CN108395475A - A kind of hirudin isolation and purification method based on affinity chromatography - Google Patents

Abstract

The invention discloses a kind of hirudin isolation and purification method based on affinity chromatography, after physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, by the leech saliva sample of acquisition, precooling trichloroacetic acid extracting solution mixing is added, it extracts, centrifuging and taking supernatant, the acetone mixing that precooling is added after adjusting pH3.0 5.0 is stood overnight, recycle acetone in supernatant, centrifugation, takes precipitation, spare；Distilled water is added in the precipitation, fully dissolves, adjusts pH 6.0 8.0, it is isolated and purified with fibrin ferment Sepharose 4FF affinity columns, high performance liquid chromatography detection leech cellulose content, fetch water leech cellulose content peak value at refined solution, above-mentioned refined solution is dialysed, and it repeats to add water 24 times in the process, continue 10 15h, the supernatant after ultrafiltration is subjected to rotary evaporation, after removing moisture removal 70 80%, it takes concentrate freeze dryer to be lyophilized, obtains hirudin product.The natural hirudin purity of the method for the present invention purifying gained is high, and activity is high, at low cost, easy to operate, easily repeats.

Description

A method for separation and purification of hirudin based on affinity chromatography

technical field

The invention relates to a method for separating and purifying hirudin, which belongs to the field of biomedical separation and purification.

Background technique

Studies have found that the secretion of leech salivary glands contains hirudin and other biologically active substances and has broad application prospects in medicine. Hirudin is an acidic polypeptide secreted by the salivary glands of leeches. It contains 65-66 amino acid residues and mainly has three isomers, HV1, HV2, and HV3. They have high structural homology. Hirudin is the strongest thrombin-specific inhibitor found so far. The activity of engineered strains expressing hirudin (HV3) protein depends on the intensity of antithrombin activity. It forms a non-covalent A stable complex with tightly bound bonds. In 1984, Haycraft found that leech extract had anticoagulant effect. In 1904, Jocoby isolated the active ingredient from medical leech for the first time and named it hirudin. In 1955, Markwardt successfully isolated and purified pure hirudin from the head of the medical leech, and began basic biochemical and pharmacological research. In 1970, he determined that hirudin was a specific inhibitor of thrombin. In 1986, Harvey et al obtained the cDNA of hirudin, and then the hirudin gene was successfully expressed in various hosts.

Hirudin is a very stable protein, which is extremely stable in a dry state, and its activity will not be affected by a simple increase in temperature (100°C water bath) or a change in pH value (1.47-12.9). It is also very stable in the presence of it, and its activity begins to decline only when the temperature and pH value increase at the same time. Generally, hirudin can exist stably in water for 6 months at room temperature, and will not be destroyed by heating at 80°C for 15 minutes. Increasing the pH of a solution decreases its stability. At 20°C, in 0.1mol/LHCl or 0.1mol/LNaOH, it can be stable for 15 minutes, and it will be completely inactivated after being treated at 80°C for 15 minutes under the condition of pH=13.

In 1955, Markwardt of Germany isolated relatively pure hirudin for the first time from medical leeches. Since then, the purification and standardization technology of hirudin has been perfected day by day. So far, there have been many related research reports and patents. Most of the work is to use the head or even the whole body of European medical leech as raw material, dehydrate with ethanol, and then add acetone to precipitate a series of steps to obtain crude hirudin. In 1970, Markwardt obtained pure hirudin by ethanol fractional precipitation, cation exchange, gel filtration and anion exchange. In 1985, WdsmaM and Marhyardt improved the purification method and obtained high-purity hirudin by combining ion exchange and affinity chromatography. In 1986, Johns et al. reported a five-step purification method in which a new technique of high performance liquid chromatography was used.

The current method for extracting hirudin is mostly to grind or dry the leeches and extract them with water, ethanol, ultrafiltration or chromatography. However, the above-mentioned method has many shortcomings, mainly because the active ingredient is not high after extraction, the activity is generally about 100-4000ATU/g, the yield is between 0.27-4.47%, and the activity varies according to different extracts, impurities The content of hirudin is high, so it is not suitable for injection medicine. Some extraction methods are complicated, and some methods are relatively slow, so they are not suitable for industrial production. Therefore, many people are still studying the extraction methods of hirudin, hoping to find high efficiency, low cost, Extraction method with high active ingredients.

