CN108840920B

CN108840920B - Human CYR61 protein Ser167 site phosphorylation antigen and antibody, and preparation method and application thereof

Info

Publication number: CN108840920B
Application number: CN201810629175.7A
Authority: CN
Inventors: 尹东; 胡开顺; 李瑜; 林乐航; 吴雯静
Original assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2018-06-19
Filing date: 2018-06-19
Publication date: 2021-09-28
Anticipated expiration: 2038-06-19
Also published as: CN108840920A

Abstract

The invention discloses a phosphorylation antigen of a Ser167 site of a human CYR61 protein, an antibody, and a preparation method and application thereof, and relates to the technical field of antibodies and preparation thereof. The antibody is produced by the amino acid sequence of SEQ ID NO: the active amino acid sequence shown in 1 is prepared by immunizing animals with antigen peptide. The phosphorylation antibody provided by the invention is beneficial to researching the kinase mediating the phosphorylation of the phosphorylation antibody and discussing various biological functions of CYR61 protein; in practical application, the method is convenient for researching the action mechanism of phosphorylation modification of a specific site of human CYR61 protein in the occurrence and development process of specific biological events or diseases, and can also be used for detecting the difference of tumor-related protein expression after drug administration, thereby providing a potential action target for diagnosis and treatment of clinical tumor diseases.

Description

Human CYR61 protein Ser167 site phosphorylation antigen and antibody, and preparation method and application thereof

Technical Field

The invention relates to the technical field of antibodies and preparation thereof, and particularly relates to a specific phosphorylation antigen of Ser167 site of human CYR61 protein, an antibody, and a preparation method and application thereof.

Background

The CCN family consists of 6 proteins with similar functional domains, and CYR61 is the first protein found in this family. This family of proteins has long been considered secreted and is well studied as a cell matrix protein. CCN family proteins are synthesized intracellularly and secreted extracellularly under the guidance of signal peptides, and they act intracellularly and extracellularly in tumor development and progression with a series of changes in the internal and external environment of the tumor. The family proteins are involved in physiological processes such as cell adhesion, division, migration, drug resistance, survival, differentiation, angiogenesis, fibrosis, osteogenesis and wound healing, many of which are associated with tumors. CYR61(CCN1), CTGF (CCN2) and CCN5 knockout mice often die at embryonic or early birth.

The biological functions exhibited by CYR61 in tumors are diverse, and CYR61 functions are also different in different tumor tissue cells. CYR61mRNA is highly expressed in malignant breast tumor cells, and can also promote the deterioration of gastric adenocarcinoma cells RF-1 cells; meanwhile, CYR61 is highly expressed in ovarian cancer and is positively correlated with lymph node metastasis. However, the expression level of CYR61 in lung cancer and leiomyoma is obviously reduced, and the CYR61 plays an important role as a tumor suppressor. For example, in non-small cell lung cancer, CYR61 is expressed less than in paracancerous normal lung tissue and is associated with factors such as the type of lung cancer pathology, lymph node metastasis, and the like.

The current research shows that: the CYR61 protein is mainly related to the proliferation and migration of tumor cells, the growth regulation of tumor cells and the formation of tumor blood vessels, and the action mechanism of the protein can be completed through related signal paths. Overexpression of CYR61 in tissues may also promote expression of its receptor AV β 3, forming a "CYR 61-AC β 3 autocrine regulatory loop" that generates cell growth and anti-apoptotic signals. In breast cancer, HRG up-regulates the level of AV beta 3 through CYR61, and CYR61 and AV beta 3 are combined to activate an ERK1/ERK2MAPK signal pathway, inhibit the aggregation of P53 and generate the effect of resisting taxol. In glioma cells, CYR61 glycogen via integrin-coupled kinase (ILK)The kinase-forming-3 beta (GSK 3-beta) is phosphorylated to activate a beta-catenin/LEF pathway, thereby promoting the expression of cell proliferation genes, and the CYR61 can also promote the activity reduction of an apoptosis protein Bad and promote the growth and migration of cells by activating a PI3K/AKT signal pathway. In lung cancer, CYR61 plays an important role as a tumor suppressor. In vitro experiments, CYR61 resulted in G₀/G₁The retardation of stage can inhibit the growth of lung cancer cells.

