CN1928556A - Reagent kit for detecting 2-diabetes serum mark in Chinese people - Google Patents
Reagent kit for detecting 2-diabetes serum mark in Chinese people Download PDFInfo
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- CN1928556A CN1928556A CN 200510098263 CN200510098263A CN1928556A CN 1928556 A CN1928556 A CN 1928556A CN 200510098263 CN200510098263 CN 200510098263 CN 200510098263 A CN200510098263 A CN 200510098263A CN 1928556 A CN1928556 A CN 1928556A
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Abstract
The invention relates to the reagent box to detect serum of II-type diabetes. Specially, it applies the serum of apolipoprotein A-I and corresponding express-level in patient to modify the box. It also relates to the application in diagnosis and treatment of apolipoprotein A-I.
Description
Technical field
The present invention relates to a kind of blood serum designated object of diabetes B, this blood serum designated object is apolipoprotein apolipoprotein A-I, i.e. the I hypotype of aPoA.The invention still further relates to the mark apolipoprotein A-I that utilization is differentiated, with and the change of the expression that in the patient body, occurs and the detection kit that designs.Further, the invention still further relates to the effect of this blood serum designated object in diabetes diagnosis, and as the target spot of Remedies for diabetes design.
Background technology
As everyone knows, diabetes B is a kind of complex character disease of controlled by multiple genes, and the obvious genetic tendency is arranged.Think that at present the absorption that participates in insulin signaling pathway, glucose transport approach, glycogen route of synthesis, fatty acid all may be relevant with the generation of diabetes with protein involved molecule synthetic, the lipocyte differentiation pathway.Research onset diabetes mechanism, the research of illustrate molecular mechanism, identifying blood glucose-control gene and function thereof all has crucial meaning to the prevention and the treatment of diabetes.Wherein, the insulin signaling pathway is the more field of studying at present, this approach is very complicated, comprises insulin receptor, IRS etc., and links together with Akt signal transduction path, MAPK signal transduction path, CAP/Cb1 signal transduction path etc.Insulin receptor belongs to receptor tyrosine kinase, comprises IGF-I and IRR.At least 9 insulin receptor kinase substrates have been identified at present, comprising 4 IRS IRS1, IRS2, IRS3 and IRS4.IRS (IRS) has high homology, but acts on different, additional mutually.The IRS-1 knock out mice shows as in utero slow with postnatal growth, insulin resistant, and glucose tolerates symptoms such as impaired.The IRS-2 knock out mice shows as at liver etc. and organizes insulin resistant and comprise a plurality of region growing defectives such as brain tissue, finally develops into diabetes B.
At present, diagnosis of diabetes mainly is to rely on to measure blood sugar level, sugar tolerance ability and insulin level.Because the early diabetes patient more than 80% does not have tangible symptom, the prevention of diabetes and treatment have in time often been caused incuring loss through delay.Therefore, the method for seeking simple and efficient, new accurately and effectively diabetes early diagnosis all has very important meaning for the prevention and the treatment of diabetes.
Diabetes B is a kind of disease of multifactorial inheritance.Be accompanied by the generation and the development of the diabetes B course of disease, various symptoms and complication occur one by one and produce.And in fact, before various symptoms were discovered by patient self, variation had just appearred in the expression that occurs relevant albumen with these symptoms.The early diagnosis of diabetes B just comprises the correct detection to these associated protein, and it can help the prevention and the treatment of diabetes effectively, improves diabetes patient's quality of life.Therefore, the protein marker of searching and evaluation diabetes B has great practical value.
Finishing of the development of protein identification techniques and genome plan, feasible " proteomics " this brand-new research field is born and is developed.Proteomics is to study the science of protein expression and function on the basis of genomics.Its whole living things system of our further understandings that appears as provides a kind of reliable method.Be used to detect the two dimensional electrophoresis technology (2DE of protein expression, two-dimensional polyacrylmide gel electrophoresis) and the mass-spectrometric technique (MS, mass spectrometry) that is used for identification of proteins proteomics has been produced crucial effects.The variation of protein expression level is the part of the reaction that produces after pathology occurring of body.This comprises the regulation and control to protein synthesis speed, at the gene expression regulation of translation skill, and the covalent modification (as phosphorylation, acyl groupization or glycosylation) after the translation of the albumen neutralization translation and the degradation speed of protein.Thereby, the research of whole protein group has just been reflected the variation of environmental baseline, comprise the variation (synthetic as mRNA, translation is modified, transhipment, proteolysis process and degraded) of the protein concentration of the multistage regulation and control of complex network.We can say that the comparative studies of protein group helps to understand the regulated and control network of cell.So it is that disease marker is differentiated, drug targets is confirmed, the behavioral mechanism research of medicine and the useful tool of pharmacology reasoning.
