CN110151977B - Method for protecting stability and activity of aFGF protein medicament - Google Patents

Method for protecting stability and activity of aFGF protein medicament Download PDF

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CN110151977B
CN110151977B CN201910458468.8A CN201910458468A CN110151977B CN 110151977 B CN110151977 B CN 110151977B CN 201910458468 A CN201910458468 A CN 201910458468A CN 110151977 B CN110151977 B CN 110151977B
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孙昌业
郭志坤
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Abstract

The invention belongs to the technical field of medicaments, and particularly discloses a method for protecting the stability and activity of an aFGF protein medicament, which comprises the steps of 1, expressing the aFGF protein; step 2, purifying aFGF protein; step 3, desalting; step 4, adjusting the concentration of aFGF protein; step 5, preparing an aFGF protein protective agent; step 6, mixing the aFGF protein and the protective agent; and 7, subpackaging and freezing for storage. The method for protecting the stability and the activity of the aFGF protein medicament can obviously improve the stability of the aFGF protein and protect the biological activity of the aFGF protein.

Description

Method for protecting stability and activity of aFGF protein medicament
Technical Field
The invention belongs to the technical field of biological medicines, and particularly discloses a method for protecting stability and activity of an aFGF protein medicine.
Background
Acidic fibroblast growth factor (aFGF), a trace active substance having a broad mitogenic action on cells derived from mesoderm and neuroectoderm, is mainly distributed in organs or tissues such as brain, pituitary, nerve tissue, retina, adrenal gland, heart and bone, and has a small content of other tissues, and exists in extremely low concentrations in serum and body fluid.
aFGF has a wide range of biological functions, and can affect the growth, differentiation and functions of a variety of cells such as endocrine cells, nerve cells and mesenchymal cells. Since aFGF has various effects of promoting repair of injury, nourishing and protecting neurons, promoting angiogenesis, etc., it has become a hot spot of current research.
However, aFGF has poor stability, and multiple studies show that the thermal stability of recombinant aFGF protein (with a melting temperature of 40-50 ℃) is far lower than that of low bFGF (with a melting temperature of 55-60 ℃), and the aFGF protein is easy to denature and degrade in a body temperature environment, so that the activity of the aFGF protein acting in a human body is influenced.
Heparin is a highly sulfated polysaccharide, which is used clinically primarily for anticoagulation, and can protect aFGF protein, increasing its stability and bioactivity. However, heparin is mostly derived from porcine intestinal mucosa, the price is relatively high, the function of protecting aFGF protein is only used for scientific research, and the heparin is not easy to popularize for practical application.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a method for protecting the stability and activity of an aFGF protein drug.
The method for protecting the stability and the activity of the aFGF protein medicament provided by the invention specifically comprises the following steps of:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence (UniProt number: P05230, selected sequence: 16F-155D) into pET-11b plasmid containing histidine tag, introducing into Escherichia coli BL21 strain, inducing expression of aFGF protein by IPTG for 12-18h at 18 ℃ when the density of bacteria grows to 600nm absorbance of 0.4-0.8, and centrifuging to collect bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution, releasing aFGF protein through ultrasonic lysis and crushing, passing the bacterial lysate through a heparin gel chromatography column, and eluting by using an eluent 1 to obtain an aFGF protein crude product; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
the eluent 1 is prepared from tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 1 is 50mmol/L, the mass concentration of NaCl in the eluent 1 is 1.5mol/L, and the pH of the eluent 1 is 7.2;
the eluent 2 is prepared from tris (hydroxymethyl) aminomethane hydrochloride buffer solution and imidazole, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 2 is 50mmol/L, the mass concentration of imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
and 3, desalting: filling the aFGF protein pure product liquid obtained in the step 2 into a dialysis bag, sealing, then placing the dialysis bag into a phosphate buffer solution, stirring for 4 hours at 4 ℃, replacing the PBS solution, and continuing stirring for 4 hours to obtain desalted aFGF protein liquid;
step 4, adjusting the concentration of the desalted aFGF protein liquid;
step 5, preparing a protective agent solution;
the protective agent solution consists of 50% of glycerol and 50% of mixed solution in percentage by volume, the mixed solution consists of phosphate buffer solution and dextran sulfate, and the concentration of the dextran sulfate in the phosphate buffer solution is 1-10 times of the concentration of the aFGF protein solution adjusted in the step 4;
step 6, mixing the aFGF protein solution desalted in the step 4 with the dextran sulfate solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of between 20 ℃ below zero and 80 ℃ below zero.
