CN108277214B - Stress phosphorylation antigen polypeptide, antibody, preparation method and application - Google Patents

Abstract

The invention provides a stress phosphorylation antigen polypeptide, an antibody, a preparation method and an application, the phosphorylation antigen polypeptide is characterized in that the antigen polypeptide comprises 15 peptides near a 458 th threonine site of human MMP9 protein as candidate polypeptides, wherein the threonine at the T184 site is in a phosphorylation state, and the C end is connected with an amino acid sequence of cysteine. The invention also provides an antibody prepared by the antigen polypeptide, a preparation method and application thereof, a kit containing the antibody and a cancer detection method. The antibody prepared by the invention can detect phosphorylation modification of MMP9 protein under a stress environment, so as to discuss the influence of MMP9 protein T458 site phosphorylation on tumor cell proliferation, migration and other processes under a stress condition, and provide a potential practical tool for clinical diagnosis and treatment of tumor diseases.

Description

Stress phosphorylation antigen polypeptide, antibody, preparation method and application

Technical Field

The invention relates to the technical field of antibody preparation, and in particular relates to a stress phosphorylation antigen polypeptide aiming at a T458 site of human MMP9 protein, an antibody, a preparation method and application thereof.

Background

Tumor metastasis is considered to be a multi-step process that requires the co-action of multiple genes to complete. Certain genes allow tumor cells to shed from a primary tumor, adhere to endothelial cells, invade the extracellular matrix (ECM), cross blood vessels, and grow rapidly, eventually forming a new tumor in distant organs. Matrix Metalloproteinases (MMPs) are important genes involved in this process. MMPs are the main physiological mediators of ECM degradation. Matrix metalloproteinases are a family of zinc-dependent endopeptidases whose primary function is to degrade the ECM. MMPs are involved in a variety of physiological and pathological processes such as morphogenesis, wound healing, tissue repair and remodeling ]. In addition, MMPs play an important role in tumor invasion and metastasis by exerting the ability to increase cell growth, migration, invasion, metastasis and angiogenesis. MMP9 is collagenase type IV, also called gelatinase, which is secreted in the form of inactive zymogen, and can degrade gelatin and various proteins in ECM after being activated by hydrolysis, playing an important role in tumor infiltration and metastasis.

Matrix metallopeptidase 9(MMP9), also known as 92kDa type IV collagenase or gelatinase b (gelb), is a member of the MMP family of enzymes responsible for degrading denatured and basement membrane collagen and promoting inflammation by processing soluble proteins including protease inhibitors, chemokines and cytokines. MMP9 also controls migration, invasion, and metastasis of tumor cells through proteolysis of membrane-bound molecules (e.g., growth factor precursors and receptors, Tyrosine Kinase Receptors (TKRs), cell adhesion molecules in diseases, MMP9 is secreted by many cell types including leukocytes, such as neutrophils, monocytes/macrophages, and lymphocytes, as well as fibroblasts, myofibroblasts, epithelial cells, smooth muscle cells, endothelial cells, osteoclasts, and tumor cells.

Clinical and experimental evidence suggests that elevated MMP9 levels are associated with cancer development, metastasis, and shortened patient survival time, as it plays a key role in tumor cell invasion and metastasis by digesting basement membrane and extracellular matrix components. Neutrophil gelatinase-associated lipocalin (NGAL), covalently linked to MMP9 in human neutrophils, protects MMP9 from proteolytic degradation and increases the enzymatic activity of MMP9 and subsequently enhances tumor invasion and spread. High concentrations of MMP9/NGAL complex in serum were associated with a short progression-free survival and a poor overall survival in clear cell renal cell carcinoma. The effects of MMP-9 are associated with colorectal, pancreatic, breast, lung, ovarian, bladder and gastric cancers.

Disclosure of Invention

The invention discloses an antigenic polypeptide aiming at a T458 phosphorylation site of MMP9 protein, a polyclonal antibody capable of specifically recognizing the T458 phosphorylation site of MMP9 protein expressed by human tumor cells and a preparation method thereof.

The first aspect of the invention provides a phosphorylated antigen polypeptide, which is characterized in that the antigen polypeptide is obtained by screening 15 peptides around the 458 th threonine position of human MMP9 protein as candidate polypeptides, wherein the threonine at the T458 th position is in a phosphorylated state, and the C end of the antigen polypeptide is connected with an amino acid sequence of cysteine.

The second aspect of the invention provides a stress phosphorylation antibody aiming at the T458 site of human MMP9 protein, which is characterized in that the antibody is an antibody aiming at the amino acid phosphorylation at the T458 site of human MMP9 protein, wherein the antibody is prepared by adopting an antigen polypeptide consisting of the following amino acid sequences, and the specific sequences are as follows: CEPRPPTTTT(p) PQPT, wherein T (p) represents the amino acid phosphorylated threonine at position T458 of human MMP9 protein, and the C terminal of the protein is connected with a cysteine.

