Parkin protein Ser65 site phosphorylated antigen peptide, antibody serum prepared by using same and application

Abstract

The invention discloses a Parkin protein Ser65 site phosphorylated antigen peptide, antibody serum prepared from the Parkin protein Ser65 site phosphorylated antigen peptide and application of the Parkin protein Ser65 site phosphorylated antigen peptide. The amino acid sequence of the Parkin protein Ser65 site phosphorylated antigen peptide is as shown in SEQ ID NO: 1, wherein the amino acid residue serine is phosphorylated. According to the antibody serum and the polyclonal antibody contained in the antibody serum, the endogenous expression pS65-Parkin protein can be detected through a Western blot experiment, the effect of phosphorylation modification of the Parkin protein in the occurrence and development process of the Parkinson's disease can be researched, and a potential action target is provided for clinical treatment of the Parkinson's disease.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a phosphorylated antigen peptide at the Ser65 site of Parkin protein, an antibody serum prepared using the same, and applications thereof. Background Art

[0002] Mutations in the Parkin gene (PARK2) are one of the major genetic factors for autosomal recessive early-onset Parkinson's disease. Parkin, an E3 ubiquitin ligase, plays a central role in maintaining cellular homeostasis by regulating key pathways such as mitophagy, the ubiquitin-proteasome system, and mitochondrial quality control. However, Parkin's functional activity is highly dependent on its post-translational modification, particularly phosphorylation at Ser65, a process considered to be the core molecular switch that activates Parkin and initiates the mitochondrial damage repair mechanism.

[0003] Recent studies have demonstrated that Parkin's Ser65 phosphorylation is specifically mediated by another Parkinson's disease-associated kinase, PINK1 (PTEN-induced putative kinase 1). In in vitro models, when mitochondria are damaged, PINK1 accumulates in the mitochondrial outer membrane and phosphorylates Parkin at Ser65, inducing a conformational change that activates Parkin's ubiquitin ligase activity, marking damaged mitochondria and initiating autophagic clearance. This mechanism is crucial for maintaining neuronal survival, as dysfunctional mitochondria lead to the accumulation of reactive oxygen species (ROS) and disrupted energy metabolism, ultimately triggering neuronal degeneration.

[0004] Previous studies by the inventors have shown that PINK1-activated Parkin phosphorylation at Ser65 significantly reduces the accumulation of toxic α-synuclein in primate models, significantly contributing to the survival and maintenance of neuronal function. Importantly, pS65-Parkin phosphorylation levels are significantly reduced in brain tissue from Parkinson's disease patients, suggesting that its dysfunction may be closely linked to disease progression. Furthermore, Ser65 phosphorylation levels are not only directly associated with the pathological progression of Parkinson's disease but also have potential implications for the regulation of mitochondrial dysfunction in various neurodegenerative diseases (such as Alzheimer's disease) and cancer.

[0005] However, current research targeting the pS65-Parkin phosphorylation site still faces key technical bottlenecks: existing antibodies generally suffer from high cross-reactivity, insufficient sensitivity, numerous nonspecific bands, or an inability to distinguish between phosphorylated and non-phosphorylated states, making it difficult to accurately detect the dynamic modification levels of this site in clinical samples or cell models. Furthermore, existing small animal models struggle to detect endogenous pS65-Parkin expression. These limitations of antibodies and the choice of animal models severely hinder in-depth research on Parkin activation mechanisms and the development of novel therapeutic strategies (such as small molecule activators or gene therapy) targeting the PINK1-Parkin pathway.

[0006] Therefore, the development of a highly specific and high-affinity pS65-Parkin phosphorylation antibody can not only provide a key tool for elucidating the molecular pathological mechanism of Parkinson's disease, but also open up new research directions for early disease diagnosis, efficacy evaluation and targeted drug screening, and has important scientific value and clinical application potential. Summary of the Invention

[0007] Based on this, the present application provides at least one Parkin protein Ser65 site phosphorylated antigen peptide, antibody serum prepared using the same, and application.

[0008] In a first aspect of the present application, a Parkin protein Ser65 phosphorylated antigen peptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1 (VQNCDLDQQSIV), wherein the amino acid residue serine (S) is phosphorylated.

[0009] In some embodiments, the antigen further comprises a carrier protein.

