Preparation method of Protobothrops mucrosquamatus venom C-type protein and its application as a diagnostic reagent

Abstract

Preparation method of the C-type protein of Protobothrops mucrosquamatus venom and its application as a diagnostic reagent. A novel target protein Protocetin is isolated and purified from domestic Protobothrops mucrosquamatus venom through three-step chromatography, which has the function of inducing platelet aggregation. Based on the fact that Protocetin can activate platelets through GPIb by acting on vWF, leading to platelet aggregation, it can be used to detect the function of vWF and the aggregation function of platelets, and thus can be used clinically to detect hemophilia and platelet-related diseases associated with abnormal structure and function of GPIb.

Description

Technical Field

[0001] The present invention relates to the venom of Protobothrops mucrosquamatus and its application fields, in particular to a method for preparing the C-type protein of Protobothrops mucrosquamatus venom and its application as a diagnostic reagent. Background Art

[0002] Snake venom is a highly complex mixture of active proteins and peptides in nature, which is an important material basis for predation and defense of venomous snakes, and also an important resource library for the research and development of various important proteases, new drugs and diagnostic reagents. The venom of Protobothrops mucrosquamatus contains a large number of active proteins acting on the blood coagulation system, among which C-type lectin-like proteins have the characteristics of high specificity and strong activity, and have important application prospects in the research and development of new anticoagulant drugs and diagnostic reagents for blood coagulation function.

[0003] The present invention relates to a novel C-type lectin-like protein isolated and purified from Protobothrops mucrosquamatus venom and its application as a reagent for detecting von Willebrand factor (vWF) and platelet aggregation function. Summary of the Invention

[0004] To solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide the preparation of the C-type of Protobothrops mucrosquamatus venom and its application as a diagnostic reagent

[0005] To achieve the above purpose, the technical solution of the present invention is as follows:

[0006] A method for preparing the C-type of Protobothrops mucrosquamatus venom, comprising the following steps:

[0007] Step 1, molecular sieve gel filtration chromatography;

[0008] A Sephadex G-75 gel chromatography column of 2.6×100 cm is equilibrated with PBS (phosphate buffered saline, 0.01 mol·L -1 Na2HPO4-NaH2PO4 buffer solution, pH 7.4) containing 0.147 mol / L NaCl, and the flow rate is 24 ml·h -1 ; Weigh 0.5 g of the freeze-dried powder of Protobothrops mucrosquamatus venom, dissolve it in 3 ml of PBS, centrifuge the dissolved venom solution at 2000 rpm for 10 min, and take the supernatant and load it onto the aforementioned Sephadex G-75 gel column equilibrated with PBS. After loading, elute with PBS, and the flow rate is 24 ml·h -1 , collect the eluate with an automatic collector, 4 ml / tube, measure the absorbance at 280 nm, and collect and combine the labeled elution parts for the next separation and purification.

[0009] Step 2, heparin affinity chromatography;

[0010] Take the active peak of Sephadex G-75 chromatography of crude Agkistrodon acutus venom. After desalting or dilution, load it onto a heparin affinity chromatography column. Equilibrate the heparin affinity chromatography with PB buffer (0.01 mol·L-1 Na2HPO4-NaH2PO4 buffer, pH 7.0), and perform gradient elution with PB buffer containing NaCl at a flow rate of 1 ml·min-1. Collect the samples at 2 ml / tube, measure the absorbance at 280 nm, and collect the active peak.

