANOS2 protein.The reagent of the specific detection NANOS2 DNA can with but it is unlimited
Then nucleic acid probe, NANOS2 DNA described in the nucleic acid probe energy specific recognition；The specific detection NANOS2 mRNA
Reagent can be, but not limited to be nucleic acid probe, NANOS2 mRNA described in the nucleic acid probe energy specific recognition；It is described special
Property detection NANOS2 protein reagent can be, but not limited to be antibody, the antibody be NANOS2 monoclonal antibody or NANOS2
Polyclonal antibody identifies the NANOS2 protein, and the sequence of the NANOS2 protein is as shown in SEQ ID NO.2.
Wherein, the in-vitro diagnosis product includes detection kit, genetic chip, solid support and test paper；It is described solid
Body support includes array, microarray or protein array.
Wherein, immunodetection detection can be used in the external diagnosis reagent, and the immunodetection includes but is not limited to
It is electrophoresis, Western blot or immunofluorescence technique；For example, the albumen encoded using immuno-fluorescence assay NANOS2 gene
When, when the level for the protein that the NANOS2 gene of test individual encodes is substantially less than Normal group, it is determined as the positive.
Preferably, the detection kit includes the primer and probe of the NANOS2 gene as shown in SEQ ID NO.1；It is excellent
Selection of land, the detection kit further comprise：Extract the kit of protein, NANOS2 antibody, as concentration reference standard
NANOS2 purifying protein and secondary antibody.It is further preferred that the detection kit includes that (100 is micro- for total protein lysate
Rise every 10 milligrams of tissues), 5x sample-loading buffer, NANOS2 antibody is (1：1000), goat-anti-rabbit horseradish peroxidase labelling
Antibody (1：5000), NANOS2 purifying protein of the NANOS2 prokaryotic expression protein as concentration reference standard.Wherein, described
NANOS2 antibody is NANOS2 monoclonal antibody or NANOS2 polyclonal antibody, and the NANOS2 antibody is anti-such as SEQ ID
The antibody of NANOS2 albumen shown in NO.2.
Wherein, the genetic chip as the NANOS2 gene as shown in SEQ ID NO.1 oligonucleotide probe point sample in
Solid support surface is prepared, wherein the oligonucleotide probe can it is sensitive and specifically with it is thin from tissue
Corresponding gene or the hybridization of its amplified production in born of the same parents' sample.
The present invention also provides a kind of primer pairs, and wherein upstream primer sequence is as shown in SEQ ID NO.3, downstream primer sequence
Column are as shown in SEQ ID NO.4.
The present invention also provides the primer pairs in preparing the product for predicting or diagnosing azoospermatism or aspermia or oligospermia
Using.
The present invention also provides a kind of NANOS2 polyclonal antibodies, pass through the protein sequence as shown in SEQ ID NO.2
Immune animal obtains.Preferably, using NANOS2-GST fusion protein as antigen immune rabbit, obtained Post-immunisation serum is i.e.
Include the NANOS2 polyclonal antibody.
It wherein, is 80%, 85%, 90%, 95%, 96%, 97%, 98% with the homology of the NANOS2 protein sequence
And 99% sequence can also be used for immune animal, to prepare NANOS2 polyclonal antibody.
In the present invention, the preparation process of the NANOS2 polyclonal antibody is as described in example 1 above.
The present invention also provides the primer pair, NANOS2 polyclonal antibodies in preparation prediction or diagnosis azoospermatism or few essence
Application in the in-vitro diagnosis product of disease.
The present invention also provides a kind of prediction or the methods of diagnosis azoospermatism or aspermia or oligospermia, by the DNA for measuring NANOS2
Sequence, the level of mRNA or protein encoded by it, predicts test individual or is diagnosed：As the NANOS2 of test individual
DNA have can the level of pathogenic mutation and/or mRNA or protein encoded by it when being substantially less than Normal group, prediction
Test individual has the high risk for suffering from azoospermatism or aspermia or oligospermia, or diagnosis test individual suffers from azoospermatism or aspermia or oligospermia.