[0010] Without wishing to be bound by any theory, it has been found that combining the aforementioned Parkin protein Ser65 phosphorylated antigen peptide with a carrier protein allows for more effective immunization.

[0011] Exemplarily, the carrier protein may be keyhole limpet hemocyanin (KLH).

[0012] In a second aspect of the present application, an antigen is provided, which comprises the Parkin protein Ser65 phosphorylated antigen peptide as described in the first aspect.

[0013] In a third aspect of the present application, a method for preparing an antibody targeting the phosphorylated antigen peptide at Ser65 of the Parkin protein as described in the first aspect and / or the antigen as described in the second aspect is provided, the preparation method comprising:

[0014] Immunizing an animal with the antigen described in the second aspect and collecting antiserum; and

[0015] Antibodies are isolated from the antiserum.

[0016] In some embodiments, the method of immunizing an animal comprises:

[0017] Using Freund's complete adjuvant to emulsify the antigen to immunize the animal for the first time;

[0018] Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant for secondary immunization;

[0019] One week later, the antigen was emulsified with Freund's incomplete adjuvant and immunized three times; and

[0020] One week later, the antigen was emulsified using Freund's incomplete adjuvant and immunization was performed four times.

[0021] In some embodiments, the antibodies isolated from the antiserum are polyclonal antibodies.

[0022] In some embodiments, the animals include but are not limited to New Zealand white rabbits, mice, rats, etc.

[0023] In a fourth aspect of the present application, an antibody is provided that targets the Parkin protein Ser65 phosphorylated antigen peptide as described in the first aspect and / or the antigen as described in the second aspect.

[0024] In some embodiments, the antibody is produced by the production method described above.

[0025] In a fifth aspect of the present application, a method for preparing an antibody serum that specifically recognizes phosphorylated Ser65 of endogenous Parkin protein in humans and / or cynomolgus monkeys is provided, comprising:

[0026] Animals are immunized with the antigen described in the second aspect, and antibody serum is collected.

[0027] Among them, the method of immunizing animals includes, for example:

[0028] Using Freund's complete adjuvant to emulsify the antigen to immunize the animal for the first time;

[0029] Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant for secondary immunization;

[0030] One week later, the antigen was emulsified with Freund's incomplete adjuvant and immunized three times; and

[0031] One week later, the antigen was emulsified using Freund's incomplete adjuvant and immunization was performed four times.

[0032] In some embodiments, the antibodies isolated from the antibody serum are polyclonal antibodies.

[0033] As mentioned above, the animals may be, for example, New Zealand white rabbits, mice, and rats.

[0034] In some embodiments, the preparation method further comprises the step of validating the antibody serum. For example, an ELISA test is used to determine whether the antibody serum can specifically recognize the aforementioned Parkin protein Ser65 phosphorylated antigen peptide and the titer against the antigen; and an immunoblotting test and immunohistochemical staining are used to determine whether the antibody serum can specifically recognize the phosphorylated Parkin protein.

[0035] In the sixth aspect of the present application, there is provided serum prepared by the method for preparing antibody serum that specifically recognizes phosphorylated Ser65 site of endogenous Parkin protein in humans and / or cynomolgus monkeys as described in the fifth aspect.

[0036] In the seventh aspect of the present application, a preparation for detecting phosphorylation of Parkin protein Ser65 is provided, which comprises the antibody according to the fourth aspect and / or the serum according to the sixth aspect.

[0037] In an eighth aspect of the present application, there is provided use of the antibody described in the fourth aspect or the serum described in the sixth aspect in preparing a preparation for detecting phosphorylation of Parkin protein Ser65.

[0038] In a ninth aspect of the present application, there is provided a use of the Parkin protein Ser65 phosphorylated antigen peptide as described in the first aspect in preparing a preparation for detecting Parkin protein Ser65 phosphorylation.

[0039] In the tenth aspect of the present application, there is provided use of the antibody according to the fourth aspect or the serum according to the sixth aspect in specifically detecting Ser65 phosphorylated Parkin protein.

[0040] This application uses a designed Parkin protein Ser65 phosphorylation antigen peptide to immunize animals. The animal serum can specifically recognize Ser65 phosphorylated Parkin without purification. The antibody has been demonstrated to be applicable to immunoblotting, immunofluorescence, and immunohistochemistry staining for the specific detection of Ser65 phosphorylated Parkin protein. This antibody can be used for the detection of Ser65 phosphorylation of Parkin and functional studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0042] Figure 1 This is a graph showing the results of ELISA titer detection of pS65-Parkin phosphorylated serum compared to serum before antigen immunization in one embodiment of the present application.

