Use of a shrimp hemocyanin as a hapten carrier protein

Abstract

The application discloses application of shrimp hemocyanin in a carrier protein of a hapten, and belongs to the technical field of biological medicines. Compared with KLH commonly used in the prior art, the shrimp hemocyanin provided in the application has good solubility, the whole antigen obtained by coupling the hapten has good solubility and is not easy to precipitate and separate out, and the immunogenicity of the shrimp hemocyanin is better than that of KLH; the whole antigen prepared by taking the shrimp hemocyanin as a carrier protein has better animal immunization activity than the whole antigen prepared by taking KLH as a carrier protein. The shrimp hemocyanin provided in the application is extracted from blood of prawns, the prawn breeding technology is mature, the shrimp hemocyanin is easy to obtain, and the purchase cost is low, so that the preparation cost of the shrimp hemocyanin is far lower than the purchase cost of KLH.

Description

[0001] This application claims priority to Chinese patent application 2023115857774, filed on 2023 / 11 / 24. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to the application of shrimp hemocyanin as a hapten carrier protein. Background Technology

[0003] Haptens (also known as incomplete antigens) are small molecules that cannot induce an immune response when present alone. Their molecular weight is generally less than 3 kDa, such as polypeptides (containing approximately 15-20 amino acids). Typically, immunizing animals with a polypeptide covalently linked to a large carrier protein can produce an effective immune response, thus preparing the target antibody. Commonly used carrier proteins include natural proteins such as BSA (bovine serum albumin), OVA (egg white albumin), KLH (keyhole limpet hemocyanin), RSA (rabbit serum albumin), and recombinant proteins such as dodecin. Among these, BSA, OVA, and KLH are the three most commonly used in traditional research. Their common characteristics are strong immunogenicity, sufficient active sites on their protein surface, and easy covalent linkage with polypeptide molecules. However, immunizing animals with a polypeptide conjugated to a carrier protein not only produces antibodies against the target polypeptide but also antibodies against the carrier protein. BSA is frequently used in many other biochemical molecular experiments, such as ELISA, Western blot (protein immunohybridization), and cell culture. Therefore, due to its broad application, BSA is generally not used as a carrier protein for peptides. Compared to BSA, OVA does not have the above-mentioned disadvantages, but its immunogenicity is slightly worse, so it is often used as a hapten carrier for subsequent antibody validation rather than as an immunization carrier. KLH protein characteristics differ greatly from mammalian proteins, thus possessing extremely strong immunogenicity and is currently the most commonly used carrier protein. However, KLH has a very large and complex molecular weight, composed of protein subunits of 350kDa and 390kDa, resulting in poor water solubility and easy precipitation after being linked to peptides. On the other hand, the production cost of KLH is relatively high. KLH is extracted from the blood of the keyhole limpet, a mollusc, and collecting this animal from nature is costly. Currently, the company capable of large-scale cultivation of keyhole limpets is Steller Biotechnologies, located in California, USA. Its market price is much higher than that of BSA and OVA, with a domestic price of around 30 RMB / mg.

[0004] Hemocyanin, also known as hemocyanin, is a multifunctional protein called a respiratory protein. It is a copper-containing respiratory protein found in the hemolymph of arthropods and mollusks. It is a copper-containing blue-green high-molecular-weight protein found in the hemolymph of arthropods and mollusks. It is an oxygen carrier and the only known copper protein that can reversibly bind to oxygen.

