A method for extracting and purifying arthropod hemocyanin

Abstract

The invention discloses a method for extracting and purifying arthropod hemocyanin, comprising the following steps: (1) extracting arthropod whole blood; (2) placing the whole blood sample on ice and letting it stand, taking the supernatant, diluting it with PBS, and filtering it with a 0.45 μm filter membrane; (3) separating and purifying the filtered sample by molecular sieve chromatography and anion exchange chromatography in sequence; (4) precipitating the purified sample with an ammonium sulfate solution having a saturation of 33-50%, and then redissolving it with PBS to obtain arthropod hemocyanin. The hemocyanin extracted and purified by the method provided by the present invention has high purity and low cost, and can be used as a protein carrier of a hapten to prepare a complete antigen, and the effect is better than the protein carrier KLH commonly used in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of animal protein extraction and purification, and in particular relates to a method for extracting and purifying arthropod hemocyanin. Background Art

[0002] Hemocyanin, also known as hemocyanin, is a multifunctional protein known as a respiratory protein. Hemocyanin is a copper-containing respiratory protein located in the hemolymph of arthropods and molluscs. It is a copper-containing blue-green high-molecular-weight protein found in the hemolymph of arthropods and molluscs. It is an oxygen carrier and the only copper protein known to reversibly bind to oxygen.

[0003] Evolutionarily, both arthropods and molluscs are very different from mammals. Mammals use hemoglobin for respiration, while arthropods and molluscs use hemocyanin for respiration. Hemocyanin exists as a single hexamer (1×6-mer) or multiples of hexamer (2×6-mer, 4×6-mer, 6×6-mer, 8×6-mer). The hemocyanin subunit (about 72 kDa) of arthropods (such as shrimp) is folded into three domains, characterized by different 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 subunit molecular weight of hemocyanin of mollusks (such as keyhole limpets) is about 350 or 400 kDa and consists of seven or eight functional units (FU) folds. Each FU consists of two distinct domains, termed α (from the α-helical domain) and β (from the β-sandwich domain), with domain α folding into a four-α-helical bundle carrying a copper active site and domain β into a six-stranded antiparallel β barrel. The α domain of molluscan hemocyanin FUs functionally corresponds to domain II and the β domain to domain III of arthropod hemocyanins, respectively. Arthropod and mollusc hemocyanins are proteins responsible for the binding, transport and storage of dioxygen, and both have several additional functions, including but not limited to enzymatic activity (i.e., phenoloxidase), hormone transport, homeostasis (molting), and hemostasis (clot formation). In addition, an important secondary function of hemocyanins involves aspects of innate immunity, such as acting as precursors for broad-spectrum antimicrobial peptides and microbial / viral agglutination.

[0004] The technicians of the present invention unexpectedly found that the hemocyanin of arthropods can be used as a protein carrier of hapten to prepare complete antigens, and the effect is better than the protein carrier KLH (keyhole limpet hemocyanin) commonly used in the prior art. More importantly, the cost of preparing hemocyanin by the method provided by the present invention is much lower than the cost of purchasing KLH. At present, there is no literature or patent reporting that the hemocyanin of arthropods can be used as a protein carrier of hapten for immunizing animals to obtain antibodies; at the same time, there is no specific prior art solution disclosing how to extract hemocyanin from arthropods. Summary of the invention

[0005] Based on the above background, one object of the present invention is to provide a method for extracting and purifying arthropod hemocyanin; another object of the present invention is to provide arthropod hemocyanin extracted according to the method.

[0006] The present invention includes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for extracting and purifying arthropod hemocyanin, the method comprising the following steps:

[0008] (1) extracting whole blood from arthropods;

[0009] (2) Place the whole blood sample on ice, take the supernatant, dilute with PBS, and filter using a 0.45 μm filter membrane;

[0010] (3) separating and purifying the filtered sample using molecular sieve chromatography and anion exchange chromatography in sequence;

[0011] (4) The purified sample is precipitated with a 33-50% saturation ammonium sulfate solution and then re-dissolved with PBS to obtain arthropod hemocyanin.

