CN106771241A - A kind of abnormal mucus cross-examination（Syndrome）Mammalian blood serum differential protein system and its screening technique and application - Google Patents
A kind of abnormal mucus cross-examination（Syndrome）Mammalian blood serum differential protein system and its screening technique and application Download PDFInfo
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Abstract
The invention provides dimension abnormal mucus cross-examination (syndrome) mammalian blood serum differential protein system of doctor, and above-mentioned differential protein system screening technique, including 1) Normal group, modeling card marquis group and disease group；2) whole blood is gathered, serum is separated；3) serum proteins are extracted；4) purified after marking；5) finger-print is obtained after Mass Spectrometer Method, proteome analysis is carried out after screening, obtain the serum proteins of the Normal group, syndrome group and disease group；6) count, compare, screening to obtain tieing up and cure abnormal mucus cross-examination (syndrome) group serum differential protein, for further the Mechanism Study of the abnormal mucus cross-examination of development and its relevant disease, drug screening, conversion are laid a good foundation with study on prevention, there is provided foundation.
Description
Technical field
The invention belongs to biological technical field, and in particular to abnormal mucus cross-examination (syndrome) the mammal difference of one kind dimension doctor
Albumen system and its screening technique and application.
Background technology
It is important component in traditional Chinese medicine that Uygur medicine (dimension doctor) is learned, and dimension medical science body fluid opinion thinks, human body by
Bile matter, blood matter, 4 kinds of body fluid matter compositions of lymphatic temperament and black courage matter, the dynamic equilibrium of above-mentioned 4 kinds of body fluid matter is body health
Physiological foundation, and the change of the amount or/and matter of a certain body fluid matter, cause body fluid matter dysequilibrium, produce a kind of abnormal humour
, there is causality with various diseases in cross-examination (syndrome).Humoral pathology (body fluid opinion) is dimension medical knowledge opinion foundation, and it is recognized
In for 4 kinds of body fluid, lymphatic temperament body fluid attribute is raw, Chang Yiyin lives, diet and environmental factor, operating pressure, shortage motion etc.
Reason causes internal isohydria to be destroyed, and lymphatic temperament body fluid changes, and produces abnormal lymphatic temperament, causes metabolic water pancake
Low, product accumulation is internal, and then causes pathology to sexually revise, and triggers relevant disease.
Clinical research confirmation, abnormal mucus cross-examination (Abnormal Phlegm) is in impotence (Erectile Dysfunction), morning
Let out, lack the metabolic diseases such as reproductive disease, obesity, the hyperlipidemia such as azoospermia, premature ovarian failure, infertile and cardiovascular disease
May be also the master of the Complex Diseases such as tumour, diabetes, hypertension generation early stage in leading position in sick generating process
Demonstrate,prove type.Although Complex Diseases dimension doctor abnormal savda card research in, genomics, metabolism group, oxidative stress,
The many-side such as Pre-thrombosis State and gut flora has carried out substantial amounts of research work, achieves breakthrough, achieves noticeable achievement, but
It is delayed always to the basic and clinic studies of abnormal mucus cross-examination and its related disease.
Therefore, inventor herein is at abnormal mucus cross-examination (syndrome) and its related disease aspect, and originality is established
Abnormal mucus cross-examination and disease combination rat model, and the model is changed from Biological Characterization, behaviouristics and development rule occur
Rule angle carries out more systematic research, it was confirmed that it has a stronger scientific, reliability and stability, and with the dimension of the syndrome
The clinical conclusion being identical of doctor, while having been carried out from Neuroendocrine net change angle to its pathogenesis more deep
Research.
The starting point and core of dimension doctor's differentiation of symptoms and signs for classification of syndrome are dimensions doctor " body fluid opinion ", and its starting point is in body fluid dynamic change in human body
External manifestation, and it is also poor according to the body fluid phenotype occurred in individual integral level to tie up the diagnosis and treatment based on an overall analysis of the illness and the patient's condition that medicine and pharmacology are praised highly
It is different to carry out, therefore, regularity of occurrence and development to open the syndrome, then need in integral level come the work that conducts a research,
Its key function molecule is found, the pathomechanism of its core mechanism and relevant disease is explored, and this integrally grinds with systems biology
Study carefully thinking perfectly in harmony.
Serum has incorporated the protein group multidate information of body multiple organ, tissue and cell, is also the close phase of various diseases
Secretory protein the only way which must be passed of pass, can reflect the spatiotemporal of the related global regulation network of the pathophysiological change of body, and
Effect of the medicine to any target organ, tissue or cell is finally also be reflected among the change of serum proteins group.Therefore, this research base
On the basis of abnormal lymphatic temperament syndrome and Disease Syndrome integrated animal model, carry out serum photeomics research, establish exception
Lymphatic temperament syndrome associated serum albumen system is further to carry out Mechanism Study, the drug sieve of abnormal mucus cross-examination relevant disease
Choosing, conversion are laid a good foundation with study on prevention.
The content of the invention
In view of this, the first object of the present invention is to provide a kind of mammal of abnormal mucus cross-examination (syndrome) of dimension doctor
Serum differential protein system, including following albumen：
Preferably, the mammalian blood serum differential protein system of abnormal mucus cross-examination (syndrome) of dimension doctor of the invention is come
Self mode animal；The model animal is rat.