The detection that proteomics is applied to disease biomarker protein has obtained the achievement that attracts people's attention.Handling discovery 28kD albumen calbindin-D continuous decrease (Benito et al., 1995 in the rat kidney that produces the kidney poison with CyA; And the increase of simultaneous UCaE thing and cortex spinal canal sodalimeization (Aicher et al.) Steiner et al.).The example of the successful discriminating of another biomarker albumen is from study for a long period of time (the Celis et al.1998) of squamous cell carcinoma of bladder process.
In clinical diagnosis, serum receives much concern as the sample of the easiest collection.More and more researchers focuses on application proteomics means with sight and seek potential disease marker in serum.Exist abundant albumen in the serum, carried very valuable information.But the albumin that exists in the serum accounts for more than 80% of all albumen, and this has just disturbed the separation and the evaluation of other low-abundance proteins.Up to now, the work of the blood serum designated object of application proteomics means searching diabetes is also very rare.
The inventor collects the sporadic diabetes B human serum and the normal human serum of clinical definite, adopt proteomics method, obtain the protein site of variant expression in diabetes B by dielectrophoresis comparison glue figure, utilize mass spectral method to determine the albumen kind then, adopt specific antibody method by enzyme even immunity in patient and normal human serum of this albumen further to verify definite at last the expression of this albumen.Finally, the inventor has determined a kind of blood serum designated object apolipoprotein A-I of diabetes B, and has finished the present invention on this basis.
Summary of the invention
According to an aspect of the present invention, the inventor provides a kit that can be used for detecting the diabetes B biomarker.This kit comprises at the antibody of apolipoprotein A-I and the normal enzyme of using and connects the contained conventional assembly of kit that immunoadsorption detects protein level in the serum.
At this, the inventor provides the blood serum designated object of a diabetes B.The present invention relates to a kind of peptide molecule, apolipoproteinA-I, i.e. the I hypotype of aPoA by name.
The invention still further relates to antibody, comprise and to discern overall molecule, all antibody of a certain domain of this molecule and some or several special antigenic determinant at this peptide molecule.
Further, the invention still further relates to apolipoprotein A-I and be used for the application of the diagnosticum of diabetes B diagnosis, and prepare application in the medicine of diabetes B as potential drug target in preparation.
Definition:
Disease blood serum mark (biomarker): be meant in the generation and evolution of disease, because the caused physiological variation of disease, as glycometabolism or lipometabolic unusual, and the abnormal expression of some molecule that causes, particularly protein molecular.These protein moleculars that abnormal expression occurs often can be used as these disease diagnosis marker, are applied to clinical.Can in serum, occur and the detected blood serum designated object that just is called as.By research to these marks, can also further get in touch the genesis mechanism of disease, study and relate to the new way of disease treatment.
Peptide quality fingerprinting collection of illustrative plates (peptide mass fingerprint): MALDI-TOF analyzes can produce peptide quality fingerprinting collection of illustrative plates.By with sequence library in theoretical peptide spectrum and the fracture spectrum relatively, can identify personal really part of institute's test sample.
(apolipoprotein Apo) is the protein that a class can combine with blood plasma lipide (mainly being meant cholesterol, triglyceride and phosphatide) to apolipoprotein, for constituting the Main Ingredients and Appearance of plasma lipoprotein.Apolipoprotein has many important physical functions in vivo, as combining, activate multiple lipoprotein metabolism enzyme etc. with lipoprotein receptor as aglucon.Now have recognized that apolipoprotein not only to the metabolism decisive role of plasma lipoprotein, and atherosclerotic generation and development are also had very big influence.
