Preparation method of circFam53b-219aa polypeptide and polyclonal antibody thereof

Abstract

The application discloses a circFam53b-219aa polypeptide and a preparation method of a polyclonal antibody thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptide can stimulate an antigen epitope of B cells, and animals are immunized to obtain a polyclonal antibody which can specifically recognize a natural circFam53b-219aa protein. The polyclonal antibody can effectively detect expression of the circFam53b-219aa in tumor tissues, can promote development of treatment aiming at the circFam53b-219aa, and has good social value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of antigen and antibody, and specifically relates to a method for preparing a circFam53b-219aa polypeptide and its polyclonal antibody. Background Technology

[0002] CircFam53b-219aa is a protein encoded by the human circular RNA circFam53b, and its amino acid sequence is shown in SEQ ID NO.1. circFam53b-219aa is highly expressed in various tumors, including breast cancer and melanoma. A search revealed that there are currently no commercially available antibodies against circFam53b-219aa, limiting in-depth research into the biological functions of this protein. Summary of the Invention

[0003] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a circFam53b-219aa polypeptide.

[0004] Another object of the present invention is to provide a method for preparing circFam53b-219aa polypeptide polyclonal antibody.

[0005] Another object of the present invention is to provide a circFam53b-219aa polypeptide polyclonal antibody obtained by the above preparation method.

[0006] The objective of this invention is achieved through the following technical solution: a circFam53b-219aa polypeptide, the amino acid sequence of which is shown below: CLKRTAHYGTGRQC.

[0007] The application of the above-mentioned circFam53b-219aa peptide in antibody preparation.

[0008] The antibody mentioned is a polyclonal antibody.

[0009] A method for preparing a circFam53b-219aa polypeptide polyclonal antibody includes the following steps:

[0010] (1) The above circFam53b-219aa polypeptide was coupled to a carrier protein to obtain circFam53b-219aa polypeptide complex protein.

[0011] (2) Immunize animals with circFam53b-219aa polypeptide complex protein to obtain serum containing circFam53b-219aa polypeptide antibody;

[0012] (3) The serum containing the circFam53b-219aa polypeptide antibody was purified to obtain the circFam53b-219aa polypeptide polyclonal antibody.

[0013] The carrier protein mentioned in step (1) is preferably keyhole hemocyanin (KLH).

[0014] The coupling steps described in step (1) are preferably as follows:

[0015] A. Dissolve the carrier protein in cross-linking buffer to obtain a carrier protein solution;

[0016] B. Dissolve the crosslinking agent in an organic solvent to obtain a crosslinking agent solution;

[0017] C. Mix the carrier protein solution and the cross-linking agent solution and react them. Purify the resulting reaction solution to obtain the activated carrier protein.

[0018] D. The circFam53b-219aa polypeptide was mixed with the activated carrier protein and reacted. The reaction solution was then dialyzed to obtain the circFam53b-219aa polypeptide complex protein.

[0019] The cross-linking buffer solution described in step (1)A has the following composition: 0.1 mol / L PB, 0.15 mol / L NaCl, and water as the solvent.

[0020] The carrier protein mentioned in step (1)A is preferably keyhole hemocyanin (KLH).

[0021] The crosslinking agent mentioned in step (1)B is preferably m-maleimide benzoyl-N-hydroxysuccinimide ester.

[0022] The organic solvent mentioned in step (1)B is preferably N,N-dimethylformamide.

[0023] In step (1)C, the carrier protein and cross-linking agent in the reaction solution are preferably mixed in a mass ratio of 1:1.

[0024] The reaction time in step (1)C is preferably 20 to 40 minutes; more preferably 30 minutes.

[0025] The purification described in step (1)C is preferably performed by an affinity chromatography column; more preferably, purification is performed using a Sephadex G-25 affinity chromatography column.

[0026] In step (1)D, the circFam53b-219aa polypeptide and the activated carrier protein in the reaction solution are preferably mixed in a mass ratio of 1:1.

[0027] The reaction time in step (1)D is preferably 2 to 4 hours; more preferably 3 hours.

[0028] The animals mentioned in step (2) include sheep, mice and rabbits; preferably rabbits.