[0005] Hemocyanin originates from arthropods and mollusks. Evolutionarily, arthropods and mollusks differ significantly from mammals; mammals use hemoglobin for respiration, while arthropods and mollusks use hemocyanin. Hemocyanin exists as a single hexamer (1×6) or multiples of hexamers (2×6, 4×6, 6×6, 8×6). In arthropods (such as shrimp), the hemocyanin subunit (approximately 72 kDa) folds into three domains characterized by distinct folding motifs: Domain I, with five or six α-helices; Domain II, with a four-α-helical bundle and two copper ion active sites; and Domain III, with seven antiparallel β-tubes. In contrast, the hemocyanin subunits of mollusks (such as keyhole limpets) have a molecular weight of approximately 350 or 400 kDa and are composed of seven or eight functional units (FUs). Each FU (Full Formation Intake) consists of two distinct domains, called α (from the α-helical domain) and β (from the β-sandwich domain); the α domain folds into a four-α-helix bundle carrying a copper active site, and the β domain forms a six-stranded antiparallel β-barrel. The α domains of mollusc hemocyanin FUs functionally correspond to domain II of arthropod hemocyanin, and the β domains correspond to domain III. Both arthropod and mollusc hemocyanins are proteins responsible for the binding, transport, and storage of oxygen, and possess several additional functions, including (but not limited to) enzymatic activity (i.e., phenol oxidase), hormone transport, homeostasis (molting), and hemostasis (clot formation). An important secondary function of hemocyanin involves innate immunity, such as serving as a precursor for broad-spectrum antimicrobial peptides and microbial / viral agglutination. Currently, no literature or patents report that shrimp hemocyanin can be used as a carrier protein of haptens for immunizing animals to obtain antibodies. Summary of the Invention

[0006] Based on the above background, this invention provides an application of shrimp hemocyanin as a hapten carrier. The shrimp hemocyanin is extracted from the blood of shrimp. Taking the prawn as an example, the prawn's circulatory system is an open-tubular system. The heartbeat causes blood to flow from the heart, along arteries and branch vessels to various organs and tissues. Blood from organs and tissues is concentrated in the thorax through interstitial spaces, then enters the gills for gas exchange, flows out of the gills, and finally returns to the heart to participate in the next cycle. The open-tubular circulation characteristic makes shrimp blood collection relatively easy; it can be easily drawn using a syringe. The inventors have found that approximately 20 mL of shrimp blood can be extracted from one pound of prawns, equivalent to 10 mL of serum, containing approximately 0.5 g of shrimp hemocyanin. Therefore, using shrimp hemocyanin as a hapten carrier protein will significantly reduce the preparation cost of hapten-carrier protein conjugates.

[0007] The inventors believe that while immunogenicity is a crucial factor in selecting a hapten carrier protein, its molecular weight, active groups, solubility, availability, and price are also essential considerations. Common arthropods, such as prawns and freshwater prawns, are staples in the Chinese market and a common food for ordinary people. Prawn farming technology is mature in China, and the price per kilogram in markets is around 30 RMB. Therefore, if haptens could be prepared using prawn hemocyanin, it would be more cost-effective than KLH.

[0008] This invention includes the following technical solutions:

[0009] In a first aspect, the present invention provides the application of shrimp hemocyanin as a hapten carrier protein.

[0010] The hapten is any hapten used in the art.

[0011] The shrimp hemocyanin is extracted from the serum of shrimp.

[0012] Furthermore, the shrimp is selected from one or more of the following: Penaeus vannamei and its subspecies, Macrobrachium rosenbergii and its subspecies, Procambarus clarkii and its subspecies, Eriocheirsinensis and its subspecies, Cherax quadricarinatus and its subspecies, Panulirus stimpsoni and its subspecies, and Homarus americanus and its subspecies.

[0013] Since shrimp farming technology is mature, raw materials are readily available, and purchase costs are low, in a specific embodiment of the present invention, the shrimp are selected from prawns.

[0014] In some embodiments, the shrimp hemocyanin is prepared by the following method:

[0015] The serum of the shrimp was separated and purified sequentially using molecular sieve chromatography and anion exchange chromatography, and then precipitated with ammonium sulfate solution and reconstituted to obtain the final product.

[0016] In some preferred embodiments, the shrimp serum is prepared by the following method: shrimp whole blood is left to stand on ice and the supernatant is collected.

[0017] In some implementations, the supernatant is also diluted with PBS.

[0018] In some implementations, filtration is also included after the dilution.