Preferably, the centrifugal speed in the step 1 is 8000r/min.
Preferably, the volume ratio of the aFGF protein pure solution to the phosphate buffer solution in the step 3 is 1.
Preferably, the concentration of the aFGF protein solution adjusted in step 4 is 5-400. Mu.g/mL.
Preferably, the phosphate buffer solution has a pH of 7.4.
Compared with the prior art, the invention has the beneficial effects that:
the method for protecting the stability and the activity of the aFGF protein drug uses dextran sulfate as a protective agent of the aFGF protein, and the disaccharide unit of the dextran sulfate contains 6-O-SO 3- When sugar chain-O-SO has a stronger electronegativity than heparin 3 - O atom of (2) and aFGF surface-NH 3 + When H atoms in the aFGF protein interact with each other, O-H hydrogen bonds are easier to form, and the effect of improving the stability of the aFGF protein is better; in addition, the length of the dextran sulfate chain in industrial production can be regulated, the dextran sulfate chain comprises 500kDa (about 500 disaccharide units) to 5kDa (about 5 disaccharide units), the average molecular weight of the heparin polysaccharide chain is 18kDa (about 50 disaccharide units), the regulation is not easy, and the dextran sulfate in industrial production is easier to standardize, so that the dextran sulfate has great application prospect as the aFGF protein protective agent.
Drawings
FIG. 1 is a graph showing the solubilization and denaturation of aFGF protein after adding different protective agents in a molecular experiment; wherein, the figure A refers to heparin, the B refers to lambda-carrageenan, the C refers to dextran sulfate, and the D refers to chondroitin sulfate; e is a graph of the denaturation temperature change of aFGF protein with increasing polysaccharide concentration; endpoint 0 refers to blank control;
FIG. 2 is a bar graph of the biological activity of aFGF proteins in cell experiments after addition of different protective agents in example 3 and comparative examples 1-3; wherein, PBS refers to a blank control group without FGF, A refers to aFGF group, and B refers to aFGF + dextran sulfate group; 0. 1 and 2 refer to incubation in a cell incubator at 37 ℃ for 0, 1 and 2 days, respectively.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
The experimental materials and instruments used in the examples and comparative examples are as follows:
1. instrument for measuring the position of a moving object
Heparin gel chromatography column: bio-Rad, USA;
nickel ion gel chromatography column: GE Healthcare, usa;
a magnetic stirrer: shanghai-Hengchang scientific instruments, inc.;
protein concentration tube, ultrafiltration centrifuging tube: merck Millipore, germany;
micro-spectrophotometer: nanodrop instruments, thermal Scientific, usa;
a low-temperature refrigerator: a medical hail refrigerator.
2. Reagents and materials
The coding sequence of aFGF protein: uniProt number: p05230, selected sequence: 16F-155D, the coding sequence being supplied by Life Technologies;
coli BL21: beijing Quanjin Biotechnology Ltd;
isopropyl thiogalactoside: isoproyl β -D-Thiogalactoside, IPTG, beijing all-grass Biotech, inc.;
Tris-Cl, naCl, imidazole, glycerol, PBS buffer solution: beijing Solaibao science and technology, inc.;
a dialysis bag: 10kDa pore size, thermal Scientific, USA;
dextran sulfate, heparin, chondroitin sulfate: sigma, USA.
