CN111826365A - Phosphorylated antigen polypeptide, antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a phosphorylated antigen polypeptide, an antibody and a preparation method thereof, and relates to the technical field of antibody preparation. The invention comprises a phosphorylation antibody aiming at threonine 202 and tyrosine 204 of human ERK protein, the amino acid sequence of which is shown as SEQ ID NO 1, wherein Thr and Tyr are mutated into Asp; the amino acid sequence of the phosphorylated antibody aiming at the 127 th serine of the YAP1 protein is shown in SEQ ID NO. 2, wherein Ser is mutated into Asp. The invention mutates phosphorylated amino acid into aspartic acid, constructs DNA of mutant antigen on pGEX-6p expression vector, uses colon bacillus to express GST fusion antigen, and can directly immunize animals after the purified antigen is mixed with Freund's adjuvant.

Description

Phosphorylated antigen polypeptide, antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of antibody preparation, and particularly relates to a preparation method of a phosphorylation antigen antibody specifically aiming at threonine 202 and tyrosine 204 of human ERK protein and serine 127 of YAP1 protein.

Background

The antibody is an important tool for protein function research, and has been widely applied to clinical applications such as diagnosis and treatment of diseases such as tumors, so that a preparation method of the antibody for specifically recognizing protein phosphorylation sites is to be further developed.

Currently, the main methods for producing phosphorylated antibodies are: solid phase synthesis of phosphorylated polypeptide antigen, and coupling of antigen to hemocyanin (KLH) or Bovine Serum Albumin (BSA) to obtain fusion antigen for immunization of animals. In the traditional method, the synthesized short peptide is unstable and has poor solubility, low fusion efficiency with carrier protein and high cost.

The invention solves the problem by using a genetic engineering method, because aspartic acid is similar to a phosphorylation group spatial structure and an antigen structure, the amino acid to be phosphorylated is mutated into aspartic acid, DNA of the mutation antigen is constructed on a pGEX-6p expression vector, escherichia coli is used for expressing a GST fusion antigen, and the purified antigen is mixed with Freund's adjuvant to directly immunize animals. The invention takes ERK and YAP1 protein as examples to illustrate the preparation process of the phosphorylated antigen antibody.

Extracellular regulated protein kinases (ERKs), including ERK1 and ERK2, are important components of the MAP kinase signal transduction pathway. When the threonine and tyrosine residues of ERK are subjected to dual-specificity phosphorylation, the ERK is activated, the activated ERK is transferred into cell nucleus from cytoplasm, and then transcription activation of ATF, Ap-1, Elk-1, c-fos, c-Jun and the like is mediated, and the activated ERK participates in various biological reactions such as cell proliferation and differentiation, cytoskeletal rearrangement, apoptosis, cell canceration and the like. Over-activation of ERK can be found in many cancers (oral cancer, melanoma, breast cancer, etc.), and therefore phosphorylated ERK can be an important target in cancer diagnosis and treatment.

Yes-associated protein 1 (YAP 1) is a core molecule in the Hippo signaling pathway of tumor suppressor pathway; normally, YAP1 in the pathway is phosphorylated, it is retained in the cytoplasm, inhibits cell proliferation, promotes apoptosis; if mutation occurs in upstream or downstream molecules in the pathway, phosphorylation of YAP1 is inhibited, and non-phosphorylated YAP1 enters the cell nucleus to be combined with TEAD1-4 or other transcription factors, so that the expression of proliferation promoting and apoptosis inhibiting genes is up-regulated, and finally, cell proliferation is promoted to generate tumors. The YAP1 protein is a key site for mediating the growth inhibition effect of Hippo pathway at serine phosphorylation site 127. The study of YAP1 helps us to understand the anti-apoptotic mechanism of tumor, and YAP1 is highly expressed in many tumor cells, so it can also be used as a tumor marker.