[0043] Figure 2 This is a diagram showing the results of alkaline phosphatase treatment to verify the specificity of pS65-Parkin phosphorylation antibody serum in one embodiment of the present application.

[0044] Figure 3A and Figure 3B This is a diagram showing the immunoblotting detection results of pS65-Parkin phosphorylation serum specifically recognizing pS65-Parkin in human and monkey brains in one embodiment of the present application.

[0045] Figure 4 This is a diagram showing the immunohistochemical staining results of pS65-Parkin phosphorylation serum in one embodiment of the present application.

[0046] Figure 5 This is a diagram showing the immunofluorescence staining detection results of S65-Parkin phosphorylation serum in one embodiment of the present application. DETAILED DESCRIPTION

[0047] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0050] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0051] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0052] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0053] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0054] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0055] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0056] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0057] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0058] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0059] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0060] Parkin is an important E3 ubiquitin ligase. Previous studies of Parkin have typically focused on two aspects: first, its ubiquitination of pathogenic substrates, which is then cleared by the ubiquitin-proteasome system, thereby maintaining neuronal survival; and second, its phosphorylation by the PINK1 kinase, which participates in mitophagy and maintains mitochondrial homeostasis. However, previous studies of pS65-Parkin phosphorylation have primarily utilized in vitro drug-treated cell models or small animal models. However, endogenous pS65-Parkin expression has been difficult to detect under physiological conditions in small animal models, including pig models. The inventors previously detected endogenous pS65-Parkin in the brains of non-human primates (cynomolgus macaques) and in postmortem human brain tissue. Notably, pS65-Parkin is specifically highly expressed in the substantia nigra pars compacta, a region of the brain most susceptible to Parkinson's disease (PD), compared to other brain regions. This suggests that studying the function of pS65-Parkin under physiological conditions in the primate brain is particularly important. The development of endogenous and highly specific pS65-Parkin antibodies provides a key tool for studying the pathogenic mechanism of PD.

[0061] The present application first provides a phosphorylated antigenic peptide at the Ser65 site of the Parkin protein. The present application finds that a phosphorylated peptide segment near the Ser65 site of the Parkin protein can be used as an immunogen to immunize animals. The active amino acid sequence of the antigenic peptide is VQNCDLDQQ(pS)IV.

[0062] This application uses a designed Parkin protein Ser65 phosphorylation antigen peptide to immunize animals. The animal serum can specifically recognize Ser65 phosphorylated Parkin without purification. The antibody has been demonstrated to be applicable to immunoblotting experiments for the specific detection of Ser65 phosphorylated Parkin. This antibody can be used for the detection of Ser65 phosphorylation of Parkin and functional studies.

[0063] In a first aspect, an embodiment of the present application provides an antigenic peptide that is resistant to phosphorylation of Ser65 site of endogenous Parkin protein in humans and cynomolgus monkeys, wherein the active amino acid sequence of the antigenic peptide is VQNCDLDQQ(pS)IV, wherein S is phosphorylated.

[0064] The present application discovered that by using a phosphorylated peptide segment near the Ser65 site of the Parkin protein (sequence: VQNCDLDQQ(pS)IV) as an immunogen to immunize animals, an antibody with specific recognition of Ser65 phosphorylated Parkin protein can be obtained without purification of the animal serum. This antibody can be used for the detection of Ser65 phosphorylation of the Parkin protein and functional research.

[0065] The present application provides an antibody that specifically recognizes the phosphorylated Ser65 site of Parkin protein, and the antibody is prepared by immunizing an animal with the phosphorylated antigen peptide (or an antigen containing the same).

[0066] In the present application, animals are immunized with the designed phosphorylated antigen peptide, and the animal serum can be used to obtain the Parkin protein that specifically detects Ser65 phosphorylation without purification.

[0067] The present application provides a method for preparing an antibody that specifically recognizes phosphorylated Ser65 of Parkin protein, comprising the following steps:

[0068] 1) coupling the phosphorylated antigen peptide with a carrier protein to obtain an antigen;

[0069] 2) Immunizing animals with the antigen described in step 1) and collecting antiserum; that is, antibodies that specifically recognize phosphorylated Parkin protein Ser65.

