CN110151977A - A method for protecting the stability and activity of aFGF protein drugs - Google Patents

Abstract

The invention belongs to the technical field of medicine, and specifically discloses a method for protecting the drug stability and activity of aFGF protein. Step 1, expression of aFGF protein; step 2, purification of aFGF protein; step 3, desalting; ; Step 5, prepare aFGF protein protective agent; Step 6, mix aFGF protein and protective agent; Step 7, subpackage, cryopreservation. The method for protecting the drug stability and activity of the aFGF protein provided by the present invention can significantly improve the stability of the aFGF protein and protect its biological activity.

Description

A method for protecting the stability and activity of aFGF protein drugs

technical field

The invention belongs to the technical field of biological drugs, and specifically discloses a method for protecting the stability and activity of aFGF protein drugs.

Background technique

Acidic fibroblast growth factor (aFGF) is a trace active substance with extensive mitogenic effect on cells derived from mesoderm and neuroectoderm, mainly distributed in the brain, pituitary, nerve tissue, In organs or tissues such as retina, adrenal gland, heart and bone, other tissues are present in very low concentrations in serum and body fluids.

aFGF has a wide range of biological functions, which can affect the growth, differentiation and functions of various cells such as endocrine cells, nerve cells and mesenchymal cells. Because aFGF has many functions such as promoting damage repair, nourishing and protecting neurons, and promoting angiogenesis, it has become a research hotspot.

However, the stability of aFGF is poor. Many studies have shown that the thermal stability of recombinant aFGF protein (melting temperature 40°C-50°C) is much lower than that of low bFGF (melting temperature 55°C-60°C), and aFGF protein is in the body temperature environment. It is easily denatured and degraded, which affects its activity in the human body.

Heparin is a highly sulfated polysaccharide, which is mainly used for anticoagulation in clinical practice, and can protect aFGF protein and improve its stability and biological activity. However, heparin is mostly derived from pig intestinal mucosa, and its price is relatively high.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a method for protecting the stability and activity of aFGF protein drugs.

The method for protecting the drug stability and activity of aFGF protein provided by the present invention specifically includes the following steps:

Step 1. Expression of aFGF protein: After inserting the aFGF protein coding sequence (UniProt number: P05230, selected sequence: 16F-155D) into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of bacteria grows to 600nm absorbance of 0.4-0.8, the aFGF protein is induced to express by IPTG at 18°C for 12-18h, and the bacteria are collected by centrifugation;

Step 2. Purification of aFGF protein: Resuspend the bacteria collected in step 1 in phosphate buffer solution, and crush the aFGF protein by ultrasonic lysis. The crude aFGF protein is obtained by elution; the crude aFGF protein is separated by a nickel ion gel chromatography column, and the eluent 2 is used to elute to obtain a pure aFGF protein solution;

Described eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of described tris(hydroxymethyl)aminomethane in described eluent 1 is 50mmol/L, and described NaCl The mass concentration in the eluent 1 is 1.5 mol/L, and the pH of the eluent 1 is 7.2;

The eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of the tris(hydroxymethyl)aminomethane in the eluent 2 is 50mmol/L, and the imidazole The mass concentration in the eluent 2 is 0.3 mol/L, and the pH of the eluent 2 is 7.2;

Step 3. Desalting: put the pure aFGF protein solution obtained in step 2 into a dialysis bag to seal, then place the dialysis bag in a phosphate buffer solution, stir at 4°C for 4 hours, replace the PBS solution, and continue stirring for 4 hours , to obtain the aFGF protein solution after desalting;

Step 4. Adjust the concentration of the desalted aFGF protein solution;

Step 5, prepare protective agent solution;

The protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction, the mixed solution is composed of phosphate buffer and dextran sulfate, and the dextran sulfate is in the phosphate buffer. The concentration in is 1-10 times the concentration of aFGF protein solution adjusted in step 4;

Step 6. Mix the desalted aFGF protein solution in Step 4 with the dextran sulfate solution prepared in Step 5 in a volume ratio of 1:1-10 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -20～-80°C refrigerator.

Preferably, the centrifugal speed in step 1 is 8000 r/min.

Preferably, in step 3, the volume ratio of the pure aFGF protein solution to the phosphate buffer is 1:50-200.

Preferably, the concentration of the aFGF protein solution adjusted in step 4 is 5-400 μg/mL.

Preferably, the pH of the phosphate buffer solution is 7.4.

