Use of shrimp hemocyanin as hapten carrier protein

Abstract

Provided is a use of shrimp hemocyanin as a hapten carrier protein, belonging to the technical field of biomedicine. Compared with KLH commonly used in the prior art, the provided shrimp hemocyanin has good solubility, a complete antigen obtained by means of coupling with a hapten has good solubility, and precipitation is not prone to occurring; in addition, the shrimp hemocyanin shows superior immunogenicity to KLH, and a complete antigen prepared by using the shrimp hemocyanin as a carrier protein has better animal immunogenic activity than a complete antigen prepared by using KLH as a carrier protein. The provided shrimp hemocyanin is extracted from prawn blood, the prawn farming technology is mature and readily available, 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

Application of shrimp hemocyanin as hapten carrier protein

[0001] This application claims the benefit of Chinese Patent Application No. 2023115857774 filed on November 24, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The invention belongs to the technical field of biomedicine, and particularly relates to an application of shrimp hemocyanin as a hapten carrier protein. Background Art

[0003] Haptens (also known as incomplete antigens) are small molecules that, when present alone, cannot induce an immune response. 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 larger carrier protein can produce an effective immune response, thereby generating the desired antibody. Commonly used carrier proteins include natural proteins such as BSA (bovine serum albumin), OVA (egg white albumin), KLH (keyhole limpet hemocyanin), and RSA (rabbit serum albumin), as well as recombinant proteins such as dodecin. BSA, OVA, and KLH are the three most commonly used in traditional research. They share a common characteristic: strong immunogenicity and abundant surface active sites, making them easy to covalently bond with polypeptide molecules. However, when peptides are conjugated to carrier proteins and immunized, animals not only produce antibodies against the target polypeptide but also against the carrier protein. BSA is commonly used in many other biochemical molecular experiments, such as ELISA, Western blot, and cell culture. Therefore, due to its broad application, BSA is generally not used as a peptide carrier protein. Compared to BSA, OVA does not have these drawbacks, but its immunogenicity is slightly lower. Therefore, it is often used as a hapten carrier for subsequent antibody validation, rather than as a carrier for immunization. KLH, whose protein properties differ significantly from those of mammalian proteins, possesses extremely strong immunogenicity and is currently the most commonly used carrier protein. However, KLH is extremely large and complex, consisting of 350 kDa and 390 kDa protein subunits. It has poor water solubility and easily precipitates after being linked to peptides. Furthermore, KLH production is relatively expensive. KLH is extracted from the blood of keyhole limpets, a mollusk that is expensive to collect from nature. Currently, the company capable of large-scale cultivation of keyhole limpets is Steller Biotechnologies in California, USA. Its market price is significantly higher than that of BSA and OVA, with a domestic price of around 30 RMB per mg. At the same time, there are few companies in China that can independently produce KLH, and KLH is largely dependent on imports. Therefore, achieving the localization of KLH or finding an excellent alternative will undoubtedly accelerate the development of China's domestic biopharmaceuticals.

[0004] Hemocyanin, also known as hemocyanin, is a multifunctional protein known as a respiratory protein. It is a copper-containing respiratory protein located 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 distinct from mammals. While mammals use hemoglobin for respiration, arthropods and mollusks use hemocyanin. Hemocyanin exists as a single hexamer (1×6-mer) or multiples of hexamers (2×6-mer, 4×6-mer, 6×6-mer, and 8×6-mer). The hemocyanin subunit (approximately 72 kDa) of arthropods (such as shrimp) 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 an active site containing two copper ions; 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 (FU). Each FU is composed of two distinct domains, designated α (derived from the α-helical domain) and β (derived from the β-sandwich domain); the α domain folds into a four-alpha-helical bundle carrying a copper active site, while the β domain folds into a six-stranded antiparallel β barrel. The α domain of molluscan hemocyanin FU functionally corresponds to domain II of arthropod hemocyanins, and the β domain corresponds to domain III, respectively. Arthropod and molluscan hemocyanins are proteins responsible for the binding, transport, and storage of dioxygen, and both possess several additional functions, including (but not limited to) enzymatic activity (i.e., phenoloxidase), hormone transport, homeostasis (molting), and hemostasis (clot formation). An important secondary function of hemocyanin involves innate immunity, for example, as a precursor of broad-spectrum antimicrobial peptides and microbial / viral agglutination. Currently, there are no literature or patents reporting the use of shrimp hemocyanin as a carrier protein for haptens for animal immunization and antibody production.