Protein group (proteome): be meant whole albumen that a cell and tissue gene group are expressed.Because the expression of same genome in different cells and different tissues has nothing in common with each other, even same cell or tissue is at different stages of development and different physiology, under the pathological conditions, or under the influence of the varying environment factor, protein expression and existence are all inequality.Therefore, protein group is the notion of a dynamic change on time and space.Proteomics is the new subject that variety of methods and means are studied protein group.Protein by dynamic change in whole angle analysis cell is formed, expression, and between modification situation and the protein and the interaction between protein and other molecules, thus the function of understanding protein and the effect in life process thereof.At present the proteomics research platform of comparative maturity is to be the isolation technics of representative with the dielectrophoresis and to be the authenticate technology of core with the mass spectrum
Embodiment
The inventor utilizes proteomics method, by the expression of albumen in comparison diabetes B patient and the normal human serum, determines that apolipoprotein AI is a kind of albumen of the differential expression relevant with diabetes B.(Apolipoprotein A-I is a kind of main secreted protein synthetic in liver and small intestine APO-AI) to apolipoprotein AI, and its size is 267 amino acid (according to the reports of the online albumen database of NCBI), belongs to aPoA/E family.APO-AI is plasma high density lipoprotein level (High-density lipoprotein, principal ingredient HDL).This albumen influences cholesterol ester from tissue flowing and discharge to liver.Simultaneously, it also is that (lecithin cholesterolacyltransferase, one of accessory factor LCAT) have helped the formation of most of cholesterol ester gruel in the blood plasma to lecithin cholesterol acyl transferase.On o.11 chromosome, this gene and other two kinds of apolipoprotein genes are closely linked, and the defective of this gene can cause numerous disease.Learn through the NCBI retrieval: the defective of APO-AI is 2 type high-density lipoprotein (HDL) defective diseases (high density lipoprotein deficiency type 2, HDLD2), also be familial hypolipoproteinemia (familial hypoalphalipoproteinemia, one of the main reasons FHA).It shows as autosomal dominant inheritance.Simultaneously, the defective of APO-AI is I type high-density lipoprotein (HDL) defective disease (high density lipoproteindeficiency type 1 clinically, HDLD 1), also be Tangier disease (Tangier disease, the HDL level main cause on the low side that TGD) is shown.HDLD 1 is a kind of with the high-density lipoprotein (HDL) shortage, the gathering of cholesterol ester, and hepatosplenomegaly, and the coronary heart disease of early onset is the recessive disorders of principal character.In HDLD 1 patient's body, the disappearance of APO-AI from HDL is likely the failure of the APO-AI precursor conversion being gone into the eubolism chain, comprises the disappearance or the damage of specific structure (as in the disease of Tangier) that are converted into normal biology enzyme ability.This shows that the lipid metabolism in APO-AI and the body is closely related, and many common complication of diabetes B are feature with lipid metabolism unusually all.To sum up, we propose as detectable secretory protein in a kind of serum, and APO-AI can be used as the blood serum designated object of diabetes B, is used to prepare a kind of novel detection and diagnostic kit.
Differentially expressed protein in embodiment 1 dielectrophoresis and mass spectrometry analysis of diabetes people and the normal human serum
1, the collection of Chinese han population diabetes B patient and normal control sample
Used diabetes B group of the present invention and normal group crowd peripheral blood sample (10ml) are mainly from the outpatient service of division of endocrinology of BJ Union Hospital.Diabetes cases can be distributes, and also can be member's (but have only in each family this moment a patient selected usually, the member without any genetic connection in the same family can select more than) of diabetic pedigree; Normal control requires the three generations of its family with the interior diabetic of not having (comprising type i diabetes) from normal population, and control group and ill group of age and sex are complementary.
The diagnosis of diabetes B is carried out in strict accordance with the standard that WHO issued in 1985.That is: fasting blood concentration of glucose 〉=7.8mmol/L or 2 hours after the meal blood sugar 〉=11.1mmol/L are diabetes.The criterion of OGTT is: back 2 hours blood glucose concentration＜7.8mmol/L of clothes sugar are for normal, and 〉=11.1mmol/L is diabetes, between 7.8-11.1mmol/L be IGT (impaired glucose tolerance, IGT).
