CN108546751A - Application of the Protein S as biomarker in preparing asthma disease diagnosis and prognosis effect appraisal reagent - Google Patents

Application of the Protein S as biomarker in preparing asthma disease diagnosis and prognosis effect appraisal reagent Download PDF

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CN108546751A
CN108546751A CN201810360210.XA CN201810360210A CN108546751A CN 108546751 A CN108546751 A CN 108546751A CN 201810360210 A CN201810360210 A CN 201810360210A CN 108546751 A CN108546751 A CN 108546751A
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pros1
asthma
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魏滨
邓力
程梦兰
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Wuhan Institute of Virology of CAS
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Abstract

The present invention relates to biomedicine fields, disclose Protein S（Protein S,Pros1）As application of the biomarker in preparing asthma disease diagnosis and prognosis effect appraisal reagent.It is found by the applicant that the rna level of the Pros1 in the peripheral blood of asthmatic patient is significantly higher than Healthy People；Further, ELISA detections are carried out to the Pros1 in mild and the peripheral blood of severe asthma patient and Healthy People, it is found that the protein level of Pros1 is significantly higher than Healthy People in the peripheral blood of severe asthma patient；Simultaneously in asthmatic patient, it is in protein level higher of the patient than the Pros1 in the peripheral blood in patients of paracmasis of acute stage, therefore Protein S can be used as biomarker for asthma disease diagnosis and prognosis effect appraisal.

Description

Protein S is preparing asthma disease diagnosis and prognosis effect appraisal as biomarker Application in reagent
Technical field
The present invention relates to biomedicine fields, and in particular, to Protein S (Protein S, Pros1) is used as biological marker Application of the object in preparing asthma disease diagnosis and prognosis effect appraisal reagent.
Background technology
Asthma is one of most common chronic immunological disorders, influences crowd and covers all age group, is referred to as seriously endangering One of four big killers of victimization class life quality, are only second to AIDS, tumour and cardiovascular and cerebrovascular disease.There are about 300,000,000 people in the whole world With asthma, at least 250,000 people die of asthma relevant disease every year.In recent years, with air pollution and environmental degradation, asthma Incidence rise year by year.The present situation of China's asthma is that illness rate is also relatively low, but illness rate growth rate is very fast at present.In Provinces and cities of covering national 8,7 areas Ge great in 2013 of asthma alliance of state, sample size are more than that the epidemiological survey of 160,000 people is aobvious Show, China's asthma total prevalence rate is 1.24%.Beijing area and District of Shanghai increase respectively compared with the investigation result before 10 years 147.9% and 190.2%.Asthma PATIENT POPULATION is increasing, has expedited the emergence of out huge pharmaceutical market.
According to coincident with severity degree of condition, asthma can be divided into mild asthma and severe asthma.And asthma is according to whether acute hair Work can be divided into acute stage and paracmasis, and patients during acute stage occurs panting, exhale since bronchial muscular spasm leads to airway constriction Difficult symptom is inhaled, enters the paracmasis after treatment symptom is relieved or disappears.There are still airway inflammations by paracmasis patient The problem of being reacted with height, such as cannot effectively control, go down will result in the irreversible reconstruction of air flue for a long time, lead to lung function forever Long property damage.The severity thus accurately to diagnose the illness and residing period, Symptomatic medicine, to the therapeutic potential weight of asthma Greatly.
All the time, people are dedicated to finding the marker for asthma disease diagnosis or prognosis effect appraisal, however Or existing some detection means sampling is difficult or correlation is poor each other, the biological marker clinically still expected The appearance of object.
We are found that a kind of potential biomarker Pros1 by screening.Pros1 is as a kind of novel dimension earliest He order K dependence glycoprotein in the 1970s by it is found that.The albumen is a kind of naturally occurring anti-coagulants, is had more Kind biological function may participate in cell Proliferation, Apoptosis, Coagulation test, adjust inflammatory factor release, angiogenesis and tumour hair The physiological reactions such as raw, up to the present there are no the relationships that people had found Pros1 and asthma.
Invention content
The object of the present invention is to provide detection Pros1 genes or the reagent of Protein S to prepare asthma disease diagnosis medicine Application in object.
It is another object of the present invention to provide detection Pros1 genes or the reagent of Protein S to prepare asthma prognosis Application in effect assessment drug.