[0070] As a preferred embodiment of the method for preparing the antibody described in the present application, the immunization method of step 2) specifically includes: using Freund's complete adjuvant to emulsify the antigen for the first immunization of the animal; two weeks later, using Freund's incomplete adjuvant to emulsify the antigen for the second immunization; one week later, using Freund's incomplete adjuvant to emulsify the antigen for the third immunization; one week later, using Freund's incomplete adjuvant to emulsify the antigen for the fourth immunization.

[0071] As a preferred embodiment of the method for preparing the antibody described in the present application, in the step 3), the antiserum is subjected to an ELISA experiment to determine the potency of the antiserum against the antigen peptide and whether it can specifically recognize the phosphorylated antigen peptide, and an immunoblotting experiment is performed to determine whether the antiserum can specifically recognize the phosphorylated Parkin protein.

[0072] The present application also provides the use of the above-mentioned Parkin protein Ser65 phosphorylation antigen peptide in the preparation of a preparation for detecting Parkin protein Ser65 phosphorylation.

[0073] The present application also provides the use of the above-mentioned antibody that specifically recognizes phosphorylation of Parkin protein Ser65 in the preparation of a preparation for detecting phosphorylation of Parkin protein Ser65.

[0074] The present application also provides the above-mentioned pharmaceutical preparation for detecting Parkin protein phosphorylated at Ser65, including the antibody that specifically recognizes Parkin protein phosphorylated at Ser65.

[0075] In this application, antibodies were generated by subcutaneous multi-point immunization of New Zealand white rabbits with a phosphorylated antigen peptide at the Ser65 site of Parkin protein. The antibodies did not require purification, and the antibody titer was detected by ELISA. Immunoblotting and immunohistochemical staining showed that the phosphorylated antibody could specifically recognize the Ser65 phosphorylated Parkin protein, while the prepared Parkin non-phosphorylated antibody (control) could not specifically recognize the Ser65 phosphorylated Parkin protein. Therefore, a rabbit polyclonal antibody against Ser65 phosphorylated Parkin was successfully prepared.

[0076] In vivo experiments confirmed that the phosphorylated antibody obtained in the present application can specifically recognize Parkin phosphorylated at Ser65.

[0077] Some examples are provided below.

[0078] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0079] Example 1 Antibodies that specifically recognize phosphorylated Parkin protein at Ser65 and their preparation method

[0080] The antibody described in the present application is prepared by immunizing an animal with the above-mentioned phosphorylated antigen peptide, specifically comprising the following steps:

[0081] (1) 5 mg of the above-mentioned phosphorylated antigen peptide was coupled with 5 mg of carrier protein KLH, and the mixture was used as the antigen;

[0082] (2) Healthy New Zealand white rabbits aged 4-5 months and weighing more than 2.5 kg were selected for immunization. The rabbits were immunized 4 times in total. The second immunization was performed two weeks after the first immunization, and then once a week. The antigen was emulsified with Freund's incomplete adjuvant.

[0083] (3) One week after the third immunization, a small amount of serum was collected for ELISA testing, and one week after the fourth booster immunization, whole blood was collected to obtain rabbit polyclonal antibody serum;

[0084] (4) The specificity of the serum obtained in step (3) for recognizing endogenous human and monkey Ser65 phosphorylated Parkin protein was verified by dephosphorylation, immunoblotting, immunofluorescence and immunohistochemistry experiments.

[0085] Example 2 Detection of an Antibody Specifically Recognizing Phosphorylation of Parkin Protein at Ser65

[0086] The antibody serum obtained by antigen immunization and the pre-immune serum are subjected to ELISA titer detection, which specifically includes the following steps:

[0087] (1) Coating and blocking: Dilute the phosphorylated antibody and pre-immune serum to 1 μg / mL in PBS coating buffer, coat the antigen in a 96-well plate at 100 μL per well, and coat overnight at 4°C. The next day, wash the plate three times with PBST, add 200 μL of 1% BSA to each well and incubate at 37°C for 1 hour. Wash the plate again three times with PBST and set aside.

[0088] (2) Incubation with primary antibody: dilute the serum serially with PBS, starting at 1:13500 and ending at 1:1093500, with 3 replicates per dilution. Set up a negative control with PBS in a 96-well plate and incubate at 37°C for 1 h.

