Preparation method and application of a succinylated keap1 antibody

Abstract

The application discloses a preparation method and application of a succinylated KEAP1 antibody, and a preparation method of a succinylated KEAP1 antibody of a lysine at a 131st site. The preparation method solves the problem that a scientist can only use a non-site-specific pan-succinylated antibody to study succinylation of a KEAP1 protein at present, and the site-specific KEAP1 antibody can only recognize a succinylation state of the KEAP1 at the 131st site, and cannot recognize an unmodified state or a succinylation modification state of other sites. The interaction between metabolism and stress reaction signals is crucial for maintaining cell homeostasis, and provides a new idea for scientists to conquer cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing and applying succinylated KEAP1 antibody. Background Technology

[0002] In 2010, the University of Chicago first disclosed lysine succinylation in Nature Chemical Biology. The study, through experimental analysis, identified that succinyl-lysine peptides are a naturally occurring lysine modification derived from proteins in vivo, which tends to be conserved in evolution and can respond to different physiological environments.

[0003] Post-translational modifications (PTMs) refer to the covalent processing of translated proteins by adding modifying groups to one or more amino acid residues. This alters the protein's physicochemical properties, thereby affecting its spatial conformation, activity state, subcellular localization, folding, stability, and protein-protein interactions. Succinylation (Ksucc) has a broad regulatory effect on cellular metabolism, and it is precisely because of this characteristic that research has shown that this modification may affect other downstream cellular biological functions.

[0004] The interaction between metabolic and stress response signaling is crucial for maintaining cellular homeostasis. This crosstalk is typically achieved through the covalent modification of proteins by endogenous reactive metabolites that regulate key stress-responsive transcription factors such as NRF2. Metabolites including methylglyoxal, glyceraldehyde-3-phosphate, fumarate, and itaconic acid covalently modify the sensor cysteine ​​residue of the NRF2 repressor KEAP1, thereby stabilizing NRF2 and activating its cellular protective transcriptional program. Here, we employed shRNA-based screening targeting enzymes of central carbon metabolism to identify other regulatory nodes bridging metabolism and NRF2 activation. Increasing succinic anhydride through gene deletion or direct administration of the TCA cycle enzyme succinyl-CoA synthase leads to N-succinylation of KEAP1 lysine 131, thereby activating NRF2 signaling. This study determines that KEAP1 can sense reactive metabolites not only through several cysteine ​​residues but also through a single conserved lysine residue, suggesting its potential to sense a wider range of reactive metabolic messengers.

[0005] After Dr. Lara Ibrahim of the United States discovered the succinylation modification at this site in August 2023, this invention produced site-specific succinylated KEAP1 site-specific antibodies, which greatly facilitated scientists around the world to further explore the succinylation modification of KEAP1. Summary of the Invention

[0006] Therefore, based on the above background and in view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing KEAP1 antibody with lysine succinylation at position 131, in order to solve the current situation where scientists do not have succinylated KEAP1 antibody available.

[0007] The technical solution of the present invention is as follows:

[0008] A polypeptide for preparing an acid-succinylated KEAP1 antibody, the amino acid sequence of said polypeptide being as shown in SEQ ID NO. 2; or a polypeptide derived from the polypeptide shown in SEQ ID NO. 2 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function.

[0009] Based on the same inventive concept, the present invention also provides a gene fragment capable of encoding the aforementioned polypeptide.

[0010] Based on the same inventive concept, the present invention also applies the above-mentioned polypeptide to the preparation of succinylated KEAP1 antibody.

[0011] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned succinylated KEAP1 antibody. The method involves immunizing animals with the polypeptide to induce the animals to produce acid succinylated KEAP1 antibody, and then collecting the animal serum to obtain serum containing acid succinylated KEAP1 antibody.

[0012] Furthermore, the animal in question is a rabbit.

[0013] Furthermore, the procedures for immunizing animals include basic immunization and three or more booster immunizations.

[0014] Furthermore, the polypeptide is used to immunize animals after being conjugated with KLH.

[0015] Furthermore, the method also includes extracting and purifying animal serum.

