WO2014075374A1

WO2014075374A1 - Agkistrodon acutus hemocoagulase-b

Info

Publication number: WO2014075374A1
Application number: PCT/CN2013/000375
Authority: WO
Inventors: 王锡娟; 孙狄
Original assignee: 北京康辰药业有限公司
Priority date: 2012-11-14
Filing date: 2013-04-01
Publication date: 2014-05-22
Also published as: CN102925422B; CN102925422A

Abstract

The present invention provides agkistrodon acutus hemocoagulase-B. The agkistrodon acutus hemocoagulase-B is high-activity hemocoagulase separated from agkistrodon acutus venom. The hemocoagulase is a single-stranded glycoprotein comprising 236 amino acids, and has an amino acid sequence shown as SEQ ID No. 1. The strand comprises 6 pairs of disulfide bonds; the molecular weight of SDS-PAGE is approximately 35kD; the molecular weight after deglycosylation is 26116.7Da; and an isoelectric point (pI) is 6.0. A hemocoagulase molecule is modified by heterozygotic polysaccharides at an Asn ^{77, 100, 229} site. The hemocoagulase is serine proteinase. The present invention further provides a separation and purification method of the hemocoagulase. The method comprise: removing undissolved substances by using pretreatment; performing anion-exchange column chromatography for two times and Sephdex-G75 molecular sieve chromatography for one time; obtaining an active elution peak; performing dialysis and lyophilization, so as to obtain the high-purity agkistrodon acutus venom hemocoagulase. The activity of the agkistrodon acutus venom hemocoagulase is not less than 2000U/mg; the purity in the HPLC analysis can exceed 95%; and the yield is 0.25% to 0.30% calculated according to the weight of an agkistrodon acutus venom raw material.

Description

Agkistrodon acutus hemagglutinase-B

The present invention relates to a serine protease, in particular to a scorpion snake venom hemagglutinase-B, and to a method for its isolation and purification. Background technique

There is a class of proteases associated with blood coagulation in Crotalinae venom, commonly referred to as "thrombin-like enzyme" (TLC). The clotting enzyme is similar to the apparent action of thrombin (thrombin), which can convert fibrinogen in plasma into fibrin and "coagulate". However, the mechanism of action of the two enzymes is different. The thrombin-like enzyme does not activate the sputum factor in the coagulation system. Its interaction with fibrinogen produces only non-crosslinked fibrin, which does not cause thrombosis. The clot formed can be dissolved by 5M urea. Thrombin activates the sputum factor in the coagulation system, which interacts with fibrinogen to form cross-linked fibrin, which can lead to thrombosis, and the clot formed can not be dissolved by 5M urea.

Scientific research in the past 50 years has found that thrombin-like components are contained in more than 40 species of snake venom, and more than 30 species have been isolated and purified, and all or part of the amino acid sequence of more than 10 species of thrombin has been elucidated. The molecular weight of TLC has been found to be between 29 and 45 kD, most of which are acidic glycoproteins.

In the snake venom TLC that has been found in the past, the primary structure of the protein is mostly single-stranded. Its commercial representative product, "Reptilase", produced by Swiss company Solco Basle, is a thrombin isolated from the venom of the Brazilian snake, i Bothmps. atrox, which consists of 255 precursors. Amino acid composition, a guide peptide formed by 24 amino acids at the N-terminus, an active enzyme containing 231 amino acids, and a SDS-PAGE molecular weight of 34 kD. Contains 6 intrachain disulfide bonds (site: 7-139, 26-42, 74-230, 118-184, 150-163, 174-199), a single-chain glycoprotein (glycosylation site is Asn) ¹⁴⁶ , ²²⁵ ), the molecular weight after removal of the glycosyl group is 25517 Da. Another product that has been commercialized is the "Botropase" produced by RAVIZZ, Italy, which is a snake from the American spear python (Bothr. Separated from the poison. The active enzyme contained 231 amino acids and differed in amino acid sequence from 14 sites in comparison with Reptilase.

In addition, domestic scholar Shen Juren et al. isolated a high-activity hemagglutinase from the Chinese snake venom Ugkistrodo " acMi ) snake venom in 2006. The enzyme is single-stranded, the molecular weight of SDS-PAGE is 36 ± 2kD, and the isoelectric point is 6.59. The specific activity is 2000U-3000U/mg, and the N-terminal 15 amino acid sequence is VIGGNECDTNEEHFL. The yield of the snake venom raw material is 0.03%.