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

In the method for protecting the drug stability and activity of aFGF protein provided by the present invention, dextran sulfate is used as a protective agent for aFGF protein, and the dextran sulfate disaccharide unit contains 6 -O-SO ^3- , which is more efficient than heparin. Stronger negative charge, when the O atom in the sugar chain -O-SO ₃ ^- interacts with the H atom in the aFGF surface -NH ₃ ⁺ , it is easier to form OH hydrogen bonds, which has a better effect on improving the stability of aFGF protein In addition, the length of the dextran sulfate chain in industrial production can be regulated, including 500kDa (about 500 disaccharide units)-5kDa (about 5 disaccharide units), while the average molecular weight of the heparin polysaccharide chain is 18kDa (about 50 Disaccharide unit), it is not easy to control, and it is easier to standardize the production of dextran sulfate in industry, so the use of dextran sulfate as a protective agent for aFGF protein has great application prospects.

Description of drawings

Figure 1 is a graph showing the dissolution and denaturation curves of aFGF protein after adding different protective agents in molecular experiments; wherein, Figure A refers to heparin, B refers to λ-carrageenan, C refers to dextran sulfate, D refers to chondroitin sulfate; With the increase of polysaccharide concentration, the denaturation temperature curve of aFGF protein; the endpoint 0 refers to the blank control group;

Figure 2 is a histogram of the biological activity of aFGF protein after adding different protective agents in Example 3 and Comparative Examples 1-3 in the cell experiment; wherein, PBS refers to the blank control group without FGF, A refers to the aFGF group, and B refers to aFGF+dextran sulfate Glycogroups; 0, 1 and 2 refer to 0, 1 and 2 days of incubation at 37°C in a cell incubator, respectively.

Detailed ways

In order to enable those skilled in the art to better understand that the technical solutions of the present invention can be implemented, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

The experimental materials and instruments used in each embodiment and each comparative example are as follows:

1. Instrument

Heparin gel column: Bio-Rad, USA;

Nickel ion gel chromatography column: GE Healthcare, USA;

Magnetic stirrer: Shanghai Yiheng Scientific Instrument Co., Ltd.;

Protein concentration tubes, ultrafiltration centrifuge tubes: Merck Millipore, Germany;

Microspectrophotometer: Nanodrop Instruments, Thermal Scientific, USA;

Low temperature refrigerator: Haier medical refrigerator.

2. Reagents and materials

aFGF protein coding sequence: UniProt number: P05230, selected sequence: 16F-155D, the coding sequence was provided by LifeTechnologies;

Escherichia coli BL21: Beijing Quanshijin Biotechnology Co., Ltd.;

Isopropylthiogalactoside: Isopropylβ-D-Thiogalactoside, IPTG, Beijing Quanshijin Biotechnology Co., Ltd.;

Tris-Cl, NaCl, imidazole, glycerol, PBS buffer solution: Beijing Soleibao Technology Co., Ltd.;

Dialysis bag: 10kDa pore size, Thermal Scientific, USA;

Dextran sulfate, heparin, chondroitin sulfate: Sigma, USA.

Example 1

A method for protecting the drug stability and activity of aFGF protein, specifically comprising the following steps:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria was grown to 600 nm and the absorbance was 0.4, at 18 The aFGF protein was induced to express by isopropyl thiogalactoside for 12 h at ℃, and the bacteria were collected by centrifugation at 4 ℃ and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in PBS buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and crushing, and the bacterial lysate was adsorbed by heparin gel chromatography column. , remove most impurity protein, and obtain aFGF protein crude product by elution 1; aFGF protein crude product is separated by nickel ion gel chromatography column, and eluted with eluent 2 to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration is 1.5mol/L, and the pH of the eluent 1 is 7.2; the specific preparation method is: weigh an appropriate amount of tris(hydroxymethyl)aminomethane, dissolve in water, add an appropriate amount of NaCl, and make the tris(hydroxymethyl)aminomethane in the The mass concentration in the entire solution system is 50 mmol/L, so that the mass concentration of NaCl in the entire solution system is 1.5 mol/L, after stirring evenly, use hydrochloric acid to adjust the pH to 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3 mol/L, and the pH of the eluent 2 is 7.2; the specific preparation method is: weigh an appropriate amount of tris(hydroxymethyl)aminomethane, dissolve in water, add an appropriate amount of imidazole, and make the tris(hydroxymethyl)aminomethane in the whole solution. The mass concentration in the system is 50 mmol/L, so that the mass concentration of imidazole in the whole solution system is 0.3 mol/L, and after stirring, the pH is adjusted to 7.2 with hydrochloric acid;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of the pure aFGF protein solution obtained in step 2 into a dialysis bag, seal the dialysis bag, and then place the dialysis bag containing the pure aFGF protein solution into a dialysis bag filled with 1 L of PBS solution (pH is 7.4). In the beaker, stir with a magnetic stirrer for 4 hours at 4°C to complete the first desalting, then replace the 1L PBS solution again, and continue stirring for 4 hours to complete the desalting of the aFGF protein solution;

