CN110151977A - A kind of protection aFGF protein medicine stability and active method - Google Patents
A kind of protection aFGF protein medicine stability and active method Download PDFInfo
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Abstract
The invention belongs to technical field of pharmaceuticals, specifically disclose a kind of protection aFGF protein medicine stability and active method, the expression of step 1, aFGF protein；The purifying of step 2, aFGF protein；Step 3, desalination；Step 4, the adjustment of aFGF protein concentration；Step 5 prepares aFGF protein protective agent；Step 6, aFGF protein and protective agent mixing；Step 7, packing, freezen protective.Protection aFGF protein medicine stability provided by the invention and active method, can significantly improve the stability of aFGF protein, and protect its bioactivity.
Description
Technical field
The invention belongs to bio-pharmaceutical technical fields, specifically disclose a kind of protection aFGF protein medicine stability and active
Method.
Background technique
Acid fibroblast growth factor (acidic fibroblast growth factor, aFGF) is a kind of right
From mesoderm and the cell of neuroderm, there is the trace activity substance for promoting splitting action extensively, is distributed mainly on brain, hangs down
In body, nerve fiber, retina, adrenal gland, heart and Gu Deng organ or tissue, other tissue contents are seldom, in serum and body
Exist in liquid with extremely low concentration.
AFGF has extensive biological function, and it is a variety of to influence endocrine cell, nerve cell and mesenchymal cell etc.
Growth, differentiation and its function of cell.Promote injury repair, nutrition and protection neuron, Angiogensis since aFGF has
Become the hot spot studied at present etc. various effects.
But aFGF stability is poor, and multinomial research all shows that the heat of recombination aFGF protein (40 DEG C -50 DEG C of melting temperature) is steady
It is qualitative to be far below low bFGF (55 DEG C -60 DEG C of melting temperature), and aFGF protein is easy denaturation degradation under body temperature environment, to it
The activity acted in human body has an impact.
Heparin is a kind of polysaccharide of height sulphation, is mainly used for anticoagulation in clinic, can protect aFGF protein, is improved
Its stability and bioactivity.But for heparin mostly from pig intestinal mucosa, price is relatively high, protects the effect of aFGF protein
It is only used for scientific research, practical application is not easy to promote.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of protection aFGF protein medicine stability and active side
Method.
Protection aFGF protein medicine stability provided by the invention and active method, specifically includes the following steps:
The expression of step 1, aFGF protein: by aFGF protein coded sequence (UniProt number: P05230, selected sequence:
After 16F-155D) being inserted into the pET-11b plasmid containing histidine tag, it imported into e. coli strain bl21, when bacterium is close
Degree grows to 600nm absorbance when being 0.4-0.8, and aFGF protein is by IPTG inducing expression 12-18h under 18 DEG C of environment, centrifugation
Collect bacterium；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution, and through super
Sound cracking is broken to discharge aFGF protein, and bacterial lysate affords aFGF egg through heparin gel chromatographic column, using eluent 1
White crude 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
Mass concentration of the aminomethane in the eluent 1 is 50mmol/L, mass concentration of the NaCl in the eluent 1
For 1.5mol/L, the pH of the eluent 1 is 7.2；
The eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and the trihydroxy methyl
Mass concentration of the aminomethane in the eluent 2 is 50mmol/L, mass concentration of the imidazoles in the eluent 2
For 0.3mol/L, the pH of the eluent 2 is 7.2；
Step 3, desalination: aFGF protein sterling liquid obtained in step 2 is fitted into bag filter and is closed, then bag filter is set
In phosphate buffer solution, after 4 DEG C are stirred 4 hours, PBS solution is replaced, continues stirring 4 hours, the aFGF after obtaining desalination
Protein liquid；
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, the mixed solution by
Phosphate buffer and dextran sulfate composition, and concentration of the dextran sulfate in the phosphate buffer is through step
1-10 times of rapid 4 aFGF protein liquid concentration adjusted；
Step 6, by after desalination in step 4 aFGF protein liquid and step 5 in the dextran sulphate solution prepared by volume
It is mixed than 1:1-10, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -20~-80 DEG C of refrigerators and saves.
Preferably, centrifugal rotational speed described in step 1 is 8000r/min.
Preferably, the volume ratio of aFGF protein sterling liquid and phosphate buffer is 1:50-200 in step 3.
Preferably, the concentration of aFGF protein liquid adjusted is 5-400 μ g/mL in step 4.
Preferably, the pH of the phosphate buffer solution is 7.4.