[0012] Preferably, the chromatographic column for molecular sieve chromatography in step (3) is a Sepharose column, the equilibration buffer and the elution buffer are 1X PBS, and the sample loading volume is less than 10% of the column volume.

[0013] Preferably, the sample collection point for molecular sieve chromatography is: when UV280>500, 1-3 peaks are collected.

[0014] In the most preferred embodiment of the present invention, the sample collection point of the molecular sieve chromatography is: when UV280 to 500, one peak is collected.

[0015] Preferably, the chromatography column for anion exchange chromatography in step (3) is a DEAE column, and the equilibrium buffer is a Tris-HCl buffer or a Hepes buffer, wherein the concentration of Tris-HCl is 10-20 mM, and the concentration of Hepes is 30-50 mM.

[0016] The elution buffer is a combination of Tris-HCl and NaCl, or a combination of Hepes and NaCl. In the combination of Tris-HCl and NaCl, the concentration of Tris-HCl is 10-20 mM, and the concentration of NaCl is 0.5-1 M; in the combination of Hepes and NaCl, the concentration of Hepes is 30-50 mM, and the concentration of NaCl is 0.5-1 M.

[0017] In a specific embodiment of the present invention, the equilibration buffer of the anion exchange chromatography is 50 mM Hepes, and the elution buffer is a combination of Hepes and NaCl, wherein the concentration of Hepes is 50 mM and the concentration of NaCl is 1 M.

[0018] In the most preferred 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.

[0019] Preferably, the anion exchange chromatography is eluted by linear gradient elution, and the sample collection point is a conductivity between 15-25 mS / cm.

[0020] The method for extracting whole blood of arthropods in step (1) of the present invention adopts a method well known to those skilled in the art to collect blood.

[0021] The arthropod 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, Eriocheirsinensis and its subspecies, Cherax quadricarinatus and its subspecies, Panulirus stimpsoni and its subspecies, and Boston lobster (Homarus americanus) and its subspecies.

[0022] In a specific embodiment of the present invention, the arthropod is a shrimp.

[0023] The method for extracting whole blood from shrimp is as follows: use a syringe to take blood from the shrimp's head, abdomen, and tail, insert the syringe needle at a 45-degree angle to a depth of about 1mm, and extract 100-400μL of shrimp blood.

[0024] Preferably, the blood sampling 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.

[0025] Preferably, the saturation of the ammonium sulfate solution used in step (4) is 33%, 40%, 45% or 50%.

[0026] More preferably, the saturation of the ammonium sulfate solution is 50%.

[0027] In a second aspect, the present invention provides an arthropod hemocyanin extracted and purified according to the above method.

[0028] The method for extracting and purifying arthropod hemocyanin provided by the present invention has the following advantages:

[0029] (1) High purity. The final purified hemocyanin is identified by Coomassie brilliant blue staining, and its purity is above 98%;

[0030] (2) The extraction rate is high. According to the method provided by the present invention, about 500 mg of high-purity hemocyanin can be obtained from 1 kg of shrimp;

[0031] (3) The extraction method is simple to operate and low in cost. Through exploration by the technicians of the present invention, the optimal blood collection point for shrimp is obtained, and the molecular sieve chromatography and anion exchange chromatography columns selected for separation and purification have low preparation costs and good separation and purification effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Diagram of the structure of shrimp; 1. ophthalmic artery, 2. anterior lateral artery, 3. hepatic artery, 4. heart, 5. dorsal and ventral artery, 6. antennal artery, 7. subthoracic artery, 8. thoracic artery, 9. subgastric artery.

[0033] Figure 2 Diagram of the molecular sieve chromatography process.

[0034] Figure 3 Diagram of the anion exchange chromatography process.

[0035] Figure 4 SDS-PAGE electrophoresis of the elution phase of anion exchange chromatography column.

[0036] Figure 5 SDS-PAGE electrophoresis of the elution phase of anion exchange chromatography column under different elution systems.