[0089] (3) Incubation with secondary antibody: Wash the plate three times with PBST, dilute the secondary antibody (goat anti-rabbit IgG-HRP) at 1:40,000 with 1% BSA, add 100 μL of the diluted secondary antibody to each well, and incubate at 37°C for 40 min.

[0090] (4) Color development and reading: Wash the plate 5 times with PBST, add 100 μL of TMB working solution to each well, incubate at 37°C for 5 min, and then add 100 μL of 1 M HCl solution to each well to terminate the reaction. Measure OD with a microplate reader. 450 The dilution corresponding to the well with an OD value 2.1 times greater than the set negative control OD value is determined as the titer of the sample.

[0091] The results of ELISA titer detection of phosphorylated antibody serum and serum before antigen immunization are shown in Figure 1 , indicating that the phospho-antibody meets the standards and can detect phospho-peptides.

[0092] Example 3 Alkaline phosphatase treatment to verify the specificity of the serum of the antibody against the phosphorylation site of Parkin protein Ser65

[0093] In order to verify the specificity and effectiveness of the Parkin protein Ser65 phosphorylation antibody serum, this application used an endogenous protein dephosphorylation experiment.

[0094] Brainstem tissue (20 mg) of adult wild type monkeys was put in 500 μL of lysis buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.5% Triton X-100, pH adjusted to 7.9) and sonicated to no tissue fragments using an ultrasonic disruptor for 30 min on ice. 20 μg of protein was added with protease inhibitors (Roche's cOmplete ULTRA EDTA-free tablet, 05892791001) and phosphatase inhibitors (Roche's PhosSTOP, 04906837001), and another 20 μg of protein was added with alkaline phosphatase (1 U / μL, FastAP, thermoscientific, EF0654) and treated at 37°C for 1 hour to remove endogenous phosphorylated proteins. Then 5x SDS loading buffer was added and verified by immunoblotting, see Figure 2 . Alkaline phosphatase almost eliminated the phosphorylation of endogenous Parkin protein at Ser65 site, while the total Parkin protein expression showed no significant difference. This indicates that the Parkin protein Ser65 site phosphorylation antibody serum can recognize phosphorylated Parkin, but not unphosphorylated Parkin total protein.

[0095] Example 4 Antibody serum specifically recognizing Parkin protein Ser65 site phosphorylation can be applied to immunoblotting experiments

[0096] The present application uses immunoblotting experiments to prove that the expression of pS65-Parkin in monkey brain tissue with Parkin or PINK1 knockdown is also reduced, proving the specificity of the antibody (as shown in Figure 3A and Figure 3B ). The inventors previously established embryonic Parkin and PINK1 targeting monkey models, and the Parkin and PINK1 KO tissues in the figure were taken from the brains of monkeys grown to 3 years old after embryonic targeting. WT is the brain tissue of a 3-year-old wild type monkey. None is the pre-immune serum of the antigen, which is set as a negative control.

[0097] The specific implementation steps of the Western blotting experiment are as follows:

[0098] (1) Take 20 mg of fresh monkey brain tissue in pre-cooled RIPA lysis buffer, use an ultrasonic disruptor to sonicate to no tissue fragments, and lyse on ice for 30 min.

[0099] (2) Put the tissue lysate in a pre-cooled centrifuge at 4°C, centrifuge at 12,000 x g for 10 min, and transfer the supernatant to a new centrifuge tube.

[0100] (3) Use the BCA kit to determine the concentration of the protein lysate, adjust the protein concentration to 2 μg / μL with RIPA lysate, add 5× SDS loading buffer, and incubate at 98°C in a metal bath for 10 min.

[0101] (4) Select a polyacrylamide gel of appropriate concentration for electrophoresis at 100 V for 90 min. Then, transfer the membrane at a constant voltage of 100 V for 1 hour to transfer the protein to the NC membrane.

[0102] (5) Block with 5% skim milk for 1 hour, then wash with TBST for 5 minutes three times. Dissolve 10 μL of antibody serum in 10 ml of 5% BSA and incubate the membrane with the diluted antibody serum at 4°C for 14 hours.