The prior art is compared, the invention has the benefit that
Protection aFGF protein medicine stability provided by the invention and active method, use dextran sulfate as aFGF
The protective agent of albumen contains 6-O-SO in dextran sulfate disaccharide unit3-, there is stronger elecrtonegativity compared to heparin, when
Sugar chain-O-SO3 -In O atom and the surface aFGF-NH3 +In H atom interaction when, O-H hydrogen bond is more likely formed, to raising
The better effect of aFGF protein stability；In addition, the length of dextran sulfate chain can regulate and control in industrial production, it include 500kDa
(about 500 disaccharide units) -5kDa (about 5 disaccharide units), and the average molecular weight of heparin polysaccharides chain is 18kDa (about 50
Disaccharide unit), not easy-regulating, production dextran sulfate is more easier to standardize in industry, therefore using dextran sulfate as
The protective agent of aFGF protein has great application prospect.
Detailed description of the invention
Fig. 1 is the dissolution denaturation curve figure of aFGF protein after different protective agents are added in molecule experiments；Wherein, figure A refers to liver
Element, B refer to that λ type-carragheen, C refer to that dextran sulfate, D refer to chondroitin sulfate；E refers to the raising with polysaccharide concentration, the change of aFGF protein
Property temperature changing curve diagram；Endpoint 0 refers to blank control group；
Fig. 2 is the bioactivity column that aFGF protein after different protective agents is added in embodiment 3 and comparative example 1-3 in cell experiment
Shape figure；Wherein, PBS refers to the blank control group of no FGF, and A refers to aFGF group, and B refers to aFGF+ dextran sulfate group；0, it 1 and 2 respectively refers to
It is incubated for 0,1 and 2 day for 37 DEG C in cell incubator.
Specific embodiment
In order to enable those skilled in the art to more fully understand, technical solution of the present invention is practiced, below with reference to specific
The invention will be further described for embodiment, but illustrated embodiment is not as a limitation of the invention.
Experimental material used in each embodiment and each comparative example and instrument are as follows:
1, instrument
Heparin gel chromatographic column: Bio-Rad, the U.S.；
Nickel ion gel chromatography column: GE Healthcare, the U.S.；
Magnetic stirring apparatus: Shanghai Yiheng Scientific Instruments Co., Ltd；
Protein concentration pipe, ultra-filtration centrifuge tube: Merck Millipore, Germany；
Micro-spectrophotometer: Nanodrop instrument, Thermal Scientific, the U.S.；
Low temperature refrigerator: Haier's Medical refrigerator.
2, reagent and material
AFGF protein coded sequence: UniProt number: P05230, selected sequence: 16F-155D, coded sequence is by Life
Technologies company provides；
E. coli bl21: Beijing Quanshijin Biotechnology Co., Ltd；
Isopropylthiogalactoside: Isopropyl β-D-Thiogalactoside, IPTG, the complete biological skill of formula gold in Beijing
Art Co., Ltd；
Tris-Cl, NaCl, imidazoles, glycerol, PBS buffer solution: Beijing Suo Laibao Science and Technology Ltd；
Bag filter: the aperture 10kDa, Thermal Scientific, the U.S.；
Dextran sulfate, heparin, chondroitin sulfate: Sigma, the U.S..
Embodiment 1
A kind of protection aFGF protein medicine stability and active method, specifically includes the following steps:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, under the conditions of bacterial density grows to 600nm absorbance be 0.4 when, at 18 DEG C
Bacterium is collected by centrifugation through 4 DEG C, 8000 revs/min by isopropylthiogalactoside, inducing expression 12h in aFGF protein under environment；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in PBS buffer solution (pH 7.4),
And release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, is removed most of
Impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product through nickel ion gel chromatography post separation,
AFGF protein sterling liquid is afforded using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；Specific preparation method are as follows: appropriate trishydroxymethylaminomethane is weighed, is dissolved in water, appropriate NaCl is added,
And makes mass concentration 50mmol/L of the trishydroxymethylaminomethane in entire solution system, make NaCl in entire solution body
Mass concentration in system is that 1.5mol/L uses hydrochloric acid tune pH to 7.2 after mixing evenly；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；Specific preparation method are as follows: appropriate trishydroxymethylaminomethane is weighed, is dissolved in water, appropriate imidazoles is added, and
Make mass concentration 50mmol/L of the trishydroxymethylaminomethane in entire solution system, makes imidazoles in entire solution system
Mass concentration be 0.3mol/L use hydrochloric acid tune pH to 7.2 after mixing evenly；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: aFGF protein sterling liquid obtained in 20mL step 2 is fitted into bag filter, closes bag filter, then aFGF protein will be housed
The bag filter of sterling liquid is placed in the beaker equipped with 1L PBS solution (pH 7.4), through magnetic stirrer in 4 DEG C of environment
4 hours, first time desalination is completed, then re-replace 1L PBS solution, continues stirring 4 hours, aFGF protein liquid can be completed
Desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by protein concentration pipe
Photometer measurement concentration, and its concentration is adjusted to 5 μ g/mL using PBS；
Step 5 prepares aFGF protein protective agent；
AFGF protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, and mixed solution is by phosphoric acid
Salt buffer (pH 7.4) and dextran sulfate composition, and concentration of the dextran sulfate in the phosphate buffer is 5 μ g/
mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
1:1 is mixed dextran sulphate solution by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -20 DEG C of refrigerators and saves.