[0016] Furthermore, the extraction and purification were performed using peptide-based sulflink gel affinity purification of animal antiserum.

[0017] Based on the same inventive concept, the above-mentioned succinylated KEAP1 antibody can be applied to immunoblotting experiments.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] As described above, the method for preparing the KEAP1 antibody with lysine succinylation at position 131 of the present invention solves the problem that scientists currently rely on non-site-specific pan-succinylation antibodies to study the succinylation of KEAP1 protein. Our invented site-specific KEAP1 antibody can only recognize the state of succinylation at position 131 of KEAP1, and does not recognize the unmodified state or the succinylation of other sites. This is crucial for maintaining cellular homeostasis through the interaction between metabolism and stress response signals, and provides scientists with new ideas for conquering cancer. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the results of immunoblotting verification of the KEAP1 antibody at lysine succinylation at position 131, as an embodiment of the present invention. Lane A is a membrane incubated together with succinylated KEPA1 and succinylated polypeptide (SEQ ID2), and lane B is a membrane incubated alone. The disappearance of the band in lane A indicates that the antibody specifically recognizes succinylated KEAP1. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to its embodiments; it should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0022] The full-length sequence information of the KEAP1 protein is shown in SEQ ID NO.4:

[0023] .

[0024] 1) Identification and optimization of the polypeptides of the present invention

[0025] The full-length amino acid sequence of KEAP1 protein was analyzed for antigenic epitopes using BepiPred 1.0Server software. Based on the succinylation modification at 131 sites, and considering factors such as sequence hydrophobicity, rigidity, specificity, and ease of synthesis, the amino acid sequence of succinylated KEAP1 was finally determined to be EGIHPK(succ)VMERL: SEQ ID NO.1 (Glu-Gly-Ile-His-Pro-Lys-Val-Met-Glu-Arg-Leu, Lys being the succinylation modification) (synthesized by Shanghai Jier Biochemical), where K amino acid is the succinylation modification site.

[0026] A cysteine ​​C-residue was added to the C-terminus of the sequence, resulting in the final polypeptide sequence CEGIHPK(succ)VMERL: SEQ ID NO.2 (Cys-Glu-Gly-Ile-His-Pro-Lys-Val-Met-Glu-Arg-Leu, where Lys represents succinylation modification). Simultaneously, an unmodified polypeptide CEGIHPKVMERL (SEQ ID NO.3: Cys-Glu-Gly-Ile-His-Pro-Lys-Val-Met-Glu-Arg-Leu) was synthesized as a control polypeptide for reverse purification of the pan keap1 antibody during affinity purification.

[0027] 2) Succinylated peptides were conjugated with KLH to prepare immunogen KLH. DMSO and cross-linking buffer were purchased from Thermo Fisher Scientific.

[0028] ① Add water to maleimide-activated mcKLH to prepare a 10 mg / mL solution. If necessary, polyethylene glycol-modified mcKLH can be reconstituted to 20 mg / mL. Note: mcKLH forms a suspension, typically translucent to bluish-white. Do not vortex or heat the suspension, as this will cause mcKLH to precipitate.

[0029] ② Dissolve the thiol-containing hapten in cross-linking buffer with a volume equal to 1.0-2.5 times the volume of recombinant mcKLH. Specifically, dissolve 2 mg of the peptide in 200-500 μL of phosphate buffer and add it to 2 mg of activated mcKLH reconstituted in 200 μL of water. Alternatively, if the peptide is freely soluble, add it as a solid to the mcKLH suspension. Note: For haptens with limited solubility, DMSO can be used for solubilization. Irreversible denaturation may occur when using ≤30% dimethyl sulfoxide or carrier protein in the final binding solution.

[0030] ③ Immediately mix the peptide and activated mcKLH and react at room temperature for 2 hours.

[0031] ④ Purify the conjugate by gel filtration or dialysis to remove EDTA. EDTA is an anticoagulant and should not be injected into laboratory animals. Desalting can be used to remove EDTA by desalting or dialysis. If DMSO is used in the conjugation, add DMSO to the purification buffer for desalting to prevent precipitation; dialysis is incompatible with DMSO. Desalting or dialysis cannot separate non-conjugated mcKLH; however, the use of a large excess of hapten in this protocol makes it unlikely that a large amount of non-conjugated carrier will be present. If the conjugate is to be injected within one week, it can be diluted with PBS.