Post-translational modifications such as phosphorylation, ubiquitination, etc., play a key role in physiological and pathological processes mediated by cellular signaling pathways. In view of the important role played by CYR61 in the development and progression of tumors, it is of great significance to further study the posttranslational modification of CYR61 protein. We found in previous researches that Ser167 site in CYR61 amino acid sequence is the site of protein phosphorylation modification, and may play an important role in regulating the stability of CYR61 protein. The antibody is an important tool for protein function research, and has been widely used in clinical applications such as diagnosis and treatment of diseases such as tumors, so that the antibody specifically recognizing the Ser167 phosphorylation site of human CYR61 is to be further developed.

Disclosure of Invention

In order to solve the problem of effective research on phosphorylation modification of CYR61 protein, the primary object of the present invention is to provide an antigenic peptide specifically aiming at Ser167 site of human CYR61 protein.

Another purpose of the invention is to provide an antibody specifically aiming at the Ser167 site phosphorylation of the human CYR61 protein.

Another object of the present invention is to provide uses of the above-mentioned antigenic peptides and antibodies.

It is still another object of the present invention to provide a method for producing the above antibody.

The purpose of the invention is realized by the following technical scheme:

the active amino acid sequence of the specific phosphorylation antigen peptide aiming at the Ser167 site of the human CYR61 protein is shown as SEQ ID NO: 1, wherein the Ser amino acid is modified by phosphorylation.

The specific phosphorylation antibody aiming at the Ser167 site of the human CYR61 protein is prepared by immunizing an animal with the specific phosphorylation antigen peptide aiming at the Ser167 site of the human CYR61 protein.

The preparation method of the specificity phosphorylation antibody aiming at the Ser167 site of the human CYR61 protein comprises the following steps:

(1) synthesis of SEQ ID NO: 1, wherein a phosphorylation group is added to amino acid Ser;

(2) coupling the N terminal of the antigen peptide synthesized in the step (1) with a carrier protein, namely hemocyanin (KLH), immunizing animals and collecting antiserum;

(3) and (3) purifying and identifying the antiserum collected in the step (2) to obtain a specific Ser167 site phosphorylation antibody of the human CYR61 protein.

The antigenic peptide described in the above step (1) can be synthesized by the following steps:

preparing a phosphorylation antigen peptide of a Ser167 site of a human CYR61 protein, taking the Ser167 site of a CYR61 protein as a center, wherein the N end is adjacent to four CYR61 amino acid sequences, and the C end is connected with five amino acid sequences, such as SEQ ID NO: 1, and synthesizing by adopting a polypeptide synthesis technology, and adding a phosphorylation group on an amino acid Ser 167.

The step of immunizing an animal as described in the above step (2) may comprise:

the antigen peptide synthesized in the step (1) is coupled with hemocyanin and adjuvant to jointly immunize a New Zealand white rabbit; the immunization mode is subcutaneous injection through two sides of a part with thinner and loose skin at the neck and the back, intramuscular injection at two sides of gluteus and thighs respectively, intradermal injection at two sides of the waist, claw pad injection and other multi-position multi-point injection; the immunization times comprise 1 priming injection, 3-4 boosting injections and the last direct antigen injection.

The purification and identification steps described in the above step (3) may include:

and (3) measuring the titer of the antiserum collected in the step (2) against the target synthetic peptide (antigenic peptide) and whether the antiserum can specifically recognize the target synthetic peptide (antigenic peptide) by using an ELISA (enzyme-linked immuno sorbent assay) experiment, purifying the antiserum by using an affinity separation-affinity purification cycle technology, and determining whether the antiserum can specifically recognize the CYR61 protein phosphorylated at the Ser167 site by using a Western Blot experiment.

