CN110187128A - It is a kind of for diagnosing the biomarker of asthénospermie - Google Patents
It is a kind of for diagnosing the biomarker of asthénospermie Download PDFInfo
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- CN110187128A CN110187128A CN201910546487.6A CN201910546487A CN110187128A CN 110187128 A CN110187128 A CN 110187128A CN 201910546487 A CN201910546487 A CN 201910546487A CN 110187128 A CN110187128 A CN 110187128A
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- asthénospermie
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- 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
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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Abstract
The present invention provides a kind of for diagnosing the biomarker of asthénospermie, belongs to biomedical and molecular diagnostic techniques field.The biomarker is TDRG1, Uniprot Serial No. Q3Y452.TDRG1 expression in the sperm of asthénospermie is significantly lowered, and can be applied to the drug that preparation has diagnosis sterility as caused by asthénospermie.
Description
Technical field
The invention belongs to biomedical and molecular diagnostic techniques fields, and in particular to a kind of for diagnosing the life of asthénospermie
Substance markers object.
Background technique
Asthénospermie (asthenospermia) refers to illness of the progressive sperm ratio less than 32% in seminal parameters, is
One of Etiological of male sterility.There are many reason of causing low sperm activity, including external environment, self-defect and heredity
Etc. factors, but the specific pathogenesis of asthénospermie is unknown, seriously hinders the progress of asthénospermie clinical diagnosis and treatment.It is existing
Document report testis specific protein content reduces related with the pathogenesis of asthénospermie.So far, asthénospermie is diagnosed
Biomarker not yet develop and clinically use, therefore, there is an urgent need to the pathogenic machines deeper into ground research asthénospermie
Reason finds new asthénospermie related protein, promotes to find male from molecular level to the understanding of asthénospermie pathogenesis
The cause of disease of sterility is truly realized the Trinity of diagnosis, treatment and prevention, provides precision individuation for male sterility patient
Treatment.
Summary of the invention
The purpose of the present invention is to provide a kind of for diagnosing the biomarker of asthénospermie, the marker and weak sperm
Disease has good correlation, can be applied to prepare the diagnostic reagent of the sterility as caused by asthénospermie.
In the sterility caused by asthénospermie, TDRG1 expression is significantly lowered, therefore, it is possible to pass through detection TDRG1
Expression, to diagnose whether patient suffers from sterility.
A kind of for diagnosing the biomarker of asthénospermie, the biomarker is protein, and the entitled testis of Chinese is sent out
Associated protein 1 is educated, full name in English Testis development-related protein 1, abridge TDRG1, Uniprot sequence
Row number is Q3Y452, and containing 100 amino acid, predicted molecular weight 11KDa, particular sequence number is as follows: MKRREAVCAH
RHFLGTGKPPHPLGRSIPVEPCPGLPAFAEVDLLSLLVPIKISSTPPSGSRLDPQIASSA FPGLGSLGGQ
DSSGSLVQRA SCELESPYEL。
It is another object of the present invention to: a kind of product for detecting above-mentioned TDRG1 expression is provided in preparation by weak sperm
Application on the diagnosis object of sterility caused by disease.
Biomarker provided by the invention will provide reliable molecular target for exploitation asthénospermie diagnostic kit, help
In the molecular diagnosis of asthénospermie, test proves that, under TDRG1 expression in the sperm of Asthenospermia is significant
It adjusts, thus, there is value for the diagnosis of azoospermia using TDRG1.
Detailed description of the invention
Fig. 1 is TDRG1 content (Relative gray value) in normal person (Norm) sperm.
TDRG1 content (Relative gray value) in Fig. 2 asthénospermie (Asth) sperm.
Specific embodiment
Combined with specific embodiments below, the present invention is further described.
When above-mentioned TDRG1 is as asthénospermie diagnosticum, application method includes the following steps:
(1) sperm total protein is extracted；
(2) sperm total protein is separated using polyacrylamide gel electrophoresis；
(3) sperm total protein is transferred on polyvinylidene fluoride film from polyacrylamide gel；
(4) after the polyvinylidene fluoride film with sperm protein is closed with skimmed milk power, TDRG1 and beta-actin are used respectively
(β-ACTIN, internal reference) monoclonal antibody is incubated for, then is incubated for sheep anti mouse secondary antibody；
(5) it is incubated for film to be developed the color with Enhanced chemiluminescence, be taken pictures with chemiluminescence gel imager；
(6) picture is analyzed with Image J, reads TDRG1 and ACTIN band gray value Int (TDRG1) and Int respectively
(ACTIN), Iht (TDRG1)/Int (ACTIN) of each sample is calculated；
(7) Int (TDRG1)/Int (ACTIN) value is the asthénospermie of TDRG1 content decline less than 0.32.
