CN108840920A - A kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application - Google Patents
A kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application, is related to antibody and its preparation technical field.The antibody is by SEQ ID NO：Active amino acid sequence shown in 1 is that Antigenic Peptide is immunized what animal was prepared.Phospho-AB provided by the invention facilitates research and mediates it that the kinases of phosphorylation occurs, and inquires into the multiple biological function of cysteine-rich 61；Mechanism of action in practical application convenient for research human CYR 61 albumen specific site phosphorylation modification during particular biological event or disease development, also it can be used for detecting the difference that tumor correlated albumen is expressed after medication, provide potential action target spot for the Clinics and Practices of clinical tumor disease.
Description
Technical field
The present invention relates to antibody and its preparation technical field, in particular to a species specificity is directed to human CYR 61 albumen
Ser167 site phosphorylation antigen, antibody and its preparation method and application.
Background technique
The albumen that CCN family possesses identity function area by 6 forms, and CYR61 is first egg being found in the family
It is white.The family protein is considered as secretory protein all the time, and is furtherd investigate as cellular matrix albumen.CCN family egg
It is white to synthesize in the cell, and be secreted into extracellularly under the guidance of signal peptide, they are in tumour occurrence and development along with swollen
A series of changes of tumor internal and external environment can in the cell and extracellularly work.The family protein takes part in cell adherence, divides
Split, migrate, drug resistance, survival, differentiation, angiogenesis, fibrosis, bone generates and the physiology courses such as wound healing, it is therein very
More phenomenons are related to tumour.CYR61 (CCN1), CTGF (CCN2), CCN5 knock out mice often die of embryonic period, embryonic phase or birth
In early days.
The biological function that CYR61 is showed in tumour is various, and in different tumor tissue cells,
The function of CYR61 is also not quite similar.CYR61mRNA high is expressed in malignant breast tumor cell, moreover it is possible to promote gastric adenocarcinoma cells RF-
1 cell deterioration；CYR61 high expression in oophoroma simultaneously, and be positively correlated with lymphatic metastasis.However CYR61 is in lung cancer, flat
Expression in sliding myomata is but substantially reduced, and is played an important role as tumor suppressor.For example, in non-small cell lung
The expression of CYR61 is lower than Ai Pang normal lung tissue in cancer, and related to the factors such as lung cancer histological type, lymphatic metastasis.
Current study show that：Cysteine-rich 61 mainly with the proliferation of tumour cell and migration, tumour cell growth regulating and
Tumor angiogenesis is related, and mechanism of action can be completed by relevant signal path.The overexpression of CYR61 in the tissue
It can promote the expression of its receptor AV β 3, be formed " 3 Autocrine regulation loop of CYR61-AC β ", generate cell growth and anti-apoptotic
Signal.In breast cancer, HRG raises the level of AV β 3 by CYR61, and CYR61 is combined activation ERK1/ERK2MAPK letter with AV β 3
Number access, inhibits the aggregation of P53, generates anti-taxol resistance effect.In neuroglial cytoma, CYR61 passes through integrin egg
White coupling kinases (ILK) makes -3 β of Glycogen synthesis kinases (GSK3- β) phosphorylation, β-catenin/LEF access is activated, to promote
The expression of cell proliferation genes, CYR61 can also promote the reduction of apoptotic proteins Bad activity by activation PI3K/AKT signal path,
Promote cell growth and migration.In lung cancer, CYR61 is played an important role as tumor suppressor.In vitro experiment,
CYR61 leads to G0/G1The retardance of phase can inhibit the growth of lung carcinoma cell.
The posttranslational modifications such as phosphorylation, ubiquitination play pass in the physiology and pathologic process that cell-signaling pathways mediate
Key effect.In view of the important function that CYR61 is played during tumour occurrence and development, thus further study cysteine-rich 61
Posttranslational modification be of great significance.We early-stage study have found that the site Ser167 in CYR61 amino acid sequence is its egg
The site of white phosphorylation modification may play an important role in regulation cysteine-rich 61 self stability.Antibody is protein
The important tool of functional study has been widely used in the clinical applications such as the medicals diagnosis on disease such as tumour, treatment, thus a kind of special knowledge
The antibody of the Ser167 phosphorylation site of others' CYR61 is up for further developing.
Summary of the invention
In order to solve the problems, such as that cysteine-rich 61 phosphorylation modification is effectively studied, for this purpose, the primary purpose of the present invention is that mentioning
Human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide is directed to for a species specificity.