In order to achieve the above object, the present invention takes following technical measures：
Applicant quantifies lots of genes after extracting RNA by acquiring the peripheral blood of asthmatic patient and Healthy People PCR is screened, and finally found that the rna level of the Pros1 in the peripheral blood of asthmatic patient is significantly higher than Healthy People；Further, right Pros1 in mild and the peripheral blood of severe asthma patient and Healthy People carries out ELISA detections, finds severe asthma patient's The protein level of Pros1 is significantly higher than Healthy People in peripheral blood；Simultaneously in asthmatic patient, patient's ratio in acute stage is in The protein level higher of Pros1 in the peripheral blood in patients of paracmasis.
The application of detection Pros1 genes or the reagent of Protein S in preparing asthma disease diagnostic medicine, including the use of ability The conventional means in domain are using mentioned reagent as sole active ingredient or are prepared into other active ingredients and examine asthma disease Disconnected kit；
According to above application, it can be used for preparing asthma disease diagnostic reagent for the quantification PCR primer of Pros1 genes design Box；
Preferably, the primer is：Forward primer 5'-TGCTGGCGTGTCTCCTCCTA-3'；Reverse primer 5'-C AGTTCTTCGATGCATTCTCTTTCA-3'。
The application of detection Pros1 genes or the reagent of Protein S in preparing asthma prognosis effect appraisal drug, including the use of The conventional means of this field are prepared into pre- to asthma progress using mentioned reagent as sole active ingredient or with other potent agents The kit of effect assessment afterwards.
Compared with prior art, the present invention has the following advantages：
1.Pros1 can secreting, expressing, in serum be Gamma Magnitude, sampling and detection operation is simple.
The percentage of protein levels and eosinophil of the 2.Pros1 in mouse asthmatic model bronchoalveolar lavage fluid has Significant correlation, the latter are the goldstandard of Diagnosing Asthma, this illustrates that Pros1 is extraordinary biomarker.
Description of the drawings
The screening of the peripheral blood from asthma patients different expression gene of Fig. 1 embodiment of the present invention 1；
The protein level testing result of Pros1 in the peripheral blood from asthma patients of Fig. 2 embodiment of the present invention 2；
Wherein A shows the protein level testing result of Pros1 in mild and severe asthma peripheral blood in patients；
B shows the protein level testing result of Pros1 in asthmatic patient acute stage and paracmasis peripheral blood；
The correlation analysis result of eosinophil and Pros1 in the mouse asthmatic model of Fig. 3 embodiment of the present invention 3；
Wherein A shows the percentage result of eosinophil in mouse asthmatic model bronchoalveolar lavage fluid；
B shows the protein level testing result of Pros1 in mouse asthmatic model bronchoalveolar lavage fluid；
C shows the correlation analysis result of eosinophil and Pros1 in mouse asthmatic model bronchoalveolar lavage fluid.
Specific implementation mode
Below in conjunction with specific embodiment detailed description of the present invention embodiment, the following examples are merely to illustrate this hair It is bright, and should not be taken as limiting the scope of the invention.Particular technique or condition are not specified in embodiment, according to text in the art It offers described technology or condition or is carried out according to product description.Reagents or instruments used without specified manufacturer, For can be with conventional products that are commercially available.
Embodiment 1：
The screening of peripheral blood from asthma patients different expression gene：
1. experiment material
1.1 asthmatic patients or healthy human peripheral blood cell
Human peripheral sample is acquired by Guangzhou Women and Children's Medical Center (GWCMC).
1.2 reagent
Human lymphocyte separating liquid Ficol is purchased from GE Healthcare companies, and RNAiso Plus are purchased from TAKARA companies, Primer entrusts the synthesis of tsingke companies, SYBR Green Real time PCR Master mix to be purchased from Quan Shi King Companies.
1.3 laboratory apparatus
Quantitative RCP instrument (Stratagene Mx3000P) is purchased from Agilent companies.
2. experimental method and result
The preparation of 2.1 peripheral blood sample cDNA
(1) asthmatic patient or healthy human peripheral blood are acquired, the separation single karyocyte of peripheral blood (PBMCs) is resuspended in 1 milli It rises in R NAiso Plus, freezes in -80 DEG C of refrigerators.
(2) simultaneously reverse transcription obtains cDNA to extraction sample rna.
