Antigen peptide of human PP1 alpha protein and Thr320 site phosphorylated protein of human PP1 alpha protein, and preparation method and application of antibody of antigen peptide

Abstract

The invention relates to the technical field of antibodies, in particular to antigen peptides of human PP1 alpha protein and Thr320 site phosphorylated protein of the human PP1 alpha protein, and a preparation method and application of an antibody of the antigen peptides of the human PP1 alpha protein and the Thr320 site phosphorylated protein. The invention provides an antigen peptide of human PP1 alpha protein and Thr320 site phosphorylated protein thereof, the antigen peptide has obvious advantages in the aspects of immunogenicity and the like, animals can be induced to generate high-level antibodies, and the generated antibodies have high specificity, affinity and titer. Based on the antigen peptide, the invention provides an anti-human PP1 alpha protein or a polyclonal antibody with phosphorylated Thr320 site thereof and a preparation method thereof, the polyclonal antibody has the characteristics of strong specificity, high affinity and high titer, and through immunoblotting and immunohistochemical verification, the polyclonal antibody can be used for preparing the anti-human PP1 alpha protein or the anti-human PP1 alpha protein or the anti-human PP1 alpha protein or the anti-human PP1 alpha protein or the anti-human PP1 alpha protein. The probe shows good specificity and sensitivity in detection of phosphorylation modification of human PP1 alpha protein and Thr320 site thereof, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to the preparation method and application of antigenic peptides of human PP1α protein and its Thr320 phosphorylated protein and their antibodies. Background Technology

[0002] Protein phosphatase 1 (PP1) is expressed at high levels in various human tissues and plays a crucial role in maintaining cellular function. PP1 is encoded by three different genes, producing four different catalytic isoforms. PP1 can bind to over 200 regulatory proteins to form highly specific holoenzymes, dephosphorylating hundreds of biological targets. Each PP1 complex controls a dephosphorylation reaction targeting only a small subset of PP1 substrates. PP1 also plays a vital role in cell cycle control. During early to metaphase of mitosis, PP1 phosphorylation inhibits enzyme activity, regulates protein phosphorylation status, and thus modulates the progression of mitosis. Furthermore, the catalytic subunit of PP1 is directly inhibited by Cdk1 phosphorylation, which is essential for the initiation and termination of normal cell division.

[0003] PP1α is an α-isomer of the catalytic subunit of PP1. It can form holoenzymes by binding to different regulatory subunits, thus exhibiting different substrate specificities and subcellular localizations. PP1α is also a key regulator of cardiac function, playing a crucial regulatory role in cardiac excitation-contraction coupling by antagonizing protein kinase function through dephosphorylation. Furthermore, the phosphorylation status of PP1α is associated with the development and progression of various diseases, such as Alzheimer's disease (AD), tumors, and abnormal neuronal differentiation; the phosphorylation status of PP1α may reveal novel therapeutic targets.

[0004] Phospho-PP1α (Thr320) is a PP1α protein phosphorylated at threonine position 320. It is of great significance for studying the role of PP1α in cardiac function regulation, cell cycle control, molecular mechanisms, and disease treatment. Research on Phospho-PP1α (Thr320) not only helps to understand the role of PP1α in physiological and pathological processes but may also provide new perspectives for the diagnosis and treatment of related diseases. In experimental studies, specific antibodies are generally used to detect the level of Phospho-PP1α (Thr320) to investigate changes in the phosphorylation state of PP1α under different physiological or pathological conditions.

[0005] Based on the research value of PP1α and Phospho-PP1α(Thr320) proteins, Western blotting (WB) and immunohistochemistry (IHC), which rely on the principle of antigen-antibody specific binding, are commonly used in the market to detect the expression levels and localization information of PP1α and Phospho-PP1α(Thr320) proteins in cells and tissues. Therefore, developing antibodies against PP1α and Phospho-PP1α(Thr320) proteins with high specificity and sensitivity is of great significance. However, currently, there are still relatively few anti-human PP1α and anti-Phospho-PP1α(Thr320) proteins on the market, and those that exist often suffer from low titers and weak specificity, which adversely affect the accuracy of the detection results. Summary of the Invention

[0006] This invention provides a method for preparing and applying antigenic peptides of human PP1α protein and its Thr320 phosphorylated protein and their antibodies.

[0007] Addressing the current state of antibody development for PP1α protein and Phospho-PP1α (Thr320) phosphorylated protein, this invention first develops human PP1α protein antigenic peptides and human Phospho-PP1α (Thr320) protein antigenic peptides with high immunogenicity. Artificial antigens based on these antigenic peptides can induce animals to produce high levels of anti-human PP1α protein antibodies and anti-human Phospho-PP1α (Thr320) protein antibodies. Based on these antigenic peptides, this invention provides polyclonal antibodies against human PP1α protein and human Phospho-PP1α (Thr320) protein, along with their preparation methods. These polyclonal antibodies exhibit high specificity and high titer, and can be used to prepare immunoassay reagents and immunohistochemical reagents or corresponding kits for human PP1α protein and Phospho-PP1α (Thr320) protein.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a human PP1α protein-associated antigenic peptide, said antigenic peptide being one of the following (1) and / or (2): (1) Human PP1α protein antigen peptide, which has the amino acid sequence shown in SEQ ID NO.2; (2) Phosphorylated antigenic peptide at Thr320 site of human PP1α protein, which has the amino acid sequence shown in SEQ ID NO.2, and the T site of the amino acid sequence shown in SEQ ID NO.2 contains phosphorylation modification.

[0010] The above-mentioned antigenic peptides were designed based on the sequence near the Thr320 site of human PP1α protein. This invention has conducted extensive analysis and screening of antigenic peptides near this site, and finally obtained the above-mentioned antigenic peptides, which have significant advantages in terms of immunogenicity, etc. They can induce animals to produce high levels of antibodies against human PP1α protein and its Thr320 site phosphorylated protein. The antibodies produced have high specificity, affinity and titer.

