Antigen peptide aiming at anti-Ran K141 Malonyl antibody and application thereof

Abstract

The invention discloses an antigen peptide aiming at an anti-Ran K141Malunyl antibody and application thereof, the amino acid sequence of the antigen peptide is VFHR-(Malunyl) K-KNLQYY C, the (Malunyl) K represents malonylation modified lysine, the (Malunyl) K represents the amino acid sequence of the anti-Ran K141Malunyl antibody, and the (Malunyl) K represents the amino acid sequence of the anti-Ran K141Malunyl antibody. And the Ran K141Mallonyl represents that the 141 lysine of the Ran protein is subjected to malonylation modification. According to the present invention, the 141 site lysine malonylation modified polypeptide is artificially synthesized, the modified polypeptide is adopted as the antigen, the animal is immunized to prepare the specific antibody, and the antibody can be used for identifying the 141 site lysine malonylation of the Ran protein so as to be used in the development of the preparation of the prostate cancer diagnostic reagent.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an antigen peptide targeting an anti-Ran K141 Malonyl antibody and an application thereof. Background Art

[0002] Prostate cancer is one of the most common cancers in men, especially in men over 50 years old. With the advancement of medical technology, early screening and diagnostic methods have been improved, and the detection rate of prostate cancer has gradually increased. Its incidence rate varies significantly in different regions. It is higher in North America, Australia and some European countries, and relatively lower in Asia and Africa. The older you are, the greater the risk of disease. Genetic factors, hormone levels, eating habits and environmental factors may affect the risk of disease. Men with direct relatives in the family have a significantly increased risk. Androgens, especially testosterone, are closely related to the occurrence of prostate cancer. A diet rich in animal fat and red meat may also increase the risk.

[0003] Prostate cancer usually has no obvious symptoms in the early stages and is often discovered accidentally during screening. As the tumor progresses, urinary tract symptoms, bone pain, weight loss, and hematuria or hematospermia may occur. Diagnostic methods include screening (such as PSA blood tests and rectal examinations), imaging tests (such as ultrasound, CT, and MRI), and tissue biopsies. Treatment options depend on factors such as the patient's age, health status, and cancer stage, and may include watchful waiting, surgery, radiotherapy, hormone therapy, and chemotherapy. In recent years, targeted therapy and immunotherapy have also attracted attention. The prognosis of prostate cancer varies from person to person. Early detection and timely treatment usually have a good prognosis. Developing a personalized treatment plan is the key to reducing the burden of the disease. Therefore, raising public awareness of prostate cancer and conducting early screening are important ways to reduce its incidence.

[0004] However, the existing prostate cancer diagnosis methods are relatively complicated, so how to develop a reagent that is convenient for diagnosis is a key issue that needs to be studied at present. Summary of the invention

[0005] The main purpose of the present invention is to provide an antigen peptide for anti-Ran K141 Malonyl antibody and its application, aiming to solve the problem that the prior art cannot specifically detect the lysine malonylation at the 141 site of the Ran protein.

[0006] To achieve the above object, the present invention proposes an antigen peptide against anti-Ran K141 Malonyl antibody, whose amino acid sequence is: VFHR-(Malonyl)K-KNLQYY C, wherein (Malonyl)K represents malonylated lysine; the Ran K141 Malonyl indicates that the 141st lysine of the Ran protein is malonylated.

[0007] The present invention also proposes the use of the antigen peptide described above in the preparation of anti-Ran K141 Malonyl antibody, wherein the anti-Ran K141 Malonyl antibody is a polyclonal antibody, which is prepared by cross-linking the antigen peptide described above with the carrier protein KLH to obtain a cross-linked polypeptide as the antigen.

[0008] The present invention also provides an anti-Ran K141 Malonyl antibody, which is prepared using the cross-linked polypeptide obtained by cross-linking the antigen peptide and the carrier protein KLH as an antigen; the anti-Ran K141 Malonyl antibody is a polyclonal antibody.

[0009] Preferably, the antibody is a rabbit polyclonal antibody, and its preparation method comprises the following steps: using the substance obtained by cross-linking the antigen peptide as described above with the carrier protein KLH as an immunogen to immunize experimental rabbits, and collecting the blood of the experimental rabbits to prepare antiserum.