2.2 quantitative PCRs (RT-PCR) testing goal gene expression
(1) cDNA that transcription obtains is negated, distilled water is used to dilute 10 times of templates reacted as PCR.5 microlitres of modulus plate, The mother liquor that 15 microlitres of primers and 2X SYBR Green mix are prepared is added, is reacted, preparation method is as follows：Distilled water 4 is micro- It rises, 10 0.5 microlitre of mmoles forward primers, 10 0.5 microlitre of mmoles reverse primers, 10 microlitres of 2X SYBR Green mix.
(2) it is directed to testing gene and designs quantification PCR primer, house-keeping gene GADPH is selected to compare base as the internal reference of correction Cause is as follows for the quantitative RCR primer sequences of people GADPH：Forward primer 5'-TGCACCACCAACTGCTTA-3'；Reverse primer 5'-GGATGCAGGGATGATGTTC-3'。
Program is run on quantitative PCR apparatus：94 DEG C 30 seconds；94 DEG C 5 seconds, 55 DEG C 15 seconds, 72 DEG C 20 seconds, 40 cycle.It is raw At solubility curve.
(3) quantitative Ct values are proofreaded by reference gene (GAPDH), as a result pass through 5 softwares of GraphPad Prism Calculate average value and standard deviation.
(4) result of calculation in step (3) is utilized to draw each horizontal testing result of gene RNA in peripheral blood from asthma patients Figure.
Finally it has been found that the rna level of Pros1 is significantly higher than Healthy People (figure in peripheral blood from asthma patients PBMCs 1), therefore Pros1 genes can be used as the marker of Diagnosing Asthma, primer for gene design or for detecting the gene Reagent can be used for being prepared into Diagnosing Asthma kit.
Quantitative RCR primers are designed for people's Pros1 gene orders, primer sequence is as follows：Forward primer 5'-TGCTGGCGT GTCTCCTCCTA-3'；Reverse primer 5'-CAGTTCTTCGATGCATTCTCTTTCA-3'.
Embodiment 2：
The protein level detection of Pros1 in peripheral blood from asthma patients：
1. experiment material
1.1 asthmatic patients or healthy human peripheral blood serum
Human peripheral sample is acquired by Guangzhou Women and Children's Medical Center (GWCMC).
1.2 reagent
Protein S (PROS) detection kit.
1.3 laboratory apparatus
Multiple labeling detecting system is purchased from EnSpire companies.
2. experimental method and result
It the acquisition of 2.1 peripheral blood samples and freezes
Asthmatic patient (including mild and severe asthma) or healthy human peripheral blood serum are acquired, -80 DEG C of preservations are placed in.
2.2 quantitative determine the content of Pros1 in human serum with double antibody sandwich ELISA
Pros1 antibody is coated in 96 orifice plates, solid phase carrier is made, standard items or sample are separately added into micropore, Wherein Pros1 is combined with the antibody being connected on solid phase carrier, and biotinylated Pros1 antibody is then added, and will be not associated with Biotinylated antibody clean after, be added HRP label Avidin, again thoroughly washing after be added tmb substrate colour developing.TM B exist Au bleu is converted under the catalysis of peroxidase, and is converted to final yellow under the action of an acid.The depth and sample of color In Pros1 concentration be proportionate.
(1) using preceding that all reagents and sample are slowly balanced to room temperature (18-25 DEG C).Standard items (dried frozen aquatic products) are taken out to add Enter 1 milliliter of standard dilutions, covers rear room temperature static about 10 minutes, gently shake simultaneously, a concentration of 4000 pik/milli It rises.Prepare the EP pipes of 7 dilution standard product, 500 microlitres of standard dilutions is added in each EP pipes, doubling dilution is at 4000 Pg/ml, 2000 pg/mls, 1000 pg/mls, 500 pg/mls, 250 pg/mls, 125 pg/mls, 62.5 pg/mls, standard dilutions are directly as blank well.Ready standard items are temporarily placed on ice.
(2) serum sample detects after diluting 50000 times.Dilution process is as follows：10 microlitres of serum are taken to be added 190 microlitres first PB S do 20 times of dilutions, then take sample 10 microlitres of additions, 490 microlitres of PBS after 20 times of dilutions, do 1000 times of dilutions, finally Sample 10 microlitres of additions, 490 microlitres of PBS after 1000 times of dilutions, diluted each step are taken all to need vortex oscillation mixing.