The mammalian blood serum differential protein that one kind dimension doctor's exception mucus cross-examination (syndrome) is also provided of the invention, including with
Lower albumen any one or a few：
Preferably, the mammalian blood serum differential protein of abnormal mucus cross-examination (syndrome) of dimension doctor of the invention comes from mould
Formula animal；The model animal is rat.
Another object of the present invention there are provided the mammalian blood serum difference of above-mentioned abnormal mucus cross-examination (syndrome) of dimension doctor
The screening technique of albumen system, comprises the following steps：
1) Normal group (N groups), abnormal lymphatic temperament syndrome group (ZM groups), abnormal lymphatic temperament disease group (BM groups) food in one's mouth are set up
Newborn animal；
2) step 1 is gathered) mammalian whole blood that obtains, the serum of mammal is obtained after separation；
3) from the step 2) protein is extracted in the serum that obtains；
4) by the step 3) in purify after the protein labeling that obtains, obtain peptide fragment after purification；
5) by the step 4) peptide fragment after purification that obtains obtains finger-print after Mass Spectrometer Method, institute after screening
State finger-print to be retrieved using proteome analysis software, carried out according to the spectrogram after the Search Results and screening for obtaining
Quantitative analysis, the analyze data that will be obtained is retrieved software and is examined in the mammalian proteins database using proteomic image
Rope, obtains the N groups, ZM groups and BM group mammalian blood serum differential proteins；
6) by the step 5) in
The BM groups obtain the serum differential protein of BM/N compared with the serum proteins of N groups；By ZM groups and N groups
Serum proteins compare, obtain the serum differential protein of ZM/N；Count the common of the BM/N and ZM/N differential proteins
The type and quantity of albumen, obtain the serum differential protein of abnormal mucus cross-examination (syndrome).
Preferably, in screening technique of the invention, the mammal is rat.
Preferably, in screening technique of the invention, the step 1) in rats in normal control group raised in conventional environment,
The environment temperature of feeding is 18~22 DEG C, and the relative humidity of the feeding environment is 40~60%, the environment of the feeding of modeling group
Temperature is 8~12 DEG C, the relative humidity 70~80% of the feeding environment；The step 1) in Normal group be common feeding
Material, modeling group feeding feed is raw property feed.Preferably, in screening technique of the invention, the step 3) described in mark
Method is marked for iTRAQ labelling kits.
Preferably, in screening technique of the invention, the step 4) in proteome analysis software be Proteome
Discoverer；The proteomic image retrieval software is mascot softwares.
Present invention also offers the mammalian blood serum differential protein system of abnormal mucus cross-examination (syndrome) of above-mentioned dimension doctor, it is
Mechanism Study, drug screening, conversion and the study on prevention for further carrying out abnormal mucus cross-examination and its relevant disease have established base
Plinth, there is provided foundation.
From the foregoing, it will be observed that the advantage of the invention is that：
1st, there is no the concept of syndrome in modern medicine, it is conventional that differential protein is found between disease and Normal group, and originally
Patent inventor, is gained knowledge based on the understanding to syndrome and disease with abundant Uygur medicine, is combined with the differentiation of disease with dialectical
Mode, by Analogy, common albumen is found between the differential protein of ZM and N groups and the differential protein of BM and N groups, sieve
The serum candidate protein markers thing of abnormal mucus cross-examination (syndrome) is found, and by checking, has established abnormal mucus cross-examination (card
Wait) mammalian blood serum protein marker, and the protein markers objects system then more accurately reflects annotation of the dimension doctor to syndrome,
Embody the essence of syndrome.
2nd, the invention provides 37 kinds of mammalian blood serum differential proteins of abnormal mucus cross-examination (syndrome) of dimension doctor, wherein on
Up to 28 kinds, (fold differences are up-regulated expression more than 1.2 times, or fold differences are less than 0.8 to lower 9 kinds of expressing protein for mileometer adjustment
Expression is lowered, p value shows its differential protein in serum in model animal such as rat less than or equal to 0.05) by embodiment
Expression is raised or lowered, shows effect of these differential proteins in the abnormal mucus cross-examination of rat and its relevant disease, most
The biomarker of abnormal mucus cross-examination (syndrome) is determined eventually, can carry out basic research, medicine for the biomarker
Screening, conversion, and for abnormal mucus cross-examination (syndrome) relevant disease of dimension doctor of preventing and treating mammal particularly people provides theory
Basis and practical basis.
Brief description of the drawings
Fig. 1 is the modeling flow chart of Normal group, abnormal mucus cross-examination marquis group and disease group rat model；
Fig. 2 is the screening process figure of the mammalian blood serum differential protein system of abnormal mucus cross-examination (syndrome) of dimension doctor；
Fig. 3 is the SDS-PAGE spectrum of the integrity detection of rat blood serum differential protein；
Fig. 4 is 8 kinds of ELISA detection figures of albumen of any selection in 37 kinds of candidate's differential proteins.
Specific embodiment
In embodiments of the present invention, experimental animal is：30 (bodies of sexual maturing period SD male rat with normal sexuality
Weight 200g or so), female rats 15 (body weight 180g or so) are provided by Xinjiang Medicine University's Experimental Animal Center；
In embodiments of the present invention, medicine and reagent：Raw property feed is by Xinjiang Medicine University's Experimental Animal Center by preceding
The requirement used in phase research is prepared；The medicinal material such as spinach reality and coriandri,fructus is purchased from Uygur medicine hospital of Xinjiang Uygur Autonomous Regions
Herbal medicine room；Yellow Jackets；Physiological saline；Apomorphine (90 μ g/kg)；Estradiol；Corpus luteum；Predominantly detect reagent：ELISA is tried
Agent box, BCA protein quantification kits etc..
