CN106749618A - A kind of new people NOTCH1 NICD proteantigens, antibody and its preparation method and application - Google Patents
A kind of new people NOTCH1 NICD proteantigens, antibody and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to application and the preparation method of above-mentioned antibody that a species specificity is directed to people's NOTCH1 NICD albumen Tyr2145 site phosphorylations Antigenic Peptide, antibody, above-mentioned Antigenic Peptide and antibody.The antibody is by active amino acid sequence such as SEQ ID NO：Shown in 1, and Tyr amino acid therein is phosphorylated what is prepared as the immune animal of Antigenic Peptide.The present invention is purified when the antibody is prepared using the affine isolation technics antagonistic Serum of affinity purification.The preparation method has the advantages such as short preparation period, the potency of antibody are high, the rate of recovery is good, the antibody of acquisition has high specific, high-affinity, the phospho-AB that the present invention is provided helps to disclose mechanism of action of the phosphorylation modification of NOTCH1 NICD albumen during the disease developments such as tumour, has extensive potential applicability in clinical practice at aspects such as diagnosing tumor, treatment and prognosis judgements.
Description
Technical field
The present invention relates to antibody and its preparing technical field, and in particular to a species specificity is directed to people's NOTCH1 NICD eggs
White Tyr2145 site phosphorylation antibody and its preparation method and application.
Background technology
NOTCH signals are a signal paths being highly conserved during evolution, be widely present in invertebrate and
Among multiple species of vertebrate, it is one of primary signal pathways of directly contact between mediated cell and cell, regulation and control
The Apoptosis of multicellular organism, propagation and break up.From fruit bat to the mankind, the hereditary feature of NOTCH signal pathways is highly protected
Keep, and its regulation process is particularly complicated in mammal.
NOTCH acceptors, NOTCH parts and intracellular effector molecule CSL collectively constitute NOTCH signal paths.NOTCH acceptors
It is single transmembrane albumen, its molecule is made up of extracellular region, transmembrane region and intracellular region (NICD), with well-conserved.Current people
4 kinds of NOTCH acceptor genes (NOTCH1, NOTCH 2, NOTCH 3, NOTCH 4) are had found in class, various Main Differences exist
Number and the length of intracellular region that EGF samples are repeated.NOTCH parts are also the single transmembrane albumen of cell surface expression, it has been found that
The NOTCH parts of people have Jagged 1, Jagged 2, Delta 1, Delta 3, Delta 4.
People's NOTCH1 signals are to be interacted to cause with part by protein receptor, and NOTCH1 protein receptors are released through 3 hydrolysis
Release intracellular section albumen (NICD), NOTCH1 NICD are transferred in nucleus, by with CBF1/Su (H)/LAG1 (CSL) phase interaction
With compound is formed, the latter is converted into activating transcription factor by transcription inhibitory factor, adjust propagation, apoptosis and the differentiation of cell
And development and the function of many organs are influenceed, the abnormal change of its signal path is more with tumour, hematological system, cardiovascular system etc.
Plant disease related.Research shows：In most of malignant tumours, NOTCH1 signals are mainly tumor promotion, the NOTCH1 of activation
Albumen can breed cell and vicious transformation is tumour, is such as sent out in cervical carcinoma, stomach cancer, kidney and various hematological system tumors
The unconventionality expression of existing NOTCH1, but discovery NOTCH1 is in the different phase of different tumours and tumour recently, its work for playing
With being also not quite similar.NOTCH1 signals have cancer suppressing action for example in SCCHN；Early stage cervical carcinoma,
NOTCH1 signals play tumor promotion, and late period then plays cancer suppressing action.Further, since NOTCH1 NICD albumen is Wnt, TGF β/BMP
With the important joint of a plurality of path such as Hedgehog, its exception may be by the influence participation tumour to other signal paths
Occur, develop, lapsing to and prognosis.
The posttranslational modifications such as phosphorylation play crucial regulation in the physiology and pathologic process that NOTCH signal paths are mediated
Effect.There are some researches show：The phosphorylation modification of NOTCH1 NICD albumen is the important regulatory mechanism of NOTCH signal paths, it
Can not only promote the activation of signal, what is more important it can degrade NOTCH1 NICD so as to end by starting ubiquitination
Stop signal, is the key of NOTCH1 signal path control accurates.The exception of phosphorylation modification, causes NOTCH1 NICD half-life period
Extension will cause the generation of leukaemia and other solid cancers.Tyr2145 sites are located in people's NOTCH1 amino acid sequences
It is a critical sites of NOTCH1 protein phosphorylations modification in NOTCH1 NICD, is lost in the activation or degraded of NOTCH1 signals
Played an important role in dynamic regulation living.
Antibody is the important tool of protein function research, has been widely used for the clinics such as the medicals diagnosis on disease such as tumour, treatment
In, the preparation of phospho-AB and application have turned into the focus that life science develops in the world, Mechanism Study in disease,
Played an important role in diagnosis and the discovery of therapy target.
The content of the invention
Phosphorylation the invention reside in the NICD albumen for solving to be played a significant role in NOTCH1 signal paths are regulated and controled is repaiied
Decorations, therefore, an object of the present invention is to provide a species specificity for people NOTCH1 NICD albumen Tyr2145 sites phosphoric acid
Change Antigenic Peptide.
People NOTCH1 NICD albumen Tyr2145 sites phosphoric acid is directed to it is a further object of the present invention to provide a species specificity
Change antibody.
It is a further object of the present invention to provide the application of above-mentioned Antigenic Peptide and antibody.
It is a further object of the present invention to provide the preparation method of above-mentioned antibody.
Realize that the technical scheme of above-mentioned purpose is as follows.
A kind of Antigenic Peptide of people NOTCH1 NICD albumen, its active amino acid sequence such as SEQ ID NO：Shown in 1, and its
In Tyr amino acid be phosphorylated.
Wherein in one embodiment, the C-terminal of the Antigenic Peptide of above-mentioned people NOTCH1 NICD albumen is connected with one and half Guangs
Propylhomoserin.
