CN108822196B

CN108822196B - Procoagulant polypeptide LGTX-F2 and application thereof

Info

Publication number: CN108822196B
Application number: CN201810571442.XA
Authority: CN
Inventors: 容明强; 刘中华; 李朋朋
Original assignee: Hunan Shengda Biotechnology Co ltd
Current assignee: Chengdu Peide Biomedical Co ltd
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2023-04-21
Anticipated expiration: 2038-06-06
Also published as: CN108822196A

Abstract

The invention discloses application of polypeptide LGTX-F2 in preparation of a blood coagulation medicament. It contains 65 amino acid residues, has a molecular weight of 7444.51Da and an isoelectric point of 7.75, and has an amino acid sequence shown in SEQ ID 1. The polypeptide LGTX-F2 is expressed by adopting escherichia coli, has very obvious coagulation activity, and can be applied to the preparation of a coagulation medicament.

Description

Procoagulant polypeptide LGTX-F2 and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of procoagulant polypeptide LGTX-F2 in preparation of procoagulant medicines.

Background

Massive hemorrhage or excessive hemorrhage is one of the causes of high death in clinical operations, and the problem of local hemostatic drugs cannot be solved in the deep hemorrhage or complex hemorrhage process, and the deep coagulation of the organism needs to be stimulated to improve the hemostatic efficiency. The data show that the bleeding patients clinically treated annually in China are approximately 1000 ten thousand, and the bleeding patients are mainly concentrated in the operation departments and part of the medical departments. The hemostatic is an indisputable product in clinic, and has the characteristic of strong clinical compliance. With the improvement of surgical operation level and surgical operation treatment coverage rate in China and the supply and demand of organ transplantation, procoagulant and hemostatic medicines are indispensable medicines. Due to the huge clinical demand, the sales volume of the global main recombinant coagulation factor products is steadily increased in 2010-2015, and reaches 78.54 hundred million dollars in 2015.

Procoagulants commonly used in clinic today can be classified into: 1) Snake venom thrombin such as styptic and bangtin; 2) Procoagulant vitamins that promote clotting factor activity in blood, lyophilized human prothrombin complex, and the like; 3) Fibrinolysis resisting procoagulant such as tranexamic acid, skin inhibiting enzyme, etc., procoagulant such as carbazochrome sodium sulfonate, phenolsulfoethylamine, etc. acting on blood vessel, and Chinese herbal medicine preparation, etc. Among these, thrombin-like drugs are most commonly used, mainly three major species, the snake venom type thrombin, thrombin and prothrombin complex. It is counted that the most commonly used in China is the snake venom thrombin, i.e. hemagglutinase. The most common of the snake venom thrombin products are the snake venom from Bothrops atrox, basel in Switzerland, and the snake venom from red-mouth Pallas pit viper, devic Kono pharmaceutical factory, germany, which represents the product as standing hemostasis. Because of the advantages of quick acting, high efficiency, long acting, safety, convenience, no influence of plasma thrombin inhibitors, etc., the instant hemostasis has been widely used for treating and preventing various hemorrhagic diseases.

Hemostasis is a complex physiological process that is primarily affected by blood coagulation, platelet aggregation, and vasoconstriction. Generally, the formation of prothrombin activator, thrombin formation and fibrinogenesis can be divided into three phases. When bleeding occurs from vascular injury, the tissue's own coagulation mechanism is fully developed, the prothrombin activator activity is enhanced, formation is increased, and then prothrombin is converted into thrombin, which in turn catalyzes the conversion of fibrinogen into fibrin. Coagulation factors play a very important role in the coagulation process, and their coagulation modes are divided into an intrinsic coagulation pathway, an extrinsic coagulation pathway and a common coagulation pathway (fig. 1).

Many factors are inactive zymogens, and when a factor is activated, it is converted to a catalytically active enzyme to activate the next corresponding factor, e.g., factor XIIa, which is an active molecule of factor XII, and one factor is activated by both the previous factor and the corresponding next factor, which in addition has the effect of accelerating and amplifying the clotting reaction, whereby the clotting process occurs in a succession of faster and faster reactions until the completion of the clotting process. In the intrinsic coagulation pathway, FXII factor is first converted to active FXIIa to further activate XI to XIa, which further activates factor X through a cascade of reactions into the common coagulation pathway, ultimately promoting fibrin production.

Fig. 1: coagulation factor-mediated coagulation pathways include the intrinsic coagulation pathway, the extrinsic coagulation pathway, and the common coagulation pathway.

