Application of shrimp hemocyanin as hapten carrier protein

Abstract

The invention discloses application of shrimp hemocyanin as hapten carrier protein, and belongs to the technical field of biological medicine. Compared with KLH commonly used in the prior art, the shrimp hemocyanin provided by the invention is good in solubility, the holoantigen obtained by coupling the shrimp hemocyanin with the hapten is good in solubility and not easy to precipitate, the immunogenicity of the holoantigen is superior to that of the KLH, and the animal immunocompetence of the holoantigen prepared by taking the shrimp hemocyanin as the carrier protein is better than that of the holoantigen prepared by taking the KLH as the carrier protein. The shrimp haemocyanin provided by the invention is extracted from shrimp blood, the shrimp culture technology is mature, the shrimp haemocyanin is easy to obtain, and the purchase cost is low, so that the preparation cost of the shrimp haemocyanin is far lower than the purchase cost of KLH.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2023115857774 with an application date of November 24, 2023. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

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

[0003] Hapten (also known as incomplete antigen) is a type of small molecule substance that cannot induce an immune response when present alone. Its molecular weight is generally less than 3 kDa, such as polypeptide molecules (with about 15 - 20 amino acids). Usually, after covalently linking a polypeptide with a macromolecular carrier protein and immunizing an animal, an effective immune response can be generated to prepare the target antibody. Commonly used carrier proteins include natural proteins such as BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin), RSA (rabbit serum albumin), and recombinant proteins such as dodecin, etc. Among them, BSA, OVA, and KLH are the three most commonly used in traditional research. Their common feature is strong immunogenicity, with a sufficient number of active sites on the protein surface, making it easy to form a covalent bond with polypeptide molecules. However, when a polypeptide is conjugated with a carrier protein and used to immunize an animal, not only antibodies against the target polypeptide are produced, but also antibodies against the carrier protein are generated. BSA is commonly used in many other biochemical molecule experiments such as ELISA, Western-blot (protein immunoblotting technique), and cell culture, etc. Therefore, due to its broad application spectrum, BSA is generally not used as a carrier protein for polypeptides. Compared with BSA, OVA does not have the above disadvantages, but its immunogenicity is slightly weaker. Therefore, it is often used as a hapten carrier for subsequent antibody verification, rather than as an immunization carrier. The protein characteristics of KLH are very different from those of mammalian proteins, so it has extremely strong immunogenicity and is currently the most commonly used carrier protein. However, KLH has an overly large and complex molecular weight, consisting of protein subunits of 350 kDa and 390 kDa. Its water solubility is poor, and it is prone to precipitation after being linked with a polypeptide. On the other hand, the production cost of KLH is relatively high. KLH is extracted from the blood of keyhole limpets, which are mollusks. The cost of collecting this animal from nature is relatively high. Currently, the company that can achieve 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, and the domestic price is about 30 RMB / mg.

[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 is the only known copper protein that can reversibly bind to oxygen.

[0005] Hemocyanin comes from within arthropods and mollusks. In terms of evolution, both arthropods and mollusks are very different from mammals. Mammals use hemoglobin for respiration, while arthropods and mollusks use hemocyanin for respiration. Hemocyanin exists as a single hexamer (1×6 oligomer) or a multiple of hexamers (2×6 oligomers, 4×6 oligomers, 6×6 oligomers, 8×6 oligomers). The hemocyanin subunit of arthropods (such as shrimp) (about 72 kDa) folds into three domains, characterized by different folding motifs: domain I with five or six α-helices; domain II with a four-α-helix bundle and an active site containing two copper ions; domain III with a seven-stranded antiparallel β-barrel. In contrast, the subunit molecular weight of hemocyanin in mollusks (such as keyhole limpets) is about 350 or 400 kDa and is folded from seven or eight functional units (FUs). Each FU consists of two different domains, called α (from the α-helical domain) and β (from the β-sandwich domain); domain α folds into a four-α-helix bundle carrying the copper active site, and domain β forms a six-stranded antiparallel β-barrel. The α-domain of the mollusk hemocyanin FU functionally corresponds to domain II of the arthropod hemocyanin, and the β-domain corresponds to domain III respectively. Hemocyanin in arthropods and mollusks is a protein responsible for the binding, transport, and storage of dioxygen and has several additional functions, including (but not limited to) enzyme activity (i.e., phenoloxidase), hormone transport, homeostasis (molting), and hemostasis (clot formation). An important secondary function of hemocyanin involves aspects of innate immunity, such as being a precursor to broad-spectrum antimicrobial peptides and microbial / viral agglutination. Currently, there is no literature or patent reporting that the hemocyanin of shrimp can be used as a carrier protein for haptens to immunize animals to obtain antibodies. Summary of the Invention