[0019] In some implementations, the filtration uses a 0.45 μm filter membrane.

[0020] In some embodiments, the ammonium sulfate solution is an ammonium sulfate solution with a saturation of 33-50%.

[0021] In some implementations, the reconstitution is performed using PBS.

[0022] In some specific implementations, the whole blood of the shrimp is extracted by the following method: using a syringe, the head, abdomen and tail of the shrimp are used as blood collection points, and the syringe needle is inserted at a 45-degree angle to a depth of about 1 mm to extract the shrimp blood.

[0023] In some specific implementations, the shrimp is selected from prawns.

[0024] In some specific implementation plans, the blood collection point for shrimp is the abdomen.

[0025] In some specific implementation schemes, the blood collection point for shrimp is along the edge of the cephalothorax and abdomen, at the blood sinuses at the base of the third and fourth walking legs.

[0026] In some implementations, the anion exchange chromatography column is a DEAE column, and the sample collection point is where the conductivity is between 15-25 mS / cm.

[0027] In other embodiments, the shrimp hemocyanin is prepared by the following method:

[0028] (1) Extract whole blood from shrimp;

[0029] (2) Place the whole blood sample on ice and let it stand. Take the supernatant, dilute it with PBS, and filter it through a 0.45 μm filter membrane.

[0030] (3) The filtered sample was separated and purified by molecular sieve chromatography and anion exchange chromatography in sequence;

[0031] (4) After purification, the sample was precipitated with ammonium sulfate solution with a saturation of 33-50%, and then reconstituted with PBS to obtain shrimp hemocyanin.

[0032] Preferably, the method for extracting whole blood from shrimp in step (1) is as follows: using a syringe, the head, abdomen and tail of the shrimp are used as blood collection points respectively. The syringe needle is inserted at a 45-degree angle to a depth of about 1 mm to extract 100-400 μL of shrimp blood.

[0033] In the most preferred embodiment of the present invention, the blood collection point of the shrimp is the abdomen, specifically along the edge of the cephalothorax and abdomen, at the blood sinuses at the base of the third and fourth walking legs.

[0034] Preferably, the molecular sieve chromatography column in step (3) is a Sepharose column, and the equilibration buffer and elution buffer are 1X PBS; the sample collection point is: when UV280>500, collect 1-3 peaks.

[0035] In the most preferred embodiment of the present invention, the sample collection point for molecular sieve chromatography is: when UV280 to 500, one peak is collected.

[0036] Preferably, in step (3), the anion exchange chromatography column is a DEAE column, and the sample collection point is where the conductivity is between 15-25 mS / cm.

[0037] In a specific embodiment of the present invention, the equilibration buffer of the anion exchange chromatography column is selected from Tris-HCl with a concentration of 10-20 mM or Hepes with a concentration of 30-50 mM.

[0038] In some embodiments, the elution buffer is selected from a combination of Tris-HCl with a final concentration of 10-20 mM and NaCl with a final concentration of 0.5-1 M; or a combination of Hepes with a final concentration of 30-50 mM and NaCl with a final concentration of 0.5-1 M.

[0039] In a specific embodiment of the present invention, the equilibration buffer for the anion exchange chromatography is 20 mM Tris-HCl, and the elution buffer is a combination of Tris-HCl and NaCl, wherein the concentration of Tris-HCl is 20 mM and the concentration of NaCl is 1 M.

[0040] Preferably, the ammonium sulfate solution used in step (4) is saturated with 33%, 40%, 45%, or 50%.

[0041] In the most preferred embodiment of the present invention, the ammonium sulfate solution has a saturation of 50%.

[0042] In a second aspect, the present invention provides a method for activating shrimp hemocyanin, the method comprising: activating shrimp hemocyanin using an activation reagent; wherein the shrimp hemocyanin is as described in the first aspect.

[0043] In some embodiments, the activating agent is selected from SMCC, EDC, glutaraldehyde, formaldehyde, sulfo-NHS, DSP, DTSSP, DSC, DMA, DMP, DDPPB, 1,4-Butanediol Diglycidyl ether, Diazotized, SPDP, MBS, SIAC, ABH, and ASBA.