A kind of antibody of people NOTCH1 NICD albumen, it is immunized by the Antigenic Peptide of above-mentioned people NOTCH1 NICD albumen
What animal prepared.
The Antigenic Peptide and/or antibody of above-mentioned people's NOTCH1 NICD albumen are preparing the diagnosis for diseases such as tumours, are controlling
Application in treatment, the medicine of prognosis judgement or reagent.
A kind of diagnosis, treatment, medicine or reagent of prognosis judgement for tumour, it includes above-mentioned people NOTCH1
The Antigenic Peptide and/or antibody of NICD albumen.
The tumour is the tumour related to people's NOTCH1 signal paths, for example cervical carcinoma, stomach cancer, carcinoma of mouth, kidney and
Various hematological system tumors.
The preparation method of the antibody of above-mentioned people NOTCH1 NICD albumen, comprises the following steps：
(1) it is such as SEQ ID NO to obtain active amino acid：The Antigenic Peptide of the amino acid sequence shown in 1, on amino acid Tyr
It is added with phosphate group and C-terminal is connected with a cysteine；
(2) using the Antigenic Peptide and carrier protein hemocyanin (KLH) or bovine serum albumin(BSA) (BSA) of step (1) synthesis
It is coupled, immune animal simultaneously collects antiserum；
(3) antiserum that step (2) is collected is purified and is identified, obtained Tyr2145, people NOTCH1 NICD albumen
The antibody of point phosphorylation.
The step of immune animal described in above-mentioned steps (2), can include：The Antigenic Peptide idol synthesized with above-mentioned steps (1)
Connection hemocyanin and adjuvant combined immunization new zealand white rabbit；Immunization wayses are through relatively thin, the lax position of neck, skin of back
Both sides hypodermic injection, it is many that gluteus distinguishes the multiple locations such as intramuscular injection, the intracutaneous injection of waist both sides, footpad injection with huckle both sides
Point injection；Immune time includes triggering injection 1 time that 3~4 booster shots and last time use antigen direct injection.
The step of purifying described in above-mentioned steps (3) and identification, can include：The antiserum that above-mentioned steps (2) are collected
The potency of above-mentioned Antigenic Peptide is directed to ELISA measurings antiserum, it is (first affine pure using affinity purification-affine isolation technics
Change, then affine separation again) antagonistic Serum purified.Can determine that whether antibody after purification can with Dot blot experiments
Specific recognition phosphorylation NOTCH1 NICD albumen.
It is above-mentioned to include the step of purified using affinity purification-affine isolation technics antagonistic Serum：Use first
Phosphorylation synthetic peptide coupling albumen is coupled with agarose carries out affinity purification as chromatographic stuffing, obtains anti-for phosphorylation
All antibody of former epitope simultaneously remove the low sequence complexity epitope antibodies of low-affinity by changing pH and ion concentration；Then
Be coupled using non-phosphorylating synthetic peptide coupling carrier protein and agarose carried out as chromatographic stuffing it is affine separate, except dephosphorization
The non-phosphorylating epitope antibodies of non-specific binding in acidifying antibody.
Present invention screening and the high specific, the people NOTCH1 NICD albumen Tyr2145 sites of high-affinity that prepare
Phospho-AB, can be used for the table of Western blot experiment detections normal cell, tumour cell and the front and rear tumour cell for the treatment of
Up to difference, can be repaiied using technique study phosphorylations such as co-immunoprecipitation combination mass-spectrometric technique (IP-MS), GST-pull down
The Protein-protein interaction of the NOTCH1 NICD of decorations；Can be using chromosome co-immunoprecipitation sequencing (ChIP-seq) research phosphorus
The DNA controlling elements that the NOTCH1 NICD of modification interact are acidified, so as to illustrate that the phosphorylation modification of NOTCH albumen exists
Function and its mechanisms during the disease developments such as tumour provide unique instrument；Further, it is also possible to pass through SABC
(IHC) and the method such as ELISA detects the phosphorylation level of people's NOTCH1 NICD albumen, it is inquired into tumour, hematological system, the heart
The relation of the diseases such as vascular system, has extensive potential applicability in clinical practice at aspects such as medical diagnosis on disease, treatment and prognosis judgements.
Advantages of the present invention and beneficial effect：(1) high specific, high-affinity are prepared by the method for the invention
People's NOTCH1 NICD albumen Tyr2145 site phosphorylation antibody；(2) in long-term experiment, the present inventor is not intended to
Middle discovery, the affinity purification-affine separation method used in purifying Tyr2145 phospho-ABs of the present invention is than existing
Affine separation-the affinity purification for having antibody purification in technology to use has that short preparation period, the potency of antibody be high, the rate of recovery is good
Etc. advantage；(3) phospho-AB that the present invention is provided can study people's NOTCH1 NICD albumen specific sites in actual applications
Phosphorylation modification changes with particular biological event such as cellular stress, genome and epigenetic group, the cell cycle is abnormal etc.
Correlation, mechanism of action of the research NOTCH signals under cell physiological and pathologic condition；(4) phosphorylation that the present invention is provided resists
Body helps to disclose mechanism of action of the phosphorylation modification of NOTCH1 NICD albumen during the disease developments such as tumour,
The relation of its disease such as with tumour, hematological system, cardiovascular system is inquired into, the aspect such as judges in medical diagnosis on disease, treatment and prognosis
With extensive potential applicability in clinical practice.
Brief description of the drawings
Preparation people's NOTCH1 NICD phospho-AB Technology Roadmaps that Fig. 1 present invention is used.
Fig. 2 obtains people's NOTCH1 NICD albumen Tyr2145 phosphorylation sites using bioinformatics screening.
Fig. 3 NOTCH1 NICD antigenicity analysis results.
Fig. 4 NOTCH1 NICD hydrophobicity analysis results.
The HPLC and mass spectroscopy result of Fig. 5 pY2145 synthetic peptides.