It is anti-for human CYR 61 Protein S er167 site phosphorylation that another object of the present invention is to provide a species specificity
Body.
Another object of the present invention is to provide the applications of above-mentioned Antigenic Peptide and antibody.
A further object of the present invention is to provide the preparation methods of above-mentioned antibody.
The purpose of the invention is achieved by the following technical solution：
One species specificity is directed to human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide, active amino acid sequence such as SEQ
ID NO：Shown in 1, and Ser amino acid therein is phosphorylated modification.
One species specificity is directed to human CYR 61 Protein S er167 site phosphorylation antibody, is directed to by above-mentioned specificity
Human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide is immunized what animal was prepared.
The specificity is directed to the preparation method of human CYR 61 Protein S er167 site phosphorylation antibody, including walks as follows
Suddenly：
(1) SEQ ID NO is synthesized：The Antigenic Peptide of active amino acid sequence shown in 1 is added with phosphoric acid on amino acid Ser
Change group；
(2) it is coupled, is immunized with carrier protein hemocyanin (KLH) using the N-terminal of the Antigenic Peptide of step (1) synthesis
Animal simultaneously collects antiserum；
(3) antiserum that step (2) are collected is purified and is identified, obtain specificity for human CYR 61 albumen
Ser167 site phosphorylation antibody.
Antigenic Peptide described in above-mentioned steps (1) can be synthesized by following steps：
Prepare human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide, centered on the site cysteine-rich 61 Ser167, N-terminal
Adjoin four CYR61 amino acid sequences, C-terminal connects five amino acid sequence, such as SEQ ID NO：Synthetic peptide shown in 1, and adopt
It is synthesized with peptide synthesis technology, adds phosphate group on amino acid Ser167.
The step of immune animal described in above-mentioned steps (2) may include：
The Antigenic Peptide coupling hemocyanin and adjuvant combined immunization new zealand white rabbit synthesized with above-mentioned steps (1)；It is immune
Mode be through neck, skin of back is relatively thin, position two sides of relaxation are subcutaneously injected, gluteus and huckle two sides difference muscle note
It penetrates, the intracutaneous injection of waist two sides, the multiple locations multi-point injection such as footpad injection；Immune time include 1 initiation injection, 3~4 times plus
Injection is penetrated and last time antigen direct injection.
The step of purifying described in above-mentioned steps (3) and identification may include：
The antiserum that above-mentioned steps (2) are collected is directed to purpose synthetic peptide (Antigenic Peptide) with ELISA measuring antiserum
Potency and its whether can specific recognition purpose synthetic peptide (Antigenic Peptide), utilize affine separation-affinity purification circulating technology
Antagonistic Serum is purified, and is tested with Western Blot and is determined whether antiserum being capable of the site specific recognition Ser167 phosphoric acid
The cysteine-rich 61 of change.
Above-mentioned the step of being purified using affine separation-affinity purification circulating technology antagonistic Serum may include：
It is carried out using the affinity chromatography of the pure albumen of synthetic peptide coupling carrier proteins Bovine (BSA) and agarose chromatography column
Antibody is affine separation and affinity purification；Made first using non-phosphorylating synthetic peptide coupling bovine serum albumin(BSA) (BSA) and agarose
The affine non-phosphorylating antibody being separated off in antiserum is carried out for the filler of chromatographic column, obtains phosphorous acidification antibody efflux；
Then phosphorylation synthetic peptide (Antigenic Peptide) coupling bovine serum albumin(BSA) (BSA) and agarose is used to carry out as the filler of chromatographic column
Affinity purification removes the low sequence complexity epitope antibodies of low-affinity in phospho-AB.
Above-mentioned specificity is for human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide and/or antibody in preparation for swelling
The application in pharmaceutical preparation that tumor, hematological system, the diagnosis of disease of cardiovascular system, treatment and prognosis determine.
Determine the present invention also provides a kind of for tumour, hematological system, the diagnosis of disease of cardiovascular system, treatment and prognosis
Pharmaceutical preparation comprising it is above-mentioned specificity be directed to human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide and/or antibody.