The above step of purifying antisera using the affinity separation-affinity purification cycling technique may comprise:

performing antibody affinity separation and affinity purification by affinity chromatography of synthetic peptide coupled carrier protein Bovine Serum Albumin (BSA) and agarose chromatographic column; firstly, adopting non-phosphorylated synthetic peptide coupled Bovine Serum Albumin (BSA) and agarose as fillers of a chromatographic column to carry out affinity separation to remove non-phosphorylated antibodies in antiserum, and obtaining effluent liquid containing phosphorylated antibodies; then, the low-affinity epitope antibody with low sequence complexity in the phosphorylated antibody is removed by affinity purification using phosphorylated synthetic peptide (antigen peptide) coupled with Bovine Serum Albumin (BSA) and agarose as the packing material of the chromatography column.

The specificity is directed at the application of the human CYR61 protein Ser167 site phosphorylation antigen peptide and/or antibody in the preparation of pharmaceutical preparations for the diagnosis, treatment and prognosis judgment of tumor, blood system and cardiovascular system diseases.

The invention also provides a pharmaceutical preparation for diagnosis, treatment and prognosis determination of diseases of tumors, blood systems and cardiovascular systems, which comprises the specific phosphorylation antigenic peptide and/or antibody of Ser167 site of human CYR61 protein.

The high-specificity phosphorylation antibody aiming at Ser167 locus of human CYR61 protein prepared by the invention can be used for detecting expression difference of normal cells, tumor cells and tumor cells after being used with drugs by Western blot experiment, is beneficial to researching the effect of phosphorylation modification of CYR61 protein in the occurrence and development process of tumor diseases, provides potential effect targets for the diagnosis or treatment of clinical tumor diseases, can also be used for detecting the phosphorylation level of human CYR61 protein by IHC, ELISA and other immunology related experiments, discusses the relation between the phosphorylation level and diseases of tumor, blood system, cardiovascular system and the like, and has wide clinical application prospect in the aspects of disease diagnosis, treatment, prognosis and the like.

Compared with the prior art, the invention has the following advantages and effects:

(1) the phosphorylation antibody provided by the invention can detect the posttranslational phosphorylation modification condition of human CYR61 protein in practical application;

(2) in the practical application of the phosphorylation antibody provided by the invention, the correlation of phosphorylation modification of a specific site of human CYR61 protein in specific biological events such as tumor cell chemotherapy resistance, DNA loss, cell cycle and the like is conveniently researched;

(3) the invention aims at the phosphorylation polyclonal antibody of the Ser167 site of the human CYR61 protein, is beneficial to researching the kinase mediating the phosphorylation of the human CYR61 protein and discussing various biological functions of the CYR61 protein;

(4) the phosphorylation antibody provided by the invention is beneficial to discussing the action mechanism of phosphorylation modification of human CYR61 protein in the occurrence and development process of tumor diseases, and can also be used for detecting the difference of tumor-related protein expression after medication, thereby providing a potential action target for the diagnosis and treatment of clinical tumor diseases.

Drawings

FIG. 1 is a map of the structural division of human CYR61 protein.

FIG. 2 is a technical scheme for preparing and purifying a highly specific human CYR61 phosphorylated antibody according to an embodiment of the present invention.

FIG. 3 shows the result of a query of the PhohosSitePlus database for Ser167 site of human CYR61 protein.

FIG. 4 is a schematic diagram of the amino acid antigenicity analysis around Ser167 site of human CYR61 protein.

FIG. 5 is a graph showing the result of Western blot screening of serum before immunization, wherein the ratio of 1: BSA standard protein (5 μ g); 2: ser167-BSA non-phosphorylated synthetic peptide (5. mu.g); 3: pSer167-BSA phosphorylated synthetic peptide (5. mu.g); a first antibody: negative serum 1:5000 dilution.