[0011] In some embodiments of the present invention, the antigenic peptide in (1) above is: GGRPITPPRNS (SEQ ID NO.2); the antigenic peptide in (2) above is: GGRPI(pT)PPRNS, wherein pT represents phosphorylated Thr.

[0012] In other embodiments of the present invention, the amino acid sequence of the human PP1α protein antigenic peptide is shown in SEQ ID NO. 5. The amino acid sequence of the human PP1α protein Thr320 phosphorylated antigenic peptide is shown in SEQ ID NO. 5, wherein the T site contains phosphorylation modification.

[0013] Compared with the sequence shown in SEQ ID NO.2, the sequence shown in SEQ ID NO.5 has an additional cysteine ​​residue at the N-terminus. The purpose of this addition is to facilitate the coupling of the polypeptide with the carrier protein. This addition does not have a substantial effect on the immunogenicity of the antigenic peptide. Therefore, those skilled in the art can expect that the antigenic peptides with and without the addition of cysteine ​​will have similar immunogenicity and the effect of inducing antibody production.

[0014] Secondly, the present invention provides a polypeptide-protein conjugate comprising the human PP1α protein-associated antigenic peptide described above and a carrier protein conjugated thereto.

[0015] The human PP1α protein antigenic peptide or its Thr320 phosphorylated antigenic peptide of the present invention can be coupled with a carrier protein to obtain an artificial antigen, further enhancing its immunogenicity. There are no particular limitations on the type of carrier protein; commonly used carrier proteins for preparing artificial antigens can be used. Preferably, the carrier protein is selected from hemocyanin KLH, bovine serum albumin BSA, and ovalbumin OVA.

[0016] In some embodiments of the present invention, the polypeptide-protein conjugate is obtained by conjugating the human PP1α protein-associated antigen peptide described above with hemocyanin KLH. The amino acid sequence of KLH is well known in the art.

[0017] Thirdly, the present invention provides the use of the human PP1α protein-associated antigenic peptide or the polypeptide-protein conjugate described above in the preparation of anti-human PP1α protein antibodies or anti-human PP1α protein Thr320 site phosphorylated protein antibodies.

[0018] The antibody may be a monoclonal antibody or a polyclonal antibody.

[0019] In some embodiments of the present invention, the antibody is a polyclonal antibody.

[0020] Fourthly, the present invention provides an antibody comprising one of the following (1) and / or (2): (1) Prepared by immunizing animals with a polypeptide-protein conjugate obtained by conjugating human PP1α protein antigen peptide with a carrier protein, wherein the human PP1α protein antigen peptide is as described in the first aspect above. (2) Prepared by immunizing animals with a phosphorylated antigenic peptide at Thr320 site of human PP1α protein coupled with a carrier protein, wherein the phosphorylated antigenic peptide at Thr320 site of human PP1α protein is as described in the first aspect above.

[0021] Preferably, the carrier protein is selected from one of hemocyanin KLH, bovine serum albumin BSA, and ovalbumin OVA.

[0022] Preferably, the antibody is a polyclonal antibody.

[0023] Preferably, the polyclonal antibody is a polypeptide-protein conjugate obtained by conjugating the human PP1α protein antigenic peptide with a carrier protein, or a polypeptide-protein conjugate obtained by conjugating the human PP1α protein Thr320 site phosphorylated antigenic peptide with a carrier protein as an antigen to immunize animals, collecting serum containing the polyclonal antibody, and purifying it.

[0024] Preferably, the animal is a rabbit. The immunization is performed by emulsifying the human PP1α protein antigen peptide with a carrier protein to obtain a polypeptide-protein conjugate, or by emulsifying the human PP1α protein Thr320 phosphorylated antigen peptide with a carrier protein with Freund's adjuvant, and then immunizing the rabbit. The immunization procedure includes 2 to 4 immunizations.

[0025] Fifthly, the present invention provides a method for preparing the antibody described in the fourth aspect above, the method comprising: emulsifying the antigen with Freund's adjuvant and then immunizing rabbits, the immunization procedure comprising 2 to 4 immunizations; after immunization, collecting serum containing polyclonal antibodies, and purifying it to obtain polyclonal antibodies against human PP1α protein or against human PP1α protein phosphorylated protein at Thr320 site.

[0026] In some embodiments of the present invention, the human PP1α protein antigen peptide or the human PP1α protein Thr320 phosphorylated antigen peptide described above is conjugated with hemocyanin KLH to obtain an antigen. The antigen is then emulsified with Freund's complete adjuvant and used to immunize rabbits. Preferably, the immunization program includes: an initial immunization, a second immunization 10-14 days after the initial immunization, a third immunization 20-22 days after the second immunization, and a fourth immunization 20-22 days after the third immunization. The preferred immunization dose is 0.3-1 mg of polypeptide antigen. The immunization method is subcutaneous injection (preferably multi-site subcutaneous injection). Immune serum is separated 6-10 days after the fourth immunization.

[0027] Preferably, the purification of the polyclonal antibody against the phosphorylated protein at Thr320 site of human PP1α protein includes: sequentially performing immunoaffinity chromatography purification using a Phospho-PP1α(Thr320) antigen peptide immunoaffinity chromatography column and a PP1α antigen peptide immunoaffinity chromatography column to obtain a purified antibody with strong affinity for Phospho-PP1α(Thr320) antigen peptide and no affinity for PP1α antigen peptide.

[0028] Sixthly, the present invention provides any of the following applications of the antibody: (1) Use in the preparation of products for detecting the presence or level of human PP1α protein and / or its Thr320 phosphorylated protein in a sample; (2) Application in detecting the presence or level of human PP1α protein and / or its Thr320 phosphorylated protein in a sample for non-disease diagnostic purposes.

[0029] In a seventh aspect, the present invention provides a detection reagent or kit comprising the antibodies described above.

[0030] In some embodiments of the present invention, the detection reagent or kit contains a polyclonal antibody against human PP1α protein or a polyclonal antibody against human PP1α protein phosphorylated at Thr320 site.