Contents of the invention

In view of the above problems, the present invention provides a method for separating and purifying hirudin, which is simple in operation and high in efficiency, and simultaneously can obtain hirudin with high purity and high activity.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for separating and purifying hirudin based on affinity chromatography, comprising the steps of:

(1) Crude extraction of hirudin: After feeding the living leech with physiological saline, physically squeeze it to make it secrete saliva and collect it, add 2-4 times (V:V) pre-cooled trichloroacetic acid extract to the obtained leech saliva sample Mix well, extract for 2-6 h, centrifuge at 8000-10000 r/min for 10-20 min, take the supernatant, adjust the pH to 3.0-5.0 with HCL, add 4 times pre-cooled acetone, mix well and let stand overnight, recover the supernatant Medium acetone, centrifuge at 8000-10000 r/min for 10-20 min, take the precipitate and set aside;

(2) Separation and purification: add the above precipitate to 4-6 times distilled water, fully dissolve, adjust the pH to 6.0-8.0 with HCL, separate and purify with thrombin Sepharose 4FF affinity column, elute, and detect the content of hirudin by high performance liquid chromatography. Take the purified solution at the peak of the hirudin content, and set aside;

(3) Desalination and concentration: dialyze the above purified solution, and add water 2-4 times during the process for 10-15 hours, and perform rotary evaporation on the supernatant after ultrafiltration (30-60 ℃, 0-180 r/min), after removing 70-80% of the water, take the concentrate for later use;

(4) Take the concentrated solution and freeze-dry it in a freeze dryer, collect the product and seal it for storage to obtain the hirudin product.

Hirudin activity detection: Markwardt thrombin direct titration method to detect its anticoagulant activity.

Crude product content detection: add 2 times distilled water to secreted saliva, vibrate and mix, and detect hirudin content by HPLC.

The carrier activator in the thrombin Sepharose 4FF affinity column in the step (2) is carbonyldiimidazole, and the carrier activation solvent includes but not limited to DMSO and dioxane, among which DMSO is preferred.

The preparation method of thrombin Sepharose 4FF medium is as follows: take Sepharose 4FF with a wet weight of 10.0 g in a Buchner funnel, wash it repeatedly with deionized water, add 25%, 50%, and 50% of 75% DMSO aqueous solutions to the medium successively. mL, then put Sepharose 4FF into an anhydrous DMSO (dimethyl sulfoxide) Erlenmeyer flask containing 20 mL, add 0.5-1.0 g CDI (carbonyldiimidazole) and shake at 100 r/min for 15 min, washed repeatedly with deionized water to obtain activated Sepharose 4FF; another 50 mg-100 mg thrombin was dissolved in 50 mL phosphate buffer (0.2mol/LK ₂ HPO ₄ -0.01 mol/L KCL, pH8. 0), and the above-mentioned activated Sepharose 4FF was added to the phosphate buffer containing thrombin, 4-8°C, 50-80 r/min, stirring and reacting for 10-15 h, followed by deionized water, 1.0 mol/ L KCL, deionized water, 0.1 mol/L HCL, deionized water (all 4-6 times the volume) for suction filtration and washing, and finally put it in phosphate buffer and store it at 4°C, the obtained thrombin Sepharose 4FF medium .

In the step (2), the column packing method is as follows: take 1/2-3/4 times the volume of the separation column of thrombin Sepharose 4FF medium and install it in the separation column, and use 15 times the volume of the column bed of phosphate with a pH of 7.4-8.4 Flush the column bed with buffer solution at a flow rate of 3-5mL/min, and wash to equilibrium.

The eluent used for elution in the step (2) is 0.01-0.03 mol/L KCL—0.1-0.3 mol/L HCL, and the elution rate is 1 ml/3 min per tube.

The detection conditions of HPLC in the step (2): chromatographic column: C18 10×200mm, detector: UV210nm, mobile phase: methanol:water=3:1, column temperature: 25°C, sample volume: 10 μL, Flow rate: 1 mL/min.