Step 4. Adjustment of aFGF protein concentration: use a protein concentration tube to measure the concentration of the desalted aFGF protein solution with a microspectrophotometer, and adjust its concentration to 5 μg/mL with PBS;

Step 5, prepare aFGF protein protectant;

The aFGF protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction. The mixed solution is composed of phosphate buffer (pH is 7.4) and dextran sulfate, and dextran sulfate is in the phosphate buffer. The concentration of 5μg/mL;

Step 6, mixing aFGF protein and protective agent: mixing the desalted aFGF protein solution in step 4 with the dextran sulfate solution prepared in step 5 in a volume ratio of 1:1 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -20°C refrigerator.

Example 2

A method for protecting the drug stability and activity of aFGF protein, specifically comprising the following steps:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria grew to the absorbance at 600 nm of 0.8, it was placed in the environment of 18°C. The aFGF protein was induced to express by IPTG for 18h, and the bacteria were collected by centrifugation at 4°C and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in phosphate buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and fragmentation, and the bacterial lysate was passed through a heparin gel chromatography column. Absorption to remove most of the impurity protein, and elution from eluent 1 to obtain aFGF protein crude product; aFGF protein crude product is separated by nickel ion gel chromatography column, and elution solution 2 is used to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration of 1.5mol/L, the pH of the eluent 1 is 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3mol/L, and the pH of the eluent 2 is 7.2;

The specific preparation method of eluate 1 and eluate 2 is the same as the preparation method of eluate 1 and eluate 2 in Example 1;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of aFGF protein pure solution into a dialysis bag, seal the dialysis bag, and then place the dialysis bag filled with aFGF protein solution in a beaker filled with 4L PBS solution (pH 7.4), at 4 ℃ environment The aFGF protein solution can be desalted after stirring with a magnetic stirrer for 4 hours to complete the first desalting, and then replace the 4L PBS solution and continue stirring for 4 hours;

Step 4. Adjustment of aFGF protein concentration: measure the concentration of the desalted aFGF protein solution with a microspectrophotometer through an ultrafiltration centrifuge tube, and adjust its concentration to 400 μg/mL with PBS;

Step 5, prepare aFGF protein protectant;

The aFGF protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction. The mixed solution is composed of phosphate buffer (pH is 7.4) and dextran sulfate, and dextran sulfate is in the phosphate buffer. The concentration of 4000μg/mL;

Step 6, mixing aFGF protein and protective agent: mixing the aFGF protein solution after desalting in step 4 and the dextran sulfate solution prepared in step 5 in a volume ratio of 1:10 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -80°C refrigerator.

Example 3

A method for protecting the drug stability and activity of aFGF protein, specifically comprising the following steps:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria grew to the absorbance at 600nm of 0.6, it was placed at 18°C. The aFGF protein was induced to express by IPTG for 16h, and the bacteria were collected by centrifugation at 4°C and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in phosphate buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and fragmentation, and the bacterial lysate was passed through a heparin gel chromatography column. Absorption to remove most of the impurity protein, and elution from eluent 1 to obtain aFGF protein crude product; aFGF protein crude product is separated by nickel ion gel chromatography column, and elution solution 2 is used to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration of 1.5mol/L, the pH of the eluent 1 is 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3mol/L, and the pH of the eluent 2 is 7.2;

The specific preparation method of eluate 1 and eluate 2 is the same as the preparation method of eluate 1 and eluate 2 in Example 1;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of pure aFGF protein solution into a dialysis bag, seal the dialysis bag, and then place the dialysis bag filled with aFGF protein solution in a beaker filled with 2L of PBS solution (pH 7.4) at 4 °C. The aFGF protein solution can be desalted after stirring with a magnetic stirrer for 4 hours. After the first desalting is completed, replace the 2L PBS solution again and continue stirring for 4 hours.