Compared to the prior art, the beneficial effects of the present invention are：Present invention firstly provides NANOS2 can be used as no essence
The marker of prediction or the diagnosis of disease or aspermia or oligospermia disease.Animal is immunized using NANOS2, NANOS2 Anti-TNF-α can be obtained
It is (outstanding to can be used for azoospermatism using the polyclonal antibody and the external diagnosis reagent prepared based on the NANOS2 marker for body
It is SCO type azoospermatism) or aspermia or oligospermia prediction or diagnosis, method provided by the invention is easy, quickly, with higher accurate
Property and specificity, can be used as extensive screening uses, and can be used as detection means clinically, also for examining for azoospermatism or aspermia or oligospermia
Disconnected detection provides a kind of new way.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of NANOS2 gene.
Fig. 2 is the electrophorogram that NANOS2-GST fusion protein is expressed in e. coli bl21.
Fig. 3 is the NANOS2 Anti-TNF-α for using the NANOS2-GST fusion protein of purifying to prepare as antigen-immunized animal
Body can detecte the NANOS2 of overexpression using the NANOS2 polyclonal antibody and using Western Blot method.
Fig. 4 is that immunofluorescence dyeing method carries out the paraffin section of normal testis and SCO testis of azoospermia disease in embodiment 2
The result of dyeing.
Fig. 5 is the comparison result of NANOS2 expression in normal testis and SCO type testis of azoospermia disease.Wherein, compareing 1 is
The statistical result of normal testis 15 lumen, the statistical result for 5 lumens that control 2 is normal testis 2, SCO1 are
The statistical result of 5 lumens of SCO type testis of azoospermia disease 1, SCO2 are the statistics of 5 lumens of SCO type testis of azoospermia disease 2
As a result, SCO3 is the statistical result of 5 lumens of SCO type testis of azoospermia disease 3, SCO4 is the 5 of SCO type testis of azoospermia disease 4
The statistical result of a lumen.
Specific embodiment
Embodiment 1：The preparation of NANOS2 polyclonal antibody
NANOS2 gene is expanded using PCR method first, then progress agarose gel electrophoresis recycling target fragment, then into
NANOS2/pGEX4T3 plasmid is transformed into large intestine by NANOS2 gene cloning to pGEX4T3 plasmid by row digestion and overnight connection
In bacillus BL21 cell, using the NANOS2-GST fusion protein of BL21 Bacillus coli cells expression and purification, using classics
IPTG inducible protein expression, induction destination protein expression.Cracking Escherichia coli GST-beads purifies NANOS2- later
Gst fusion protein.Specificity NANOS2 polyclonal antibody is prepared using the NANOS2-GST fusion protein immunization rabbit, and is carried out
External protein function detection.Wherein, preparing NANOS2 albumen used in NANOS2 polyclonal antibody is full-length proteins.
Wherein, the synthetic method of NANOS2/pGEX4T3 recombinant plasmid is：
PGEX-4T3 is purchased from Amersham Biosciences core gene company, according to NANOS2 full-length gene order,
The special primer of design amplification NANOS2 gene.
Upstream primer (SEQ ID NO.3)：5'-GACGCGTGGATCCCATGCAGCTGCCACCCTTCGA-3'
Downstream primer (SEQ ID NO.4)：5'-GTCCGCCGAATTCTCAGCGCTTGACCCTGCGTC-3'
Using upstream primer, limit can be added by the site of IPTG inducing expression, the end of downstream primer 5 ' by being added at its 5 ' end
Property restriction endonuclease EcoRI restriction enzyme site processed.Truncated NANOS2 genetic fragment is gone out using above-mentioned primer amplification, 5 μ l is taken to carry out 1% fine jade
Sepharose electrophoresis.The gel containing target fragment is cut from Ago-Gel, is operated and is walked according to DNA gel purification kit
Rapid purification and recovery target fragment.The target gene positive fragment of recovery purifying connects through NEB T4DNA ligase and pGEX-T carrier
Inducing expression is connect, full-length gene transformed competence colibacillus DH5 α filters out positive colony, after extracting plasmid, through the bis- enzymes of BamHI/EcoRI
Reaction identification is cut, the expression product positive colony containing target gene fragment is filtered out, after sending company to be sequenced, obtains positive colony i.e.
For purpose plasmid.