[0006] Summary of the Invention

[0007] Based on the above background, the present invention provides the use of shrimp hemocyanin as a hapten carrier. The shrimp hemocyanin is extracted from shrimp blood. For example, shrimp have an open circulatory system. The beating heart causes blood to flow from the heart, flowing along arteries and branching blood vessels to various organs and tissues. Blood from the organs and tissues collects in the chest through the 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 circulation. The characteristics of the open circulation make shrimp blood collection relatively easy, and shrimp blood can be easily drawn with a syringe. The present inventors have found that approximately 20 mL of shrimp blood can be extracted from one kilogram of shrimp, 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.

[0008] The present inventors believe that while the immunogenicity of the carrier protein is a crucial factor when selecting a hapten carrier protein, the molecular weight, active groups, solubility, availability, and price are also crucial considerations. Common arthropods, such as shrimp and Macrobrachium, are common agricultural products in the Chinese market and a staple on the dining tables of ordinary people. Shrimp farming technology is mature in China, and the price per kilogram at the market is around 30 RMB. Therefore, if haptens could be prepared using shrimp hemocyanin, it would undoubtedly be more cost-effective than KLH.

[0009] The present invention includes the following technical solutions:

[0010] In a first aspect, the present invention provides a use of shrimp hemocyanin as a hapten carrier protein.

[0011] The hapten is any hapten used in the art.

[0012] The shrimp hemocyanin is extracted from the serum of shrimp.

[0013] Furthermore, the shrimp is selected from one or a combination of two or more of the following: penaeus vannamei and its subspecies, macrobrachium rosenbergii and its subspecies, crayfish (Procambarus clarkii) and its subspecies, hairy crab (Eriocheir sinensis) and its subspecies, Australian lobster (Cherax quadricarinatus) and its subspecies, green lobster (Panulirus stimpsoni) and its subspecies, Boston lobster (Homarus americanus) and its subspecies.

[0014] Since the breeding technology of prawns is mature, the raw materials are easily available and the purchase cost is low, in a specific embodiment of the present invention, the prawns are selected from prawns.

[0015] In some embodiments, the shrimp hemocyanin is prepared by the following method:

[0016] The shrimp serum is separated and purified by molecular sieve chromatography and anion exchange chromatography in sequence, and then precipitated with ammonium sulfate solution and redissolved to obtain the product.

[0017] In some preferred embodiments, the shrimp serum is prepared by the following method: the shrimp whole blood is placed on ice and the supernatant is collected.

[0018] In some embodiments, the method further comprises diluting the supernatant with PBS.

[0019] In some embodiments, filtering is further included after the dilution.

[0020] In some embodiments, the filtration uses a 0.45 μm filter membrane.

[0021] In some embodiments, the ammonium sulfate solution is an ammonium sulfate solution with a saturation of 33-50%.

[0022] In some embodiments, the reconstitution is with PBS.

[0023] In some specific embodiments, the whole shrimp blood is extracted by the following method: using a syringe, the shrimp head, abdomen, and tail are used as blood sampling points, and the syringe needle is inserted at a 45-degree angle to a depth of about 1 mm to extract the shrimp blood.

[0024] In some embodiments, the shrimp is selected from shrimp.

[0025] In some embodiments, the bleeding point of the shrimp is the abdomen.

[0026] In some specific embodiments, the bleeding points of the shrimp are along the edge of the cephalothorax and abdomen, and at the blood sinuses at the base of the third and fourth walking legs.

[0027] In some embodiments, the chromatography column for anion exchange chromatography is a DEAE column, and the sample collection point has a conductivity between 15-25 mS / cm.

[0028] In other embodiments, the shrimp hemocyanin is prepared by the following method:

[0029] (1) Extracting shrimp whole blood;

[0030] (2) Place the whole blood sample on ice, take the supernatant, dilute with PBS, and filter using a 0.45 μm filter membrane;

[0031] (3) Separating and purifying the filtered sample using molecular sieve chromatography and anion exchange chromatography in sequence;

[0032] (4) The purified sample was precipitated with 33-50% saturation ammonium sulfate solution and then redissolved with PBS to obtain shrimp hemocyanin.