The third aspect of the invention provides a preparation method of polyclonal antibody aiming at T458 site stress phosphorylation of human MMP9 protein, which is characterized by comprising the steps of (1) analyzing the secondary structure, immunogenicity, hydrophilicity and hydrophobicity, surface accessibility and the like of an amino acid sequence near 458 site of MMP9 protein, determining a proper peptide sequence and artificially synthesizing; (2) coupling the synthesized polypeptide with maleimide activated carrier mcKLH, desalting the coupling product with desalting column and immunizing New Zealand rabbit; (3) detecting the titer of the antibody by using the rabbit serum subjected to the four immunizations by using an ELISA method, collecting the immune rabbit serum after the titer reaches an ideal value, and purifying the antibody by using a polypeptide-coated cyanogen bromide-activated agarose affinity purification column; (4) the purified antibody was subjected to ELISA, western bot assay.

The fourth aspect of the present invention provides a use of a stress phosphorylated antibody against T458 site of human MMP9 protein, wherein the use specifically recognizes T458 site of human MMP9 protein expressed by cancer cells using the antibody prepared according to claim 2 or claim 3. Wherein the cancer cell is preferably human nasopharyngeal carcinoma, cervical carcinoma, gastric cancer, and glioma cell.

The invention provides a cancer cell detection kit in a fifth aspect, which comprises the stress phosphorylation antibody aiming at the T458 site of the human MMP9 protein, antigen retrieval liquid, PBS buffer solution, enzyme blocking agent 3% H2O2, horseradish enzyme labeled goat anti-mouse/rabbit IgG polymer, DAB color developing agent, hematoxylin staining solution, ethanol, environment-friendly clearing agent, 0.5% ammonia water and ultrapure water.

The invention also provides a method for detecting cancer cells, which is characterized in that the cancer cell detection kit is used, and comprises the following steps:

(1) slicing the tissue to be detected with conventional paraffin, respectively dewaxing by a table concentrator through an environment-friendly transparent agent and ethanol, and rinsing with ultrapure water;

(2) heating the antigen retrieval liquid to boiling, putting the paraffin sections into the boiling antigen retrieval liquid, performing medium-high grade microwave treatment, cooling at room temperature, putting the paraffin sections into ultrapure water, soaking, and then shaking and washing the ultrapure water with PBS for 3 times;

(3) placing the sample in an endogenous peroxidase blocking agent 3% H2O2, incubating the sample at room temperature in a dark place, and shaking and washing the sample by using a PBS buffer solution;

(4) taking out the slices, dropwise adding the diluted stress phosphorylation antibody aiming at the T458 site of the human MMP9 protein, putting the slices into an incubation box, putting the slices into a refrigerator at 4 ℃ for overnight, and putting the slices into PBS buffer solution for full washing;

(5) wiping off liquid around the tissues, dripping horseradish enzyme-labeled goat anti-mouse/rabbit IgG polymer, incubating at room temperature, washing with PBS buffer solution, dripping a proper amount of DAB color developing agent which is prepared in situ, developing at room temperature, stopping developing with tap water, dyeing in hematoxylin dye solution, washing with water, soaking the slices in 0.5% ammonia water, and washing with water;

(6) and (3) sequentially placing the slices into ethanol, taking out the slices, placing the slices into a clearing agent, sealing the slices with neutral gum, and observing under an optical microscope.

According to the results of previous work, the inventors predicted that threonine 458 (T458) of MMP9 protein is a potential phosphorylation site, and may be involved in the stabilization and activation functions of the protein. The invention adopts artificial design and synthesis of a section of MMP9 protein polypeptide (pT458) containing the phosphorylation site, couples with Keyhole Limpet Hemocyanin (KLH) activated by maleic amide, immunizes New Zealand rabbits after desalination and purification, collects rabbit serum after four times of immunization and ELISA titer detection, and purifies the rabbit serum by an agarose gel purification column activated by cyanogen bromide coated with the polypeptide. The polyclonal antibody can specifically recognize MMP9 protein pT458 site expressed by human nasopharyngeal carcinoma CNE1 cells through identification of ELISA, western blot and the like.

The invention selects 15 peptides near the 458 th threonine site (T458) of MMP9 protein as candidate polypeptides, and artificially synthesizes the polypeptide containing pT458 and prepares complete antigen. Analyzing the secondary structure, immunogenicity, hydrophilicity and hydrophobicity, surface accessibility and the like of an amino acid sequence near the 458 th site of the MMP9 protein, and determining a proper peptide sequence for artificial synthesis; coupling the synthesized polypeptide with maleimide activated carrier mcKLH, desalting the coupling product with desalting column and immunizing New Zealand rabbit; detecting the titer of the antibody by ELISA method of the rabbit serum after four times of immunization, collecting the immune rabbit serum after the titer reaches an ideal value, and purifying the antibody by a polypeptide-coated cyanogen bromide activated agarose (CNBr-activated sepharose) affinity purification column; the purified antibody was identified by ELISA, western bot, etc. The identification result shows that the polyclonal antibody can specifically recognize MMP9 protein pT458 site, can be used for detecting the phosphorylation level of the site of the tumor cell, provides a tool for exploring the research of tumor cell proliferation and metastasis mechanisms, provides help for tumor diagnosis and can guide the clinical prognosis judgment of the tumor cell.

