CN110082539A - A kind of gestational diabetes auxiliary early diagnosis reagent - Google Patents
A kind of gestational diabetes auxiliary early diagnosis reagent Download PDFInfo
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- CN110082539A CN110082539A CN201910351324.2A CN201910351324A CN110082539A CN 110082539 A CN110082539 A CN 110082539A CN 201910351324 A CN201910351324 A CN 201910351324A CN 110082539 A CN110082539 A CN 110082539A
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- gdm
- early diagnosis
- serum
- gestational diabetes
- polypeptide
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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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- 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
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Abstract
The invention discloses a kind of gestational diabetes to assist early diagnosis reagent.Serum/plasma polypeptide marker relevant to gestational diabetes auxiliary early diagnosis, it is characterised in that the marker is selected from P32004 or P32004 and any one polypeptide or two polypeptides in Q6PFW1, P35658, Q9Y4H2.Serum/plasma polypeptide marker of the present invention is preparing the application in gestational diabetes auxiliary early diagnosis reagent as detection target spot.Above-mentioned marker representation amount can be used for GDM auxiliary early diagnosis in detection pregnancy serum/blood plasma.For the GDM morbidity final result of clinician Accurate Prediction pregnant woman, takes the control prece of more personalized to provide support in time, to reduce the disease incidence of GDM to greatest extent, reduce Averse pregnancy outcomes.
Description
Division explanation
The application be the applying date be on September 6th, 2018, it is entitled " with gestation application No. is 2018110390312
Point of the Chinese invention patent application of the relevant serum/plasma polypeptide marker of phase diabetes auxiliary early diagnosis and its application "
Case application.
Technical field
The invention belongs to proteomics fields, are related to a kind of gestational diabetes auxiliary early diagnosis reagent.
Background technique
Gestational diabetes (gestational diabetes mellitus, GDM) refer to that the gestational period occurs or sends out for the first time
Existing different degrees of impaired glucose tolerance, if occurring to be not excluded for impaired glucose tolerance before gestation just in First Trimester
Through there are a possibility that.GDM is one of most common complication of pregnancy 2222, and there are about the pregnant woman of 3%-8% during gestation to occur
GDM, especially it is worth noting that, with the increase of women of child-bearing age's obesity rates, the incidence of GDM constantly rises.
GDM can carry out a series of complication to parent and fetal zone, and it is comprehensive such as to increase spontaneous abortion incidence, the hypertension of pregnancy
Simulator sickness, hydramnion etc. also increase macrosomia, shoulder dystocia, maternal infuries, premature labor, abnormal development of fetus, fetal distress in uterus, dead
Tire, stillbirth incidence；And make newborn Yi Fasheng hypoglycemia, hyperbilirubinemia, Respiratory Distress Syndrome(RDS), erythremia
Disease etc., while increasing the risk that diabetes B, metabolic disease and cardiovascular disease occur for pregnant and lying-in women.If carried out not in time
Diagnosing and treating, not only pregnant and lying-in women and tire baby illness rate and case fatality rate will obviously rise, puerpera and its offspring's long term diabetes
Illness rate will also rise.Therefore, early diagnosis, early intervention, timely rational therapy are prevention GDM, reduce mother and baby's complication
Key is important scientific problems urgently to be resolved.
The screening of GDM and diagnostic method and standard are not completely consolidated in the world at present, usual GDM need to be in the 2nd pregnancy period
Advanced stage or the 3rd pregnancy period just can be carried out diagnosis, according to American Diabetes Association (American Diabetes Association,
ADA) and U.S.'s gynecotokology meeting (American College of Obstetricians and Gynecologists,
ACOG the serological screening of) guide, GDM is carried out in 24-28 pregnant week, and it is fewer and fewer to can be used for intervention time in this way.Therefore, we
Discovery definitely effective biomarker is needed, auxiliary early diagnosis is made to GDM, this will be helpful to early intervention,
Reduce Averse pregnancy outcomes.
For many years, people are studying always gene and proteomics, to find the biomarker of various diseases.However
Due to the characteristic of gene and proteomics, they are not conventional childbirth into biological fluids, so the exploration of biomarker
Road it is extremely difficult.And low molecular weight peptide and the proteolysis fragment of molecular mass are continually secreted into serum, it is therefore, more
Emphasis of the peptide group just as the research of serum biomarkers.
