Antigenic Peptides, Antibodies Targeting Phosphorylation of STAT3 Protein at Ser701 and Their Applications

Abstract

The present application discloses antigenic peptides targeting phosphorylation of STAT3 protein at Ser701 and their applications. The antigenic peptides have amino acid sequences shown in SEQ ID NO.1-4, or homologous sequences having the same functions as those sequences. The antibodies prepared by using the antigenic peptides can detect the phosphorylation level of STAT3 protein at Ser701 in cells and tissues, which helps to study the molecular mechanism and biological functions of phosphorylation of STAT3 protein at Ser701; and provides assistance for studying the roles of STAT3 in cell signal pathways such as cell cycle progression, autophagy, apoptosis, metabolism, inflammation, invasion and angiogenesis. The present application provides a method for preparing an antibody against phosphorylation of STAT3 protein at Ser701. The method is simple to operate, and the antibody prepared by this method has strong specificity, high purity and good stability.

Description

Technical Field

[0001] This application belongs to the technical field of STAT3 protein phosphorylation detection. This application discloses an antigenic peptide targeting STAT3 protein Ser701 phosphorylation and its application. Specifically, it involves using the peptide as an antigen to prepare an antibody that specifically recognizes STAT3 protein Ser701 phosphorylation and its application for detecting the phosphorylation level of STAT3 protein Ser701. Background Art

[0002] Signal transducer and activator of transcription (STAT) is a protein with DNA-binding activity, including STAT1-6. Among them, STAT3 protein is one of the most important members of the STAT family. As a prominent nuclear transcription factor, it is related to the expression of more than 1000 gene products.

[0003] The STAT3 protein can be structurally divided into the following functional regions: N-terminal domain, DNA-binding domain, coiled-coil domain, SH2 domain, and C-terminal transcriptional activation domain. Among them, the SH2 domain is the most conserved and the most important region for its function. Phosphorylation of the conserved Tyr705 residue located between the SH2 and C-terminal transcriptional activation domains mediates the interaction between two STAT3 molecules, forming a homodimer.

[0004] Post-translational modification of proteins is crucial for the regulation of STAT3 protein activity, and phosphorylation modification is the most important one. Phosphorylation of STAT3 at Tyr705 can be activated by a variety of cytokines and growth factors, including those that utilize the IL-6 signal transduction receptor chain gp130 (such as interleukin-6, oncostatin M, interleukin-11) or homodimeric cytokine receptors (such as granulocyte colony-stimulating factor, G-CSF), as well as those that act through protein tyrosine kinase receptors (such as epidermal growth factor, EGF). After the cytokine binds to the receptor, it activates the phosphorylation of specific tyrosine residues at the receptor complex. The SH2 domain of the STAT3 protein interacts with the specific receptor phosphotyrosine sequence, recruiting the STAT3 protein into the receptor signal complex, phosphorylating the Tyr705 residue of the STAT3 protein, and causing it to dissociate from the cell membrane. Phosphorylated STAT3 at Tyr705 forms a homodimer with DNA-binding activity in the cytoplasm, which can translocate into the nucleus and bind to specific sites on the promoter sequence of target genes, participating in the transcriptional regulation of target genes. In addition, phosphorylation at Ser727 can cause STAT3 to translocate to the mitochondria, where it can enter the mitochondria and bind to mtDNA to regulate gene transcription and expression, increase the activity of electron transport chain complex enzymes under ischemic injury, reduce the production of ROS, increase the production of ATP, and reduce tissue damage. Summary of the Invention

[0005] Through in-depth research on the structure of STAT3 protein, the inventors of the present application found that there is a possibility of phosphorylation of the 701st amino acid Ser in STAT3 protein, and phosphorylation at this position may affect the activation of the classical signaling pathway of STAT3 protein. Therefore, exploring relevant technologies and methods for effectively detecting the phosphorylation of Ser701 in STAT3 protein has positive significance for exploring the mechanism of STAT3 Ser701 phosphorylation in cells, as well as its biological functions in animals and humans and related drug development. For this reason, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the embodiments of the present application disclose an antigenic peptide, which is a peptide or its functional fragment that mimics the phosphorylation of STAT3 protein at Ser701. This peptide or its functional fragment can generate an immune response in vivo, thereby generating an antibody that can specifically bind to the phosphorylation of STAT3 at Ser701. Thus, using this antibody, the phosphorylation level of Ser701 in STAT3 protein in cells can be effectively detected, and the biological processes such as cell growth, proliferation, metabolism, and inflammation can be indirectly explored.

[0007] The antigenic peptide has immunogenicity targeting the Ser701 phosphorylation site of STAT3 protein, wherein the antigenic peptide is at least one of (I) to (IV):

[0008] (I). The amino acid sequences shown in SEQ ID NOs. 1 to 4;

[0009] (II). An amino acid sequence having at least 75% similarity to the sequence described in (I);

[0010] (III). An amino acid sequence obtained by substituting, deleting or adding one or more amino acid sequences to the amino acid sequence shown in (I) or (II), and having the same or similar function as the amino acid sequence shown in (I) or (II);

[0011] (IV). The peptide described in (I), (II) or (III) or is modified at some or all of the amino acid residues in its amino acid sequence.