[0031] In other embodiments of the present invention, the detection reagent or kit comprises a polyclonal antibody against human PP1α protein and a polyclonal antibody against human PP1α protein phosphorylation at Thr320. These two polyclonal antibodies can be used as an antibody pair to detect human PP1α protein and its Thr320 phosphorylation level.

[0032] The beneficial effects of this invention include at least the following: This invention provides antigenic peptides of human PP1α protein and its Thr320 phosphorylated protein. These antigenic peptides have significant advantages in terms of immunogenicity, etc. Specifically, the human PP1α protein antigenic peptide can induce animals to produce high levels of antibodies against human PP1α protein, and the Thr320 phosphorylated protein antigenic peptide can induce animals to produce high levels of antibodies against the Thr320 phosphorylation of human PP1α protein. Furthermore, the produced antibodies exhibit high specificity, affinity, and titer. Based on the above antigenic peptides, this invention provides polyclonal antibodies against human PP1α protein or its Thr320 phosphorylation and their preparation method. These polyclonal antibodies are characterized by high specificity, high affinity, and high titer. Verification by Western blotting (WB) and immunohistochemistry (IHC) shows that these polyclonal antibodies exhibit good specificity and sensitivity in detecting human PP1α protein and its Thr320 phosphorylation modification, demonstrating promising application prospects. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is an HPLC detection chromatogram of the Phospho-PP1α(Thr320) antigen peptide (SEQ ID NO.5, with T site phosphorylation modification) in Example 1 of the present invention.

[0035] Figure 2 This is the mass spectrum of the Phospho-PP1α(Thr320) antigen peptide (SEQ ID NO.5, with T site phosphorylation modification) in Example 1 of the present invention.

[0036] Figure 3 This is an HPLC detection chromatogram of PP1α antigenic peptide (SEQ ID NO.5) in Example 1 of the present invention.

[0037] Figure 4 This is the mass spectrum of the PP1α antigen peptide (SEQ ID NO.5) in Example 1 of the present invention.

[0038] Figure 5The electrophoretic detection results of the purified PP1α polyclonal antibody and Phospho-PP1α(Thr320) polyclonal antibody in Example 3 of the present invention are shown; where A is the purified PP1α polyclonal antibody and B is the purified Phospho-PP1α(Thr320) polyclonal antibody.

[0039] Figure 6 This is the Western Blot verification result of the PP1α polyclonal purified antibody in Example 6 of the present invention.

[0040] Figure 7 The results of Western Blot verification of the Phospho-PP1α(Thr320) polyclonal purified antibody in Example 6 of this invention are shown; where A is the detection of Phospho-PP1α(Thr320) protein expression level and B is the detection of PP1α protein expression level.

[0041] Figure 8 The results of IHC detection of breast cancer and gastric cancer tissues in Example 7 of this invention are shown.

[0042] Figure 9 The results of IHC detection of thyroid cancer and colon cancer tissues in Example 7 of this invention are shown.

[0043] Figure 10 The results of epitope identification and prediction of the Phospho-PP1α(Thr320) polyclonal purified antibody in Example 8 of this invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Example 1: Preparation and identification of PP1α antigenic peptide and Phospho-PP1α(Thr320) antigenic peptide 1.1 Basic Scheme for Information Acquisition of PP1α Protein and Design of Antigenic Peptides The amino acid sequence of human PP1α protein was obtained from the NCBI database, as follows (SEQ ID NO.1): MSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDIHGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHECASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIV DEKIFCCHGGLSPDLQSMEQIRRIMRPTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEVVAKFLHKHDLDLICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSGLNPGGRPITPPRNSAKAKK.

[0046] Bioinformatics databases such as uniprot and PhosphoSitePlus confirmed that Thr320 in the human PP1α protein is a phosphorylation site located in the C-terminal regulatory region of PP1α. Phosphorylation may inhibit its enzymatic activity or regulate subunit binding. Therefore, it is necessary to first screen antigenic peptides with good specificity and antigenicity as PP1α antigenic peptides. After phosphorylation modification of the PP1α antigenic peptide, PP1α antigenic peptide and Phospho-PP1α(Thr320) antigenic peptide are obtained for subsequent antibody development and antibody validation. The Phospho-PP1α(Thr320) antigenic peptide is mainly used for immunovalidation and purification of phosphorylated antibodies, while the PP1α antigenic peptide is mainly used for the preparation and validation of PP1α antibodies, as well as the purification and standardization validation of Phospho-PP1α(Thr320) antibodies.

[0047] 1.2 Antigen epitope prediction analysis and screening of antigen sequence segments Linear B-cell epitopes near the Thr320 site in human PP1α protein were predicted and analyzed. Sequence conservation was analyzed using multiple sequence alignment, hydrophilicity was analyzed using Kyte-Doolittle, secondary structure was predicted using Karplus-Schulz, surface accessibility was analyzed using Emini, and antigen index was analyzed using Jameson-Wolf. Considering the above parameters and the physicochemical properties of amino acids, several polypeptide antigen sequences with a length of 7-20 amino acids were initially selected as candidates.

[0048] In further analysis, based on the above indicators, priority was given to sequences with high antigenic index, greater flexibility, and higher surface accessibility. Species cross-reactivity was also considered, prioritizing sequences covering human, mouse, rat, and monkey antigens and those with low synthetic difficulty as the final selected sequences. The following three sequences are examples of the final selected sequences for illustration: 1) Antigen peptide sequence 1: GGRPITPPRNS (SEQ ID NO.2) The antigen peptide sequence contains the Thr320 phosphorylation site and its core flanking sequences (GGRPI and PPRNS, as shown in SEQ ID NO. 6 and 7, respectively), focusing on the direct surrounding environment of the phosphorylation site Thr320, avoiding immune dispersion that may be caused by excess amino acids, ensuring that the antibody specifically recognizes the phosphorylation state, and the short peptide is easier to synthesize.