[0032] ⑤ During desalting, add 60 mL of degassed ultrapure water to the bottle to dissolve the contents of one bottle of purification buffer salt. Store excess buffer at 4°C.

[0033] ⑥ Remove the top and bottom caps from the desalting column in sequence to drain the storage solution. Use one desalting column for every 0.5 mL of sample.

[0034] ⑦ Wash the column with 3-5 column volumes (15-25 mL) of purification buffer. Spread 0.5 mL of the hapten vector mixture directly onto the center of the column pan. Add 8-10 aliquots of 0.5 mL purification buffer and collect each fraction in a separate tube.

[0035] ⑧ Measure absorbance at 280 nm to locate the component containing the conjugated compound. The hapten carrier conjugated compound will be detected in the first absorbance peak. Mix all fractions containing acceptable levels of conjugation together.

[0036] ⑨ After the conjugated hapten appears, the non-conjugated hapten can be recovered by continuing to add buffer to the column and collecting additional components.

[0037] ⑩ If the immunogen needs to be stored for more than a few days, the binding portion should be sterile filtered and stored in a sterile container at 4°C or -20°C.

[0038] 3) Animal immunization and serum collection

[0039] Select two healthy 6-week-old New Zealand white rabbits (approximately 2 kg) and allow them to acclimatize to their new living environment for at least a few days before the first blood draw.

[0040] Pre-collected blood (for negative reference)

[0041] ① Carefully place the rabbit in the designated shelf and allow it to calm down;

[0042] ② Carefully shave the fur off the rabbit's ears to make the blood vessels clearly visible (optional);

[0043] ③ If necessary, apply alcohol to the blood vessels with a small cotton ball to make them dilate.

[0044] ④ Draw approximately 10 ml of blood (approximately 5 ml of serum) from the ear vein using a syringe;

[0045] ⑤ Carefully remove the needle, apply appropriate pressure to the wound to prevent bleeding, and then disinfect the wound with an alcohol swab.

[0046] ⑥ Place the collected blood at 37°C for 30 minutes to inactivate it, and finally place it at 4°C overnight to allow it to coagulate and release serum;

[0047] ⑦ Centrifuge the coagulated blood at 10000r / min for 10min; h. Collect the supernatant, which is the serum.

[0048] Rabbit's immunity

[0049] ① Inject 1 ml of antigen into two rabbits. The antigen buffer solution must not contain any chemicals that are harmful to rabbits. For the initial immunization of each rabbit, 400 μg of antigen is suitable, but it can be reduced appropriately to obtain better results. Subsequent immunizations should be 100 μg each time.

[0050] ② Mix 1 ml of Freund's complete adjuvant with 1 ml of prepared antigen thoroughly until it turns milky white;

[0051] ③ Carefully remove the rabbit from the cage. Vaccinate each rabbit at 4 sites (back and groin are acceptable), 250ul at each site. Insert the needle at a 45-degree angle 1-2cm under the skin and leave it for a few seconds after injection to prevent antigen leakage.

[0052] ⑤ The immunization cycle is 20 days. Blood is collected 7-10 days after the immunization is completed (including blood collection for testing during the process and final blood collection). A total of 4-5 immunizations are performed.

[0053] Valence testing

[0054] ① Two references: The negative reference is pre-collected serum, diluted with the initial concentration of primary antibody (blocking diluent); the positive reference is previously positive serum.

[0055] ② Add 100 μL of antigen (1 μg / ml) to each well of a 96-well plate and incubate overnight at 4°C, or incubate at 37°C for 2 hours.

[0056] ③ Discard the antigen solution, add 200 μL of blocking solution to each well, and incubate overnight at 4°C or for 2 hours at 37°C.

[0057] ④ Pour off the sealing solution, pat the plate on absorbent paper to absorb as much residual liquid as possible, wash the plate three times with detergent, patting as much residual liquid as possible each time, and if you need to leave it for a period of time, dry it at 37°C and seal it in a sealed bag and store it at -20°C.