The prior patent 201611096418.2 discloses a novel human NOTCH1NICD protein antigen, antibody, preparation method and application thereof. The provided phosphorylation antibody can study the correlation between phosphorylation modification of specific sites of human NOTCH1NICD protein and specific biological events such as cell stress, genome and epigenetic group change, cell cycle abnormality and the like in practical application, and study the action mechanism of NOTCH signals under the conditions of cell physiology and pathology, and the main technical scheme is to prepare the phosphorylation antibody of the Tyr2145 sites of the human NOTCH1NICD protein with high specificity and high affinity.

Disclosure of Invention

The invention aims to provide a phosphorylated antigen polypeptide, an antibody and a preparation method thereof, wherein phosphorylated amino acid is mutated into aspartic acid, DNA of the mutated antigen is constructed on a pGEX-6p expression vector, escherichia coli is used for expressing GST fusion antigen, and the purified antigen can be mixed with Freund's adjuvant to directly immunize animals, so that the problems of unstable synthesized short peptide, poor solubility, low fusion efficiency with a carrier protein and high cost in the conventional method are solved.

In order to solve the technical problems, the invention is realized by the following technical scheme: a phosphorylated antigen polypeptide, which is a point mutation antigen peptide at Thr202 and Tyr204 sites of ERK protein, or a point mutation antigen peptide at Ser127 site of YAP1 protein; the amino acid sequence of the phosphorylated antibody for Thr202 and Tyr204 sites of human ERK protein is shown in SEQ ID NO 1, wherein Thr and Tyr are mutated into Asp. The phosphorylation antibody aiming at the Ser127 site of the human YAP1 protein is the phosphorylation antibody aiming at the serine 127 site of the YAP1 protein, the amino acid sequence of the phosphorylation antibody is shown as SEQ ID NO:2, wherein Ser is mutated into Asp.

A method for preparing a phosphorylated antibody, comprising the steps of:

(1) obtaining an antigenic peptide with active amino acid as an amino acid sequence shown in SEQ ID NO. 1, and point mutation antigenic peptides of the sites Thr202 and Tyr204 of the ERK protein, wherein Thr and Tyr are mutated into Asp; and an antigen peptide with active amino acid of an amino acid sequence shown in SEQ ID NO. 2, and a point mutation antigen peptide at a Ser127 site of YAP1 protein, wherein Ser is mutated into Asp;

(2) amplifying partial gene fragments of ERK and YAP1, cloning the partial gene fragments into a pGEX-6P expression vector, and expressing proteins by escherichia coli;

(3) amplifying partial gene fragments of ERK and YAP1, cloning the partial gene fragments into a pcDNA-GFP expression vector, and transfecting HEK293T cells to obtain ERK and YAP1 over-expression cell samples;

(4) obtaining fusion protein through affinity purification;

(5) immunizing a mouse or a New Zealand rabbit with the fusion protein, and taking serum;

(6) the phosphorylated antibody is obtained by means of affinity purification.

Further, the specific process of the step (2) is as follows:

constructing recombinant plasmids pGEX-6p-ERK-pTY and pGEX-6p-YAP1-p 127;

primers were designed and the restriction sites EcoRI and Xho I were introduced:

YAP1-p127-5:AATTCACTCCACAGCATGTTCGAGCTCATGACTCTCCAGCTTCTCTGCAGTTGTGAC；

YAP1-p127-3:TCGAGTCACAACTGCAGAGAAGCTGGAGAGTCATGAGCTCGAACATGCTGTGGAGTG；

ERK-pTY-5:AATTCGACCATGATCACACAGGGTTCCTGGACGAAGACGTGGCCACACGTTGGTAGC；

ERK-pTY-3:TCGAGCTACCAACGTGTGGCCACGTCTTCGTCCAGGAACCCTGTGTGATCATGGTCG；

annealing primers to form dimers, said primers annealing to form dimers under conditions: denaturation at 94 deg.C for 4min, binding at 75 deg.C for 5min, binding at 65 deg.C for 15min, and binding at 25 deg.C for 20min to form stable dimer; connecting the primer dimer with a precutting vector pGEX-6P at 22 ℃ for 2h, converting a connecting product into escherichia coli DH5 alpha, correctly sequencing, extracting a plasmid, converting escherichia coli BL21, selecting a single clone, inoculating the single clone into an LB liquid culture medium containing ampicillin sodium, and carrying out shaking culture at 37 ℃ overnight.