[0044] In some embodiments, the mass-to-volume ratio of the activating agent to the shrimp hemocyanin is 2:1.

[0045] In some embodiments, the method further includes a step of desalting and purifying the activated shrimp hemocyanin.

[0046] In some preferred embodiments, the desalting purification includes desalting the shrimp hemocyanin by column chromatography using a balancing buffer and an elution buffer selected from ultrapure water, PBS, and MES.

[0047] Thirdly, the present invention provides an activated shrimp hemocyanin prepared by the method described in the second aspect.

[0048] Fourthly, the present invention provides a method for preparing a hapten-shrimp hemocyanin conjugate using shrimp hemocyanin as a carrier, the method comprising the following steps:

[0049] (1) Activate shrimp hemocyanin using an activating reagent;

[0050] (2) The activated shrimp hemocyanin was desalted and purified;

[0051] (3) The activated and desalted shrimp hemocyanin was coupled with the hapten to obtain the hapten-shrimp hemocyanin conjugate.

[0052] The hapten is any hapten used in the art.

[0053] Preferably, the activating agent mentioned in step (1) is a coupling agent with activation function commonly used in the art, including but not limited to SMCC and NHS. In a specific embodiment of the present invention, the activating agent used is SMCC.

[0054] Preferably, the desalting purification method in step (2) is selected from one or a combination of two of desalting column elution and dialysis desalting. In a preferred embodiment of the present invention, the desalting purification uses a desalting column, and the equilibration buffer and elution solution are ultrapure water.

[0055] In some implementations, the activation is as described in the second aspect.

[0056] In some implementations, in step (3), the mass-to-volume ratio of shrimp hemocyanin to hapten is 1:1 during the coupling.

[0057] Fifthly, the present invention provides a hapten-shrimp hemocyanin conjugate prepared according to the method described above.

[0058] The application of shrimp hemocyanin as a hapten carrier protein provided by this invention has the following advantages:

[0059] 1. Compared with the commonly used KLH in the prior art, the shrimp hemocyanin prepared by the present invention has good solubility, and the whole antigen obtained by coupling with the hapten has good solubility and is not easy to precipitate.

[0060] 2. The immunogenicity of the shrimp hemocyanin prepared by this invention is superior to that of KLH;

[0061] 3. The full antigen prepared using shrimp hemocyanin as a carrier protein has better animal immune activity than the full antigen prepared using KLH as a carrier protein.

[0062] 4. The shrimp hemocyanin provided by this invention is extracted from shrimp blood. Shrimp farming technology is mature, readily available, and has low purchase cost, making the preparation cost of shrimp hemocyanin much lower than the purchase cost of KLH. Attached Figure Description

[0063] Figure 1 : Schematic diagram of the process of preparing hapten conjugates from shrimp hemocyanin.

[0064] Figure 2 SDS-PAGE electrophoresis image of the hapten-shrimp hemocyanin conjugate.

[0065] Figure 3 : Serum titer results of hapten-shrimp hemocyanin conjugate.

[0066] Figure 4 Figure: Comparison of immunogenicity between shrimp hemocyanin and KLH.

[0067] Figure 5 Figure: Comparison of the immunogenicity of antigens prepared from shrimp hemocyanin and KLH. Detailed Implementation

[0068] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0069] The process for preparing the shrimp hemocyanin hapten conjugate in this embodiment is as follows: Figure 1 As shown.

[0070] Extraction of shrimp hemocyanin

[0071] S1: Extraction of shrimp blood

[0072] The shrimp to be blooded are selected from prawns. Hold the shrimp with its abdomen facing up in your left hand, and hold the syringe in your right hand. Insert the syringe along the edge of the carapace and abdomen into the blood sinus at the base of the third and fourth walking legs. Insert the syringe needle into the shrimp abdomen at a 45-degree angle to a depth of about 1 mm. Slowly pull the syringe back to extract 400 μL of shrimp blood. There are about 50 shrimp in 1 kg of prawns, and a total of 20 mL of whole shrimp blood can be extracted from 1 kg of shrimp.