Example 1
A method for protecting the stability and the activity of an aFGF protein medicament specifically comprises the following steps:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing a histidine tag, introducing the aFGF protein coding sequence into an escherichia coli BL21 strain, inducing and expressing the aFGF protein for 12 hours by isopropyl thiogalactoside at the temperature of 18 ℃ when the absorbance is 0.4 under the condition that the bacterial density grows to 600nm, and centrifuging at the temperature of 4 ℃ and 8000 rpm to collect bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a PBS buffer solution (pH 7.4), releasing aFGF protein through ultrasonic cracking and crushing, adsorbing the bacterial lysate through a heparin gel chromatography column, removing most impurity proteins, and eluting by an eluent 1 to obtain a crude product of aFGF protein; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of the trihydroxymethyl aminomethane in the eluent 1 is 50mmol/L, the mass concentration of the NaCl in the eluent 1 is 1.5mol/L, and the pH value of the eluent 1 is 7.2; the preparation method comprises the following steps: weighing a proper amount of tris (hydroxymethyl) aminomethane, adding water to dissolve, adding a proper amount of NaCl, enabling the mass concentration of tris (hydroxymethyl) aminomethane in the whole solution system to be 50mmol/L and the mass concentration of NaCl in the whole solution system to be 1.5mol/L, stirring uniformly, and adjusting the pH value to 7.2 by using hydrochloric acid;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2; the preparation method comprises the following steps: weighing a proper amount of tris (hydroxymethyl) aminomethane, adding water to dissolve the tris (hydroxymethyl) aminomethane, adding a proper amount of imidazole to ensure that the mass concentration of tris (hydroxymethyl) aminomethane in the whole solution system is 50mmol/L and the mass concentration of imidazole in the whole solution system is 0.3mol/L, stirring uniformly, and then adjusting the pH value to 7.2 by using hydrochloric acid;
and 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: putting 20mL of aFGF protein pure liquid obtained in the step 2 into a dialysis bag, sealing the dialysis bag, then placing the dialysis bag filled with the aFGF protein pure liquid into a beaker filled with 1L of PBS solution (the pH value is 7.4), stirring for 4 hours in a magnetic stirrer in the environment of 4 ℃, completing primary desalination, replacing 1L of PBS solution again, and continuing stirring for 4 hours, thus completing desalination of the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a micro spectrophotometer through a protein concentration tube, and adjusting the concentration of the aFGF protein solution to 5 mu g/mL by using PBS;
step 5, preparing an aFGF protein protective agent;
the aFGF protective agent solution consists of 50% of glycerol and 50% of mixed solution in percentage by volume, the mixed solution consists of phosphate buffer solution (the pH is 7.4) and dextran sulfate, and the concentration of the dextran sulfate in the phosphate buffer solution is 5 mu g/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the dextran sulfate solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of-20 ℃.
Example 2
A method for protecting the stability and the activity of an aFGF protein medicament specifically comprises the following steps:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing histidine tag, introducing into Escherichia coli BL21 strain, inducing and expressing aFGF protein by IPTG (isopropyl-beta-thiogalactoside) for 18h at 18 ℃ when the density of bacteria grows to 600nm absorbance of 0.8, and centrifuging at 4 ℃ and 8000 rpm to collect bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution (pH 7.4), releasing aFGF protein through ultrasonic cracking and crushing, adsorbing the bacterial lysate through a heparin gel chromatography column, removing most impurity proteins, and eluting by an eluent 1 to obtain a crude product of aFGF protein; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of the trihydroxymethyl aminomethane in the eluent 1 is 50mmol/L, the mass concentration of the NaCl in the eluent 1 is 1.5mol/L, and the pH value of the eluent 1 is 7.2;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
the concrete preparation method of eluent 1 and eluent 2 is the same as that of eluent 1 and eluent 2 in example 1;
and 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: putting 20mL of aFGF protein pure product liquid into a dialysis bag, sealing the dialysis bag, then putting the dialysis bag filled with the aFGF protein liquid into a beaker filled with 4L of PBS (pH 7.4), stirring for 4 hours by a magnetic stirrer in the environment of 4 ℃, completing primary desalination, replacing 4L of PBS solution again, and continuing stirring for 4 hours, thus desalting the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a micro spectrophotometer through an ultrafiltration centrifugal tube, and adjusting the concentration of the aFGF protein solution to 400 mu g/mL by using PBS;
step 5, preparing an aFGF protein protective agent;
the aFGF protective agent solution consists of 50% of glycerol and 50% of mixed solution in percentage by volume, the mixed solution consists of phosphate buffer solution (the pH is 7.4) and dextran sulfate, and the concentration of the dextran sulfate in the phosphate buffer solution is 4000 mu g/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the dextran sulfate solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of-80 ℃.