Further, the process of the step (3) is as follows:

constructing recombinant plasmids pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP 1-pep;

designing a primer, introducing enzyme cutting sites EcoRI and Xho I,

ERK-pep-5:AATTCGACCATGATCACACAGGGTTCCTGACTGAATACGTGGCCACACGTTGGTAGC；

ERK-pep-3:TCGAGCTACCAACGTGTGGCCACGTATTCAGTCAGGAACCCTGTGTGATCATGGTCG；

YAP1-pep-5:AATTCACTCCACAGCATGTTCGAGCTCATTCGTCTCCAGCTTCTCTGCAGTTGTGAC；

YAP1-pep-3:TCGAGTCACAACTGCAGAGAAGCTGGAGACGAATGAGCTCGAACATGCTGTGGAGTG；

annealing primers to form dimers, said primers annealing to form dimers under conditions: annealing primers to form dimers, said primers annealing to form dimers under conditions: denaturation at 94 deg.C for 4min, binding at 75 deg.C for 5min, binding at 65 deg.C for 15min, and binding at 25 deg.C for 20min to form stable dimer; and (3) connecting the primer dimer with the precut vector pcDNA-GFP for 2h at 22 ℃, converting a connecting product into escherichia coli DH5 alpha, and extracting the plasmid after the sequencing is correct.

2-3 mu g of the constructed weightThe plasmids pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP1-pep were transfected into HEK293T cells using calcium phosphate kit in CO₂After culturing for 48 hours in an incubator, observing fluorescence under a fluorescence microscope, and determining transfection efficiency and intensity; cells were then collected for use.

Further, the process of the step (4) is as follows: expression and purification of the fusion proteins GST-YAP1-p127 and GST-ERK-pTY.

Further, the steps specifically include:

ultrasonic thallus breaking: adding 40ml of PBS and 400 mu l of PMSF into the thalli, and carrying out ultrasonic cracking; ultrasonic conditions are as follows: the amplitude transformer phi 6, the power 80%, the total ultrasonic time 4min, the ultrasonic time 3s and the gap 3 s;

uniformly mixing the lysed thallus at 4 ℃ for 30min in a rotating manner, centrifuging at 10000rpm for 20min, taking the supernatant, adding the supernatant into a new centrifugal tube, repeatedly centrifuging once, and collecting the supernatant;

add 300. mu.l GST agarose gel to the lysis supernatant;

rotating and shaking for 1-2 h in a chromatography cabinet at 4 ℃; centrifuging at 4 deg.C and 2000rpm for 2min, and removing supernatant; adding PBS into the centrifuge tube with gel to 40ml, shaking at 4 deg.C for 30min, centrifuging at 4 deg.C and 2000rpm for 2min, discarding supernatant, and adding PBS for 2 times; transferring the gel into 1.5ml EP tube, centrifuging at 2000rpm for 2min, discarding supernatant, adding 1ml eluent, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, and collecting supernatant as E1; adding 1ml of eluent into the gel, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, collecting supernatant, and labeling as E2; 1ml of the eluate was added to the gel, shaken at 4 ℃ for 30min in a chromatography cabinet, centrifuged at 2000rpm for 2min and the supernatant was taken and labelled E3.

E1, E2 and E3 were mixed together, dialyzed 3 times against 1 XPBS for 4 hours each, and the protein was concentrated to 1mg/ml using ultrafiltration tube centrifugation.

Further, antibody activity assays are also included.

The invention has the following beneficial effects:

the invention mutates phosphorylated amino acid into aspartic acid, constructs DNA of mutant antigen on pGEX-6p expression vector, uses colon bacillus to express GST fusion antigen, and can directly immunize animals after the purified antigen is mixed with Freund's adjuvant.

Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows a sample of cells transfected with pcDNA-GFP-ERK-pTY in lane 1, and a sample of cells transfected with pcDNA-GFP-ERK-pTY in lane 2: pcDNA-GFP-ERK-pep transfected cell samples, lane 3: HEK293T cell-like; the purified ERK-pTY antibody only specifically recognizes the phosphorylated short peptide and does not recognize the non-phosphorylated short peptide;

FIG. 2 is lane 4: pcDNA-GFP-YAP1-p127, lane 5: pcDNA-GFP-YAP 1-pep; the purified YAP1-p127 antibody only specifically recognizes the phosphorylated short peptide and does not recognize the non-phosphorylated short peptide;

fig. 3 and 4 are lanes 6: insulin stimulated HEK293T cell sample, lane 7: HEK293T cell-like; the ERK-pTY antibody only recognizes endogenous phosphorylated proteins and does not recognize endogenous non-phosphorylated proteins;

fig. 5 and 6 are lanes 8: TGF- β stimulated HepG2 cell-like, lane 9: HepG2 cell-like; the YAP1-p127 antibody recognizes only endogenous phosphorylated proteins and does not recognize endogenous non-phosphorylated proteins;

FIG. 7 is a flow chart of a method for preparing phosphorylated antibodies.

FIG. 8 is a detailed flowchart of the method for preparing phosphorylated antibodies.

Detailed Description

In order to facilitate an understanding of the present invention, a more complete description of the present invention is provided below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1: constructing recombinant plasmids pGEX-6p-ERK-pTY, pGEX-6p-YAP1-p127, pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP 1-pep;

primers were designed and the restriction sites EcoRI and Xho I were introduced:

ERK-pep-5:AATTCGACCATGATCACACAGGGTTCCTGACTGAATACGTGGCCACACGTTGGTAGC；

ERK-pep-3:TCGAGCTACCAACGTGTGGCCACGTATTCAGTCAGGAACCCTGTGTGATCATGGTCG；

YAP1-pep-5:AATTCACTCCACAGCATGTTCGAGCTCATTCGTCTCCAGCTTCTCTGCAGTTGTGAC；

YAP1-pep-3:TCGAGTCACAACTGCAGAGAAGCTGGAGACGAATGAGCTCGAACATGCTGTGGAGTG；

YAP1-p127-5:AATTCACTCCACAGCATGTTCGAGCTCATGACTCTCCAGCTTCTCTGCAGTTGTGAC；

YAP1-p127-3:TCGAGTCACAACTGCAGAGAAGCTGGAGAGTCATGAGCTCGAACATGCTGTGGAGTG；

ERK-pTY-5:AATTCGACCATGATCACACAGGGTTCCTGGACGAAGACGTGGCCACACGTTGGTAGC；

ERK-pTY-3:TCGAGCTACCAACGTGTGGCCACGTCTTCGTCCAGGAACCCTGTGTGATCATGGTCG；

annealing primers to form dimers, said primers annealing to form dimers under conditions: denaturation at 94 deg.C for 4min, binding at 75 deg.C for 5min, binding at 65 deg.C for 15min, and binding at 25 deg.C for 20min to form stable dimer; and (3) connecting the primer dimer with a precut vector pGEX-6P or pcDNA-GFP at 22 ℃ for 2h, converting a connecting product into escherichia coli DH5 alpha, and extracting a plasmid after correct sequencing.

The constructed pGEX-6P recombinant plasmid was transformed into BL21(DE3), and the selected single clone was inoculated into LB liquid medium containing ampicillin sodium (final concentration: 50. mu.g/ml) and cultured with shaking overnight at 37 ℃. The following day is as follows: 200, inoculating the overnight activated bacterial liquid into a fresh LB resistant culture medium, carrying out amplification culture, carrying out shaking culture at 37 ℃ until the OD600 of the bacterial liquid is 0.6-0.7, adding IPTG (isopropyl-beta-thiogalactoside) into the bacterial liquid until the final concentration is 1mmol/L, continuing the shaking culture at 37 ℃ for 4 hours, finishing the induction, and centrifuging at 8500rpm for 2min to collect thalli.