[0049] 2) Antigen peptide sequence 2: PITPPRNSAKAKK (SEQ ID NO.3) The antigen peptide sequence contains the Thr320 upstream and downstream sequences, and has better surface accessibility and higher surface exposure, making it easier for antibodies to recognize and bind to it, and also easier for it to come into contact with the immune system, thereby stimulating a stronger immune response.

[0050] 3) Antigen peptide sequence 3: KNKGKYGQFSGLNPGGRPIT (SEQ ID NO.4) Compared with the amino acid sequences of PP1 isoforms (α / β / γ), selecting this sequence can reduce the risk of cross-reactivity with other PP1 isoforms (β / γ); moreover, the longer sequence has better immunogenicity.

[0051] The three candidate sequences were then evaluated and scored using the IEBDB (Bepipred Linear Epitope) antigenicity prediction tool for reference. Antigen peptide sequence 2 received the lowest score. After comprehensive evaluation, antigen peptide sequence 1 and antigen peptide sequence 3 were used for subsequent synthesis. In the actual synthesis process, it was found that antigen peptide sequence 1 had better synthesis effect, while antigen peptide sequence 3 had poor synthesis effect and could not obtain a high-purity product. Therefore, antigen peptide sequence 1 was finally used for subsequent synthesis, immunization and application verification.

[0052] 1.3 Synthesis of Phospho-PP1α (Thr320) antigenic peptide and PP1α antigenic peptide (1) The synthesis of Phospho-PP1α(Thr320) antigen peptide and PP1α antigen peptide requires the addition of a cysteine-containing sequence (SEQ ID NO.5: CGGRPITPPRNS) to the N-terminus of the selected PP1α antigen peptide sequence (antigen peptide sequence 1) for peptide-carrier protein coupling; Phospho-PP1α(Thr320) antigen peptide further requires phosphorylation modification of threonine (Thr) at position 320 of the selected PP1α antigen peptide (CGGRPI(pT)PPRNS, i.e., phosphorylation modification of the T site of SEQ ID NO.5); The following antigenic peptides were thus obtained: PP1α antigenic peptide: CGGRPITPPRNS (SEQ ID NO.5) Phospho-PP1α(Thr320) antigenic peptide: CGGRPI(pT)PPRNS (SEQ ID NO.5, with phosphorylation modification at the T site) (2) Synthesis and identification of Phospho-PP1α(Thr320) antigenic peptide and PP1α antigenic peptide The designed antigenic peptide was synthesized using a solid-phase synthesis method. First, the C-terminal amino acid of the desired peptide chain was attached to an insoluble polymer resin. Then, starting from this amino acid, the remaining amino acids were sequentially attached from right to left through repeated operations of deprotection, activation, and coupling until the desired peptide chain length was reached. Finally, the peptide chain was cleaved from the resin, and the side chain protecting groups of each amino acid were removed to obtain the crude peptide.

[0053] The crude peptide was purified by HPLC to obtain a liquid pure product, which was then freeze-dried to obtain a solid pure powder. Finally, the purity of the pure product was detected by HPLC and its molecular weight was detected by mass spectrometry to ensure sample quality.

[0054] The results of polypeptide synthesis are shown in Table 1.

[0055] Table 1

[0056] The HPLC chromatogram of the Phospho-PP1α(Thr320) antigen peptide (SEQ ID NO.5, with T-site phosphorylation modification) is shown below. Figure 1 As shown, the mass spectrometry detection spectrum is as follows: Figure 2 As shown, the HPLC chromatogram of the PP1α antigenic peptide (SEQ ID NO.5) is as follows. Figure 3 As shown, the mass spectrometry detection spectrum is as follows: Figure 4 As shown.

[0057] (3) Phospho-PP1α(Thr320) antigenic peptide is coupled to the carrier protein of PP1α antigenic peptide. Chemically synthesized peptides are small molecules and act as haptens. Although they possess reactivity, they do not inherently possess strong immunogenicity, and therefore generally require cross-linking with carrier proteins. Carrier proteins contain many antigenic determinants that can stimulate T helper cells, thereby inducing a B cell response. The most commonly used carrier proteins include hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA), with KLH being the most frequently used.

[0058] The peptide synthesized in (2) above is coupled with KLH using a two-step coupling method. First, KLH is linked to Sulfo-SMCC, and then linked to the peptide. The specific method is as follows: Prepare reagents according to the mass ratio (peptide:KLH:Sulfo-SMCC=10:10:5); dissolve KLH in PBS solution to a concentration of 10 mg / mL, dissolve Sulfo-SMCC in pure water to a concentration of 5 mg / mL, mix the two and react at room temperature for 2 hours, then dialyze the reaction product using 1×PBS solution. The peptide was dissolved in 1×PBS solution to a concentration of 5 mg / mL, mixed with the coupling products of KLH and Sulfo-SMCC, and reacted at room temperature for 2 hours. The reaction product was then dialyzed using 1×PBS solution.

[0059] The conjugated peptides prepared above were used as immunogenic antigens for the subsequent preparation of polyclonal antibodies.

[0060] Example 2: Preparation of polyclonal antibodies against human PP1α antigenic peptide and Phospho-PP1α (Thr320) Polyclonal antibodies were prepared using the PP1α antigenic peptide (SEQ ID NO.5) and KLH conjugate peptide synthesized in Example 1, as well as the Phospho-PP1α(Thr320) antigenic peptide (SEQ ID NO.5, with T-site phosphorylation modification) and KLH conjugate peptide as immunogenic antigens. The specific methods and results are described below.

[0061] 1. Animal immunization Three-month-old male New Zealand white rabbits were selected. Human PP1α antigen peptide conjugated with KLH and Phospho-PP1α (Thr320) antigen peptide were emulsified with an equal volume of Freund's complete adjuvant at a 1:1 ratio for immunization. Each immunization consisted of 0.5 mg of the peptide antigen, administered via subcutaneous injection at multiple sites. The immunization schedule was one, two, and three immunizations, with intervals of 12 and 21 days, respectively. Blood was collected after three immunizations to measure titer. Once the titer reached the target level, a final booster immunization was administered, with a 21-day interval between the third and fourth immunizations. Eight days after the fourth immunization, blood was collected from the heart to separate and collect immune serum.