Step 4. Adjustment of aFGF protein concentration: use a protein concentration tube to measure the concentration of the desalted aFGF protein solution using a microspectrophotometer, and adjust its concentration to 200 μg/mL using PBS;

Step 5, prepare aFGF protein protectant;

The aFGF protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction. The mixed solution is composed of phosphate buffer (pH is 7.4) and dextran sulfate, and dextran sulfate is in the phosphate buffer. The concentration of 2000μg/mL;

Step 6, mixing aFGF protein and protective agent: mixing the desalted aFGF protein solution in step 4 with the dextran sulfate solution prepared in step 5 in a volume ratio of 1:5 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -50°C refrigerator.

Comparative Example 1

Using heparin as a protective agent for aFGF protein, the specific operation steps are as follows:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria grew to the absorbance at 600nm of 0.6, it was placed at 18°C. The aFGF protein was induced to express by IPTG for 16h, and the bacteria were collected by centrifugation at 4°C and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in phosphate buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and fragmentation, and the bacterial lysate was passed through a heparin gel chromatography column. Absorption to remove most of the impurity protein, and elution from eluent 1 to obtain aFGF protein crude product; aFGF protein crude product is separated by nickel ion gel chromatography column, and elution solution 2 is used to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration of 1.5mol/L, the pH of the eluent 1 is 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3mol/L, and the pH of the eluent 2 is 7.2;

The specific preparation method of eluate 1 and eluate 2 is the same as the preparation method of eluate 1 and eluate 2 in Example 1;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of pure aFGF protein solution into a dialysis bag, seal the dialysis bag, and then place the dialysis bag filled with aFGF protein solution in a beaker filled with 2L of PBS solution (pH 7.4) at 4 °C. The aFGF protein solution can be desalted after stirring with a magnetic stirrer for 4 hours. After the first desalting is completed, replace the 2L PBS solution again and continue stirring for 4 hours.

Step 4. Adjustment of aFGF protein concentration: measure the concentration of the desalted aFGF protein solution with a microspectrophotometer through an ultrafiltration centrifuge tube, and adjust its concentration to 200 μg/mL with PBS;

Step 5, prepare heparin protective agent solution;

The heparin protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction, the mixed solution is composed of phosphate buffer (pH is 7.4) and heparin, and the concentration of heparin in the phosphate buffer is 2000μg/mL ;

Step 6, mixing aFGF protein and protective agent: mix the aFGF protein solution after desalting in step 4 with the heparin protective agent solution prepared in step 5 in a volume ratio of 1:5 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -50°C refrigerator.

Comparative Example 2

Using chondroitin sulfate as a protective agent for aFGF protein, the specific operation steps are:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria grew to the absorbance at 600nm of 0.6, it was placed at 18°C. The aFGF protein was induced to express by IPTG for 16h, and the bacteria were collected by centrifugation at 4°C and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in phosphate buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and fragmentation, and the bacterial lysate was passed through a heparin gel chromatography column. Absorption to remove most of the impurity protein, and elution from eluent 1 to obtain aFGF protein crude product; aFGF protein crude product is separated by nickel ion gel chromatography column, and elution solution 2 is used to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration of 1.5mol/L, the pH of the eluent 1 is 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3mol/L, and the pH of the eluent 2 is 7.2;

The specific preparation method of eluate 1 and eluate 2 is the same as the preparation method of eluate 1 and eluate 2 in Example 1;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of pure aFGF protein solution into a dialysis bag, seal the dialysis bag, and then place the dialysis bag filled with aFGF protein solution in a beaker filled with 2L of PBS solution (pH 7.4) at 4 °C. The aFGF protein solution can be desalted after stirring with a magnetic stirrer for 4 hours. After the first desalting is completed, replace the 2L PBS solution again and continue stirring for 4 hours.

Step 4. Adjustment of aFGF protein concentration: use a protein concentration tube to measure the concentration of the desalted aFGF protein solution using a microspectrophotometer, and adjust its concentration to 200 μg/mL using PBS;

Step 5, prepare chondroitin sulfate protective agent solution;

Chondroitin sulfate protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction, and the mixed solution is composed of phosphate buffer (pH is 7.4) and chondroitin sulfate, and chondroitin sulfate is in the phosphate buffer. The concentration of 2000μg/mL;

Step 6, mixing aFGF protein and protective agent: mix the aFGF protein solution desalted in step 4 with the chondroitin sulfate protective agent solution prepared in step 5 in a volume ratio of 1:5 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -50°C refrigerator.