Among them, chemically synthesized polypeptide antigens are small molecules, which hardly have good antigenicity per se and induce only weak immune reactions in animals, and thus it is important to crosslink them with carrier proteins. The carrier protein contains many epitopes and is capable of stimulating T helper cells and thereby inducing B cell responses. There are many carrier proteins for cross-linking with polypeptides, the most commonly used carriers being Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA) and bovine Thyroglobulin (THY). KLH is more antigenic and is the most commonly used polypeptide cross-linking vehicle. BSA is also commonly used as a polypeptide carrier, but since BSA is often used as a blocking agent in detection assays, the antibodies produced by this method have certain limitations in application.

Advantageous effects of the invention

The invention adopts artificial design and synthesis of an antigen polypeptide containing a fragment of MMP9 protein pT458 site of the phosphorylation site, and prepares a corresponding polyclonal antibody. The polyclonal antibody can specifically recognize MMP9 protein pT458 phosphorylation sites through identification of ELISA, western blot and the like, and is highly expressed in various cancer tissue cells relative to a cancer adjacent tissue, and the difference has statistical significance. The phosphorylation polyclonal antibody can specifically recognize MMP9 protein pT458 phosphorylation site, can be used for detecting the phosphorylation level of the site of tumor cells, provides a tool for exploring the research of tumor cell proliferation and metastasis mechanisms, provides help for tumor diagnosis and can guide the clinical prognosis judgment.

Drawings

FIG. 1 is a graph of human MMP9 protein characterization using DNAstar software. The sequences marked in the box are selected polypeptide sequences which are positioned near threonine 458 of MMP9 protein and have stronger antigenicity, hydrophilicity and surface accessibility.

FIG. 2 shows the OD values of pMMP9-Thr458 antibody and non-phosphorylated MMP9-Thr458 antibody, and the pMMP9-Thr458 antibody is detected as a specific phosphorylation site antibody (. about.P < 0.05) by ELISA.

FIG. 3 is the result of western-blot identification of the anti-MMP 9(pT458) polyclonal antibody of the present invention. Loading CNE1 cell (human nasopharyngeal carcinoma cell line) lysate. The cells were transiently transfected with pcDNA6.0/myc-His-MMP9 wild-type and pcDNA6.0/myc-His-MMP9(T458A) mutant plasmids, respectively.

FIG. 4 is a preparation process of the present invention.

FIG. 5 is the result of cytoimmunohistochemical identification of an anti-MMP 9(pT458) polyclonal antibody; the cells are nasopharyngeal carcinoma cells CNE2, liver cancer cells HepG2 and stomach cancer cells MGC 803.

FIG. 6 is the immunohistochemical identification result of the anti-MMP 9(pT458) polyclonal antibody of the present invention. The specimen used is a pathological section of cervical cancer cells.

FIG. 7 shows the immunohistochemical identification of the anti-MMP 9(pT458) polyclonal antibody of the present invention. The specimens used were pathological sections of gastric cancer and glioma cells.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Any modifications that can be easily made by a person skilled in the art to the present invention without departing from the technical solutions of the present invention will fall within the scope of the claims of the present invention.

Example 1

Step 1: design and synthesis of MMP9 protein pT458 polypeptide

1.1 MMP9 amino acid sequence

The amino acid sequence of human MMP9 protein was obtained from GenBank (NP _ 004985):

1 mslwqplvlv llvlgccfaa prqrqstlvl fpgdlrtnlt drqlaeeyly rygytrvaem

61 rgeskslgpa llllqkqlsl petgeldsat lkamrtprcg vpdlgrfqtf egdlkwhhhn

121 itywiqnyse dlpravidda farafalwsa vtpltftrvy srdadiviqf gvaehgdgyp

181 fdgkdgllah afppgpgiqg dahfdddelw slgkgvvvpt rfgnadgaac hfpfifegrs

241 ysacttdgrs dglpwcstta nydtddrfgf cpserlytqd gnadgkpcqf pfifqgqsys

301 acttdgrsdg yrwcattany drdklfgfcp tradstvmgg nsagelcvfp ftflgkeyst

361 ctsegrgdgr lwcattsnfd sdkkwgfcpd qgyslflvaa hefghalgld hssvpealmy

421 pmyrftegpp lhkddvngir hlygprpepe prppttttpq ptapptvcpt gpptvhpser

481 ptagptgpps agptgpptag pstattvpls pvddacnvni fdaiaeignq lylfkdgkyw

541 rfsegrgsrp qgpfliadkw palprkldsv feerlskklf ffsgrqvwvy tgasvlgprr

601 ldklglgadv aqvtgalrsg rgkmllfsgr rlwrfdvkaq mvdprsasev drmfpgvpld

661 thdvfqyrekayfcqdrfywrvssrselnqvdqvgyvtydilqcped（SEQ ID NO：3）

as a result: the human MMP9 protein contains 707 amino acids.

1.2 analysis of human MMP9 protein properties using DNAstar software (Table 1):

TABLE 1

As a result: the molecular weight of the human MMP9 protein is 784580.47 daltons, the isoelectric point is 5.82, and the protein is acidic protein.