In embodiments of the present invention, term is following meanings：
Term APO:Apomorphine (Apomorphine)；
Term iTRAQ：Isotope marks are relative and absolute quantitation (isobaric tags for relative and
absolute quantitation)
Term HPLC:High performance liquid chromatography (High Performance Liquid Chromatography)
Term SDS：Lauryl sodium sulfate (sodium dodecyl sulfate, sodium salt)
Biological Characterization is observed：
Qualitative index is observed：The qualitative index is observed skin and hair and observes in the sunlight, and emotional reactions are with a cage
Each rat is observed 30 minutes based on reacting to each other, and behavior state is based on activity interior during this, 30 points of observation under quiet environment
Clock, excitement degree with press from both sides tail test when reaction based on, tongue picture tongue fur is observed, recorded and takes pictures in the sunlight, diet water state
With the same time feeding observation diet water state of each group, when urine was just urinated with each group rat same day based on color, state of defecating
Based on form of being defecated during bowel movement positive with the same day.
Quantitative target is fixed on 20 daily to avoid circadian influence:00-22:00 collects every data, wherein body
Weight：Weigh in required time and record daily, with every cage TBW divided by the cage rat number of elements, draw average weight, and press
Formula is stated to draw each cage body anharmonic ratio value of model group and record, body weight ratio=model group average weight-model group original body mass/just
Often organize average weight-normal group original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food surplus,
The corresponding dietary amount of 100g body weight is drawn according to following formula and record；Amount of drinking water：Daily 20:00 feedwater 500ml, next day
20:00 amount surplus water, calculates the corresponding amount of drinking water of 100g body weight and records；Urine volume：First day record of modeling is given well
Dry dunnage weight, the weight of the wet bedding and padding containing urine and stool is claimed within second day in required time and is recorded, then in constant temperature
Weighed again after drying in 2 hours at 100 DEG C of air dry oven, calculate each group urine volume；Stool amount：Calculate 100g body weight corresponding
Stool is measured and records.
APO erective tests or APO experimental techniques are
With reference to the method for Heaton etc., experiment rat is placed in 10~15min in inspection box and adapts to environment, kept quite,
Light is dimmed, (0.5mg/kg vitamin Cs are in middle dissolving Apo in rat neck injection of skin apomorphine (APO) 90 μ g/kg
After coffee, mixed with 5mL/kg physiological saline), observed at once after injection, during timer, observe and record rat in 1800s
Erect incubation period and erection number of times.The standard of telotism：Rat crouches position in crouching during erection, and phallosome end is stretched out, bows and lick
Lick glans penis.Erection incubation period is the time erected to first time after injecting.Erection number of times is rat appearance in 1800s after injection
The number of times of telotism.If having no erection in 1800s, the rat erection number of times is designated as 0, and incubation period of erecing is designated as 1800s.
Mating test method：
Male mouse and raettin (having carried out oestrus preparation) are respectively placed in and environment is adapted in different inspection boxes, 10~15 points
Female-male proportion 1 is pressed after clock:2 are put into same inspection box, and the climb back of the body, insertion and the ejaculation of male rat are hidden in observation 1800s
Phase and climb the back of the body, insertion and number of times.If male rat does not carry out climbing the back of the body in 1800s, the rat climb the back of the body incubation period be designated as
1800s, climbs back of the body number of times and is designated as 0.Insertion is with ejaculation behavior similarly.
In embodiments of the present invention, Normal group (N), abnormal lymphatic temperament syndrome group (ZM) and abnormal mucus are built first
Matter disease group (BM) rat.In embodiments of the present invention, the acquisition methods to the N groups, ZM groups and BM group rats are not special
Limitation, using the technical scheme for setting up abnormal lymphatic temperament syndrome animal model well known to those skilled in the art；At this
In inventive embodiments, following steps are preferably specifically included：
Selection sexual maturing period raettin, raises under castration Post operation normal condition, and the raettin is entered in oestrus with male mouse
Row mating test and APO experiment tests, obtain the male mouse of normal sexual function.The present invention is tested to the mating test and APO
Time sequencing be not particularly limited.The mating test and the object of APO experiments are that the Normal group and modeling group are big
Mouse.
In embodiments of the present invention, the specific steps of the mating are preferably the male mouse and raettin point that will carry out oestrus
It is not placed in and environment is adapted in different inspection boxes, female-male proportion 1 is pressed after 10~15min:2 are put into same inspection box, observation
In 1800s male rat climb the back of the body, insertion and ejaculation latency and climb the back of the body, insert and number of times, if male rat is not in 1800s
Carry out climbing the back of the body, then the rat climb the back of the body incubation period be designated as 1800s, climb the back of the body number of times be designated as 0；Insertion is with ejaculation behavior similarly.
Male mouse with normal mating ability is included Normal group and modeling by the present invention according to the result of the mating
Group, to being eliminated without normal mating ability hero mouse.