Coagulation factors have a very important role in coagulation, but most clinically used drugs are thrombin (snake venom factor), inhibiting activation of plasmin (6-aminocaproic acid) or increasing the number of platelets, enhancing platelet activity (phenolsulfoethylamine). The kaolin is a nonmetallic mineral product, and is clay and clay rock mainly comprising clay minerals of the kaolinite family. It has been found that kaolin can be directly contacted with activated FXII or FXI to initiate the intrinsic clotting pathway and thereby stop bleeding. Animal experiments prove that the animal treated by the kaolin hemostatic material has the characteristics of quick hemostasis, less side effects and high survival rate, so that the kaolin gradually occupies a place in the hemostatic material field. The kaolin hemostatic material is mainly from the U.S. Z-medical company, which utilizes the characteristic that kaolin contacts and activates FXII to activate an internal coagulation pathway, and develops a series of kaolin hemostatic materials aiming at different bleeding amounts and bleeding sites, including kaolin war wound gauze, kaolin intervention hemostatic bandages, kaolin tonsil hemostatic sponge, kaolin hemostatic pads and the like. The action mechanism and the good application of the kaolin also disclose the good application prospect of the blood coagulation factor XII in hemostasis. However, no molecules specifically enhancing the activity of factor II have been found so far, resulting in a lack of clinical understanding of the important role of factor XII in the clotting process, and a lack of hemostatic agents targeting factor XII. The spider is a large spider distributed in southwest of China, and the active ingredients in venom of the spider have not been intensively studied. The project discovers a polypeptide LGTX-F2 for promoting the activity of a coagulation factor from the spider venom, and successfully expresses the polypeptide in an escherichia coli system with high efficiency. LGTX-F2 can obviously shorten the blood coagulation time in the test of recalcification and APTT, and simultaneously has good hemostatic effect on a tail bleeding model of a mouse.

Disclosure of Invention

The invention aims at providing a procoagulant polypeptide LGTX-F2 and application thereof in preparing a coagulation promoting medicine.

The aim of the invention is achieved by the following technical scheme: the small molecule polypeptide AKK8 comprises 65 amino acid residues, has a molecular weight of 7444.51Da, an isoelectric point of 7.75, and an amino acid sequence shown as SEQ ID 1 of AKACTPRLHDCSHDRHSCCRGELFKDVCYCFYPEGEDKTEVCSCQQPKSHKYIEKVVDKTKTLVG.

The beneficial effects of the invention are as follows: the procoagulant polypeptide LGTX-F2 is expressed by escherichia coli, so that the procoagulant effect is obvious, and the procoagulant polypeptide LGTX-F2 can be applied to preparation of procoagulant medicines.

Drawings

FIG. 2 is the effect of procoagulant polypeptide LGTX-F2 on plasma re-calcifying experiments. (A) Plasma recalcification time gradually decreases with increasing dose of procoagulant polypeptide LGTX-F2, dosing gradients 0.75, 1.5 and 3 μm; bar graph of different dosing amounts. Physiological saline was used as a negative control and T-test was used for the difference significance analysis.

FIG. 3 is the effect of procoagulant polypeptide LGTX-F2 on APTT experiments. (A) LGTx-F2 dosing concentrations in the APTT experiments were 0.03, 0.3 and 3 μm and at doses of 0.3 and 3 μm were significantly different from the negative control. (B) APTT experimental histogram for different dosing amounts of LGTx-F2. FIG. 4 LGTx-F2 was effective in reducing bleeding in tail bleeding and liver bleeding models. (A) In the liver hemorrhage model, LGTx-F2 significantly reduced the amount of hemorrhage relative to the negative control group, while the positive group EACA did not significantly differ when administered at 5 mg/kg. (B) Liver hemorrhage model, negative control group and LGTx-F2 administration group (5 mg/kg). (C) In the tail bleeding model, LGTx-F2 (administered at a dose of 1.25 mg/kg) showed better hemostatic effect than the positive control EACA (administered at a dose of 5 mg/kg).