[0012] In a second aspect, an antigen construct, the antigen construct comprising the antigen peptide of the first aspect and a carrier for loading or conjugating the antigen peptide of the first aspect.

[0013] In a third aspect, the embodiments of the present application also disclose the applications of the antigen peptide described in the first aspect and the antigen construct described in the second aspect, and the applications include at least one of the following:

[0014] (1) Preparing a Ser701 phosphorylation-specific antibody of STAT3 protein;

[0015] (2) Preparing a vaccine for diseases related to Ser701 phosphorylation of STAT3 protein;

[0016] (3) An immune activator.

[0017] In a fourth aspect, the embodiments of the present application disclose an antibody, the antibody immunoreacts with the antigen peptide defined in the first aspect or with the construct described in the second aspect.

[0018] In a fifth aspect, the embodiments of the present application disclose a method for preparing the antibody described in the fourth aspect, including the steps of immunizing an animal with the antigen peptide described in the first aspect and the construct described in the second aspect, collecting antiserum and purification.

[0019] In a sixth aspect, the embodiments of the present application disclose an ELISA kit, the ELISA kit includes the antibody described in the fourth aspect, and the ELISA kit is used to detect the phosphorylation level of the Ser701 site of STAT3 protein.

[0020] In a seventh aspect, the embodiments of the present application disclose a pharmaceutical composition, including the antibody described in the fourth aspect or the antibody obtained by the preparation method described in the fifth aspect, and a pharmaceutically acceptable excipient or carrier.

[0021] Compared with the prior art, the present application has at least one of the following beneficial effects:

[0022] The present application provides an antigenic peptide targeting phosphorylation of STAT3 protein at Ser701 and an antibody prepared therefrom, which helps to study the role of STAT3 in cell signaling pathways such as cell cycle progression, autophagy, apoptosis, metabolism, inflammation, invasion and angiogenesis, as well as the mechanism of action of STAT3 in various diseases such as tumors, metabolic diseases, inflammatory bowel disease, cardiovascular diseases, etc., and provides potential action targets for the treatment of clinical diseases; the antibody against phosphorylation of STAT3 protein at Ser701 site can be used in the prognosis determination of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a mass spectrometry identification diagram of phosphorylation of STAT3 protein at Ser701 site provided by an embodiment of the present application.

[0024] Figure 2 It is a HPLC purity determination result diagram of phosphorylated (upper figure) antigenic peptide and non-phosphorylated (lower figure) antigenic peptide provided by an embodiment of the present application.

[0025] Figure 3 It is an electrophoresis diagram (a), sequencing alignment diagram (b) of expression plasmids of different STAT3 protein mutants and a Western Blot detection diagram (c) of the homemade antibody used to detect the background of STAT3 protein in cells and the phosphorylation signal at Ser701 site upon IL-6 stimulation provided by an embodiment of the present application;

[0026] Figure 3 In a, the lanes from left to right are Marker, pCDNA3.1-GFP, pCDNA3.1-GFP-STAT3-FL and pCDNA3.1-GFP-STAT3-S701A in sequence; Figure 3 In b, it is a sequencing alignment result diagram of pCDNA3.1-GFP-STAT3-FL and pCDNA3.1-GFP-STAT3-S701A; Figure 3 In c, the vertically arranged lanes 1, 2, and 3 are all Western Blot detection results of cell lysates obtained by transfecting Hela cells with pCDNA3.1-GFP-STAT3-FL as samples, and the lane 4 shows the Western Blot detection results of cell lysates obtained by transfecting Hela cells with pCDNA3.1-GFP-STAT3-S701A as samples.

[0027] Figure 4 It is a Western Blot detection diagram of the homemade antibody used to detect the phosphorylation signal of STAT3 protein at Ser701 site in mouse colon tissue provided by an embodiment of the present application. Detailed implementation manners

[0028] In order to make the objectives, technical solutions and advantages of the present application more clear and understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. Reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0029] When the inventors of the present application studied the signal pathway related to STAT3 and its biological functions, they focused on serine at position 701 of the STAT3 protein. Serine has a hydroxyl group and can dehydrate with a phosphate group to form a phosphate ester, that is, phosphorylation can occur. Phosphorylation can change the steric hindrance, charge and stability of amino acids, thereby controlling the activity, localization, stability and interaction of proteins. In the prior art, the three-dimensional structure analysis of the STAT3 protein has obtained the general structure of STAT3 including Tyr705, however, the structure at the position where Ser701 is located still cannot be resolved, indicating that the structure of this part is unstable or flexible and may have a regulatory function. Due to the nature and structural flexibility of serine at position 701 of STAT3, it is suggested that it has the possibility of phosphorylation.