(3) gauge orifice, sample to be tested hole, blank well are set respectively on 96 orifice plates (pre-coated), repeating hole is done per hole.Bidding 7 hole of quasi- hole sequentially adds the standard items of 100 microlitres of various concentrations, and 100 microlitres of standard dilutions are added in blank well, remaining Hole adds 100 microlitres of sample to be tested, in addition overlay film, 37 DEG C are incubated 1 hour.To prevent sample from evaporating, 96 orifice plates are placed in by when incubation In wet box.
(4) 96 orifice plate liquid are discarded, are dried, washing is not had to.Add 100 microlitres of detection solution A working solution (new before use per hole Fresh preparation detects solution A, according to 1:Such as 990 microlitres of detection diluent As are added, fully in 10 microlitres of detection solution As by 100 dilutions Mixing), in addition overlay film, 37 DEG C are incubated 1 hour.
(5) 96 orifice plate liquid are discarded, add 300 microlitres of (Fresh cleaning solutions before use, according to 1 of cleaning solution per hole:30 is dilute Release, 43.5 milliliters of distilled water such as be added in 1.5 milliliters of cleaning solutions, are mixed well), it impregnates 1-2 minutes, drying.Repeat board-washing 3 Secondary, after last time is washed, 96 orifice plates are tipped upside down on blotting paper, the liquid remained in hole are all blotted by drying.Per hole Add 100 microlitres of detection solution B working solution (Fresh detection solution B before use, according to 1:100 dilutions, such as detect 10 microlitres 990 microlitres of detection dilution B are added in solution B, mix well), in addition overlay film, 37 DEG C are incubated 30 minutes 1 hour.
(6) 96 orifice plate liquid are discarded, are dried, board-washing 5 times, the same step of method (5).Add 90 microlitres of tmb substrate solution per hole, In addition overlay film, 37 DEG C are protected from light colour developing (reaction time control at 10 minutes or so, and the holes 3-4 have apparent gradient blue before gauge orifice Color, when rear 3-4 gradient pores unobvious, you can terminate).Add 50 microlitres of terminate liquid per hole, terminates reaction, blue is changed into Huang at this time Color gently shakes 96 orifice plates so that solution is uniformly mixed.
(7) OD value (O.D. values) in each hole is measured in 450 nano wave lengths with microplate reader.
(8) regression equation that standard curve is calculated according to the concentration of standard items and OD value, by sample to be tested OD value substitutes into equation, calculates concentration of specimens, is multiplied by extension rate 50000, the as actual concentrations of sample to be tested.
(9) result of calculation in step (8) is utilized to draw the protein level testing result of Pros1 in peripheral blood from asthma patients Figure.The results are shown in Figure 2.
A results show that the protein level of Pros1 in severe asthma peripheral blood in patients is significantly higher than Healthy People in Fig. 2.In Fig. 2 B results are shown in asthmatic patient, are in albumen water of the patient than the Pros1 in the peripheral blood in patients of paracmasis of acute stage Flat higher, therefore, Pros1 can be used as the marker of asthma prognosis effect appraisal, and the reagent for detecting Pros1 can be used for being prepared into heavy breathing Breathe heavily the kit of prognosis effect appraisal.
Embodiment 3:
The relationship of the protein level of Pros1 and eosinophil percentage in mouse asthmatic model bronchoalveolar lavage fluid
1. experiment material
1.1 mouse asthmatic model
6~8 week old wild type C57BL/6 mouse are purchased from Beijing dimension tonneau China, in Wuhan Virology Institute,Chinan academy of Sciences The barrier environment of animal house SPF cleaning grades is raised.50 micrograms of intraperitoneal injection were carried out at the 0th, 7,14 day to be emulsified with aluminium adjuvant in advance Chicken egg white sensitization, 50 microgram chicken egg white of Nasal immunization was carried out continuously at the 21st, 22,23 day, is put to death after 1 day small Mouse collects bronchoalveolar lavage fluid (BAL).
1.2 reagent
Mouse vitamin k-dependent Pros1 (PROS1) enzyme linked immunological kit is purchased from the limited public affairs of the magnificent bioengineering in Wuhan Department, antibody Silgec-F-APC, CD11c-PE, Fixable Viability Dye506 is public purchased from eBioscience Department, FcR Block are purchased from Biolegend companies.