In embodiments of the present invention, APO experiment specific steps be preferably experimental rat is placed in 10 in inspection box~
15min adapts to environment, keeps quite, and dims light, in the μ g/kg apomorphine APO of rat neck injection of skin 90, is stood after injection
Observation is carved, during timer, erection incubation period and the erection number of times of rat in 1800s is observed and record；The standard of the erection
For rat crouches position in crouching when erecing, phallosome end is stretched out, bows to lick and lick glans penis；The erection incubation period for after injection to the
The time once erected；The erection number of times is that the number of times of telotism occurs in rat in 1800s after injecting；If in 1800s not
See erection, then the rat erection number of times is designated as 0, incubation period of erecing is designated as 1800s.
The result tested according to the APO in the embodiment of the present invention includes just the male mouse with normal penile erection function
Normal control group and modeling group, to being eliminated without normal penile erection function hero mouse.
In embodiments of the present invention, the 90 μ g/kg apomorphine solution preferably includes 90 μ g apomorphines and is dissolved in
In 0.5mg/kg vitamin Cs and physiological saline, adjustment volume is 5ml/kg.
In embodiments of the present invention, the Normal group is raised in conventional environment, the environment temperature of feeding for 18~
22 DEG C, the relative humidity of the feeding environment is 40~60%；The environment temperature of the feeding of modeling group is 8~12 DEG C, described to feed
Eat the relative humidity 70~80% of environment；Normal group is normal diet, and modeling group feeding feed is raw property feed.
In embodiments of the present invention, the Biological Characterization observation refers to qualitative and quantitative observation and record.Qualitative index
Observation：The qualitative index is observed skin and hair and observes in the sunlight, emotional reactions with each rat in a cage react to each other for
Subjectivity is examined 30 minutes, and behavior state is observed 30 minutes based on interior activity during this, under quiet environment, and excitement degree is pressing from both sides tail
Based on reaction during experiment, tongue picture tongue fur is observed, recorded and takes pictures in the sunlight, and diet water state is fed with each group same time
Observation diet water state is big during stool positive with the same day bowel movement of state when urine was just urinated with each group rat same day based on color
Just based on form；Quantitative target is fixed on 20 daily to avoid circadian influence:00-22:00 collects every data, its
Middle body weight：Weigh in required time and record daily, with every cage TBW divided by the cage rat number of elements, draw average weight, and
Each cage body anharmonic ratio value of model group is drawn by following formula and record, the first initial body of body weight ratio=model group average weight-model group
Weight/normal group average weight-normal group original body mass；Dietary amount：Daily 20:00 addition feed 300g, next day 20:00 claims food
Surplus, draws the corresponding dietary amount of 100g body weight and records according to following formula；Amount of drinking water：Daily 20:00 feedwater 500ml,
Next day 20:00 amount surplus water, calculates the corresponding amount of drinking water of 100g body weight and records；Urine volume：Modeling is recorded for first day
The good dry dunnage weight given, claims the weight of the wet bedding and padding containing urine and stool for second day and records in required time, Ran Hou
Weighed again after drying in 2 hours at 100 DEG C of constant temperature blast drying oven, calculate each group urine volume；Stool amount：Calculate 100g body weight relative
The stool for answering is measured and records.
It is of the invention by having for obtaining after obtaining the male mouse of normal sexual function (normal mating ability and penile erectile function)
The male mouse of normal performance is divided into Normal group and modeling group, and the Normal group is raised under conventional environment, the modeling
Group is raised with raw property forage feed in raw environment, and the Biological Characterization for observing and recording normal group and modeling group is seen.Bag
Include qualitative index and quantitative target.Qualitative index observes all groups of skin and hairs of rat, emotional reactions, excitement degree, sleeps
State, drinking-water state, urine volume property, stool state and tongue picture tongue fur.Quantitative target is to record all groups of body weight of rat, diet
Amount, amount of drinking water, urine volume and stool amount.Find that rat skin hair is slightly dim during modeling, it is matt；Burnout, curls up sleeping few
It is dynamic；Sensitive to stimulating, happiness flocks together, and burnout is drowsiness；Nothing strives water phenomenon；Urine volume is clear, and color is light, measures many；It is half congealed when soft during stool；Tongue fur
Secretly, white and greasy fur.And as the extension of modeling time, modeling group body weight increase are slow, dietary amount increases, and amount of drinking water is reduced, greatly
Urine amount significantly increases, and above-mentioned Biological Characterization meets the clinical card Hou Tedian of dimension doctor, big so as to obtain abnormal mucus cross-examination marquis
Mouse model, and the model has good science, reliability and stability.By APO and mating test from syndrome model group
In filter out abnormal lymphatic temperament type impotence disease binding model (Erectile Dysfunction and amixia ability), so as to obtain different
Normal lymphatic temperament disease animal model.The present invention to the syndrome into mould rate up to 100%, Syndrome model into mould rate reach 50% with
On, the cycle of Syndrome model is preferably 22~25 weeks.
In embodiments of the present invention, the rat quantity of the N groups is preferably 10.
In embodiments of the present invention, the method raised under conventional environment conventional environment raising side ibid in step
Method.In the present invention, the feeding volume of the N groups is preferably the normal diet of daily 300g.The normal diet is to include 500ml water
With 80g bedding and padding.The present invention is not particularly limited to the bedding and padding, using bedding and padding well-known to those skilled in the art.
In the present invention, the quantity of the rat of described experimental group is preferably 20.