[0030] Therefore, the inventors of the present application conducted the following experiments:

[0031] Inoculate Hela cells into a 10 cm 2 cell culture dish and culture them in DMEM medium containing 10% fetal bovine serum. When the cell density grows to about 60% - 70% in a 5% CO 2 incubator, take the STAT3 expression vector pCDNA3.1-Flag-STAT3-FL (constructed by the method shown in "Construction of eukaryotic expression vector of PCDNA3.1-GFP-LC3B [J]. Journal of Anhui Agricultural Sciences, 2015, issue 6.") and transfect Hela cells by the conventional liposome transfection method. After 24 hours of transfection, use trypsin to digest and collect the cells. After lysing the cells with RIPA lysis buffer, perform immunoprecipitation using agarose gel microspheres conjugated with Flag antibody (GenScript Biotech, Cat. L00432) to obtain purified STAT3 protein. Perform SDS-PAGE and Coomassie Brilliant Blue staining on the purified product, cut out the target protein band, and perform biomass spectrometry on the cut gel. As Figure 1 shown in the mass spectrometry results, serine at position 701 of the human STAT3 protein can be phosphorylated.

[0032] Therefore, phosphorylation of Ser701 of STAT3 can indeed occur. Whether phosphorylation of Ser701 of STAT3 will affect the classical signal pathway activation sites Tyr705 / Ser727 and how it specifically affects are of great significance for filling the current research gap in STAT3, signal pathway transduction of STAT3, and biological functions. As a brand-new phosphorylation site of STAT3, there is currently a lack of STAT3 antibodies that can be used to study the phosphorylation of Ser701 in the domestic and international markets, so related research and applications cannot be carried out. Therefore, it is necessary to develop an antibody against the phosphorylation of Ser701 of STAT3 protein to explore the mechanism of STAT3 Ser701 phosphorylation in cells, as well as its biological functions in animals and humans and related drug development, etc.

[0033] Antigenic peptide

[0034] For this purpose, the embodiments of the present application disclose an antigen peptide, and the antigen peptide has immunogenicity targeting the phosphorylation site of Ser701 of STAT3 protein, wherein the antigen peptide is at least one of (I) to (IV):

[0035] (I). The amino acid sequences shown in SEQ ID NO.1 - 4;

[0036] (II). An amino acid sequence having at least 75% similarity to the sequence described in (I);

[0037] (III). An amino acid sequence obtained by substituting, deleting, or adding one or more amino acid sequences to the amino acid sequence shown in (I) or (II), and having the same or similar function as the amino acid sequence shown in (I) or (II);

[0038] (IV). The antigen peptide described in (I), (II), or (III) or being modified at some or all amino acid residues in its amino acid sequence.

[0039] The terms "polypeptide", "peptide", and "protein" used herein are interchangeable and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0040] The term "peptide" refers to a chain of amino acids (usually L-amino acids) in which the α-carbons of the amino acids are linked by peptide bonds formed by a condensation reaction between the carboxyl group of the α-carbon of one amino acid and the amino group of the α-carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxyl terminus) has a free carboxyl group. Thus, the term "amino terminus" (abbreviated as N-terminus) refers to the free α-amino group on the amino-terminal amino acid of a peptide, or to the α-amino group of an amino acid at any other position within the peptide (which is an imino group when participating in a peptide bond). Similarly, the term "carboxyl terminus" (abbreviated as C-terminus) refers to the free carboxyl group on the carboxyl-terminal amino acid of a peptide, or to the carboxyl group of an amino acid at any other position within the peptide.

[0041] In the present application, "being modified at some or all of the amino acid residues in its amino acid sequence", such modified peptides can be prepared by any method known in the art. For example, modified peptides can be prepared by modifying the functional groups of the side chains of the amino acid residues constituting the peptide, such as esterification, alkylation, halogenation, phosphorylation, sulfonation, or amidation. Moreover, various substances can be bound to the peptide at the N- and / or C-terminals. For example, an amino acid, a peptide, or an analogue thereof can be bound to the peptide. When such a substance is bound to the "antigenic peptide as described in (I), (II), or (III)" above, this substance can be removed by any method, such as by an in vivo enzymatic reaction or by intracellular processing, so that the antigenic peptide is ultimately produced. The purpose of such "modification" can be to regulate the solubility of the peptide; to improve the stability of the peptide, such as protease resistance; to deliver the peptide to a specific tissue or organ; or to increase the uptake of the peptide by antigen-presenting cells. For example, the purpose of such "modification" can also be to enhance the immunogenicity of the antigenic peptide and to promote the specific binding of the antigenic peptide to the STAT3 protein Ser701 phosphorylation epitope.

[0042] The term "amino acid sequence having the same or similar function" or "functional fragment" as used herein refers to a functional peptide fragment having substantially the same (biological) activity as the peptide defined herein (e.g., as shown in SEQ ID NOs. 1 to 4 respectively), that is, this fragment can still cause a highly specific immune response (i.e., having immunogenic activity) in an organism, especially in an animal, especially a mammal or a human, to produce antibodies that can specifically recognize and bind to the STAT3 protein Ser701 phosphorylation epitope.