Example 3
A method for protecting the stability and the activity of an aFGF protein medicament specifically comprises the following steps:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing histidine tag, introducing into Escherichia coli BL21 strain, inducing and expressing aFGF protein by IPTG (isopropyl-beta-thiogalactoside) for 16h at 18 ℃ when the density of bacteria grows to 600nm absorbance of 0.6, and centrifuging at 4 ℃ and 8000 rpm to collect bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution (pH 7.4), releasing aFGF protein through ultrasonic lysis and crushing, adsorbing the bacterial lysate by a heparin gel chromatography column, removing most of impurity protein, and eluting by an eluent 1 to obtain an aFGF protein crude product; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of the trihydroxymethyl aminomethane in the eluent 1 is 50mmol/L, the mass concentration of the NaCl in the eluent 1 is 1.5mol/L, and the pH value of the eluent 1 is 7.2;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
the specific preparation method of eluent 1 and eluent 2 is the same as the preparation method of eluent 1 and eluent 2 in example 1;
and 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: filling 20mL of aFGF protein pure product liquid into a dialysis bag, sealing the dialysis bag, then placing the dialysis bag filled with the aFGF protein liquid into a beaker filled with 2L of PBS (pH 7.4), stirring for 4 hours by a magnetic stirrer in the environment of 4 ℃, replacing 2L of PBS solution again after primary desalination is finished, and continuously stirring for 4 hours to desalt the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a micro spectrophotometer through a protein concentration tube, and adjusting the concentration of the aFGF protein solution to 200 mu g/mL by using PBS;
step 5, preparing an aFGF protein protective agent;
the aFGF protective agent solution consists of 50% of glycerol and 50% of mixed solution according to volume fraction, the mixed solution consists of phosphate buffer solution (the pH value is 7.4) and dextran sulfate, and the concentration of the dextran sulfate in the phosphate buffer solution is 2000 mu g/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the dextran sulfate solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of 50 ℃ below zero.
Comparative example 1
The heparin is used as a protective agent of aFGF protein, and the specific operation steps are as follows:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing a histidine tag, introducing the aFGF protein coding sequence into an escherichia coli BL21 strain, inducing expression of the aFGF protein by IPTG for 16h at the environment of 18 ℃ when the density of bacteria grows to the condition that the absorbance at 600nm is 0.6, and centrifuging at the temperature of 4 ℃ and 8000 rpm to collect the bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution (pH 7.4), releasing aFGF protein through ultrasonic lysis and crushing, adsorbing the bacterial lysate by a heparin gel chromatography column, removing most of impurity protein, and eluting by an eluent 1 to obtain an aFGF protein crude product; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 1 is 50mmol/L, the mass concentration of NaCl in the eluent 1 is 1.5mol/L, and the pH of the eluent 1 is 7.2;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
the concrete preparation method of eluent 1 and eluent 2 is the same as that of eluent 1 and eluent 2 in example 1;
and 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: filling 20mL of aFGF protein pure product liquid into a dialysis bag, sealing the dialysis bag, then placing the dialysis bag filled with the aFGF protein liquid into a beaker filled with 2L of PBS (pH 7.4), stirring for 4 hours by a magnetic stirrer in the environment of 4 ℃, replacing 2L of PBS solution again after primary desalination is finished, and continuously stirring for 4 hours to desalt the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a microspectrophotometer through an ultrafiltration centrifugal tube, and adjusting the concentration of the aFGF protein solution to 200 mu g/mL by using PBS;
step 5, preparing a heparin protectant solution;
the heparin protectant solution consists of 50% of glycerol and 50% of mixed solution in volume fraction, the mixed solution consists of phosphate buffer (pH is 7.4) and heparin, and the concentration of the heparin in the phosphate buffer is 2000 mug/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the heparin protectant solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of 50 ℃ below zero.