The Markwardt thrombin direct titration method detects its anticoagulant activity principle as follows:

The component with the highest anticoagulant activity in leech saliva is generally considered to be hirudin; hirudin can specifically bind to thrombin, and its binding ratio is 1:1. The principle of inactivating thrombin is to use Matkwardt thrombin The direct titration method can detect the activity of natural hirudin; the measurement unit of hirudin is the international unit, expressed in ATU, and the international unit of thrombin activity is NIH, that is, 1 ATU is equal to the amount of hirudin that neutralizes 1 NIH thrombin.

The Markwardt thrombin direct titration method detects its anticoagulant activity and the specific operations are:

Take about 0.1 g of the powder of this product, weigh it accurately, accurately add 500 μL of 0.9% normal saline solution, stir and dissolve thoroughly, prepare a 0.2 g/mL test solution, accurately measure 100 μl of the supernatant, put it in a 1.5 mL test tube, and add 0.5 Tris hydrochloride buffer solution of pig fibrinogen (take 25ml of 0.2m0l/L tris solution and about 40ml of 0.1m0l/L hydrochloric acid solution, add water to 100ml, adjust the pH value to 7.4) 200μl , shake well, place in a water bath (37°C±0.5°C) and slowly add thrombin solution containing 40U (unit) per 1ml (middle) dropwise (5μl/min, shake gently while adding) until solidified, record The volume of thrombin solution consumed is calculated according to the following formula:

U=C1V1/C2V2W

In the formula: U---unit of thrombin activity per 1g, i.e. hirudin content, U/g;

C1---concentration of thrombin solution, μ/ml;

C2---the concentration of the test solution, g/ml;

V1---consumed volume of thrombin solution, μl;

V2---the amount of the test solution added, μl;

The above method for detecting the content of hirudin is also applicable to the quality monitoring of various preparations made from blood-sucking leeches as the main raw material.

Beneficial effect:

The purified natural hirudin obtained by the method of the invention has high purity, high activity, low cost, simple operation and easy repetition, and can be industrialized. The natural hirudin products obtained by the process can be used as raw materials for food, health care products, medicines or cosmetics. They have anticoagulant and thrombolytic effects, improve blood circulation, and promote metabolism. They are clinically used to prevent and treat cardiovascular and cerebrovascular diseases, such as stroke , coronary heart disease, hyperlipidemia, etc.

Detailed ways

The present invention can be better understood from the following examples. Then, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention.

Example 1

In this example, the preparation method of the thrombin adsorbent in the thrombin Sepharose 4FF affinity column is as follows: take Sepharose 4FF (purchased from Shanghai Rongjun Biomedical Technology Co., Ltd.) with a wet weight of 10.0 g in a Buchner funnel, Wash repeatedly with deionized water, add 25%, 50%, and 75% DMSO aqueous solutions to the medium in turn, each 50 mL, then put Sepharose4FF into a 20 mL anhydrous DMSO (dimethyl sulfoxide) Erlenmeyer flask, add Shake 0.5-1.0 g CDI (carbonyldiimidazole) at 30°C with a constant temperature oscillator at 100 r/min for 15 min, and wash repeatedly with deionized water to obtain activated Sepharose4FF; another 50 mg-100 mg thrombin (purchased (Lanzhou Institute of Biological Products Co., Ltd.) was dissolved in 50 mL of phosphate buffer (0.2mol/L K ₂ HPO ₄ -0.01 mol/L KCL, pH8.0), and the activated Sepharose 4FF was added to the thrombin-containing In the phosphate buffer solution, 4-8 ℃, 50-80 r/min speed, stirring reaction for 10-15 h, according to deionized water, 1.0 mol/L KCL, deionized water, 0.1 mol/L HCL, deionized water (All 4-6 times the volume) were filtered and washed, and finally stored in phosphate buffer at 4°C, the obtained thrombin Sepharose 4FF medium.

The column packing method is as follows: take 2/3 times the volume of the separation column of Sepharose 4FF medium and install it on a separation column with a size of 20 x 100 mm, and use 15 times the volume of the column bed with pH 8.0 phosphate buffer at 3-5mL/min Flush the column bed at the flow rate and wash to equilibrium.