[0010] The present invention also proposes the use of the above-mentioned antibody in the preparation of a reagent for detecting the level of Ran K141 Malonyl.

[0011] The present invention also provides the use of the above-mentioned antibody in preparing a reagent for diagnosing prostate cancer.

[0012] The present invention also provides a reagent for detecting the level of Ran K141 Malonyl, comprising the antibody described above.

[0013] The present invention also provides a diagnostic reagent for prostate cancer, comprising the antibody described above.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] In the prior art, no antibody that specifically recognizes lysine malonylation at position 141 of the Ran protein has been developed and applied. The present invention artificially synthesizes a polypeptide modified by lysine malonylation at position 141, uses the modified polypeptide as an antigen, and prepares a specific antibody by immunizing animals. The antibody can be used to recognize lysine malonylation at position 141 of the Ran protein, and can thus be used in the development of diagnostic reagents for prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a diagram showing the mass spectrometry detection results of Ran K141ma modified polypeptide A in Example 1 of the present invention;

[0018] Figure 2 This is a diagram showing the mass spectrometry detection result of Ran K141ma modified polypeptide B in Example 1 of the present invention;

[0019] Figure 3 This is a graph showing the mass spectrometry detection result of the Ran K142ma modified control polypeptide C in Example 1 of the present invention;

[0020] Figure 4 This is a graph showing the mass spectrometry results of the Ran non-modified polypeptide in Example 1 of the present invention;

[0021] Figure 5 (a) and (b) are diagrams showing the serum WB preliminary screening results in Example 4 of the present invention;

[0022] Figure 6 This is a flowchart of antibody purification in Example 5 of the present invention;

[0023] Figure 7 This is a graph showing the antibody Dotblot test results in Example 6 of the present invention;

[0024] Figure 8 This is a graph showing the Western blot test results of the antibody in Example 6 of the present invention;

[0025] Fig. 9 This is a diagram of the Western blot experimental results in Example 7 of the present invention;

[0026] Fig.10 This is a graph showing the degree of malonylation of Ran K141 in prostate cancer tissue and adjacent tissue in Example 7 of the present invention;

[0027] Fig.11 This is a graph showing the degree of Ran K141 malonylation in castration-resistant prostate cancer tissue and paired androgen-sensitive prostate cancer tissue in Example 7 of the present invention;

[0028] Fig.12 This is a survival analysis diagram in Example 7 of the present invention.

[0029] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0030] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0032] Example 1 Synthesis of polypeptide

[0033] According to the protein sequence and lysine site of Ran protein Uniprot ID: P62826, the following four antigenic peptides, i.e., immunogens, were designed and synthesized, including two peptides with lysine at position 141 being malonylated, one modified control peptide (lysine at position 142 being malonylated), and one non-modified peptide (Table 1).

[0034] Table 1 Peptide information

[0035]

[0036] The four synthesized peptides were detected by immunogen mass spectrometry, and the results are as follows Figure 1-4 As shown in the figure, the sequences of the four peptides are accurate. The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the ratio of mass to charge. The difference between the measured mass and the theoretical mass of the four peptides is within 10ppm, and the sequence is correct.

[0037] Example 2 Preparation of Antibodies

[0038] The four antigenic peptides synthesized above were coupled to the carrier protein KLH to obtain antigenic peptide-KLH conjugates, which were used as immunogens to immunize experimental rabbits to prepare polyclonal antibodies. The specific steps are as follows:

[0039] (1) Primary immunization: Each rabbit was injected with 1 mL of a mixture of emulsified antigen peptide-KLH conjugate and Freund's complete adjuvant (volume ratio 1:1) at two points under the skin of both shoulders and at two points in the muscles of both hind legs, wherein the antigen peptide content was 0.5 mg.

[0040] (2) One week after the initial immunization, a second booster immunization was performed according to the method of the initial immunization.

[0041] (3) One week after the second booster immunization, the third booster immunization was carried out according to the method of the initial immunization.

[0042] (4) One week after the fifth booster immunization, peripheral blood of the experimental rabbits was collected, the supernatant was taken after centrifugation, and sent to the laboratory for serum screening and testing, so that the serum with positive test results could be selected for purification.