The high specific that the present invention is prepared can be used for human CYR 61 Protein S er167 site phosphorylation antibody
The differential expression of tumour cell, helps to study after the detectable normal cell of Western blot experiment, tumour cell and medication
Effect of the phosphorylation modification of cysteine-rich 61 during tumor disease occurrence and development is the diagnosis of clinical tumor disease or is controlled
It treats and potential action target spot is provided, the phosphoric acid of human CYR 61 albumen can also be detected with the immunologys such as IHC, ELISA related experiment
Change level, inquire into the relationship of the diseases such as itself and tumour, hematological system, cardiovascular system, determines in medical diagnosis on disease, treatment and prognosis
Etc. have extensive potential applicability in clinical practice.
The present invention has the following advantages and effects with respect to the prior art：
(1) phospho-AB provided by the invention is able to detect phosphoric acid after the translation of human CYR 61 albumen in practical applications
Change modification situation；
(2) convenient for research human CYR 61 albumen specific site phosphorylation in phospho-AB practical application provided by the invention
Modify the correlation in particular biological event such as tumour cell chemotherapy resistance, DNA loss, cell cycle etc.；
(3) the present invention be directed to human CYR 61 Protein S er167 site phosphorylation polyclonal antibodies, facilitate research and mediate it
The kinases that phosphorylation occurs, inquires into the multiple biological function of cysteine-rich 61；
(4) phospho-AB provided by the invention helps to inquire into the phosphorylation modification of human CYR 61 albumen in tumor disease
Mechanism of action during occurrence and development also can be used for detecting the difference of tumor correlated albumen expression after medication, be clinical tumor
The Clinics and Practices of disease provide potential action target spot.
Detailed description of the invention
Fig. 1 is human CYR 61 protein structure block plan.
Fig. 2 is the human CYR 61 phospho-AB technology path of preparation and purification high specific according to an embodiment of the present invention
Figure.
Fig. 3 is the human CYR 61 site Protein S er167 in PhosohoSitePlus database query result.
Fig. 4 is human CYR 61 Protein S er167 location proximate amino acid antigenicity analysis schematic diagram.
Fig. 5 is preimmune serum screening Western blot result figure, wherein 1：BSA standard protein (5 μ g)；2：
Ser167-BSA non-phosphorylating synthetic peptide (5 μ g)；3：PSer167-BSA phosphorylation synthetic peptide (5 μ g)；Primary antibody：Negative serum 1:
5000 dilutions.
Fig. 6 is special disposition of the serum after Western blot detection before purification to Ser167 site phosphorylation synthetic peptide
Condition；Wherein, scheme in A, 1：BSA standard protein (5 μ g)；2：PSer167-BSA synthetic peptide (5 μ g)；3：Ser167-BSA synthetic peptide
(5μg)；Primary antibody：167 site antiserums before purification 1:5000 dilutions.Scheme in B, 1：BSA standard items (5 μ g)；2：Ser167-
BSA synthetic peptide (5 μ g)；3：PSer167-BSA synthetic peptide (5 μ g)；Primary antibody：167 site antiserums after purification 1:500 dilutions.
Fig. 7 is that Ser167 site phosphorylation antibody after purification (is named as：P-CYR61-S167 antibody) it is anti-to phosphorylation
Former and non-phosphorylating antigen ELISA detection data figure.
Fig. 8 is the specificity of cellular level verifying p-CYR61-S167 antibody.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Human CYR 61 protein structure block plan, as shown in Figure 1.