[0029] The preferred steps for immunization in step (2) are as follows: the circFam53b-219aa polypeptide complex protein is emulsified with Freund's complete adjuvant and then injected subcutaneously; three booster immunizations are performed after the initial immunization; more preferably, the following specific steps are included: the circFam53b-219aa polypeptide complex protein is dissolved in PBS solution to obtain a complex protein solution with a concentration of 1 mg / mL; then it is thoroughly emulsified with an equal volume of Freund's complete adjuvant to obtain an immunizing agent; the initial immunization is completed by subcutaneous injection at multiple points, with 1 mL of immunizing agent per animal; the first booster immunization is performed 2 weeks later with 1 mL of immunizing agent per animal, followed by the second and third booster immunizations every 3 weeks.

[0030] The purification described in step (3) is performed by affinity chromatography, and the preferred specific steps are as follows:

[0031] A. Wash the chromatography column containing the activated gel with binding buffer; then add the circFam53b-219aa peptide dissolved in coupling buffer and react; then wash with binding buffer to obtain the circFam53b-219aa peptide-bound chromatography column.

[0032] B. Dilute the serum containing circFam53b-219aa polypeptide antibody with binding buffer, then load it onto the circFam53b-219aa polypeptide binding chromatography column. After sufficient binding, wash with washing buffer and then elute with elution buffer to obtain circFam53b-219aa polypeptide polyclonal antibody.

[0033] The gel mentioned in step (3)A is preferably Sephadex G-200.

[0034] The preferred composition of the binding buffer in steps (3) A and B is: 3 mol / L NaCl, 100 mmol / L citrate / phosphate buffer, pH 9.0, and water as the solvent.

[0035] The coupling buffer described in steps (3) A and B has the following composition: 0.1 mol / L PB, 0.15 mol / L NaCl, and water as the solvent.

[0036] The reaction conditions described in step (3)A are preferably overnight at 2–8°C; more preferably overnight at 4°C.

[0037] The binding time described in step (3)B is preferably 1.5 to 2.5 hours; more preferably 1 hour.

[0038] The composition of the rinsing buffer in step (3)B is as follows: 1 mol / L Tris, with water as the solvent.

[0039] The elution buffer described in step (3)B has the following composition: 100 mmol / L citrate / phosphate buffer, pH 3.0.

[0040] A polyclonal antibody containing circFam53b-219aa peptide was prepared by the above method; it has good specificity, high purity, and can specifically recognize the natural circFam53b-219aa protein in tissues or cells.

[0041] The above-mentioned circFam53b-219aa polypeptide polyclonal antibody is used in the preparation of diagnostic tumor agents or in the study of the biological function of circFam53b-219aa.

[0042] The tumors mentioned are solid tumors and non-solid tumors expressing circFam53b-219aa.

[0043] This invention offers the following advantages and effects compared to existing technologies: CircFam53b-219aa is a polypeptide encoded by circFam53b that has been shown to be highly expressed in various tumors. CircFam53b-219aa can serve as a novel target for tumor therapy, particularly tumor immunotherapy. To achieve precision treatment, it is necessary to detect whether circFam53b-219aa is expressed in the patient's tissues before initiating treatment targeting circFam53b-219aa. Currently, there are no commercially available antibodies against circFam53b-219aa, and this invention fills this gap. The circFam53b-219aa polypeptide provided by this invention can stimulate B cells, sensitizing immunized animals (such as rabbits) to obtain a polyclonal antibody that specifically recognizes natural circFam53b-219aa. This polyclonal antibody can effectively detect the expression of circFam53b-219aa in tumor tissues, promoting the development of treatments targeting circFam53b-219aa, and possesses significant social value and market potential. Attached Figure Description

[0044] Figure 1 This is a Western blotting image; the target band in the immunoblotting image is approximately 24 kDa, which is consistent with the theoretical molecular weight (24 kDa) of the circFam53b-219aa protein.

