Polypeptide antigen designed based on guidance of molecular docking algorithm and application of polypeptide antigen in preparation of polyclonal antibody

Abstract

The invention discloses a polypeptide antigen designed based on the guidance of a molecular docking algorithm and an application of the polypeptide antigen in preparation of a polyclonal antibody, and the antigen aims at an R346 site of a nuclear receptor Nur77 protein and is used for preparing the polyclonal antibody for specifically recognizing the symmetric double methylation state of the site. The design method comprises the steps of structural analysis, molecular docking simulation, polypeptide sequence optimization, synthesis and modification and purity structure verification. The optimized polypeptide sequence is C-SLKGRR (me2s) GRLPS, and the polypeptide is coupled with keyhole limpet hemocyanin (KLH) and then injected into a rabbit body to induce generation of an antibody. The preparation method comprises the steps of antigen preparation, animal immunization, multi-antibody serum detection, antibody purification and detection and the like. And the specificity of the antibody is verified through Dot Blot detection. The polyclonal antibody prepared by the invention provides a powerful tool for deeply researching the methylation function of the Nur77 protein R346 site, and has important scientific research and clinical application values.

Description

Technical Field

[0001] The invention belongs to the field of immunology, and specifically discloses a polypeptide antigen designed under the guidance of a molecular docking algorithm and its application in preparing a polyclonal antibody. Background Art

[0002] Nuclear receptor Nur77, also known as nerve growth factor-induced gene B (NGFI-B), is a protein encoded by the early gene NR4A1 and belongs to the orphan nuclear receptor family [1]. The NR4A family includes three members: Nur77 (NR4A1), Nurr1 (NR4A2) and Nor-1 (NR4A3) [2]. Nur77 not only acts as a transcription factor to regulate the transcription and expression of downstream target genes, but also acts as a transcription-independent regulatory factor, exerting its effects through protein interactions and changes in subcellular localization, playing an important role in cell proliferation, differentiation, apoptosis, metabolism, immunity and development. Our previous studies have shown that the lack of Nur77 in stromal cells impairs uterine decidualization in mice and stimulates stromal cell fibrosis in endometriotic mice [3]; Mst1-mediated phosphorylation of Nur77 in epithelial cells participates in the regulation of embryo-epithelial interactions [4].

[0003] However, although we have gained some understanding of the function of Nur77 and the importance of arginine methylation modification at R346, the specific molecular mechanism of this modification site and its detailed role in vivo and in vitro are still poorly understood. In particular, the lack of a specific antibody that can effectively recognize and detect the symmetrical dimethylation state of Nur77 R346 has greatly limited our in-depth study of this modification in physiological and pathological processes.

[0004] Traditional antibody preparation methods often rely on random screening and a large number of experimental verifications, which are not only time-consuming and labor-intensive, but also difficult to ensure the specificity and affinity of the antibody. Therefore, developing a peptide antigen based on precise design and preparation to induce the production of specific antibodies targeting specific modification sites has become the key to solving this problem.

[0005] References

[0006] 1.Hazel TG.et al.Agene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily.Proc Natl AcadSci US A. 1988;85(22):8444-8448.

[0007] 2.Hedvat CV.et al.The isolation and characterization of MINOR,a novelmitogen-inducible nuclear orphan receptor.Mol Endocrinol.1995;9(12):1692-1700.

[0008] 3.Jiang,Y.et al.Decreased expression of NR4Anuclear receptors inadenomyosis impairs endometrial decidualization.Mol.Hum.Reprod.2016;22:655–668

[0009] 4.Cai X.et al.Mst1-mediated phosphorylation of Nur77 improves theendometrial receptivity in human and mice.EBioMedicine.2023;88:104433. Summary of the invention

[0010] To solve the above problems, the present invention proposes a polypeptide antigen designed under the guidance of a molecular docking algorithm and a preparation method thereof, aiming to provide an effective solution to the problem of the lack of specific antibodies against the symmetrical dimethylation state of the Nur77 R346 site, and to provide a powerful tool for in-depth research on the methylation modification function of Nur77 and its role in endometrial stromal cell decidualization and embryo implantation.