Referring to fig. 2, the preparation side provided in an embodiment of the present invention for human CYR 61 Protein S er167 site phosphorylation antibody
Method and application, generally comprise following steps：
Step 1：The position that phosphorylation may occur in human CYR 61 amino acid sequence is screened by bioinformatics software first
Point, and the site Ser167 (Fig. 3) of human CYR 61 albumen has been determined by mass spectrum and database, analyze the antigenicity (figure in the site
4) corresponding Antigenic Peptide, is designed, and analyzes its homology；
Step 2：Synthesize the Antigenic Peptide of the phosphorylation site containing Ser167：Step 1 is respectively synthesized using peptide synthesis technology
10 amino acid Antigenic Peptides of the designed phosphorylation site containing Ser167, wherein amino acid Ser167 adds phosphate group, and
It is coupled with carrier protein hemocyanin (KLH), prepares phospho-AB as immunogene；With the pure albumen of carrier proteins Bovine
(BSA) filler that coupling is used as affinity purification carries out affinity purification；It corresponds, is respectively synthesized non-phosphorylating modification synthetic peptide
It is used as affine isolated filler with the pure albumen of carrier proteins Bovine (BSA) coupling, all synthetic peptides are all through the purifying of HPLC；
Step 3：Holoantigen is immunized animal and collects antiserum：Ser167 site phosphorylation in step 2 is synthesized respectively
Peptide (Antigenic Peptide)-KLH is coupled holoantigen and SPF grades of new zealand white rabbits of adjuvant combined immunization, using through neck, skin of back compared with
Thin, relaxation position two sides subcutaneous (s.c) injection, gluteus and huckle two sides difference muscle (i.m) injection, waist two sides carry out
Intradermal (i.d) injection, the multiple locations multi-point injection antigen emulsion such as rabbit palmula position injection.Immune time includes 1 initiation
Injection, 3~4 booster shots and last time antigen direct injection are directed to the effect of purpose synthetic peptide with ELISA measurement antiserum
Valence；
Step 4：Purify simultaneously surveyor's cysteine-rich 61 Ser167 locus specificity phospho-AB：Using synthetic peptide coupling
The affinity chromatography of bovine serum albumin(BSA) (BSA) and agarose chromatography column carries out that antibody is affine to separate-affinity purification circulatory purification
Technology.Use the corresponding non-phosphorylating synthetic peptide coupling bovine serum albumin(BSA) (BSA) in the site Ser167 and agarose respectively first
Filler as chromatographic column carries out the affine non-phosphorylating antibody being separated off in antiserum, obtains phosphorous acidification antibody outflow
Liquid；Second use phosphorylation synthetic peptide (Antigenic Peptide) be coupled bovine serum albumin(BSA) (BSA) with agarose filling out as chromatographic column
Material carries out affinity purification, the low sequence complexity epitope antibodies of low-affinity in phospho-AB is removed, with Western blot
It tests and determines antiserum specific recognition phosphorylation synthetic peptide (Antigenic Peptide)；The anti-p-Ser167 phosphoric acid of purifying is finally obtained respectively
Change antibody, be named as p-CYR61-S167 antibody, with 2.5% (wt/vol) BSA, 0.01% (vol/vol) Tween-20 and
The mixed liquor of 25% (vol/vol) glycerol saves, and detects purified antibodies potency again with ELISA and is directed to phosphorylation purpose
Synthetic peptide (Antigenic Peptide), the identity of non-phosphorylating synthetic peptide are finally tested with Western blot and carry out Identification of the antibodies.
Below in conjunction with specific embodiment referring to attached drawing, the site human CYR 61 Protein S er167 phosphorus is directed to provided by the invention
It is acidified the technology of preparing route of antibody, further progress illustration and description.
Embodiment 1 determines human CYR 61 protein phosphorylation site
1.1 screened first by bioinformatics software NetPhorest 2.0 and NetPhos 2.0 can in human CYR 61 albumen
The site of phosphorylation can occur, then pass through mass spectrum Literature Consult and protein phosphorylation site database
The lookup of PhosohoSitePlus (Fig. 3), confirmation Ser167 are a phosphorylation sites in human CYR 61 amino acid sequence；Together
Shi Liyong software CLC Protein Workbench 5 calculates the antigenicity (Fig. 4) of human CYR 61 amino acid sequence, final to determine
Human CYR 61 protein-specific phosphorylation site Ser167.
1.2 designer's cysteine-rich 61 Ser167 site phosphorylation Antigenic Peptides.In being with the site human CYR 61 Protein S er167
The heart, N-terminal adjoin four CYR61 amino acid sequences, and C-terminal connects five CYR61 amino acid sequences, the synthetic peptide (Antigenic Peptide) of design
Sequence, adds phosphate group on amino acid Ser167, and peptide section sequence is：CDEDS(p)IKDPM.
The synthesis of embodiment 2 includes the synthetic peptide (Antigenic Peptide) of Ser167 phosphorylation site
According to the design of hapten synthesis peptide, a phosphate group is added on the site Ser167 respectively and obtains phosphorylation
Synthetic peptide (Antigenic Peptide), and holoantigen is obtained for rabbit immunization (pSer167-KLH) with hemocyanin (KLH) coupling.Furthermore
Phosphorylation synthetic peptide (Antigenic Peptide) is used as affinity purification chromatographic column by glutaraldehyde method and bovine serum albumin(BSA) (BSA) coupling
Filler (pSer167-BSA).It corresponds, synthesizes the corresponding non-phosphorylating synthetic peptide of one section of Ser167 and bovine serum albumin(BSA)
(BSA) coupling is used as the filler (Ser167-BSA) of affine separation chromatographic column.