1.3 analysis of human MMP9 protein immunogenicity, hydrophobicity and surface accessibility with DNAstar software (FIG. 1):

as a result: the protein MMP9 has 14 amino acids from 449 to 462, strong antigenicity, hydrophilicity and surface accessibility, and threonine 458 is contained.

1.4 MMP9 synthetic polypeptide sequence:

through the above analysis, the selected polypeptide sequence is CEPRPPTTTTPQPT (SEQ ID NO: 1).

Step 2: polypeptide synthesis and conjugation to carrier proteins

2.1 polypeptide Synthesis

To facilitate coupling to the carrier protein, the synthetic polypeptide has a cysteine added at the C-terminus and threonine 458 is in a phosphorylated state: CEPRPPTTTT(p) PQPT (SEQ ID NO: 2). Simultaneously synthesizing a polypeptide sequence containing non-phosphorylated threonine 458: CEPRPPTTTTPQPT are provided. Polypeptides were synthesized by pocky biotechnology (suzhou) limited.

The polypeptide synthesis process comprises the following steps:

A. polypeptide synthesis: the solid phase synthesis process includes connecting the hydroxyl group of the hydroxyl terminal amino acid of the peptide chain to be synthesized with insoluble polymer resin in covalent bond structure, eliminating amino protecting group from the amino group of the amino acid combined on the solid phase carrier, reacting with excessive active carboxyl group, lengthening the peptide chain, repeating the operation until reaching the length of the peptide chain, cracking the peptide chain from the resin, purifying and other treatment to obtain the polypeptide.

B. And (3) purification: RP-HPLC purification

1) HPLC conditions:

mobile phase: A) 0.1% aqueous TFA; B) 0.1% TFA acetonitrile solution

Gradient: A/B (90/40) to A/B (40/90) for 30min

Flow rate: 1ml/min

Temperature: room temperature (23 ℃ C.)

And (3) detection: ultraviolet at 214nm

Sample preparation: lyophilized crude product

2) The method comprises the following steps:

a. dissolving the crude product in a mobile phase

b. Injecting 20-30mg (2-2.5 ml) of sample

c. Major peaks were collected in 50ml tubes

d. Freeze-drying

3) And (3) identification: LC/MS conditions:

mobile phase: A) 0.05% aqueous TFA B) 0.1% TFA in acetonitrile

Gradient: A/B (90/10) to A/B (40/60) 15min

Flow rate: 1ml/min

Temperature: room temperature (23 ℃ C.)

And (3) detection: ultraviolet at 214nm

MS API：ESI

TABLE 2 results of polypeptide Synthesis

Polypeptide synthesis number	Polypeptide sequence	Polypeptide purity (%)
			150803001	NH2-CEPRPPTTTp[Thr]PQPT-CONH2	>90%
150803002	NH2-CEPRPPTTTTPQPT-CONH2	>90%

2.2 conjugation of Polypeptides to Carrier proteins

Chemically synthesized polypeptide antigens are small molecules, which hardly have good antigenicity and can induce animals to have weak immune reactions, so that crosslinking with carrier proteins is important. The carrier protein contains many epitopes and is capable of stimulating T helper cells and thereby inducing B cell responses. There are many carrier proteins for cross-linking with polypeptides, the most commonly used carriers being Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA) and bovine Thyroglobulin (THY). KLH is more antigenic and is the most commonly used polypeptide cross-linking vehicle. BSA is also commonly used as a polypeptide carrier, but since BSA is often used as a blocking agent in detection assays, the antibodies produced by this method have certain limitations in application.

Polypeptide coupling process:

a. solution preparation: the coupling buffer solution comprises Na2HPO4, NaH2PO4, NaCl and EDTA, and the pH is adjusted to 7.2;

b. the experimental steps are as follows:

1) preparing a column bed: washing the column bed with pure water and coupling buffer solution;

2) polypeptide preparation: dissolving polypeptide with a small amount of DMF, standing for half an hour until no particulate insoluble substances exist in the solution, adding a proper amount of AH solution to prepare a polypeptide solution of 6mg/ml, and separating the amount to be coupled from the polypeptide solution;

3) KLH, Sulfo-SMCC preparation: according to the mass ratio, the total amount of the conjugated polypeptide: pure KLH = 1: 1, calculating the amount of pure KLH, wherein the mass ratio of pure KLH: Sulfo-SMCC = 10: 1, calculating the amount of Sulfo-SMCC;

4) KLH and Sulfo-SMCC reaction and collection of the reaction: dissolving weighed KLH in an appropriate amount of AH solution to prepare a solution with a final concentration of 10mg/ml, dissolving Sulfo-SMCC in DMSO to prepare a solution with a final concentration of 100mg/ml, mixing and shaking the solution and the solution uniformly, reacting for 4 hours at room temperature, intermittently mixing and shaking the solution to enable the solution to react sufficiently, and separating a sample by using a chromatographic column;

5) the KLH reactant with Sulfo-SMCC was conjugated to a polypeptide: adding corresponding amount of KLH and Sulfo-SMCC reactants into each tube of polypeptide to be coupled, reacting for 2h at room temperature or overnight at room temperature, uniformly mixing by using a vertical mixer, and storing the coupled polypeptide at-20 ℃.

Note: KLH carrier protein is used for coupling synthesis of polypeptide, and the obtained coupled peptide is used as an immunizing antigen.