[0033] Preferably, the method for extracting whole shrimp blood in step (1) is as follows: using a syringe, the head, abdomen, and tail of the shrimp are used as blood sampling points, the syringe needle is inserted at a 45-degree angle to a depth of about 1 mm, and 100-400 μL of shrimp blood is extracted.

[0034] In the most preferred embodiment of the present invention, the bleeding point of the shrimp is the abdomen, specifically along the edge of the cephalothorax and the abdomen, and at the blood sinus at the base of the third and fourth walking legs.

[0035] Preferably, the molecular sieve chromatography column in step (3) is a Sepharose column, the equilibration buffer and the elution buffer are 1X PBS; and the sample collection point is: when UV280>500, 1-3 peaks are collected.

[0036] In the most preferred embodiment of the present invention, the sample collection point for molecular sieve chromatography is: when UV is between 280 and 500, one peak is collected.

[0037] Preferably, the chromatography column for anion exchange chromatography in step (3) is a DEAE column, and the sample collection point has a conductivity between 15-25 mS / cm.

[0038] 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.

[0039] 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.

[0040] In a specific embodiment of the present invention, the equilibration buffer of 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.

[0041] Preferably, the saturation of the ammonium sulfate solution used in step (4) is 33%, 40%, 45% or 50%.

[0042] In the most preferred embodiment of the present invention, the saturation of the ammonium sulfate solution is 50%.

[0043] 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.

[0044] In some embodiments, the activating reagent is selected from the group consisting of SMCC, EDC, glutaraldehyde, formaldehyde, sulfo-NHS, DSP, DTSSP, DSC, DMA, DMP, DDPPB, 1,4-Butanediol Diglycidyl ether, Diazotized, SPDP, MBS, SIAC, ABH, and ASBA.

[0045] In some embodiments, the mass-to-volume ratio of the activation reagent to the shrimp hemocyanin is 2:1.

[0046] In some embodiments, the method further comprises the step of desalting and purifying the activated shrimp hemocyanin.

[0047] In some preferred embodiments, the desalting purification comprises desalting the shrimp hemocyanin by column using an equilibration buffer and an eluent, wherein the equilibration buffer and the eluent are selected from ultrapure water, PBS and MES.

[0048] In a third aspect, the present invention provides an activated shrimp hemocyanin prepared by the method described in the second aspect.

[0049] In a fourth aspect, 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:

[0050] (1) activating shrimp hemocyanin using an activation reagent;

[0051] (2) desalting and purifying the activated shrimp hemocyanin;

[0052] (3) coupling the activated and desalted shrimp hemocyanin with a hapten to obtain a hapten-shrimp hemocyanin conjugate.

[0053] The hapten is any hapten used in the art.

[0054] Preferably, the activation reagent 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 activation reagent used is SMCC.

[0055] Preferably, the desalting and purification method in step (2) is selected from one or a combination of desalting column elution desalting and dialysis desalting. In a preferred embodiment of the present invention, the desalting and purification uses a desalting column for desalting, and the equilibration buffer and eluent are ultrapure water.

[0056] In some embodiments, the activation is as described in the second aspect.

[0057] In some embodiments, in step (3), in the coupling, the mass volume ratio of shrimp hemocyanin to hapten is 1:1.

[0058] In a fifth aspect, the present invention provides a hapten-shrimp hemocyanin conjugate prepared according to the method described above.

[0059] The use of shrimp hemocyanin as a hapten carrier protein provided by the present invention has the following advantages:

[0060] 1. Compared with KLH commonly used 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;

[0061] 2. The immunogenicity of the shrimp hemocyanin prepared by the present invention is better than that of KLH;

[0062] 3. The whole antigen prepared with shrimp hemocyanin as the carrier protein has better animal immune activity than the whole antigen prepared with KLH as the carrier protein;

[0063] 4. The shrimp hemocyanin provided by the present invention is extracted from shrimp blood. Shrimp breeding technology is mature, easy to obtain, and has low purchase cost, making the preparation cost of shrimp hemocyanin much lower than the purchase cost of KLH. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1: Schematic diagram of the process of preparing hapten conjugates from shrimp hemocyanin.

[0065] Figure 2: SDS-PAGE electrophoresis of hapten-shrimp hemocyanin conjugate.