Polypeptide group is defined as comprehensive qualitative and quantitative analysis to peptide in biological sample.In body fluid or tissue
The circulating protein fragment of generation can reflect biological event, and provide library abundant for diagnosis biomarker.Now, many peptides are all
It is used as the biomarker of cancer.For example, plasma fibrin 1 can be used as one kind of screening and Diagnosis of renal cell carcinoma
Means, early stage clear-cell carcinoma, plasma fibrin 1 is horizontal to be risen.
Existing mature polypeptide technology, it is raw using serum peptide as molecule including showing to the qualitative of peptide and quantitative determination
The method of object marker will be more efficient than traditional differential protein molecule labelling method, has opened up new ground for biomarker
Boundary.
However, there is presently no the reports of the relatively stable biomarker for GDM auxiliary early diagnosis, if can sieve
It selects GDM specifically or the serum/plasma peptide of unconventionality expression is as biomarker, and develop corresponding auxiliary early diagnosis reagent
Box, the diagnosis status to China GDM will be primary strong promotions.
Summary of the invention
The purpose of the present invention is in view of the above technical problems, propose a kind of serum/blood relevant to GDM auxiliary early diagnosis
Starch peptide marker.
It is a further object of the present invention to provide the applications of the marker.
It is yet another object of the invention to provide a kind of GDM to assist early diagnosis kit.
The purpose of the present invention can be achieved through the following technical solutions:
Serum/plasma polypeptide marker relevant to gestational diabetes auxiliary early diagnosis, it is characterised in that the mark
Any one polypeptide or multiple polypeptides of object in Q6PFW1, P35658, P32004 and Q9Y4H2；The wherein amino of Q6PFW1
Acid sequence is as shown in SEQ ID NO.1, and the amino acid sequence of P35658 is as shown in SEQ ID NO.2, the amino acid sequence of P32004
Column are as shown in SEQ ID NO.3, and the amino acid sequence of Q9Y4H2 is as shown in SEQ ID NO.4.
Wherein, the preceding albumen of Q6PFW1 is the own diphosphonic acid of inositol and inositol diphosphate/five phosphokinases, 1 (Inositol
hexakisphosphate and diphosphoinositol-pentakisphosphate kinase 1)；Before P35658
Albumen is nucleopore compound protein (Nuclear pore complex protein Nup214)；The preceding albumen of P32004 is nerve
Cell adhesion molecule L1 (Neural cell adhesion molecule L1)；The preceding albumen of Q9Y4H2 is insulin receptor bottom
Object 2 (Insulin receptor substrate 2).
Serum/plasma polypeptide marker of the present invention is preparing gestational diabetes auxiliary early stage as detection target spot
Application in diagnostic reagent.
A kind of gestational diabetes auxiliary early diagnosis kit, includes to detect the serum/plasma polypeptide marker
Reagent；
The described gestational diabetes auxiliary early diagnosis reagent, preferably comprise Q6PFW1, P35658, P32004 and
The ELISA detection reagent of any one polypeptide or multiple polypeptides in Q9Y4H2.
The utility model has the advantages that
The present invention uses strict design and appraisement system, and mature polypeptide experimental method obtains that disease is special and exception table
The serum/plasma polypeptide reached, and select polypeptide at random and verified, guarantee the stability of experimental result.
The present invention, to the influence factor of disease development, studies serum/plasma polypeptide marker in GDM by control age etc.
The application prospect of early diagnosis illustrates the influence that the polypeptide marker of unconventionality expression is in progress for GDM, discloses its screening and early stage
Diagnostic value.Therefore, present invention obtains GDM morbidity specific serum/plasma polypeptide expression database and Specific markers；
By measuring serum/plasma polypeptide marker, it may make the early diagnosis of GDM more convenient and easy, be that clinician is accurately pre-
The GDM morbidity final result for surveying pregnant woman, takes the control prece of more personalized to provide support, to reduce to greatest extent in time
The disease incidence of GDM reduces Averse pregnancy outcomes.The present invention helps clinician quick and precisely to grasp conditions of patients simultaneously, to face
Bed treatment effectiveness evaluation lays the foundation, and provides help to be found to have the new small molecule drug targets of potential treatment value.
Detailed description of the invention
The discovery route map of Fig. 1 polypeptide marker of the present invention
This figure is the Technology Roadmap of this research, and the screening of acquisition, differential peptides including early stage blood serum sample, difference are more
Selectivity verifying of peptide etc..