[0073] S2: Shrimp blood pretreatment

[0074] Place the shrimp blood on ice and let it stand for 2 hours. Take the supernatant to obtain 10 mL of serum. Dilute it 10 times with 1×PBS (pH 7.2-7.4) and filter it once through a 0.45 μm filter membrane.

[0075] S3: Isolation and purification of shrimp hemocyanin

[0076] A: Molecular sieve chromatography

[0077] The chromatography column was a Sepharose-G25 column. The equilibration buffer was 1×PBS. At least two column volumes were equilibrated first. The sample was loaded at a low flow rate. The elution buffer was 1×PBS. The flow rate was 10 mL / min throughout the entire process, including the sample loading. 10 mL of serum was loaded. A peak was collected when the UV280 was 500.

[0078] B: Anion exchange chromatography

[0079] The chromatography column was DEAE sephadex A-25, the flow rate was 2 mL / min, the equilibration buffer was 20 mM Tris-HCl pH 8.0, the elution buffer was 20 mM Tris-HCl + 1 M NaCl pH 8.0, and linear elution was performed (the elution buffer was increased linearly from 0% to 100% within 30 min). When the conductivity was 15-25 mS / cm, the sample was collected and the collected sample was run on an SDS-PAGE gel and stained with Coomassie Brilliant Blue.

[0080] S4: After purification, the sample was precipitated by adding a 50% saturated ammonium sulfate solution and then reconstituted using 1×PBS.

[0081] During long-term experiments, the inventors discovered the following phenomena: 1. Different blood collection sites from shrimp not only significantly affect the amount of blood collected, but also the quality and purity of the final extracted and purified shrimp hemocyanin. The optimal shrimp blood extraction site is the blood sinus at the base of the third and fourth pereiopods along the edge of the cephalothorax and abdomen; 2. When using DEAEsephadexA-25 for anion exchange chromatography separation and purification, different equilibration and elution systems also affect the quality and purity of the final obtained shrimp hemocyanin. The optimal equilibration system is 20mM Tris-HCl, and the optimal elution system is 20mM Tris-HCl + 1M NaCl.

[0082] Preparation of hapten-shrimp hemocyanin conjugate (SMCC method)

[0083] S1: Reagent Preparation

[0084] The purified shrimp hemocyanin was quantified by cosmography. A 10 mg / mL shrimp hemocyanin stock solution was prepared with pure water. SMCC (succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate) was prepared with 1×PBS (pH 7.2-7.4) to a stock solution concentration of 2 mg / mL. The concentration was determined using Nanodrop.

[0085] S2: Shrimp hemocyanin activation

[0086] Add SMCC to shrimp hemocyanin at a concentration of 1 mg / ml to a final concentration of 2 mg / ml, invert at room temperature for 1 hour, centrifuge and collect the supernatant;

[0087] S3: Remove coupling agent

[0088] SMCC was removed using a desalting column (NW75, column volume 20 ml, maximum loading of 20% of column volume), and ultrapure water was used as the equilibration buffer and elution buffer. Protein peak samples were collected, SDS-PAGE gels were run, and Coomassie Brilliant Blue staining was used for quantification.

[0089] S4: Conjugated polypeptide

[0090] Take activated and desalted shrimp hemocyanin (1 mg / mL), add 1 mg / mL of peptide (hapten, diluted with PBS to a stock solution of 10 mg / mL), invert and couple at room temperature for 1 hour, then dialyze overnight to remove excess salt, obtaining the hapten-shrimp hemocyanin conjugate. Add 50% glycerol (using 5× loading) and 0.1 M DTT (using 10× loading) to run gel electrophoresis, and photograph fluorescence.