Concrete application for example under:
The screening of asthénospermie relevant biomarkers object:
1, the extraction of human spermatogoa total protein
(1) the RIPA buffer (Thermo Scientific) containing protease inhibitors that 250 μ l pre-cooling is added is split
Cell is solved into the Sperm pellets of 1ml Seminal plasma；
(2) it after piping and druming mixes, sets and cracks 3min in Ultrasonic Cell Disruptor, then be placed on shaking table and rock cracking 1h, whole process is all
It operates on ice；
(3) cracked albumen is placed in temperature in advance to be adjusted in 4 DEG C of centrifuge, 12000g is centrifuged 10min；
(4) supernatant of the transfer containing albumen is into new centrifuge tube；
(5) according to green skies BCA method, the concentration of albumen is detected；
(6) it is being collected into be proportionally added into SDS sample-loading buffer (Thermo Scientific) in protein liquid, boil
10min albuminate.
2, glue (separation gel and concentration glue)
(1) gel mold (operating according to BioRad MP4mini gel mold specification) is assembled；
(2) separation gel of preparation 10% (pays attention to cannot thering is bubble when between separation gel implantation glass plate, avoids influencing in proportion
Experimental result.)；
(3) the concentration glue that 5% is prepared by certain formula, adds on separation gel, then plug comb after mixing；Gelling to be concentrated
After Gu, slotting comb is pulled out.
3, loading electrophoresis
(1) glass plate is removed, the electrophoresis inside groove to match with glue is selected and is assembled, confirms that interior tank liquid does not leak outside；
(2) 1 × SDS electrophoretic buffer (health is reagent) is poured into inside groove, and liquid level covers loading hole, has cleared up in glue hole
Broken glue, every 50 μ g protein sample of hole loading；
(3) upper excellent inside groove is put into electrophoresis outer groove, adds 1 × SDS electrophoretic buffer into outer groove；
(4) install electrode, constant current 25mA, the bottom for going to glass plate to bromophenol blue stops electrophoresis, with about when 90min.
4, transferring film
(1) after electrophoresis, gel is taken out, removes concentration glue, glue is immersed in transfering buffering liquid；It cuts one big with glue
Small comparable pvdf membrane (Thermo Scientific) prepares 6 filter paper (Millipore) more bigger than film.
(2) it is immersed in transfering buffering liquid again after pvdf membrane is impregnated with through methanol, distilled water embathes.
(3) first soak fiber mat and filter paper with transfering buffering liquid, then successively will transfer folder black side, filter paper (three layers), glue,
The sequence installation transfer of film, filter paper (three layers), transfer folder white face is pressed from both sides, and putting into electrophoresis tank after mounted transfer folder, is added
Improvement liquid relief, 50V constant pressure transferring film 1h；
(4) after transferring film, pvdf membrane is taken out, taking-up drying at room temperature 15min after methanol is impregnated with is put into, marks, it is standby
With.
5 closings and antibody incubation
(1) above-mentioned pvdf membrane is placed in TBST (health is reagent) solution of 5%BSA, room temperature shaker closes 1h；
(2) TBST is washed film 5 times, every minor tick 3min；
(3) the TDRG1 monoclonal antibody (Hunan ProMab Biotech Inc.'s preparation) that addition has been diluted with confining liquid
With ACTIN monoclonal antibody (Proteintech company) on pvdf membrane, 4 DEG C of shaking tables be incubated overnight (dilute primary antibody with TBST,
1: 1000 dilution of TDRG1,1: 100000 dilution of ACTIN)；
(4) TBST is washed film 5 times, every minor tick 3min；
(5) sheep anti mouse secondary antibody (1: 5000, Proteintech company) is diluted with confining liquid, then film is placed in secondary antibody dilution
In liquid, room temperature shaker is incubated for 1h；
(6) it is first washed film 3 times with the TS of 0.05%Tween-20, is spaced 3min, finally washes film twice with TBS, it is spare.
6, enhanced chemical luminous (ECL) colour developing and imaging
The A liquid in ECL colour reagent box (Proteintech company) is taken to mix with B liquid 1: 1, by mixed liquor uniform fold
In film surface, it is protected from light colour developing 3min, the film after colour developing is put into exposure in chemiluminescence gel imaging system (Biorad company) and claps
According to.
7, TDRG1 relative quantification
ECL develop the color picture with Image J analyze, respectively read TDRG1 and ACTIN band gray value Int (TDRG1) and
Int (ACTIN) calculates Int (TDRG1)/Int (ACTIN) of each sample；Iht (TDRG1)/Int (ACTIN) value is less than
0.32 asthénospermie declined for TDRG1 content.