[0011] Step 3: Cation exchange chromatography;

[0012] Equilibrate the HiTrap SP cation exchange pre-packed column with PB (0.01 mol·L-1 Na2HPO4-NaH2PO4 buffer, pH 7.0). After loading the active peak obtained from heparin affinity chromatography and fully eluting, perform gradient elution with PB buffer containing 1 mol NaCl at a flow rate of 1 ml·min-1. Collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

[0013] Further, Step 3 can be replaced by:

[0014] Step 3: Gel filtration chromatography

[0015] Dissolve the freeze-dried powder of the active peak obtained from heparin affinity chromatography in 1.5 ml of PBS, centrifuge at 2000 rpm for 10 min, take the supernatant and load it onto a Sephacrycl S-100 gel chromatography column (1.0×70 cm), elute with PBS buffer at a flow rate of 30 ml / h, collect the samples at 2 ml / tube, and measure the absorbance at A 280 nm. Collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

[0016] Furthermore, a Protobothrops mucrosquamatus venom C-type protein is characterized in that: the molecular weight of the target protein is determined by 12% SDS-PAGE under reducing and non-reducing conditions, the working voltage is 160 V, and Coomassie Brilliant Blue R-250 staining is used after electrophoresis. The molecular weights of the standard protein markers are phosphatase b (97.2 kDa), bovine serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29.0 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa); Sephacrycl S-100 gel (1.0 cm × 60 cm) is used for molecular weight determination, and four standard proteins (Albumin (67.0 kDa), Ovalbumin (43.0 kDa), Chymotrypsinogen A (25.0 kDa), Ribonuclease A (13.7 kDa)) are used. MALDI / TOF is used to determine the exact molecular mass of the target protein;

[0017] Isoelectric focusing (IEF) is used to determine the isoelectric point of the target protein under denaturing conditions. The pH range of the ampholyte Ampholine is 3.5 - 10, and the pH gradient of the gel after electrophoresis is determined by the KCl soaking method.

[0018] Furthermore, the Protobothrops mucrosquamatus venom C-type protein can be used in the application of a kit for diagnosing hemophilia and platelet-related diseases associated with abnormal GPIb structure and function.

[0019] Furthermore, the specific application is that: the Protobothrops mucrosquamatus venom C-type protein can activate platelets through GPIb by acting on vWF, leading to platelet aggregation, so that it can be used to detect the function of vWF and the aggregation function of platelets.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The present invention separates and purifies a novel target protein Protocetin from domestic Protobothrops mucrosquamatus venom by three-step chromatography, which has the function of inducing platelet aggregation. Based on the fact that Protocetin can activate platelets through GPIb by acting on vWF, leading to platelet aggregation, it can be used to detect the function of vWF and the aggregation function of platelets, and thus can be clinically used to detect hemophilia and platelet-related diseases associated with abnormal GPIb structure and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the Sephadex G-75 gel filtration chromatography map of Protobothrops mucrosquamatus venom;

[0023] Figure 2 Chromatogram of the crude venom Sephadex G-75 peak 2 purified by Heparin HP affinity chromatography;

[0024] Figure 3 Chromatogram of the HiTrap Peparin HP affinity chromatography active peak by Hitrap SP chromatography;

[0025] Figure 4 Chromatogram of the HiTrap Peparin HP affinity chromatography active peak by gel filtration chromatography;

[0026] Figure 5 Electrophoresis of the target protein; among them Figure 5 A, 12% SDS-PAGE. From left to right, low molecular weight Marker, Protocetin (non-reduced), Protocetin (reduced). Figure 5 B, denaturing isoelectric focusing electrophoresis;

[0027] Figure 6 Protocetin induced human platelet aggregation in a dose-dependent manner; **P<0.01 vs control(0);

[0028] Figure 7 Protocetin could not effectively induce human washed platelet aggregation *P<0.05, **P<0.01 vs control(PBS);

[0029] Figure 8 Protocetin could induce human washed platelet aggregation by adding plasma **P<0.01 vs control(PBS);

[0030] Figure 9 GPIb monoclonal antibody inhibited Protocetin-induced platelet aggregation **P<0.01 vs control(0). Detailed implementation manners

[0031] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners:

[0032] Test examples:

[0033] 1. Materials and methods

[0034] 1.1 Main materials

[0035] Protobothrops mucrosquamatus venom; Protein separation media: Sephadex G-75 gel, Sephacrycl S-100 gel, Heparin gel, Gel filtration molecular weight determination kit, Ampholine (pH 3.5 - 10) were purchased from GE Healthcare; Low molecular weight protein standard Marker was a product of TaKaRa; Thrombin, N,N'-Methylenebisacrylamide, Sodium dodecyl sulfate (SDS) were purchased from Sigma; GPIb monoclonal antibody was a product of BD; Thrombin Time (TT) assay kit, Prothrombin Time (PT) assay kit, Activated partial thromboplastin Time (APTT) assay kit, Fibrinogen (FIB) assay kit were all purchased from Shanghai Sunbiotech Co., Ltd.; Enhanced BCA protein quantification kit was purchased from Jiangsu Beyotime Biotechnology Institute; Low molecular weight standard protein Marker was purchased from Dalian TaKaRa; Other reagents were all of analytical grade, imported or domestic.

[0036] 1.2 Methods

[0037] 1.2.1 Isolation and purification of target protein

[0038] 1.2.1.1 Molecular sieve gel filtration chromatography

[0039] Sephadex G-75 gel chromatography column (2.6×100 cm) was equilibrated with PBS (phosphate buffered saline, 0.01 mol·L -1 Na2HPO4-NaH2PO4 buffer, pH 7.4) at a flow rate of 24 ml·h -1 . Weigh 0.5 g of freeze-dried Protobothrops mucrosquamatus venom, dissolve it in 3 ml of PBS, centrifuge the dissolved venom solution at 2000 rpm for 10 min, and take the supernatant to load onto the previously PBS-equilibrated Sephadex G-75 gel column. After loading, elute with PBS at a flow rate of 24 ml·h -1 , collect the eluate with an automatic collector, 4 ml / tube, measure the absorbance at 280 nm, and collect and combine the labeled elution fractions for the next step of isolation and purification.

[0040] 1.2.1.2 Heparin affinity chromatography

[0041] Take the Sephadex G-75 chromatography active peak of Protobothrops mucrosquamatus venom, desalt or dilute it, and load it onto the heparin affinity chromatography column. Use PB buffer (0.01 mol·L-1 Equilibrate heparin affinity chromatography with Na2HPO4-NaH2PO4 buffer (pH 7.0), and perform gradient elution with PB buffer containing NaCl at a flow rate of 1 ml·min -1 , collect the samples at 2 ml / tube, measure the absorbance at 280 nm, and collect the active peak.

[0042] 1.2.1.3 Cation exchange chromatography

[0043] Equilibrate the HiTrap SP cation exchange pre-packed column with PB (0.01 mol·L -1 Na2HPO4-NaH2PO4 buffer, pH 7.0). After loading the active peak obtained from heparin affinity chromatography and fully eluting, perform gradient elution with PB buffer containing 1 mol NaCl at a flow rate of 1 ml·min -1 , collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

[0044] Or

[0045] 1.2.1.3 Gel filtration chromatography

[0046] Dissolve the freeze-dried powder of the active peak obtained from heparin affinity chromatography in 1.5 ml of PBS, centrifuge at 2000 rpm for 10 min, take the supernatant and load it onto a Sephacrycl S-100 gel chromatography column (1.0×70 cm), elute with PBS buffer at a flow rate of 30 ml / h, collect the samples at 2 ml / tube, and measure the absorbance at A 280 nm. Collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

[0047] 1.2.2 Molecular weight and isoelectric point

[0048] The molecular weight of the target protein was determined by 12% SDS-PAGE under reducing and non-reducing conditions. The working voltage was 160 V, and Coomassie Brilliant Blue R-250 staining was used after electrophoresis. The molecular weights of the standard protein markers were phosphatase b (97.2 kDa), bovine serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29.0 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa). Sephacrycl S-100 gel (1.0 cm × 60 cm) was used for molecular weight determination, and four standard proteins were used: Albumin (67.0 kDa), Ovalbumin (43.0 kDa), Chymotrypsinogen A (25.0 kDa), and Ribonuclease A (13.7 kDa). The exact molecular mass of the target protein was determined by MALDI / TOF.

[0049] Isoelectric focusing (IEF) was used to determine the isoelectric point of the target protein under denaturing conditions. The pH range of the ampholyte Ampholine was 3.5 - 10, and the pH gradient of the gel after electrophoresis was determined by the KCl soaking method.