[0091] Preparation of hapten-shrimp hemocyanin conjugate (EDC method)

[0092] S1: Reagent Preparation

[0093] The purified shrimp hemocyanin was quantified by cosmography. A 20 mg / mL shrimp hemocyanin stock solution was prepared with pure water. EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) was prepared with 1×PBS (pH 7.2) or MES (pH 4.0) with a stock solution concentration of 10 mg / mL. The concentration was determined using Nanodrop.

[0094] S2: Shrimp hemocyanin activation

[0095] Add EDC to a final concentration of 2 mg / ml in shrimp hemocyanin at a concentration of 1 mg / ml, invert at room temperature for 1 h (activate for 30-60 min when using MES solution with EDC), centrifuge and collect the supernatant.

[0096] S3: Remove coupling agent

[0097] EDC was removed using a desalting column (NW75, 20 ml column volume, maximum loading of 20% of column volume), with equilibration buffer and elution buffer of PBS or MES, at a flow rate of 2 mL / min and a pressure less than 0.5 MPa. 1 ml of sample was loaded onto a sample loop, and protein peak samples were collected based on the UV280 elution position. The samples were then run on an SDS-PAGE gel and quantified using Coomassie Brilliant Blue staining.

[0098] S4: Conjugated polypeptide

[0099] Take activated and desalted shrimp hemocyanin (1 mg / mL), add 1 mg / mL of peptide (hapten, diluted to 5 mg / mL stock solution using PBS pH=7.2 or MES pH=4.0), invert and couple at room temperature for 1 h, then dialyze overnight to remove excess salt, obtaining the hapten-shrimp hemocyanin conjugate. Add 50% glycerol (for 5× loading) and 0.1 M DTT (for 10× loading) to run gel electrophoresis, and photograph fluorescence.

[0100] Specifically, in the hapten-shrimp hemocyanin conjugate prepared by the SMCC method described above in this embodiment of the invention, the hapten consists of: 126-A-3, 129-P-3, 130-L-3, 131-Z-3, 132-M-3, 161-C-3, 162-H-3, 163-T-3, 164-C-3, 165-T-3, 116-G-3, PDI, Cytokeratin 10, Desmin, α-Tubulin, and NF-κB. The amino acid sequence of the hapten is shown in the table below.

[0101] Table 1 shows the amino acid sequences of the haptens used in the examples.

[0102]

[0103]

[0104] The prepared hapten-shrimp hemocyanin conjugates were analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The results showed that all the above haptens could be conjugated with shrimp hemocyanin to obtain hapten-shrimp hemocyanin conjugates. Due to the large number of haptens, this invention provides SDS-PAGE electrophoresis images of some conjugates. The results are as follows... Figure 2 As shown, lane 1 is the corresponding hapten, and lane 2 is the hapten-shrimp hemocyanin conjugate. The electrophoresis results show that haptens 161-C-3, 162-H-3, 126-A-3, 130-L-3, and 132-M-3 can all be successfully conjugated with shrimp hemocyanin by the SMCC method to obtain conjugates.

[0105] Animal immunological activity assay of hapten-shrimp hemocyanin conjugate

[0106] The hapten-shrimp hemocyanin conjugates to be tested are: α-Tubulin-shrimp hemocyanin, Cytokeratin 10-shrimp hemocyanin, Desmin-shrimp hemocyanin, and NF-κB-shrimp hemocyanin.

[0107] I. Immunizing Rabbits

[0108] First immunization: Collect negative serum as a blank control, mix 500 μg of each of the above conjugates with an equal volume of Freund's complete adjuvant, and inject subcutaneously at multiple points on the dorsal side;

[0109] Secondary immunization: Mix 500 μg of each of the above conjugates with an equal volume of Freund's incomplete adjuvant and inject subcutaneously at multiple points on the dorsal side;

[0110] Enhanced immunity: 1 mg of each of the above conjugates were injected subcutaneously at multiple points on the dorsal side for shock immunization. The rabbits were euthanized three days later and the serum was collected.