Each selected member record generalized case, comprise projects such as sex, date of birth, native place, height, body weight, heart rate, breathing, blood pressure, the investigation and the special examined of going where necessary are associated with diseases such as hypertension, heart disease, atherosclerotic with eliminating.Except that above-mentioned project, the diabetic is also write down projects such as age of onset, the highest body weight of the past, blood fat, glycosylated hemoglobin and diabetic complication, normal control group is also measured fasting blood-glucose and got rid of diabetes family history person.
Guarantee before the blood sample collection that all members understand the blood sampling purpose, right to know is promptly arranged, and go up signature at " Informed Consent Form ".The data of collecting is holded in close confidence, externally do not announce all data such as name, age, disease of blood supply member.
2. albumin and Ig-G in the serum are removed in the processing of blood serum sample
The blood serum sample of collecting is crossed post, remove albumin and Ig-G in the serum.At first, with 10mM Tris-HCl pH 7.4 (serum bind buffer) solution equilibria post material.After the drying of the solution composition in the post material, in the eppendorf pipe, take by weighing the dried post material of 0.02 gram.Then, with the serum of the 25ul ratio with 1: 3, with 10mM Tris-HCl pH 7.4 solution dilutions, join in the eppendorf pipe that fills the post material, vibration is 40 minutes on the shaking table.Then, centrifugal collection supernatant.Simultaneously,, take by weighing the dried post material Protein A of 0.04 gram after centrifugal, mixes, vibrate in conjunction with 40min on the shaking table with above-mentioned serum with 10mM Tris-HCl pH 7.4 solution equilibria Protein A.Centrifugal once more collection supernatant can be measured protein concentration, prepares to carry out follow-up dielectrophoresis.
Blood serum sample carry out first to isoelectric focusing (IEF)
In advance the strip holder of Len req and quantity is thoroughly cleaned with soft bristle tooth brush and special-purpose washing lotion and dry.Prepare swelling liquid: take out the swelling liquid of 1ml packing, add therein the IPG Buffer 5ul identical with the pH gradient of selected strip (final concentration: 0.5%) and DTT 3mg, mixing.Then, select applied sample amount by the concrete condition of selected strip length, pH gradient scope and sample; Determine the total amount of sample and swelling liquid sample solution to be dropped to evenly carefully among the strip holder by selected strip length,, note trying not to produce bubble the two abundant mixing.Then; take out the IPG strip of a Len req and pH scope; peel off the diaphragm of glue face from acidic terminal (promptly most advanced and sophisticated); with tweezers or with handing the strip two ends; slowly put into strip holder; and front and back move up and down several times, are evenly distributed in holder to guarantee sample solution, will avoid producing bubble in whole process as far as possible.After putting strip well, cover one deck cover fluid in the above, separate out with evaporation and the urea of avoiding sample solution.Build the holder loam cake, holder is placed on the battery lead plate of IPGphor, keep the holder direction parallel by ruler, and guarantee that two electrodes of holder all contact with battery lead plate with electrode.At last, set IEF electrophoresis parameter, carry out isoelectric focusing according to length and the pH scope of strip.The situation of monitoring voltage at any time in electrophoresis carries out if voltage does not reach the regulation requirement, is then wanted the proper extension electrophoresis time, to reach desired volt hourage.After electrophoresis finishes, strip is taken out, carry out balance or place-80 ℃ of preservations in the plastic tube.
After the IEF electrophoresis finishes, strip is taken out, glue faces up and places on the dry filter paper.Other gets a wetting slightly thick filter paper, covers on the strip, and flicking is several down, inhales and removes the oil droplet that adsorbs above.Simultaneously, get volume required equilibrium liquid, every 10ml adds 100mg DTT, and it is fully dissolved.The strip bar is put into the pipe that fills volume required equilibrium liquid, support face to paste tube wall, jolting 15min in shaking table.Then, take out the strip bar, several times with distilled water flushing.Then, get volume required equilibrium liquid, every 10ml adds the 250mg iodo-acetamide, and it is fully dissolved.The strip bar is put into the pipe that fills volume required equilibrium liquid, support face to paste tube wall, jolting 15min in shaking table.Strip is taken out, and glue faces up and places on the dry filter paper, inhales the moisture that goes on the supporting film, and the strip side is dipped on filter paper, inhales the moisture of the face that removes photoresist.At this moment just can prepare to carry out second to polyacrylamide gel electrophoresis (SDS-PAGE).