[0103] (6) Recover the diluted antibody serum and wash with TBST for 5 minutes. Dilute the secondary antibody conjugated with horseradish peroxidase with milk at a ratio of 1:7000 and incubate on a shaker at room temperature for 1 hour. Then wash with TBST for 5 minutes three times. Mix the luminol chemiluminescent reagent solution A and solution B in the same ratio, evenly cover the NC membrane, place it in a chemiluminescent imaging instrument for detection, and develop and analyze it on a developer.

[0104] Figure 3A The results showed that pS65-Parkin expression was reduced in both Parkin- and PINK1-depleted monkey brains. Furthermore, no specific reduced bands were observed in the pre-immune serum, indicating that the antibody serum specifically recognizes endogenous pS65-Parkin expression in monkey brains.

[0105] Figure 3B The results showed that the present application used the antibody serum to conduct immunoblotting experiments on the postmortem human cerebral cortex and substantia nigra brain regions, and detected the expression of endogenous pS65-Parkin, which was consistent with the band pattern in the monkey brain, indicating that the antibody serum can specifically recognize the phosphorylation of the Ser65 site of the endogenous Parkin protein in human and monkey brains.

[0106] Example 5 Antibody serum that specifically recognizes phosphorylated Parkin protein Ser65 can be used in immunohistochemistry and immunofluorescence experiments

[0107] This application uses immunohistochemistry to demonstrate the specificity of pS65-Parkin antibody serum ( Figure 4 ). Figure 4 Center AC is the results of serum immunohistochemical staining of pS65-Parkin antibody under different magnifications. Figure 4 Middle D shows the immunohistochemical staining results of serum before antigen immunization.

[0108] pS65-Parkin antibody serum immunofluorescence staining was performed on the substantia nigra brain sections of monkeys with PINK1 knockdown. The inventors previously constructed an adult monkey model for PINK1 targeting by injecting AAV9-PINK1 gRNA / Cas9 into the substantia nigra of the monkey brain using stereotaxic technology. The sensitivity and specificity of the antibody serum immunostaining were tested using monkey brain sections from this model ( Figure 5 ).

[0109] The specific steps of immunofluorescence staining are as follows:

[0110] (1) After returning the monkey brain slices to room temperature, wash them with PBS three times, each time for 5 minutes.

[0111] (2) Soak the slide in 0.3% Triton X-100 solution for 1 hour to permeabilize the membrane. Then, place the slide in blocking permeabilization solution (3% BSA, 2% NGS, 0.3% Triton X-100) and block at room temperature for 1 hour.

[0112] (3) Prepare the primary antibody with blocking solution and incubate overnight at 4°C on a shaker. Then wash three times with PBS, 5 minutes each time.

[0113] (4) Prepare secondary antibody using blocking solution and wash with PBS three times, 5 min each time.

[0114] (5) Add anti-fluorescence quenching sealing agent on the tissue, seal the slice and observe the image.

[0115] The specific steps of immunohistochemical staining are as follows:

[0116] (1) Return the monkey brain slices to room temperature, wash them with PBS three times, 5 minutes each time, and remove the embedding agent.

[0117] (2) Treat the slices with 0.3% H2O2 until there are no bubbles in the slices to fully remove endogenous peroxidase. Wash with PBS three times, 5 minutes each time. Then place the slices in a blocking permeabilization solution (3% BSA, 2% NGS, 0.3% Triton X-100) and block at room temperature for 1 hour.

[0118] (3) Prepare the primary antibody with the blocking solution and incubate overnight on a shaker at 4°C. Then wash three times with PBS, 5 minutes each time.

[0119] (4) Use the secondary antibody from the immunohistochemical staining kit that is consistent with the primary antibody species and add it to the tissue section. Incubate at room temperature for 20 minutes. Wash with PBS three times, 5 minutes each time. Add horseradish peroxidase for 10 minutes. Wash with PBS three times, 10 minutes each time. Add DAB colorimetric reagent. The reaction time depends on the thickness of the section and the specificity of the antibody. When the color develops to the appropriate depth, immediately rinse with PBS to remove the DAB colorimetric solution. Wash three times, 10 minutes each time.

[0120] (5) Dehydrate using a gradient of ethanol: 50% ethanol, 75% ethanol, 90% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol, with each concentration dehydrating for 10 minutes. Immediately after dehydration, place the slide in a xylene solution for transparency. The xylene treatment time is determined based on the degree of transparency of the tissue. Finally, before the xylene is dry, seal the slide with neutral gum and observe the image under a microscope after drying.