[0111] II. Serum titer testing

[0112] Rabbits were immunized with conjugates prepared using α-Tubulin, Cytokeratin 10, Desmin, and NF-κB as haptens. The corresponding rabbit serum was then analyzed using Western blotting. The antigen samples used for testing were cell lines (including HEK293T, HeLa, HepG2, SCC-9, RD, and NCI-H2347). The specific method was as follows: The protein loading amount was determined, and an equal volume of 2× diluted Laemmli sample buffer was added. During sample reduction and denaturation, the cell lysis buffer in the sample buffer was boiled at 100°C for 5 minutes. The lysis buffer could be aliquoted and stored at -20°C for later use. Equal amounts of protein and molecular weight markers were loaded into the wells of an SDS-PAGE gel. The total protein loading amount from cell lysis buffer or tissue homogenate was 20-30 μg, and the purified protein loading amount was 10-100 ng. The gel was run at 100V for 1-2 hours. Proteins were transferred from the gel to a membrane, which could be nitrocellulose. The membrane was blocked with blocking buffer for 1 hour at room temperature or overnight at 4°C. Rabbit serum was diluted 1:5000 and incubated overnight at 4°C. The membrane was washed three times with TBST for 5 minutes each time, and then incubated with 1:5000 HRP-conjugated anti-rabbit secondary antibody at room temperature for 1 hour. The membrane was washed three times with TBST for 5 minutes each time. Excess reagents were removed, and the membrane was covered with a transparent plastic film. Colorimetric images were acquired using standard image scanning methods. Results are as follows: Figure 3 As shown in the results, the target bands could be detected in rabbit serum immunized with the above four hapten-shrimp hemocyanin conjugates in the corresponding cell lines, indicating that the whole antigen prepared by shrimp hemocyanin as a carrier can effectively induce an immune response in animals.

[0113] Comparison of solubility of shrimp hemocyanin and KLH

[0114] The molecular weight and solubility of shrimp hemocyanin and KLH prepared in the embodiments of the present invention were determined respectively. The solubility of SMCC-activated shrimp hemocyanin and KLH was further determined, wherein the SMCC activation method is as shown in the specific embodiments of the present invention. α-Tubulin was used as a hapten to conjugate α-Tubulin-shrimp hemocyanin and α-Tubulin-KLH, and the solubility of the hapten conjugates was determined. The results are shown in the table below:

[0115] Table 2 Comparison of the solubility of shrimp hemocyanin and KLH.

[0116] molecular weight solubility Shrimp hemocyanin 70kDa 300mg / mL KLH 360kDa 200mg / mL SMCC-activated shrimp hemocyanin Not determined 100mg / mL SMCC-activated KLH Not determined 50mg / mL α-Tubulin-shrimp hemocyanin Not determined 50mg / mL α-Tubulin-KLH Not determined 25mg / mL

[0117] As can be seen from the results in the table above, the solubility of shrimp hemocyanin prepared by the method provided in this invention is 300 mg / mL, which is significantly higher than that of KLH (200 mg / mL). Furthermore, the solubility of shrimp hemocyanin activated with SMCC is also significantly higher than that of KLH activated with SMCC. Hapten-carrier conjugates were prepared using shrimp hemocyanin and KLH prepared in this invention as carriers, respectively. Taking α-Tubulin as an example, those skilled in the art found that the solubility of the whole antigen prepared using shrimp hemocyanin as a carrier is significantly higher than that of the whole antigen prepared using KLH as a carrier.

[0118] Comparison of the immunogenicity of shrimp hemocyanin and KLH

[0119] Experimental objective: To compare the immunogenicity of shrimp hemocyanin prepared in this invention with that of KLH commonly used in existing technologies.

[0120] Experimental Methods: Mice were immunized in two groups of three, one with shrimp hemocyanin and the other with KLH. The immunization dose was 10 μg of shrimp hemocyanin or 10 μg of KLH. After emulsifying the shrimp hemocyanin or KLH, the mice were injected intraperitoneally. A second immunization was performed on day 9 after the first immunization. Blood was collected from the tail of the mice on the first day of immunization, and then every 3 days thereafter for subsequent ELISA detection.