[0006] Based on the above background, the present invention provides an application of shrimp hemocyanin as a hapten carrier. The shrimp hemocyanin is extracted from the blood of shrimp. Taking penaeid shrimp as an example, the circulatory system of penaeid shrimp is an open circulatory system. The beating of the heart causes the blood to flow out of the heart, along the arteries and branched blood vessels to various organs and tissues. The blood from the organs and tissues is concentrated in the thorax through the tissue 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 characteristics of the open circulatory system make it relatively easy to collect the blood of penaeid shrimp, and the shrimp blood can be easily extracted with a syringe. The technical personnel of the present invention found that about 20 mL of shrimp blood can be extracted from one catty of penaeid shrimp, which is equivalent to 10 mL of serum and contains about 0.5 g of shrimp hemocyanin. Therefore, using the hemocyanin of penaeid shrimp as the carrier protein of hapten will greatly reduce the preparation cost of hapten-carrier protein conjugates.

[0007] The technical personnel of the present invention believe that when selecting a hapten carrier protein, although the immunogenicity of the carrier protein is a very important factor, the molecular weight, active groups, solubility, availability, and price of the carrier protein are all key factors that have to be considered. Common arthropods, such as penaeid shrimp and Macrobrachium rosenbergii, are common agricultural products in the Chinese market and common foods on the tables of ordinary people. The breeding technology of penaeid shrimp is mature in China, and the price per catty in the market is about 30 RMB. Therefore, if the hemocyanin of penaeid shrimp can be used to prepare hapten, it will surely be more cost-effective than KLH in terms of cost.

[0008] The present invention includes the following technical solutions:

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

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

[0011] The shrimp hemocyanin is obtained by extracting from the serum of shrimp.

[0012] Furthermore, the shrimp is selected from one or a combination of two or more of 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.

[0013] Since the farming technology of penaeid shrimp is mature, the raw materials are easily available, and the purchase cost is low, in the specific embodiments of the present invention, the shrimp selected is penaeid shrimp.

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

[0015] The serum of the shrimp 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 hemocyanin.

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

[0017] In some embodiments, it further includes diluting the supernatant with PBS.

[0018] In some embodiments, it further includes filtering after the dilution.

[0019] In some embodiments, the filtering 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 embodiments, the redissolution uses PBS.

[0022] In some specific embodiments, 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 blood sampling points respectively, inserting the syringe needle at an angle of 45 degrees into a depth of about 1 mm, and extracting the shrimp blood.

[0023] In some specific embodiments, the shrimp selected is penaeid shrimp.

[0024] In some specific embodiments, the blood sampling point of the penaeid shrimp is the abdomen.

[0025] In some specific embodiments, the blood sampling point of the penaeid shrimp is at the blood sinus at the edge of the cephalothorax and abdomen, and at the base of the third and fourth walking legs.

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

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

[0028] (1) Extract the whole blood of the shrimp;

[0029] (2) Leave the whole blood sample standing on ice, take the supernatant, dilute it with PBS, and filter it using a 0.45 μm filter membrane;

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

[0031] (4) The purified sample is precipitated with ammonium sulfate solution with a saturation of 33 - 50%, and then redissolved with PBS to obtain shrimp hemocyanin.