In the present invention, described raw property feed mixture real for spinach and coriandri,fructus and conventional feed.In the present invention
In embodiment, the raw property feed is preferably：Include 100~200g coriandri,fructuses and 100~200g spinach per 1kg conventional feeds
Dish reality, more preferably includes 150g coriandri,fructuses and 150g spinach realities per 1kg conventional feeds.The present invention is not special to conventional feed
Limitation, using the feed for raising rat well-known to those skilled in the art.The present invention is to the dish reality and coriander
Real source is also not particularly limited, using dish well-known to those skilled in the art reality and coriandri,fructus.Of the invention real
Apply in example, the real source with coriandri,fructus of the dish is purchased from Uygur medicine hospital of Xinjiang Uygur Autonomous Regions herbal medicine room.
In the present invention, the feeding volume of described raw property feed is preferably daily 300g.
In the present invention, the temperature of the raw environment is preferably 8~12 DEG C, and the humidity of the raw environment is preferably 70~
80%.The present invention is not particularly limited to the facility for providing the raw environment, is carried using well-known to those skilled in the art
For the facility of raw environment.In the present invention in example, there is provided the facility of raw environment is growth cabinet.
In the present invention, the time of the raw property forage feed was preferably in Beijing time 09:00 to 21:00 is put into, its
Its time is positioned over raising in control rats identical feeding environment.
In the present invention, the method and the result of the APO experiments and mating test are tested and mating test with above-mentioned APO
Method and the result.
The present invention is anaesthetized to preferred pair rat before the blood sampling.The present invention does not have special limit to the method for the anesthesia
System, using anesthesia well-known to those skilled in the art.In the embodiment of the present invention, described anesthesia is abdominal cavity
Injection.Anesthetic preferably yellow Jackets to being used in the anaesthesia process of the invention.Described yellow Jackets
Source is purchased from and medicine company Co., Ltd of field Uygur.
In embodiments of the present invention, the specific method of the collection peripheral blood is preferably postanesthetic rat is quick from abdomen
Sustainer takes blood.
In embodiments of the present invention, the method for separating serum is preferably and for fresh rat whole blood to put 4 DEG C of environment immediately,
With the 5~10min of centrifugation of 3000~3500rpm after 30~40min, the supernatant that will be obtained under the conditions of 4 DEG C, with 3000
The speed of~3500rpm is centrifuged again, obtains serum, and the serum for obtaining will be finally centrifuged, and to be positioned over -80 DEG C of ultra low temperature freezers standby
With.4 DEG C of described environment are preferably 4 DEG C of refrigerators.
After obtaining serum, the embodiment of the present invention is detected to extracting protein in serum, obtains concentration known haemocyanin
Matter.
In the embodiment of the present invention, sample mixing is preferably carried out respectively according to group before albumen is extracted, obtain three groups of blood serum samples.
The embodiment of the present invention is not particularly limited to the method for extracting proteins, ripe using those skilled in the art institute
The method for extracting proteins known.In the embodiment of the present invention, the method for extracting proteins is TCA- acetone precipitations.
In the embodiment of the present invention, the detection preferably includes the detection to protein content and the inspection to protein integrity
Survey.In the embodiment of the present invention, the detection method to protein content is preferably Bradford methods, described complete to protein
The detection method of property is preferably SDS-PAGE methods.
After obtaining the protein of concentration known, the embodiment of the present invention is marked to the protein with iTRAQ labelling kits,
The protein for being marked, it is purified, obtain peptide fragment after purification.
In embodiments of the present invention, the protein of the concentration known that will preferably be obtained before the mark is digested.This
Inventive embodiments are not particularly limited to the method for the digestion, using protein digestibility side well-known to those skilled in the art
Method.Trypsinization is used in digestion described in the embodiment of the present invention.
The embodiment of the present invention is originated without special limitation to the iTRAQ labelling kits, using art technology
The commercial goods of iTRAQ labelling kits known to personnel.The kit that the embodiment of the present invention is used is
Reagent-8Plex Multiplex Kit (Applied Biosystem) 8 mark iTRAQ reagents, including 8 kinds of equivalent
Isotope reagent (reporter group quality 113-121, not including that 120), can be marked to peptide fragment after protein digestion, so as to tie
The methods such as tandem mass spectrum are closed, accurate identification can be carried out to peptide fragment and is quantified.
In embodiments of the present invention, it is described to purify the purifying that peptide fragment is preferably carried out using HPLC.The instrument for being purified
Device uses model (inventor's offer) Agilent conventional liquid phase.SCX strong cat ion exchange columns point are used in the embodiment of the present invention
From, in pH=3 phosphate buffers, using 20%-30% gradient eluent (10mM KH2PO4,2M KCl, 25%ACN,
PH=3), elution requirement and gradient reference instrument and consumptive material concrete operations explanation, elute postdigestive peptide fragment.After wash-out
Jump section and use C18 reverse-phase chromatography desalting processings.
After obtaining purifying peptide fragment, the embodiment of the present invention obtains mass spectrum total figure to the peptide fragment of the purifying after Mass Spectrometer Method,
The finger-print is screened in PD softwares, in embodiments of the present invention, the instrument of the Mass Spectrometer Method uses model
The mass spectrograph of Thermo fisher Q-Exactive.
In embodiments of the present invention, the version of the PD softwares is Proteome Discoverer 1.3, purchased from the U.S.
Thermo companies.
In the embodiment of the present invention, the parameter of mass spectrum screening is as follows：
Spectrogram after PD extractions, is searched for mascot, Search Results is obtained, using PD softwares according to mascot Search Results
Quantitative analysis is carried out with the spectrogram after first step screening.