Example 2: expression and purification of fusion proteins GST-YAP1-p127 and GST-ERK-pTY

Ultrasonic thallus breaking: adding 40ml of PBS and 400 mu l of PMSF into the thalli, and carrying out ultrasonic cracking; ultrasonic conditions are as follows: the amplitude transformer phi 6, the power 80%, the total ultrasonic time 4min, the ultrasonic time 3s and the gap 3 s. And (3) uniformly mixing the cracked thalli at 4 ℃ for 30min, centrifuging at 10000rpm for 20min, taking the supernatant, adding the supernatant into a new centrifugal tube, centrifuging once again, and collecting the supernatant. To the lysis supernatant was added 300. mu.l of GST agarose gel. Rotating and shaking for 1-2 h in a chromatography cabinet at 4 ℃. Centrifuge at 2000rpm for 2min at 4 deg.C, and discard the supernatant. PBS was added to 40ml in the centrifuge tube with gel, shaken at 4 ℃ for 30min in a chromatography cabinet, centrifuged at 4 ℃ and 2000rpm for 2min, the supernatant was discarded, and PBS was added thereto and repeated 2 times. Transferring the gel into 1.5ml EP tube, centrifuging at 2000rpm for 2min, discarding supernatant, adding 1ml eluent, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, and collecting supernatant as E1; adding 1ml of eluent into the gel, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, collecting supernatant, and labeling as E2; 1ml of the eluate was added to the gel, shaken at 4 ℃ for 30min in a chromatography cabinet, centrifuged at 2000rpm for 2min and the supernatant was taken and labelled E3. E1, E2 and E3 were mixed together, dialyzed 3 times against 1 XPBS for 4 hours each, and the protein was concentrated to 1mg/ml using ultrafiltration tube centrifugation.

Example 3: animal immunization

3.1 mixing the dialyzed antigen and Freund's complete adjuvant or Freund's incomplete adjuvant according to the volume ratio of 1:1, and uniformly mixing for 2 hours at 4 ℃ until water-in-oil droplets are formed.

3.2 select healthy New Zealand white rabbits of about 7 weeks, and breed in animal house for 4-6 days to adapt to environment.

In the first week, 1mg of antigen GST-YAP1-p127 and Freund's complete adjuvant mixed solution is injected into the left thigh and the right thigh of the rabbit at 4 points;

at the fourth week, 1mg of the mixed solution of the antigen GST-YAP1-p127 and Freund's incomplete adjuvant is injected;

in the sixth week, 1mg of the antigen GST-YAP1-p127 and Freund's incomplete adjuvant mixture was injected;

in the eighth week, 1mg of the antigen GST-YAP1-p127 and Freund's incomplete adjuvant mixed solution is injected;

on the ninth week, carotid blood was taken. Incubating the collected blood at 37 ℃ for 1 hour, and then standing at 4 ℃ overnight; serum was collected by centrifugation the next day under the conditions: 4 ℃, 6000rpm, 10 min.

3.3 healthy 7 weeks old female ICR mice were selected and acclimatized in the animal housing for 4 to 6 days.

In the first week, the mice were injected with 0.2mg of antigen GST-ERK-pTY and Freund's complete adjuvant mixture intraperitoneally;

in the third week, 0.2mg of antigen GST-ERK-pTY and Freund's incomplete adjuvant mixed solution is injected into the abdominal cavity;

in the fifth week, 0.2mg of antigen GST-ERK-pTY and Freund's incomplete adjuvant mixed solution is injected into the abdominal cavity;

seventhly, injecting 0.2mg of antigen GST-ERK-pTY and Freund incomplete adjuvant mixed solution into the abdominal cavity;

in the eighth week, blood was collected from the eyeball. Incubating the collected blood at 37 ℃ for 1 hour, and then standing at 4 ℃ overnight; serum was collected by centrifugation the next day under the conditions: 4 ℃, 6000rpm, 10 min.