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

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

[0034] Preferably, in step (3), the chromatography column for molecular sieve chromatography 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 reaches 500, collect 1 peak.

[0036] Preferably, in step (3), the chromatography column for anion exchange chromatography is a DEAE colume, 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 for 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 the combination of Tris-HCl with a final concentration of 10 - 20 mM and NaCl with a final concentration of 0.5 - 1 M; or the 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 saturation of the ammonium sulfate solution used in step (4) is 33%, 40%, 45% or 50%.

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

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

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

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

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

[0046] In some preferred embodiments, the desalting and purification includes passing the shrimp hemocyanin through a column for desalting using a balance buffer and an elution buffer, and the balance buffer and the elution buffer are selected from ultrapure water, PBS and MES.

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

[0048] 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:

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

[0050] (2) Desalting and purifying the activated shrimp hemocyanin;

[0051] (3) Coupling the activated and desalted shrimp hemocyanin with a hapten to obtain a hapten-shrimp hemocyanin conjugate.

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

[0053] Preferably, the activating reagent in step (1) is a coupling agent having an activating function conventionally used in the art, including but not limited to SMCC, NHS. In the specific embodiment of the present invention, the activating reagent used is SMCC.

[0054] Preferably, the desalting and purification method in step (2) is selected from one or a combination of two 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 elution buffer are ultrapure water.

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

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

[0057] Fifth aspect, the present invention provides a hapten-hemocyanin conjugate prepared by the method as described above.

[0058] The application of using hemocyanin as a carrier protein for haptens provided by the present invention has the following advantages:

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

[0060] 2. The immunogenicity of the hemocyanin prepared in the present invention is superior to KLH;

[0061] 3. The whole antigen prepared with hemocyanin as the carrier protein has better animal immunization activity than the whole antigen prepared with KLH as the carrier protein;

[0062] 4. The hemocyanin provided by the present invention is extracted from shrimp blood. The shrimp farming technology is mature, it is easy to obtain, and the purchase cost is low, so that the preparation cost of hemocyanin is much lower than the purchase cost of KLH. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 : Process diagram of preparing hapten conjugate with hemocyanin.

[0064] Figure 2 : SDS-PAGE electrophoresis diagram of hapten-hemocyanin conjugate.

[0065] Figure 3 : Detection result diagram of serum titer of hapten-hemocyanin conjugate.

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

[0067] Figure 5 : Comparison result diagram of immunization activity of antigens prepared with hemocyanin and KLH. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0068] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0069] The process of preparing the hemocyanin hapten conjugate in this embodiment is as Figure 1 shown.

[0070] Extraction of shrimp hemocyanin

[0071] S1: Extraction of shrimp blood

[0072] The shrimp for blood collection is selected from penaeid shrimp. Hold the shrimp with the left hand, making its abdomen face upwards. Hold a syringe with the 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 walking 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, and extract 400 μL of shrimp blood. There are about 50 shrimps in 1 kg of penaeid shrimp, and a total of 20 mL of whole shrimp blood can be extracted from 1 kg of penaeid shrimp.

[0073] S2: Pretreatment of shrimp blood

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

[0075] S3: Isolation and purification of shrimp hemocyanin

[0076] A: Molecular sieve chromatography

[0077] The chromatography column is a Sepharose-G25 column. The equilibration buffer is 1×PBS. At least equilibrate for 2 column volumes first, and keep the sample loading at a low flow rate. The elution buffer is 1×PBS, and the flow rate of 10 mL / min is maintained throughout the process including sample loading. Take 10 mL of serum for sample loading, and collect a peak when UV280 is at 500.

[0078] B: Anion exchange chromatography

[0079] The chromatography column is DEAE sephadexA-25, with a flow rate of 2 mL / min. The equilibration buffer is 20 mM Tris-HCl pH 8.0, and the eluent is 20 mM Tris-HCl + 1 M NaCl pH 8.0 with a final concentration. Linear elution (within 30 minutes, the eluent increases linearly from 0% to 100%). Start collecting samples when the conductivity is between 15 - 25 mS / cm. Run the collected samples on an SDS-PAGE gel and stain with Coomassie Brilliant Blue.