[0058] ⑤ Take the coated 96-well plate, add 100 μL of negative control solution to the first well, 100 μL of positive control solution to the second well, and add 1:500 of the antiserum to be tested to the third well. Serially dilute the solution in each subsequent well and incubate at 37°C for 1 hour.

[0059] ⑥ Discard the liquid, wash the plate three times with washing solution, and pat dry. Add 100 μL of HRP-labeled mouse anti-rabbit IgG (1:2000 dilution) to each well and incubate at 37°C for 45 minutes.

[0060] ⑦ Discard the liquid, wash the plate three times with washing solution, and pat dry. Add 100 μL of TMB substrate (Sigma single-component TMB) to each well and incubate at 37°C for 5-20 minutes (the development time depends on the color intensity).

[0061] ⑧ Add 50 μL of stop solution (2N H2SO4) to each well and read the absorbance at 450 nm on a microplate reader.

[0062] Once the antibody titer reaches 10,000 or higher, the rabbits can undergo final bloodletting. Antibody purification follows.

[0063] 4) Antibody purification

[0064] Preparation of affinity chromatography columns:

[0065] ① Column: Select a glass or plastic column suitable for the SulfoLink Resin volume. A disposable column trial kit (product number 29925) includes accessories plus two of each of three different column sizes, suitable for resin bed volumes from 0.5 to 10 mL. Alternatively, several centrifuge-prepared thermal science Pierce columns can be used for resin bed volumes from 25 μl to 10 mL. Coupling buffer: 50 mM Tris, 5 mM EDTA-Na; pH 8.5. Prepare a quenching agent equal to 20 times the volume of SulfoLink Resin. Wash buffer: 1 M sodium chloride (NaCl). Storage buffer: phosphate-buffered saline (PBS) or other suitable buffer containing 0.05% sodium azide (NaN3).

[0066] ②Preparation of SulfoLink chromatography column

[0067] 1. Equilibrate the SulfoLink coupling resin and all other reagents to room temperature.

[0068] 2. Stir or rotate the bottle to suspend the resin evenly, then use a wide-mouth pipette to transfer an appropriate amount of 50% resin slurry into the empty column. For example, transfer 2 mL of resin slurry to obtain a 1 mL resin bed.

[0069] 3. The balance column is coupled with the four resin beds to buffer the volume. Replace the bottom column cap.

[0070] Note: When using a gravity flow column, never allow the resin bed to dry out at any point during the process. Add more solution or replace the bottom cap on the column whenever the buffer reaches the top of the resin bed.

[0071] ③ Couple succinylated and non-succinylated peptides to SulfoLink resin

[0072] 1. Dissolve the prepared (i.e., reduced) peptide / protein in cross-linking buffer and add it to the column. Use 1-2 mL of peptide or protein solution per mL of SulfoLink coupling resin. If necessary, reserve a small amount of peptide or protein solution for later comparison, using the coupling reaction flow fraction to estimate coupling efficiency.

[0073] 2. Replace the top lid and stir for 15 minutes at room temperature (shaking or mixing end to end).

[0074] 3. Stand the column upright and incubate at room temperature for another 30 minutes. Do not mix.

[0075] 4. Remove the top and bottom column caps in sequence, allowing the solution to drain from the column into a clean tube.

[0076] 5. Place the column on a new collection tube and wash the column with three resin bed coupling buffers.

[0077] 6. Determine the coupling efficiency decoupling fraction (step 4) to the starting sample (step 1) by comparing protein / peptide concentrations (e.g., by absorbance at 280 nm).

[0078] ④ Blocking non-specific binding sites on the resin

[0079] 1. Replace the bottom cap on the column. Prepare a 50 mM l-cysteine·HCl solution in the coupling buffer.

[0080] 2. Add a layer of 50 mM cysteine ​​solution to the chromatographic column. Stir at room temperature for 15 minutes, then incubate the reaction for another 30 minutes without mixing.

[0081] ⑤ Washing pillars

[0082] 1. Remove the top and bottom caps in sequence to allow the chromatographic column to drain.