4. blood serum sample carries out second to polyacrylamide gel electrophoresis (SDS-PAGE)
The preparation of polyacrylamide gel: mould is tilted to place, required glass plate is put into wherein, fill with baffle plate all the other positions, each is to separating with the plastics partition between the glass plate, at last put into several partitions again, cover enclosing cover, screw tightening fully to occupy the space of mould.The glue of preparation q.s.Get the replacement liquid of capacity in advance ready.On the encapsulating mouth, insert funnel, glue is poured into near desired height, pour replacement liquid rapidly into, make replacement liquid not have the V-type groove.Add the water saturated normal butyl alcohol of skim with liquid-transfering gun in the glass plate upper end.Treat glue polymerization (after about 1 hour) fully, mould is taken apart, carefully take out glass plate, remove the gel that is bonded at the glass plate outer wall, discard the normal butyl alcohol of upper end, with distilled water flushing repeatedly, blot unnecessary water, can carry out electrophoresis with filter paper.
Carry out second to polyacrylamide gel electrophoresis: the toggle switch of electrophoresis tank rear end is allocated to circulation position, about 10 liters of electrophoretic buffers are injected in the electrophoresis tank.To fill it up with the sealing liquid of thawing on the glass plate, the IPG adhesive tape that balance is good is put into, and it is fully contacted with the glue upper end.Treat that sealing liquid solidifies fully, glass plate and baffle plate are put into electrophoresis tank, preferably earlier that glass plate is lubricated once with electrophoresis liquid when putting into.Next the electrophoresis parameter is set, generally adopts permanent power electrophoresis, be about to voltage and electric current and all be made as maximum, the size of a power controlling.The loam cake that closes then, the beginning electrophoresis generally needs 5-7 hour approximately.After treating that electrophoresis finishes, glass plate is taken out, carefully open glass plate, take out gel, dye with getting the glue device.
5. the dyeing of glue figure and analysis
Glue figure dyeing-Yin dyes: carefully take off running gel, fix 30 minutes or the longer time.Immobile liquid is 40% ethanol, 10% glacial acetic acid.Sensitization is 30 minutes then, and solution is 75ml ethanol, and 17g sodium acetate, 28.2g sodium acetate trihydrate, 0.5g sodium thiosulfate add water to final volume 250ml.Then, washing gel 3 times, each 10 minutes.Get 0.625g AgNO
3, 100ul 37% formaldehyde adds water and mixes to final volume 250ml.Carrying out silver in dye liquor dyed 20 minutes.Wash twice then, noted the assurance time in each 1 minute, the washing time, long color speed was slow, and the color of point is yellow partially.Colour developing liquid is 6.25g Na
2CO
3, 50ul 37% formaldehyde adds water to final volume 250ml.Note during colour developing observing, determine developing time according to the situation of colour developing.At last, cessation reaction 10 minutes in stop buffer.Stop buffer is 3.65g EDTA.Na
2.2H
2O or 1g glycocoll add water to final volume 250ml.
The glue map analysis: scanning glue figure, according to the size of the big or small Treatment Analysis expressing quantity of the depth of dyeing and protein site.The difference of the protein site that comparison diabetes B patient and normal control group obtain defines the protein site that expression difference changes.At this, the inventor finds that the marked change of expression has appearred in a protein site in diabetes patient and normal person's running gel figure, and expression raises in diabetes patient's serum, is more than 4 times of expression in diabetes patient's serum.