The method for inducing destination protein expression：
The BL21 bacterial strain of the recombinant expression plasmid NANOS2/pGEX4T3 containing target gene is inoculated in containing 100 μ g/ml
In the 2YT culture medium of ampicillin, after 37 DEG C of overnight incubations, by 1：100 ratio transferred speciess continue in fresh 2YT culture medium
Culture adds IPTG to final concentration of 1mmol/L, induces 4~5h, thallus is taken to analyze for SDS-PAGE to logarithmic growth phase.
Resulting fusion protein calculates after SDS-PAGE electrophoresis and coomassie brilliant blue staining according to newborn stripe size
NANOS2 molecular weight is consistent with the molecular weight calculated according to NANOS2 eukaryotic expression band, as shown in Figure 2.Fig. 2 is in large intestine
The NANOS2-GST fusion protein (being the SDS-PAGE glue through coomassie brilliant blue staining in figure) expressed in bacillus BL21；Wherein,
1-3 column are respectively that IPTG (0.1mM) induces 2h, and the purifying NANOS2-GST fusion protein obtained after 3h, 4h, the 4th is classified as egg
White marker.
Use the NANOS2-GST fusion protein as antigen immune rabbit, obtained Post-immunisation serum is contaminated with immunofluorescence
Color, the NANOS2 that can detecte endogenous expression by Western Blot.Wherein, the preparation method of NANOS2 polyclonal antibody
It makes reference to the text-book with step：《Immunology》, sharp enlightening Ya De (Peter M Lydyard), Alex Whelan, Michael W
Fanger, translator：Lin Weici, Wei Xuetao, Xue Bin, 2010 year, Science Press, page 134 page -137.
Resulting NANOS2 polyclonal serum antibody can also identify the NANOS2 albumen of heterogenous expression, as shown in Figure 3；Figure
3 detect the case where NANOS2 polyclonal antibody identifies albumen for Western Blot method, and Fig. 3 is the warp after secondary antibody is incubated for
Figure after SDS-PAGE glue transferring film；Wherein, upper figure, which is represented, identifies external source NANOS2 protein expression situation, the following figure using serum antibody
It represents and identifies external source NANOS2 protein expression situation using FLAG tag antibody, from left to right respectively control group (no external source egg
White expression) and experimental group (NANOS2 protein expression).Western Blot method can detecte the NANOS2 of overexpression, it was demonstrated that this
The NANOS2 polyclonal antibody of invention preparation has specificity.
Embodiment 2：Low expression of the NANOS2 in people's SCO type azoospermatism
The present invention is contaminated the paraffin section of normal testis and SCO testis of azoospermia disease using immunofluorescence dyeing method
Color, coloration result is as shown in figure 4, there is 3 to 6 not equal cell expression around each lumen in normal testis (left figure)
NANOS2 albumen, i.e. green fluorescence are the cell (as shown by arrows in figure) of positive signal；And the testis of SCO azoospermatism is (right
Figure) the absolutely not albumen of cell expression NANOS2 in lumen, i.e. green fluorescence is all negative signal.
Normal testis and SCO type testis of azoospermia disease are divided into six groups, as shown in Figure 5；Wherein, totally two groups of normal testis group,
Respectively control 1 and control 2, SCO azoospermia testis group is four groups, respectively SCO1~4.Every group take 5 seminiferous tubules into
Row counts, expression of the NANOS2 in protein level in detection normal testis and SCO type testis of azoospermia disease；Wherein, normal testis
2 groups of ball (compares 1 and control 2, wherein statistics knot of the control 1 for normal testis 15 lumen (seminiferous tubule) in such as Fig. 5
Fruit, control 2 for normal testis 25 lumens statistical result), 4 groups of SCO azoospermia testis (SCO1~4 in such as Fig. 5,
Wherein SCO1 is the statistical result of 5 lumens of SCO type testis of azoospermia disease 1, and SCO2 is 5 of SCO type testis of azoospermia disease 2
The statistical result of lumen, SCO3 are the statistical result of 5 lumens of SCO type testis of azoospermia disease 3, and SCO4 is SCO type azoospermatism
The statistical result of testis 45 lumens).NANOS2 positive cell number in each lumen is counted, statistical result is such as
Shown in Fig. 5.Wherein, the corresponding lumen of each histogram in every group.As shown in Figure 5, NANOS2 is in SCO type testis of azoospermia disease
Expression be substantially less than normal testis (P < 0.001).