So far, there are few varieties of commercial snake venom hemagglutinase, so it is particularly necessary to find a new high-activity and high-yield snake venom hemagglutinase suitable for industrialization.

The inventor from me

Acutus, commonly known as the five-step snake, is isolated and purified to obtain a highly active hemagglutinase-B. Summary of the invention

It is an object of the present invention to provide a snake venom hemagglutinase-B which is a thrombin-like enzyme (TLC) isolated from the snake venom of Agkistrodon acutus.

Another object of the present invention is to provide a method for isolating and purifying the above hemagglutinase-B.

It is also an object of the present invention to provide the use of the above-mentioned Agkistrodon acutus hemagglutinase-B in the preparation of a hemostatic drug.

The hemagglutinase-B of the present invention is a highly active hemagglutinase (designated "hemagglutinase-B") isolated from the snake venom of Agkistrodon acutus, which has the SEQ ID No. Amino acid sequence. The enzyme has the following characteristics: 1 a single chain consisting of 236 amino acids, SDS-PAGE molecular weight is about 35kD, isoelectric point pi is 6.0, 2 contains 6 intrachain disulfide bonds (site: 7-141, 28- 44, 78-234, 120-188, 152-167, 178-203).

3 There are heteropolysaccharide modifications consisting of 5 different monosaccharides at Asn ⁷⁷ , ¹⁰⁰ ' ²²⁹ .

4 Enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating that it is a serine protease. 5 has good thermal stability, 60 ° C constant temperature 60 days of enzyme activity can be maintained at 90%.

It will be understood that one skilled in the art can according to the amino acid sequences disclosed herein, The mutated sequence of the protein is obtained by substituting, deleting and/or adding one or several amino acids without affecting its activity. Accordingly, the protein of the present invention further comprises a protein derived from the protein having the amino acid sequence of SEQ ID No. 1 substituted, substituted and/or increased by one or several amino acids and having an equivalent activity. A person skilled in the art can design a synthetic oligonucleotide probe according to the N-terminal amino acid sequence of hemagglutinase-B of the present invention, extract mRNA from the tissue of Agkistrodon acutus, reverse transcription and construct a cDNA library through the above probe. The cDNA library is screened, and the selected positive clones are separately subjected to sequencing and cloning analysis to obtain a gene encoding the protein of the present invention. The gene encoding the protein of the present invention can also be directly designed and synthesized according to the amino acid sequence of hemagglutinase-B of the present invention.

The present invention also provides a method for purifying the above hemagglutinase-B, which comprises the following steps:

1), snake venom pretreatment;

2), pre-treated the snake venom solution on a pre-equilibrated DEAE-Sephrose FF anion exchange chromatography column, wash the column with 0.01M PBS pH 7.0 ~ 7.5, and then use 0.01M pH7 containing 0.02 and 0.06M NaCl .0 - 7.5 PBS was eluted in sections, and the elution peak of 0.06 M NaCr solution was collected;

3), the elution peak collected in step 2) is concentrated by ultrafiltration to a small volume, and then dialyzed to remove NaCl;

4), the dialysis solution was loaded again onto a pre-equilibrated DEAE-Sephrose FF column, washed with 0.01 M PBS pH 7.0 ~ 7.5, and then 0.01 M with 0.02, 0.04, 0.06 M NaCl The PBS was fractionally eluted with pH 7.0 - 7.5, and the third elution peak in the eluate of 0.06 M NaCl solution was collected;

5), the eluted peak collected in the above step 4) was concentrated by ultrafiltration to a small volume, precipitated with 80% ammonium sulfate, and centrifuged at 12000 g for 30 minutes to separate the precipitate.

6) Dissolve the pellet in an appropriate amount with PBS, further purify it with Sephadex-G75, and then freeze-dry by dialysis and desalting.

Among them, step 1) snake venom pretreatment method is to dissolve the snake venom with an appropriate amount of pre-cooled 0.01M pH7.0 - 7.5 PBS, centrifuge to take the supernatant for dialysis (dialysis bag molecular weight cut-off is 7,000D~10,000D). Pretreatment can remove insoluble impurities as well as small molecule polypeptides and reduce solution ionic strength.