6. the mass spectrophotometry of differential protein spot is identified among the glue figure
Digestion in the glue: carefully extract the protein adhesive point of expression difference, place clean Eppendorf pipe, prepare the yin and yang attribute contrast simultaneously.With 50 μ l Milli-Q H
2O washes twice, each 5 minutes, discards supernatant liquor.Dye destainer with silver and wash twice, each 30 minutes, discard supernatant liquor, disappear up to color.Be the 30mM potassium ferricyanate, it is 100mM sodium thiosulfate that silver dyes destainer B.With 5 * silver dye destainer A 50ml and 5 * silver dye destainer B 50ml and mix the back moisturizing to 500ml, mixing is standby.Then, with 50 μ l ACN dehydration 20 minutes, it is opaque that glue is become; Discard supernatant liquor, concentrate dry adhesive tape in the hydro-extractor in vacuum.Next, add an amount of 25mM NH that contains 10mM DTT
4HCO
3, be not as the criterion to have adhesive tape, place 56 ℃ of water-baths 1 hour.Take out adhesive tape, make the slow room temperature that drops to of its temperature.Discard upper strata liquid, add an amount of 25mM NH that contains 55mM IAM
4HCO
3The submergence adhesive tape placed dark place 45 minutes.Discard upper strata liquid once more, use 25mMNH successively
4HCO3,50%ACN, ACN wash once, each 10 minutes, discard upper strata liquid.Repeat this step operation once.Again next, concentrate dry adhesive tape in the hydro-extractor in vacuum.Simultaneously, according to the experiment expense, get an amount of Trypsin and be dissolved as Trypsin stoste by operation instructions.According to the quantity of sample digestion, Trypsin stoste (0.1mg/mL) is by 1: 40 (mass ratio) (trypsin: protein content) add, use 25mM NH again
4HCO
3Dilution is the Trypsin working fluid.Every pipe adds an amount of (not being as the criterion to have micelle) Trypsin working fluid, places 20 minutes, and Trypsin is infiltrated in the glue fully.Then, add 25mM NH
4HCO
310-20 μ l makes the complete submergence glue of solution; Placed 37 ℃ of water enzyme digestion 4-6 hours.Xiang Guanzhong adds 3 times of digestive juice volume 67%ACN, makes final concentration reach 50%ACN, 2.5%TFA, and water-bath 30 minutes-1 hour, vibration in per 10 minutes is once.At last, sample is concentrated into 5-10 μ l in the concentrated hydro-extractor of vacuum, preserves down for-20 ℃ or-80 ℃.
MALDI-TOF mass spectrophotometry: draw 0.5ul matrix solution point on MALDI sample target, before it is not dried, add 0.5ul sample or standard items immediately in matrix.Made solvent evaporates at least 15 minutes under the room temperature, then the sample target is changed in the mass spectrometric vacuum chamber.Then, earlier obtain preliminary mass spectrum, reduce laser intensity then and adjust series of parameters such as voltage simultaneously and make the mass spectrum result optimizing, and proofread and correct mass spectrometer with standard items with surpassing the more laser intensity of ionization thresholding.Repeat above operation, obtain the mass spectrogram of unknown sample, and further use the pancreatin peak (2163.05,2273.15) in the mass spectrogram to do the interior positive mass spectrogram of calibration.Thus, the result of peptide quality fingerprinting collection of illustrative plates (the peptide mass fingerprint) searching database that obtains according to mass spectrum, the inventor determines that the protein site of the differential expression that occurs is apolipoprotein A-I, i.e. the I hypotype of aPoA in dielectrophoresis.
The method that embodiment 2 uses enzyme linked immunological absorption (ELISA) detects the expression of this albumen in diabetes B patient and normal human serum
1) the serum bag is added 100 μ l/ holes in the enzyme mark hole by the serum with PBS dilution (1: 1000 to 1: 10000), and 4 ℃ of bags are spent the night
2) the standard protein bag is spent the night 4 ℃ of bags in 100 μ l/ holes in the standard protein adding enzyme mark hole of PBS gradient dilution
3) sealing is dissolved in PBS with skimmed milk power and is made into 5% confining liquid, outwells coating buffer, adds confining liquid, 37 ℃, 2h by 200 μ l/ holes.Outwell confining liquid, can in super-clean bench, dry up, seal 4 ℃ of preservations with adhesive tape as do not use at once.
4) will dilute good anti-apolipoprotein A-I antibody (IgG of purifying, serum, ascites etc.) and add enzyme mark hole, 100 μ l/ holes, 4 ℃ are spent the night, or 37 ℃, 3-4h.
5) outwell one and resist,, add corresponding two anti-(diluting) by manufacturer's recommended with the HRP mark of PBS-T preparation with PBST (PBS that promptly contains 0.1% Tween 20) hole flushing 3 times, 37 ℃, 1-2h.
6) the PBS-T hole flushing is 3 times, adds o-phenylenediamine colour developing liquid, 100 μ l/ holes, and 37 ℃, 30min adds 2M sulfuric acid cessation reaction, measures OD with microplate reader
492 (490)
7) do typical curve according to the resulting OD value of standard protein, calculate the protein content of each blood serum sample.