Comparative Example 3

Using λ-carrageenan as a protective agent for aFGF protein, the specific operation steps are:

Step 1. Expression of aFGF protein: After inserting the coding sequence of aFGF protein into the pET-11b plasmid containing histidine tag, it was introduced into Escherichia coli BL21 strain. When the density of the bacteria grew to the absorbance at 600nm of 0.6, it was placed at 18°C. The aFGF protein was induced to express by IPTG for 16h, and the bacteria were collected by centrifugation at 4°C and 8000 rpm;

Step 2. Purification of aFGF protein: The bacteria collected in step 1 were resuspended in phosphate buffer solution (pH 7.4), and the aFGF protein was released by ultrasonic lysis and fragmentation, and the bacterial lysate was passed through a heparin gel chromatography column. Absorption to remove most of the impurity protein, and elution from eluent 1 to obtain aFGF protein crude product; aFGF protein crude product is separated by nickel ion gel chromatography column, and elution solution 2 is used to obtain aFGF protein pure product liquid;

Wherein, eluent 1 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and NaCl, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 1 is 50mmol/L, and NaCl is in eluent 1 The mass concentration of 1.5mol/L, the pH of the eluent 1 is 7.2;

Eluent 2 is prepared from tris(hydroxymethyl)aminomethane hydrochloric acid buffer and imidazole, and the mass concentration of tris(hydroxymethyl)aminomethane in eluent 2 is 50mmol/L, and the mass of imidazole in eluent 2 is 50 mmol/L. The concentration is 0.3mol/L, and the pH of the eluent 2 is 7.2;

The specific preparation method of eluate 1 and eluate 2 is the same as the preparation method of eluate 1 and eluate 2 in Example 1;

Step 3. Desalting: The purified aFGF protein eluate generally contains high concentrations of imidazole and inorganic salts. It is necessary to replace the imidazole and inorganic salts in the aFGF protein eluate with PBS by dialysis bag dialysis. The specific operation The method is as follows: put 20 mL of pure aFGF protein solution into a dialysis bag, seal the dialysis bag, and then place the dialysis bag filled with aFGF protein solution in a beaker filled with 2L of PBS solution (pH 7.4) at 4 °C. The aFGF protein solution can be desalted after stirring with a magnetic stirrer for 4 hours. After the first desalting is completed, replace the 2L PBS solution again and continue stirring for 4 hours.

Step 4. Adjustment of aFGF protein concentration: measure the concentration of the desalted aFGF protein solution with a microspectrophotometer through an ultrafiltration centrifuge tube, and adjust its concentration to 200 μg/mL with PBS;

Step 5, prepare λ type-carrageenan protective agent solution;

The λ-type-carrageenan protective agent solution is composed of 50% glycerol and 50% mixed solution by volume fraction, and the mixed solution is composed of phosphate buffer (pH is 7.4) and λ-type-carrageenan, and the λ-type-carrageenan is in The concentration in the phosphate buffer is 2000 μg/mL;

Step 6, mixing aFGF protein and protective agent: mix the desalted aFGF protein solution in step 4 with the λ-carrageenan protective agent solution prepared in step 5 in a volume ratio of 1:5 to obtain a mixed solution;

Step 7. Divide the mixed solution in step 6 and store it in a -50°C refrigerator.

The following methods are provided for each embodiment of the present invention and each comparative example to detect the protective effect of dextran sulfate on the stability and activity of aFGF protein.

1. Molecular experiments to detect the protective effect of dextran sulfate on the thermal stability of aFGF protein

1. Test method

The thermal stability test of aFGF protein mainly detected the change of the melting temperature of aFGF protein in heparin, λ-carrageenan, dextran sulfate and chondroitin sulfate in the concentration range of 2.5-2000μg/mL. The aFGF protein was heated by a real-time quantitative PCR instrument, the melting curves of the aFGF protein in the four solutions were detected, and the changes in the melting temperature of the aFGF protein were evaluated.

2. Experimental results

The results are shown in Figure 1.

It can be seen from Figure 1 that the thermal stability of aFGF protein is significantly improved when dextran sulfate is added. After the polysaccharide with gradient concentration was mixed with aFGF protein, the thermal stability of the protein was detected by fluorescence quantitative technology. Among them, 40 μg/mL heparin, 200 μg/mL λ-carrageenan, and 10 μg/mL dextran sulfate could significantly bind to aFGF protein and improve protein thermal stability. When the polysaccharide concentration exceeds 5μg/mL, the protective ability of the same concentration of polysaccharide on aFGF protein is that dextran sulfate is better than heparin, and heparin is better than λ-carrageenan. When the polysaccharide concentration reached 2000μg/mL, these three polysaccharides could improve the thermal stability of aFGF protein to above 60℃, and dextran sulfate improved the thermal stability of aFGF protein most significantly. However, the protective effect of low sulfated chondroitin sulfate on the thermostability of aFGF protein was not obvious.