[0043] The rabbits immunized with Ran K141ma modified polypeptide A were labeled R1 and R2, and the rabbits immunized with Ran K141ma modified polypeptide B were labeled R3 and R4.

[0044] Example 3 ELISA detection

[0045] (1) Antigen coating:

[0046] Dilute the antigen (i.e., antigen-modified polypeptide and control polypeptide) with the coating solution, add 50ug / well of the ELISA plate in sequence, and incubate in a refrigerator at 4°C overnight or in a 37°C oven for 2h.

[0047] (2) Washing plate:

[0048] Take out the ELISA plate coated the day before, add 1×TBST and wash three times.

[0049] (3) Closure:

[0050] The washed ELISA plate was added with 1% BSA blocking solution, incubated at 37°C for 1 hour, and then washed 1-3 times.

[0051] (4) Primary antibody incubation:

[0052] The antibody (i.e. the serum of the experimental rabbit) was diluted 3 times starting from 1:1K. The dilution volume was adjusted according to the actual situation. The samples were added to the ELISA plate in sequence, incubated at 37℃ for 1.5h, and then washed 1-3 times.

[0053] (5) Secondary antibody incubation:

[0054] Dilute the secondary antibody to 1:10K with 1% BSA blocking solution, incubate at room temperature or 37°C for 45 min, and then wash 1-3 times.

[0055] (6) Color rendering:

[0056] After adding TMB colorimetric solution for 5-10 min, 1 M sulfuric acid was used to terminate the display reaction, and the data was read using an enzyme reader.

[0057] The ELISA test results are shown in Table 2. The titers of R2, R3, and R4 recognizing the modified peptides are all 1:10K (OD>1).

[0058] Table 2 Serum ELISA test results

[0059]

[0060] Example 4 Western blot detection

[0061] (1) Lysis of cells:

[0062] Select cells or tissues for testing according to experimental requirements, select different lysis methods according to different samples to obtain the total protein of the sample, and determine the protein concentration by the BCA method.

[0063] (2) Protein electrophoresis and membrane transfer:

[0064] Protein electrophoresis: According to the molecular weight of the target protein, select the corresponding concentration of separation gel. When preparing the gel, add 10% ammonium persulfate and TEMED at the end, 4.7ml of separation gel per plate. Load 20-40ug sample per well and perform electrophoresis; the electrophoresis conditions are: 80v for concentrated gel and 120v for separation gel.

[0065] Wet transfer: Cool the transfer solution before transfer, and lay the gel, membrane, and filter paper in the transfer solution to form a sandwich structure to avoid bubbles. The transfer voltage is 80v-120v.

[0066] (3) Closure:

[0067] Add blocking solution to the transferred membrane and block at room temperature for 60 minutes. After incubation, wash with 1×TBST for 10 minutes.

[0068] (4) Primary antibody incubation:

[0069] The antibody (ie, serum of the experimental rabbit) was diluted with 2.5-5% skim milk powder, incubated at room temperature for 2 hours, and then washed three times with 1×TBST, each time for 5-10 minutes.

[0070] (5) Secondary antibody incubation:

[0071] Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45min-1h, and wash 3 times with 1×TBST, each time for 5-10min.

[0072] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0073] The endogenous RanK141ma protein was detected by WB in the immunized rabbit serum, and the results are shown in Figures 5(a) and (b).

[0074] WB results showed that R2 and R3 could detect a band with a theoretical molecular weight of about 24 kDa for the target protein.

[0075] Based on the results of ELISA and WB, sera from two rabbits, R2 and R3, were selected for antibody purification.

[0076] Example 5 Antibody Purification

[0077] Antibody purification flow chart Figure 6 shown.

[0078] (1) Prepare protein A affinity column:

[0079] Protein A is derived from a strain of Staphylococcus aureus. It contains 5 domains that can specifically bind to the Fc segment of antibody IgG molecules. Protein A is coupled to the agarose matrix as an affinity ligand. It can specifically bind to the antibody molecules in the sample, while allowing other impurities to flow through, and has extremely high selectivity. One Protein A molecule can bind to at least 2 IgGs.

[0080] Select 5mL or 10mL protein A filler, mix and stir equal volumes of filler and PBS buffer solution, and remove bubbles in the filler by vacuuming.