[0062] 2. Preliminary ELISA identification of PP1α polyclonal immune serum and Phospho-PP1α (Thr320) polyclonal immune serum The titer of PP1α-enzyme-coated serum was determined by ELISA. The titer of PP1α-enzyme-coated serum was determined by cross-identification of PP1α-enzyme-coated serum with PP1α-enzyme-coated serum and PP1α-enzyme-coated serum with ELISA. The corresponding titer was determined and whether the serum bound to both antigen peptides was determined. 1) Antigen coating: Using ELISA coating buffer (Solarbio), ELISA detection plates were coated with 2 replicates of Phospho-PP1α (Thr320) antigen peptide and 4 replicates of PP1α antigen peptide, respectively, at a coating concentration of 1 μg / mL. 100 μL was added to each well and incubated at 37°C for 2 h.

[0063] 2) Discard the coating solution, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat twice.

[0064] 3) Block the coated ELISA detection plate with blocking solution (0.01M PBS solution containing 2% BSA), add 200μL to each well, and incubate at 37℃ for 2h.

[0065] 4) Discard the blocking solution, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 3 times.

[0066] 5) Dilute the PP1α polyclonal immune serum starting at 1:5000 using antibody dilution buffer (0.01M PBS containing 1% BSA, pH 7.4 solution), serially diluting it into wells corresponding to two replicates of the PP1α antigen peptide, adding 100 μL to each well; dilute the Phospho-PP1α(Thr320) polyclonal immune serum starting at 1:500, serially diluting it into wells coated with two additional replicates of the PP1α antigen peptide and two replicates of the Phospho-PP1α(Thr320) antigen peptide, adding 100 μL to each well, and incubate at 37°C for 1 h.

[0067] 6) Discard the immune serum diluent, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 4 times.

[0068] 7) Dilute goat anti-rabbit IgG-HRP (Solarbio) 1:5000 with antibody dilution buffer (0.01M PBS containing 1% BSA, pH 7.4 solution). After dilution, add the secondary antibody to the ELISA detection plate using a pipette, adding 100 μL to each well and incubating at 37°C for 45 min.

[0069] 8) Discard the secondary antibody, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 6 times.

[0070] 9) Develop color using the TMB colorimetric kit (Solarbio), 100 μL / well, incubate at 25℃±2℃ in the dark for 3-5 min, then add ELISA stop solution (Solarbio), 50 μL / well.

[0071] 10) The colorimetric values ​​were read using an ELISA reader at 450 nm, and the results are shown in Tables 2 and 3. The binding titer of the PP1α immune serum corresponding to the PP1α antigen peptide was positive at 1:16W (Table 2, where K represents thousands and W represents tens of thousands). The binding titers of Phospho-PP1α(Thr320) immune serum to the PP1α antigen peptide and Phospho-PP1α(Thr320) antigen peptide showed significant differences, and the immune serum still showed high OD values ​​for Phospho-PP1α(Thr320) antigen peptide at titers above 1:3.2W (Table 3, where K represents thousands and W represents tens of thousands). The results indicate that this invention yielded high-titer PP1α polyclonal immune serum and crude Phospho-PP1α(Thr320) polyclonal immune serum.

[0072] Table 2

[0073] Table 3

[0074] Example 3: Preparation of PP1α polyclonal purified antibody and Phospho-PP1α (Thr320) polyclonal purified antibody 1. Preparation of PP1α polyclonal purified antibody The PP1α polyclonal immune serum was purified using the rProtein A (Solarbio) method. By binding Protein A to the FC fragment of mammalian IgG, IgG in the immune serum was captured, thereby obtaining purified PP1α polyclonal antibodies.

[0075] 2. Preparation of Phospho-PP1α (Thr320) polyclonal purified antibody 1) Construction of immunoaffinity chromatography column Phospho-PP1α(Thr320) antigenic peptide and PP1α antigenic peptide were coupled with Sulfolink CouplingResin pre-activated resin (Solarbio) to immobilize the antigen through reaction with the amino group of the antigenic peptide, thus obtaining Phospho-PP1α(Thr320) antigenic peptide immunoaffinity chromatography column and PP1α antigenic peptide immunoaffinity chromatography column respectively.

[0076] 2) Two-step immunoaffinity chromatography purification of Phospho-PP1α (Thr320) polyclonal immune serum First, the Phospho-PP1α(Thr320) polyclonal immune serum was purified using a Phospho-PP1α(Thr320) antigen peptide immunoaffinity chromatography column. The eluent was then equilibrated and dialyzed to obtain purified antibody 1, which has a strong affinity for Phospho-PP1α(Thr320) antigen peptide. Next, purified antibody 1 with a strong affinity for Phospho-PP1α(Thr320) antigen peptide was purified using a PP1α antigen peptide immunoaffinity chromatography column. The flow-through was purified antibody 2, which has a strong affinity for Phospho-PP1α(Thr320) antigen peptide but no affinity for PP1α antigen peptide. After concentration and dialyzed, purified antibody 2 was obtained as Phospho-PP1α(Thr320) polyclonal antibody.

[0077] The SDS-PAGE purity verification graphs of the polyclonal purified antibody PP1α and the polyclonal purified antibody Phospho-PP1α(Thr320) are shown below. Figure 5 As shown.

[0078] Example 4: Titer verification of PP1α polyclonal purified antibody and ELISA cross-validation of Phospho-PP1α (Thr320) polyclonal purified antibody. The purified PP1α polyclonal antibody obtained in Example 3 was used to verify its titer. Phospho-PP1α(Thr320) polyclonal antibody was cross-validated using ELISA. The specific methods are as follows: 1) Antigen coating: Using ELISA coating buffer (Solarbio), ELISA detection plates were coated with 2 replicates of Phospho-PP1α (Thr320) antigen peptide and 4 replicates of PP1α antigen peptide, respectively, at a coating concentration of 1 μg / mL. 100 μL was added to each well and incubated at 37°C for 2 h.