2. Cell experiments to detect the protective effect of dextran sulfate on the thermal stability of aFGF protein

1. Method

According to the method provided in each comparative example, heparin, λ-carrageenan, chondroitin sulfate and protected aFGF protein were mixed uniformly and then placed in a cell culture incubator for 2 days and 1 day incubation at 37°C, provided according to Example 3 (Because the protective effects of the methods provided in Examples 1-3 on the aFGF protein are basically parallel, only the method provided in Example 3 was used for detection in this experiment) The dextran sulfate was mixed evenly and placed in a cell incubator for testing. 2 days and 1 day of incubation at 37°C, and the biological activity of the protein after incubation was detected by cells.

2. Results

The results are shown in Figure 2.

As can be seen from Figure 2, the aFGF protein activity was severely lost after 24 hours and 48 hours of incubation. The aFGF protected by heparin, λ-carrageenan and dextran sulfate had higher activity, while chondroitin sulfate had no obvious protective effect on the activity of aFGF. Therefore, dextran sulfate can be used as a protective agent for aFGF protein for long-term preservation of aFGF protein.

It should be noted that when the claims of the present invention relate to numerical ranges, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. 3 is the same, in order to avoid redundant description, the present invention describes the preferred embodiments, but those skilled in the art can make additional changes and modifications to these embodiments once the basic inventive concept is known. Therefore, the appended claims are intended to be construed to include the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (5)

1. a kind of protection aFGF protein medicine stability and active method, which comprises the following steps:

The expression of step 1, aFGF protein: aFGF protein amino acid coding is inserted into the pET-11b matter containing histidine tag It after in grain, imported into e. coli strain bl21, when it is 0.4-0.8 that bacterial density, which grows under 600nm absorbance, 18 12-18h is expressed by IPTG induction aFGF protein under DEG C environment, bacterium is collected by centrifugation；

The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution, and is split through ultrasound Solution is broken to discharge aFGF protein, and it is thick that bacterial lysate through heparin gel chromatographic column, using eluent 1 affords aFGF protein Product；AFGF protein crude product affords aFGF protein sterling liquid through nickel ion gel chromatography post separation, using eluent 2；

The eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and the trihydroxy methyl amino Mass concentration of the methane in the eluent 1 is 50mmol/L, and mass concentration of the NaCl in the eluent 1 is 1.5mol/L, the pH of the eluent 1 are 7.2；

The eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and the trihydroxy methyl amino Mass concentration of the methane in the eluent 2 is 50mmol/L, and mass concentration of the imidazoles in the eluent 2 is 0.3mol/L, the pH of the eluent 2 are 7.2；

Step 3, desalination: aFGF protein sterling liquid obtained in step 2 is fitted into bag filter, then bag filter is placed in phosphate Desalination in buffer solution, the aFGF protein liquid after obtaining desalination；

The concentration of aFGF protein liquid after step 4, adjustment desalination；

Step 5 prepares protection agent solution；

The protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, and the mixed solution is by phosphoric acid Salt buffer and dextran sulfate composition, and concentration of the dextran sulfate in the phosphate buffer is through step 4 1-10 times of aFGF protein liquid concentration adjusted；

Step 6, by after step 4 adjusts concentration aFGF protein liquid and step 5 in the dextran sulphate solution prepared by volume Than mixing for 1:1-10, mixed liquor is obtained；

Step 7 dispenses the mixed liquor in step 6, is placed in -20~-80 DEG C of refrigerators and saves.

2. protection aFGF protein medicine stability according to claim 1 and active method, which is characterized in that step 1 Described in centrifugal rotational speed be 8000r/min.

3. protection aFGF protein medicine stability according to claim 1 and active method, which is characterized in that step 3 The volume ratio of middle aFGF protein sterling liquid and phosphate buffer is 1:50-200.

4. protection aFGF protein medicine stability according to claim 1 and active method, which is characterized in that step 4 In the concentration of aFGF protein liquid adjusted be 5-400 μ g/mL.

5. protection aFGF protein medicine stability according to claim 1 and active method, which is characterized in that the phosphorus The pH of hydrochlorate buffer solution is 7.4.