A method for separating and purifying hirudin, the concrete steps are as follows:

(1) Crude extraction of hirudin: After feeding the living leech with physiological saline, physically squeeze it to make it secrete saliva and collect it, add 3 times (V:V) pre-cooled trichloroacetic acid extract to the obtained leech saliva sample and mix well , extract for 2 h, centrifuge at 10,000 r/min for 20 min, take the supernatant, adjust the pH to 4.0 with HCL, add 4 times pre-cooled acetone, mix and let stand overnight, recover the acetone in the supernatant, centrifuge at 10,000 r/min for 20 min, and take the precipitate ,spare;

(2) Separation and purification: add the above precipitate to 5 times of distilled water, fully dissolve, adjust the pH to 7.0 with HCL, separate and purify with thrombin Sepharose 4FF affinity column, and elute with 0.025 mol/L KCL-0.2 mol/L HCL. The size can not only maintain the activity of hirudin and the stability of the affinity medium, but also effectively separate and elute hirudin. The elution speed is 1ml/3 min per tube. Purified solution, spare;

(3) Desalination and concentration: Dialyze the above-mentioned purified solution, the dialysis membrane is a dialysis bag with a molecular weight cut-off of 7000 or less, and add water 4 times during the process for 10 hours, and perform a rotary evaporation operation on the supernatant after ultrafiltration ( 30-60 ℃, 0-180 r/min), after removing 70% of the water, take the concentrated solution for later use;

(4) Freeze-dry the concentrated liquid in a freeze dryer, collect the product and store it in a sealed container;

(5) Activity detection: Markwardt thrombin direct titration method was used to detect its anticoagulant activity.

(6) Detection of crude product content: secrete saliva, add 2 times of distilled water, vibrate and mix, and detect the content of hirudin by HPLC.

The hirudin purified product obtained by the method is detected by the Markwardt thrombin direct titration method to have an activity of 7350 ATU/g of the hirudin, and the yield is 7.51%, and the hirudin with high purity and activity is obtained.

Example 2

The present invention provides the influence of different carrier activators on the separation and purification of hirudin, and the specific steps are as follows:

On the basis of Example 1, different carrier activators (CDI, cyanogen bromide, epichlorohydrin) were used to treat and activate Sepharose 4FF.

This method gains: 0.8 g CDI activates Sepharose 4FF and carries out the activity of the hirudin obtained in the next step experiment is 6937ATU/g, and the yield is 7.37%; 1.0 g cyanogen bromide (optimum addition amount) carries out the next step after activating Sepharose 4FF The activity of the hirudin obtained in the experiment was 4219ATU/g, and the yield was 2.19%; the activity of the hirudin obtained in the next experiment after 130 μmol/L epichlorohydrin activation (optimum concentration) Sepharose 4FF was 5231ATU/g, yielding The rate is 3.51%. Therefore, the carrier activator CDI used in this experiment is an activator with good effect, which can provide accurate scientific basis for the activation of agarose gel.

Example 3

The present invention provides the influence of different carrier activator solvents on the separation and purification of hirudin, and the specific steps are as follows:

On the basis of Example 1, different carrier activation solvents (DMSO, dioxane, acetone, ethanol, isopropanol) were used to treat and activate Sepharose 4FF.

The result of this example: the activity of the hirudin obtained in the next experiment after using DMSO as the solvent is 7524 ATU/g, and the yield is 7.28%; the activity of the hirudin obtained in the next experiment after using dioxane as the solvent is 6424 ATU/g, the productive rate is 3.18%; the activity of the hirudin obtained in the next step experiment after using acetone as the solvent is 3018 ATU/g, and the productive rate is 2.31%; the next step experiment obtained after using ethanol and isopropanol The activities of hirudin were 4359 ATU/g and 4893 ATU/g, and the yields were 2.09% and 1.68%, respectively. Therefore, the carrier activator and dissolving agent DMSO used in this experiment is a kind of activator with good effect, which can provide accurate scientific basis for the activation of agarose gel.