1.3 laboratory apparatus
Multiple labeling detecting system is purchased from EnSpire companies, and flow cytometer LSR Fortessa are purchased from BD companies.
2. experimental method and result
2.1 bronchoalveolar lavage fluid sample collections and processing
(1) anesthetized mice, after plucking eyeball blood sampling, exposure mouse tracheae is inserted into remaining needle, and 500 are pushed into 1 milliliter of syringe Microlitre fresh PBS slowly withdraws, is again pushed into, and withdraws, and is transferred to clean EP pipes, the as bronchoalveolar lavage fluid of first time.With 1 Milliliter syringe is pushed into 500 microlitres of fresh PBS, slowly withdraws, and is transferred to clean EP pipes, 500 microlitres are pushed into 1 milliliter of syringe Fresh PBS is slowly withdrawn, and is transferred to and is managed with EP, as secondary bronchoalveolar lavage fluid.
(2) 3000 leave the heart 3 minutes, retain the bronchoalveolar lavage fluid supernatant of first time, the as bronchoalveolar lavage fluid of the mouse Supernatant, be stored in -80 DEG C it is to be detected.It will merge for the first time with secondary bronchoalveolar lavage fluid cell when washing, as the mouse Bronchoalveolar lavage fluid cell.
The detection of acidophic cell percentage in 2.2 bronchoalveolar lavage fluids
(1) PBS+2%FBS washs cell, and 50 microlitres of PBS+2%FBS, which are added, in 0.5 microlitre of FcR Block prepares closing Liquid is resuspended cell precipitation with confining liquid, is incubated 10~20 minutes on ice.
(2) PBS+2%FBS washs cell, by 0.5 microlitre Siglec-F-APC/0.1 microlitres CD11c-PE/0.1 microlitres FVD506 is added 30 microlitres of PBS and matches antibody processed mixed liquor, and cell precipitation is resuspended with antibody mixed liquor, is incubated 30 minutes on ice.
(3) PBS+2%FBS washs cell, and cell precipitation is resuspended with 250 microlitres of PBS+2%FBS.With flow cytometer into Row detection, analyzes Siglec-F+CD11c-The ratio of group's cell is the percentage of eosinophil in bronchoalveolar lavage fluid.Knot Fruit is such as
In Fig. 3 shown in A.
A results are shown in Fig. 3：The percentage of eosinophil is significantly higher than P in mouse bronchoalveolar lavage fluid immune OVA Control mice immune BS.
The protein level of Pros1 in 2.3 flow cytometer detection bronchoalveolar lavage fluid supernatants
It is coated in 96 orifice plates with the mouse Pros1 of purifying, solid phase carrier is made, standard items are separately added into 96 orifice plates Or the anti-mouse Pros1 antibody of sample, HRP label, with substrate TMB colour developings after thoroughly washing.TMB is in peroxidase Catalysis is lower to convert au bleu, and is converted to final yellow under the action of an acid.Mouse Pros1 in the depth and sample of color Concentration is negatively correlated.
(1) using preceding that all reagents and sample are slowly balanced to room temperature (18-25 DEG C).Standard items (dried frozen aquatic products) are taken out, The heart is left in 6000~10,000 30 seconds, 1 milliliter of standard dilutions are added, and is used in combination pipette tips alignment to freeze bottom of the tube and inhale repeatedly and is made a call to 5 It is secondary to be mixed well with hydrotropy solution, a concentration of 400 nanograms/milliliter.Prepare the EP pipes of 7 dilution standard product, adds in each EP pipes Entering 150 microlitres of Sample dilutions, doubling dilution is at 400 nanograms/milliliters, 200 nanograms/milliliters, 100 nanograms/milliliters, 50 nanograms/ Milliliter, 25 nanograms/milliliters, 12.5 nanograms/milliliters, 6.25 nanograms/milliliters, Sample dilution is directly as blank well.It is ready to Standard items be temporarily placed on ice.
(2) it is detected after 10 times of bronchoalveolar lavage fluid Sample Dilution, takes 15 microlitres of bronchoalveolar lavage fluids that 135 microlitres of Sample Dilutions are added Liquid, vortex oscillation mixing.