[0066] Figure 3: Serum titer test results of hapten-shrimp hemocyanin conjugate.

[0067] Figure 4: Comparison of the immunogenicity of shrimp hemocyanin and KLH.

[0068] Figure 5: Comparison of the immunoreactivity of antigens prepared with shrimp hemocyanin and KLH. DETAILED DESCRIPTION

[0069] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0070] The process of preparing the shrimp hemocyanin hapten conjugate in this example is shown in FIG1 .

[0071] Extraction of shrimp hemocyanin

[0072] S1: Extraction of shrimp blood

[0073] Select the shrimp to be blooded. Hold the shrimp in your left hand with its abdomen facing up. Hold the syringe in your right hand and insert it along the edge of the cephalothorax and abdomen into the blood sinus at the base of the third and fourth ambulacral legs. Insert the syringe needle at a 45-degree angle to the shrimp abdomen to a depth of about 1 mm. Slowly pull back the syringe to extract 400 μL of shrimp blood. There are about 50 shrimps in 1 kg, so a total of 20 mL of shrimp whole blood can be extracted from 1 kg of shrimp.

[0074] S2: Shrimp blood pretreatment

[0075] Place the shrimp blood on ice and let it stand for 2 hours. Take the upper liquid to obtain 10 mL of serum, add 1× PBS (pH 7.2-7.4) to dilute it 10 times, and filter it once using a 0.45 μm filter membrane;

[0076] S3: Isolation and purification of shrimp hemocyanin

[0077] A: Molecular sieve chromatography

[0078] The chromatography column was a Sepharose-G25 column, and the equilibration buffer was 1× PBS. At least 2 column volumes were equilibrated first, and the sample was loaded at a low flow rate. The elution buffer was 1× PBS, and the flow rate was 10 mL / min throughout the entire process including the sample loading. 10 mL of serum was loaded, and a peak was collected when UV280 was 500.

[0079] B: Anion exchange chromatography

[0080] The chromatography column was DEAE sephadex A-25, with a flow rate of 2 mL / min. The equilibration buffer used was 20 mM Tris-HCl, pH 8.0. The eluent used a final concentration of 20 mM Tris-HCl + 1 M NaCl, pH 8.0. Linear elution (the eluent increased linearly from 0% to 100% within 30 min) was used. Samples were collected when the conductivity was 15-25 mS / cm. The collected samples were run on SDS-PAGE gels and stained with Coomassie Brilliant Blue.

[0081] S4: After purification, the sample was precipitated by adding 50% saturated ammonium sulfate solution and re-dissolved in 1× PBS.

[0082] During long-term experiments, the inventors discovered the following: 1. Different blood collection sites on shrimp significantly affect not only the blood volume collected, but also the quality and purity of the hemocyanin ultimately extracted and purified. The most preferred site for blood extraction is the sinusoidal region at the base of the third and fourth ambulacral legs, along the edge of the cephalothorax and abdomen. 2. When using DEAE sephadex A-25 for anion exchange chromatography separation and purification, different equilibration and elution systems also affect the quality and purity of the hemocyanin ultimately obtained. The most preferred equilibration system is 20 mM Tris-HCl, and the most preferred elution system is 20 mM Tris-HCl + 1 M NaCl.

[0083] Preparation of Hapten-Shrimp Hemocyanin Conjugate (SMCC Method)

[0084] S1: Reagent Preparation

[0085] The purified shrimp hemocyanin was stained and quantified. A 10 mg / mL shrimp hemocyanin stock solution was prepared with pure water. SMCC (succinimidyl 4-(N-maleimidomethyl)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 a Nanodrop.

[0086] S2: Activation of shrimp hemocyanin

[0087] Add SMCC to a final concentration of 2 mg / ml to shrimp hemocyanin at a concentration of 1 mg / ml, invert at room temperature for 1 h, and centrifuge to obtain the supernatant;

[0088] S3: Removal of coupling agent

[0089] SMCC was removed by desalting column (NW75, column volume 20 ml, maximum 20% column volume could be loaded), ultrapure water was used as equilibration buffer and eluent, protein peak samples were collected, SDS-PAGE gel was run, and Coomassie Brilliant Blue staining was used for quantification;

[0090] S4: coupled peptides

[0091] To activated, desalted shrimp hemocyanin (1 mg / mL), add 1 mg / mL of the peptide (hapten, diluted to a 10 mg / mL stock solution in PBS). Incubate the mixture at room temperature for 1 hour, then dialyze overnight to remove excess salts to obtain the hapten-shrimp hemocyanin conjugate. Add 50% glycerol (based on a 5× loading) and 0.1 M DTT (based on a 10× loading) to the gel and capture the fluorescence.