Specifically, the pretreatment of the snake venom can be carried out by the following steps: weighing a few grams of snake venom, using a venom weight of 5 to 10 times the volume of 0.01 M pH 7.0 ~ 7.5 PBS in a 4 to 8 ° C chromatographic cabinet Stir for 30~60 minutes, centrifuge at 4~8°C, 5,000-10,000g for 10~20 minutes, pour the supernatant into the dialysis bag, and add the venom weight 5~10 times the volume of pre-cooled PBS. Stir in suspension and centrifuge again. Combine the two centrifugation supernatants into a dialysis bag and dialyze on 0.01 M pH 7.0 ~ 7.5 PBS for 12 to 24 hours at 4~8 °C, and change the solution 2~4 times to remove small peptides and reduce the solution. Ionic strength.

Among them, step 2) and step 4) pre-equilibrated DEAE-Sephrose FF column with 0.01M pH 7.0 ~ 7.5 PBS, and then loaded.

Among them, step 3) the purpose of dialysis is to remove the NaCl present in the solution. Step 3) and step 5) The large volume eluent is concentrated by ultrafiltration (membrane cutoff value 5000-10000Da)

The purpose of using Sephade _X- G75 molecular sieve in step 6) is to further separate and purify the protein in the two anion column chromatography collections by molecular weight. Specifically, the precipitate was dissolved in PBS, and the column was eluted with a 0.01 M pH 7.4 eluate to collect the first peak of the eluate.

The specific activity of hemagglutinase-B purified by the method of the present invention is not less than 2000 U/mg, and both reduction and non-reduction SDS-PAGE are one band; HPLC analysis has a purity of over 95%. The enzyme has good thermal stability and can maintain 90% enzyme activity at 40 Ό constant temperature for 60 days. Based on the weight of the venom of the snake venom, the purification yield of the method is 0.25% - 0.3%.

The hemagglutinase-B of the present invention has good blood coagulation activity and can be used as a raw material medicine for preparing various hemostatic drugs, that is, the present invention also includes a hemostatic drug containing the scorpion snake blood coagulase-B. The medicament may be a lyophilized powder, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostatic spray for various medical conditions requiring hemostasis to reduce the amount of bleeding. Can also be used to prevent bleeding, avoid or reduce the surgical site and surgery After bleeding. DRAWINGS

Figure 1 shows the SDS-PAGE-band of hemagglutinase-B after purification.

Figure 2 shows the results of HPLC analysis of hemagglutinase-B after purification.

Figure 3 Schematic diagram of hemagglutinase-B heterozygous glycosylation structure. detailed description

The following examples are intended to further illustrate the invention, but are not to be construed as limiting the invention. Modifications or substitutions of the methods, steps or conditions of the invention are intended to be within the scope of the invention.

The technical means employed in the examples are conventional techniques well known to those skilled in the art unless otherwise specified.

The percent sign "%" referred to in the present invention means mass percentage unless otherwise specified; however, the percentage of the solution, unless otherwise specified, means that the solution contains several grams of solute in 100 ml; the percentage between the liquids, It refers to the ratio of capacity at 20 °C. In the present invention, it is similar to the expression "pre-cooled with a venom weight of 10 times by volume", wherein the units of weight and volume are g and ml, respectively. Example 1 Purification of snake venom hemagglutinase-B

Take 30g of Agkistrodon acutus venom lyophilized powder (batch number: 20090701, Guangxi Snake Venom Research Institute), stir and dissolve in a 4°C chromatography cabinet with 10 times volume of venom weighted 0.01M pH 7.4 in a chromatography cabinet for 30 minutes. After centrifugation at 1000 °C for 15 minutes at 4 ° C, the centrifugation supernatant was poured into a dialysis bag, and the pellet was centrifuged again to add 10 volumes of pre-cooled PBS with a snake venom weight, and the mixture was again centrifuged. The two supernatants were combined and centrifuged in a dialysis bag (molecular weight cutoff of 10,000 D), and dialyzed against 0.01 M pH 7.4 PBS for 24 hours in a 4 ° C chromatography cabinet, during which time the solution was changed three times. The pretreated snake venom solution was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column pre-equilibrated with 0.01 M pH 7.4 PBS, first washed with 0.01 M pH 7.4 PBS, and then with 0.02 M, 0.06 M NaCl, respectively. 0.01M pH 7.4 The PBS was eluted in sections, and the elution peak of 0.06 M NaCl solution was collected to obtain a total of 1960 ml.