Comparative example 2
The chondroitin sulfate is used as a protective agent of aFGF protein, and the specific operation steps are as follows:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing a histidine tag, introducing the aFGF protein coding sequence into an escherichia coli BL21 strain, inducing expression of the aFGF protein by IPTG for 16h at the environment of 18 ℃ when the density of bacteria grows to the condition that the absorbance at 600nm is 0.6, and centrifuging at the temperature of 4 ℃ and 8000 rpm to collect the bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution (pH 7.4), releasing aFGF protein through ultrasonic lysis and crushing, adsorbing the bacterial lysate by a heparin gel chromatography column, removing most of impurity protein, and eluting by an eluent 1 to obtain an aFGF protein crude product; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of the trihydroxymethyl aminomethane in the eluent 1 is 50mmol/L, the mass concentration of the NaCl in the eluent 1 is 1.5mol/L, and the pH value of the eluent 1 is 7.2;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
the specific preparation method of eluent 1 and eluent 2 is the same as the preparation method of eluent 1 and eluent 2 in example 1;
step 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS (phosphate buffer solution) in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: filling 20mL of aFGF protein pure product liquid into a dialysis bag, sealing the dialysis bag, then placing the dialysis bag filled with the aFGF protein liquid into a beaker filled with 2L of PBS (pH 7.4), stirring for 4 hours by a magnetic stirrer in the environment of 4 ℃, replacing 2L of PBS solution again after primary desalination is finished, and continuously stirring for 4 hours to desalt the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a micro spectrophotometer through a protein concentration tube, and adjusting the concentration of the aFGF protein solution to 200 mu g/mL by using PBS;
step 5, preparing a chondroitin sulfate protective agent solution;
the chondroitin sulfate protective agent solution consists of 50% of glycerol and 50% of mixed solution according to volume fraction, the mixed solution consists of phosphate buffer solution (the pH is 7.4) and chondroitin sulfate, and the concentration of the chondroitin sulfate in the phosphate buffer solution is 2000 mu g/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the chondroitin sulfate protective agent solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of 50 ℃ below zero.