[0045] Figure 2 This is an immunohistochemical image; the stained areas in the image represent positive signals from antibody reactions. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0047] Example 1: Analysis of the circFam53b-219aa sequence and design and synthesis of the circFam53b-219aa peptide

[0048] Based on the circFam53b-219aa sequence, the protein sequence is known to contain 219 amino acids. By analyzing the antigenicity, hydrophilicity, and surface probability of this protein sequence, and using DNAstar software for prediction, a peptide with the sequence CLKRTAHYGTGRQ was screened. To increase immunogenicity, a cysteine ​​residue was added to the C-terminus, resulting in the final peptide: CLKRTAHYGTGRQC. The peptide was synthesized using an automated peptide synthesizer. The purity of the synthesized peptide was determined by high-performance liquid chromatography (HPLC), and the purity was 95%. The amino acid sequence of the peptide was confirmed to be correct by MS mass spectrometry.

[0049] Example 2: Crosslinking of peptides with carrier protein KLH

[0050] MBS (m-maleimide benzoyl-N-hydroxysuccinimide ester) was used as a cross-linking agent to cross-link the carrier protein KLH (keyhole hemocyanin) with the synthetic peptide of Example 1. The specific steps are as follows:

[0051] (1) KLH was dissolved in cross-linking buffer (0.1M PB, 0.15M NaCl) to obtain a KLH solution with a concentration of 10 mg / mL;

[0052] (2) MBS was dissolved in DMF (N,N-dimethylformamide) to obtain an MBS solution with a concentration of 10 mg / mL;

[0053] (3) Mix the dissolved KLH solution and MBS solution at a ratio of 10:1 (w / w), activate KLH at room temperature for 30 minutes to obtain solution A; purify the activated KLH solution (i.e., solution A) with Sephadex G-25. The Sephadex G-25 treatment steps are as follows: Weigh 3-4g of Sephadex G-25, add about 50mL of water, swell in a boiling water bath for 2 hours, pour off the excess water and fine particles from the upper layer of the fully swollen gel, repeat this washing process 2-3 times, place it in a vacuum bottle to remove air bubbles, and set aside for use; wash with distilled water until its pH value is consistent with distilled water, pack the column, and equilibrate the gel column after packing. Load the sample, elute (the eluent consists of 100mmol / L citrate / phosphate buffer, pH 3.0), and control the flow rate at 3mL / 13min.

[0054] (4) The purified activated KLH solution (5 mg / mL) and the peptide solution (5 mg peptide added to 1 mL cross-linking buffer, concentration is 5 mg / mL) were mixed at a ratio of 1:1 (W / W) and reacted at room temperature for 3 hours to obtain solution B; the above reaction solution was dialyzed overnight in PBS (0.01 M, pH 7.4) at 4°C to obtain peptide-KLH complex protein.

[0055] Example 3: Animal Immunization

[0056] Suitable-age (approximately 2-2.5 kg, purchased from Guangdong Provincial Experimental Animal Center) New Zealand male rabbits were selected as immunization animals. Before immunization, 2-3 mL of blood was collected from the ear vein as a negative control for subsequent ELISA testing. For the first immunization, 0.5 mg of the peptide-KLH complex protein was dissolved in 0.5 mL of PBS solution, thoroughly emulsified with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple sites, 1 mL per rabbit. Two weeks later, a first booster immunization was performed. 0.5 mg of the peptide-KLH cross-linked complex protein was dissolved in 0.5 mL of PBS solution, thoroughly emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously at multiple sites. Thereafter, booster immunizations were performed every 3 weeks, for a total of 3 times. One week after each booster immunization, a small amount of blood was collected from the rabbit's ear vein, and the serum titer was detected using an indirect ELISA method. When the titer reached 1:20000 or higher, blood was collected from the rabbit's carotid artery to prepare serum. The serum and binding buffer were mixed to obtain a binding buffer containing serum containing circFam53b-219aa antibody.