[0080] S4: Add ammonium sulfate solution with a saturation of 50% to the purified sample for precipitation, and redissolve it with 1×PBS.

[0081] During the long-term experiment, the inventors found the following phenomena: 1. Different blood collection sites of prawns not only have a significant impact on the blood collection volume, but also affect the quality and purity of the finally extracted and purified hemocyanin. The most preferred blood extraction site of prawns is the blood sinus where the third walking leg and the base of the fourth walking leg are inserted along the edge of the cephalothorax and the abdomen; 2. When using DEAEsephadex A-25 for anion exchange chromatography separation and purification, different equilibration and elution systems also affect the quality and purity of the finally obtained hemocyanin. The most preferred equilibration system is 20 mM Tris-HCl, and the most preferred elution system is 20 mM Tris-HCl + 1 M NaCl.

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

[0083] S1: Reagent preparation

[0084] Stain and quantify the purified hemocyanin of prawns above, and prepare a stock solution of hemocyanin at 10 mg / mL with pure water; prepare SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) with 1×PBS (pH 7.2 - 7.4), and the stock solution concentration is 2 mg / mL; measure the concentration with Nanodrop;

[0085] S2: Activation of hemocyanin

[0086] Add SMCC with a final concentration of 2 mg / mL to the hemocyanin with a concentration of 1 mg / ml, invert at room temperature for 1 h, and centrifuge to take the supernatant;

[0087] S3: Removal of coupling agent

[0088] Remove SMCC through a desalting column (NW75, column volume 20 ml, and the maximum sample loading volume is only 20% of the column volume), use ultrapure water as the equilibration buffer and eluent, collect the protein peak sample, run SDS-PAGE gel, and stain and quantify with Coomassie Brilliant Blue;

[0089] S4: Coupling of polypeptide

[0090] Take the activated and desalted hemocyanin of prawns (1 mg / mL), add 1 mg / mL of polypeptide (hapten, dilute the stock solution to 10 mg / mL with PBS), invert and couple at room temperature for 1 h, perform overnight dialysis to remove excess salts, and obtain a hapten-hemocyanin conjugate. Add 50% glycerol (used according to 5×loading) and 0.1 M DTT (used according to 10×) to run the gel and take fluorescence pictures.

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

[0092] S1: Reagent Preparation

[0093] Quantify the purified shrimp hemocyanin by Coomassie staining, and prepare a 20 mg / mL shrimp hemocyanin stock solution with pure water; prepare EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) with 1×PBS (pH 7.2) or MES (pH 4.0), and the stock solution concentration is 10 mg / mL; measure the concentration with Nanodrop;

[0094] S2: Activation of Shrimp Hemocyanin

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

[0096] S3: Removal of Coupling Agent

[0097] Remove EDC through a desalting column (NW75, column volume 20 ml, and the maximum sample loading volume is only 20% of the column volume). Accordingly, the equilibration buffer and elution buffer are PBS or MES, the flow rate is 2 mL / min, and the pressure is less than 0.5 Mpa. Load 1 ml of the sample loop, collect the protein peak sample according to the elution peak position of UV280, run SDS-PAGE gel, and quantify by Coomassie Brilliant Blue staining;

[0098] S4: Coupling of Polypeptide

[0099] Take the activated and desalted shrimp hemocyanin (1 mg / mL), add 1 mg / mL of the polypeptide (hapten, dilute the stock solution to 5 mg / mL with PBS pH = 7.2 or MES pH = 4.0), couple by inversion at room temperature for 1 h, perform overnight dialysis to remove excess salts, and obtain the hapten-shrimp hemocyanin conjugate. Add 50% glycerol (used according to 5×loading) and 0.1 M DTT (used according to 10×) to run the gel and take fluorescence pictures.