[0079] 2) Discard the coating solution, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat twice.

[0080] 3) Block the coated ELISA detection plate with blocking solution (0.01M PBS solution containing 2% BSA), add 200μL to each well, and incubate at 37℃ for 2h.

[0081] 4) Discard the blocking solution, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 3 times.

[0082] 5) Dilute the purified PP1α polyclonal antibody starting at 1:2500 with antibody dilution buffer (0.01M PBS containing 1% BSA, pH 7.4 solution), serially diluting it into wells corresponding to two replicates of the PP1α antigenic peptide, adding 100 μL to each well; dilute the Phospho-PP1α(Thr320) polyclonal immune serum starting at 1:500, serially diluting it into wells coated with two more replicates of the PP1α antigenic peptide and two replicates of the Phospho-PP1α(Thr320) antigenic peptide, adding 100 μL to each well, and incubate at 37°C for 1 h.

[0083] 6) Discard the purified antibody diluent, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 4 times.

[0084] 7) Dilute Solarbio goat anti-rabbit IgG-HRP1:5000 with antibody dilution buffer (0.01M PBS containing 1% BSA, pH 7.4 solution). After dilution, add the secondary antibody to the ELISA detection plate using a pipette, adding 100 μL to each well and incubating at 37°C for 45 min.

[0085] 8) Discard the secondary antibody, wash each coated well thoroughly with 0.01M PBST (pH 7.4), discard the washing solution, and repeat 6 times.

[0086] 9) Develop color using the TMB colorimetric kit (Solarbio), 100 μL / well, incubate at 25℃±2℃ in the dark for 3-5 min, then add ELISA stop solution (Solarbio), 50 μL / well.

[0087] 10) The colorimetric values ​​were read at 450 nm using an ELISA reader. The differences in the values ​​of the Phospho-PP1α(Thr320) antigenic peptide and the PP1α antigenic peptide detected by the Phospho-PP1α(Thr320) polyclonal antibody were compared. The results are shown in Table 4 (where K represents thousands and W represents tens of thousands, and the two columns of OD values ​​for each antigenic peptide corresponding to the antibody are from two parallel wells). The results show that the present invention successfully obtained a Phospho-PP1α(Thr320) polyclonal antibody that specifically binds to the Phospho-PP1α(Thr320) antigenic peptide. Meanwhile, the binding titer of the purified PP1α polyclonal antibody to the PP1α antigenic peptide is also relatively high. As shown in Table 5, a positive detection was still observed at a dilution ratio of 1:4W, indicating that the present invention also successfully obtained a purified PP1α polyclonal antibody that specifically binds to the PP1α antigenic peptide.

[0088] Table 4

[0089] Table 5

[0090] Example 5: Affinity verification of PP1α polyclonal purified antibody and Phospho-PP1α (Thr320) polyclonal purified antibody. Following the indirect ELISA method in Example 4 above, 1 μg / mL PP1α antigen peptide (1 strip) and Phospho-PP1α(Thr320) antigen peptide (1 strip) were coated, respectively. After washing, blocking, and washing again, the corresponding PP1α polyclonal purified antibody (dilution ratio of 1 / 2500) and Phospho-PP1α(Thr320) polyclonal purified antibody (dilution ratio of 1 / 5000) were added, 100 μL / well, and incubated at 37℃ for 1 h for antigen binding. After washing with PBST, the plates were eluted with sodium thiocyanate of different concentrations, and 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mol / L sodium thiocyanate solutions were added sequentially, 60 μL / well, and the plates were incubated at room temperature for 15 min. After washing with PBST, goat anti-rabbit IgG-HRP secondary antibody (1:5000) was added for colorimetric detection. Result determination: OD after elution 450The sodium thiocyanate concentration corresponding to a decrease to 50% of the uneluted amount is the relative affinity constant of the antibody, expressed in mol / L. The results are shown in Table 6. The relative affinity constants of the PP1α polyclonal purified antibody and the Phospho-PP1α(Thr320) polyclonal purified antibody are 4.75 and 2.75, respectively.

[0091] Table 6

[0092] Example 6: Western Blot (WB) Validation of PP1α Polyclonal Purified Antibody and Phospho-PP1α (Thr320) Polyclonal Purified Antibody 1. Western Blot validation of routine samples of PP1α polyclonal purified antibody 1) Sample preparation: Protein extraction was performed on various samples, including human cells HEK293, MCF7, HepG2, HeLa, A431, MDA-MB-231, Jurkat, A549, monkey cells COS7, rat and mouse cells NIH3T3 and PC12, and rat and mouse tissues Mousebrain and Rat brain. The sample concentration was adjusted to 2.5 mg / mL, and the samples were boiled and denatured with loading buffer to obtain the loading samples.

[0093] 2) Gel preparation, sample loading and gel running, transfer, and blocking. Prepare an 11% separating gel (lower layer) and a 5% stacking gel (upper layer). After gel preparation, load the gels into the electrophoresis apparatus. Add electrophoresis buffer to both inner and outer tanks, and begin sample loading at 25 μg for each. After replenishing the electrophoresis buffer, connect the electrophoresis apparatus and run the gel under the following conditions: 80V, 20 min, 120V, 1 h. Perform a membrane transfer operation using a "sandwich" structure. Fill the electrophoresis tank with transfer working solution, maintaining a low temperature during the transfer process. Transfer at a constant current of 200 mA for 60 min. After transfer, place the membrane in a blocking working solution and incubate on a shaker at room temperature for 60 min.

[0094] 3) Incubation with primary and secondary antibodies, followed by development and exposure with ECL chemiluminescence solution. The PP1α polyclonal purified antibody was diluted 1:1000 and incubated overnight at 2-8°C. The next day, the membrane was removed and washed 5 times with Western blotting buffer for 5 minutes each. Afterward, the goat anti-rabbit IgG-HRP secondary antibody was diluted 1:5000 and incubated at room temperature for 1 hour. Afterward, the membrane was washed 5 times with Western blotting buffer for 5 minutes each.