[0050] 1.2.3 Peptide fingerprint analysis

[0051] After the purified sample was subjected to reduced SDS-PAGE electrophoresis, the gel strip containing the protein was cut out. First, it was decolorized (50% acetonitrile + 50 mmol ammonium bicarbonate 100 μL, 20 minutes, remove the solution, repeat twice), dried (100% acetonitrile 100 μL, 10 minutes, remove the acetonitrile), and then placed in an oven at 37 °C for 5 - 10 minutes to ensure complete drying of the gel). Then, it was enzymatically digested (add 5 μL - 10 μL of Trypsin enzyme solution with a concentration of 12.5 ng·μL -1 and place it in a refrigerator at 4 °C for about 30 minutes to ensure that the gel particles can well absorb the enzyme solution. After taking it out, it was enzymatically digested overnight in an oven at 37 °C), and the peptide segments were extracted (50% acetonitrile + 0.1% TFA 60 μL, after 30 - 40 minutes, transfer the solution to a new 96-well plate, repeat 2 - 3 times). The peptide segment solution was dried and concentrated under a stream of N2 for mass spectrometry identification.

[0052] The sample was hydrolyzed with trypsin, and the completely dried peptide segments were redissolved in 0.7 μL of 0.5 g·L -1In CHCA solution (0.1% TFA + 50% ACN), all of it was spotted onto a stainless steel MALDI target plate and air-dried at room temperature. The sample was analyzed by a 4700 Proteomics Analyzer (TOF / TOF™) (Applied Biosystems, USA). The laser source was an Nd:YAG laser with a wavelength of 355 nm, the acceleration voltage was 20 kV, and data were acquired in positive ion mode and automatic data acquisition mode. The PMF mass scanning range was 700 - 4000 Da, and tandem mass spectrometry analysis was performed on the 10 peaks with the highest intensity; the spectrum was externally calibrated with myoglobin digested peptides. The obtained results were searched against a database using GPS (Applied Biosystems, USA)–MASCOT (Matrix Science, London, UK). Search parameters were set as follows: the database was NCBInr; the data search method was Combined; the maximum allowed missed cleavage sites was 1; the mass error range was set as: PMF ±100 ppm, MS / MS ±0.6 Da.

[0053] 1.2.4 Mechanism of protocetin-induced platelet aggregation

[0054] 1.2.4.1 Preparation of platelet-rich plasma

[0055] Blood was collected by cardiac puncture in rabbits or from the forearm vein of healthy volunteers. The blood was anticoagulated by mixing with 0.129 mol·L -1 sodium citrate solution (1:9) and centrifuged at 1500 or 500 r·min -1 for 10 min to obtain platelet-rich plasma (PRP); then centrifuged at 3600 r·min -1 for 15 min to obtain platelet-poor plasma (PPP). The platelet count in PRP was adjusted to 3×10 11 per L with PPP. The prepared PRP should be used up within 2 h. 1.2.4.2 Preparation of washed platelets

[0056] Blood was drawn from the forearm vein of healthy volunteers. The blood was anticoagulated by mixing with 0.129 mol·L -1 sodium citrate solution (1:9) and centrifuged at 500 r·min -1Centrifuge for 10 min to obtain PRP. Suspend the PRP with platelet washing solution A (113 mM NaCl, 4.3 mM K2HPO4, 24.4 mM NaH2PO4, 4.3 mM Na2HPO4, 5.5 mM glucose, pH 6.5), then centrifuge at 250×g for 10 min. Discard the supernatant, and wash the platelets with solution A again. Suspend the washed platelets with solution B (20 mM Hepes, 140 mM NaCl, 4 mM KCl, 5.5 mM glucose, pH 7.4), and adjust the platelet count to 3×10 11 cells / L using solution B. Before the platelet aggregation experiment, add 2 mM CaCl2 and pre-incubate with the platelets at 37°C for 2 min.