The method that holoantigen described in embodiment 3 routinely prepares polyclonal antibody prepares antiserum
3.1 prepare negative serum：From the new zealand white rabbit for injection, (2~3kg, female is healthy and strong, is purchased from the Chinese Academy of Sciences
This Lake Experimental Animal Center) ear vein take 3mL blood in heparin tube, with cotton balls hemostasis by compression.Blood is set into room temperature
1h or so waits for blood to solidify to form clot, and 2h is placed at 4 DEG C is precipitated serum, and 2500g is centrifuged 10min, draws supernatant, label
For negative control sera, dispense and be stored in -20 DEG C it is to be measured.
3.2 immune preceding screening-Western blot：Each 5 μ of BSA standard items, Ser167-BSA, pSer167-BSA is taken respectively
5 × SDS sample-loading buffer appropriate is added in g, and boiling water bath, which boils 10min, makes protein denaturation, and 10000 × g is centrifuged 1min；
SDS-PAGE electrophoretic separation glue is 8%, and concentration glue is 5%；Sample loading gun loading is used in a predetermined order, in unused sample well
Add isometric 1 × sds gel sample-loading buffer；It is changed to 120V about 50min after 80V20min electrophoresis, divides until bromophenol blue reaches
Power supply is closed in bottom from glue.Transferring film condition：Constant current 300mA, time 120min.The closing of 5% skimmed milk power, 37 DEG C of shaking table
1h.Transfer film is put into primary antibody dilution buffer by 1:5000 prepare immune preceding antiserum dilution, and level slowly shakes up, and 4
DEG C overnight.Next day washes film 10min using 1 × PBST, is repeated 4 times.Film is placed in 1 × PBST by 1:5000 diluted HRP marks
In the secondary antibody diluent of the goat anti-rabbit igg of note, 37 DEG C of shaking table, 60min.Two corresponding anti-solution is abandoned, 1 × PBST washes film 10min, repeats 3
It is secondary.Developed according to producer's explanation using ECL kit, is photographed to record.As a result such as Fig. 5：Do not occur purpose band, i.e., does not occur needle
It is gedanken experiment animal to the antibody of purpose tissue or cell extract.
3.3 animal immune：About 73 days
3.3.1 pSer167-KLH synthetic peptide powder is dissolved respectively with 1 sterile × PBS, drawn with an asepsis injector
Antigenic solution, another syringe draws equal amounts Freund's complete adjuvant (CFA), is connected with plastic tube therebetween, back and forth
PSer167-KLH peptide fragment is mixed with CFA respectively, until forming the emulsion emulsified completely, drips the indiffusion in water by suction.
3.3.2 first immunisation is respectively through neck, position two sides subcutaneous (s.c) injection that skin of back is relatively thin, loose, gluteus
With huckle two sides difference muscle (i.m) injection, waist two sides carry out intradermal (i.d) injection, and rabbit palmula position injection etc. is more
Position multi-point injection antigen emulsion.The first immunisation total amount of two kinds of antigens is about 0.61mg.
3.3.3 first time booster immunization is carried out after first immunisation 20 days, replaces CFA to make with incomplete Freund's adjuvant (IFA)
Antigen emulsion is prepared for immunologic adjuvant and is injected by first immunisation mode, and this time the antigen total amount of booster immunization is each about
0.9mg。
3.3.4 it is carried out after being immunized 12 days plus second strong immune, incomplete Freund's adjuvant (IFA) is used to replace CFA as exempting from
Epidemic disease adjuvant prepares antigen emulsion and is injected by first immunisation mode, and this time the antigen total amount of booster immunization is each about
0.9mg。
3.3.5 after last time booster immunization 3 days, rabbit carries out the big blood sampling of abdominal aorta, largely collects serum.It will collect
Room temperature is stood overnight after the beaker closing of blood, makes clot contraction.The serum of precipitation is sub-packed in 50mL by next day sterile working
In centrifuge tube, 4000g is centrifuged 10min, takes supernatant, packing 1mL/ pipe, labeled as antiserum after being immunized (about 51mL altogether), storage
In -20 DEG C.