Retrieval by header parameter is as follows：
Note：Fixed modification are to fix modification；Carbamidomethyl (C) be reductive alkylation treatment after,
The urea that cysteine is produced methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；When Gln → Pyro-Glu (N-term Q) refers to that peptide fragment N-terminal has glutamine, recirculation may be produced, become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in diverse location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MS tol are the precision of second order mses.Max missed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is the used enzyme class of experiment.Database is retrieval
The database for using.Time files compressed are the Database time.Number of sequences are data
Sequence number in storehouse.
Quantitative search argument is as follows：
| Parameter name | Experiment option |
| Protein ratio Type | median |
| Minimum peptides | 1 |
| Normalisation method | median |
| P-value | <0.05 |
Note:Protein ratio Type：For quantitative peptide fragment obtaining value method, median refers to taking the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choosing in one group of sample all can the median of Quantitative Western correct.P-value albumen is credible
Degree assessment, albumen confidence level is represented less than 0.05 more than 95%.
According to the spectrogram quantitative analysis after the Search Results and screening, the analyze data for obtaining is in rat database
(Uniprot-2014-rattus) retrieval in, it is 318 kinds, FDR to obtain total serum protein<1%, specifically see the table below.
| Parameter name | Experiment option |
| All spectra | 342077 |
| Matched spectra | 31609 |
| Peptide | 2758 |
| Protein Group | 318 |
Note：All spectra are whole spectrogram numbers；Matched spectra are the spectrogram number for matching；Peptide
It is peptide fragment species number；Protein Group are the protein total for matching；FDR is false positive rate, and the present embodiment uses vacation
Positive rate carrys out filter result less than 1%.
According to software operational procedure, using identical parameters and method, evaluation and screening obtains 318 kinds of rat blood serum protein.
After three groups of (N, the ZM and BM) serum proteins for obtaining, the embodiment of the present invention carries out dimension doctor to the three histones matter
The screening of the mammalian blood serum differential protein of abnormal mucus cross-examination (syndrome).
The embodiment of the present invention is specifically to the screening technique of abnormal mucus cross-examination (syndrome) the group differential protein：
The BM groups obtain the serum differential protein of BM/N compared with the serum proteins of N groups；By ZM groups and N groups
Serum proteins compare, obtain the serum differential protein of ZM/N；The differential protein for counting the BM/N and ZM/N is total to
With the type and quantity of albumen, the serum differential protein of abnormal mucus cross-examination (syndrome) group is obtained.
Technical scheme is further illustrated below by way of specific embodiment.
Embodiment 1
1st, modeling and packet
From sexual maturing period raettin 15, raised under castration Post operation normal condition, mating test is standby.By male rat
Through with oestrus raettin carry out mating test and with reference to APO erect experiment confirm its there is normal sexual function (without normal sexual function
Person eliminates), take 30 and only enter experiment, 10 rats are therefrom randomly selected again is set to Normal group, raise under conventional environment,
18~22 DEG C of temperature, temperature is fed, envionmental humidity 40~60%, remainings 20 for modeling group, with raw property forage feed, is pressed
Uygur's medical science carries out modeling to the view of environment using growth cabinet, sets temperature：8~12 DEG C, humidity is 70~80%,
Day alternates with night within 12/12 hour raises, and other time is positioned over raising in normal rats identical feeding environment.When modeling group mouse
There is skin and hair slightly dim, it is matt；Burnout, curls up sleeping few dynamic；Sensitive to stimulating, happiness flocks together, and burnout is drowsiness；Show without water is striven
As；Urine volume is clear, and color is light, measures many；It is half congealed when soft during stool；Tongue fur is dark, white and greasy fur.And with the extension of modeling time, and compare
Group is compared, and modeling group body weight increase is slow, and dietary amount increases, and amount of drinking water is reduced, and stool and urine amount significantly increases, above-mentioned biology table
Levy and meet the clinical card Hou Tedian of dimension doctor, so as to obtain abnormal mucus cross-examination marquis's rat model.By APO and mating test from card
Filtered out in time model group without penile erectile function and mating ability male rat, obtain abnormal lymphatic temperament type impotence disease animal
Model.The present invention reaches 50% to the mould rate, and the cycle of above Syndrome model is preferably 22~25 weeks.Opened within first week from modeling
Beginning records weekly the Biological Characterization of rat, and abnormal mucus cross-examination (syndrome) model is determined into mould situation according to Biological Characterization,
The present invention reaches for 100% preferably 8-10 weeks cycle to the syndrome mould rate.And on this basis, monthly carry out an APO
Experiment and mating test, APO experiments judge penile erectile function by counting the number of times of erection and erecing incubation period, and mating is real
Test to be climbed by statistics and rat is judged by incubation period/number of times, insertion incubation period/number of times and ejaculation latency/index of number of times this several
Mating ability, reached after 50% into mould rate when there is Erectile Dysfunction and mating ability obstacle, i.e. impotence disease,
Tested and mating test screening impotence disease rat by APO from abnormal mucus cross-examination (syndrome) model group, and include BM groups,
Remaining is ZM groups.
2nd, peripheral blood collection, tissue sample materials and preservation：After yellow Jackets intraperitoneal anesthesia, quickly taken from abdominal aorta
Blood, 4 DEG C of refrigerators are put by fresh rat whole blood immediately, with the centrifugation 5min of 3000rpm after 30 minutes, Aspirate supernatant, then
Put high speed low temperature centrifugal machine, under the conditions of 4 DEG C, with the centrifugation of 3000rpm again Aspirate supernatant after, -80 DEG C of separating device is low
Temperature refrigerator is saved backup.