FIG. 6 shows the specificity of serums before and after being purified by Western blot detection on synthetic peptide phosphorylated at Ser167 site; in fig. a, 1: BSA standard protein (5 μ g); 2: pSer167-BSA synthetic peptide (5. mu.g); 3: ser167-BSA synthetic peptide (5. mu.g); a first antibody: the 167 th antiserum was diluted 1:5000 before purification. In fig. B, 1: BSA standard (5 μ g); 2: ser167-BSA synthetic peptide (5. mu.g); 3: pSer167-BSA synthetic peptide (5. mu.g); a first antibody: the 167 th antiserum was purified in 1:500 dilution.

FIG. 7 is a diagram showing the data of ELISA detection of a purified Ser167 site phosphorylated antibody (designated as p-CYR61-S167 antibody) on a phosphorylated antigen and a non-phosphorylated antigen.

FIG. 8 is a cell level demonstration of the specificity of the p-CYR61-S167 antibody.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The structural partition map of human CYR61 protein is shown in figure 1.

Referring to fig. 2, the preparation method and application of the antibody for phosphorylation of Ser167 site of human CYR61 protein provided by the embodiment of the present invention generally include the following steps:

the method comprises the following steps: firstly, screening a site which is possibly phosphorylated in an amino acid sequence of human CYR61 through biological information software, determining a Ser167 site of human CYR61 protein through mass spectrum and a database (figure 3), analyzing the antigenicity of the site (figure 4), designing a corresponding antigenic peptide, and analyzing the homology;

step two: synthesizing an antigen peptide containing a Ser167 phosphorylation site: respectively synthesizing 10 amino acid antigen peptides containing Ser167 phosphorylation sites designed in the first step by adopting a polypeptide synthesis technology, wherein a phosphorylation group is added to the Ser167 of the amino acid, and the amino acid is coupled with carrier protein hemocyanin (KLH) to be used as immunogen for preparing a phosphorylation antibody; coupling with Bovine Serum Albumin (BSA) as carrier protein for affinity purification; correspondingly, non-phosphorylation modified synthetic peptides are respectively synthesized and coupled with a carrier protein Bovine Serum Albumin (BSA) to be used as a filler for affinity separation, and all the synthetic peptides are purified by HPLC;

step three: immunizing animals with whole antigens and collecting antiserum: and (2) respectively carrying out combined immunization on the Ser167 site phosphorylation synthetic peptide (antigenic peptide) -KLH coupled holoantigen and the adjuvant in the second step on the SPF-level New Zealand white rabbit, and carrying out subcutaneous (s.c) injection on two sides of a part with thinner and loose skin on the neck and back, intramuscular (i.m) injection on two sides of the gluteus and the thigh, intradermal (i.d) injection on two sides of the waist, and multi-site multi-point injection of the rabbit paw pad, and the like. The immunization times comprise 1 priming injection, 3-4 boosting injections and the last direct antigen injection, and the titer of the antiserum for the target synthetic peptide is determined by ELISA;

step four: purifying and identifying a Ser167 site-specific phosphorylation antibody of the human CYR61 protein: the antibody affinity separation-affinity purification cycle purification technology is carried out by adopting an affinity chromatography method of synthetic peptide coupled Bovine Serum Albumin (BSA) and an agarose chromatographic column. Firstly, respectively using non-phosphorylation synthetic peptide corresponding to Ser167 locus coupled Bovine Serum Albumin (BSA) and agarose as fillers of a chromatographic column to carry out affinity separation to remove non-phosphorylation antibody in antiserum, and obtaining effluent containing phosphorylation antibody; performing affinity purification by using phosphorylated synthetic peptide (antigenic peptide) coupled Bovine Serum Albumin (BSA) and agarose as a filler of a chromatographic column for the second time, removing low-affinity low-sequence complex epitope antibodies in the phosphorylated antibodies, and determining the specificity recognition of the phosphorylated synthetic peptide (antigenic peptide) by antiserum through a Western blot experiment; finally, respectively obtaining purified anti-p-Ser 167 phosphorylation antibodies, which are named as p-CYR61-S167 antibodies, preserving the antibodies by using a mixed solution of 2.5% (wt/vol) BSA, 0.01% (vol/vol) Tween-20 and 25% (vol/vol) glycerol, detecting the antibody titer after purification and the identification of synthetic peptides (antigen peptides) and non-phosphorylation synthetic peptides aiming at phosphorylation by ELISA, and finally carrying out antibody identification by using Western blot experiment.