[0043] The term "residue" as used herein refers to an amino acid incorporated into a peptide through an amide bond. Thus, the amino acid can be a naturally occurring amino acid, or, unless otherwise restricted, can include known analogues of naturally occurring amino acids that function in a manner similar to naturally occurring amino acids (i.e., amino acid analogues).

[0044] To maintain the immunogenicity of the STAT3 protein Ser701 phosphorylation epitope, a few (e.g., 1, 2, or several) or a small percentage of amino acids can be modified (added, deleted, and / or substituted). Here, the term "several" means less than 7 amino acids, such as less than 6 or 5. The percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, more preferably 10% or less, and even more preferably 1 - 5%.

[0045] Those skilled in the art should understand that the individual addition, deletion, or substitution of a single amino acid or a small percentage of amino acids in an amino acid sequence will result in the retention of the characteristics of the original amino acid side chain. Therefore, it is called "conservative substitution" or "conservative modification", and this change means replacing an amino acid with a chemically similar amino acid, where the change to the protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following items 1) - 8) each contain amino acids that are conservative substitutions for each other:

[0046] 1) Alanine (A), Glycine (G);

[0047] 2) Serine (S), Threonine (T);

[0048] 3) Aspartic acid (D), Glutamic acid (E);

[0049] 4) Asparagine (N), Glutamine (Q);

[0050] 5) Arginine (R), Lysine (K);

[0051] 6) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0052] 7) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and

[0053] 8) Cysteine (C), Methionine (M).

[0054] Such conservatively modified peptides are also regarded as the peptides of the present invention. However, the peptides of the present invention are not limited thereto and may include non-conservative modifications as long as the peptide retains the immunogenicity of phosphorylation at the STAT3 protein Ser701 site of the original peptide.

[0055] In certain embodiments of the present application, the amino acid sequences of the antigen peptides are as shown in SEQ ID NOs. 1 - 4, as shown in Table 1. Table 1 also shows the amino acid sequences of the control peptides for comparative experiments as shown in SEQ ID NOs. 5 - 8. In Table 1, "p-" indicates that the amino acid residue at this site is phosphorylated, and "-NH 2 " indicates that the amino acid residue at this site is amidated.

[0056] Table 1

[0057] Antigenic peptide Sequence information Phosphorylated peptide 1 <![CDATA[CPEADPG(p-S)AAPYL-NH 2 , as shown in SEQ ID NO.1]]> Phosphorylated peptide 2 CEHPEADPG(p-S)AAPY, as shown in SEQ ID NO.2 Phosphorylated peptide 3 <![CDATA[HPEADPG(p-S)AAPYLKT-NH 2 , as shown in SEQ ID NO. 3]]> Phosphorylated peptide 4 CHPEADPG(p-S)AAPYLK, as shown in SEQ ID NO.4 Control peptide 1 <![CDATA[CPEADPGSAAPYL-NH 2 , as shown in SEQ ID NO.5]]> Control peptide 2 CEHPEADPGSAAPY, as shown in SEQ ID NO.6 Control peptide 3 <![CDATA[HPEADPGSAAPYLKT-NH 2 , as shown in SEQ ID NO.7]]> Control peptide 4 CHPEADPGSAAPYLK, as shown in SEQ ID NO.8

[0058] On the other hand, embodiments of the present application disclose an antigen construct, which comprises the antigen peptide of the first aspect and a carrier for loading or conjugating the antigen peptide of the first aspect. In particular, the carrier is also a carrier having the functionality of an adjuvant for generating a supramolecular antigen construct. In certain embodiments, the antigen peptide according to the first aspect is modified by attachment to or reconstitution in, for example, liposomes to produce the "supramolecular antigen construct" described in WO publication WO2005 / 081872, the description of which WO publication is hereby incorporated by reference in its entirety. The thus obtained "supramolecular antigen construct" enables a unique antigen peptide presentation on its surface, which presentation results in enhanced antigen exposure and ultimately leads to the production of antibodies showing high conformational sensitivity. Specifically, the antigen peptide according to the present application or its functional fragment is modified by binding to a lipophilic or hydrophobic moiety facilitating insertion into the lipid bilayer of the liposome carrier / immunoadjuvant, in particular by a lipophilic or hydrophobic moiety acting as an anchor of the peptide in the lipid bilayer and having a size that results in the localization and stabilization of the peptide close to the liposome surface.

[0059] In some embodiments, the lipophilic or hydrophobic moiety is a fatty acid, triglyceride or phospholipid, in particular a fatty acid, triglyceride or phospholipid containing a carbon chain between C12 and C24, but especially palmitic acid.

[0060] In some embodiments, the antigen peptide of the present application is modified by covalently binding at least two molecules of palmitic acid to the N-terminal and C-terminal ends of the antigen peptide and by reconstitution in a liposome carrier.