3rd, the preparation of protein group sample
Protein extracting method uses TCA- ice acetone extractions.
4th, iTRAQ marks and HPLC
(1) iTRAQ marks：Each group rat blood serum is taken, after group sample mixing, each group protein is extracted, egg is detected by SDS
White integrality and albumen is quantified using Bradford methods.
Protein through Trypsin digest after with ITRAQ labelling kits ( Reagent-8Plex
Multiplex Kit (Applied Biosystem)) it is marked, carry out peptide fragment by HPLC (Agilent conventional liquid phase)
Purifying；The concentration of three groups of serum proteins of rat is as shown in table 1.From accompanying drawing 3 from the point of view of PAGE gel electrophoresis picture, carry
The three histone matter integralities for taking preferably, can be used for downstream experiment.
The Bradford methods of table 1 carry out quantitative result to rat blood serum albumen
| Sample ID | N | BM | ZM |
| Concentration (μ g/ μ L) | 0.26 | 0.39 | 0.42 |
After quantitative, by each group protein digestibility after, use ITRAQ labelling kits.Liquid phase peptide fragment after purification is through mass spectrum
Finger-print is obtained after (Thermo fisher Q-Exactive mass spectrographs) detection, mass spectrogram is input to PD (Proteome
Discoverer 1.3, thermo) after software, software first carries out Screening and Identification, species, title, after PD is extracted to mass spectrogram
Spectrogram scanned for mascot, search terminate after, PD softwares according to mascot Search Results and the first step screening after spectrum
Figure carries out quantitative analysis, finally retrieves rat database, and finally identifying rat blood serum protein amounts to 318 kinds for acquisition, specifically
Parameter is as follows.
In the embodiment of the present invention, the parameter of mass spectrum screening is as follows：
| Parent ion mass range | 350Da-6000Da |
| Minimum peak number in second order mses figure | 10 |
| Signal to noise ratio S/N thresholdings | 1.5 |
Spectrogram after PD extractions, is searched for mascot, Search Results is obtained, using PD softwares according to mascot Search Results
Quantitative analysis is carried out with the spectrogram after first step screening.
Retrieval by header parameter is as follows：
Note：Fixed modification are to fix modification；Carbamidomethyl (C) be reductive alkylation treatment after,
The urea that cysteine is produced methylates.Variable modification are variable modifications；Oxidation (M) is methionine
Oxidation；When Gln → Pyro-Glu (N-term Q) refers to that peptide fragment N-terminal has glutamine, recirculation may be produced, become
Glutamic acid；ITRAQ 8plex (K), iTRAQ 8plex (Y), iTRAQ 8plex (N-term) are iTRAQ reagents in diverse location
Combination.Peptide tol are the precision of first mass spectrometric.MS/MS tol are the precision of second order mses.Max missed
Cleavages is maximum allowable when being enzymolysis to leak the number cut.Enzyme is the used enzyme class of experiment.Database is retrieval
The database for using.Time files compressed are the Database time.Number of sequences are data
Sequence number in storehouse.
Quantitative search argument is as follows：
| Parameter name | Experiment option |
| Protein ratio Type | median |
| Minimum peptides | 1 |
| Normalisation method | median |
| P-value | <0.05 |
Note:Protein ratio Type：For quantitative peptide fragment obtaining value method, median refers to taking the side of middle position
Method.Minimum peptides：For quantitative minimum unique peptide number Normalisation method：Correction
Method, median refer to choosing in one group of sample all can the median of Quantitative Western correct.P-value albumen is credible
Degree assessment, albumen confidence level is represented less than 0.05 more than 95%.
5th, the screening technique of abnormal mucus cross-examination (syndrome) the rat model serum differential protein of dimension doctor
(1) screening technique of abnormal mucus cross-examination (syndrome) the rat model serum differential protein of dimension doctor
BM groups are compared with the serum proteins of N groups (fold differences are taken more than 1.2 times, or fold differences are less than
0.8, p value obtains the serum differential protein of BM/N using above-mentioned comparison less than or equal to 0.05)；By ZM groups and the blood of N groups
Albumin matter compares and (takes fold differences more than 1.2 times, or fold differences are less than 0.8, p value is used less than or equal to 0.05)
Above-mentioned software obtains the serum differential protein of ZM/N；Count the species of the common albumen of the differential protein of the BM/N and ZM/N
And quantity, the serum differential protein for obtaining abnormal mucus cross-examination (syndrome) amounts to 37 kinds, as shown in table 2 below, wherein upper mileometer adjustment
Up to protein 28 kind (fold differences are more than 1.2 times), lower 9 kinds of expressing protein (fold differences are less than 0.8)；Therefore the present invention is obtained
Abnormal mucus cross-examination (syndrome) the serum mark albumen system of dimension doctor, it is as shown in table 3 below：
Abnormal mucus cross-examination (syndrome) the rat model serum differential protein of table 2
Abnormal mucus cross-examination (syndrome) the serum mark albumen system of table 3
6th, the ELISA detections of abnormal mucus cross-examination (syndrome) rat model serum candidate's differential protein
8 kinds of serum candidate's differential proteins of random selection, enter to abnormal mucus cross-examination (syndrome) rat model group with control group
Row ELISA is detected, to verify whether serum candidate differential protein of the present invention can be as the mark of abnormal mucus cross-examination (syndrome)
Will thing：
That is random selection detects No. 10 albumen P48199 (c reactive proteins)；No. 12 albumen P26644 (β -2- glycoprotein
1)；No. 14 albumen P20059 (Hemopexin)；No. 21 albumen P06238 (α -2- macroglobulin)；No. 24 albumen
P04916 (retinol-binding proteins)；No. 25 albumen P02767 (transthyretin)；No. 26 albumen P02764
(α -1- acidoglycoproteins) and No. 28 albumen P01015 (proangiotensin).