It will be evident for a person skilled in the art that not departing from disclosed by appended claims of the present invention
Scope and spirit under the premise of, can be with numerous modifications and variations may be made, and these modifications and variations each fall within power of the present invention
In the protection scope that benefit requires.
Sequence table
<110>Foresight biotechnology（Shanghai）Co., Ltd
<120>Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1577
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 1
gggaaacgcc tgctgctctt gcccttgccc acgggccccc agccccagcc cctctcctgt 60
ggcccaccac ccctgctttg gtgccatgca gctgccaccc ttcgacatgt ggaaggacta 120
cttcaacctg agccaggtgg tgtgggcgct gatcgcaagt cggggtcaaa ggctggagac 180
ccaagagatt gaggagccaa gtcccgggcc tccgctgggg caggatcagg ggctgggggc 240
gccaggggcc aacgggggcc tggggaccct gtgcaacttc tgcaagcaca acggggagtc 300
ccgccacgtc tactcctcac accagctgaa gacaccggat ggcgtggtgg tgtgtcccat 360
cctgaggcac tacgtgtgtc ccgtgtgcgg ggccaccggt gaccaggccc atacgctcaa 420
gtactgcccg cttaacggtg gccagcagtc cctctaccgc cgcagcgggc gcaactcggc 480
cggacgcagg gtcaagcgct gagcatcgtg agatgctcac cacccgcacc cctgaccctc 540
ctggtccatc ccacccaagt ccctaaactc tctcttccag tcacccagcc ctttggaatt 600
tctggccccc agaactccac cctggctgga tcctgaaaca gtggaggctg ctgggagcca 660
ggccagcccc gcccccttgg acctgtcagc cctcggagct ctggcctgtg ggcgttggcc 720
tgagggtcct tggtgccgcg ggaacccttc ctgcacggcc ctgtagcgcc ctctgggccc 780
cggttcatgg atcttttgtc ctcgtggggc ctgctggtga ctggagccct gccccttcga 840
cttctctgtt ccttgtggag ttcttggatt tttgaacctc caggattcca cggtgctgtc 900
ccaagccagc ccttaggaga tactgaacaa ggctggctga gaagtgccta ctcaagaact 960
tcaagatggg atgtcgactt aagaccagcc ttctccatct tcagcctcct gcagaaccac 1020
ggtgtcatcg tggaccacgc ctgttcattc cttccctgac ctcagtaccc atccgtcagg 1080
agcggagtag gactctggag agcaccgtat cactggaggg caccgtgtct tcgcaggctc 1140
acctgtggat ggagagccgt ggggtgagct gacacctttc aatgccccct ggttgttctt 1200
ccccttgttc agcccattgc acccggtacc tgcccccatc cccaaatcac cgatgggtcc 1260
tttcccccag cgagaagcat atctctcgac cacctcccgt ccgacccccc aaatcgtgat 1320
agttgactaa aggacatttt tttttcctcc tgtgtttttc aacctccaac gttcaagtca 1380
aggaaccgcc gataagaagg gggtttgagt acccggatgg gaatggcaat ggcctagctt 1440
ttattttagg ggcaaagaga agaagggccc ccgtcagatt ttaagttgtt cagtttttgg 1500
cacagggtcg ggaccctgag tattgagctc tggtgaataa aaaggttttc tagtcgtctt 1560
tgaaaaaaaa aaaaaaa 1577
<210> 2
<211> 138
<212> PRT
<213>Artificial sequence (artificial sequence)
<400> 2
Met Gly Leu Pro Pro Pro Ala Met Thr Leu Ala Thr Pro Ala Leu Ser
1 5 10 15
Gly Val Val Thr Ala Leu Ile Ala Ser Ala Gly Gly Ala Leu Gly Thr
20 25 30
Gly Gly Ile Gly Gly Pro Ser Pro Gly Pro Pro Leu Gly Gly Ala Gly
35 40 45
Gly Leu Gly Ala Pro Gly Ala Ala Gly Gly Leu Gly Thr Leu Cys Ala
50 55 60
Pro Cys Leu His Ala Gly Gly Ser Ala His Val Thr Ser Ser His Gly
65 70 75 80
Leu Leu Thr Pro Ala Gly Val Val Val Cys Pro Ile Leu Ala His Thr
85 90 95
Val Cys Pro Val Cys Gly Ala Thr Gly Ala Gly Ala His Thr Leu Leu
100 105 110
Thr Cys Pro Leu Ala Gly Gly Gly Gly Ser Leu Thr Ala Ala Ser Gly
115 120 125
Ala Ala Ser Ala Gly Ala Ala Val Leu Ala
130 135
<210> 3
<211> 34
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 3
gacgcgtgga tcccatgcag ctgccaccct tcga 34
<210> 4
<211> 33
<212> DNA
<213>Artificial sequence (artificial sequence)
<400> 4
gtccgccgaa ttctcagcgc ttgaccctgc gtc 33
Claims (13)
1. a kind of for predicting or diagnosing the NANOS2 marker of azoospermatism or aspermia or oligospermia, which is characterized in that the NANOS2 mark
Will object is DNA, mRNA or protein encoded by it of NANOS2；Wherein, 453 bases of the NANOS2 full length gene, such as
Shown in SEQ ID NO.1, the protein sequence of the NANOS2 is as shown in SEQ ID NO.2.