[0037] Figure 6Grayscale scan of shrimp hemocyanin in elution system group 1.

[0038] Figure 7 Grayscale scan of shrimp hemocyanin in elution system group 2. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Preparation of 100% saturated ammonium sulfate solution

[0041] Slowly add 767g (NH4)2SO4 to 1L distilled water while stirring, and adjust the pH of the solution to 7.0 with ammonia water or sulfuric acid to obtain a 100% saturated ammonium sulfate solution (4.1mol / L, 25°C).

[0042] Extraction of shrimp hemocyanin

[0043] Example 1

[0044] S1: Extraction of shrimp blood

[0045] 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 into the blood sinus at the base of the third and fourth walking legs along the edge of the cephalothorax and abdomen. Figure 1 Insert the syringe needle into the shrimp abdomen at a 45-degree angle to a depth of about 1 mm near the position shown in 7, slowly pull back the syringe, and extract 400 μL of shrimp blood. There are about 50 shrimps per kg, and a total of 20 mL of whole blood can be extracted from 1 kg of shrimp.

[0046] S2: Shrimp blood pretreatment

[0047] Place the shrimp blood on ice and let it stand for 2 hours. Take the supernatant. 10 mL of serum can be obtained from 1 kg of shrimp. Add 1X PBS (pH 7.2-7.4) to dilute 10 times, and filter once using a 0.45 μm filter membrane.

[0048] S3: Isolation and purification of shrimp hemocyanin

[0049] A: Molecular sieve chromatography

[0050] The chromatography column is Sepharose-G25 column, and 1X PBS is used as the equilibration buffer. At least 2 column volumes are equilibrated first, and the sample is loaded at a low flow rate. 1X PBS is used as the elution buffer. The flow rate is 10 mL / min throughout the whole process including loading. 10 mL of serum is loaded, and a peak is collected when UV280 is 500. The process is as follows Figure 2 As shown;

[0051] B: Anion exchange chromatography

[0052] The column was DEAE sephadex A-25, with a flow rate of 2 mL / min, an equilibrium buffer of 20 mM Tris-HCl pH 8.0, an eluent of 20 mM Tris-HCl + 1 M NaCl pH 8.0 at a final concentration, and a linear elution (within 30 min, the eluent increased linearly from 0% to 100%). Samples were collected when the conductivity was 15-25 mS / cm. The process was as follows: Figure 3 As shown, the sample tubes are numbered A01-A12, and the collected samples are run on SDS-PAGE gel and stained with Coomassie Brilliant Blue;

[0053] S4: After purification, the sample was precipitated by adding 50% saturated ammonium sulfate solution and reconstituted with 1X PBS.

[0054] In step S3B, the eluted phase of the anion exchange chromatography column is subjected to SDS-PAGE electrophoresis, and then the finally purified shrimp hemocyanin is identified by Coomassie brilliant blue staining. Figure 4 As shown, the target protein band is scanned in grayscale. According to the signal distribution of the image, it can be calculated that the purity of the target protein (shrimp hemocyanin) is 98.35%, and the mass of the obtainable shrimp hemocyanin is about 500 mg.

[0055] Optimization test

[0056] 1. Effect of different blood collection sites on the extraction of shrimp hemocyanin

[0057] Purpose of the experiment: To explore whether different blood sampling sites of shrimp have an effect on the purity and extraction amount of hemocyanin.

[0058] Test method: The shrimp blood extraction, pretreatment, separation and purification operations were the same as in Example 1, the only difference being the blood sampling site. Three shrimps were randomly selected from each group, and the results were averaged, as follows:

[0059] Group 1: Blood sinuses at the base of the third and fourth stepping legs ( Figure 1 The position shown in 7 );

[0060] Group 2: Shrimp eye position ( Figure 1 The position shown in 1);

[0061] Group 3: Shrimp Back ( Figure 1 5).