The HPLC and mass spectrographic measurement result of Fig. 6 Y2145 synthetic peptides.
The SDS-PAGE qualification results of Fig. 7 synthetic peptide coupling albumen：Wherein, M1, M2：Protein Marker
(Broad)；1：Y2145-BSA；2：pY2145-BSA；3：pT2145-KLH；4：pY2145-KLH.
Fig. 8 is immunized with the screening (Western blot) of rabbit：Wherein, 1：pY2145-BSA(5μg)；2：Y2145-BSA
(5μg)；3：BSA standard proteins (5 μ g)；Primary antibody：Negative serum 1:5000 dilutions.
The result of antibody activity in Fig. 9 Dolt blot identification eluents, wherein, 1：Eluent 1-5 is managed；2：Eluent 6-
10 pipes；3：Eluent 11-15 is managed.
Figure 10 Dot blot detect the phosphoric acid peptide specific of purified antibodies, wherein, 1：The concentration gradient of BSA albumen is dilute
Release；2：The concentration gradient dilution of Y2145-BSA synthetic peptides；3：The concentration gradient dilution of pY2145-BSA synthetic peptides；A schemes：Primary antibody
It is negative serum 1:5000 dilutions；B schemes：Primary antibody is antiserum 1 before purification:5000 dilutions；C schemes：Purified antibodies 1:
1000 dilutions.
Phospho-AB prepared by Figure 11 present invention is used to detect people in normal gastric mucosa epithelial cell and stomach cancer cell
The Western blot results of NOTCH NICD albumen, wherein, 1：Ags cell RIPA lysates；2：MKN-45 cells RIPA splits
Solution thing；3：BGC823 cell RIPA lysates；Primary antibody is antibody 1 prepared by the present invention:1000 dilutions；B schemes：Primary antibody be β-
Actin antibody 1:2000 dilutions.
Figure 12 is the SABC that phospho-AB prepared by the present invention is used to detect oral cavity cancerous tissue and its cancer beside organism
As a result, wherein, a and b figures are oral cavity cancer beside organism and the carcinoma of mouth histogenic immunity histochemical staining result of patient A16361；C and d schemes
Oral cavity cancer beside organism and the immunohistochemical staining result of cancerous tissue for patient CRY；Primary antibody is antibody 1 prepared by the present invention:100
Dilution；Multiplication factor is 200 times.
Specific embodiment
For the ease of understanding the present invention, the present invention is described more fully below.But, the present invention can be with many
Different form is realized, however it is not limited to embodiment described herein.On the contrary, the purpose that these embodiments are provided be make it is right
The understanding of the disclosure is more thorough comprehensive.
Unless otherwise defined, all of technologies and scientific terms used here by the article with belong to technical field of the invention
The implication that technical staff is generally understood that is identical.The term for being used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all of combination of the Listed Items of pass.
It is provided in an embodiment of the present invention for people's NOTCH1 NICD albumen Tyr2145 site phosphorylation antibody referring to Fig. 1
Preparation method with application, generally comprise following steps：
Step one：Epitope is designed and screened：By long-term experiment screening, intracellular in people's NOTCH1 albumen is have selected
Section albumen (NICD) has can induced animal generation high antigenic, the SEQ ID NO of the ability of high-hydrophilic antibody：1 polypeptide, and
Phosphorylation modification is carried out to 2145 site tyrosine (Tyr) therein.
Step 2：It is prepared by synthetic peptide and coupling protein：Using peptide synthesis technology synthesis such as SEQ ID NO：It is anti-shown in 1
Former peptide, adds phosphate group on amino acid Tyr2145, and C-terminal is connected with a cysteine and (obtains the antigen
Peptide), it is coupled with hemocyanin (KLH) and bovine serum albumin(BSA) (BSA) respectively after being identified through the purifying of HPLC and mass spectrum (MS), it is preceding
Person prepares phospho-AB as immunogene, and the filler that the latter is used as affinity purification carries out affinity purification；Correspond, use
Peptide synthesis technology synthesis such as SEQ ID NO：Synthetic peptide shown in 1, synthesizes on amino acid Tyr2145 without phosphate group
Non-phosphorylating modified peptides, C-terminal is connected with a cysteine, with bovine serum albumin(BSA) after the purifying of HPLC and Mass Spectrometric Identification
(BSA) filler of the coupling as affine separation.
Step 3：Animal is immunized and antiserum is prepared.Tyr2145 site phosphorylations synthetic peptide-KLH in step 2 is coupled
The holoantigen of formation and SPF grades of new zealand white rabbit of adjuvant combined immunization, injected using subcutaneous (s.c) injection, muscle (i.m),
The multiple location multi-point injection antigen emulsion such as intracutaneous (i.d) injection and rabbit palmula injection location.Immune time includes 1 initiation
Injection, 3~4 booster shots and last time antigen direct injection, effect of the antiserum for purpose synthetic peptide is determined with ELISA
Valency；
Step 4：Purify and surveyor's NOTCH1 NICD albumen Tyr2145 specific phosphoantibodies.Phosphoric acid is used first
Change Antigenic Peptide-bovine serum albumin(BSA) and agarose is coupled and carries out affinity purification as chromatographic stuffing, obtain and be directed to phosphorylation
All antibody of epitope simultaneously remove the low sequence complexity epitope antibodies of low-affinity by changing ion concentration and pH value；
Be then used by non-phosphorylating synthetic peptide-bovine serum albumin(BSA) and agarose be coupled as chromatographic stuffing carry out it is affine separate,
Remove the non-phosphorylating epitope antibodies of non-specific binding in phospho-AB.Obtain Tyr2145, people NOTCH1 NICD albumen
The antibody of point phosphorylation.
Antiserum specific recognition phosphorylation synthetic peptide, ELISA detection purified antibodies effects are determined with Dot blot experiments
Valency, the final anti-pY2145 phospho-ABs for obtaining purifying.Antibody after purification is after centrifugal concentrating with 0.1% (wt/vol)
The mixed liquor of BSA/PBST and 40% (vol/vol) glycerine is preserved.