Example 4: antibody purification

4.1 mixing antigen and 5 xSDS-PAGE sample buffer proportionally, heating at 95 deg.C for 5 min; adding antigen into all loading wells of 12% SDS-PAGE gel, and performing 90V electrophoresis for 40 min; adding antigen into all sample loading holes of the gel again, and performing 90V electrophoresis for 40 min; antigen was added to all wells of this gel for a third time and electrophoresed at 90V for 80 min. And (5) after electrophoresis is finished, performing membrane rotation at 90V and 90 mim. And (3) dyeing the nylon membrane after the membrane transfer is finished with ponceau red, cutting off the antigen bands, and washing the antigen bands with pure water until the antigen bands are colorless.

4.2 GST antibody in serum: adding a GST antigen membrane strip into prepared serum, rotating for 120min at 4 ℃, and taking out the GST antigen membrane strip; sterile PBS was added to the membrane strip, spun at 4 ℃ for 20min, PBS was discarded, and the procedure was repeated 2 times. Placing the GST antigen membrane strip in a 2ml EP tube, adding 1ml of 0.2M glycine eluent (pH 2.4), rotating and shaking at room temperature for 8-9 min, immediately adding 150 μ l of 1M Tris-HCl buffer (pH 8), and mixing; the membrane strips were removed and washed 3 times with sterile PBS.

4.3 purification of antibody: removing GST antibody from the serum for 2 times, adding corresponding antigen membrane strip into the serum, rotating at 4 deg.C for 120min, and taking out the antigen membrane strip; sterile PBS was added to the membrane strip, spun at 4 ℃ for 20min, PBS was discarded, and the procedure was repeated 2 times. Placing the antigen membrane strip into a 2ml EP tube, adding 1ml of 0.2M glycine eluent (pH 2.4), rotating and shaking at room temperature for 8-9 min, immediately adding 150 μ l of 1M Tris-HCl buffer (pH 8), and mixing; the liquid is the purified target antibody. The membrane strips were removed and washed 3 times with sterile PBS.

Example 5: cell transfection

2-3 mu g of the constructed recombinant plasmids pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP1-pep are transfected into HEK293T cells by using a calcium phosphate kit, and then the cells are subjected to CO reaction₂After culturing for 48 hours in an incubator, observing fluorescence under a fluorescence microscope, and determining transfection efficiency and intensity; cells were then collected for use.

Example 6: determination of antibody Activity

Respectively taking transfected cell samples of pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP1-pep, HEK293T cell samples, HepG2 cell samples, insulin-stimulated HEK293T cell samples and TGF-beta-stimulated HepG2 cell samples, adding a proper 1 xSDS-PAGE sample loading buffer solution, and boiling for 5min at 95 ℃; SDS-PAGE electrophoresis separated gel is 12 percent, concentrated gel is 5 percent, and the gel is loaded by using a pipette gun according to the preset sequence and is subjected to 90V electrophoresis for 2 hours. Film transferring conditions: constant pressure of 90V and time of 90 min. Blocking 5% skimmed milk powder at room temperature for 2h, adding purified antibodies ERK-pTY (FIG. 1 and FIG. 4), YAP1-p127 (FIG. 2 and FIG. 6), ERK (FIG. 3) and YAP1 (FIG. 5) at a ratio of 1:2000, incubating overnight at 4 deg.C, washing membrane with TBST 3 times the next day, each for 20 min; adding HRP (horse radish peroxidase) to obtain a goat anti-rabbit IgG (immunoglobulin G) secondary antibody at a ratio of 1:5000, incubating at room temperature for 1 hour, removing the secondary antibody, washing the membrane with TBST (tert butyl Steel) for 3 times, and each time for 20 min; develop using ECL kit, take photograph and record. In FIG. 1, lane 1, pcDNA-GFP-ERK-pTY transfected cell sample, lane 2: pcDNA-GFP-ERK-pep transfected cell samples, lane 3: HEK293T cell-like; in FIG. 2, lane 4: pcDNA-GFP-YAP1-p127, lane 5: pcDNA-GFP-YAP 1-pep; in fig. 3 and 4, lane 6: insulin stimulated HEK293T cell sample, lane 7: HEK293T cell-like; in fig. 5 and 6, lane 8: TGF- β stimulated HepG2 cell-like, lane 9: HepG2 cell-like. FIG. 1 shows that the purified ERK-pTY antibody specifically recognizes only phosphorylated short peptides and does not recognize non-phosphorylated short peptides; FIG. 2 shows that purified YAP1-p127 antibody specifically recognizes only phosphorylated short peptides and does not recognize non-phosphorylated short peptides; FIGS. 3 and 4 show that the ERK-pTY antibody recognizes only endogenous phosphorylated proteins and does not recognize endogenous non-phosphorylated proteins; FIGS. 5 and 6 show that the YAP1-p127 antibody recognizes only endogenous phosphorylated proteins and does not recognize endogenous non-phosphorylated proteins.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Sequence listing