[0057] 1.2.4.3 Induce platelet aggregation experiment

[0058] Take 270 μl of the adjusted PRP or washed platelets, pre-incubate at 37°C for 5 min, add thrombin to detect the platelet aggregation function. The PRP and washed platelets with normal aggregation function are used for the experiment. For 270 μl of PRP or washed platelets, pre-incubate at 37°C for 5 min, then add 30 μl of Protocetin at different concentrations respectively, and measure the maximum aggregation rate within 5 min using a platelet aggregometer.

[0059] 1.2.4.4 Effect of GPIb monoclonal antibody on Protocetin-induced platelet aggregation

[0060] Take 170 μl of normal human washed platelets and pre-incubate with 30 μl of membrane glycoprotein Ib (GPIb) monoclonal antibody (final concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml) at 37°C for 30 min. The control group is incubated with 30 μl of PBS buffer for the same time. Then add 50 μl of platelet washing solution B and 20 μl of PPP, and add 30 μl of Protocetin with a final concentration of 60 μg / ml. Measure the maximum aggregation rate within 5 min using a platelet aggregometer.

[0061] 2. Results

[0062] 2.1 Isolation and purification of Protocetin

[0063] 2.1.1 Sephadex G-75 gel filtration chromatography

[0064] The crude venom of Protobothrops mucrosquamatus is subjected to Sephadex G-75 gel filtration chromatography to obtain the following peak pattern ( Figure 1 ), take the active part indicated by the horizontal line, concentrate it, and then desalt it using Sephadex G-25 gel filtration chromatography.

[0065] 2.1.2 Heparin Affinity Chromatography

[0066] The sample obtained from Sephadex G-75 gel was concentrated, desalted, and then loaded onto a 1 ml Heparin HP affinity chromatography pre-packed column via AKTA Primer for further purification. Gradient elution was performed to obtain the following peak pattern ( Figure 2 ). After electrophoresis detection, the protein peak obtained by 50% gradient elution was taken, concentrated, desalted, and further purified.

[0067] 2.1.3 Cation Exchange Chromatography

[0068] The sample obtained by 50% gradient elution from Heparin HP affinity chromatography was concentrated, desalted, and then loaded onto a 1 ml Hitrap SP pre-packed column via AKTA Primer. Linear gradient elution was set to obtain the purified target protein ( Figure 3 ).

[0069] Or:

[0070] 2.1.3 Gel Filtration Chromatography

[0071] The freeze-dried powder solution of the active peak obtained from heparin affinity chromatography was loaded onto a Sephacrycl S-100 gel chromatography column, and the active peak was collected to obtain the target protein ( Figure 4 ).

[0072] 2.2 Molecular Weight and Isoelectric Point

[0073] Detected by 12% SDS-PAGE, the molecular weight of Protocetin was 25.4 kDa under non-reducing conditions, and two polypeptide chains with molecular weights of 16.8 kDa and 15.8 kDa respectively were presented under reducing conditions ( Figure 5 ). Determined by Sephacryl S-100 gel filtration, the molecular weight of Protocetin was 40.9 kDa. The isoelectric points of the two peptide chains of Protocetin were 9.1 and 8.2 respectively determined by isoelectric focusing electrophoresis ( Figure 5 ). The determination result of MALDI / TOF showed that the exact molecular mass of the target molecule was 24.98 kDa.

[0074] Among them Figure 5 A, 12% SDS-PAGE. From left to right, low molecular weight Marker, Protocetin (non-reducing), Protocetin (reducing). Figure 5 B, denaturing isoelectric focusing electrophoresis.

[0075] 2.3 Peptide Fingerprint Analysis

[0076] After obtaining the peptide fingerprint map and peptide sequence information, NCBI BLAST analysis showed that Protocetin was partially identical to some peptides of C-type lectin (Trimecetin) from the venom of Protobothrops mucrosquamatus in Taiwan (Tables 1 and 2). There was homology between them, but there were also significant evolutionary differences. Peptide fingerprint mapping also indicated that the α and β polypeptide chains in Protocetin were heterologous.