After the enzyme activity assay (refer to the method of Appendix 1 or 7) and electrophoresis analysis, the target appeared in the elution peak collection solution of 0.06 M NaCl solution. The mixture was concentrated to 200 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M ² cut off 10 k membrane), and the 200 ml ultrafiltration concentrate was poured into a dialysis bag (10,000 D) using 2000 ml of 0.01 M pH 7.4. The PBS was dialyzed at 4 ° C for 24 hours, during which time the solution was changed 3 times. The dialyzed enzyme solution was applied to a pre-equilibrated DEAE-Sepharose Fast Flow anion exchange chromatography column, washed with 0.01 M pH 7.4 PBS, and then 0.02 M, 0.04 M, 0.06 M NaCl 0.01 M, respectively. The PBS of pH 7.4 was eluted in sections, and the third eluting peak of the eluate of 0.06 M NaCl solution was collected, and a total of 660 ml was obtained. The mixture was concentrated to 120 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M ² cut off 5k membrane), and the 120 ml was precipitated by 80% ammonium sulfate at 4 ° C overnight, and centrifuged at 12,000 g for 4 minutes at 4 ° C for 30 minutes. The pellet was suspended and dissolved in 10 ml of M pH 7.4 PBS. Immediately applied to a Sephdex-G75 column and eluted with 0.01 M pH 7.4 eluate. The first peak of the eluate was collected in 65 ml. The measurement confirmed the presence of the objective product. The 65 ml concentrate was placed in a dialysis bag, and dialyzed against ion-free water for 24 hours, during which time the solution was changed three times, 2000 ml each time. After dialysis, the volume was 73 ml and directly freeze-dried. 81 mg of the lyophilizate was obtained, and the specific activity of the enzyme was determined to be 2500 U/mg, and the final yield was 0.27%. Both reduced and non-reduced SDS-PAGE are banded (see Figure 1) and the molecular weight of the electrophoresis is approximately 35kD. The HPLC purity was 96.2% (see Figure 2). The isoelectric point pi of the enzyme was determined by isoelectric focusing electrophoresis to be 6.0.

The amino acid and glycosylation assays were carried out by Edman digestion and peptide mapping mass spectrometry, and the amino acid sequence thereof is shown in SEQ ID No. 1. The hemagglutinase contains 236 amino acids, and the molecular weight is only 26116.7Dalton based on amino acid; the chain contains 6 disulfide bonds, and the sites are: Cys ⁷ -Cys ¹⁴¹ , Cys ²⁸ -Cys ⁴⁴ , Cys ⁷⁸ -Cys ²³⁴ , Cys ^120- Cys' ⁸⁸ , Cys ^{, 52} -Cys ¹⁶⁷ , Cys ¹⁷⁸ -Cys ²⁰³ _o glycosylation sites are Asn ⁷⁷ - G2F4S, Asn ¹⁰⁰ - Hybrid, Asn ²²⁹ - G2F4S, glycosylation structure consists of 5 different monosaccharides The heterozygous polysaccharide is composed (see Figure 3). Example 2 Purification of snake venom hemagglutinase-B