SEQ ID NO:1 DHDHTGFLDEDVATRW

SEQ ID NO:2 TPQHVRAHDSPASLQL

SEQUENCE LISTING

<110> Danhuaneng Biotech Co., Ltd

<120> phosphorylated antigen polypeptide, antibody and preparation method thereof

<130>2

<160>1

<170>PatentIn version 3.3

<210>1

<211>16

<212>PRT

<213> human ERK protein

<400>1

Thr Pro Gln His Val Arg Ala His Asp Ser Pro Ala Ser Leu Gln Leu

1 5 10 15

SEQUENCE LISTING

<110> Danhuaneng Biotech Co., Ltd

<120> phosphorylated antigen polypeptide, antibody and preparation method thereof

<130>1

<160>1

<170>PatentIn version 3.3

<210>1

<211>16

<212>PRT

<213> human ERK protein

<400>1

Asp His Asp His Thr Gly Phe Leu Asp Glu Asp Val Ala Thr Arg Trp

1 5 10 15

Claims (10)

1. A phosphorylated antigen polypeptide, which is characterized by: the antigenic polypeptide is

Point mutation of antigenic peptides at the Thr202 and Tyr204 positions of the ERK protein, or

The YAP1 protein Ser127 site point mutation antigen peptide.

2. The phosphorylated antibody at positions Thr202 and Tyr204 of the human ERK protein according to claim 1, wherein the antibody is a phosphorylated antibody against threonine at position 202 and tyrosine at position 204 of the human ERK protein, and the amino acid sequence is shown in SEQ ID No. 1, wherein Thr and Tyr are mutated to Asp.

3. The phosphorylated antibody against Ser127 of human YAP1 according to claim 1, wherein the antibody is a phosphorylated antibody against serine 127 of YAP1 protein, the amino acid sequence of which is shown in SEQ ID No. 2, wherein Ser is mutated to Asp.

4. A method for preparing a phosphorylated antibody, comprising the steps of:

(1) obtaining an antigenic peptide with active amino acid as an amino acid sequence shown in SEQ ID NO. 1, and point mutation antigenic peptides of the sites Thr202 and Tyr204 of the ERK protein, wherein Thr and Tyr are mutated into Asp; and an antigen peptide with active amino acid of an amino acid sequence shown in SEQ ID NO. 2, and a point mutation antigen peptide at a Ser127 site of YAP1 protein, wherein Ser is mutated into Asp;

(2) amplifying partial gene fragments of ERK and YAP1, cloning the partial gene fragments into a pGEX-6P expression vector, and expressing proteins by escherichia coli;

(3) amplifying partial gene fragments of ERK and YAP1, cloning the partial gene fragments into a pcDNA-GFP expression vector, and transfecting HEK293T cells to obtain ERK and YAP1 over-expression cell samples;

(4) obtaining fusion protein through affinity purification;

(5) immunizing a mouse or a New Zealand rabbit with the fusion protein, and taking serum;

(6) the phosphorylated antibody is obtained by means of affinity purification.