Below in conjunction with specific embodiment referring to the drawings, to present invention offer for people's NOTCH1 NICD albumen Tyr2145
The technology of preparing route of site phosphorylation antibody, is further illustrated and illustrates.
The epitope of embodiment 1 is designed and screened
The present inventor screens and is produced for Tyr2145, people NOTCH1 NICD albumen with energy induced animal
High specific, the Antigenic Peptide of high-hydrophilic antibody of point phosphorylation, this section of epitope include 9 amino acid (aa), and sequence is such as
SEQ ID NO：Shown in 1：N-Ser Pro Asn Gly p(Tyr)Leu Gly Ser Leu–C；Finally using NCBI websites
Blastp carries out homology analysis to the synthetic peptide, as a result shows the epitope peptide sequence and rabbit without homology, and calculate
The antigenicity of people's NOTCH1 amino acid sequences, hydrophily (Fig. 3, Fig. 4).
Embodiment is 2-in-1 to be prepared into peptide and coupling protein
According to the design of the epitope peptide of embodiment 1, a phosphate group is added on Tyr2145 sites and in sequence
C-terminal add cysteine and obtain phosphorylation synthetic peptide (SEQ ID NO：2), reached through the purity of acquisition after purification of HPLC
96% synthetic peptide, the synthetic peptide is shown in design and is consistent (Fig. 5) through Mass Spectrometric Identification, then by the cysteine and blood of C-terminal
Azurin (KLH) coupling obtains holoantigen (pY2145-KLH, Tyr abbreviation Y) will be used for animal immune；Meanwhile, the synthetic peptide is led to
The cysteine for crossing C-terminal is separated (pY2145-BSA) with after bovine serum albumin(BSA) (BSA) coupling for phospho-AB.Equally,
Using peptide synthesis technology synthesis such as SEQ ID NO：Synthetic peptide shown in 1, without phosphate group on amino acid Tyr2145
Synthesis non-phosphorylating modified peptides, in SEQ ID NO：1 C-terminal is connected with cysteine, is reached through the purity of acquisition after purification of HPLC
96% synthetic peptide, the synthetic peptide is shown in design and is consistent (Fig. 6) through Mass Spectrometric Identification, then the cysteine by C-terminal and ox
Seralbumin (BSA) is coupled for the affine separation of phospho-AB (Y2145-BSA).
Embodiment 3 is immunized animal and sero-fast preparation
The dissolving and identification of 3.1 synthetic peptide coupling albumen：
The concentration of the synthetic peptide coupling albumen dissolved with 1 × PBS of sterilizing is determined by BCA protein concentrations detection kit,
The concentration of Y2145-BSA, pY2145-BSA is respectively 1.1mg/ml, 0.845mg/ml (coefficient R 2=of standard curve
0.991), the concentration of pY2145-KLH is 0.571mg/ml (R2=0.997), through SDS-PAGE qualification results such as Fig. 7：
PY2145-BSA molecular weight is slightly above Y2145-BSA, and pY2145-KLH has obvious purpose band in more than 200KD.
The 3.2 immune screenings with animal：Immune preceding 1 week from for immune new zealand white rabbit (2~3kg, it is female,
Stalwartness, Nanfang Hospital Experimental Animal Center is provided) ear vein take 3mL blood, serum is separated, labeled as negative control blood
Clearly, dispense and be stored in -80 DEG C it is to be measured.The synthetic peptide coupling albumen of dissolving is carried out into SDS-PAGE electrophoresis, after electrophoresis terminates
200mA constant currents 90min goes to pvdf membrane, is sealed with TBST (pH7.5, the 0.08%Tween-20) room temperature containing 5% skimmed milk power
1h is closed, the negative control sera of collection is added as primary antibody (1:5000) incubation at room temperature 30min after 4 DEG C overnight, washed away with TBST
Uncombined primary antibody, adds goat anti-rabbit igg-HRP secondary antibodies, is incubated at room temperature 1h；TBST is sent out after washing away remaining secondary antibody with EZ-ECL
Light reagent testing result.
Result shows (see Fig. 8)：PY2145-BSA, Y2145-BSA and BSA swimming lane do not occur purpose band, show this
Invention is immunized with the antibody not occurred in new zealand white rabbit body for these three antigens, is preferably to be immunized to use animal.
3.3 animal immunes：
3.3.1 first immunisation is former prepares：The pY2145-KLH solution that will have been dissolved is mixed with equivalent Freund's complete adjuvant (CFA)
Close and first immunisation original is prepared after emulsifying completely.
3.3.2 first immunisation：Using subcutaneous (s.c) injection, muscle (i.m) injection, intracutaneous (i.d) injection and rabbit palmula
The multiple location multi-point injection such as injection location first immunisation is former.The antigen total amount of first immunisation is about 0.62mg.
3.3.3 follow-up immunization：First immunisation carries out first time booster immunization after 20 days, with incomplete Freund's adjuvant (IFA)
As immunologic adjuvant antigen emulsion being prepared instead of CFA and being injected by first immunisation mode, antigen total amount is each about
0.9mg；Carried out after 12 days plus strong second immune, antigen total amount is each about 0.9mg；Carried out after 12 days plus third time be strong immune,
Antigen total amount is each about 0.9mg；After 10 days, from ear vein take a blood sample 5mL, separate serum dispense and be stored in -20 DEG C it is to be measured.
3.3.4 Efficacy evaluation：By antiserum to be measured in proportion 1:1000、1:5000 and 1:10000 dilutions, pass through
ELISA detection antibody potency.Result shows：The OD values of testing sample more than 0.3, the to be measured sero-fast P/ of each dilution factor
N values difference 4.0,3.8,3.9, its serum antibody titer>1:10000, reach expected requirement.