And step 3: preparation of rabbit polyclonal antibody for resisting MMP9 polypeptide

3.1 immunization and blood sampling:

3.2 antibody titer ELISA detection method

3.2.1 serum ELISA assay:

a. solution preparation:

coating liquid: 50mM Na2CO3 (pH 9.6), 20mM Tris-HCl (pH 8.5) or 10mM PBS (pH 7.4)

Sealing liquid: typically, BSA, skim milk powder, casein, gelatin, etc. are used for blocking

Washing liquid: PBST or pure water

b. The experimental steps are as follows:

1) dissolving the antigen in the coating liquid according to a proper concentration;

2) add 100ul antigen to the corresponding well, 4 degrees C overnight;

3) emptying the liquid and beating the residual liquid, and washing the washing liquid for 3 times;

4) adding 200ul of blocking solution into each well, and incubating for 1 hour at 37 ℃;

5) emptying the liquid and beating the residual liquid, and washing the washing liquid for 3 times;

6) adding 100ul primary antibody to each well, and incubating for 1 hour at 37 ℃;

7) emptying the liquid and beating the residual liquid, and washing the washing liquid for 3 times;

8) adding 100ul of secondary antibody to each well, and incubating for 1 hour at 37 ℃;

9) emptying the liquid and beating the residual liquid, and washing the washing liquid for 5 times;

10) patting off residual liquid in the holes, adding 100ul of color development liquid into each hole, and developing for 10min at 37 ℃ in a dark place;

11) the color development was stopped by adding 50ul 2M H2SO4 to each well and the OD 450nm was read immediately.

c. Serum ELISA test results:

note: RB55631-55632 serum ELISA assays were positive-converting (1: 32000, P/N values > 2.1), two-step affinity purification was scheduled and ELISA antibody identification was performed.

3.2.2 antibody affinity purification results:

3.2.3 antibody ELISA results

And (4) conclusion:

the RB55631-55632 serum ELISA of the item immunity is positive; the antibody was purified by two-step affinity method to obtain 3.89mg of phosphorylated antibody.

Step 4 identification of anti-MMP 9(pT458) polyclonal antibody

4.1 ELISA identification of anti-MMP 9(pT458) polyclonal antibodies

A) ELISA procedure

Polypeptide MMP9-Thr458 and pMMP9-Thr458 are dissolved into 0.2 mug/100 ul by 1x CBS coating liquid, the solution is spread in the holes of each elisa plate for 100ul, and the elisa plate is coated overnight at 4 ℃.

And taking out the coated ELISA plate, drying the coating liquid and beating the plate in the next day.

And adding confining liquid (3% BSA), adding 200ul of blocking liquid into each hole, and incubating at 37 ℃ for 2 h.

After sealing is finished, the ELISA plate is taken out, the plate is washed for three times, 300 ul of PBST is added into each hole, the plate is kept stand for 2 min, then the plate is dried in a drying mode (pollution between the holes is avoided), the plate is patted on clean gauze, and liquid in the holes is preferably patted dry.

The experiment of the next step can be directly carried out, and the product can also be packaged by a self-sealing bag and stored at 4 ℃ for later use.

Sixthly, adding 100ul of MMP9 primary antibody into each hole at a dilution concentration of 1:4000, and incubating for 1 h at 37 ℃.

Taking out the ELISA plate after the self-skin is 1 h, washing the plate for three times, adding a secondary HRP antibody, and incubating for 30min at 37 ℃.

Taking out the enzyme label plate after 30min, washing the plate for three times, adding TMB color developing agent, and incubating for 15min in a dark place at 37 ℃.

Taking out the ELISA plate (changing blue) after 15min, immediately adding the stop solution, oscillating the ELISA reader for 30s, reading at 450nm, and analyzing the result.

B) ELISA experiment detection of pMMP9-458 antibody as specific phosphorylation site antibody

Since phosphorylation of MMP9-Thr458 site is not reported, the inventor entrusts companies to customize pMMP9-Thr458 antibody, and tests whether the pMMP9-Thr458 antibody is a specific phosphorylation site antibody through ELISA experiments. The results showed that the OD value of pMMP9-Thr458 antibody bound to pMMP9-Thr458 antigen was significantly greater than that of pMMP9-Thr458 antibody bound to non-phosphorylated MMP9-Thr458 antigen (see FIG. 2), and that the OD value of pMMP9-Thr458 antibody bound to pMMP9-Thr458 antigen decreased with dilution of the antibody concentration, suggesting that the pMMP9-Thr458 antibody is a specific antibody at pMMP9-Thr458 site.

4.2 Western-Blot identification of polyclonal antibodies against MMP9(pT458)

A) Transient transfection was carried out using the transfection reagent Jetpei from the company Polyplus, taking a6 cm dish as an example:

(1) when the cells grow to 80% -90% and the cell state is good, counting the cells after cell digestion, spreading 5 × 105-1 × 106 cells per 6 cm culture dish, and the total volume of cell suspension and culture medium is 5ml

(2) Taking 3 mu g DNA, calculating the sum of the volume of the obtained plasmid and the volume of NaCl to be 250 ul, then taking 6 ul of transfection reagent jetpei, 244 ul of NaCl, fixing the volume to 250 ul, mixing uniformly, adding the mixed transfection reagent into the mixed DNA, mixing uniformly, centrifuging simply, and standing at room temperature for 15-30 min.