[0057] Example 4: Purification of antibody using circFam53b-219aa peptide affinity chromatography column

[0058] 1 mL of activated gel suspension (Sephadex G-200, Sigma-Aldrich, catalog number: 37224-29-6) was injected into the chromatography column. After the liquid in the column had dried out, 5 mL of binding buffer (3 mol / L NaCl, 100 mmol / L citrate / phosphate buffer, pH 9.0) was added to wash the column. 2 mg of the synthesized circFam53b-219aa peptide was dissolved in 1 mL of coupling buffer (0.1 M PB, 0.15 M NaCl) and added to the chromatography column. Another 1 mL of coupling buffer was added to the column, and the mixture was incubated overnight at 4°C by rotation. The column was washed three times with binding buffer until the liquid in the column had dried out, thus preparing the circFam53b-219aa peptide binding chromatography column. Add the binding buffer containing circFam53b-219aa antibody serum prepared in Example 3 to the chromatography column, mix well at room temperature for 1 hour, and wash the column with 30 mL of washing buffer (1 mol / L Tris) until the A280 nm absorption peak of the eluent is stable. Elute the column with 15 mL of elution buffer (100 mmol / L citrate / phosphate buffer, pH 3.0) to obtain purified circFam53b-219aa polyclonal antibody.

[0059] Example 5: Determination of antibody titer by indirect ELISA

[0060] The circFam53b-219aa modified peptide-KLH complex was coated onto an ELISA plate overnight at 4°C, followed by blocking with 5% (w / w) skim milk powder at room temperature for 2 hours. The circFam53b-219aa antibody obtained in Example 4 was diluted with 5% BSA (bovine serum albumin) solution at different concentrations: 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000, and incubated at room temperature for 2 hours or overnight at 4°C. Horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added, and incubation was continued at room temperature for 2 hours. TMB was added for colorimetric reaction, and the reaction was terminated with sulfuric acid. The absorbance at 450 nm was measured. The antibody titer was calculated when the ratio to negative serum was greater than 2.1. The purified circFam53b-219aa antibody was tested and found to have a titer of 1:512,000.

[0061] Example 6: Western Blot analysis of the specificity of circFam53b-219aa antibody

[0062] SDS-PAGE gels with a stacking gel concentration of 5% and a separating gel concentration of 12.5% ​​were prepared according to standard methods. 20 μL of lysate from normal breast tissue or breast cancer tissue with a protein concentration of 5 mg / mL was added to the sample wells of a vertical electrophoresis tank. Electrophoresis was performed at a constant voltage of 80V. When the sample passed through the stacking gel in a straight line, the voltage was changed to 120V. Electrophoresis was stopped when the bromophenol blue indicator completely ran off the separating gel. Wet transfer was then performed at a constant voltage of 100V for 90 minutes to transfer the protein to a PVDF membrane. The circFam53b-219aa antibody obtained in Example 4 was used as the primary antibody, diluted 1:500 (v / v), and incubated with the wet-transferred PVDF membrane at room temperature for 2 hours. Hybridization was then performed with HRP-labeled secondary antibody at room temperature for 2 hours. The immunoblot results were obtained using chemiluminescence staining. Figure 1 As shown: Figure 1 In this context, N represents normal breast tissue and T represents breast cancer tissue. This result indicates that circFam53b-219aa is highly expressed in breast cancer tissue.

[0063] Example 7: Immunohistochemical analysis of circFam53b-219aa antibody to detect the expression of circFam53b-219aa protein in tissues

[0064] Human breast cancer tissue (obtained from the Breast Cancer Center of Sun Yat-sen Memorial Hospital, Sun Yat-sen University; all samples were obtained with patient consent) was fixed with 4% formaldehyde solution and then blocked with goat serum blocking solution. The circFam53b-219aa antibody obtained in Example 4 was used as the primary antibody, diluted 1:100 (v / v), and incubated overnight at 4°C. Then, HRP-labeled goat anti-rabbit fluorescent secondary antibody was used as the secondary antibody, diluted 1:50 (v / v), and incubated for 15 min. Unreacted secondary antibody was washed off, and DAB staining was performed. Fluorescence was observed and photographed. The results are as follows: Figure 2 As shown in the figure. The experimental results of this embodiment indicate that circFam53b-219aa is expressed in breast cancer.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A circFam53b-219aa polypeptide, the amino acid sequence of which is shown below: CLKRTAHYGTGRQC.

2. The application of the circFam53b-219aa polypeptide according to claim 1 in the preparation of antibodies, characterized in that: The antibody mentioned is a polyclonal antibody.