The selectivity of Fig. 2 serum polypeptide marker of the present invention is verified
On the basis of the screening of serum differential peptides, 7 are selected to lapse to group high expression in GDM or only lapse to group in GDM
The differential peptides of expression, acquire normal group and GDM lapses to early stage blood serum each 30 of group, are verified using mass spectrographic method.A
Figure is the expression comparison diagram for normally lapsing to group and GDM and lapsing to P35658 in group, and B figure is normally to lapse to group and GDM to lapse in group
The expression comparison diagram of Q6PFW1, C figure are the expression comparison diagram for normally lapsing to group and GDM and lapsing to P32004 in group, and D figure is normal turns
It is grouped into the expression comparison diagram that Q9Y4H2 in group is lapsed to GDM.
Specific embodiment
Embodiment 1, the collection of sample and sample data arrangement
Inventor has collected the outer of a large amount of 14-18 pregnant week pregnant woman from Nanjing Women and Children Healthcare Hospital in April, 2015 to August
All blood samples (sample for research is collected for the same period, sample, dispense, preservation condition it is uniform), by the whole of sample data
Reason, inventor have therefrom selected 200 samples for meeting following standard as laboratory sample:
It 1, is 14-18 pregnant week healthy pregnant women when taking a blood sample
2, the pregnant woman that the studies above object is diagnosed as GDM through OGTT in 24-28 pregnant week GDM screening is defined as case
3, in 24-28 pregnant week GDM screening GDM does not occur for the studies above object, matched with case age, BMI, pregnant week
Healthy pregnant women is defined as situations such as control and system acquisition demographic data of these samples, clinical data.
Embodiment 2, sample preparation, proteomics (polypeptide group) screening and bioinformatic analysis
By above-mentioned qualified 100 GDM cases and 100 normal healthy controls, it is real to carry out ultrafiltration, TMT, LC-MS/MS etc.
It tests, specific steps are as follows:
The acquisition preparation and ultrafiltration of sample in serum/plasma
1, sample is centrifuged 15 minutes under 4 DEG C of environment with 3000 turns of speed per minute, upper layer sample is stored in -20
DEG C, wait further analysis.Usually lead to 200-250ul serum or blood plasma.
2, sample is centrifuged 15 minutes under 4 DEG C of environment with 12000 turns of speed per minute, the upper of centrifugation is added in propylene cyanogen
In layer clear liquid, of short duration vortex is then carried out, is placed 20 minutes at room temperature, is then used according to specification, use 10kDa
MWCO filter (Millipore, USA) obtains 10kDa peptide below and is lyophilized.
3, the concentration of albumen is measured by using BCA (Pierce, Rockford, IL, USA).
4, it carries out peptide desalination and is marked using TMT (TMT 6-plex Label Reagent, Thermo Scientific)
Note.100ug polypeptide below will be reduced, and clean 1 hour by using 60 10mMDTT, and use at room temperature
55mMIAA is alkylated 45 minutes, finally in vacuo desalination and drying.Based on total tire amount, AF is marked with isobaric label at room temperature
Peptide needs a hour.According to TMT operation instructions, TMT's by the analysis to isobaric tag combination the result is that obtained.Three
A normal AF sample is marked as reagent 126,127 and 128, and the AF sample volume of three ventricular septal defects is marked as
129,130 and 131.
6. use liquid chromatography tandem mass spectrometry (Thermofisher Scientific Inc., Easy-nLC,
Thermofisher Scientific Inc., San Jose, CA) peptide is analyzed.Using buffer solution A (2% acetonitrile,
0.5% acetic acid) and buffer solution B (80% acetonitrile, 0.5% acetic acid) with gradient carry out peptide reverse phase separation.The peptide of elution is existed
It is ejected under 1.8kV voltage and is configured to utilize generation during the ion (m/z 445.12002) generated during electron spray
The lock mass feature of polydimethylcyclosil.xane collects high-resolution (R=60,000 at m/z 400) broadband mass spectrum (m/z
Mass spectrograph 375-1800).20 be dynamically determined using 35% opposite collision induced dissociation (CID) energy selection from MS scanning
The most abundant peptide molecule ion of kind is used for tandem mass spectrum.Most strong product ion is touched for higher energy in selection MS2 step
Hit induction dissociation (HCD) MS3 fragmentation.
7. using PEAKS software (version 7.0, Bioinformatics Solutions, Waterloo, Canada) to peptide
It is identified.Using PEAKS software (version 7.0, Bioinformatics Solutions, Waterloo, Canada), to packet
The MS/MS spectrum that database search containing 20,194 people's protein sequences (in May, 2015 publication) extracts.Merge target-bait
Method be used to estimate false discovery rate (FDR), and can be set to that≤1% in protein and peptide level (- 10logP >=
20.0).Only when at least there are two spectrum, there are just think that peptide is correctly identified when peptide peak for each sample.