3.3.6 sero-fast titration after being immunized, specific step is as follows：
3.3.6.1 with antigen coat liquid (CBS) respectively by phosphorylation antigen pSer167-BSA and non-phosphorylating antigen
Ser167-BSA coating, addition is into 96 hole elisa Plates, every hole 0.1mL, vibrates mixing after covering titer plate, 4 DEG C are coated with overnight
(12h or more).
3.3.6.2 coating finishes, and discards the liquid in hole, 1 × PBST sufficiently washs each coating hole of titer plate, discards and wash
Liquid is washed, is repeated 3 times, buckles dry residual liquid on filter paper each time after washing.Block buffer (0.25% is added in each coating hole
BSA/PBST), 200 hole μ L/, 37 DEG C of incubation 2h, 1 × PBST are washed titer plate 3 times, buckles dry remain on filter paper each time after washing
Liquid.
3.3.6.3 antiserum is by 1 after being immunized with 1 × PBST:1000,1:3000,1:9000,1:27000,1:81000,
1:243000,1:729000 dilution proportion is added separately to 96 hole elisa Plates using 1 × PBS buffer solution as blank control,
Lid envelope titer plate, 37 DEG C of incubation 1h.
3.3.6.4 liquid in hole is discarded, is washed titer plate 3 times with 1 × PBST.It is added and presses 1 with 1 × PBST:5000 dilutions
HRP mark goat anti-rabbit igg secondary antibody diluent, 100 holes μ L/, 37 DEG C of incubation 1h.Lid envelope titer plate, 37 DEG C of incubation 1h.With 1 ×
PBST is washed titer plate 5 times, and button is dry.The TMB developing solution of Extemporaneous, 100 holes μ L/ is added, room temperature is protected from light 30min.Add
Enter 2M H2SO4Terminate reaction, 50 holes μ L/.Microplate reader 450nm surveys each hole OD value.
3.3.6.5 antiserum dilution is 1 after Ser167 antigen is immunized:1000,1:3000,1:9000 it is immune after anti-blood
Clear ELISA detected value is in 0.5 or more (see the table below 1)；
The OD value of antiserum ELISA detection after Ser167 antigen is immunized in table 1
| Former serum dilution | Ser167-BSA | pSer167-BSA |
| 1:1000 | 2.319 | 2.357 |
| 1:3000 | 1.152 | 1.677 |
| 1:9000 | 0.553 | 1.391 |
| 1:27000 | 0.071 | 0.452 |
| 1:81000 | 0.02 | 0.059 |
| 1:243,000 | 0.005 | 0.023 |
| 1:729000 | 0.004 | 0.011 |
| BLANK | 0.003 | 0.01 |
The purifying of embodiment 4 and surveyor's cysteine-rich 61 Ser167 specific phosphorylation site
The preparation of 4.1 phosphorylation synthetic peptide chromatographic columns and non-phosphorylating synthetic peptide chromatographic column.
Synthetic peptide coupling chromatographic column is using Thermo scientificCoupling Resin reagent
Box preparation, specific step is as follows：
4.1.1 Ser167-BSA, pSer167-BSA are dissolved respectively with coupling buffer, concentration of ordinary dissolution is 0.7mg/mL.
It takes 7mg Ser167-BSA, 6mg pSer167-BSA lysate to be added to 5mL resin respectively, is added separately to chromatograph after mixing
Column compartment temperature mixes 15 minutes, and chromatographic column room temperature is just being set 30 minutes, removes the cap of chromatographic column upper and lower ends respectively, collects outflow
Liquid, and rinse pillar with the coupling buffer of 3 times of volume of resins.
4.1.2 chromatographic column bottom end lid is covered, 50mM L-CysteineHCl is added into coupling buffer, after mixing
It takes isometric buffer in chromatographic column, after room temperature mixes 15 minutes, stands 30 minutes.
4.1.3 bottom end lid is removed, coupling buffer is discharged, cleans chromatography with the tears liquid (1M NaCl) of washing of 6 times of volumes
Column, then chromatographic column is rinsed with 2 times of storage volumetric buffers, it closes the lid, the store buffer liquid of 1 times of volume is added, 4 degree of preservations are standby
With.
The purifying of 4.2 phospho-ABs
Antibody purification is using Thermo scientificThe preparation of Coupling Resin kit,
Specific step is as follows：
4.2.1 chromatographic column bottom end lid is removed, store buffer liquid is discharged, the combination buffer that 6mL is added washes tears chromatographic column.