Double crush syndrome kit (rat c reactive protein (CRP) double antibodies sandwich kit is used respectively：Yi Lairuite
Bio tech ltd；Rat beta2 Glycoprotein 1 (APOH) double antibodies sandwich kit：The excellent Er Sheng commerce and trade Co., Ltd in Wuhan；Greatly
Mouse Hemopexin (HPx) double antibodies sandwich kit：Kit：Abcam Ai Bo resist (Shanghai) trade Co., Ltd；Rat
Alpha2 Macroglobulin (α 2MG) double antibodies sandwich kit：Kit：The excellent Er Sheng commerce and trade Co., Ltd in Wuhan；Rat retinol combination egg
White 4 (RBP4) double antibodies sandwich kit：Yi Lairuite bio tech ltd；Rat thyroid element transporter (TTR) is double
Resist sandwich kit：Kit：Abnova Yanuofa Biotechnology Co., Ltd.；The acidoglycoproteins of rat α 1 (α 1AG) dual anti-folder
Heart kit：Abcam Ai Bo resist (Shanghai) trade Co., Ltd；Rat angiotensin original (AGT) double antibodies sandwich kit：It is military
The excellent Er Sheng commerce and trade Co., Ltd of the Chinese；ELSA inspections are carried out to Normal group and syndrome group rat blood serum according to kit specification
Survey.
Measurement result as shown in Fig. 4 A-H, as shown in Figure 4, each abnormal mucus cross-examination (syndrome) rat model serum candidate
Protein content is above control group, and this is consistent with iTRAQ results, illustrates that the inventive method acquisition protein marker can be as different
The biomarker of normal mucus cross-examination (syndrome) serum, concrete outcome see the table below：
Protein content in each group rat blood serum ( s)
The * P compared with N groups<0.05
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of dimension cures abnormal mucus cross-examination (syndrome) mammalian blood serum differential protein system, it is characterised in that the albumen
System includes following albumen：
2. mammalian blood serum differential protein system according to claim 1, it is characterised in that the albumen system comes from
Model animal；The model animal is rat.
3. a kind of mammalian blood serum differential protein of abnormal mucus cross-examination (syndrome) of dimension doctor, it is characterised in that the albumen is
Any one or a few in following albumen system：
4. mammalian blood serum differential protein system according to claim 3, it is characterised in that the albumen system comes from
Model animal；The model animal is rat.
5. a kind of dimension cures the mammalian blood serum differential protein System For Screening method of abnormal mucus cross-examination (syndrome), and its feature exists
In comprising the following steps：
1) Normal group (N groups), abnormal lymphatic temperament syndrome group (ZM groups), abnormal lymphatic temperament disease group (BM groups) lactation is set up to move
Thing；
2) step 1 is gathered) mammalian whole blood that obtains, the serum of mammal is obtained after separation；
3) from the step 2) protein is extracted in the serum that obtains；
4) by the step 3) in purify after the protein labeling that obtains, obtain peptide fragment after purification；
5) by the step 4) peptide fragment after purification that obtains obtains finger-print after Mass Spectrometer Method, the finger after screening
Line collection of illustrative plates is retrieved using proteome analysis software, is quantified according to the spectrogram after the Search Results and screening for obtaining
Analysis, the analyze data that will be obtained retrieves software retrieval in the mammalian proteins database using proteomic image,
Obtain the serum proteins of the N groups, ZM groups and BM group mammals；
6) by the step 5) in
The BM groups obtain the serum differential protein of BM/N compared with the serum proteins of N groups；By ZM groups and the blood of N groups
Albumin matter compares, and obtains the serum differential protein of ZM/N；Count the common egg of the differential protein of the BM/N and ZM/N
White type and quantity, obtain the serum differential protein of abnormal mucus cross-examination (syndrome).
6. screening technique according to claim 5, it is characterised in that the mammal is rat.
7. screening technique according to claim 5, it is characterised in that the step 1) in Normal group in conventional environment
Middle raising, the environment temperature of feeding is 18~22 DEG C, and the relative humidity of the feeding environment is 40~60%；The feeding of modeling group
Environment temperature be 8~12 DEG C, the relative humidity 70~80% of the feeding environment；The step 1) in Normal group for general
Logical feed, modeling group feeding feed is raw property feed.
8. screening technique according to claim 5, it is characterised in that the step 3) described in labeling method be iTRAQ
Labelling kit is marked.
9. screening technique according to claim 5, it is characterised in that the step 4) in proteome analysis software be
Proteome Discoverer；The proteomic image retrieval software is mascot softwares.
10. proteosome according to claim 1 and 2 ties up to grinding for mammal exception mucus cross-examination (syndrome) relevant disease
Study carefully, drug screening, conversion and preventing and treating in application.
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| CN105467022A (en) * | 2014-09-05 | 2016-04-06 | 天士力制药集团股份有限公司 | Analysis method for neuron injury related differentially expressed protein |
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