2. the detection reagent of NANOS2 marker as described in claim 1 is in preparation for predicting or diagnosing azoospermatism or few essence
Application in the product of disease, which is characterized in that the detection reagent of the NANOS2 marker includes specific detection NANOS2
The reagent of the reagent of DNA and/or the reagent of specific detection NANOS2 mRNA and/or specific detection NANOS2 protein.
3. the in-vitro diagnosis product of a kind of prediction or diagnosis azoospermatism or aspermia or oligospermia, which is characterized in that the in-vitro diagnosis product
The reagent of reagent and/or specific detection NANOS2 mRNA including specific detection NANOS2 DNA, and/or specificity inspection
Survey the reagent of NANOS2 protein.
4. in-vitro diagnosis product as claimed in claim 3, which is characterized in that the in-vitro diagnosis product includes detection reagent
Box, genetic chip, solid support and test paper；The solid support includes array, microarray or protein array.
5. in-vitro diagnosis product as claimed in claim 4, which is characterized in that the detection kit includes such as SEQ ID
The primer and probe of NANOS2 gene shown in NO.1.
6. in-vitro diagnosis product as claimed in claim 5, which is characterized in that the detection kit further comprises：It extracts
Kit, NANOS2 antibody, the NANOS2 purifying protein and secondary antibody as concentration reference standard of protein.
7. in-vitro diagnosis product as claimed in claim 6, which is characterized in that in the detection kit, total protein lysate
For 100 microlitres of every 10 milligrams of tissues, 5x sample-loading buffer, NANOS2 antibody is 1：1000, goat-anti-rabbit horseradish peroxidase
Labelled antibody is 1：NANOS2 purifying protein of 5000, the NANOS2 prokaryotic expression proteins as concentration reference standard；Wherein, described
NANOS2 antibody is NANOS2 monoclonal antibody or NANOS2 polyclonal antibody, and the NANOS2 antibody is anti-such as SEQ ID
The antibody of NANOS2 albumen shown in NO.2.
8. in-vitro diagnosis product as claimed in claim 4, which is characterized in that the genetic chip is as shown in SEQ ID NO.1
The oligonucleotide probe point sample of NANOS2 gene be prepared in solid support surface.
9. the primer pair for detecting the NANOS2 gene expression as shown in SEQ ID NO.1, which is characterized in that upstream primer sequence
Column are as shown in SEQ ID NO.3, and downstream primer sequence is as shown in SEQ ID NO.4.
10. primer pair as claimed in claim 9 is preparing answering in the product for predicting or diagnosing azoospermatism or aspermia or oligospermia
With.
11. NANOS2 marker as described in claim 1 or application or such as claim 3 as described in claim 2 or 10
~8 described in any item in-vitro diagnosis products or primer pair as claimed in claim 9, which is characterized in that the azoospermatism
For SCO azoospermatism.
12. a kind of NANOS2 polyclonal antibody, which is characterized in that it is immune by the protein sequence as shown in SEQ ID NO.2
Animal obtains.