[0083] 2. Wash the column with at least 6 resin layer volumes of washing buffer (1M NaCl).

[0084] 3. Clean the chromatographic column with a two-layer resin degassing storage buffer.

[0085] 4. Replace the bottom cap and add an extra storage buffer for the resin bed capacity.

[0086] 5. Covalently coupled ligands can be used for affinity purification. Replace the top cap and store the column upright at 4°C, or remove the bottom cap and proceed according to the general affinity purification procedure.

[0087] 5) Affinity purification of succinylated antibodies

[0088] 1. Pour the serum to be precipitated into a beaker equipped with a stir bar and place it on a stirring plate at 4°C. Stir slowly to avoid foaming. Add (NH4)2SO4 to the serum very slowly until 40% saturation (2.26 g / 10 mL).

[0089] 2. Wait until (NH4)2SO4 is completely dissolved, and allow stirring for another 30 minutes.

[0090] 3. Centrifuge at 10,000×g for 20 minutes.

[0091] 4. Discard the supernatant and resuspend the microspheres in 10 ml of PBS.

[0092] 5. If the serum is frozen, thaw it or precipitate it with ammonium sulfate. If the serum appears cloudy without precipitation, vortex it at 20,000 x g for 10 minutes to clarify.

[0093] 6. If using unprecipitated serum, filter it into a clean tube using a 1.0 mm Whatman filter, and dilute with 500 × Pefabloc to a final concentration of 0.2 mm - 2 mL / 1 ml. Add 100 × Benzamidine to a final concentration of 2 mm - 10 mL / 1 ml.

[0094] 7. Remove sodium azide storage buffer from the previously prepared succinylated or non-succinylated affinity column and wash the column with at least 25 ml of PBS.

[0095] 8. Circulate the serum three times on the column and save the collected flow rate (Note 6). This flow rate can be used to purify desuccinylated antibodies (Note 7).

[0096] 9. Wash with 100 ml of BBS, then wash with 50 ml of PBS.

[0097] 10. Elute the antibodies on the column. For a 1 ml column bed, use 1 ml of elution buffer for every 1 ml of serum placed on the column (Note 8-11).

[0098] 11. Wash the column with 50 ml PBS and 5 ml column buffer. Cap the bottom of the column, add 2 ml of column buffer, and cap the top of the column (Note 12). Store at 4°C.

[0099] 12. Place the eluent in a dialysis tube and dialyze overnight at 4°C with 2L of buffer.

[0100] 13. Concentrate the antibody in the dialysis tube to approximately 1-3 mL using polyethylene glycol M8000. Rinse the tubing with water and perform a second dialysis buffering with 2 L of dialysis buffer for at least 3 hours, or overnight at 4°C.

[0101] 14. Remove the antibody from the dialysis tube and place it into a conical tube. If purifying PAN KEAP1 antibody, the purification process is complete. If purifying a succinylated antibody, proceed to the next step.

[0102] 15. Remove the sodium azide storage buffer from the non-succinylated column. Wash with 25 ml of PBS.

[0103] 16. Transfer the dialyzed antibody to a non-succinylated column (Note 13). Collect and discard the first milliliters of liquid that flow through (for columns with a 1-milliliter bed). This is PBS that has shifted in the column bed. Collect the remaining flow. Push the remaining antibody on the column into a collection tube by running 1 ml of dialysis buffer over the column. What flows through is affinity-purified succinylated antibody.

[0104] 17. Elute the pan antibody from the non-succinylated column into a separate tube. Dialyze the antibody if necessary.

[0105] 18. Wash the column with 25 ml PBS and 5 ml column buffer. Cap the column bottom, add 2 ml of column buffer, and cap the column top. Store at 4°C (Note 12).

[0106] 19. Results: The results were obtained by using an enzyme-linked immunosorbent assay (ELISA) reader and are shown in Table 3.

[0107]

[0108] ELISA results show that succinylated KEAP1 has excellent peptide-level specificity, does not react with non-succinylated peptides, and only recognizes peptides in the succinylated state.

[0109] 6) Immunoblotting verification of succinylated KEAP1 antibody

[0110] 1. Clean all glassware and utensils with ethanol.