8. carrying out relative quantification to peptide using TMT labelling method in PEAKS Q template.Use the TMT in PEAKS Q module
The relative quantification of labeling method progress serum peptide.Feature is executed to each sample respectively using expectation-maximization algorithm to detect.Make
It is compared with feature of the high-performance retention time alignment algorithm to the identical peptide from different samples.When p value < 0.05 and multiple
When variation > 1.5, it is believed that peptide and protein significantly change between blood serum sample.It is quantitative that determining result is included in TMT label
Final step in.
9. calculating isoelectric point (pI) (http://web.expasy.org/ of every kind of peptide using pI/Mw online tool
compute__pi/)。
10. being determined using path and Gene Ontology (GO) analysis (http://geneontology.org) potential raw
Manage function.GO result includes three classifications: cell component, bioprocess and molecular function.
11. before IPA software v7.1 (Ingenuity Systems, Mountain View, CA) further analysis
The path, further to inquire into the meaning of difference peptide and preceding albumen.
12. generating GAP-associated protein GAP functional network using Ingenuity Systems Knowledge Base.
13. being the searching of specific spilting of an egg point using MEROPS database (http://merops.sanger.ac.uk)
Candidate enzyme.
According to LC-MS/MS, the present inventor detects to be organized in " gestational diabetes example " group and " healthy women control "
The peptide of expression totally 297 (being shown in Table 1) is had differences in serum, and these difference peptides share 228 preceding albumen, the results are shown in Table 1.
1 297 difference peptides of table
Remarks: protein sequence number derives from database SWISS-PROT (Protein Sequence Database)
(http://www.expasy.ch/sprot/sprot-top.html).C: control group；G:GDM group, G/C:GDM group expression quantity
With the ratio of control group expression quantity；The T value that T:T is examined
Embodiment 3, accidental validation
According to the above results, random selection 7 lapse to the significantly high expression of group in GDM or only lapse to group expression in GDM
Differential peptides are as verifying target, and in the pregnant 14-18 weeks Serum Bank that we establish in early days, selection normally lapses to group and GDM turns
Each 30 of the serum sample of grouping (two groups of ages, BMI, pregnant weeks are mutually matched), further verify our primary dcreening operations as a result, research
Confirm have 4 (Q6PFW1, P35658, P32004 and Q9Y4H2) expression patterns and primary dcreening operation result almost the same in 7 polypeptides
(table 2), there are significant differences for the expression between two groups, and prompt is associated with gestational diabetes mellitus, they have can
The molecular marked compound (Fig. 2) of GDM early prediction and diagnosis can be become.
The polypeptide of 27, table verifyings
Note: 1. bolded sections are verification result and 4 almost the same polypeptides of primary dcreening operation result
2. protein sequence number derives from database SWISS-PROT (Protein Sequence Database)
(http://www.expasy.ch/sprot/sprot-top.html).C: control group；G:GDM group, G/C:GDM group expression quantity
With the ratio of control group expression quantity；The T value that T:T is examined.
Sequence table
<110>Nanjing Women and Children Healthcare Hospital
<120>a kind of gestational diabetes auxiliary early diagnosis reagent
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213>mankind (HomoSapiens)
<400> 1
Lys Lys Leu Pro Pro Ala Ser
1 5
<210> 2
<211> 8
<212> PRT
<213>mankind (HomoSapiens)
<400> 2
Phe Ser Ser Pro Asn Lys Thr Gly
1 5
<210> 3
<211> 5
<212> PRT
<213>mankind (HomoSapiens)
<400> 3
Arg Pro Gln Gly Thr
1 5
<210> 4
<211> 7
<212> PRT
<213>mankind (HomoSapiens)
<400> 4
Arg Thr Tyr Ser Leu Thr Thr
1 5
Claims (4)
1. serum/plasma polypeptide marker relevant to gestational diabetes auxiliary early diagnosis, it is characterised in that the marker
Group selected from P32004 or P32004 with any one polypeptide or two polypeptides in Q6PFW1, P35658, Q9Y4H2
It closes；Wherein the amino acid sequence of Q6PFW1 is as shown in SEQ ID NO.1, the amino acid sequence of P35658 such as SEQ ID NO.2 institute
Show, the amino acid sequence of P32004 is as shown in SEQ ID NO.3, and the amino acid sequence of Q9Y4H2 is as shown in SEQ ID NO.4.