[0011] Specifically, the present invention provides a highly sensitive Nur77-R346 site symmetrical dimethylation-specific rabbit polyclonal antibody Nur77-R346me2S. The antibody can well detect the level of symmetrical dimethylation modification of the R346 site of Nur77 protein in human and mouse endometrial tissue, and can be applied to detection and screening fields such as immunohistochemistry (IHC) and Western blotting. The present invention also relates to antigen preparation for immunization, antibody purification methods, and the use of anti-Nur77-R346me2S rabbit polyclonal antibodies in protein detection methods.

[0012] The technical solution adopted by the present invention is as follows:

[0013] A polypeptide antigen designed under the guidance of a molecular docking algorithm, characterized in that the polypeptide antigen is designed for the Nur77-R346 site of the nuclear receptor Nur77 protein, and the polypeptide antigen can be used to prepare a polyclonal antibody, and the polyclonal antibody can specifically recognize the symmetrical dimethylation state of the Nur77-R346 site of the nuclear receptor protein.

[0014] Furthermore, the above polypeptide antigen is characterized by comprising the following steps:

[0015] a) Structural analysis of the nuclear receptor Nur77 protein, including analysis of the structural features of the Nur77-R346 site and its surrounding environment;

[0016] b) Using molecular docking algorithms, the designed peptide sequences were docked with Nur77 protein to simulate and evaluate the binding affinity and specificity between the peptide and protein;

[0017] c) optimizing the polypeptide sequence according to the docking results to improve its binding specificity and affinity to the Nur77-R346 site;

[0018] d) synthesizing the optimized polypeptide sequence and performing symmetrical dimethylation modification on the R346 site;

[0019] e) Verify the purity and structural correctness of the polypeptide antigen.

[0020] Furthermore, in the above polypeptide antigen, in step c), the optimization of the polypeptide sequence includes adjusting the amino acid composition, introducing amino acids that can specifically recognize dimethylated arginine or chemical modification.

[0021] The invention discloses a polypeptide antigen, the sequence of which is C-SLKGRR(me2s)GRLPS.

[0022] The invention discloses the application of the polypeptide antigen in preparing a polyclonal antibody which specifically recognizes the methylation state of the nuclear receptor protein Nur77-R346 site.

[0023] Furthermore, in the above application, the methylation state is a symmetrical dimethylation state.

[0024] The invention discloses a polyclonal antibody for species-specifically recognizing the symmetrical double methylation state of the nuclear receptor protein Nur77-R346 site, which is characterized in that the polyclonal antibody is induced to be produced by injecting the above polypeptide antigen into an experimental animal.

[0025] Furthermore, in the above-mentioned polyclonal antibody, the polypeptide antigen is modified by KLH, and the experimental animal is a rabbit.

[0026] The present invention also discloses a method for preparing the above-mentioned polyclonal antibody, comprising the following steps:

[0027] 1). Antigen preparation

[0028] A. Peptide Synthesis

[0029] A modified peptide P1 was synthesized, and its sequence was C-SLKGRR(me2s)GRLPS;

[0030] B. Coupling carrier

[0031] The modified polypeptide P1 is coupled to keyhole limpet hemocyanin (KLH) to obtain a modified polypeptide P1-KLH conjugate, which is used as an immunogen for animal immunization;

[0032] C. Dosage for immunization

[0033] Rabbits were immunized with modified peptide P1-KLH conjugate;

[0034] D. Antigen affinity purification

[0035] preparing modified polypeptide P1 and non-modified polypeptide for subsequent antigen affinity purification steps;

[0036] 2). Animal immunization and polyclonal antibody serum testing

[0037] A. Immunity

[0038] Performing routine immunization on the rabbits for multiple times to stimulate the rabbits to produce antibodies against the modified polypeptide P1;