The technical route for preparing the phosphorylated antibody of Ser167 site of human CYR61 protein provided by the invention is further illustrated and explained by referring to the attached drawings in combination with the specific examples.

EXAMPLE 1 determination of phosphorylation sites of human CYR61 protein

1.1 firstly screening sites which are possibly phosphorylated in human CYR61 protein by bioinformatics software Netphorest 2.0 and Netphos 2.0, and then confirming that Ser167 is a phosphorylation site in the amino acid sequence of human CYR61 by mass spectrometry literature reference and search of protein phosphorylation site database PhohosiTeplus (figure 3); meanwhile, the antigenicity of the amino acid sequence of human CYR61 is calculated by using software CLC Protein Workbench 5 (figure 4), and finally the specific phosphorylation site Ser167 of human CYR61 Protein is determined.

1.2 designing a Ser167 site phosphorylation antigen peptide of the human CYR61 protein. Taking a Ser167 site of human CYR61 protein as a center, wherein the N end is adjacent to four CYR61 amino acid sequences, the C end is connected with five CYR61 amino acid sequences, a designed synthetic peptide (antigen peptide) sequence is obtained, a phosphorylation group is added on the amino acid Ser167, and the peptide segment sequence is as follows: CDEDS (p) IKDPM.

Example 2 Synthesis of synthetic peptide (antigenic peptide) comprising Ser167 phosphorylation site

According to the design of hapten synthetic peptide, a phosphorylation group is added on a Ser167 site to obtain phosphorylation synthetic peptide (antigen peptide), and the phosphorylation synthetic peptide is coupled with hemocyanin (KLH) to obtain a holoantigen for rabbit immunization (pSer 167-KLH). The phosphorylated synthetic peptide (antigenic peptide) was further coupled with Bovine Serum Albumin (BSA) by the glutaraldehyde method and used as a filler for an affinity purification column (pSer 167-BSA). Correspondingly, a section of non-phosphorylated synthetic peptide corresponding to Ser167 was synthesized and coupled with Bovine Serum Albumin (BSA) to be used as a packing material for an affinity separation chromatography column (Ser 167-BSA).

Antiserum was prepared from the whole antigen described in example 3 by conventional methods for polyclonal antibody preparation

3.1 preparation of negative sera: 3mL of blood was collected from the auricular vein of New Zealand white rabbits (2-3 kg, female, healthy, purchased from Central institute of sciences, Silake laboratory animals) for injection into a blood collection tube, and hemostasis was performed by compression with a cotton ball. Standing at room temperature for about 1h until blood clots are formed, standing at 4 deg.C for 2h to precipitate serum, centrifuging at 2500g for 10min, collecting supernatant, marking as negative control serum, subpackaging and storing at-20 deg.C for testing.