[0092] Preparation of Hapten-Shrimp Hemocyanin Conjugate (EDC Method)

[0093] S1: Reagent Preparation

[0094] The purified shrimp hemocyanin was stained and quantified. 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) to a stock solution concentration of 10 mg / mL. The concentration was measured using a Nanodrop.

[0095] S2: Activation of shrimp hemocyanin

[0096] Add EDC to a final concentration of 2 mg / ml to 1 mg / ml shrimp hemocyanin, incubate at room temperature for 1 hour (activate for 30-60 minutes when using MES solution of EDC), and centrifuge to obtain the supernatant.

[0097] S3: Removal of coupling agent

[0098] EDC was removed by desalting the sample using a NW75 desalting column (20 mL column volume, with a maximum loading of 20% column volume). The equilibration buffer and eluent were PBS or MES, respectively, at a flow rate of 2 mL / min and a pressure of less than 0.5 MPa. A 1 mL sample loop was loaded, and protein peaks were collected based on the UV280 peak position. The protein samples were run on an SDS-PAGE gel and quantified using Coomassie Brilliant Blue staining.

[0099] S4: coupled peptides

[0100] Take activated and desalted shrimp hemocyanin (1 mg / mL) and add 1 mg / mL of the peptide (hapten, diluted to a 5 mg / mL stock solution using PBS pH 7.2 or MES pH 4.0). Incubate the mixture at room temperature for 1 hour, then dialyze overnight to remove excess salts to obtain the hapten-shrimp hemocyanin conjugate. Add 50% glycerol (based on a 5× loading) and 0.1 M DTT (based on a 10× loading) and run the gel, capturing fluorescence.

[0101] Specifically, the hapten-shrimp hemocyanin conjugate prepared by the SMCC method as described above in the embodiment of the present invention comprises haptens: 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-kB. The amino acid sequences of the haptens are shown in the following table:

[0102] Table 1 Hapten amino acid sequences used in the examples

[0103] The prepared hapten-shrimp hemocyanin conjugates were analyzed by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. The results showed that all of the above haptens could be conjugated with shrimp hemocyanin to obtain hapten-shrimp hemocyanin conjugates. Due to the large number of haptens, the present invention provides SDS-PAGE electrophoresis images of some of the conjugates. The results are shown in Figure 2, where lane 1 shows the corresponding hapten and lane 2 shows the hapten-shrimp hemocyanin conjugate. From the electrophoresis results, it can be seen 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 via the SMCC method to obtain conjugates.

[0104] Detection of animal immune activity of hapten-shrimp hemocyanin conjugate

[0105] The hapten-shrimp hemocyanin conjugates to be detected are: α-Tubulin-shrimp hemocyanin, Cytokeratin 10-shrimp hemocyanin, Desmin-shrimp hemocyanin, and NF-kB-shrimp hemocyanin.

[0106] 1. Immunization of Rabbits

[0107] First immunization: first collect negative serum as blank control, add 500 μg of each of the above conjugates to an equal volume of Freund's complete adjuvant and mix evenly, then inject subcutaneously at multiple points on the dorsal flank;

[0108] Secondary immunization: 500 μg of each of the above conjugates was added to an equal volume of Freund's incomplete adjuvant and mixed evenly, and injected subcutaneously at multiple points on the dorsal flank;

[0109] Enhanced immunity: 1 mg of each of the above conjugates was injected subcutaneously at multiple points on the dorsal flank for shock immunization. Three days later, the rabbits were killed and serum was collected.