Embodiment one
TDRG1 content in 27 normal persons and 25 asthénospermie human spermatogoas has been screened, has been obtained through relative quantitative assay
Linear regression analysis normally is carried out with the TDRG1 content of illness group and propulsion rate, as a result respectively such as Fig. 1 (r2=0.7463,
P < 0.0001, Fig. 1), shown in Fig. 2.In Fig. 1, discovery TDRG1 content is positively correlated with propulsion rate.In Fig. 2, normal person's essence
The minimum relative amount of TDRG1 is 0.32 in son.
Therefore, the expression of TDRG1 shows the correlation of height with asthénospermie in sperm, can be used as biomarker
Object is applied to diagnosis asthénospermie, specifically, the asthénospermie that the relative amount of TDRG1 is lower than 0.32 can be defined as TDRG1
The asthénospermie of content decline.
Sequence table
<110>healthcare hospital for women & children, Jiangxi Province
<120>a kind of for diagnosing the biomarker of asthénospermie
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 100
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Lys Arg Arg Glu Ala Val Cys Ala His Arg His Phe Leu Gly Thr
1 5 10 15
Gly Lys Pro Pro His Pro Leu Gly Arg Ser Ile Pro Val Glu Pro Cys
20 25 30
Pro Gly Leu Pro Ala Phe Ala Glu Val Asp Leu Leu Ser Leu Leu Val
35 40 45
Pro Ile Lys Ile Ser Ser Thr Pro Pro Ser Gly Ser Arg Leu Asp Pro
50 55 60
Gln Ile Ala Ser Ser Ala Phe Pro Gly Leu Gly Ser Leu Gly Gly Gln
65 70 75 80
Asp Ser Ser Gly Ser Leu Val Gln Arg Ala Ser Cys Glu Leu Glu Ser
85 90 95
Pro Tyr Glu Leu
100
Claims (3)
1. a kind of for diagnosing the biomarker of asthénospermie, which is characterized in that the biomarker is TDRG1, Uniprot
Serial No. Q3Y452.
2. according to claim 1 a kind of for diagnosing the biomarker of asthénospermie, which is characterized in that the biology
Marker is TDRG1, Uniprot Serial No. Q3Y452, is used to prepare the product of diagnosis sterility as caused by asthénospermie.
3. detecting application of the product of TDRG1 expression in sperm in preparation diagnosis asthénospermie product, it is characterised in that:
The biomarker is TDRG1, Uniprot Serial No. Q3Y452.
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| CN116814573A (en) * | 2023-07-24 | 2023-09-29 | 南昌大学 | Immunogen for preparing succinylation site specific antibody of lactate dehydrogenase C, and preparation method and application thereof |
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| CN101255426A (en) * | 2008-02-28 | 2008-09-03 | 蒋先镇 | Novel human testicle specificity gene TDRG1 |
| CN106872630A (en) * | 2017-03-29 | 2017-06-20 | 山东大学 | The screening of the biomarker related to severe teen bra and application |
| WO2018185322A1 (en) * | 2017-04-06 | 2018-10-11 | Universität Zu Köln | A method for determining the fertility of spermatozoa |
| KR20180138021A (en) * | 2017-06-20 | 2018-12-28 | 이화여자대학교 산학협력단 | Composition for diagnosing male reproductive organ disease, composition for preventing or treating male reproductive organ disease, and use thereof |
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| CN101255426A (en) * | 2008-02-28 | 2008-09-03 | 蒋先镇 | Novel human testicle specificity gene TDRG1 |
| CN106872630A (en) * | 2017-03-29 | 2017-06-20 | 山东大学 | The screening of the biomarker related to severe teen bra and application |
| WO2018185322A1 (en) * | 2017-04-06 | 2018-10-11 | Universität Zu Köln | A method for determining the fertility of spermatozoa |
| KR20180138021A (en) * | 2017-06-20 | 2018-12-28 | 이화여자대학교 산학협력단 | Composition for diagnosing male reproductive organ disease, composition for preventing or treating male reproductive organ disease, and use thereof |
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| HOUYANG CHEN等: "Expression and Localization of Testis Developmental Related Gene 1 (TDRG1) in Human Spermatozoa", 《THE TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116814573A (en) * | 2023-07-24 | 2023-09-29 | 南昌大学 | Immunogen for preparing succinylation site specific antibody of lactate dehydrogenase C, and preparation method and application thereof |
| CN116814573B (en) * | 2023-07-24 | 2024-05-14 | 南昌大学 | Immunogen for preparing succinylation site specific antibody of lactate dehydrogenase C, and preparation method and application thereof |
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