3.2 screening before immunization-Western blot: respectively taking 5 μ g of BSA standard, Ser167-BSA, and pSer167-BSA, adding appropriate 5 xSDS loading buffer, boiling in boiling water bath for 10min to denature protein, and centrifuging at 10000 × g for 1 min; 8% of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation gel and 5% of concentrated gel; loading by using a loading gun according to a preset sequence, and adding an equal volume of 1 xSDS gel loading buffer solution into the unused sample holes; after electrophoresis at 80V for 20min, changing the voltage to 120V for about 50min until bromophenol blue reaches the bottom of the separation gel, and turning off the power supply. Film transferring conditions: constant current 300mA, time 120 min. 5% skimmed milk powder is sealed, and the shaking table is at 37 ℃ for 1 h. And (3) placing the transfer printing film into an antiserum diluent prepared by using an anti-dilution buffer solution according to the ratio of 1:5000, horizontally and slowly shaking up, and standing overnight at 4 ℃. The following day the membrane was washed with 1 XPBST for 10min and repeated 4 times. The membrane was placed in secondary antibody dilution of HRP-labeled goat anti-rabbit IgG diluted 1:5000 in 1 XPBST for 60min at 37 ℃ on a shaker. Discard the secondary antibody solution, wash the membrane for 10min with 1 XPBST, repeat 3 times. ECL kit was used for development and recorded by photography according to the manufacturer's instructions. The results are shown in FIG. 5: no target band, that is, no antibody against the target tissue or cell extract, appears, and this is an ideal experimental animal.

3.3 animal immunization: about 73 days

3.3.1 separately dissolving pSer167-KLH synthetic peptide powder in sterile 1 XPBS, sucking antigen solution with one sterile syringe and equal amount of Freund's complete adjuvant (CFA) with the other syringe, connecting the two syringes with a plastic tube, repeatedly sucking back and forth, and mixing pSer167-KLH peptide fragments with CFA until completely emulsified emulsion is formed, and the emulsion is dropped in water without diffusion.

3.3.2 for the first immunization, subcutaneous (s.c) injection is respectively carried out on two sides of the part with thin and loose skin on the neck and the back, muscle (i.m) injection is respectively carried out on two sides of the gluteus and the thigh, intradermal (i.d) injection is carried out on two sides of the waist, and antigen emulsion is injected into multiple parts and multiple points such as rabbit paw pad part injection. The total amount of first immunization for both antigens was about 0.61 mg.

3.3.3 first immunization 20 days later, first booster immunization was performed, and antigen emulsion was prepared using Freund's incomplete adjuvant (IFA) instead of CFA as an immune adjuvant and injected in the first immunization mode, and the total amount of antigen in this booster immunization was about 0.9 mg.

3.3.4 immunization 12 days later, a second booster immunization was performed, and an antigen emulsion was prepared using Freund's incomplete adjuvant (IFA) instead of CFA as an immunoadjuvant and injected in the primary immunization mode, and the total amount of the antigen in this booster immunization was about 0.9 mg.

3.3.5 after 3 days of the last booster immunization, the rabbits were subjected to abdominal aorta large blood collection and a large amount of serum was collected. The beaker with the collected blood was closed and allowed to stand overnight at room temperature to shrink the clot. The next day, the precipitated serum was aseptically dispensed into 50mL centrifuge tubes, centrifuged at 4000g for 10min, the supernatant was collected and dispensed into 1 mL/tube, labeled as immunized antiserum (total about 51mL), and stored at-20 ℃.

3.3.6 measurement of the titer of antiserum after immunization, the specific steps are as follows:

3.3.6.1 phosphorylated antigen pSer167-BSA and non-phosphorylated antigen Ser167-BSA were coated with antigen coating solution (CBS), added to 96-well microplate, each well was 0.1mL, the plate was covered and shaken well, and coated overnight (12h or more) at 4 ℃.

3.3.6.2 the wells were discarded after coating, and each well of the plate was washed thoroughly with 1 XPBST, discarded and the wash was repeated 3 times, each wash being followed by draining the residual liquid on the filter paper. Blocking buffer (0.25% BSA/PBST), 200. mu.L/well, incubation at 37 ℃ for 2h, washing the plate 3 times with 1 XPBST, and draining the residual liquid on the filter paper after each wash.

3.3.6.3 the immune antiserum was diluted with 1 XPBST at a ratio of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, 1:729000, and added to a 96-well microplate with 1 XPBS buffer as a blank, the plate was sealed and incubated at 37 ℃ for 1 h.