[0061] In some embodiments, each peptide in the conjugate is conjugated to four molecules of palmitic acid; thus they are tetra-palmitoylated.

[0062] In some embodiments, two molecules of palmitic acid are conjugated to the N-terminal end of the antigen peptide and two molecules of palmitic acid are conjugated to the C-terminal end of the peptide or fragment.

[0063] In some embodiments, the antigen peptide provided by the present application is modified by binding to a lipophilic or hydrophobic moiety, such as palmitic acid, and reconstituted in a liposome, wherein the liposome preparation may further comprise an adjuvant for generating a supramolecular antigen construct, such as lipid A, alum, calcium phosphate, interleukin 1 and / or microcapsules of polysaccharides and proteins, in particular detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum.

[0064] In one embodiment of the present application, the supramolecular construct involved in the present application, each carrier molecule of which contains one or more antigenic peptides as described in the present application, especially two or more antigenic peptides.

[0065] In one embodiment of the present application, the carrier molecule is a liposome.

[0066] In some embodiments, the supramolecular constructs involved in the present application and the supramolecular constructs as described herein, each carrier molecule of which contains a combination of two or more antigenic peptides shown in any one of SEQ ID NOs. 1 to 4.

[0067] In the "supramolecular antigen construct" of the present application, the liposome can have a dual function. It can be used as a carrier containing the supramolecular construct described previously herein, and at the same time act as an adjuvant to increase or stimulate the immune response in the target animal or human to be treated with the therapeutic vaccine of the present application. It should also be understood that the supramolecular antigen construct composition of the present application can further contain other adjuvants, including but not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA), bovine thyroglobulin (THY), and other adjuvants such as lipid A, alum, calcium phosphate, interleukin 1, and / or microcapsules of polysaccharides and proteins, but especially detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, or alum; other preservatives; diluents; emulsifiers; stabilizers; and other components known and used in vaccines of the prior art. In addition, any adjuvant system known in the art can be used in the composition of the present application. Such adjuvants include but not limited to Freund's incomplete adjuvant; Freund's complete adjuvant; polydisperse β-(1,4)-linked acetylated mannan; polyoxyethylene-polyoxypropylene copolymer adjuvant; modified lipid adjuvant; saponin derivative adjuvant; inactivated pertussis toxin; hemocyanin; outer membrane protein of Neisseria meningitidis group B; Pseudomonas aeruginosa exotoxin A; cholera toxin B subunit; bacterial outer membrane protein; Escherichia coli heat-labile enterotoxin; pneumococcal hemolysin; gonococcal pilin; lipopolysaccharide (LPS) of Gram-negative bacteria; large polymeric anions such as dextran sulfate; and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate.

[0068] In certain embodiments of the present application, the peptides described in the first aspect can be synthesized according to the synthetic methods commonly used in the chemical field and / or biosynthesized.

[0069] The term "isolated" herein refers to a substance that is substantially or essentially free from the components that normally accompany it in its natural state. Thus, the peptides described herein do not contain substances that are normally bound to their in situ environment. Generally, by measuring the band intensity on a silver-stained gel, the isolated immunogenic peptides described herein are at least about 90% (pure) by HPLC.

[0070] Protein purity or homogeneity can be shown by many methods well known in the art, such as by staining after polyacrylamide gel electrophoresis of a protein sample. For certain purposes, high resolution will be required and purification will be carried out using HPLC or similar means.

[0071] When the length of the immunogenic peptide is relatively short (i.e., less than about 50 amino acids), they are usually synthesized using standard chemical peptide synthesis techniques. For example, solid-phase synthesis methods well known to those skilled in the art are used. Solid-phase synthesis is the preferred method for chemically synthesizing the immunogenic peptides described herein, in which the C-terminal amino acid of the sequence is linked to an insoluble support and then the remaining amino acids in the sequence are added sequentially.

[0072] For example, in one embodiment, referring to the classical Fmoc solid-phase synthesis method provided in "Study on the Chemical Synthesis of O-Phosphorylated Polypeptides Based on the Fmoc Strategy [J]. Chemical Journal of Chinese Universities, Special Issue 1, 2001", phosphoramidite was used as the phosphorylation reagent, and the phosphorylated amino acids in the amino acid sequences shown in SEQ ID NOs. 1-8 were synthesized by the monomer phosphorylation method. And referring to "New Process for the Synthesis of L-Prolinamide [J]. Shandong Industrial Technology, 2016, Issue 9", the amide amino acids at the carbon terminus were synthesized, and then the polypeptides with the amino acid sequences shown in SEQ ID NOs. 1-8 were synthesized in sequence by the Fmoc solid-phase synthesis method. The freeze-dried products with HPLC purity greater than 90% were obtained after purification (the results are as Figure 2 )

[0073] Alternatively, a method combining recombinant expression biosynthesis methods well known to those skilled in the art with chemical modification can be used to obtain the antigenic peptides or their functional fragments described herein. Generally speaking, this involves generating a nucleic acid sequence encoding the peptide, placing the nucleic acid in an expression cassette under the control of a specific promoter, expressing the peptide in a host, isolating the expressed peptide, and refolding the peptide as needed. Techniques sufficient to guide a person skilled in the art to complete such methods are found in the literature. As is well known in the art, immunogenic peptides expressed by a host can be purified by standard methods including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. For use as a therapeutic agent, a substantially pure composition with a homogeneity of preferably about 50% to 95%, and most preferably 80% to 95% or greater is preferred.