3.3.5 final boost：Last time is carried out with the direct intramuscular injection rabbit of pY2145-KLH antigenic solutions to add
It is strong immune, amount of antigen about 0.9mg.After 3 days, rabbit carries out the big blood sampling of abdominal aorta, largely collects whole blood.The whole blood chamber that will be collected
Temperature stands overnight, 4000g centrifugation 10min, takes supernatant, and packing 1ml/ pipes, labeled as immune rear antiserum, are stored in -80 DEG C.
3.3.6 antiserum titre is determined：With 1 μ g/mL, 2 μ g/mL, 4 μ g/mL that antigen coat liquid (pH9.0) dilutes
PY2145-BSA, with 1:5000、1:10000、1:20000、1:40000、1:80000、1:The antiserum of 16000 dilutions is one
It is anti-, 1:The HRP mark goat anti-rabbit igg dilutions of 5000 dilutions carry out ELISA for secondary antibody.Result shows：Each dilution factor it is immune
(see 1 table 1) more than 0.5, the ratio between antiserum and negative serum (P/N values) are more than 2.1 to sero-fast OD values after being immunized, and exempt from afterwards
Anti serum potency is 1 after epidemic disease:More than 160000.
Sero-fast OD values after table 1 is immune
The purifying and identification of the people's NOTCH1 albumen Tyr2145 specific phosphoantibodies of embodiment 4
The preparation of 4.1 phosphorylation synthetic peptide chromatographic columns and non-phosphorylating synthetic peptide chromatographic column.
4.1.1 Y2145-BSA and pY2145-BSA, BCA protein quantification kit measurement are dissolved respectively with aseptic 1 × PBS
Its concentration is respectively 1.13mg/ml, 0.867mg/ml (R2=0.9922).Y2145-BSA and pY2145-BSA synthesis is taken respectively
Peptide lysate, pH value is adjusted to 9.0 with coupling buffer, 4 DEG C of preservation solution for standby.
4.1.2 0.3g CNBr- agaroses are claimed to be added in 15mL centrifuge tubes, for the chromatographic column of 1mL posts bed capacity, tool
Body usage ratio see the table below 2.7mL 10mM HCl are added to CNBr- agarose powders, is mixed using reversion well distributing rocker at room temperature
60min。
The 2-in-1 agarose usage ratio table into peptide-BSA, CNBr activation of table
4.1.3 10mM HCl are filled it up with to Bio-Rad Econo-Pac posts, allows it to flow out naturally, be repeated 3 times.Keep piston
Close, the swelling CNBr- agarose resins of step 4.1.2 are added in post, rinse centrifuge tube with 10mM HCl and will rinse
Liquid is added in post and allows it to flow out naturally.Fill it up with 10mM HCl and allow it to flow out naturally, be repeated 3 times.Added to chromatographic column
10mL coupling buffers simultaneously allow it to flow out naturally (activation resin).Before last drop buffer solution outflow, received with 1.5mL EP pipes
Intensive 500 μ l effluxes, labeled as liquid is retained 1., are imitated by the coupling of SDS-PAGE electrophoresis detections synthetic peptide-BSA and agarose
Rate.
4.1.4 to the synthetic peptide-BSA mixed liquors that pH in chromatographic column addition step 4.1.1 is 9.0.To being filled it up with chromatographic column
Coupling buffer, gently reversion shakes up the content 1h of chromatographic column at room temperature after sealing, and 4 DEG C are overnight continued to mix post content.
4.1.5 bottom piston release synthetic peptide-BSA mixed liquors are opened, the μ l of efflux 500 are collected with 1.5mL EP pipes mark,
Labeled as liquid is retained 2., by SDS-PAGE electrophoresis detections synthetic peptide-BSA and the coupling efficiency of agarose.
4.1.6 after synthetic peptide-BSA is finished with the coupling efficiency detection of agarose, delay to Alkaline Elution is filled it up with chromatographic column
Fliud flushing, allows its to flow out naturally and discard.To acid dcq buffer liquid is filled it up with chromatographic column, its is allowed to flow out naturally and discard, by upper
Operation is stated to be repeated 5 times.
4.1.7 to Block buffer is filled it up with chromatographic column, allow its to flow out naturally and discard, be repeated 5 times.Close slow with 10mL
Fliud flushing adds chromatographic column for preserving, and can be preserved 3 months at 4 DEG C.
4.1.8 pY2145-BSA phosphorylations chromatographic column and Y2145-BSA non-phosphorylatings layer are prepared by above-mentioned steps
Analysis post, the former is used for the affinity purification of phospho-AB；The latter is used for the affine separation of phospho-AB.
The purifying of 4.2 phospho-ABs
4.2.1. sample preparation：The antiserum for after defrosting 1mL is immune on ice separate, 10000g, 4 DEG C of centrifugation 5min, removal
Precipitation；Using the 100mM NaCl of refrigeration with 1：10 dilute serums, operate on ice.
4.2.2 affinity purification
4.2.2.1 take out and prepared phosphoeptide chromatographic column by 4.1, open the lid and piston of chromatographic column, make storage molten
Liquid flows out.To 100mM NaCl are filled it up with chromatographic column, it is allowed to flow out naturally.Closure piston, the sample for adding step 4.2.1 to prepare
Product, add 100mM NaCl to ensure chromatographic column top without unnecessary room, seal whole chromatographic column, and 4 DEG C of overnight gentle reversions are shaken
Even chromatographic column.Next day, chromatographic column collects efflux after shaking up 1h again at room temperature, labeled as liquid is retained 3., is preserved at 4 DEG C.
4.2.2.2, chromatographic column is added the mixed liquor of 10mM Tris (pH7.5) and 0.5M NaCl, is flowed out naturally, repeat 3
It is secondary.To alkaline dcq buffer liquid (pH9.5) is added in chromatographic column, it is allowed to flow out naturally.Delay to acid flushing is added in chromatographic column
Fliud flushing (pH4.0), allows it to flow out naturally.By alkaline dcq buffer liquid-acidity dcq buffer liquid order repeated washing chromatographic column 3
It is secondary.