Comparative example 3
The method takes lambda-carrageenan as a protective agent of aFGF protein, and comprises the following specific operation steps:
step 1, expression of aFGF protein: inserting an aFGF protein coding sequence into pET-11b plasmid containing histidine tag, introducing into Escherichia coli BL21 strain, inducing and expressing aFGF protein by IPTG (isopropyl-beta-thiogalactoside) for 16h at 18 ℃ when the density of bacteria grows to 600nm absorbance of 0.6, and centrifuging at 4 ℃ and 8000 rpm to collect bacteria;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution (pH 7.4), releasing aFGF protein through ultrasonic cracking and crushing, adsorbing the bacterial lysate through a heparin gel chromatography column, removing most impurity proteins, and eluting by an eluent 1 to obtain a crude product of aFGF protein; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
wherein the eluent 1 is prepared from tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 1 is 50mmol/L, the mass concentration of NaCl in the eluent 1 is 1.5mol/L, and the pH of the eluent 1 is 7.2;
the eluent 2 is prepared from trihydroxymethyl aminomethane hydrochloric acid buffer solution and imidazole, the mass concentration of the trihydroxymethyl aminomethane in the eluent 2 is 50mmol/L, the mass concentration of the imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
the concrete preparation method of eluent 1 and eluent 2 is the same as that of eluent 1 and eluent 2 in example 1;
and 3, desalting: the purified aFGF protein eluent generally contains high-concentration imidazole and inorganic salt components, the imidazole and the inorganic salt in the aFGF protein eluent are replaced by PBS in a dialysis bag dialysis mode, and the specific operation method comprises the following steps: filling 20mL of aFGF protein pure product liquid into a dialysis bag, sealing the dialysis bag, then placing the dialysis bag filled with the aFGF protein liquid into a beaker filled with 2L of PBS (pH 7.4), stirring for 4 hours by a magnetic stirrer in the environment of 4 ℃, replacing 2L of PBS solution again after primary desalination is finished, and continuously stirring for 4 hours to desalt the aFGF protein liquid;
step 4, adjusting the concentration of aFGF protein: measuring the concentration of the desalted aFGF protein solution by using a micro spectrophotometer through an ultrafiltration centrifugal tube, and adjusting the concentration of the aFGF protein solution to 200 mu g/mL by using PBS;
step 5, preparing a lambda-carrageenan protective agent solution;
the lambda-carrageenan protective agent solution consists of 50 percent of glycerin and 50 percent of mixed solution according to volume fraction, the mixed solution consists of phosphate buffer solution (pH is 7.4) and lambda-carrageenan, and the concentration of the lambda-carrageenan in the phosphate buffer solution is 2000 mug/mL;
step 6, mixing the aFGF protein and the protective agent: mixing the aFGF protein solution desalted in the step 4 with the lambda-carrageenan protective agent solution prepared in the step 5 according to a volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of 50 ℃ below zero.
The following methods provided in the examples and comparative examples of the present invention were used to examine the protective effect of dextran sulfate on the stability and activity of aFGF protein.
1. Molecular experiment detection of protective effect of dextran sulfate on thermal stability of aFGF protein
1. Test method
The aFGF protein thermal stability experiment mainly detects the change of the melting temperature of aFGF protein in heparin, lambda-carrageenan, dextran sulfate and chondroitin sulfate with the concentration range of 2.5-2000 mug/mL. Heating the aFGF protein by a real-time quantitative PCR instrument, detecting the melting curves of the aFGF protein in the four solutions, and evaluating the change of the melting temperature of the aFGF protein.
2. Results of the experiment
The results are shown in FIG. 1.
As can be seen from fig. 1, the thermal stability of aFGF protein was significantly improved when dextran sulfate was added. After polysaccharide with gradient concentration is mixed with aFGF protein, the thermal stability of the protein is detected by using a fluorescence quantitative technology. Wherein 40 mug/mL heparin, 200 mug/mL lambda-carrageenan and 10 mug/mL dextran sulfate can be obviously combined with aFGF protein, and the thermal stability of the protein is improved. When the concentration of the polysaccharide exceeds 5 mu g/mL, the protection capability of the polysaccharide with the same concentration on aFGF protein is that dextran sulfate is superior to heparin, and heparin is superior to lambda-carrageenan. When the concentration of the polysaccharides reaches 2000 mug/mL, the three polysaccharides can improve the thermal stability of the aFGF protein to be more than 60 ℃, and the degree of improvement of the thermal stability of the aFGF protein by the dextran sulfate is most obvious. However, the protective effect of low sulfated chondroitin sulfate on the thermal stability of aFGF protein is not significant.
2. Cell experiment detection of protective effect of dextran sulfate on thermal stability of aFGF protein
1. Method of producing a composite material
Heparin, lambda-carrageenan, chondroitin sulfate and protected aFGF protein are uniformly mixed and then placed in a cell culture box for incubation at 37 ℃ for 2 days and 1 day respectively according to the methods provided by each proportion, dextran sulfate is uniformly mixed and then placed in the cell culture box for incubation at 37 ℃ for 2 days and 1 day according to the method provided by the example 3 (because the protection effects of the methods provided by the examples 1-3 on the aFGF protein are basically parallel, the method provided by the example 3 is only selected for detection in the experiment), and the biological activity of the incubated protein is detected through cells.