[0111] 2. Determine the percentage of acrylamide required for effective protein separation based on the molecular weight of the protein of interest, and pour it into a low-lying (dissolved) SDS-PAGE gel. Add water on top of the gel and polymerize for 45 minutes.

[0112] 3. Remove the water, pour (stack) the gel, and insert the desired comb. Polymerize for 20-30 minutes.

[0113] 4. Remove the comb and excess polymerized gel, and attach the gel to the running gel apparatus as directed by the manufacturer. Fill the chamber with 1x running buffer.

[0114] 5. Add 4 times the sample buffer to prepare the lysis buffer sample, and boil at 100°C for 5 minutes. Remove and cool for 5 minutes.

[0115] 6. Load the sample and molecular weight marker into the gel. Refer to the manufacturer's recommended labeling amount.

[0116] 7. Connect the electrode to the power supply, turn on the power, and run the gel according to the manufacturer's recommended voltage and time, or until the dye tip is at the bottom of the gel.

[0117] 8. Turn off the power, remove the gel rack, remove the tray from the gel rack, and then pull the glass apart to hold the gel to one side. Trim off any excess gel, carefully remove the gel of interest, and place it in 1x transfer buffer until ready for placement in the transfer device.

[0118] 9. Cut the membrane to gel size and activate it according to the manufacturer's recommendations. Rinse with water and then place in 1x transfer buffer until transfer time.

[0119] 10. Prepare the transfer device according to the manufacturer's recommendations, ensuring the membrane and gel are added in the correct orientation. Add 1 times the transfer buffer.

[0120] 11. Connect the electrodes to the transfer device. Set the voltage and runtime to the manufacturer's recommended settings. Turn on the power.

[0121] 12. Turn off the power and remove the stain. Rinse the stain with water. Let it air dry overnight to allow the protein to bind to the membrane.

[0122] 13. Reactivate the membrane, rinse with water, seal the membrane in TTBS with 5% milk for 30 minutes at room temperature, and shake.

[0123] 14. Prepare primary antibody solution: Dilute the antibody in 1% milk in TTBS.

[0124] 15. Remove the blocking solution from the membrane and add the primary antibody solution (succinylated Keap1 antibody). Incubate the membrane overnight with shaking at 4°C.

[0125] 16. Remove the primary antibody solution, add enough washing buffer to cover the membrane while shaking, and wash the membrane three times, 5 minutes each time.

[0126] 17. Incubate the membrane in the secondary antibody solution (goat anti-rabbit HRP) and shake at room temperature for 1 hour.

[0127] 18. While shaking, add enough washing solution to cover the membrane, and wash the membrane three times, 5 minutes each time.

[0128] 19. Add ECL detection solution to the membrane and incubate the blot for the time recommended by the manufacturer. Remove the solution and develop the membrane with film or a developing film.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide for preparing succinylated KEAP1 polyclonal antibodies, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

2. A gene characterized in that, The gene can encode the polypeptide as described in claim 1.

3. The use of the polypeptide of claim 1 in the preparation of succinylated KEAP1 polyclonal antibody.

4. The application according to claim 3, characterized in that, The polypeptide was used to prepare succinylated KEAP1 polyclonal antibody after coupling.

5. A method for preparing the succinylated KEAP1 polyclonal antibody according to claim 3, characterized in that, The method involves immunizing animals with the polypeptide to induce the production of succinylated KEAP1 polyclonal antibodies, and then collecting the animal serum to obtain serum containing succinylated KEAP1 polyclonal antibodies.

6. The method for preparing a succinylated KEAP1 polyclonal antibody according to claim 5, characterized in that, The animal in question is a rabbit.

7. The method for preparing a succinylated KEAP1 polyclonal antibody according to claim 5, characterized in that, Immunization procedures for animals include basic immunization and three or more booster immunizations.

8. The method for preparing a succinylated KEAP1 polyclonal antibody according to claim 5, characterized in that, The polypeptide, after being conjugated with KLH, is used to immunize animals.

9. The method for preparing a succinylated KEAP1 polyclonal antibody according to claim 4, characterized in that, The method also includes extracting and purifying animal serum.

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