13. primer pair as claimed in claim 9 or polyclonal antibody as claimed in claim 12 are in preparation prediction or diagnosis
Application in the in-vitro diagnosis product of azoospermatism or aspermia or oligospermia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810503358.4A CN108828228A (en) | 2018-05-23 | 2018-05-23 | Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810503358.4A CN108828228A (en) | 2018-05-23 | 2018-05-23 | Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108828228A true CN108828228A (en) | 2018-11-16 |
Family
ID=64148582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810503358.4A Pending CN108828228A (en) | 2018-05-23 | 2018-05-23 | Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108828228A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577991A (en) * | 2019-09-23 | 2019-12-17 | 中国人民解放军陆军军医大学 | Diagnosis box for non-obstructive azoospermia |
-
2018
- 2018-05-23 CN CN201810503358.4A patent/CN108828228A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| K.M. KUSZ，ET AL.: "The highly conserved NANOS2 protein: testis-specific expression and significance for the human male reproduction", 《MOLECULAR HUMAN REPRODUCTION》 * |
| KHALID A. FAKHRO, ET AL.: "Point-of-care whole-exome sequencing of idiopathic male infertility", 《 GENETICS IN MEDICINE》 * |
| NCBI: "Homo sapiens nanos C2H2-type zinc finger 2（NANOS2）,mRNA, Reference Sequence：NM_001029861.2", 《NCBI》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110577991A (en) * | 2019-09-23 | 2019-12-17 | 中国人民解放军陆军军医大学 | Diagnosis box for non-obstructive azoospermia |
| CN110577991B (en) * | 2019-09-23 | 2022-05-27 | 中国人民解放军陆军军医大学 | Diagnosis box for non-obstructive azoospermia |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1820020B (en) | SARS-coronavirus virus-like particles and methods of use | |
| CN108586618B (en) | Preparation and application of porcine epidemic diarrhea subunit vaccine | |
| JPH08505045A (en) | Method for detecting tagA gene and predisposition to peptic ulcer formation | |
| JP7231665B2 (en) | Compositions and methods for diagnosing Lyme disease and predicting Lyme disease spirochete clearance after treatment | |
| KR101528169B1 (en) | Method and kit for detection of anti-avibacterium paragallinarum antibody | |
| JP2001508649A (en) | Novel coding sequence from herpes simplex virus type 2 | |
| JPH10509864A (en) | TagA gene and method for detecting predisposition to peptic ulcer and gastric cancer | |
| JP2013507103A (en) | Cytolytic RTX toxin derived from Gallibacterium mutanitis | |
| CN111840530A (en) | Preparation method of Eimeria tenella recombinant polypeptide vaccine VNQS and application method thereof in chicken coccidiosis resistance | |
| JP2010535030A (en) | Novel genes and proteins of Brachyspira hyodysenteriae and their use | |
| KR20140064884A (en) | Improved vaccine diagnostics | |
| CN111596070B (en) | Application of portunus trituberculatus tropomyosin allergy detection reagent | |
| KR20210123234A (en) | Recombinant nucleocapsid protein for diagnosis and vaccine of COVID-19 and use thereof | |
| CN108828228A (en) | Prediction or NANOS2 marker and its application of diagnosis azoospermatism or aspermia or oligospermia | |
| BRPI0708959A2 (en) | brachyspira hyodysenteriae genes and proteins and use for diagnosis and therapy. | |
| Padmalayam et al. | Molecular cloning, sequencing, expression, and characterization of an immunogenic 43-kilodalton lipoprotein of Bartonella bacilliformis that has homology to NlpD/LppB | |
| CN108624602B (en) | anti-Nipah virus G protein monoclonal antibody with blocking activity and application thereof | |
| CN110133290B (en) | ELISA kit for diagnosing heartworm disease | |
| CN108588096B (en) | Babesia orientalis spheroid protein gene 4 and protein coded by same | |
| CN113341160A (en) | ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep | |
| MX2010009516A (en) | Novel sequences of brachyspira, immunogenic compositions, methods for preparation and use thereof. | |
| JPH11511032A (en) | IceA gene and related methods | |
| US6514697B1 (en) | Methods for detection of Crytosporidium species and isolates and for diagnosis of Cryptosporidium infections | |
| CN110133291B (en) | Application of heartworm small heat shock protein | |
| CN106636138A (en) | DNA (Deoxyribonucleic Acid) vaccine aiming at sheep breeding type 3 brucella infection and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181116 |
|
| RJ01 | Rejection of invention patent application after publication |