Detailed Description

The invention is further described below with reference to the accompanying drawings, the scope of the invention not being limited to the following:

example 1: preparation of procoagulant polypeptide LGTX-F2

Construction of prokaryotic expression vectors

Appropriate prokaryotic expression vectors are selected according to the size of the target protein to facilitate the subsequent isolation and purification steps. Finally, the expression of the protein using a PET-32a prokaryotic expression vector was determined. Considering that the expression efficiency of the vector is high, and the molecular weight difference between the linker protein and the target protein is large after the fusion protein (the linker protein 18 KDa+the target protein 7 KDa) is subjected to TEV enzyme digestion, the subsequent separation and purification are easy. Finally, the dual restriction sites of the prokaryotic expression vector are EcoR I (-GG A TTCC-) and Hind III (-CA A GACTTG-) and the rTEV restriction site (-ENLYFQ G-) are used for determining the restriction sites, and the plasmid of the prokaryotic expression vector is synthesized by Nanjing Jinsrey company.

Transformation of prokaryotic expression vector plasmid

The synthesized plasmid was transformed into BL21 competent cells for subsequent inducible expression. The conversion steps are as follows:

1. competent cells were thawed on ice for about 30min. 1 μl of the constructed prokaryotic expression vector plasmid was added to the competent cell suspension, gently beaten with a pipette to mix the contents, and left in an ice bath for 30 minutes.

2. The centrifuge tube was quickly transferred to a 42 ℃ water bath for 90 seconds, kept stable, and then quickly transferred to an ice bath for 2-3 minutes, taking care not to shake the centrifuge tube.

3. 1ml of sterile and antibiotic-free LB medium was added to the centrifuge tube, and the culture was performed at 37℃for 1 hour with shaking at 180rpm, to activate the cells.

4. After 4000g of the activated cells, a part of the supernatant was centrifuged for 5 minutes, and about 100. Mu.l of the supernatant was kept, and the pellet was blown and suspended by a pipette. Spread on LB plate containing AMP resistance, and cultured upside down at 37℃for 16-18 hours.

Prokaryotic expression of polypeptide LGTx-F2

1. Induction of prokaryotic protein expression

And (3) picking a plurality of single colonies obtained after transformation into a culture medium by using an inoculating loop, and sending the single colonies to Boshang sequencing company for sequencing to confirm that the transformed plasmids are error-free, so as to ensure that the subsequent experiments are successful. Single colonies sequenced correctly were inoculated into LB medium supplemented with AMP (final concentration 100. Mu.g/ml) and shake cultured overnight at 37℃at 180 rpm.

After overnight culture of the bacterial liquid, examination of prokaryotic expression induction conditions was performed. The induction temperature was set at three concentration gradients of 20 ℃,28 ℃ and 37 ℃ and the inducer IPTG concentration was three concentration gradients of 0.5mM, 1mM and 1.5 mM. The induction temperature of 28℃and the final concentration of the inducer of 1mM were finally determined as the optimum induction conditions. The method comprises the following specific steps:

the bacterial liquid after overnight culture is inoculated into 1L of fresh LB culture medium (AMP resistance) according to the proportion of 1:100, shake culture is carried out for about 5-6 hours at 37 ℃ and 180rpm, a small amount of bacterial liquid is taken during shake culture, a spectrophotometer is used for detecting OD600, after the bacterial liquid reaches 0.8-1.0, IPTG (the final concentration is 1 mM) is added for induction expression, and the bacterial liquid is induced overnight at 28 ℃ (about 10-12 hours). 1ml of bacterial liquid is respectively taken before and after induction, 5000g is centrifuged for 5min, the supernatant is discarded, 100 mu l of water is added for heavy suspension, 20 mu l of 5 XLoding Buffer is added for blowing and mixing uniformly, boiling water bath is carried out at 100 ℃ for 5min, 5-10 mu l is taken for 12% SDS-PAGE electrophoresis, and whether the protein is successfully expressed by induction is detected.

2. Ultrasonic disruption of prokaryotic expression of proteins

Centrifuging overnight induced expression thallus at 8000 rpm for 10 min, enriching thallus, discarding supernatant, re-suspending and rinsing twice with ultrapure water, centrifuging to remove supernatant, re-suspending thallus precipitate with Binding Buffer (1L thallus is re-suspended by about 100 ml BindingBuffer), performing ultrasonic pyrolysis (ultrasonic 3sec, stopping for 3sec, 10 min, standing on ice for 30min, repeating the above ultrasonic steps once), centrifuging at 12000 rpm for 10 min, collecting supernatant, and filtering the collected supernatant with 0.45 μm filter membrane to prevent macromolecular particles from blocking the column. All the above steps were performed on ice or at 4 ℃. Taking a small amount of thallus lysate supernatant and sediment, adding Loading Buffer, boiling water bath for 5min, performing 12% SDS-PAGE electrophoresis, detecting whether the target protein is expressed in the supernatant or in thallus sediment, if a large amount of target protein exists in the supernatant, discarding a small amount of protein in thallus sediment, if a large amount of target protein exists in thallus sediment, re-suspending and splitting by using Binding Buffer containing 6M urea, ice-bathing for 1h, centrifuging at 16000 rpm for 30min, and filtering by a 0.45 μm filter membrane.