3.3.6.4 the wells were discarded and the plate was washed 3 times with 1 XPBST. A dilution of HRP-labeled goat anti-rabbit IgG secondary antibody diluted 1:5000 in 1 XPBST was added, 100. mu.L/well and incubated at 37 ℃ for 1 h. The plate was capped and incubated at 37 ℃ for 1 h. The plate was washed 5 times with 1 XPBST and dried. Adding the temporarily prepared TMB color developing solution, 100 mu L/hole, and reacting for 30min at room temperature in a dark place. 2M H was added₂SO₄The reaction was stopped, 50. mu.L/well. And measuring the OD value of each hole by a microplate reader at 450 nm.

3.3.6.5 the ELISA detection values of antiserum after immunization with the Ser167 antigen are all above 0.5 (see Table 1 below) with the antiserum dilution of 1:1000, 1:3000 and 1:9000 after immunization;

TABLE 1 OD values of antiserum ELISA detection after Ser167 antigen immunization

Dilution of original serum	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

Example 4 purification and identification of Ser167 specific phosphorylation site of human CYR61 protein

4.1 preparation of phosphorylated synthetic peptide chromatography columns and non-phosphorylated synthetic peptide chromatography columns.

The synthetic peptide coupling chromatographic column is prepared by using Thermo scientific

The preparation method of the applying Resin kit comprises the following specific steps:

4.1.1 the Ser167-BSA and pSer167-BSA were dissolved in coupling buffer, respectively, at concentrations of 0.7 mg/mL. Adding 7mg of Ser167-BSA and 6mg of pSer167-BSA dissolving solutions into 5mL of resin, mixing uniformly, adding into a chromatographic column at room temperature, mixing uniformly for 15 minutes, rightly placing the chromatographic column at room temperature for 30 minutes, taking down caps at the upper end and the lower end of the chromatographic column respectively, collecting effluent liquid, and washing the column by using a coupling buffer solution with 3 times of volume of resin.

4.1.2 cover the bottom of the chromatographic column, add 50mM L-Cysteine & HCl to the coupling buffer solution, mix well, take the buffer solution of the same volume to the chromatographic column, mix well for 15 minutes at room temperature, then stand for 30 minutes.

4.1.3 remove the bottom cover, release the coupling buffer, wash the column with 6 volumes of nasal wash (1M NaCl), then wash the column with 2 volumes of storage buffer, cover the cover, add 1 volume of storage buffer, store at 4 degrees for use.

4.2 purification of phosphorylated antibodies

The antibody was purified using Thermo scientific

4.2.1 remove the bottom cap of the column, release the storage buffer, add 6mL of binding buffer to wash the column. Adding the Ser167 antiserum into the corresponding non-phosphorylation chromatographic column, collecting the effluent liquid, repeating twice to obtain Ser167 effluent liquid, then washing the chromatographic column with 12mL of nasal wash liquid, retaining the eluent reserved at the bottom end, and finally eluting the chromatographic column with the eluent to obtain the corresponding non-phosphorylation antibody.

4.2.2 adding the effluent collected in 4.2.1 to the corresponding washed phosphorylation column, washing the column with 12mL of nasal wash, eluting the column with the eluent to obtain the corresponding Ser167 phosphorylation antibody, dissolving the antibody in 1 XPBS, adding 0.1% NaN₃And (5) standby.

Detecting the specificity condition of serum to Ser167 locus phosphorylation synthetic peptide before and after purification by Western blot; as shown in fig. 6; in fig. a, 1: BSA standard protein (5 μ g); 2: pSer167-BSA synthetic peptide (5. mu.g); 3: ser167-BSA synthetic peptide (5. mu.g); a first antibody: the 167 th antiserum was diluted 1:5000 before purification. In fig. B, 1: BSA standard (5 μ g); 2: ser167-BSA synthetic peptide (5. mu.g); 3: pSer167-BSA synthetic peptide (5. mu.g); a first antibody: the 167 th antiserum was purified in 1:500 dilution. The result shows that the purified Ser167 locus antiserum can specifically recognize the phosphorylated Ser167 peptide segment and not recognize the non-phosphorylated Ser167 peptide segment.