(3) And (3) slightly adding the prepared transfection system into a corresponding marked culture dish, uniformly mixing, and placing the mixture into a constant-temperature incubator at 37 ℃ and 5% CO2 for cell culture.

(4) Cells can be identified by Western-Blot after 24-48 h.

B) Western-Blot identification of anti-MMP 9(pT458) polyclonal antibody

After CNE1 cells are transiently transfected with cDNA, MMP9-WT and MMP9-Thr458Ala, whether Thr458 is phosphorylated is detected by Western blot using pMMP9-Thr458 antibody, and the result shows that the phosphorylation level of MMP9-Thr458 of wild type MMP9 (WT) is obviously higher than that of MMP9-Thr458 of MMP9-Thr458 mutation point (T458A) (see figure 3). The results suggest that MMP9Thr458Ala is the site of phosphorylation.

4.3 cellular immunohistochemical identification of anti-MMP 9(pT458) polyclonal antibodies

A) Cellular immunohistochemistry procedure for anti-MMP 9(pT458) polyclonal antibody:

(1) and placing the special cell slide in a 6-well plate culture dish, inoculating the cells in the culture dish according to the cell density of 2 x 104/ml, and performing immunohistochemical staining identification after the cells grow to be full of 50-70%.

(2) The medium in the six well plates was aspirated and the specimens were shaken 3 times for 1min each with PBS.

(3) Fixing in the fixing solution for 15 min.

(4) The specimens were shaken 1 time with PBS for 5 min.

(5) 0.1% Triton X-100 (DPBS conjugate) was incubated for 1 time 20 min.

(6) The specimens were shaken 3 times with PBS for 3 min each.

(7) Blocking with 2% BSA was incubated for 30 min.

(8) The PBS shakes and washes the specimen for 1 time for 5min

(9) Primary antibody (p-MMP 9)) (PBS formulation, titer 1:200, wet box) 4OC overnight or 37OC 60 min. The negative control is preferably an antiserum, or PBS

(10) The specimens were washed 4 times with PBS for 5min each.

(11) The secondary antibody working solution was incubated (wet box) for 37OC 30 min.

(12) The specimens were shaken 4 times with PBS for 5min each. 15C solution (wet box) 37OC 30 min.

(13) The specimens were washed 3 times for 5min each with PBS.

(14) DAB color development (dark, brown under the lens) for about 1-5 min.

(15) Washing with distilled water for 2 times for 1 min.

(16) And (3) counterstaining with hematoxylin for 0.5-1 min.

(17) Washing with tap water.

(18) 8% ammonia water for 30 s.

(19) Glycerin or neutral gum.

(20) And (4) observing under a mirror.

B) Cellular immunohistochemical identification of anti-MMP 9(pT458) polyclonal antibody:

selecting nasopharyngeal carcinoma cell CNE2, liver cancer cell HepG2 and stomach cancer cell MGC803 to perform a cell immunohistochemical experiment, wherein the result (shown in figure 6) shows that the anti-MMP 9(pT458) experimental group can be stained brown yellow in cytoplasm or nucleus and shows a positive result, and the control group can be stained blue in cytoplasm or nucleus and shows a negative result. Repeated for a plurality of times, and the result is consistent with the previous result. It was suggested that the anti-MMP 9(pT458) polyclonal antibody can be used for tumor cell level detection.

4.4 tissue immunohistochemical identification of anti-MMP 9(pT458) polyclonal antibodies

A) The experimental steps are as follows: the immunohistochemistry of 50 cases of cervical cancer, gastric cancer and glioma patients is carried out to verify the expression difference of the P-MMP9 (T458) antibody in the tissues beside the cancer.

1, organizing conventional paraffin sections with the thickness of 3-5um, taking the anti-dropping sheets out, airing, and putting into an oven at 65 ℃ for baking the sheets for 2 hours.

2 section Dewaxing hydration procedure

Carrying out table dewaxing on environment-friendly transparent agents I, II and III for 12 minutes respectively;

100% -95% -80% -70% -50% of ethanol for 3 minutes respectively, and then shaking tables are used.

Rinsing with ultrapure water for 5 minutes, and washing is required to be sufficient.

4, antigen heat repair: heating EDTA (1X) antigen repairing liquid and a microwave box to boil, putting the paraffin section into the boiling antigen repairing liquid, and performing medium-high grade microwave treatment for 20-30 minutes.

5 stopping heating, and cooling for 20-30 minutes at room temperature.

6 the sections after antigen retrieval were immersed in ultrapure water 2 times for 5 minutes each, and then shaken with PBS 3 times for 5 minutes each.

7 the samples were incubated in 3% H2O2 as endogenous peroxidase blocker for 15min at room temperature protected from light. The PBS buffer was shaken 3 times for 5 minutes each.

8 taking out the slices, dripping 60ul of primary antibody (concentration ratio, PBS dilution 1: 200), putting the primary antibody in a special incubation box after dripping, and keeping the primary antibody in a refrigerator at 4 ℃ overnight.