(3) gauge orifice, sample to be tested hole, blank well are set respectively on 96 orifice plates (pre-coated), repeating hole is done per hole.Bidding 7 hole of quasi- hole sequentially adds the standard items of 50 microlitres of various concentrations, and blank well is not added with any solution, and remaining hole adds sample to be tested 50 microlitres.50 microlitres of HRP markers working solution is added immediately, and (Fresh horseradish peroxidase marker works before use Liquid, according to 1:Such as 990 microlitres of horseradish peroxidase-labeleds are added in 10 microlitres of horseradish peroxidase markers by 100 dilutions Object dilution, gently mixing).Mixing is gently shaken, in addition overlay film, 37 DEG C are incubated 30 minutes.
(4) 96 orifice plate liquid are discarded, are dried, 200 microlitres of washing lotion working solution is added per hole, and (Fresh washing lotion works before use Liquid, according to 1:2 milliliters of cleaning solutions are such as added 48 milliliters of distilled water, mixed well by 25 dilutions), it impregnates 2 minutes, drying.It repeats Board-washing 5 times, after last time is washed, drying tips upside down on 96 orifice plates on blotting paper, and the liquid remained in hole is all inhaled It is dry.Add 90 microlitres of tmb substrate solution per hole, add overlay film, 37 DEG C be protected from light colour developing (reaction time control at 20 minutes or so, when There is an apparent gradient blue in the holes 3-4 before gauge orifice, when rear 3-4 gradient pores unobvious, you can terminate).Add terminate liquid 50 micro- per hole It rises, terminates reaction, blue is changed into yellow at this time, gently shakes 96 orifice plates so that solution is uniformly mixed.
(5) OD value (O.D. values) in each hole is measured in 450 nano wave lengths with microplate reader.
(6) regression equation that standard curve is calculated according to the concentration of standard items and OD value, by sample to be tested OD value substitutes into equation, calculates concentration of specimens, is multiplied by extension rate 10, the as actual concentrations of sample to be tested.As a result As shown in B in Fig. 3.
B results are shown in Fig. 3：The protein level of Pros1 is significantly higher than PBS and exempts from mouse bronchoalveolar lavage fluid immune OVA The control mice of epidemic disease.
2.4 draw the protein level and eosinophil percentage of Pros1 in mouse asthmatic model bronchoalveolar lavage fluids Correlation results figure
The line of the protein level and eosinophil percentage of Pros1 is calculated by 5 softwares of GraphPad Prism Property regression correlations R2With P values, linear regression graph is drawn.As a result as shown in C in Fig. 3.
C results are shown in Fig. 3：The protein level and eosinophil of Pros1 in mouse asthmatic model bronchoalveolar lavage fluid The notable positive correlation of percentage.
Generally speaking, Pros1 is expressed variant in severe asthma patient and healthy human peripheral blood, acute in asthmatic patient Expressed in phase and paracmasis peripheral blood it is variant, while in Pros1 expressions and bronchoalveolar lavage fluid eosinophil hundred The apparent positive correlation of score can be used as the index of clinically asthmatic patient diagnosis and prognosis.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example, " " are specifically shown Example, " or the description of " some examples " etc. mean specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, changes, replacing and modification.
Sequence table
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Claims (5)

  1. The application of 1.Pros1 genes or Protein S in being prepared into Diagnosing Asthma marker.
  2. 2. the application of detection Pros1 genes or the reagent of Protein S in preparing asthma disease diagnostic medicine.
  3. 3. being directed to application of the quantification PCR primer of Pros1 genes design in preparing asthma disease diagnostic kit.
  4. 4. application according to claim 3, the primer are：Forward primer 5'-TGCTGGCGTGTCTCCTCCTA- 3'；Reverse primer 5'-CAGTTCTTCGATGCATTCTCTTTCA-3'.
  5. 5. the application of detection Pros1 genes or the reagent of Protein S in preparing asthma prognosis effect appraisal drug.
CN201810360210.XA 2018-04-20 2018-04-20 Application of the Protein S as biomarker in preparing asthma disease diagnosis and prognosis effect appraisal reagent Pending CN108546751A (en)

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CN109439744A (en) * 2018-12-24 2019-03-08 常州市第二人民医院 Molecular marker is used in severe asthma diagnosis

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Application publication date: 20180918