4.3 the purified phosphorylated antibody titer and the recognition of the phosphorylated target synthetic peptide (antigen peptide) and non-phosphorylated synthetic peptide were measured again by ELISA. Firstly, artificially synthesized phosphorylated synthetic peptide antigen-BSA and non-phosphorylated synthetic peptide antigen-BSA are respectively coated, diluted serum before purification and purified antiserum in different proportions are added for incubation, an HRP-labeled goat anti-rabbit IgG diluent is added after washing, washing is carried out after incubation, TMB is developed, and OD450 is measured after reaction termination. The results are shown in FIG. 7: when the dilution of the Ser167 phosphorylated antibody is 1:9000, the OD450 value aiming at the phosphorylated antigen is more than 0.75, the OD450 value aiming at the non-phosphorylated antigen is less than 0.25, and the P/N value is more than 3, the titer of the Ser167 phosphorylated antibody after purification is at least 1: 9000.

Example 5 application of Ser167 site phosphorylation antibody of human CYR61 protein in tumor

5.1 the phosphorylation antibody of Ser167 site of human CYR61 protein prepared by the invention with high specificity can be used for detecting the difference of cell phosphorylation levels by Western blot experiment, as shown in FIG. 8, 293T cells (sold in the market) are transfected with Flag-CYR61-WT (the protein coding sequence of CYR61 gene of human is cloned to overexpression Vector pcDNA3.1(+) Vector [ invitrogen ], a recombinant Vector Flag-CYR61-WT) and a Flag-CYR61-S167A plasmid (a plasmid obtained by mutating 167Ser of Flag-CYR61-WT plasmid to alanine) are obtained, after 24h, cell protein is extracted, in immunoprecipitation Flag products, the phosphorylation antibody of Ser167 site (named as p-CYR61-S167) can recognize CYR61 protein with Ser167 site in a phosphorylation state, but cannot recognize CYR61 protein with Ser167 site in a non-phosphorylation state, and the phosphorylation antibody is proved to have good specificity.

5.2 the phosphorylation antibody provided by the invention can be applied to WB (Western blot), ELISA and other immunological experiments to detect the phosphorylation modification condition of human CYR61 protein after transcription, thereby discussing the significance of the phosphorylation modification of human CYR61 protein in tumor-related diseases.

5.3 degradation of human CYR61 protein depends on phosphorylation modification, and the prolongation of half-life thereof can lead cells to continuously proliferate and differentiate, so that the effect of the phosphorylation modification of Ser167 site of human CYR61 protein on specific biological events such as cell proliferation and cell differentiation can be conveniently researched in practical application of the phosphorylated antibody, thereby discussing the effect of the phosphorylated antibody in the process of generating and developing diseases such as tumor and the like.

5.4 the invention is a phosphorylated polyclonal antibody prepared aiming at the specific Ser167 site of the human CYR61 protein, which is helpful for researching the kinase action for mediating the phosphorylation of the human CYR61 protein and discussing the potential cell signal path of the human CYR61 protein, thereby finding the potential action target spot for the diagnosis and treatment of clinical tumor diseases.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> grand era memorial Hospital of Zhongshan university

<120> human CYR61 protein Ser167 site phosphorylation antigen and antibody, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> phosphorylation antigen peptide aiming at Ser167 site of human CYR61 protein

<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

1. A specific phosphorylation antigen peptide aiming at Ser167 site of human CYR61 protein, which is characterized in that: the active amino acid sequence is CDEDS (p) IKDPM, and the amino acid sequence is shown as SEQ ID NO: 1, wherein the Ser amino acid is modified by phosphorylation.

2. The use of the specific peptide for phosphorylation of Ser167 site of human CYR61 protein according to claim 1 in the preparation of a formulation for detecting phosphorylation of Ser167 site of human CYR61 protein.