The 9 sections were washed 3 times for 5 minutes each in PBS buffer and washed thoroughly to prevent nonspecific staining due to incomplete washing. (first two PBS's were poured off)

After 10 wiping off the liquid around the tissue, 70 ul of horseradish enzyme-labeled goat anti-mouse/rabbit IgG polymer (covering the sample according to the actual condition) is added dropwise, the sample is incubated for 30 minutes at room temperature, and the sample is washed for 3 minutes by 3 times by PBS buffer.

11, developing, dripping in the ready-prepared DAB color developing agent, developing at room temperature for 5-20 minutes. The tap water stopped developing color. The first tap water was treated centrally and the tap water was washed for 3 minutes.

12 counterstaining, staining in hematoxylin staining solution for 40 seconds, washing with water for 1 minute, soaking the slices in 0.5% ammonia water for about 30 seconds, and continuing to wash with water for 5-10 minutes.

13 placing the slices in 70% ethanol-80% ethanol-5% ethanol-100% ethanol for 3 minutes each.

14 taking out the slices, sequentially placing the slices in clearing agents I, II and III for 5 minutes respectively

15 neutral gum was encapsulated and observed under an optical microscope.

B) Tissue immunohistochemical identification of anti-MMP 9(pT458) polyclonal antibody

When cervical cancer, gastric cancer and glioma sections after immunohistochemical experiments are observed under a microscope (as shown in figure 7), and the expression of the anti-MMP 9(pT458) polyclonal antibody cancer and the expression of the anti-MMP 9 polyclonal antibody in a para-carcinoma tissue are compared, the cancer tissue can be seen to be brownish yellow in cytoplasm or nucleus and is positive. The tissue beside the cancer is blue in cytoplasm or nucleus, and is negative. The results suggest that the anti-MMP 9(pT458) polyclonal antibody was expressed in cancer tissues and not expressed in paracarcinoma tissues, and there was a significant difference between them.

The test results prove that the phosphorylated polyclonal antibody can specifically recognize the phosphorylation site of the MMP9 protein pT458, can be used for detecting the phosphorylation level of the site of tumor cells such as gastric cancer, cervical cancer, nasal cancer and the like, provides a tool for researching the proliferation and metastasis mechanisms of the tumor cells, provides help for tumor diagnosis and can guide the clinical prognosis judgment of the tumor cells.

SEQUENCE LISTING

<110> Guangdong university of medicine

<120> stress phosphorylation antigen polypeptide, antibody, preparation method and application

<130> 2018

<160> 3

<170> PatentIn version 3.3

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<212> PRT

<213> Artificial sequence

<400> 1

Cys Glu Pro Arg Pro Pro Thr Thr Thr Thr Pro Gln Pro Thr

1 5 10

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<212> PRT

<213> Artificial sequence

<400> 2

Cys Glu Pro Arg Pro Pro Thr Thr Thr Thr Pro Pro Gln Pro Thr

1 5 10 15

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<211> 707

<212> PRT

<213> human MMP9 protein

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Met Ser Leu Trp Gln Pro Leu Val Leu Val Leu Leu Val Leu Gly Cys