4.2.2.3 15 EP pipes of 1.5mL are taken, 100 μ l 1M Tris alkali are separately added into, sequence number is put on.To in chromatographic column
Glycine buffer wash-out (pH2.2) of 1mL is added, eluent is collected using EP pipes, upset shakes up neutralization sample and is placed in ice
On.It is repeated 14 times, 15 pipe eluents is collected altogether.
4.2.2.4 take whether 1 μ l neutralize for pH detection papers from each EP pipe, 1M Tris are used if neutrality is not shown
In alkali 1~No. 15 eluent is respectively labeled as with eluent to neutrality.Identified by Nucleolar skeleton (Dolt blot) and washed
The activity of antibody in de- liquid, Sepharose G25 desalinations are passed through after active antibody is merged.
The activity of antibody in 4.3 Nucleolar skeletons (Dolt blot) identification eluent：1 μ is respectively taken from 15 pipe eluents
L in order o'clock on a nitrocellulose membrane paper, with 1:The goat anti-rabbit igg of the HRP marks of 5000 dilutions is secondary antibody, as a result as schemed
Shown in 9：3-5 eluents have specific antibody positive spots, collect 3-6 pipes eluent and are mixed labeled as phospho-AB is positive
Close liquid.
4.4 affine separation：Take out by the 4.1 non-phosphopeptide chromatographic columns for having prepared, to adding 100mM in chromatographic column
NaCl, allows it to flow out naturally.Valve is closed, 100mM NaCl are added after adding the sample of 4.2.2.4 preparations, it is ensured that chromatography capital
Portion seals whole chromatographic column without unnecessary room, and overnight gentle reversion shakes up at 4 DEG C.Next day collects the efflux mark of chromatographic column
It is designated as purified antibodies, 4 DEG C of preservations.
4.5 phosphospecifics that purified antibodies are detected using Dolt blot：Y2145-BSA (1.13mg/ml) is matched somebody with somebody
Be set to 56ng/ μ l, then carry out gradient dilution i.e. 28.3ng/ μ l, 14.13ng/ μ l, 7.06ng/ μ l, 3.5306ng/ μ l and
1.77ng/ μ l, are equally configured to 43.35ng/ μ l by pY2145-BSA (0.867mg/ml), then carry out gradient dilution i.e.：
21.68ng/μl、10.84ng/μl、5.42ng/μl、2.71ng/μl、1.35ng/μl；Prepare with the BSA albumen of Gradient：
1mg/ml, 100ng/ μ l, 10ng/ μ l, 1ng/ μ l and 0.1ng/ μ l.5 concentration points of the above-mentioned 3 kinds of albumen of 1 μ l are respectively taken in order
Sample on same nitrocellulose membrane, three, concurrent sample.Three nitrocellulose membranes are successively respectively with 1:The negative blood of 5000 dilutions
Clearly, 1:Immune rear antiserum, 1 of 5000 dilutions:The phospho-AB mixed liquor of 500 dilutions is primary antibody, and secondary antibody is by 1:
The dilution of the HRP mark goat anti-rabbit iggs of 5000 dilutions carries out Dot blot experiments, as a result such as Figure 10：Before a figures primary antibody is to be immunized
The serum dilution of negative rabbit, BSA, Y2145-BSA and pY2145-BSA are without there are positive spots in figure；B figure primary antibodies are
The antiserum gathered after pY2145-KLH immunizing rabbits, in figure each concentration gradients of BSA without there is specific positive spot,
Y2145-BSA and pY2145-BSA synthetic peptides appear above specific positive spot in 7.06ng and 5.42ng respectively；C figure primary antibodies
Be antibody that the antiserum gathered after pY2145-KLH immunizing rabbits is prepared by affinity purification-affine separation, in figure BSA and
Each concentration gradient points of Y2145-BSA do not occur specific positive spot, and pY2145-BSA synthetic peptides are in 5.42ng levels
Specific positive spot is appeared above, shows that the present invention has obtained people's NOTCH1 albumen Tyr2145 specificity phosphorus of high specific
Acidifying antibody.
The titration of 4.6 phospho-ABs after purification：Y2145-BSA and pY2145-BSA two are coated with 1ug/ holes respectively
Kind of antigen, the negative serum of different proportion dilution, before purification serum and after affinity purification-affine separation antibody as primary antibody
It is incubated, HRP mark goat anti-rabbit iggs dilution (1:5000) ELISA experiments are carried out as secondary antibody, as a result such as table 3, dilution factor is 1:
When 8000, for phosphorylation antigen OD450 values more than 0.4 and purified antibodies be more than with the P/N values of the ratio between negative serum
2.1, the OD450 values for non-phosphorylating antigen are below 0.3, then purified antibodies potency>1:8000.And use prior art
In the potency of antibody for preparing of affine separation-affinity purification method (identical with method of the present invention step, order is different) be 1:
2000 (being shown in Table 4).
The OD values of the purified antibodies of table 3
The OD values of the purified antibodies that table 4 is prepared using affine separation in the prior art-affinity purification method
The replacing of the ultrafiltration concentration and buffer solution of 4.7 antibody：By all antibody purifications using Amicon Ultra-410K from
Heart filter is concentrated by ultrafiltration, and the solvent of antibody purification is replaced with into PBS, and concentration is measured with BCA protein quantification determination methods
The concentration of antibody purification prepared by the present invention is 0.3776mg/ml afterwards, and volume is 1.05 milliliters；And use affine in the prior art
Antibody purification concentration prepared by separation-affinity purification method is 0.17096mg/ml, and volume is 0.85 milliliter.
Phospho-AB potency and the rate of recovery prepared by 4.8 two methods compares
By comparing 4.6 and 4.7 results, method (being named as method 1) prepared by the phospho-AB that the present invention is provided exists
The aspects such as manufacturing cycle, antibody titer and the rate of recovery (are named as method better than affine separation-affinity purification method in the prior art
2)
Table 5：Phospho-AB potency and the rate of recovery prepared by two methods compares
The preservation of 4.9 antibody：Final concentration of 1% (wt/vol) BSA/PBST and 40% is added to antibody purification after concentration
(vol/vol) mixed liquor of glycerine, is packed as 100 μ l/ pipes, and -80 DEG C long-term preserve.