[0074] Antibody

[0075] On the other hand, the embodiments of the present application also disclose an antibody that can specifically immunoreact with the antigenic peptides disclosed in the above embodiments. Specifically, the antibody can be prepared by injecting the antigen peptide or the antigen construct as an antigen into the body to cause an immune response.

[0076] In some embodiments, the preparation of the antibody comprises the following process:

[0077] 1. Preparation of antigens

[0078] In this embodiment, phosphorylated peptides 1-4 shown in Table 1 are respectively conjugated with keyhole limpet hemocyanin (KLH) (the coupling agent is Sulfo-SMCC) as immunogens. Phosphorylated peptides 1-4 and control peptides are respectively conjugated with bovine serum albumin (BSA) (the coupling agent is glutaraldehyde) as detection antigens; the conjugation method refers to "Study on the immunogenicity and arthritis-inducing properties of synthetic cyclic citrullinated protein peptides [J]. Chinese Journal of Immunology, 2017, Issue 1". The prepared antigens are diluted to 1 mg / mL with phosphate-buffered saline (PBS) respectively, aliquoted and stored frozen at -20 °C in a refrigerator.

[0079] 2. Animal immunization

[0080] On the 1st, 15th, 29th, and 43rd days respectively, 1 mL of each immunogen is taken and added to 1 mL of Freund's complete adjuvant, emulsified (to check the emulsification degree: drop a drop of the emulsified antigen solution into physiological saline, if it does not disperse, it indicates that the requirement has been met), and immunized subcutaneously at multiple points on the back of the neck (at least 8 points). Two New Zealand white rabbits (Jiangsu Ailingfei) are immunized with each antigen. On the 53rd day, blood is collected from the carotid artery, and a large amount of antiserum is collected. The rabbit blood is left standing overnight in a 4 °C refrigerator. The next day, under aseptic conditions, the rabbit blood is aliquoted into 50 mL centrifuge tubes, centrifuged at 4 °C, 10,000 rpm / min for 30 minutes, and the supernatant is collected, which is the antiserum after immunization and stored at -20 °C.

[0081] 3. Antibody purification

[0082] (1) Affinity purification columns are respectively prepared using phosphorylated peptides 1-4 shown in SEQ ID NO. 1-4 to affinity purify phosphorylated specific antibodies. The polypeptide is conjugated to activated Sulfolink Resin (product number 20401, Thermo Fisher Scientific) to prepare an antigen affinity column, and 1 mg of polypeptide is conjugated to 1 mL of Sulfolink Resin.

[0083] (2) The affinity column is equilibrated with 10 column volumes of PBS, and the solution is drained; the rabbit serum is filtered through a 0.45 μm filter membrane.

[0084] (3) The serum is passed through the antigen affinity column, the solution is drained, and the flow-through is collected.

[0085] (4) Equilibrate with 10 column volumes of PBS, and drain the solution.

[0086] (5) Add 5 mL of antibody eluent, collect the eluent in separate tubes, 1 mL per tube.

[0087] (6) Detect the absorbance of the collected eluate at 280 nm, combine the components with an absorbance greater than 1.0, and dialyze against PBS.

[0088] 4. Identification of the antibody after dialysis

[0089] The protein concentration was detected by the ultraviolet absorption method, and the antibody titer was detected by the enzyme-linked immunosorbent assay. The test samples were antibodies obtained after immunizing New Zealand white rabbits with antigens prepared from the phosphorylated peptides 1 to 4 shown in SEQ ID NO.1 to 4 above and through the above purification steps. The antibodies were diluted with the coating diluent at ratios of 1:1250, 1:2500, 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000, and the following steps (1) to (6) were carried out.

[0090] (1) Coating: Dilute the coating antigen with the coating buffer to 1 μg / ml, add 100 μl to each well of the enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4°C.

[0091] (2) Blocking: Discard the coating solution, add 200 μl of the blocking solution (5% skim milk powder) to each well, and incubate statically at 37°C for 1.5 hours.

[0092] (3) Add the test sample: Discard the blocking solution, add the sample (serum or antibody), add 100 μl to each well of the ELISA plate, and incubate statically at 37°C for 1 hour; wash 10 times with the washing buffer, and pat dry the liquid in the wells.

[0093] (4) Incubation with the secondary antibody: Dilute the enzyme-labeled goat anti-rabbit secondary antibody to the working concentration with the blocking solution, add 100 μl to each well of the ELISA plate, and incubate statically at 37°C for 30 minutes; wash 10 times with the washing buffer, and pat dry the liquid in the wells.