[0062] Shrimp blood was extracted from the above-mentioned parts, and pre-treated and separated and purified according to the method described in Example 1. The blood collection amount, hemocyanin purity and extraction amount of Groups 1 to 3 are shown in the following table:

[0063] Table 1 Effects of different blood collection sites on the extraction of shrimp hemocyanin

[0064]

[0065] According to the data in the table above, different blood collection sites have a significant effect on the amount of blood collected. Figure 1 In addition, it can be seen from the data in the table that the amount of hemocyanin that can be extracted from shrimp blood obtained from different parts is different, and the purity of hemocyanin is also different. The technicians believe that due to the different types of cells contained in different tissue parts and the differences in the distribution of substances in the intercellular matrix, the content and purity of hemocyanin in the blood of different parts are different.

[0066] 2. Effect of the equilibrium and elution system of anion exchange chromatography column on the extraction of shrimp hemocyanin

[0067] Purpose of the experiment: Anion exchange chromatography is an important step in the separation and purification of hemocyanin. This study aimed to explore whether different equilibrium buffers and elution buffers have an effect on the purity and extraction amount of hemocyanin.

[0068] Experimental method: The shrimp blood extraction, pretreatment, separation and purification operations are the same as in Example 1, except that the equilibration buffer and elution buffer used in the anion exchange chromatography are different, as follows:

[0069] Table 2 Elution system of anion exchange chromatography column

[0070]

[0071] The shrimp blood was extracted and pretreated according to the method described in Example 1, and combined with the separation and purification method described above, the purity and extraction amount of hemocyanin in Group 1 and Group 2 are shown in the following table:

[0072] Table 3 Effect of different elution systems on the extraction of shrimp hemocyanin

[0073]

[0074] The shrimp hemocyanin extracted from group 1 and group 2 was run on SDS-PAGE gel and stained with Coomassie brilliant blue. Figure 5As shown, the shrimp hemocyanin purity test results obtained in Group 1 and Group 2 are as follows Figure 6 and 7 As shown. According to the above results, it can be seen that in the anion exchange chromatography step, different elution systems have an impact on the purity and extraction amount of hemocyanin. Compared with Hepes, selecting Tris-HCl as the equilibrium buffer and a mixture of Tris-HCl and NaCl solution as the elution buffer are more conducive to the purification and separation of hemocyanin. This is because the charged characteristics and strength of ions affect the binding of proteins to the chromatography column. The technicians of the present invention found that impurities are more easily separated from the target protein in Tri-HCl.

[0075] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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.

Claims

1. A method for extracting and purifying arthropod hemocyanin, the method comprising the following steps: (1) extracting whole blood from an arthropod, wherein the arthropod is a shrimp Penaeus vannamei, and the whole blood extraction method is as follows: using a syringe, inserting the needle of the syringe at a 45-degree angle to a depth of about 1 mm along the edge of the cephalothorax and the abdomen, and at the blood sinus at the base of the third and fourth walking legs as the blood sampling point, to extract the shrimp blood; (2) Place the whole blood sample on ice, take the supernatant, dilute with PBS, and filter using a 0.45 μm filter membrane; (3) Separating and purifying the filtered sample by molecular sieve chromatography and anion exchange chromatography in turn, the chromatographic column of the molecular sieve chromatography is a Sepharose-G25 column, the equilibrium buffer and the elution buffer are 1XPBS, and the sample loading volume is less than 10% of the column volume; the sample collection point is: when UV280 to 500, collect 1 peak; the chromatographic column of the anion exchange chromatography is a DEAE sephadexA-25column, and the elution is performed by linear gradient elution, and the sample collection point is when the conductivity is between 15-25mS / cm, the equilibrium buffer of the anion exchange chromatography is 20mM Tris-HCl, pH 8.0, and the elution buffer is a combination of Tris-HCl and NaCl, pH 8.0, Tris-HCl concentration is 20mM, and NaCl concentration is 1M; (4) The purified sample was precipitated with 50% saturated ammonium sulfate solution and then re-dissolved with PBS to obtain arthropod hemocyanin.

Images