Application of the people's NOTCH1 NICD albumen Tyr2145 site phosphorylation antibody of embodiment 5 in lesion detection
5.1 present invention prepare the people NOTCH1 NICD albumen Tyr2145 sites phosphoric acid of high specific, high-affinity
Change antibody, can be used for the detection of expression of people phosphorylation NOTCH1 NICD in cell and tissue.The present embodiment is made using the present invention
The table of phosphorylated human NOTCH1 NICD albumen in standby phospho-AB detection normal gastric mucosa epithelial cell and stomach cancer cell
Reach.
5.1.1 cell culture and total protein extraction：Normal human gastric mucosa's cell GES1 and gastric carcinoma cells MKN45,
BGC823 carries out cellar culture in 1640 complete mediums (10%FBS), passes on 1 time within every 2~3 days.When cell state is good with
2.5×106The cell concentration of individual/ml is inoculated into six orifice plates, when cell growth is to 80~90% abundance on 1640 culture mediums
After culture 24h, digestion each group cell simultaneously removes supernatants collection cells with PBS 3 times, by 50~1,000,000 cell/100~
200 μ L add RIPA protein lysates.(12000rpm, 20min) is centrifuged after cell lysis 20min on ice and takes supernatant measure albumen
After concentration, by 5:1 ratio adds albumen sample-loading buffer 5 × loading buffer, and after boiling water bath 7min, -20 DEG C long-term are protected
Deposit.
5.1.2Western blot：Gel electrophoresis are separated using 10% concentration SDS-PAGE, albumen applied sample amount is about every hole 16
μ g, electrophoresis terminates rear 200mA constant currents 90min and goes to pvdf membrane, with TBST (pH7.5,0.08% containing 5% skimmed milk power
Tween-20) room temperature closing 1h, adds antibody prepared by the present invention as primary antibody (1:1000) 4 DEG C of mistakes after incubation at room temperature 30min
At night, uncombined primary antibody is washed away with TBST, add goat anti-rabbit igg-HRP secondary antibodies, be incubated at room temperature 1h；TBST washes away remaining two
EZ-ECL luminescence reagent testing results are used after anti-.
5.1.3 expressions of results of the phosphorylated human NOTCH1 NICD on three kinds of cells：The specificity prepared using the present invention
Antibody shows as the Western blot results that primary antibody is completed：Gastric carcinoma cells MKN45 (swimming lane 2), BGC823 (swimming lane 3) are big
About there is specific band in 110kD or so, and normal human gastric mucosa's cell GES1 (swimming lane 1) has no any positive band
(see Figure 11).As can be seen here, the antibody that prepared by the present invention can detect phosphorylated human in normal gastric mucosa cell and stomach cancer cell
NOTCH1 NICD protein expression differences.
The phospho-AB that 5.2 present invention are prepared, can be used for people's phosphorylation NOTCH1 NICD eggs in detection tissue
White positioning and differential expression are detected.The present embodiment passes through immunohistochemical assay using phospho-AB prepared by the present invention
(IHC) phosphorylated human NOTCH1 NICD albumen positioning and expression in the cancer beside organism of detection oral cavity cancerous tissue and its pairing is poor
It is different.
5.2.1 paraffin section de-waxing is to water：Successively by 2 groups of carcinoma of mouth patients (sample number A16361 and sample numbers
CRY) paraffin section of cancerous tissue and corresponding cancer beside organism is put into the 15min- absolute ethyl alcohols I of I 15min- dimethylbenzene of dimethylbenzene II
The 5min-85% alcohol 5min-75% alcohol 5min- of 5min- absolute ethyl alcohols II distillation washings.
5.2.2 antigen retrieval and closing：Above-mentioned paraffin tissue sections are placed in and fill with EDTA antigen retrieval buffer solutions
(pH8.0) in antigen retrieval is carried out in micro-wave oven in reparation box, to boiling, truce 8min is incubated low fire in turning again to moderate heat 8min
7min, should prevent buffer solution excessive vaporization during this, be sure not dry plate.Slide is placed in PBS (pH7.4) after natural cooling
Washing 3 times, each 5min are rocked on decolorization swinging table.Section is put into 3% hydrogenperoxide steam generator (hydrogen peroxide：Pure water=1:9), room temperature
Lucifuge is incubated 25min, slide is placed in PBS (pH7.4) washing 3 times, each 5min are rocked on decolorization swinging table.Section is slightly got rid of
Drawn a circle (prevent antibody from flowing away) around tissue with groupization pen after dry, be added dropwise in circle with the confining liquid of 3%BSA.
5.2.3 antibody is added and developed the color：Confining liquid is gently got rid of, PBS is added dropwise in section and is pressed 1:100 ratios add this to send out
The antibody of bright preparation lies against 4 DEG C of overnight incubations in wet box as primary antibody, section.Slide is placed in PBS (pH7.4) and is decolourizing to shake
Washing 3 times, each 5min are rocked on bed.Section is added dropwise secondary antibody (HRP marks) covering group of goat antirabbit in circle after slightly drying
Knit, be incubated at room temperature 50min.Slide is placed in PBS (pH7.4) and washing 3 times, each 5min is rocked on decolorization swinging table.Section is slightly
The DAB nitrite ions of Fresh are added dropwise after drying in circle, developing time is controlled under microscope, the positive is brown color, running water
Rinse section color development stopping.
5.2.4 nucleus is redyed：Harris haematoxylins redye 3min or so, originally wash, 1% hydrochloride alcohol differentiation number
Second, running water is rinsed, and ammoniacal liquor returns indigo plant, and flowing water is rinsed.
5.2.5 mounting and microscopy are dehydrated：Section is sequentially placed into 75% alcohol 6min-85% alcohol 6min-- absolute ethyl alcohols
It is dehydrated transparent in the 5min of I 6min- absolute ethyl alcohols, II 6min- dimethylbenzene I, section is taken out from dimethylbenzene and slightly dried, neutral tree
Glue mounting.Sediments microscope inspection, IMAQ analysis.