[0121] Figure 4 The results showed that the pre-immune serum had no specific signal in immunohistochemical staining, while the antibody serum had a sensitive immunostaining reaction and could specifically recognize the expression of pS65-Parkin.

[0122] Figure 5 The results showed that in glial cells infected with the PINK1 gRNA / Cas9 virus, the fluorescence signal of pS65-Parkin was specifically reduced as PINK1 was knocked down. In the control gRNA / Cas9, the serum immunofluorescence staining effect of this antibody showed high sensitivity and strong specificity.

[0123] In summary, the phosphorylated antigen peptide of the present application has the characteristics of short amino acid sequence and strong immunogenicity. The obtained antibody serum has strong immunity and high specificity. The antibody serum can be used for ELISA, immunoblotting, immunofluorescence and immunohistochemistry to specifically detect Ser65 phosphorylated Parkin protein endogenously expressed in human and cynomolgus macaque brain, which is of great significance in the study of the phosphorylation function of Parkin.

[0124] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0125] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. Antigenic peptide phosphorylated at Ser65 of Parkin protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 1, wherein the amino acid residue serine is phosphorylated.

2. An antigen, characterized in that The method comprises the Parkin protein Ser65 phosphorylated antigen peptide as claimed in claim 1.

3. The antigen according to claim 2, wherein The antigen further comprises a carrier protein.

4. The antigen according to claim 3, wherein The carrier protein optionally comprises keyhole limpet hemocyanin.

5. A method for preparing an antibody targeting the Parkin protein Ser65 phosphorylated antigen peptide according to claim 1 and / or the antigen according to any one of claims 2 to 4, characterized in that: The preparation method comprises: Immunizing an animal with the antigen according to any one of claims 2 to 4 and collecting antiserum; and Antibodies are isolated from the antiserum.

6. The preparation method according to claim 5, wherein The preparation method satisfies one or more of the following conditions (1) to (3): (1) Methods for immunizing animals include: Using Freund's complete adjuvant to emulsify the antigen to immunize the animal for the first time; Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant for secondary immunization; One week later, the antigen was emulsified with Freund's incomplete adjuvant and immunized three times; and One week later, the antigen was emulsified with Freund's incomplete adjuvant and immunized four times; (2) the antibody is a polyclonal antibody; and (3) The animals include New Zealand white rabbits, mice and rats.

7. An antibody targeting the Parkin protein Ser65 phosphorylated antigen peptide according to claim 1 and / or the antigen according to any one of claims 2 to 4.

8. The antibody according to claim 7, wherein The antibody is prepared using the preparation method according to claim 5 or 6.

9. A method for preparing an antibody serum that specifically recognizes phosphorylated Ser65 of endogenous Parkin protein in humans and / or cynomolgus monkeys, characterized in that: It includes: An animal is immunized with the antigen according to any one of claims 2 to 4, and antibody serum is collected.

10. The preparation method according to claim 9, characterized in that The animals include New Zealand white rabbits, mice and rats.

11. The preparation method according to claim 9, wherein A method for immunizing an animal as defined in claim 6.

12. The preparation method according to any one of claims 9 to 11, characterized in that The preparation method further comprises a verification step, which comprises: ELISA test is used to determine whether the antibody serum can specifically recognize the Parkin protein Ser65 phosphorylated antigen peptide according to claim 1 and the titer against the antigen; Western blotting and immunohistochemical staining were used to determine whether the antibody serum could specifically recognize phosphorylated Parkin protein.

13. Serum prepared by the method for preparing an antibody serum that specifically recognizes phosphorylated Ser65 of endogenous Parkin protein in humans and / or cynomolgus monkeys according to any one of claims 9 to 12.

14. A preparation for detecting phosphorylation of Parkin protein Ser65, characterized in that It comprises the antibody according to claim 7 or 8 and / or the serum according to claim 13.

15. Use of the antibody according to claim 7 or 8 or the serum according to claim 13 in the preparation of a preparation for detecting phosphorylation of Parkin protein Ser65.

16. Use of the Parkin protein Ser65 phosphorylated antigen peptide according to claim 1 in preparing a preparation for detecting Parkin protein Ser65 phosphorylation.

17. Use of the antibody according to claim 7 or 8 or the serum according to claim 13 for specifically detecting Parkin protein phosphorylated at Ser65.

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