3. A method for preparing a circFam53b-219aa polypeptide polyclonal antibody, characterized in that... Includes the following steps: (1) The circFam53b-219aa polypeptide of claim 1 is coupled to a carrier protein to obtain the circFam53b-219aa polypeptide complex protein; (2) Immunize animals with circFam53b-219aa polypeptide complex protein to obtain serum containing circFam53b-219aa polypeptide antibody; (3) The serum containing the circFam53b-219aa polypeptide antibody was purified to obtain the circFam53b-219aa polypeptide polyclonal antibody.

4. The preparation method according to claim 3, characterized in that: The carrier protein mentioned in step (1) is keyhole hemocyanin (KLH); The animals mentioned in step (2) include sheep, mice, and rabbits; The immunization steps described in step (2) are as follows: after mixing and emulsifying circFam53b-219aa polypeptide complex with Freund's complete adjuvant, it is injected subcutaneously; after the initial immunization, three booster immunizations are performed. The purification described in step (3) is performed by affinity purification using an affinity chromatography column.

5. The preparation method according to claim 3, characterized in that: The coupling steps described in step (1) are as follows: A. Dissolve the carrier protein in cross-linking buffer to obtain a carrier protein solution; B. Dissolve the crosslinking agent in an organic solvent to obtain a crosslinking agent solution; C. Mix the carrier protein solution and the cross-linking agent solution and react them. Purify the resulting reaction solution to obtain the activated carrier protein. D. The circFam53b-219aa polypeptide of claim 1 is mixed and reacted with the activated carrier protein, and then the reaction solution is dialyzed to obtain the circFam53b-219aa polypeptide complex protein.

6. The preparation method according to claim 5, characterized in that: The cross-linking buffer solution described in step (1)A has the following composition: 0.1 mol / L PB, 0.15 mol / L NaCl, and water as the solvent; The carrier protein mentioned in step (1)A is keyhole hemocyanin (KLH); The crosslinking agent mentioned in step (1)B is m-maleimide benzoyl-N-hydroxysuccinimide ester; The organic solvent mentioned in step (1)B is N,N-dimethylformamide; In step (1) C, the carrier protein and cross-linking agent in the reaction solution are mixed in a mass ratio of 1:1; The reaction time described in step (1) C is 20–40 min; The purification described in step (1) C is performed by affinity chromatography. In step (1) D, the circFam53b-219aa polypeptide and the activated carrier protein are mixed in a mass ratio of 1:1 in the reaction solution. The reaction time described in step (1) D is 2 to 4 hours.

7. The preparation method according to claim 3, characterized in that: The specific purification steps described in step (3) are as follows: A. Wash the chromatography column containing the activated gel with binding buffer; then add the circFam53b-219aa peptide dissolved in coupling buffer and react; then wash with binding buffer to obtain the circFam53b-219aa peptide-bound chromatography column. B. Dilute the serum containing circFam53b-219aa polypeptide antibody with binding buffer, then load it onto the circFam53b-219aa polypeptide binding chromatography column. After sufficient binding, wash with washing buffer and then elute with elution buffer to obtain circFam53b-219aa polypeptide polyclonal antibody.

8. The preparation method according to claim 7, characterized in that: The gel mentioned in step (3)A is Sephadex G-200; The composition of the binding buffer described in steps (3) A and B is: 3 mol / L NaCl, 100 mmol / L citrate / phosphate buffer, pH 9.0, and water as the solvent; The coupling buffer solution described in step (3)A has the following composition: 0.1 mol / L PB, 0.15 mol / L NaCl, and water as the solvent; The reaction conditions described in step (3)A are to react overnight at 2–8°C; The binding time described in step (3)B is 1.5 to 2.5 hours; The composition of the rinsing buffer described in step (3)B is as follows: 1 mol / L Tris, with water as the solvent; The elution buffer described in step (3)B has the following composition: 100 mmol / L citrate / phosphate buffer, pH 3.

0.

9. A circFam53b-219aa polypeptide polyclonal antibody, characterized in that: It is obtained by the preparation method according to any one of claims 3 to 8.

10. The application of the circFam53b-219aa polypeptide polyclonal antibody according to claim 9 in the preparation of diagnostic tumor agents, characterized in that: The tumor in question is a circFam53b-219aa positive tumor.

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