Example 4

A kind of method for separating and purifying hirudin provided by the present invention, concrete steps are as follows

(1) Crude extraction of hirudin: After feeding the living leech with physiological saline, physically squeeze it to make it secrete saliva and collect it, add 3 times (V:V) pre-cooled trichloroacetic acid extract to the obtained leech saliva sample and mix well , extract for 2 h, centrifuge at 10,000 r/min for 20 min, take the supernatant, adjust the pH to 4.0 with HCL, add 4 times pre-cooled acetone, mix and let stand overnight, recover the acetone in the supernatant, centrifuge at 10,000 r/min for 20 min, and take the precipitate ,spare;

(2) Separation and purification: add the above precipitate to 5 times of distilled water, fully dissolve, adjust the pH to 6.5 with HCL, separate and purify with thrombin Sepharose 4FF affinity column, elute with 0.025 mol/L KCL-0.2 mol/L HCL, and elute The speed is 1 ml/3 min per tube, the collected liquid is detected by high performance liquid chromatography for hirudin content, and the purified liquid at the peak of hirudin content is taken for later use;

(3) Desalination and concentration: dialyze the above purified solution, and add water 4 times during the process for 10 hours, and perform rotary evaporation on the supernatant after ultrafiltration (30-60 ℃, 0-180 r/min) , after removing 75% of the water, take the concentrated solution for subsequent use;

(4) Freeze-dry the concentrated liquid in a freeze dryer, collect the product and store it in a sealed container;

(5) Activity detection: Markwardt thrombin direct titration method was used to detect its anticoagulant activity.

(6) Detection of crude product content: secrete saliva, add 2 times of distilled water, vibrate and mix, and detect the content of hirudin by HPLC.

The hirudin purification product that this method obtains is through adopting Markwardt thrombin direct titration method to detect that the activity of this product hirudin is 7518ATU/g, and productive rate is 6.79%, compared with prior art, the product yield of the present invention and final Activity has been greatly improved.

Claims (8)

1. a hirudin separation and purification method based on affinity chromatography, is characterized in that, comprises the following steps: 1) Crude extraction of hirudin: After feeding the living leech with physiological saline, physically squeeze it to make it secrete saliva and collect it, extract the obtained leech saliva sample, centrifuge, take the precipitate, and set aside; 2) Separation and purification: Add the precipitate described in step 1) into distilled water, fully dissolve, adjust the pH to 6.0-8.0, separate and purify with thrombin Sepharose 4FF affinity column, detect the hirudin content by high performance liquid chromatography, and take the peak value of hirudin content Purified solution, spare; 3) Desalination and concentration: Dialyze the above purified solution, and add water 2-4 times during the process, last for 10-15 hours, perform rotary evaporation on the supernatant after ultrafiltration, remove 70-80% of water, and take the concentrated solution spare; 4) Freeze-dry the concentrated solution in a freeze dryer to obtain the hirudin product. 2. A method for separating and purifying hirudin based on affinity chromatography according to claim 1, characterized in that in step 1), HCL is used to adjust the pH to 3.0-5.0 to inactivate some impurity proteins without affecting activity of the target product. 3. A method for separating and purifying hirudin based on affinity chromatography as claimed in claim 1, characterized in that step 1) the extraction step is: adding pre-cooled trichloroacetic acid extract and mixing, extraction, centrifugation Take the supernatant, adjust the pH to 3.0-5.0, add pre-cooled acetone, mix and let stand overnight, recover the acetone in the supernatant, centrifuge, and take the precipitate. 4. A method for separating and purifying hirudin based on affinity chromatography according to claim 1, characterized in that, in step 2) the carrier activator in the thrombin Sepharose 4FF affinity column is carbonyldiimidazole. 5. A method for separating and purifying hirudin based on affinity chromatography as claimed in claim 1, characterized in that the column packing method in step 2) is: take 1/2-3/4 times the column volume of thrombin The Sepharose 4FF medium is installed in the separation column, and the column bed is washed with 15 times the volume of the column bed volume of pH 7.4-8.4 phosphate buffer at a flow rate of 3-5mL/min, and washed to equilibrium. 6. A method for separating and purifying hirudin based on affinity chromatography as claimed in claim 1, wherein the eluent in step 2) is (0.01-0.03) mol/L KCL—(0.1-0.3) mol/L HCL, the elution rate is 1 ml/3min per tube. 7. A method for separating and purifying hirudin based on affinity chromatography as claimed in claim 2, characterized in that, the detection conditions of high performance liquid chromatography in step (2): chromatographic column: C18 10×200mm, detector: UV 210nm, mobile phase: methanol: water = 3:1, column temperature: 25°C, injection volume: 10μL, flow rate: 1mL/min. 8 . The method for separating and purifying hirudin based on affinity chromatography according to claim 1 , wherein the dialysis membrane used in the dialysis in step 3) is a dialysis bag with a molecular weight cut-off of 7,000 or less.