1 5 10 15

Cys Phe Ala Ala Pro Arg Gln Arg Gln Ser Thr Leu Val Leu Phe Pro

20 25 30

Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln Leu Ala Glu Glu Tyr

35 40 45

Leu Tyr Arg Tyr Gly Tyr Thr Arg Val Ala Glu Met Arg Gly Glu Ser

50 55 60

Lys Ser Leu Gly Pro Ala Leu Leu Leu Leu Gln Lys Gln Leu Ser Leu

65 70 75 80

Pro Glu Thr Gly Glu Leu Asp Ser Ala Thr Leu Lys Ala Met Arg Thr

85 90 95

Pro Arg Cys Gly Val Pro Asp Leu Gly Arg Phe Gln Thr Phe Glu Gly

100 105 110

Asp Leu Lys Trp His His His Asn Ile Thr Tyr Trp Ile Gln Asn Tyr

115 120 125

Ser Glu Asp Leu Pro Arg Ala Val Ile Asp Asp Ala Phe Ala Arg Ala

130 135 140

Phe Ala Leu Trp Ser Ala Val Thr Pro Leu Thr Phe Thr Arg Val Tyr

145 150 155 160

Ser Arg Asp Ala Asp Ile Val Ile Gln Phe Gly Val Ala Glu His Gly

165 170 175

Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe

180 185 190

Pro Pro Gly Pro Gly Ile Gln Gly Asp Ala His Phe Asp Asp Asp Glu

195 200 205

Leu Trp Ser Leu Gly Lys Gly Val Val Val Pro Thr Arg Phe Gly Asn

210 215 220

Ala Asp Gly Ala Ala Cys His Phe Pro Phe Ile Phe Glu Gly Arg Ser

225 230 235 240

Tyr Ser Ala Cys Thr Thr Asp Gly Arg Ser Asp Gly Leu Pro Trp Cys

245 250 255

Ser Thr Thr Ala Asn Tyr Asp Thr Asp Asp Arg Phe Gly Phe Cys Pro

260 265 270

Ser Glu Arg Leu Tyr Thr Gln Asp Gly Asn Ala Asp Gly Lys Pro Cys

275 280 285

Gln Phe Pro Phe Ile Phe Gln Gly Gln Ser Tyr Ser Ala Cys Thr Thr

290 295 300

Asp Gly Arg Ser Asp Gly Tyr Arg Trp Cys Ala Thr Thr Ala Asn Tyr

305 310 315 320

Asp Arg Asp Lys Leu Phe Gly Phe Cys Pro Thr Arg Ala Asp Ser Thr

325 330 335

Val Met Gly Gly Asn Ser Ala Gly Glu Leu Cys Val Phe Pro Phe Thr

340 345 350

Phe Leu Gly Lys Glu Tyr Ser Thr Cys Thr Ser Glu Gly Arg Gly Asp

355 360 365

Gly Arg Leu Trp Cys Ala Thr Thr Ser Asn Phe Asp Ser Asp Lys Lys

370 375 380

Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala

385 390 395 400

His Glu Phe Gly His Ala Leu Gly Leu Asp His Ser Ser Val Pro Glu

405 410 415

Ala Leu Met Tyr Pro Met Tyr Arg Phe Thr Glu Gly Pro Pro Leu His

420 425 430

Lys Asp Asp Val Asn Gly Ile Arg His Leu Tyr Gly Pro Arg Pro Glu

435 440 445

Pro Glu Pro Arg Pro Pro Thr Thr Thr Thr Pro Gln Pro Thr Ala Pro

450 455 460

Pro Thr Val Cys Pro Thr Gly Pro Pro Thr Val His Pro Ser Glu Arg

465 470 475 480

Pro Thr Ala Gly Pro Thr Gly Pro Pro Ser Ala Gly Pro Thr Gly Pro

485 490 495

Pro Thr Ala Gly Pro Ser Thr Ala Thr Thr Val Pro Leu Ser Pro Val

500 505 510

Asp Asp Ala Cys Asn Val Asn Ile Phe Asp Ala Ile Ala Glu Ile Gly

515 520 525

Asn Gln Leu Tyr Leu Phe Lys Asp Gly Lys Tyr Trp Arg Phe Ser Glu

530 535 540

Gly Arg Gly Ser Arg Pro Gln Gly Pro Phe Leu Ile Ala Asp Lys Trp

545 550 555 560

Pro Ala Leu Pro Arg Lys Leu Asp Ser Val Phe Glu Glu Arg Leu Ser

565 570 575

Lys Lys Leu Phe Phe Phe Ser Gly Arg Gln Val Trp Val Tyr Thr Gly

580 585 590

Ala Ser Val Leu Gly Pro Arg Arg Leu Asp Lys Leu Gly Leu Gly Ala

595 600 605

Asp Val Ala Gln Val Thr Gly Ala Leu Arg Ser Gly Arg Gly Lys Met

610 615 620

Leu Leu Phe Ser Gly Arg Arg Leu Trp Arg Phe Asp Val Lys Ala Gln

625 630 635 640

Met Val Asp Pro Arg Ser Ala Ser Glu Val Asp Arg Met Phe Pro Gly

645 650 655

Val Pro Leu Asp Thr His Asp Val Phe Gln Tyr Arg Glu Lys Ala Tyr

660 665 670

Phe Cys Gln Asp Arg Phe Tyr Trp Arg Val Ser Ser Arg Ser Glu Leu

675 680 685

Asn Gln Val Asp Gln Val Gly Tyr Val Thr Tyr Asp Ile Leu Gln Cys

690 695 700

Pro Glu Asp

705

Claims (5)

1. A phosphorylated antigen polypeptide having the sequence CEPRPPTTTT(p) PQPT, wherein T (p) represents the amino acid phosphorylated threonine at position T458 of human MMP9 protein.

2. A polyclonal antibody directed against the phosphorylated antigen polypeptide of claim 1.

3. The method for producing a polyclonal antibody as set forth in claim 2, comprising: (1) artificially synthesizing the phosphorylated antigen polypeptide according to claim 1; (2) coupling the synthesized polypeptide with maleimide activated carrier mcKLH, desalting the coupling product with desalting column and immunizing New Zealand rabbit; (3) detecting the titer of the antibody by using the rabbit serum subjected to the four immunizations by using an ELISA method, collecting the immune rabbit serum after the titer reaches an ideal value, and purifying the antibody by using a polypeptide-coated cyanogen bromide-activated agarose affinity purification column; (4) the purified antibody was subjected to ELISA, western bot assay.

4. Use of the polyclonal antibody of claim 2 in the preparation of a reagent or kit for detecting human MMP9 protein in which phosphorylation at T458 occurs.

5. A detection kit comprising the polyclonal antibody of claim 2, an antigen retrieval solution, a PBS buffer solution, and an enzyme blocking agent of 3% H₂O₂The kit comprises horseradish enzyme-labeled goat anti-mouse/rabbit IgG polymer, a DAB color developing agent, hematoxylin dye solution, ethanol, an environment-friendly transparent agent, 0.5% ammonia water and ultrapure water.

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