3. His-Tag affinity chromatography of prokaryotic expression of protein

The protein solution filtered by the 0.45 mu m filter membrane is uniformly flowed through a His Bind Resin affinity column, and the target protein is enriched and purified. The method comprises the following specific steps:

and (3) column loading: a volume of about 6ml of the affinity Resin suspension was applied to the column with a pipette, and after naturally draining the liquid therein, a volume of about 3ml of His Bind Resin affinity column remained.

Rinsing: the affinity column was washed with 5-10 volumes of deionized water.

If a newly prepared nickel column is adopted, a Binding Buffer balancing affinity column with 5-10 times of column volume is directly used; if the regenerated nickel column is adopted, 5-10 times of column volume Strip Buffer is added for removing nickel, 5-10 times of column volume deionized water is added for rinsing, 5-10 times of column volume Charge Buffer is added for re-hanging nickel, and the affinity gel is washed to be obvious blue.

Balance: the affinity column was equilibrated with 5-10 column volumes Binding Buffer.

Loading: after the affinity column is well balanced, loading the sample at a flow rate of one drop every 6-8 seconds, enabling the expressed protein solution to uniformly flow through the affinity column, and collecting the protein solution after passing through the affinity column for electrophoresis detection.

Eluting: washing the affinity column with 10-20 times of column volume Binding Buffer to wash off the impurity protein, and collecting eluent; then, washing Buffer with 5-10 times of column volume is used for further removing the foreign proteins, and eluent is collected; finally, the target protein hung on the nickel column is eluted by using an ElutingBuffer, 10 parts of the target protein are added for ml Eluting Buffer elution, and the target protein is collected by a separate tube, and one tube is 2ml. The eluate from each step was taken in small amounts and subjected to 12% SDS-PAGE to trace the location of the target protein.

Mu.l of target protein eluent is taken, protein concentration is detected by Nano Drop (alternatively protein quantification is carried out by BCA protein quantification kit), and an Eluting Buffer is used as a Blank control.

4. rTEV cleavage of fusion proteins

rTEV cleavage of the eluted target protein was performed according to the following system:

fusion protein: 1000 mug

250×rTEV Protease Buffer 4μl

rTEV protease 2. Mu.l

Constant volume to 1000. Mu.l

Enzyme cutting conditions: the mixture was digested overnight in a water bath at 30 ℃. After digestion, a small amount of protein samples before and after digestion were subjected to 12% SDS-PAGE to observe digestion effect.

5. Separation and purification of target protein

Predicting the target protein PI value according to software EditSeq as follows: 7.72, rapid protein liquid chromatography (FPLC) separation and purification using Resource S ion exchange column (GE) was determined. The buffer used for FPLC to Resource S ion exchange column was:

Buffer A：20 mM MES PH=6.0

Buffer B：20 mM MES,1M NaCL PH=6.0

flow rate: elution gradient of 1.5 ml/min: 1min/gradient detection wavelength: 280/215 nm

All using buffers were filtered through 0.22 μm filters and sonicated to remove bubbles.

And (3) carrying out gradient dialysis and desalination on the prokaryotic expression protein solution after enzyme digestion, filtering by a 0.22 mu m filter membrane, and then injecting the filtered prokaryotic expression protein solution into FPLC, collecting each elution peak, and detecting and confirming the peak of the target protein.

Example 2: procoagulant polypeptide LGTX-F2 for shortening recalcitrant and APTT time

APTT test method:

the APTT reagent is balanced to room temperature, the APTT reagent is mixed by gently reversing, and simultaneously the CaCl2 solution is placed in a water bath kettle or a constant temperature incubator to be preheated to 37 ℃. The final concentration of the incubated samples was 0nM, 30nM, 0.3. Mu.M, 3. Mu.M.