[0121] Experimental results: The results are as follows Figure 4 As shown, 18 days after immunization, both shrimp hemocyanin and KLH stimulated mice to produce antibodies against their respective antigens, with no significant difference in the immune responses. However, the immune response induced by shrimp hemocyanin was more stable, while the immune response induced by KLH increased and then decreased between days 18 and 27 after immunization, exhibiting poor stability. Technicians believe this result is because KLH is more prone to precipitation in mice than shrimp hemocyanin, reducing the uptake and presentation of antigens by immune cells.

[0122] Effects of shrimp hemocyanin and KLH as hapten carrier proteins on the immunogenicity of whole antigens

[0123] Experimental objective: To select PDI as a hapten polypeptide, control the hapten to remain unchanged, and conjugate it with shrimp hemocyanin, KLH, and polylysine to prepare conjugates as whole antigens, and to detect the animal immune activity of the three whole antigens.

[0124] Experimental Methods: Following the method described in this embodiment, the PDI peptide was ligated and purified with the three carrier proteins mentioned above, and then used to immunize rabbits. The immunization method was the same as described previously. Western blot analysis was performed on the collected serum. The Western blot analysis used real cell line antigens HEK293T, HeLa, and HepG2. The three antigens were loaded with equal cell numbers (10,000 cells per well). Serum was diluted 1:5000, and the secondary antibody anti-Rabbit IgG was diluted 1:10000. Images were acquired on all three membranes with the same exposure time of 10 seconds. The specific detection method was the same as described previously.

[0125] Experimental results: Western blot detection results are as follows Figure 5 As shown, both pDI peptides conjugated to shrimp hemocyanin and KLH can induce an immune response in rabbits, stimulating the production of antibodies against the PDI antigen at the cell border. However, pDI peptides conjugated to poly-L-lysine as a carrier cannot elicit a corresponding immune response. Furthermore, comparing the pDI-pDI and KLH-pDI profiles, it can be seen that immunizing rabbits with pDI-pDI as the antigen yields antibodies with higher purity and fewer non-specific bands. This indicates that compared to KLH, pDI's sequence and structure are more evolutionarily distant from mammals, making it more suitable as a carrier protein for haptens.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0127] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. The application of a shrimp hemocyanin as a hapten carrier protein; in, The shrimp hemocyanin was prepared by the following method: (1) Extracting whole blood from shrimp: The whole blood extraction method is as follows: Using a syringe, along the edge of the cephalothorax and abdomen, the blood sinus at the base of the third and fourth walking legs is the blood collection point. Insert the syringe needle at a 45-degree angle to a depth of about 1 mm and extract the shrimp blood. (2) Place the whole blood sample on ice and let it stand. Take the supernatant, dilute it with PBS, and filter it through a 0.45 μm filter membrane. (3) The filtered sample was separated and purified by molecular sieve chromatography and anion exchange chromatography in sequence. The molecular sieve chromatography column was a Sepharose-G25 column, and the equilibration buffer and elution buffer were 1×PBS. The sample loading volume was less than 10% of the column volume. The sample collection point was: when the UV value was 280 to 500, one peak was collected. The anion exchange chromatography column was a DEAE sephadexA-25 column, and linear gradient elution was used. The sample collection point was when the conductivity was between 15-25 mS / cm. The equilibration buffer for the anion exchange chromatography was 20 mM Tris-HCl with a pH of 8.

0. The elution buffer was a combination of Tris-HCl and NaCl with a pH of 8.

0. The concentration of Tris-HCl was 20 mM and the concentration of NaCl was 1 M. (4) After purification, the sample was precipitated with a 50% saturated ammonium sulfate solution and then reconstituted with PBS to obtain the shrimp hemocyanin. The shrimp in question is the prawn (Penaeus vannamei) or its subspecies.

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