[0110] 2. Serum titer test

[0111] Rabbits were immunized with conjugates prepared with α-tubulin, cytokeratin 10, desmin, and NF-kB as haptens. Serum titers were determined by Western blot. Antigen samples tested were from cell lines (including HEK293T, HeLa, HepG2, SCC-9, RD, and NCI-H2347). The specific method is as follows: Determine the protein loading amount and add an equal volume of 2× diluted Laemmli sample buffer. To reduce and denature the sample, boil the cell lysate in the sample buffer at 100°C for 5 minutes. The lysate can be aliquoted and stored at -20°C until needed. Equal amounts of protein and molecular weight markers were loaded onto SDS-PAGE gel wells. The loading amount for cell lysate or tissue homogenate was 20-30 μg of total protein, and for purified protein, 10-100 ng. Run the gel at 100V for 1-2 hours. The proteins were transferred from the gel to a nitrocellulose membrane and 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 and incubated with a 1:5000 HRP-conjugated anti-rabbit secondary antibody for 1 hour at room temperature. The membrane was washed three times with TBST for 5 minutes each. Excess reagents were removed and the membrane was covered with transparent plastic film. Colorimetric detection images were acquired using conventional image scanning. The results are shown in Figure 3. Rabbit serum immunized with the four hapten-shrimp hemocyanin conjugates detected the target bands in the corresponding cell lines, demonstrating that the full antigen prepared using shrimp hemocyanin as a carrier is highly effective in eliciting an immune response in animals.

[0112] Comparison of solubility between shrimp hemocyanin and KLH

[0113] The molecular weight and solubility of shrimp hemocyanin and KLH prepared in the examples of the present invention were tested. The solubility of shrimp hemocyanin and KLH activated with SMCC was further tested, wherein the SMCC activation method is as described in the specific examples of the present invention. α-Tubulin was used as a hapten for conjugation to produce α-Tubulin-shrimp hemocyanin and α-Tubulin-KLH, and the solubility of the hapten conjugates was tested. The results are shown in the following table:

[0114] Table 2 Comparison of solubility of shrimp hemocyanin and KLH

[0115] As can be seen from the results in the table above, the solubility of shrimp hemocyanin prepared by the method provided by the present invention is 300 mg / mL, which is significantly higher than KLH (200 mg / mL). Furthermore, the solubility of shrimp hemocyanin activated with SMCC is also significantly higher than that of SMCC-activated KLH. Hapten-carrier conjugates were prepared using shrimp hemocyanin prepared by the present invention and KLH as carriers. Taking α-tubulin as an example, the present inventors found that the solubility of the whole antigen prepared using shrimp hemocyanin as a carrier was significantly higher than that prepared using KLH as a carrier.

[0116] Comparison of immunogenicity between shrimp hemocyanin and KLH

[0117] Experimental purpose: To compare the immunogenicity of the shrimp hemocyanin prepared by the present invention with that of KLH commonly used in the prior art.

[0118] Experimental Methods: Mice were immunized in two groups of three mice each and immunized with either 10 μg of shrimp hemocyanin or 10 μg of KLH, respectively. Emulsified shrimp hemocyanin or KLH was injected intraperitoneally into the mice. A second immunization was performed nine days after the first immunization. Blood was collected from the tail of the mice on the first day of immunization and then every three days for subsequent ELISA testing.

[0119] Test Results: As shown in Figure 4, 18 days after immunization, both shrimp hemocyanin and KLH stimulated the production of antibodies against their respective antigens in mice. There was no significant difference in the immune responses between the two groups. However, the immune response induced by shrimp hemocyanin was more stable, while the immune response induced by KLH increased initially and then decreased between days 18 and 27 of immunization, demonstrating poor stability. Technicians believe this result is due to the fact that KLH precipitates more readily in mice than shrimp hemocyanin, reducing the uptake and presentation of antigens by immune cells.

[0120] Effects of shrimp hemocyanin and KLH as hapten carrier proteins on the immune activity of whole antigens

[0121] Purpose of the experiment: PDI was selected as the hapten polypeptide, and the hapten was controlled to be unchanged. It was coupled with shrimp hemocyanin, KLH, and polylysine to prepare conjugates as full antigens, and the animal immune activity of the three full antigens was detected.

[0122] Experimental Method: After purification by conjugating the PDI polypeptide to the three aforementioned carrier proteins according to the methods described in the Examples of the present invention, rabbits were immunized using the same immunization method as previously described, and the collected serum was subjected to Western blot analysis. Western blot analysis was performed using authentic HEK293T, HeLa, and HepG2 cell line antigens. Equal cell counts (10,000 cells per well) were used for the three antigens, serum was diluted 1:5000, and the secondary anti-Rabbit IgG was diluted 1:10,000. Images were captured using the same exposure time of 10 seconds across the three membranes, using the same detection method as previously described.