[0081] Slowly add protein A filler into the glass column to prepare the chromatography column. Avoid column drying during this process. After filling, balance the column with 10 times the volume of pre-cooled PBS buffer solution.

[0082] (2) Protein A affinity chromatography:

[0083] After filtering the serum with a filter, load it onto the balanced protein A chromatography column. In order to detect the binding efficiency of the antiserum and the filler, the load effluent needs to be retained.

[0084] The column was washed with PBS buffer solution and then eluted with 150 mM glycine buffer solution. The eluate was collected and adjusted to pH 7 by adding neutralization buffer solution.

[0085] (3) Enrichment of target antibodies:

[0086] The crude pure IgG obtained after protein A purification is loaded onto the balanced antigen polypeptide affinity chromatography column to specifically enrich the target antibody.

[0087] (4) Removal of non-specific antibodies:

[0088] The target antibody obtained in the previous step is loaded onto a non-modified affinity chromatography column, and the flow-through (FT3) is directly collected to remove non-specific antibody components.

[0089] (5) Antibody storage:

[0090] The protein content was determined. 10% glycerol was added to preserve the antibody, and the purified antibody was aliquoted and stored at -20°C.

[0091] Example 6 Antibody Quality Control

[0092] Sufficient amount of rabbit serum was taken for affinity purification of Protein A and immunogenic polypeptide column. The antibodies purified from R2 and R3 were labeled as Ab2 and Ab3, respectively, and the purified antibodies were subjected to ELISA, Dot Blot and Western detection.

[0093] Antibody ELISA test:

[0094] In a 96-well ELISA plate coated with antigen-modified peptides and control peptides, antibodies were added for incubation at different dilution ratios (e.g. 1:54K, 1:162K). Then, enzyme-labeled secondary antibodies and TMB substrates were applied to detect the binding of peptides and antibodies. The ELISA test results showed that Ab3's recognition ability for at least one modified peptide was not less than 1:50K dilution, and was more than 10 times stronger than the ability to recognize non-modified negative peptides.

[0095] Table 3 Antibody ELISA test results

[0096]

[0097] Antibody Dotblot detection:

[0098] (1) Sample:

[0099] The antigen peptides were spotted onto the PVDF membrane according to the gradient of 1ng, 4ng, 16ng, and 64ng.

[0100] (2) Closure:

[0101] After the membrane surface is dry, add blocking solution and block at room temperature for 60 minutes.

[0102] (3) Washing:

[0103] Wash with 1× TBST for 10 min.

[0104] (4) Primary antibody incubation:

[0105] The purified antibody was diluted with 2.5-5% skim milk powder, incubated at room temperature for 2 h, and then washed three times with 1×TBST, each time for 5-10 min.

[0106] (5) Secondary antibody incubation:

[0107] Select the corresponding rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45min-1h, and wash 3 times with 1×TBST, each time for 5-10min.

[0108] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0109] Modified peptides with different doses (such as 4ng, 16ng) and non-modified peptides were fixed on the solid phase membrane, incubated with antibodies, and then enzyme-labeled secondary antibodies and chemiluminescent substrates were added to detect the binding of peptides and antibodies. Figure 7 The Dotblot results showed that Ab3 had a strong binding to the modified peptide and had almost no binding to the negative non-modified peptide and the modified control peptide.

[0110] Antibody Western blot detection:

[0111] The positive antibody Ab3 from Dot / ELISA was tested by Western Blot in HeLa, HeLa+Sodium malonylate (20mM, 72h), Human glioma, and Human prostate cancer lysates to obtain Figure 8 .Depend on Figure 8 It can be seen that Ab3 can detect a band of about 24 kDa in HeLa and HeLa+Sodiummalonylate (20 mM, 72 h) lysates, and this band is the target protein RanK141ma.

[0112] Based on the above antibody detection analysis, Ab3 is recommended as the specific antibody for Anti-RanK141ma. The results of Ab3 antibody are as follows:

[0113] (1) When the dilution of the ELISA-positive (OD450>1.0) antibody was greater than 54,000, the recognition ability of at least one modified peptide was 10 times stronger than that of the negative non-modified peptide.

[0114] (2) The positive detection limit of Dotblot is 4 ng, and the signal is 10 times stronger than that of the unmodified peptide.