[0095] Development and exposure using ECL luminescent solution: Mix solutions A and B of ECL luminescent solution (Solarbio, product number PE0010) in a 1:1 ratio. Thoroughly saturate the film with the luminescent solution, then place it onto the imaging area of ​​the exposure unit and run the program for automatic exposure. Western blotting results are as follows: Figure 6 As shown, all samples exhibited a relatively single target band at 34kDa, consistent with the estimated protein size, demonstrating high specificity and the ability to cover cross-reactivity in different species such as humans, monkeys, rats, and mice.

[0096] 2. Western Blot validation of Phospho-PP1α (Thr320) polyclonal purified antibody Untreated Jurkat samples were used as controls, while Jurkat samples treated with Calyculin A were used as samples with enhanced phosphorylation.

[0097] The expression of PP1α protein and Phospho-PP1α(Thr320) protein was verified using the obtained polyclonal purified antibody and Phospho-PP1α(Thr320) polyclonal purified antibody, respectively. At the same time, β-tubulin internal reference protein was detected as a control for sample consistency.

[0098] 1) Sample processing: Prepare Jurkat cell samples treated with Calyculin A and untreated Jurkat cell protein samples. When the Jurkat cell density reaches 70-80%, starve serum overnight. Add Calyculin A to the experimental group at a final concentration of 100 nM and stimulate for 30 min. No treatment is given to the control group. Collect cells and place them in 1.5 mL centrifuge tubes.

[0099] 2) Gel preparation, sample loading and gel running, transfer, and blocking: Prepare an 11% separating gel (lower layer) and a 5% stacking gel (upper layer). After gel preparation, load the gels into the electrophoresis apparatus. Add electrophoresis buffer to both inner and outer tanks, and begin sample loading at 25 μg for each. After replenishing the electrophoresis buffer, connect the electrophoresis apparatus and run the gel under the following conditions: 80V, 20 min, 120V, 1 h. Perform a membrane transfer operation using a "sandwich" structure. Fill the electrophoresis tank with transfer working solution, maintaining a low temperature during the transfer process. Transfer at a constant current of 200 mA for 60 min. After transfer, place the membrane in a blocking working solution and incubate on a shaker at room temperature for 60 min.

[0100] 3) Incubation with primary and secondary antibodies, followed by development and exposure with ECL chemiluminescence solution. The purified polyclonal antibody against PP1α and the polyclonal antibody against Phospho-PP1α(Thr320) were diluted 1:1000, and β-tubulin was diluted 1:5000 (Solarbio, catalog number K200059M). The membranes were incubated overnight at 2-8°C. The next day, the membranes were removed and washed 5 times with Western blotting buffer for 5 minutes each. Afterward, the membranes were incubated 1 hour at room temperature with secondary antibodies against goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (PP1α and Phospho-PP1α(Thr320) primary antibodies correspond to goat anti-rabbit IgG-HRP, and β-tubulin primary antibody corresponds to goat anti-mouse IgG-HRP). The membranes were washed 5 times with Western blotting buffer for 5 minutes each. Development and exposure using ECL luminescent solution: Mix solutions A and B of ECL luminescent solution (Solarbio, product number PE0010) in a 1:1 ratio. Thoroughly saturate the film with the luminescent solution, then place it onto the imaging area of ​​the exposure unit and run the program for automatic exposure. Western blotting results are as follows: Figure 7 As shown, the molecular weight positions of the obtained bands all met expectations. In samples treated with Calyculin A, the β-tubulin internal reference protein levels were basically consistent. Verification using a polyclonal purified antibody against Jurkat showed that the PP1α protein expression level was significantly reduced before and after Jurkat treatment. Figure 7 (B), and the expression level of Phospho-PP1α(Thr320) protein was significantly increased in samples before and after Jurkat treatment when the Phospho-PP1α(Thr320) polyclonal purified antibody was used to verify the results. Figure 7 The results of the verification (A) were as expected, thus proving that the Phospho-PP1α(Thr320) polyclonal purified antibody can sensitively recognize the expression of Phospho-PP1α(Thr320) protein and almost no normal PP1α protein expression can be observed, which shows good specificity.

[0101] Example 7 Immunohistochemical detection of Phospho-PP1α (Thr320) protein expression in tissues Immunohistochemistry (IHC) was performed using the prepared Phospho-PP1α(Thr320) polyclonal antibody to detect the expression of Phospho-PP1α(Thr320) protein in tissues. The specific method is as follows: 1) Sample preparation: Paraffin sections with a thickness of about 3 μm were baked in an oven at 60℃ for 1 hour.

[0102] 2) Dewaxing: Immerse the paraffin slices in xylene for 5 minutes each time, 4 times.

[0103] 3) Hydration: Transfer the paraffin slices in sequence to 100%, 100%, 95%, 85%, and 75% ethanol, soaking for 3 minutes in each solution, and then rinse with pure water.

[0104] 4) Antigen retrieval: Use 0.01 mol / L citrate buffer (pH 6.0) for autoclaving and heat retrieval for 2.5 min, then allow to cool naturally to room temperature.

[0105] 5) Washing: Wash 3 times with 1×PBS buffer, 3 minutes each time.

[0106] 6) Blocking: 3% hydrogen peroxide, incubate at room temperature for 10 minutes.

[0107] 7) Washing: Wash 3 times with 1×PBS buffer, 3 minutes each time.

[0108] 8) Sealing: Add sealing solution and incubate at 37°C for 10 minutes.

[0109] 9) Incubation of primary antibody: Dilute Phospho-PP1α(Thr320) protein antibody to working concentration with antibody dilution buffer and prepare fresh for use; add the diluted Phospho-PP1α(Thr320) protein to the slide and incubate overnight at 4°C in a humidified chamber.

[0110] 10) Rewarming: Take it out of the refrigerator and let it return to room temperature for 10 minutes.

[0111] 11) Washing: Wash 3 times with 1×PBS buffer, 3 min each time.

[0112] 12) Incubation of secondary antibody: Add secondary antibody dropwise and incubate at 37℃ for 30 min.