5.2.6 ImmunohistochemistryResults Results：As shown in figure 12, sample number is the cancer beside organism of A16361 carcinoma of mouth patients to result
(Figure 12-a) and cancerous tissue (Figure 12-b) do not find the expression of phosphorylated human NOTCH1 NICD albumen；But sample number is
The table of phosphorylated human NOTCH1 N1ICD albumen in the cancer beside organism (Figure 12-c) of CRY carcinoma of mouth patients and cancerous tissue (Figure 12-d)
Up to there is significant difference, cancerous tissue is in obvious nuclei staining positive, shows that tumour cell is in m period, and match
Cancer beside organism does not detect any positive staining but.As can be seen here, the antibody that prepared by the present invention can detect different patients'
The differential expression of people NOTCH1 N1ICD albumen and cellular localization change in tumor tissues, while after pointing out this two classes patient more
There may be difference.
5.3 present invention provide phospho-AB and can be applied to the side such as ELISA, GST-pull down in actual applications
Method detection people NOTCH1 NICD protein phosphorylation modification situations, so as to inquire into the modification of people NOTCH1 NICD protein phosphorylations swollen
Effect and mechanism in the relevant diseases such as knurl.
The phosphorylation modification of 5.4 people's NOTCH1 NICD albumen is to carry out ubiquitination degraded and to NOTCH1 signal paths essence
The key of quasi- regulation and control.The exception of phosphorylation modification, causes the extension of NOTCH1 NICD half-life period to cause cell can be made persistently to increase
Stopping differentiation being grown, therefore phospho-AB is easy to research people NOTCH1 NICD albumen Tyr21451 sites phosphorus in actual applications
Acidifying modification is bred for particular biological event such as cell, the influence of cell differentiation, so as to inquire into it be sent out in diseases such as tumours
Effect during hair tonic exhibition.
5.5 the present invention be directed to people NOTCH1 NICD albumen specific T yr2145 sites prepare phosphorylation Anti-TNF-α
Body, contributes to the zymogenesis of its generation phosphorylation of research mediation, inquires into people's NOTCH1 NICD albumen various biological functions and exists
There is evolution in tumor disease in NOTCH1 cell-signaling pathways and albumen/nucleic acid interaction network and its phosphorylation modification
In mechanism of action, so as to find the latent effect target spot of diagnosis and the treatment of clinical tumor disease.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Nanfang Medical Univ
Zhuhai south biological medicine public service platform Co., Ltd of medical university
<120>A kind of new people NOTCH1 NICD proteantigens, antibody and its preparation method and application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> Protein
<213>Artificial sequence
<400> 1
Ser Pro Asn Gly Tyr Leu Gly Ser Leu
1 5
<210> 2
<211> 10
<212> Protein
<213>Artificial sequence
<400> 2
Ser Pro Asn Gly Tyr Leu Gly Ser Leu Cys
1 5 10
Claims (10)
1. a kind of Antigenic Peptide of people NOTCH1 NICD albumen, it is characterized in that, its active amino acid sequence such as SEQ ID NO：1 institute
Show, and Tyr amino acid therein is phosphorylated.
2. the Antigenic Peptide of people NOTCH1 NICD albumen according to claim 1, it is characterized in that, the people NOTCH1 NICD
The C-terminal of the Antigenic Peptide of albumen is connected with a cysteine.
3. a kind of antibody of people NOTCH1 NICD albumen, it is characterized in that, it is by the people NOTCH1 described in claim 1 or 2
Immune animal prepares the Antigenic Peptide of NICD albumen.
4. the Antigenic Peptide described in claim 1 or 2 is preparing the medicine or reagent of the diagnosis for tumour, treatment, prognosis judgement
In application.
5. the antibody of the people's NOTCH1 NICD albumen described in claim 3 is sentenced in preparation for diagnosis, treatment and the prognosis of tumour
Application in fixed medicine or reagent.
6. a kind of diagnosis for tumour, treatment, prognosis judge medicine or reagent, it is characterized in that, it includes claim 1
Or the antibody described in the Antigenic Peptide and/or claim 3 of the people's NOTCH1 NICD albumen described in 2.
7. the application according to claim 4 or 5, medicine or reagent described in claim 6, it is characterized in that, the tumour
It is the tumour related to people's NOTCH1 signal paths.
8. a kind of preparation method of the antibody of people NOTCH1 NICD albumen, it is characterized in that, comprise the following steps：
(1) active amino acid is obtained for SEQ ID NO：The Antigenic Peptide of the amino acid sequence shown in 1, phosphorus is added on amino acid Tyr
It is acidified group and C-terminal is connected with a cysteine；
(2) Antigenic Peptide is coupled with carrier protein hemocyanin, and immune animal simultaneously collects antiserum；
(3) antiserum that step (2) is collected is purified and is identified, obtained people NOTCH1 NICD albumen Tyr2145 sites phosphorus
The antibody of acidifying.
9. preparation method according to claim 8, it is characterized in that, the step that the antiserum described in step (3) is purified
Suddenly include：Determine potency of the antiserum for Antigenic Peptide described in claim 1 or 2；Then after first affinity purification, then affine point
From realizing that antagonistic Serum is purified.
10. preparation method according to claim 9, it is characterized in that, after the first affinity purification, then affine separation, including：
Be coupled using phosphorylation synthetic peptide coupling albumen and agarose first carries out affinity purification as chromatographic stuffing, and acquisition is directed to
All antibody of phosphorylation epitope simultaneously remove the low sequence complexity epitope of low-affinity by changing pH and ion concentration
Antibody；It is then used by non-phosphorylating synthetic peptide coupling carrier protein and agarose is coupled and carries out affine point as chromatographic stuffing
From the non-phosphorylating epitope antibodies of non-specific binding in removing phospho-AB.
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