[0077] Table 1 Analysis of matching peptide segments between the α-chain of Protocetin and the α-chain of Trimecetin

[0078]

[0079] The actual peptide segment molecular weight calculation is based on the isotopic peptide segment molecular weight. Assuming the molecular weight of this peptide segment is [actual molecular weight + H + molecular weight].

[0080] Table 2 Analysis of matching peptide segments between the β-chain of Protocetin and the β-chain of Trimecetin

[0081]

[0082] The actual peptide segment molecular weight calculation is based on the isotopic peptide segment molecular weight. Assuming the molecular weight of this peptide segment is [actual molecular weight + H + molecular weight].

[0083] Peptide fingerprint mapping analysis of Protocetin showed that there were some similar peptide segments between Protocetin and C-type lectin (Trimecetin) from the venom of Protobothrops mucrosquamatus in Taiwan. The similarity of the corresponding α and β peptide chains of the two proteins was approximately 34% and 30% respectively. This indicated that although Protocetin had a certain degree of homology with C-type lectin Trimecetin from the venom of Protobothrops mucrosquamatus in Taiwan, they were clearly different protein molecules.

[0084] 2.4 Mechanism of Protocetin-induced platelet aggregation in vitro

[0085] 2.4.1 Protocetin-induced aggregation of human PRP

[0086] Protocetin could induce the aggregation of human PRP. There was a good dose-effect relationship between the platelet aggregation rate and the concentration of Protocetin. When the concentration of Protocetin was 1.0 μg / ml, platelet aggregation could be induced. When the concentration of Protocetin was 60 μg / ml, the platelet aggregation rate could reach 73.42% ( Figure 6 ). Protocetin could hardly induce the aggregation of washed platelets (Figure 7 )。

[0087] 2.4.2 Platelet aggregation induced by Protocetin through plasma components

[0088] Protocetin cannot induce aggregation of washed platelets. After adding a certain amount of PPP, the platelet aggregation activity induced by Protocetin can be restored, and the aggregation rate increases with the increase in the amount of plasma added ( Figure 8 )。

[0089] 2.4.3 Inhibitory effect of GPIb monoclonal antibody on Protocetin-induced platelet aggregation

[0090] After incubating GPIb monoclonal antibody with human washed platelets at 37 °C for 30 min, the aggregation of reconstituted plasma platelets induced by Protocetin (60 μg / ml) was inhibited to varying degrees. Within a certain concentration range, there was an inverse correlation between the platelet aggregation rate and the concentration of GPIb monoclonal antibody ( Figure 9 )。

[0091] 3. Discussion

[0092] In the present invention, a novel target protein Protocetin was isolated and purified from the venom of domestic Protobothrops mucrosquamatus by three-step chromatography, and it has the function of inducing platelet aggregation.

[0093] Protocetin can induce aggregation of human PRP, and within a certain concentration range, the platelet aggregation rate increases with the increase in concentration. However, it cannot induce aggregation of human washed platelets. When the PPP reconstituted plasma system is added, its platelet-inducing activity can gradually recover, and within a certain range, the aggregation rate has a dose-effect relationship with the amount of PPP added, indicating that Protocetin-induced platelet aggregation requires the participation of plasma components. Blocking the GPIb site on the platelet membrane with the monoclonal antibody HIP1 inhibits the platelet-inducing activity of Protocetin, indicating that Protocetin-induced platelet aggregation is related to the GPIb receptor. The research results show that Protocetin acts on vWF and then activates platelets through GPIb, thereby inducing platelet aggregation.

[0094] Platelets are important members of the blood system, and their main physiological function is to participate in hemostasis and thrombus formation. GPIb is an important receptor on the platelet membrane and plays an important role in the process of platelets performing physiological functions. vWF is an important plasma component and is an important member in the coagulation function. GPIb is the receptor of vWF. Under physiological conditions of coagulation, the conformation of vWF changes and binds to GPIb, leading to platelet activation and aggregation, and then generating a procoagulant physiological function. Therefore, based on the fact that Protocetin can activate platelets through vWF and then through GPIb to cause platelet aggregation, it can be used to detect the function of vWF and the aggregation function of platelets, and thus can be clinically used to detect hemophilia and platelet-related diseases associated with abnormal GPIb structure and function.