[0113] 13) Washing: Wash 3 times with 1×PBS buffer, 3 min each time.

[0114] 14) Color development: DAB color development (select the appropriate color development time according to the color development of the section).

[0115] 15) Counterstain: Counterstain with hematoxylin for 2 min, then rinse with pure water for 15 min.

[0116] 16) Dehydration: Soak in 75%, 85%, 95%, 100%, and 100% ethanol for 2 minutes each.

[0117] 17) Transparent: Soak in xylene for 2 minutes twice.

[0118] 18) Mounting: Add neutral resin and cover with a coverslip to seal the slide.

[0119] 19) Microscopic examination: Observe and judge the stained sections under an optical microscope.

[0120] IHC results as follows Figure 8 and Figure 9 As shown, according to information from uniprot and The Human Protein AtLas websites, the expression of Phospho-PP1α(Thr320) protein in tissues is mainly localized in the cytoplasm and nucleus. In four randomly selected human tissues (breast cancer, gastric cancer, thyroid cancer, and colon cancer), the protein was accurately localized in both the cytoplasm and nucleus. Microscopic examination showed that the protein exhibited moderate (++) expression in the cytoplasm and nucleus in the tissues. The results indicate that the Phospho-PP1α(Thr320) polyclonal antibody of this invention passed the immunohistochemical (IHC) verification.

[0121] Example 8 Epitope Identification and Prediction of Phospho-PP1α (Thr320) Polyclonal Purified Antibody Epitope identification and analysis of polyclonal antibodies are of great significance. They can provide insights into the key binding sequence epitopes that generate antibodies and can be used for subsequent antibody performance optimization, such as antibody epitope optimization, improving antibody sensitivity and affinity, reducing cross-reactivity, and screening for conserved epitopes. At the same time, epitope identification and analysis can also reduce the difficulty of preparation to some extent.

[0122] To further identify the obtained Phospho-PP1α(Thr320) polyclonal purified antibody, the recognized antigenic epitope combinations were analyzed to screen which antigenic epitopes the antibody showed strong affinity and high sensitivity to, i.e., to identify the recognition epitopes for which the antibody mainly functions. Five to eleven consecutively overlapping short peptides containing phosphorylation sites were synthesized and reacted one by one with the target Phospho-PP1α(Thr320) polyclonal purified antibody. The binding titer was detected using ELISA to verify the antibody's affinity for the target epitope region, thus determining the epitope region recognized by the antibody. The detection results are as follows: Figure 10 As shown, the key binding epitopes of the Phospho-PP1α(Thr320) polyclonal purified antibody are mostly concentrated in the first 6-8 amino acids. For peptides containing only the last 5-7 amino acids, the average OD values ​​of the Phospho-PP1α(Thr320) polyclonal purified antibody at a 1:3.2W dilution are all below 0.5, and the affinity at a 1:2500 dilution is also below 1. In contrast, the ELISA efficacy and affinity values ​​of short peptides containing the first 6-8 amino acid sequences are 2 times or more higher than those of peptides containing only the last 5-7 amino acids.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human PP1α protein-associated antigen peptide, characterized in that, The antigenic peptide is one of the following (1) and / or (2): (1) Human PP1α protein antigen peptide, which has the amino acid sequence shown in SEQ ID NO.2; (2) Phosphorylated antigenic peptide at Thr320 site of human PP1α protein, which has the amino acid sequence shown in SEQ ID NO.2, and the T site of the amino acid sequence shown in SEQ ID NO.2 contains phosphorylation modification.

2. The human PP1α protein-associated antigen peptide according to claim 1, characterized in that, The amino acid sequence of the human PP1α protein antigen peptide is shown in SEQ ID NO.

5.

3. The human PP1α protein-associated antigen peptide according to claim 1, characterized in that, The amino acid composition of the phosphorylated antigenic peptide at Thr320 site of the human PP1α protein is shown in SEQ ID NO.5, and the T site contains phosphorylation modification.

4. A polypeptide-protein conjugate, characterized in that, The polypeptide-protein conjugate comprises the human PP1α protein-associated antigenic peptide as described in any one of claims 1 to 3 and the carrier protein conjugated thereto.

5. The polypeptide-protein conjugate according to claim 4, characterized in that, The carrier protein is selected from one of hemocyanin KLH, bovine serum albumin BSA, and ovalbumin OVA.

6. The use of the human PP1α protein-associated antigenic peptide according to any one of claims 1 to 3 or the polypeptide-protein conjugate according to claim 4 or 5 in the preparation of anti-human PP1α protein antibody or anti-human PP1α protein Thr320 site phosphorylated protein antibody.

7. An antibody, characterized in that, The antibody comprises one of the following (1) and / or (2): (1) Prepared by immunizing animals with a polypeptide-protein conjugate obtained by conjugating human PP1α protein antigen peptide with a carrier protein, wherein the human PP1α protein antigen peptide is as described in claim 1 or 2. (2) Prepared by immunizing animals with a polypeptide-protein conjugate obtained by conjugating a human PP1α protein phosphorylated antigenic peptide at Thr320 site with a carrier protein, wherein the human PP1α protein phosphorylated antigenic peptide at Thr320 site is as described in claim 1 or 3.

8. The antibody according to claim 7, characterized in that, The antibody is a polyclonal antibody.

9. The method for preparing the antibody according to claim 7 or 8, characterized in that, The method includes: emulsifying the antigen with Freund's adjuvant and then immunizing rabbits, the immunization program including 2 to 4 immunizations; after immunization, collecting serum containing polyclonal antibodies and purifying it to obtain polyclonal antibodies against human PP1α protein or phosphorylated protein at Thr320 site of human PP1α protein.

10. Any of the following applications of the antibody according to claim 7 or 8: (1) Use in the preparation of products for detecting the presence or level of human PP1α protein and / or its Thr320 phosphorylated protein in a sample; (2) Application in detecting the presence or level of human PP1α protein and / or its Thr320 phosphorylated protein in a sample for non-disease diagnostic purposes.

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