APTT (advanced paratactic test) experiment method

Table 3-1 The method of APTT experiment

Figure CZASJCELN6XQ1CY4OKSUV4CO7HGTZJIAKFRWYVHS

Plasma decalcification experimental method:

mu.L of normal human plasma (anticoagulated with 3% sodium citrate) was combined with 3. Mu.L of sample (final incubation concentrations of 0. Mu.M, 0.75. Mu.M, 1.5. Mu.M, 3. Mu.M). Mix in Hepes buffer (150 mM NaCl,pH7.5) to a final volume of 70. Mu.L. Then incubated in a constant temperature incubator at 37℃for 10 min. After the incubation, 50. Mu.L of a pre-warmed CaCl2 solution (25 mM) at 37℃was added to initiate the reaction. Enzymatic kinetics of the clotting reactions OD650nm,20 min, 47s apart, three replicates were detected using an Epoch (BioTek) microplate reader, GEN CHS 1.09 software.

Then incubated in a constant temperature incubator at 37℃for 10 min. After the incubation, 50. Mu.L of a pre-warmed CaCl2 solution (25 mM) at 37℃was added to initiate the reaction. Enzymatic kinetics of the clotting reactions OD650nm,20 min, 47s apart, three replicates were detected using an Epoch (BioTek) microplate reader, GENCHS 1.09 software.

Example 3: procoagulant polypeptide LGTX-F2 for shortening tail bleeding time

Experimental method for mouse tail hemorrhage model:

20-25g of Kunming mice were grouped into negative control groups (sample), dosing groups (0.625 mg/kg, 1.25mg/kg, 2.5 mg/kg) and positive control groups (5 mg/kg EACA), 6 each, randomly allocated, male and female halves. 15min before tail shearing, 100. Mu.l of saline, 100. Mu.l of sample (0.625 mg/kg, 1.25mg/kg, 2.5 mg/kg) and 100. Mu.l of EACA (5 mg/kg) were injected intravenously at the tail, respectively. After the dosing treatment, the tail is cut by a blade to be immediately in 37 ℃ constant temperature physiological saline solution, the tail bleeding starts to time, the bleeding is stopped, the time is stopped, the bleeding is again generated within 120 seconds, the time is continued, the process is repeated until the bleeding is stopped, the experiment is finished, and only the accumulated total time of the bleeding is recorded. Experimental data were statistically analyzed and the difference significance was analyzed using SPSS software.

Example 4: procoagulant polypeptide LGTX-F2 for shortening liver bleeding time

The experimental method of the mouse liver hemorrhage model comprises the following steps: 20-25g of Kunming mice were grouped into negative control groups (sample), dosing groups (1.25 mg/kg, 2.5mg/kg,5 mg/kg) and positive control groups (5 mg/kg EACA), each group of 7 animals were randomly assigned, and each male and female were half. After anesthetizing the mice with 3% sodium pentobarbital solution, 100 μl of sample (1.25 mg/kg, 2.5mg/kg,5 mg/kg) and 100 μl of EACA (5 mg/kg) were injected into tail veins, respectively, after 15min of drug addition treatment, the mice were opened in the abdominal cavity by surgical scissors, the liver lobes were exposed, 5mg of liver tissue was cut at the lower edge of the right lobe of the liver, after 20min at room temperature, the abdominal cavity of the mice was sequentially lavaged with 4ml of physiological saline solution, and the lavage fluid was sucked with a pipette, and after combining, the number of red blood cells was recorded with a blood cell counting plate. Experimental data were statistically analyzed and the difference significance was analyzed using SPSS software.

Sequence listing

<110> Hunan university of teachers and students

<120> a procoagulant polypeptide LGTX-F2 and uses thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 65

<212> PRT

<213> Lycosa grahami

<400> 1

Ala Lys Ala Cys Thr Pro Arg Leu His Asp Cys Ser His Asp Arg His

1 5 10 15

Ser Cys Cys Arg Gly Glu Leu Phe Lys Asp Val Cys Tyr Cys Phe Tyr

20 25 30

Pro Glu Gly Glu Asp Lys Thr Glu Val Cys Ser Cys Gln Gln Pro Lys

35 40 45

Ser His Lys Tyr Ile Glu Lys Val Val Asp Lys Thr Lys Thr Leu Val

50 55 60

Gly

65

Claims

1. The procoagulant polypeptide LGTX-F2 is characterized in that the procoagulant polypeptide LGTX-F2 comprises 65 amino acid residues, has a molecular weight of 7444.51Da and an isoelectric point of 7.75, and has an amino acid sequence shown as SEQ ID: 1.

2. Use of a procoagulant polypeptide LGTX-F2 according to claim 1 for the preparation of a procoagulant medicament.