[0095] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any change or replacement that can be thought of without creative work should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.

Claims

1. Preparation method of Protobothrops mucrosquamatus venom C-type protein, characterized in that, It includes the following steps: Step 1, molecular sieve gel filtration chromatography; The Sephadex G-75 gel chromatography column is 2.6×100 cm, balanced with PBS, phosphate buffered saline, 0.01 mol·L-1 Na2HPO4-NaH2PO4 buffer containing 0.147 mol / L NaCl, pH 7.4, and the flow rate is 24 ml·h-1; Weigh 0.5 g of the freeze-dried venom of Protobothrops mucrosquamatus, dissolve it in 3 ml of PBS, centrifuge the dissolved venom solution at 2000 rpm for 10 min, take the supernatant and load it onto the Sephadex G-75 gel column that has been balanced with PBS; After loading, elute with PBS at a flow rate of 24 ml·h-1, collect the eluate with an automatic collector, 4 ml / tube, measure the absorbance at 280 nm, and collect and combine the labeled eluted parts for the next separation and purification; Step 2, heparin affinity chromatography; Take the Sephadex G-75 chromatography active peak of Protobothrops mucrosquamatus venom, after desalting or dilution, load it onto the heparin affinity chromatography column, use PB buffer: 0.01 mol·L-1 Na2HPO4-NaH2PO4 buffer, pH 7.0, to balance the heparin affinity chromatography, perform gradient elution with PB buffer containing NaCl, the flow rate is 1 ml·min-1, collect the samples in 2 ml / tubes, measure the absorbance at 280 nm, and collect the active peak; Step 3, cation exchange chromatography; The HiTrap SP cation exchange pre-packed column is balanced with PB. After loading the active peak obtained by heparin affinity chromatography, after sufficient elution, perform gradient elution with PB buffer containing 1 mol NaCl, the flow rate is 1 ml·min-1, collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

2. The method according to claim 1, wherein The said Step 3 can be replaced with: Step 3, gel filtration chromatography Dissolve the freeze-dried powder of the active peak obtained by heparin affinity chromatography in 1.5 ml of PBS, centrifuge at 2000 rpm for 10 min, take the supernatant and load it onto the Sephacrycl S-100 gel chromatography column, elute with PBS buffer, the flow rate is 30 ml / h, collect the samples in 2 ml / tubes, and measure its absorbance at A280 nm; Collect the active peak, detect the purity by SDS-PAGE, perform protein quantification with a BCA protein quantification kit, and then store it in a -80 °C ultra-low temperature refrigerator for standby.

3. The preparation method according to claim 1 or 2, characterized in that, The original Protobothrops mucrosquamatus venom C-type protein: The molecular weight of the target protein was determined by 12% SDS-PAGE under reducing and non-reducing conditions, with a working voltage of 160 V. After electrophoresis, Coomassie Brilliant Blue R-250 staining was used; the molecular weights of the standard protein markers were phosphatase b - 97.2 kDa, bovine serum albumin - 66.4 kDa, ovalbumin - 44.3 kDa, carbonic anhydrase - 29.0 kDa, trypsin inhibitor - 20.1 kDa, and lysozyme - 14.3 kDa; Sephacrycl S-100 gel (1.0 cm × 60 cm) was used for molecular weight determination, and four standard proteins were used: Albumin - 67.0 kDa, Ovalbumin - 43.0 kDa, Chymotrypsinogen A - 25.0 kDa, and Ribonuclease A - 13.7 kDa; MALDI / TOF was used to determine the exact molecular weight of the target protein; Isoelectric focusing was used to determine the isoelectric point of the target protein under denaturing conditions. The pH range of the ampholyte Ampholine was 3.5 - 10, and the pH gradient of the gel after electrophoresis was determined by the KCl soaking method.

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