5. The method for preparing phosphorylated antibody according to claim 4, wherein the step (2) comprises the following steps:

constructing recombinant plasmids pGEX-6p-ERK-pTY and pGEX-6p-YAP1-p 127;

designing a primer, introducing enzyme cutting sites EcoRI and Xho I, annealing the primer to form a dimer, and annealing the primer to form the dimer under the following conditions: denaturation at 94 deg.C for 4min, binding at 75 deg.C for 5min, binding at 65 deg.C for 15min, and binding at 25 deg.C for 20min to form stable dimer; connecting the primer dimer with a precutting vector pGEX-6P at 22 ℃ for 2h, converting a connecting product into escherichia coli DH5 alpha, correctly sequencing, extracting a plasmid, converting escherichia coli BL21, selecting a single clone, inoculating the single clone into an LB liquid culture medium containing ampicillin sodium, and carrying out shaking culture at 37 ℃ overnight.

6. The method for preparing phosphorylated antibody according to claim 4, wherein the step (3) comprises:

constructing recombinant plasmids pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP 1-pep;

designing a primer, introducing enzyme cutting sites EcoRI and Xho I,

annealing primers to form dimers, said primers annealing to form dimers under conditions: denaturation at 94 deg.C for 4min, binding at 75 deg.C for 5min, binding at 65 deg.C for 15min, and binding at 25 deg.C for 20min to form stable dimer; connecting the primer dimer with a precutting vector pcDNA-GFP for 2h at 22 ℃, converting a connecting product into escherichia coli DH5 alpha, and extracting plasmids after correct sequencing;

2-3 mu g of the constructed recombinant plasmids pcDNA-GFP-ERK-pTY, pcDNA-GFP-YAP1-p127, pcDNA-GFP-ERK-pep and pcDNA-GFP-YAP1-pep are transfected into HEK293T cells by using a calcium phosphate kit, and then the cells are subjected to CO reaction₂After culturing for 48 hours in an incubator, observing fluorescence under a fluorescence microscope, and determining transfection efficiency and intensity; cells were then collected for use.

7. The method for preparing phosphorylated antibody according to claim 4, wherein the step (4) comprises: expression and purification of the fusion proteins GST-YAP1-p127 and GST-ERK-pTY.

8. The method for preparing a phosphorylated antibody according to claim 7, wherein the steps specifically comprise:

ultrasonic thallus breaking: adding 40ml of PBS and 400 mu l of PMSF into the thalli, and carrying out ultrasonic cracking; ultrasonic conditions are as follows: the amplitude transformer phi 6, the power 80%, the total ultrasonic time 4min, the ultrasonic time 3s and the gap 3 s;

uniformly mixing the lysed thallus at 4 ℃ for 30min in a rotating manner, centrifuging at 10000rpm for 20min, taking the supernatant, adding the supernatant into a new centrifugal tube, repeatedly centrifuging once, and collecting the supernatant;

add 300. mu.l GST agarose gel to the lysis supernatant;

rotating and shaking for 1-2 h in a chromatography cabinet at 4 ℃; centrifuging at 4 deg.C and 2000rpm for 2min, and removing supernatant; adding PBS into the centrifuge tube with gel to 40ml, shaking at 4 deg.C for 30min, centrifuging at 4 deg.C and 2000rpm for 2min, discarding supernatant, and adding PBS for 2 times; transferring the gel into 1.5ml EP tube, centrifuging at 2000rpm for 2min, discarding supernatant, adding 1ml eluent, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, and collecting supernatant as E1; adding 1ml of eluent into the gel, shaking at 4 deg.C for 30min, centrifuging at 2000rpm for 2min, collecting supernatant, and labeling as E2; 1ml of the eluate was added to the gel, shaken at 4 ℃ for 30min in a chromatography cabinet, centrifuged at 2000rpm for 2min and the supernatant was taken and labelled E3.

9. The method of claim 8, wherein the phosphorylated antibody is produced,

e1, E2 and E3 were mixed together, dialyzed 3 times against 1 XPBS for 4 hours each, and the protein was concentrated to 1mg/ml using ultrafiltration tube centrifugation.

10. The method of claim 4, further comprising measuring the activity of the phosphorylated antibody.

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