[0100] Specifically, the haptens in the hapten-shrimp hemocyanin conjugate prepared by the SMCC method as described above in the embodiments of the present invention are: 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, NF-kB, and the amino acid sequences of the haptens are shown in the following table:

[0101] Table 1 Amino Acid Sequences of Haptens Used in the Embodiments

[0102]

[0103]

[0104] The prepared hapten-keyhole limpet hemocyanin conjugates were analyzed by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. The results showed that all the above haptens could be conjugated with keyhole limpet hemocyanin to obtain hapten-keyhole limpet hemocyanin conjugates. Since there were a large number of haptens, the SDS-PAGE electrophoresis patterns of some conjugates were provided in the present invention. The results were as Figure 2 shown. Lane 1 was the corresponding hapten, and lane 2 was the hapten-keyhole limpet hemocyanin conjugate. From the electrophoresis results, it could be seen that haptens 161-C-3, 162-H-3, 126-A-3, 130-L-3, and 132-M-3 could all be successfully conjugated with keyhole limpet hemocyanin by the SMCC method to obtain conjugates.

[0105] Detection of the animal immunological activity of hapten-shrimp hemocyanin conjugate

[0106] The hapten-keyhole limpet hemocyanin conjugates to be detected were: α-Tubulin-keyhole limpet hemocyanin, Cytokeratin 10-keyhole limpet hemocyanin, Desmin-keyhole limpet hemocyanin, and NF-kB-keyhole limpet hemocyanin.

[0107] I. Immunizing rabbits

[0108] Primary immunization: First, 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 evenly and inject it 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 evenly and inject it subcutaneously at multiple points on the dorsal side.

[0110] Booster immunization: Inject 1 mg of each of the above conjugates subcutaneously at multiple points on the dorsal side for booster immunization. Kill the rabbits three days later and collect the serum.

[0111] II. Detection of serum titer

[0112] After immunizing rabbits with conjugates prepared using α-Tubulin, Cytokeratin 10, Desmin, and NF-kB as haptens, the corresponding rabbit sera were taken, and the serum titers were detected by Western-blot. The antigen samples for detection were cell line samples (including HEK293T, HeLa, HepG2, SCC-9, RD, NCI-H2347). The specific method is as follows: Determine the protein loading amount and add an equal volume of 2× diluted Laemmli sample buffer. When reducing and denaturing the sample, boil the cell lysate in the sample buffer at 100 °C for 5 minutes. The lysate can be aliquoted equally and stored at -20 °C for later use. Load an equal amount of protein and molecular weight markers into the wells of the SDS-PAGE gel. The total protein loading amount of the cell lysate or tissue homogenate is 20 - 30 μg, and the protein loading amount of the purified protein is 10 - 100 ng. Run the gel at 100 V for 1 - 2 hours. Transfer the protein from the gel to the membrane, which can be nitrocellulose. Block the membrane with the blocking buffer at room temperature for 1 hour or overnight at 4 °C. Dilute the rabbit serum at 1:5000 and incubate it overnight at 4 °C; wash the membrane 3 times with TBST, 5 minutes each time, and incubate the membrane with the anti-rabbit secondary antibody conjugated with HRP at 1:5000 at room temperature for 1 hour. Wash the membrane 3 times with TBST, 5 minutes each time. Remove the excess reagent and cover the membrane with a transparent plastic film. Collect the colorimetric detection images using the conventional image scanning method. The results are as Figure 3 shown. The results show that the rabbit sera immunized with the above four hapten-keyhole limpet hemocyanin conjugates can all detect the target bands in the corresponding cell lines, indicating that the whole antigen prepared using keyhole limpet hemocyanin as a carrier can well induce an immune response in animals.