[0094] (5) Color development: Add the TMB color development substrate: Add 100 μl to each well of the ELISA plate, and incubate statically at 37°C for 15 minutes.

[0095] (6) Termination and reading: Add 50 μl of 2M H 2 SO 4 to terminate the reaction, and use an ELISA reader to read the value at OD450nm.

[0096] 5. Results

[0097] The antibody titers generated by detecting the detection antigens prepared by conjugating bovine serum albumin with the peptides shown in SEQ ID NOs. 1 to 8 were respectively detected, and the results are shown in Table 2. Among them, Ag-1 to 4 are detection antigens prepared by conjugating BSA with the phosphorylated peptides shown in SEQ ID NOs. 1 to 4, and Ag-control-1 to 4 are detection antigens prepared by conjugating BSA with the control peptides shown in SEQ ID NOs. 5 to 8 respectively. Ab-1 to 4 are phosphorylated antibodies obtained by conjugating hemocyanin with the phosphorylated peptides 1 to 4 shown in SEQ ID NOs. 1 to 4 through steps such as animal immunization and antibody purification. The results show that the ELISA titer of the Ser701 phosphorylation-specific antibody of the STAT3 protein against the phosphorylated polypeptide is greater than 1:80000.

[0098] Table 2 OD450nm values

[0099]

[0100] Application

[0101] Therefore, the embodiments of the present application essentially also disclose the applications of the antigen peptides described in the first aspect and the antigen constructs described in the second aspect. The applications include at least one of the following:

[0102] (1) Preparing a Ser701 phosphorylation-specific antibody of the STAT3 protein;

[0103] (2) Preparing a drug for a disease related to Ser701 phosphorylation of the STAT3 protein;

[0104] (3) Acting as an immune activator.

[0105] In addition, in order to detect the Ser701 phosphorylation level of the STAT3 protein in some cells, tissues or organisms, the embodiments of the present application also disclose a kit for detecting the Ser701 phosphorylation level of the STAT3 protein, and the kit includes the antibody disclosed in the above embodiments. By targeting and recognizing the Ser701 site of the STAT3 protein in cells, tissues or organisms with this antibody, an immunoblot kit or an ELISA kit is prepared to achieve detection.

[0106] In some embodiments of the present application, the detection kit includes an enzyme-linked immunosorbent assay (ELISA) plate coated with the Ser701 phosphorylation antibody of the STAT3 protein, a washing buffer, a blocking solution, a coating buffer, an enzyme-labeled antibody, a TMB chromogenic solution, and a termination solution.

[0107] In one embodiment, the ELISA detection kit for detecting the Ser701 phosphorylation of the STAT3 protein in the present application specifically includes the following components:

[0108] The STAT3 protein Ser701 phosphorylation antibody provided by the above embodiments;

[0109] Enzyme-labeled antibody;

[0110] Washing buffer: PBST buffer: 1000 mL of 0.01 mol / L PBS + 0.5 mL of Tween-20;

[0111] Blocking solution: PBS buffer containing 5% non-fat milk powder;

[0112] Coating buffer: containing 0.015 M Na 2 CO 3 and 0.035 M NaHCO 3 , pH 9.6;

[0113] TMB stock solution: 10 mg of TMB is fully dissolved in 5 mL of absolute ethanol;

[0114] TMB chromogenic solution: containing 0.5 mL of TMB stock solution, 10 mL of substrate buffer and 2.1 μL of 30% mass percentage of H 2 O 2 aqueous solution, freshly prepared when in use; and

[0115] Stop solution: 2 M H 2 SO 4 .

[0116] The above kit can be used to identify the phosphorylation of STAT3 protein Ser701, for example, to detect the Ser phosphorylated STAT3 protein or its peptide segment at position 701 using the Elisa method. In one embodiment, the preparation method of the detection kit further includes the step of coating the enzyme-labeled plate with the STAT3 protein Ser701 phosphorylation antibody.

[0117] In one embodiment, the steps for detecting the phosphorylation of STAT3 protein at the Ser701 site using the antibody prepared above include:

[0118] (1) Construct expression vectors

[0119] Referring to the method shown in "Construction of eukaryotic expression vector PCDNA3.1-GFP-LC3B [J] Journal of Anhui Agricultural Sciences, 2015, Issue 6.", construct the STAT3 protein expression vector pCDNA3.1-GFP-STAT3-FL and the STAT3 protein expression vector pCDNA3.1-GFP-STAT3-S701A with Ser701 dephosphorylation respectively. The agarose gel electrophoresis and sequencing results of these expression plasmids are respectively as Figure 3 a and Figure 3 b shown.