30 g of the lyophilized powder of Agkistrodon acutus venom (batch number: 20090701, Guangxi Snake Venom Research Institute) was pretreated in the same manner as in Example 1. The pretreated snake venom solution was subjected to the first DEAE-Sephrose FF column chromatography in the same manner as in Example 1. The target substance appeared in the elution peak of 0.06 M NaCl by enzyme activity measurement and electrophoresis analysis, and the eluted collection solution was combined. A total of 2000ml was prepared and concentrated to 210ml by Millipore Pellicon 2 tangential flow ultrafilter (0.1M ² cut off 10k membrane). The 210ml ultrafiltration concentrate was poured into a dialysis bag (10,000D) with 2000ml 0.01 M pH 7.4 PBS was dialyzed against 4'C for 24 hours, during which time the solution was changed 3 times. The dialyzed enzyme solution was applied to a DEAE-Sephrose FF column, and a second chromatography was carried out in the same manner as in Example 1. Hemagglutinase appeared in the third elution peak of the 0.06 M NaCl solution eluate, and the peak eluate was collected to obtain 648 ml. The mixture was concentrated to 130 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M ² cut off 5k membrane), and the 130 ml was passed through 80 °/. Ammonium sulfate was precipitated at 4 ° C overnight, 12,000 g of 4 . After centrifugation for 30 minutes, the pellet was suspended and dissolved in 10 ml of 0.01 M pH 7.4 PBS, and immediately applied to a Sephdex-G75 column to collect 68 ml of the first peak of the eluate, which was confirmed by enzyme activity measurement. The 68 ml was dialyzed in the same manner as in Example 1 to a volume of 78 ml, which was directly freeze-dried. 85 mg of the lyophilizate was obtained, and the specific activity of the enzyme was determined to be 2460 U/mg, and the final yield was 0.28%. SDS-PAGE is a band with an electrophoretic molecular weight of approximately 35 kD. The HPLC purity was 96.0%. Example 3 Serine protein property test of blood clot-B in Agkistrodon acutus

The hemagglutinase-B isolated in Example 1 was diluted with physiological saline to an enzyme activity of 1 U/ml. A 1% solution of bovine fibrinogen (Sigma) was prepared in physiological saline.

Dissolve phenylmethylsulfonyl fluoride (PMSF, Merck) with isopropanol at a solution concentration of mg/mL

The experimental steps are as follows:

(1) Take 1 ml of 1% bovine fibrinogen solution and incubate at 37 °C for 5 minutes.

(2) Take three small test tubes, respectively labeled W, 2#, 3#, and add 200 μl of lU/ml hemagglutinase-B solution to each tube. (3) Add ΙΟμΙ distilled water to the 1# tube, add ΙΟμΙ isopropanol to the 2# tube, add lC^l PMSF to the 3# tube, and incubate for 5 minutes at 37 °C in a water bath.

(4) The agglutination test was separately performed in the order of the test tube numbers. Add a constant temperature of 1% bovine fibrinogen solution 200μ1 to the test tube, immediately time the mixture, gently shake and mix, and let stand in 37 C water, observe the agglutination reaction in the tube. The timing is terminated when the solution is completely solidified.

The results are shown in Table 1.

Table 1. Effect of PMSF on agglutination

Based on the experimental results in Table 1, the following conclusions were drawn: PMSF at a concentration of 1 OO ppm completely inhibited the activity of hemagglutinase-B, demonstrating that the blood coagulase-B is a serine protease. 2 Trace isopropanol has no effect on the agglutination reaction. Example 4 Thermal stability test of blood clot 晦-B of Agkistrodon acutus

The freeze-dried pure enzyme (without adding any lyoprotectant) obtained in column 1 and column 2 was dispensed into a plurality of ampoules and sealed in a plurality of ampoules, and placed in a 40 ° C incubator for 30 days respectively. The enzyme activity was measured and taken at 60 days.

The results showed that the specific activity of the two batches of enzymes maintained 95% of the original enzyme activity for 30 days, and remained at 90% of the original enzyme activity for 60 days. This result indicates that the isolated hemagglutinase has good thermal stability. The results are shown in Table 2.

Thermal stability study enzyme activity measurement results (40Ό)

appendix

Definition of hemagglutinase unit of Agkistrodon acutus and method for measuring activity

1 Bovine fibrinogen assay 1.0% bovine fibrinogen (Sigma) solution prepared in physiological saline was placed in a small test tube, kept in a 37±0.5°C water bath for 3 minutes, and preheated at 37±0.5°C. The enzyme solution is 1 ml, immediately timed, and the fibrinogen solution shakes white flocs in 120±30 seconds, and the enzyme solution is 1 U/ml.

2 Standard human plasma assay Take standard human plasma 1ml in a small test tube, preheat for 3 minutes in a 37 ° C ± 0.5 ° water bath, add 37 ± 0.5 ° C preheated enzyme solution 1 ml, immediately timed, human plasma When the white floc appeared in shaking within 60 ± 20 seconds, the enzyme solution was 1 U/ml.