[0039] B. Dot Blot Detection

[0040] Dot Blot assay was performed using sera from multiple immunizations to preliminarily verify the reactivity of the antibody to the modified peptide P1 and the non-modified peptide;

[0041] 3). Antibody purification and detection

[0042] A. Purification

[0043] Step 1: affinity purifying the serum using an affinity purification column coupled to the modified polypeptide P1 to specifically capture antibodies against the modified polypeptide P1;

[0044] Step 2: Further purification using a non-modified peptide-coupled affinity purification column to remove possible non-specific antibodies;

[0045] B. Animals

[0046] Purifying the rabbit serum respectively to obtain specific antibodies against the modified polypeptide P1;

[0047] C. Dot Blot Detection

[0048] The purified antibodies were tested by Dot Blot to verify their specificity.

[0049] The present invention also discloses the use of the modified polypeptide P1 or the polyclonal antibody prepared therefrom in preparing a kit for diagnosing infertility.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1) Improved antibody specificity and affinity: Through the design of polypeptide antigens guided by the molecular docking algorithm, the present invention can accurately design antigens for the R346 site of the nuclear receptor Nur77 protein, ensuring that the prepared polyclonal antibodies can specifically recognize the symmetrical dimethylation state of the site. This precise design method significantly improves the specificity and affinity of antibodies compared to traditional random screening and experimental verification methods.

[0052] 2) Accelerate the antibody preparation process: Using molecular docking algorithms for antigen design reduces the number and time of experimental verification, thereby accelerating the antibody preparation process. This not only saves time and resources, but also improves the efficiency of antibody preparation.

[0053] 3) Provides a powerful tool for in-depth research: The specific polyclonal antibody prepared by the present invention provides a powerful tool for in-depth research on the methylation modification function of Nur77 and its role in the decidualization of endometrial stromal cells and embryo implantation. This antibody can be used in the detection and screening fields such as immunohistochemistry and immunoblotting, which helps to reveal the important role of Nur77 methylation modification in physiological and pathological processes. At the same time, it can also play a role in the preparation of a kit for diagnosing infertility.

[0054] In summary, the present invention provides a specific polyclonal antibody targeting the symmetrical dimethylation state of the Nur77-R346 site through precise antigen design and preparation, which provides a powerful tool for in-depth research on the function and methylation modification mechanism of Nur77, and also contributes to the development of the field of immunology. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Antiserum Dot Blot detection image;

[0056] Figure 2 Antigen Dot Blot test image;

[0057] Figure 3 Western blot results of Nur77 R346 site-specific methylation antibody Nur77-R346me2s. Lane 1: Nur77 and Nur77-R346me2s with Flag tags can be detected in the cell lysate of Nur77 overexpression. Lane 2: Nur77 mutant protein with Flag tags can be detected in the cell lysate of Nur77 with R346 site mutation (Nur77-R346K), but Nur77-R346me2s cannot be detected.

[0058] Figure 4 Western blot experiment of endometrial tissue detected by Nur77-R346me2s antibody, lanes 1-3: Nur77-R346me2s is highly expressed in the endometrial tissue of the control group; lanes 4-6: Nur77-R346me2s is lowly expressed in the endometrial tissue of infertile patients;

[0059] Figure 5 Immunohistochemistry experiment of endometrial tissue detected by Nur77-R346me2s antibody. Left: Nur77-R346me2s was highly expressed in the nuclei of endometrial tissue of the control group; Right: Nur77-R346me2s was lowly expressed in the nuclei of endometrial tissue of infertile patients. DETAILED DESCRIPTION

[0060] The technical scheme in the embodiment of the present invention is clearly and completely described below, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The reagent or instrument used in the embodiment of the present invention do not indicate the manufacturer, and are all conventional reagent products that can be obtained by commercial purchase.