[0120] (2) Construction of eukaryotic expression cells

[0121] Human Hela cells (ATCC cell bank) were inoculated into 6-well plates, cultured in DMEM medium containing 10% fetal bovine serum, and placed in a 5% CO 2 incubator for culture; when the cell density reached about 60-70%, the above two vectors were transfected into Hela cells by the conventional liposome transfection method respectively; 24 hours after transfection, the cells were collected without using or after stimulating with IL-6 at a concentration of 10 ng / ml for 30 min, and the cells overexpressing STAT3 FL type and Ser701A mutant were obtained respectively.

[0122] (3) Immunoblotting detection

[0123] Collect the above various cell lysates, use 500 μl of RIPA lysis buffer (Beyotime Biotechnology, Cat.P0013C) added with protease inhibitor to lyse the cells, and prepare samples for immunoblotting detection according to the method described in the PPase (New England Biolabs, NEB, Cat.P0753S) instruction manual. Control group: The cell samples digested with PPase were used for immunoblotting method (Western Blot, hereinafter referred to as WB) detection after digestion.

[0124] The results are as Figure 3 shown in c. The self-made Ser701 antibody can recognize the phosphorylation signal of serine at position 701 of the STAT3 protein in cells, that is, the Ser701 site.

[0125] In one embodiment, the present application also discloses a method for detecting the phosphorylation of the Ser701 site of STAT3 protein in the tissues of colitis mice, and the method includes:

[0126] (1) Construction of colitis model mice: SPF-grade male C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.) at 8-10 weeks old were induced to establish a colitis model according to the method described in "Zhang HX, Xu ZS, Lin H, Li M, Xia T, Cui K, Wang SY, Li Y, Shu HB, Wang YY. TRIM27 mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis. Nat Commun. 2018 Aug 24;9(1):3441. IF = 11.878".

[0127] (2) Preparation of the sample to be detected

[0128] Take C57BL / 6J mice as the control group and the colitis model mice as the experimental group. Take the colon tissues of the mice in the control group and the experimental group respectively and put them into a pre-cooled mortar for grinding with liquid nitrogen. Add PMSF (Beyotime Biotechnology, Cat.ST506) to the lysis buffer within several minutes before use to make the final concentration of PMSF 1 mM. Add the pre-cooled lysis buffer (Beyotime Biotechnology, Cat.P0013C) for lysis according to the ratio of adding 150 - 250 μl of lysis buffer per 20 mg of tissue. After sufficient lysis, centrifuge at 10000 - 14000 g for 3 - 5 minutes, transfer the supernatant to a new centrifuge tube, and the sample is used for detection by immunoblotting method after the treatment is completed.

[0129] The results are as Figure 4 shown that the self-made Ser701 antibody can recognize the Ser701 site of STAT3 protein in mouse tissues, that is, the phosphorylation signal of serine at position 701.

[0130] Pharmaceutical composition

[0131] Therefore, the embodiment of the present application also discloses a pharmaceutical composition, which comprises the antibody provided in the above embodiment and a pharmaceutically acceptable excipient or carrier.

[0132] The term "pharmaceutically acceptable" means approved by the relevant regulatory agency or listed in recognized pharmacopoeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvants), excipient, or vehicle with which the agent is administered. Such carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. When administering a pharmaceutical and / or diagnostic composition intravenously, water is a commonly used carrier. Aqueous solutions of saline, as well as aqueous solutions of glucose and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, ethylene glycol, water, ethanol, and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions forming salts such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICSTM, as known in the art. In addition to the above components, the pharmaceutical and / or diagnostic compositions of the present invention may also include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, and preservatives. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

[0133] In summary, the present application discloses a STAT3 protein Ser701 phosphorylation antigenic peptide and its application, and the antibody can be used to detect the phosphorylation level of STAT3 protein Ser701 in cells and mouse tissues; the method for preparing the antibody provided in the present application is simple to operate, and the antibody prepared by this method has strong specificity, high purity, and good stability.

[0134] As described above, the above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application.

Claims

1. An isolated antigenic peptide, the antigenic peptide having immunogenicity targeting the Ser701 phosphorylation site of the STAT3 protein, the antigenic peptide having an amino acid sequence as shown in any one of SEQ ID NO.1 to 4.

2. An antigen construct, the antigen construct comprising the antigenic peptide according to claim 1 and a carrier for loading or conjugating the antigenic peptide according to claim 1.

3. The antigen construct according to claim 2, wherein, the carrier is a liposome.

4. The antigen construct according to claim 2, wherein, the carrier is selected from one of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA), bovine thyroglobulin (THY), inactivated pertussis toxin, group B meningococcal outer membrane protein, Pseudomonas aeruginosa exotoxin A, cholera toxin B subunit, bacterial outer membrane protein, Escherichia coli heat-labile enterotoxin, pneumococcal hemolysin, gonococcal pili protein, and lipopolysaccharide of Gram-negative bacteria.

5. Use of the antigenic peptide according to claim 1 and the antigen construct according to claim 2, the use comprising at least one of the following: (1) being used for preparing a Ser701 phosphorylation-specific antibody of the STAT3 protein.

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