2. serum/plasma polypeptide marker described in claim 1 is preparing gestational diabetes auxiliary early as detection target spot
Application in phase diagnostic reagent.
3. a kind of gestational diabetes auxiliary early diagnosis reagent, it is characterised in that comprising P32004 or P32004 be selected from
The detection reagent of any one polypeptide or two polypeptides in Q6PFW1, P35658, Q9Y4H2.
4. a kind of gestational diabetes auxiliary early diagnosis reagent according to claim 3, it is characterised in that include
The ELISA of P32004 or P32004 and any one polypeptide or two polypeptides in Q6PFW1, P35658, Q9Y4H2
Detection reagent.
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| CN201910351324.2A CN110082539B (en) | 2018-09-06 | 2018-09-06 | Auxiliary early diagnosis reagent for gestational diabetes |
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| CN201910351324.2A CN110082539B (en) | 2018-09-06 | 2018-09-06 | Auxiliary early diagnosis reagent for gestational diabetes |
| CN201811039031.2A CN108957011B (en) | 2018-09-06 | 2018-09-06 | Serum/plasma polypeptide marker relevant to gestational diabetes auxiliary early diagnosis and its application |
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| CN201811039031.2A Active CN108957011B (en) | 2018-09-06 | 2018-09-06 | Serum/plasma polypeptide marker relevant to gestational diabetes auxiliary early diagnosis and its application |
| CN201910351324.2A Active CN110082539B (en) | 2018-09-06 | 2018-09-06 | Auxiliary early diagnosis reagent for gestational diabetes |
| CN201910350484.5A Active CN110221076B (en) | 2018-09-06 | 2018-09-06 | Serum/plasma polypeptide marker related to early diagnosis assisted by gestational diabetes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110387414B (en) * | 2019-07-19 | 2022-09-30 | 广州市达瑞生物技术股份有限公司 | Model for predicting gestational diabetes by using peripheral blood free DNA |
| CN112904014B (en) * | 2019-12-04 | 2023-01-13 | 张曼 | Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes |
| CN112924691B (en) * | 2019-12-06 | 2022-05-31 | 中国科学院大连化学物理研究所 | Polypeptide combination marker for early diagnosis of diabetes, detection kit and method |
| CN112924692B (en) * | 2019-12-06 | 2022-05-31 | 中国科学院大连化学物理研究所 | Diabetes diagnosis kit based on polypeptide quantitative determination and method thereof |
| CN112924689B (en) * | 2019-12-06 | 2022-05-31 | 中国科学院大连化学物理研究所 | Diabetes diagnosis kit based on quantitative determination of polypeptide combined marker and method thereof |
| CN112924687B (en) * | 2019-12-06 | 2022-05-31 | 中国科学院大连化学物理研究所 | Diabetes diagnosis kit based on polypeptide combined marker and method thereof |
| CN111751529A (en) * | 2020-05-12 | 2020-10-09 | 陕西中医药大学 | Serum polypeptide marker P for tic disorder and detection method thereof |
| CN113009162B (en) * | 2021-02-26 | 2022-07-12 | 南芯芯仪(广州)制造有限公司 | Serum metabolic marker for diagnosing gestational diabetes and application thereof |
| CN113092777B (en) * | 2021-03-26 | 2023-11-14 | 泰达国际心血管病医院 | Method for screening severe preeclampsia patients in early gestation period |
| CN113607960A (en) * | 2021-07-19 | 2021-11-05 | 重庆医科大学附属第一医院 | Application of FGL2 in preparation of reagent for diagnosis and prognosis of gestational diabetes |
| CN114034781A (en) * | 2021-09-16 | 2022-02-11 | 中日友好医院 | Biomarker, method and early warning model for predicting gestational diabetes in early pregnancy |
| CN114703272B (en) * | 2022-02-11 | 2022-12-23 | 南京市妇幼保健院 | Screening or early diagnosis kit for pregnant women with high risk of gestational diabetes mellitus and giant children |
| CN114517229B (en) * | 2022-02-22 | 2022-12-02 | 南京市妇幼保健院 | Application of serum exosome RNA in preparation of pregnancy diabetic giant screening or early diagnosis reagent |
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| Publication number | Publication date |
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| CN108957011B (en) | 2019-06-07 |
| CN108957011A (en) | 2018-12-07 |
| CN110082539B (en) | 2020-02-21 |
| CN110221076A (en) | 2019-09-10 |
| CN110221076B (en) | 2020-02-21 |
| CN110018319A (en) | 2019-07-16 |
| CN110018319B (en) | 2020-02-21 |
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