Ser167 antiserum is added to corresponding non-phosphorylating and chromatographs pillar, efflux is collected, is repeated twice, obtains Ser167 outflow
Then liquid washes tears liquid with 12mL and cleans chromatographic column, retain the eluent that bottom end reserves, finally obtained with elution chromatographic column
Corresponding non-phosphorylating antibody.
4.2.2 the efflux collected in 4.2.1 is added to the phosphorylation pillar after corresponding flushing, is washed later with 12mL
Tears liquid cleans chromatographic column, finally obtains corresponding Ser167 phospho-AB with elution chromatographic column, and antibody is dissolved in 1 ×
0.1%NaN is added in PBS3It is spare.
Serum after Western blot detection before purification is to the special implementations of Ser167 site phosphorylation synthetic peptide；Such as
Shown in Fig. 6；Wherein, scheme in A, 1：BSA standard protein (5 μ g)；2：PSer167-BSA synthetic peptide (5 μ g)；3：Ser167-BSA is closed
At peptide (5 μ g)；Primary antibody：167 site antiserums before purification 1:5000 dilutions.Scheme in B, 1：BSA standard items (5 μ g)；2：
Ser167-BSA synthetic peptide (5 μ g)；3：PSer167-BSA synthetic peptide (5 μ g)；Primary antibody：167 site antiserums after purification 1:500
Dilution.The result shows that the Ser167 peptide fragment of the site Ser167 antiserum energy specific recognition phosphorylation after purification, and nonrecognition
The Ser167 peptide fragment of non-phosphorylating.
4.3 detect the potency of phospho-AB after purification again with ELISA and are directed to phosphorylation purpose synthetic peptide (antigen
Peptide), the identity of non-phosphorylating synthetic peptide.It is coated with artificial synthesized phosphorylation antigenic synthetic peptide-BSA and non-phosphoric acid respectively first
It is combined to peptide antigen-BSA, the diluted serum before purification of different proportion is added and antiserum is incubated for after purification, is added after washing
HRP marks goat anti-rabbit igg dilution, washs after incubation, and TMB colour developing, reaction terminating measures OD450.As a result as shown in Figure 7：
Ser167 phospho-AB dilution is 1:When 9000, it is greater than 0.75 for the OD450 value of phosphorylation antigen, for non-phosphorylating
The OD450 value of antigen is greater than 3 lower than 0.25 and P/N value, then Ser167 phospho-AB potency is at least 1 after purification:9000 with
On.
Application of the 5 human CYR 61 Protein S er167 site phosphorylation antibody of embodiment in tumour
The human CYR 61 Protein S er167 site phosphorylation antibody that high specific is prepared in 5.1 present invention can use Western
Blot experiment detection cells phosphorylation level difference, as shown in figure 8, (being incited somebody to action in (commercially available) the transfection Flag-CYR61-WT of 293T cell
The albumen coded sequence of the CYR61 gene of people is cloned into over-express vector pcDNA3.1 (+) Vector [invitrogen], obtains
Recombinant vector Flag-CYR61-WT), Flag-CYR61-S167A plasmid (sports the 167Ser of Flag-CYR61-WT plasmid
The obtained plasmid of alanine), extract cell protein afterwards for 24 hours, in immunoprecipitation Flag product, Ser167 site phosphorylation is anti-
Body (is named as：P-CYR61-S167 it) can identify that the site Ser167 is in the cysteine-rich 61 of phosphoric acid state, and cannot identify
The cysteine-rich 61 of the site Ser167 non-phosphorylating state, it was demonstrated that phospho-AB specificity is good.
5.2 phospho-ABs provided by the invention can be applied to WB (Western blot), ELISA in practical applications
Phosphorylation modification situation after the transcription of detection human CYR 61 albumen in equal immunological experiments, to inquire into human CYR 61 protein phosphorylation
Modify the meaning in tumor-related illness.
The degradation of 5.3 human CYR 61 albumen depends on phosphorylation modification, and the extension of half-life period can make cell continuous proliferation point
Change, therefore phospho-AB is convenient for studying in practical applications er167 phosphorylation modifications of human CYR 61 Protein S for specific life
The influence of object event such as cell Proliferation, cell differentiation, to inquire into its effect during the disease developments such as tumour.