[0113] Comparison of the solubility of shrimp hemocyanin and KLH

[0114] The molecular weights and solubilities of the keyhole limpet hemocyanin and KLH prepared in the examples of the present invention were detected respectively, and the solubilities of SMCC-activated keyhole limpet hemocyanin and KLH were further detected, where the SMCC activation method is as shown in the specific examples of the present invention; α-Tubulin was used as a hapten to conjugate α-Tubulin-keyhole limpet hemocyanin and α-Tubulin-KLH, and the solubilities of the hapten conjugates were detected. The results are shown in the following table:

[0115] Table 2 Comparison results of the solubilities of keyhole limpet hemocyanin and KLH

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

[0117] As can be seen from the results in the above table, the solubility of the hemocyanin prepared by the method provided by the present invention is 300 mg / mL, which is significantly higher than that of KLH (200 mg / mL). Further, the solubility of the hemocyanin activated by SMCC is also significantly higher than that of KLH activated by SMCC. Taking the hemocyanin and KLH prepared by the present invention as carriers respectively, hapten-carrier conjugates are prepared. Taking α-Tubulin as an example, the inventors of the present invention found that the solubility of the complete antigen prepared with the hemocyanin as the carrier is significantly higher than that of the complete antigen prepared with KLH as the carrier.

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

[0119] Test objective: To compare the immunogenicity of the hemocyanin prepared by the present invention with KLH commonly used in the prior art.

[0120] Test method: Mice were immunized and divided into two groups, with 3 mice in each group. They were immunized with hemocyanin and KLH respectively. The immunization dose was 10 μg of hemocyanin or 10 μg of KLH. After emulsifying the hemocyanin or KLH, the mice were injected intraperitoneally. A booster immunization was carried out on the 9th day after the first immunization. Blood was collected from the tails of the mice on the first day of immunization, and then blood was taken every 3 days for subsequent Elisa detection.

[0121] Test results: As shown in Figure 4 , after 18 days of immunization, both hemocyanin and KLH can stimulate mice to produce antibodies against their respective antigens, and there is no significant difference in the immune response between the two. However, the immune response caused by hemocyanin is more stable. The immune response produced by KLH first increases and then decreases between the 18th and 27th days of immunization, with poor stability. The technicians analyzed that the reason for this result is that KLH is more likely to form precipitates in mice than hemocyanin, reducing the uptake and presentation effect of immune cells on antigens.

[0122] Effect of shrimp hemocyanin and KLH as hapten carrier proteins on the immunological activity of whole antigen

[0123] Test objective: Select PDI as the hapten polypeptide, keep the hapten unchanged, and conjugate hemocyanin, KLH, and polylysine respectively to prepare conjugates as complete antigens, and detect the animal immunological activities of the three complete antigens.

[0124] Test method: After connecting and purifying the PDI polypeptide with the above three carrier proteins according to the method described in the embodiments of the present invention, immunize rabbits in the same manner as described above, and perform Western-blot detection on the collected serum. The real cell line antigens HEK293T, HeLa, and HepG2 are used for Western-blot detection. The three antigens are loaded with an equal number of cells (10,000 cells per well), the serum is diluted 1:5000, the secondary antibody anti-Rabbit IgG is diluted 1:10,000, and image acquisition is performed on three membranes with the same exposure time of 10 s. The specific detection method is the same as described above.

[0125] Test results: The results of Western-blot detection are as Figure 5 shown. The PDI polypeptides conjugated with both hemocyanin and KLH can induce immune responses in rabbits and can stimulate rabbits to produce antibodies against the PDI antigen on the inner edge of cells, while the PDI polypeptide conjugated with polylysine as a carrier cannot induce the corresponding immune response. And by comparing the figures of hemocyanin-PDI and KLH-PDI, it can be seen that the antibody obtained by immunizing rabbits with hemocyanin-PDI as an antigen has higher purity and fewer non-specific bands, indicating that compared with KLH, the sequence and structure of hemocyanin itself are further from mammals in evolution and are 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 are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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.

[0127] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples. Without departing from the principles and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope 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 equilibrium 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-ButanediolDiglycidylether, 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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