Sequence table
＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
＜120〉detection kit of Chinese's diabetes B blood serum designated object
<160>1
<170>PatentIn?version?3.1
<210>1
<211>267
<212>PRT
<213>Homo?sapiens
<400>1
Met?Lys?Ala?Ala?Val?Leu?Thr?Leu?Ala?Val?Leu?Phe?Leu?Thr?Gly?Ser
1 5 10 15
Gln?Ala?Arg?His?Phe?Trp?Gln?Gln?Asp?Glu?Pro?Pro?Gln?Ser?Pro?Trp
20 25 30
Asp?Arg?Val?Lys?Asp?Leu?Ala?Thr?Val?Tyr?Val?Asp?Val?Leu?Lys?Asp
35 40 45
Ser?Gly?Arg?Asp?Tyr?Val?Ser?Gln?Phe?Glu?Gly?Ser?Ala?Leu?Gly?Lys
50 55 60
Gln?Leu?Asn?Leu?Lys?Leu?Leu?Asp?Asn?Trp?Asp?Ser?Val?Thr?Ser?Thr
65 70 75
80
Phe?Ser?Lys?Leu?Arg?Glu?Gln?Leu?Gly?Pro?Val?Thr?Gln?Glu?Phe?Trp
85 90 95
Asp?Asn?Leu?Glu?Lys?Glu?Thr?Glu?Gly?Leu?Arg?Gln?Glu?Met?Ser?Lys
100 105 110
Asp?Leu?Glu?Glu?Val?Lys?Ala?Lys?Val?Gln?Pro?Tyr?Leu?Asp?Asp?Phe
115 120 125
Gln?Lys?Lys?Trp?Gln?Glu?Glu?Met?Glu?Leu?Tyr?Arg?Gln?Lys?Val?Glu
130 135 140
Pro?Leu?Arg?Ala?Glu?Leu?Gln?Glu?Gly?Ala?Arg?Gln?Lys?Leu?His?Glu
145 150 155
160
Leu?Gln?Glu?Lys?Leu?Ser?Pro?Leu?Gly?Glu?Glu?Met?Arg?Asp?Arg?Ala
165 170 175
Arg?Ala?His?Val?Asp?Ala?Leu?Arg?Thr?His?Leu?Ala?Pro?Tyr?Ser?Asp
180 185 190
Glu?Leu?Arg?Gln?Arg?Leu?Ala?Ala?Arg?Leu?Glu?Ala?Leu?Lys?Glu?Asn
195 200 205
Gly?Gly?Ala?Arg?Leu?Ala?Glu?Tyr?His?Ala?Lys?Ala?Thr?Glu?His?Leu
210 215 220
Ser?Thr?Leu?Ser?Glu?Lys?Ala?Lys?Pro?Ala?Leu?Glu?Asp?Leu?Arg?Gln
225 230 235
240
Gly?Leu?Leu?Pro?Val?Leu?Glu?Ser?Phe?Lys?Val?Ser?Phe?Leu?Ser?Ala
245 250 255
Leu?Glu?Glu?Tyr?Thr?Lys?Lys?Leu?Asn?Thr?Gln
260 265
Claims (3)
1. a peptide species, it is full-length proteins or its fragment with biologic activity with complete amino acid sequence of Apolipoprotein A-1.
2. the antibody of the polypeptide of anti-claim 1 comprises and can discern overall molecule, all antibody of a certain domain of molecule and some or several special antigenic determinant.
3. claim 1 or 2 peptide molecule are as the purposes of the blood serum designated object of diabetes diagnosis.
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| CN 200510098263 CN1928556A (en) | 2005-09-05 | 2005-09-05 | Reagent kit for detecting 2-diabetes serum mark in Chinese people |
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| CN102565416A (en) * | 2010-12-27 | 2012-07-11 | 中国科学院上海生命科学研究院 | Application of apolipoprotein Al as diabetes mellitus marker |
| CN105648074A (en) * | 2016-02-29 | 2016-06-08 | 北京泱深生物信息技术有限公司 | Application of FAN1 genes in II-type diabetes diagnosis |
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