[0115] (3) Other application results showed that Ab3 antibody could recognize endogenous RanK141ma by WB, and the band size was accurate.

[0116] Example 7 Application in prostate cancer

[0117] (1) Western blot experiments were used to detect the degree of Ran K141 malonylation (RanK141mal) in prostate cancer tissue (T) and paired adjacent normal tissue (N), castration-resistant prostate cancer tissue (C) and paired androgen-sensitive prostate cancer tissue (H), castration-resistant prostate cancer tissue that developed resistance after enzalutamide treatment (CR) and matched castration-resistant prostate cancer tissue before treatment (C). The results showed that compared with the paired adjacent normal tissue (N), the degree of RanK141 malonylation (RanK141mal) in prostate cancer tissue (T) was significantly increased; compared with the paired androgen-sensitive prostate cancer tissue (H), the degree of RanK141 malonylation (RanK141mal) in castration-resistant prostate cancer tissue (C) was significantly increased; compared with the matched castration-resistant prostate cancer tissue before treatment (C), the degree of RanK141 malonylation (RanK141mal) in castration-resistant prostate cancer tissue (CR) that developed resistance after enzalutamide treatment was significantly increased. The degree of K141 malonylation (RanK141mal) was significantly increased ( Fig. 9 ).

[0118] (2) Immunohistochemistry was used to detect the degree of RanK141 malonylation in prostate cancer tissues (n=180) and adjacent tissues (n=130) using prostate cancer tissue microarrays. The results showed that the degree of RanK141 malonylation (RanK141mal) in prostate cancer tissues was significantly increased compared with adjacent tissues. Further analysis also showed that the degree of Ran K141 malonylation (RanK141mal) was positively correlated with the clinical T stage, Gleason score, and clinical grade (Grade) of prostate cancer tissues. Fig.10 ).

[0119] (3) The puncture biopsy specimens were further used to detect the level of Ran K141 malonylation (RanK141mal) in castration-resistant prostate cancer tissue (CRPC) and paired androgen-sensitive prostate cancer tissue (HSPC). The results showed that the level of Ran K141 malonylation (RanK141mal) in castration-resistant prostate cancer tissue (C) was significantly increased ( Fig.11 ).

[0120] (4) Survival analysis found that compared with prostate cancer patients with lower Ran K141 malonylation levels (RanK141mal), prostate cancer patients with higher Ran K141 malonylation levels (RanK141mal) had shorter disease-free survival (DFS) and overall survival (OS). Fig.12 ).

[0121] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. An antigen peptide against anti-Ran K141 Malonyl antibody, characterized in that: The amino acid sequence is: VFHR-(Malonyl)K-KNLQYY C, wherein (Malonyl)K represents lysine modified by malonylation; the Ran K141Malonyl represents that the 141st lysine of the Ran protein is modified by malonylation.

2. Use of the antigen peptide according to claim 1 in the preparation of anti-Ran K141 Malonyl antibodies, characterized in that: The anti-Ran K141 Malonyl antibody is a polyclonal antibody, which is prepared using the cross-linked polypeptide obtained by cross-linking the antigen peptide according to claim 1 and the carrier protein KLH as an antigen.

3. An antibody against Ran K141 Malonyl, characterized in that: The antigen is prepared by using the cross-linked polypeptide obtained by cross-linking the antigen peptide according to claim 1 and the carrier protein KLH as the antigen; the anti-Ran K141 Malonyl antibody is a polyclonal antibody.

4. The anti-Ran K141 Malonyl antibody according to claim 3, characterized in that The antibody is a rabbit polyclonal antibody, and its preparation method comprises the following steps: using the substance obtained by cross-linking the antigen peptide according to claim 1 with the carrier protein KLH as an immunogen to immunize experimental rabbits, and collecting the blood of the experimental rabbits to prepare antiserum.

5. Use of the antibody according to claim 3 or 4 in the preparation of a reagent for detecting the degree of Ran K141 Malonyl.

6. Use of the antibody according to claim 3 or 4 in the preparation of a reagent for diagnosing prostate cancer.

7. A reagent for detecting the degree of Ran K141 Malonyl, characterized in that: Comprising the antibody according to claim 3 or 4.

8. A prostate cancer diagnostic reagent, characterized in that: Comprising the antibody according to claim 3 or 4.

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