[0061] The technical solution adopted by the present invention is as follows:

[0062] 1) Antigen preparation:

[0063] A. Peptide synthesis:

[0064] 1. Modified polypeptide P1: (polypeptide sequence, C-SLKGRR(me2s)GRLPS=SEQ ID No.1), wherein R(me2s) represents two symmetrically connected methyl groups on the arginine at this site, synthesized by Wuhan Abclone Company, a total of 19 mg (3 rabbits), with a purity of 85%.

[0065] 2. Modified polypeptide P2: (polypeptide sequence, C-SLKGRR(me1)GRLPS=SEQ ID No.2), total 14 mg (detection), purity 85%.

[0066] 3. Modified polypeptide P3: (polypeptide sequence, C-SLKGRR(me2a)GRLPS=SEQ ID No.3), total 14 mg (tested), purity 85%.

[0068] 4. Non-modified polypeptide: (polypeptide sequence, C-PKLASDDRPS=SEQ ID No. 4), total 14 mg (purified and tested), purity 85%.

[0069] Note: Peptides 2, 3, and 4 are used to detect whether the serum antibodies produced by rabbits immunized with peptide 1 are specific. They are not used to immunize rabbits to produce antibodies, so they do not need to be modified with KLH. KLH modification is to make it easier for rabbits to produce antibodies. Peptide 2 is a methyl group added to the R site, peptide 3 is a methyl group added to the R site, and peptide 4 is an unrelated peptide used for negative detection.

[0070] B. Conjugated carrier: modified polypeptide P1, KLH, for immunization.

[0071] C. Dose for immunization: 7.5 mg / 3 rabbits.

[0072] D Antigen affinity purification: used for antigen affinity purification.

[0073] 1. Modified polypeptide P1: 8 mg naked peptide.

[0074] 2. Non-modified peptide: 8 mg naked peptide.

[0075] 2) Animal immunization and polyclonal antibody serum testing

[0076] A. Immunization: 3 rabbits were immunized, 5 routine immunizations.

[0077] B. Dot Blot test: five immune sera, targeting modified P1 and unmodified naked peptide.

[0078] 3) Antibody purification and detection

[0079] A. Purification: Antigen affinity purification.

[0080] 1. Step 1: Affinity purification column coupled with modified polypeptide P1.

[0081] 2. Second step: affinity purification column coupled with non-modified peptide.

[0082] B. Animals: Three rabbits were purified separately.

[0083] C. Dot Blot detection: purified antibody, P antibody,

[0084] It is necessary to test naked peptide modified peptide P1, modified peptide P2, modified peptide P3 and non-modified peptide C separately. Note: The naked peptide refers to the peptide without KLH coupling.

[0085] 1.P antibody: modified antibody.

[0086] 2.C antibody: non-modified antibody.

[0087] Example

[0088] (1) Antigen preparation

[0089] The peptide C-SLKGRR(me2s)GRLPS was dissolved in PBS buffer solution; the sulfhydryl group was coupled to KLH for subsequent immunization process.

[0090] (2) Immunization process

[0091] Three healthy 6-week-old New Zealand white rabbits were selected, and blood was collected from the ear vein to collect serum as a negative reference. Antigens containing complete / incomplete adjuvants were injected subcutaneously on days 1, 7, 19, 40, and 61; blood was collected from the ear vein on day 73 to collect serum, and the serum titer was determined. The Dot Blot test results of the antiserum are shown in Figure 1 , wherein the polypeptide coating amount is 100 ng, and the serum dilution ratios are 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000. WG-01377P: modified polypeptide; WG-01377C: control polypeptide. The immunization process is shown in Table 1.

[0092] Table 1 Immunization process

[0093]

[0094] (3) Purification of antiserum

[0095] The antiserum was diluted with an equal volume of PBS, centrifuged at 5000-10000 r / min for 15 minutes, and the supernatant was taken. At the same time, the prepared antigen affinity column was washed with 10 times the volume of PBS to balance the column; then the diluted antiserum was added to the balanced column, gently mixed and shaken at 4°C overnight, and the antigen column was washed with 10 times the volume of PBS to wash away the foreign proteins bound to the column; the column was washed with 2 times the volume of antibody elution solution to obtain specific antibodies, and their concentrations were detected.