5.4, the present invention be directed to the phosphorylation polyclonal antibody of the specific site the Ser167 preparation of human CYR 61 albumen, facilitate
Research mediates it that the zymogenesis of phosphorylation occurs, and the potential cell-signaling pathways of human CYR 61 albumen is inquired into, to find clinic
The latent effect target spot of the diagnosing and treating of tumor disease.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Sun Yat-sen Memorial Hospital
<120>A kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>For human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide
<220>
<221> NP_BIND
<222> (5)..(5)
<223>Phosphorylation modification
<400> 1
Cys Asp Glu Asp Ser Ile Lys Asp Pro Met
1 5 10
Claims (9)
1. a species specificity is directed to human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide, it is characterised in that：Its active amino acid
Sequence such as SEQ ID NO：Shown in 1, and Ser amino acid therein is phosphorylated modification.
2. a species specificity is directed to human CYR 61 Protein S er167 site phosphorylation antibody, it is characterised in that：It is wanted by right
Specificity described in asking 1 is immunized what animal was prepared for human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide.
3. the preparation method that a species specificity is directed to human CYR 61 Protein S er167 site phosphorylation antibody, it is characterised in that：Including
Following steps：
(1) SEQ ID NO is synthesized：The Antigenic Peptide of active amino acid sequence shown in 1 is added with phosphorylation base on amino acid Ser
Group；
(2) it is coupled using the N-terminal of the Antigenic Peptide of step (1) synthesis with carrier protein hemocyanin, immune animal is simultaneously received
Collect antiserum；
(3) antiserum that step (2) are collected is purified and is identified, obtain specificity for human CYR 61 Protein S er167
Point phospho-AB.
4. preparation method according to claim 3, it is characterised in that：
Antigenic Peptide described in step (1) is synthesized by following steps：
Human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide is prepared, centered on the site cysteine-rich 61 Ser167, N-terminal adjoins
Four CYR61 amino acid sequences, C-terminal connects five amino acid sequence, such as SEQ ID NO：Synthetic peptide shown in 1, and using more
Peptide symthesis technology is synthesized, and adds phosphate group on amino acid Ser167.
5. preparation method according to claim 3, it is characterised in that：
The step of immune animal described in step (2) includes：
The Antigenic Peptide coupling hemocyanin and adjuvant combined immunization new zealand white rabbit synthesized with above-mentioned steps (1)；Immunization ways
To be subcutaneously injected through neck, the position two sides that skin of back is relatively thin, loose, intramuscular injection, waist are distinguished in gluteus and huckle two sides
The intracutaneous injection of portion two sides, the multiple locations multi-point injection such as footpad injection；Immune time is injected including 1 initiation, 3~4 reinforcements note
It penetrates and last time antigen direct injection.
6. preparation method according to claim 3, it is characterised in that：
The step of purifying described in step (3) and identification includes：
Whether the antiserum that step (2) are collected for the potency of purpose antigen peptide and its can with ELISA measuring antiserum
Enough specific recognition purpose antigen peptides, are purified using affine separation-affinity purification circulating technology antagonistic Serum, with
Western Blot tests the cysteine-rich 61 for determining whether antiserum is capable of specific recognition Ser167 site phosphorylation.
7. preparation method according to claim 6, it is characterised in that：
Described the step of being purified using affine separation-affinity purification circulating technology antagonistic Serum includes：
It is affine that antibody is carried out using the affinity chromatography of the pure albumen of synthetic peptide coupling carrier proteins Bovine and agarose chromatography column
Separation and affinity purification；First using non-phosphorylating synthetic peptide coupling bovine serum albumin(BSA) and filler of the agarose as chromatographic column
The affine non-phosphorylating antibody being separated off in antiserum is carried out, phosphorous acidification antibody efflux is obtained；Then phosphorylation is used
The filler progress affinity purification of synthetic peptide coupling bovine serum albumin(BSA) and agarose as chromatographic column, removes low in phospho-AB
The low sequence complexity epitope antibodies of affinity.
8. specificity described in claim 1 is for human CYR 61 Protein S er167 site phosphorylation Antigenic Peptide in preparation for swelling
The application in pharmaceutical preparation that tumor, hematological system, the diagnosis of disease of cardiovascular system, treatment and prognosis determine.
9. it is as claimed in claim 2 specificity for human CYR 61 Protein S er167 site phosphorylation antibody preparation for tumour,
The application in pharmaceutical preparation that hematological system, the diagnosis of disease of cardiovascular system, treatment and prognosis determine.
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