[0123] Test Results: Western blot analysis, as shown in Figure 5, shows that both shrimp hemocyanin and KLH-conjugated PDI peptides elicited immune responses in rabbits, stimulating the production of antibodies against the PDI antigen at the intracellular rim. However, the PDI peptide conjugated to a polylysine carrier failed to elicit a corresponding immune response. Comparing the shrimp hemocyanin-PDI and KLH-PDI images, we can see that the antibodies obtained from rabbits immunized with shrimp hemocyanin-PDI were purer and showed fewer nonspecific bands. This suggests that, compared to KLH, shrimp hemocyanin's sequence and structure are more evolutionarily distant from mammals, making it more suitable as a hapten carrier protein.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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.

[0125] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may 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. An application of shrimp hemocyanin as a hapten carrier protein.

2. The use according to claim 1, characterized in that: The shrimp hemocyanin is extracted from the serum of shrimp, and the shrimp is selected from one or a combination of two or more of the following: Penaeus vannamei and its subspecies, Macrobrachium rosenbergii and its subspecies, Procambarus clarkii and its subspecies, Eriocheir sinensis and its subspecies, Cherax quadricarinatus and its subspecies, Panulirus stimpsoni and its subspecies, and Homarus americanus and its subspecies.

3. The use according to claim 2, characterized in that: The shrimp hemocyanin is prepared by the following method: Separating and purifying the shrimp serum by molecular sieve chromatography and anion exchange chromatography in sequence, and re-dissolving after precipitation with ammonium sulfate solution to obtain the serum; Preferably, the shrimp serum is prepared by the following method: The whole shrimp blood is placed on ice and then the supernatant is collected, preferably the supernatant is diluted with PBS, and more preferably the supernatant is filtered after dilution; More preferably, the filtration uses a 0.45 μm filter membrane; and / or the ammonium sulfate solution is an ammonium sulfate solution with a saturation of 33-50%; And / or, PBS is used for the reconstitution.

4. The use according to claim 3, characterized in that: The whole blood of the shrimp is extracted by the following method: using a syringe, taking the head, abdomen and tail of the shrimp as the blood sampling points, inserting the syringe needle at a 45-degree angle to a depth of about 1 mm to extract the shrimp blood; Preferably, the shrimp is selected from prawns, and the blood sampling point of the prawns is preferably the abdomen, for example, along the edge of the cephalothorax and the abdomen, and at the blood sinus at the base of the third and fourth walking legs.

5. The use according to claim 3, characterized in that: The molecular sieve chromatography column is a Sepharose column, the equilibration buffer and the elution buffer are 1×PBS; the sample collection point is: when UV280>500, 1-3 peaks are collected; the anion exchange chromatography column is a DEAE colume, and the sample collection point is when the conductivity is between 15-25 mS / cm; Preferably, 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; 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; More preferably, the equilibration buffer of 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.

6. A method for activating shrimp hemocyanin, characterized in that: The method comprises: activating shrimp hemocyanin using an activation reagent; wherein the shrimp hemocyanin is defined as the use according to any one of claims 1 to 5; Preferably, the activation reagent 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; and / or, the mass volume ratio of the activation reagent to the shrimp hemocyanin is 2:1; and / or, the method further comprises the step of desalting and purifying the activated shrimp hemocyanin; More preferably, the desalting purification comprises desalting the shrimp hemocyanin by column using an equilibrium buffer and an eluent, wherein the equilibrium buffer and the eluent are selected from ultrapure water, PBS and MES.

7. An activated shrimp hemocyanin prepared by the method according to claim 6.

8. A method for preparing a hapten-shrimp hemocyanin conjugate using shrimp hemocyanin as a carrier, the method comprising the following steps: (1) activating shrimp hemocyanin using an activation reagent; (2) desalting and purifying the activated shrimp hemocyanin; (3) coupling the activated and desalted shrimp hemocyanin with a hapten to obtain a hapten-shrimp hemocyanin conjugate.

9. The method according to claim 8, characterized in that The activation is performed using the method as claimed in claim 6; And / or, in step (3), in the coupling, the mass volume ratio of shrimp hemocyanin to hapten is 1:

1.

10. A hapten-shrimp hemocyanin conjugate prepared according to the method of claim 8 or 9.

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