[0096] After the antiserum was purified by antigen affinity using modified peptide and control peptide, the concentrated antibody was obtained (P: modified peptide, C: control peptide):

[0097] E26937(P) concentration: 0.58 mg / mL; E26937(C) concentration: 2.60 mg / mL; E26938(P) concentration: 0.39 mg / mL; E26938(C) concentration: 2.39 mg / mL; E26939(P) concentration: 2.07 mg / mL; E26939(C) concentration: 2.59 mg / mL.

[0098] (4) Antigen Dot Blot Detection

[0099] The obtained antigen was subjected to Dot Blot detection, and the results were as follows Figure 2 The amount of peptide coating was 100ng, and the antibody dilution ratio was 1000ng / ml, 333ng / ml, 111ng / ml, 37ng / ml, 12ng / ml, and 4ng / ml. P: modified antibody. WG-01377P1: modified peptide 1, WG-01377P2: modified peptide 2, WG-01377P3: modified peptide 3, WG-01377C: control peptide.

[0100] The test results showed that the E26937 batch of antibodies could not distinguish between R346me2s and R346me1 or R346me2a at a concentration of 111 ng / ml, the E26938 batch of antibodies could not distinguish between R346me2s and R346me2a at a concentration of 111 ng / ml, while E26939 could effectively distinguish between R346me2s and R346me1 or R346me2a at a concentration of 111 ng / ml. E26939(P) was subsequently selected for detection experiments.

[0101] (5) Western blot experiment

[0102] Human endometrial stromal cells overexpressing Nur77-wt and Nur77-R346K were prepared using an SDS-PAGE gel rapid preparation kit.

[0103] hESCs total protein was used as antigen, and anti-Nur77-R346me2s antibody was used as primary antibody to perform western blot experiments. The results are shown in Figure 3 . It was demonstrated that the Nur77-R346me2s antibody can only recognize symmetrical dimethylation at the R346 site, and does not recognize the R346K mutation at the R346 site or the monomethylation or asymmetrical dimethylation at the R346 site; it has a high degree of specificity.

[0104] (6) Select human endometrial tissue specimens for immunohistochemistry experiments

[0105] The tissue sections were baked at 65°C for 30 min; then the sections were dewaxed in xylene solution, soaked in gradient alcohol, and rinsed with distilled water;

[0106] The slides were repaired with sodium citrate antigen repair solution for 2.5 min, cooled to room temperature and then rinsed with PBS three times; treated with endogenous peroxidase blocker for 10 min, rinsed with PBS three times; blocked with goat serum blocking solution for 1 h; anti-Nur77-R346me2s antibody was used as the primary antibody, diluted at a ratio of 1:200, and incubated at 4°C overnight; after incubation with secondary antibody, DAB was used for color development and hematoxylin was used for nuclei staining, and the slides were rinsed with running water; dehydrated with gradient alcohol, dried and sealed with neutral resin, and observed under a microscope (see the results in the table). Figure 4 , Figure 5 The results showed that Nur77-R346me2s was highly expressed in the nuclei of the endometrial tissue of the control group, while Nur77-R346me2s was lowly expressed in the nuclei of the endometrial tissue of the infertile patients.

[0107] In summary: The present invention discloses a polypeptide antigen designed under the guidance of a molecular docking algorithm and its application in the preparation of polyclonal antibodies. The methylation modification of the R346 site of the nuclear receptor Nur77 is involved in regulating the decidualization of endometrial stromal cells and the embryo implantation process, and may be an important indicator of endometrial function, but there is currently no available antibody to detect its expression level. The present invention obtains an antibody that specifically detects the symmetrical arginine dimethylation modification of the Nur77 R346 site by antigen immunization and affinity purification, and detects that it can effectively distinguish other methylation modifications of the Nur77 R346 site and identify clinical infertile endometrial samples with low expression levels.