Note: When measuring the unknown high enzyme activity solution, it needs to be diluted with ion-free water until it reaches lU/ml for determination; the dilution factor is the number of enzyme activity units per ml of the original enzyme solution. Industrial applicability

The blood cobase-B obtained by the purification method of the invention has good coagulation activity, high enzyme specific activity and good thermal stability, and the enzyme has good thermal stability. . Therefore, the hemagglutinase-B of the present invention can be used as a raw material medicine for preparing various hemostatic drugs, and the medicine can be a lyophilized powder original medicine, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostasis. Liquid spray for various medical conditions where hemostasis is required to reduce the amount of bleeding. It can also be used to prevent bleeding and to avoid or reduce bleeding at the surgical site and after surgery.

Claims

claims

1. Agkistrodon hemagglutinin-B, which has the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence with equivalent functions obtained by replacing, deleting and/or adding one or more amino acids to this sequence.

2. The adder adder hemocoagulase-B according to claim 1, characterized in that the chain contains 6 pairs of disulfide bonds and has a hybrid polysaccharide modification at the Asn ⁷⁷ ' ^{, o 229} position.

3. A medicine containing the adder adder hemagglutinin-B described in claim 1 or 2.

4. The drug according to claim 3, which is a freeze-dried powder original drug, a freeze-dried powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic ointment or a hemostatic body spray.

5. The purification method of hemagglutinin-B according to claim 1 or 2, which includes the following steps:

1), snake venom pretreatment;

2). Place the pretreated snake venom solution on the pre-equilibrated DEAE-Sepharose Fast Flow anion exchange chromatography column, wash the column with 0.01M pH7.0-7.5 PBS, and then use 0.01MpH7.0 containing 0.2 and 0.6M NaCl. Stepwise elution with PBS of ~7.5, collect the elution peak of 0.06M NaCl solution;

3). Concentrate the elution peak collection liquid obtained in step 2) above by ultrafiltration and then dialyze to remove NaCl;

4). Put the dialyzed solution on the pre-equilibrated DEAE-Sepharose Fast Flow chromatography column again, wash the column with 0.01M PBS pH7.0~7.5, and then use 0.01M pH7.0 containing 0.02, 0.04, and 0.06M NaCl. Stepwise elution with PBS of ~7.5, collect the third elution peak of 0.06M NaCl solution;

5). Concentrate the elution peak collection solution obtained in step 4) above by ultrafiltration and precipitate with 80% ammonium sulfate, and centrifuge to collect the precipitate;

6) Dissolve the precipitate with an appropriate amount of PBS, further purify it with Sephadex-G75, remove salt through dialysis, and freeze-dry.

6. The method of claim 5, wherein step 1) snake venom pretreatment method is to dissolve the snake venom with an appropriate amount of pre-cooled 0.01M PBS pH 7.0-7.5, and centrifuge to take out the venom. The serum is dialyzed.

7. The method of claim 5, wherein step 1) snake venom pretreatment method is: weigh several grams of snake venom, and add pre-cooled 0.01 M pH 7.0 to 7.5 PBS with a volume of 5 to 10 times the weight of the snake venom. Stir and dissolve in a chromatography cabinet at 4 to 8°C for 30 to 60 minutes, centrifuge at 4°C and 5,000 to 10,000g for 10 to 20 minutes, pour the centrifugal supernatant into a dialysis bag, centrifuge and weigh the snake venom. Stir and suspend 5 to 10 times the volume of pre-cooled PBS, centrifuge again, combine the two centrifugation supernatants and put them into a dialysis bag, and dialyze against 0.01M PBS of pH 7.0 to 7.5 for 12 to 24 hours at 4 to 8°C. The solution was replaced 2 to 4 times during this period.

8. The method of claim 5, wherein step 2) and step 4) use 0.01M PBS of pH 7.0 ~ 7.5 to pre-equilibrate the DEAE-Sepharose Fast Flow chromatography column, and then load the sample.

9. The method according to any one of claims 5 to 8, wherein step 3) uses an ultrafiltration membrane with a molecular cutoff value of 5,000 to 10,000D for ultrafiltration concentration.

10. The method according to any one of claims 5 to 8, wherein the purification method in step 6) is to dissolve the precipitate collected in step 5) with PBS, then put it on a Sephadex-G75 chromatography column, and use 0.01M pH7. 4 PBS elution, collect the first peak of the eluate.