2. Results
The results are shown in FIG. 2.
As can be seen from fig. 2, aFGF protein activity was severely lost after 24 hours and 48 hours of incubation. Heparin, lambda-carrageenan and dextran sulfate protected aFGF all have higher activity, and chondroitin sulfate has no obvious protection effect on the activity of aFGF. Thus, 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 following claims refer to numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods used are the same as those in embodiments 1 to 3, the present invention describes preferred embodiments in order to prevent redundant description, but once the basic inventive concept is known, other variations and modifications can be made to the embodiments by those skilled in the art. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.

Claims (4)

1. A method for protecting the stability and activity of an aFGF protein drug, comprising the steps of:
step 1, expression of aFGF protein: inserting an amino acid coding sequence of aFGF protein into a pET-11b plasmid containing a histidine tag, introducing the aFGF protein coding sequence into a escherichia coli BL21 strain, inducing the expression of the aFGF protein for 12-18h by IPTG (isopropyl thiogalactoside) in an environment of 18 ℃ when the absorbance is 0.4-0.8 when the bacterial density grows to 600nm, and centrifuging to collect bacteria; the aFGF protein amino acid coding sequence is derived from a UniProt database, and is numbered as P05230, and the selected sequence is 16F-155D;
step 2, purification of aFGF protein: resuspending the bacteria collected in the step 1 in a phosphate buffer solution, releasing aFGF protein through ultrasonic cracking and crushing, and eluting a bacterial lysate through a heparin gel chromatography column by using an eluent 1 to obtain a crude product of the aFGF protein; separating the aFGF protein crude product by a nickel ion gel chromatographic column, and eluting by using an eluent 2 to obtain an aFGF protein pure product liquid;
the eluent 1 is prepared from tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution and NaCl, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 1 is 50mmol/L, the mass concentration of NaCl in the eluent 1 is 1.5mol/L, and the pH of the eluent 1 is 7.2;
the eluent 2 is prepared from tris (hydroxymethyl) aminomethane hydrochloride buffer solution and imidazole, the mass concentration of tris (hydroxymethyl) aminomethane in the eluent 2 is 50mmol/L, the mass concentration of imidazole in the eluent 2 is 0.3mol/L, and the pH value of the eluent 2 is 7.2;
and 3, desalting: filling the aFGF protein pure product liquid obtained in the step 2 into a dialysis bag, and desalting the dialysis bag in a phosphate buffer solution to obtain desalted aFGF protein liquid;
step 4, adjusting the concentration of the desalted aFGF protein liquid, wherein the adjusted concentration of the aFGF protein liquid is 5-400 mu g/mL;
step 5, preparing a protective agent solution;
the protective agent solution consists of 50% of glycerol and 50% of mixed solution in percentage by volume, the mixed solution consists of phosphate buffer solution and dextran sulfate, and the concentration of the dextran sulfate in the phosphate buffer solution is 1-10 times of the concentration of the aFGF protein solution adjusted in the step 4;
step 6, mixing the aFGF protein solution with the concentration adjusted in the step 4 with the dextran sulfate solution prepared in the step 5 according to the volume ratio of 1;
and 7, subpackaging the mixed solution obtained in the step 6, and storing in a refrigerator at the temperature of between 20 ℃ below zero and 80 ℃ below zero.
2. The method for protecting the stability and activity of aFGF protein drugs according to claim 1, wherein the centrifugation speed in step 1 is 8000r/min.
3. The method for protecting the stability and the activity of the aFGF protein drug according to claim 1, wherein the volume ratio of the aFGF protein pure solution to the phosphate buffer solution in step 3 is 1.
4. The method of claim 1, wherein the phosphate buffered saline has a pH of 7.4.
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