[0108] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A polypeptide antigen designed under the guidance of a molecular docking algorithm, characterized in that: The polypeptide antigen is designed for the Nur77-R346 site of the nuclear receptor Nur77 protein, and the polypeptide antigen can be used to prepare a polyclonal antibody, wherein the polyclonal antibody can specifically recognize the symmetrical double methylation state of the Nur77-R346 site of the nuclear receptor protein.

2. The polypeptide antigen according to claim 1, characterized in that The design guided by the molecular docking algorithm comprises the following steps: a) Structural analysis of the nuclear receptor Nur77 protein, including analysis of the structural features of the Nur77-R346 site and its surrounding environment; b) Using molecular docking algorithms, the designed peptide sequences were docked with Nur77 protein to simulate and evaluate the binding affinity and specificity between the peptide and protein; c) optimizing the polypeptide sequence according to the docking results to improve its binding specificity and affinity to the Nur77-R346 site; d) synthesizing the optimized polypeptide sequence and performing symmetrical dimethylation modification on the R346 site; e) Verify the purity and structural correctness of the polypeptide antigen.

3. The polypeptide antigen according to claim 2, characterized in that In step c), the optimization of the polypeptide sequence includes adjusting the amino acid composition, introducing amino acids that can specifically recognize dimethylated arginine or chemical modification.

4. A polypeptide antigen, characterized in that Its sequence is C-SLKGRR(me2s)GRLPS.

5. Use of the polypeptide antigen as claimed in claim 4 in the preparation of a polyclonal antibody that specifically recognizes the methylation status of the nuclear receptor protein Nur77-R346 site.

6. The use according to claim 5, characterized in that: The methylation state is a symmetrical dimethylation state.

7. A polyclonal antibody that specifically recognizes the symmetrical dimethylation state of the nuclear receptor protein Nur77-R346 site, characterized in that: The polypeptide antigen as claimed in claim 4 is injected into the body of an experimental animal to induce production.

8. The polyclonal antibody according to claim 7, characterized in that The polypeptide antigen is modified by KLH, and the experimental animal is a rabbit.

9. The method for preparing a polyclonal antibody according to claim 7, characterized in that: The following steps are involved: 1). Antigen preparation A. Peptide Synthesis A modified peptide P1 was synthesized, and its sequence was C-SLKGRR(me2s)GRLPS; B. Coupling carrier The modified polypeptide P1 is coupled to keyhole limpet hemocyanin (KLH) to obtain a modified polypeptide P1-KLH conjugate, which is used as an immunogen for animal immunization; C. Dosage for immunization Rabbits were immunized with modified peptide P1-KLH conjugate; D. Antigen affinity purification preparing modified polypeptide P1 and non-modified polypeptide for subsequent antigen affinity purification steps; 2). Animal immunization and polyclonal antibody serum testing A. Immunity Performing routine immunization on the rabbits for multiple times to stimulate the rabbits to produce antibodies against the modified polypeptide P1; B. Dot Blot Detection Dot Blot assay was performed using sera from multiple immunizations to preliminarily verify the reactivity of the antibody to the modified peptide P1 and the non-modified peptide; 3). Antibody purification and detection A. Purification Step 1: affinity purifying the serum using an affinity purification column coupled to the modified polypeptide P1 to specifically capture antibodies against the modified polypeptide P1; Step 2: Further purification using a non-modified peptide-coupled affinity purification column to remove possible non-specific antibodies; B. Animals Purifying the rabbit serum respectively to obtain specific antibodies against the modified polypeptide P1; C. Dot Blot Detection The purified antibodies were tested by Dot Blot to verify their specificity.

10. Use of the polypeptide antigen according to any one of claims 1 to 4 or the polyclonal antibody according to any one of claims 7 to 8 in the preparation of a kit for diagnosing infertility.

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