Embodiment 2
A kind of protection aFGF protein medicine stability and active method, specifically includes the following steps:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, when bacterial density grow to 600nm absorbance be 0.8 when, under 18 DEG C of environment
Bacterium is collected by centrifugation by IPTG inducing expression 18h, through 4 DEG C, 8000 revs/min in aFGF protein；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution (pH 7.4)
In, and release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, removes big portion
Divide impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product is through nickel ion gel chromatography column point
From affording aFGF protein sterling liquid using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；
The specific preparation method of eluent 1 and eluent 2 and the preparation method phase of eluent 1 and eluent 2 in embodiment 1
Together；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: 20mL aFGF protein sterling liquid is fitted into bag filter, closes bag filter, then the bag filter equipped with aFGF protein liquid is set
In the beaker equipped with 4L PBS solution (pH 7.4), through magnetic stirrer 4 hours in 4 DEG C of environment, complete for the first time
Desalination, then 4L PBS solution is re-replaced, continue stirring 4 hours, it can be to aFGF protein liquid desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by ultra-filtration centrifuge tube
Photometer measurement concentration, and its concentration is adjusted to 400 μ g/mL using PBS；
Step 5 prepares aFGF protein protective agent；
AFGF protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, and mixed solution is by phosphoric acid
Salt buffer (pH 7.4) and dextran sulfate composition, and concentration of the dextran sulfate in the phosphate buffer is 4000 μ
g/mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
1:10 is mixed dextran sulphate solution by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -80 DEG C of refrigerators and saves.
Embodiment 3
A kind of protection aFGF protein medicine stability and active method, specifically includes the following steps:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, when bacterial density grow to 600nm absorbance be 0.6 when, under 18 DEG C of environment
Bacterium is collected by centrifugation by IPTG inducing expression 16h, through 4 DEG C, 8000 revs/min in aFGF protein；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution (pH 7.4)
In, and release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, removes big portion
Divide impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product is through nickel ion gel chromatography column point
From affording aFGF protein sterling liquid using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；
The specific preparation method of eluent 1 and eluent 2 and the preparation method phase of eluent 1 and eluent 2 in embodiment 1
Together；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: 20mL aFGF protein sterling liquid is fitted into bag filter, closes bag filter, then the bag filter equipped with aFGF protein liquid is set
In the beaker equipped with 2L PBS solution (pH 7.4), through magnetic stirrer 4 hours in 4 DEG C of environment, complete for the first time
After desalination, then 2L PBS solution is re-replaced, continues stirring 4 hours, it can be to aFGF protein liquid desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by protein concentration pipe
Photometer measurement concentration, and its concentration is adjusted to 200 μ g/mL using PBS；
Step 5 prepares aFGF protein protective agent；
AFGF protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, and mixed solution is by phosphoric acid
Salt buffer (pH 7.4) and dextran sulfate composition, and concentration of the dextran sulfate in the phosphate buffer is 2000 μ
g/mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
1:5 is mixed dextran sulphate solution by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -50 DEG C of refrigerators and saves.
Comparative example 1
Using heparin as the protective agent of aFGF protein, concrete operation step are as follows:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, when bacterial density grow to 600nm absorbance be 0.6 when, under 18 DEG C of environment
Bacterium is collected by centrifugation by IPTG inducing expression 16h, through 4 DEG C, 8000 revs/min in aFGF protein；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution (pH 7.4)
In, and release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, removes big portion
Divide impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product is through nickel ion gel chromatography column point
From affording aFGF protein sterling liquid using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；
The specific preparation method of eluent 1 and eluent 2 and the preparation method phase of eluent 1 and eluent 2 in embodiment 1
Together；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: 20mL aFGF protein sterling liquid is fitted into bag filter, closes bag filter, then the bag filter equipped with aFGF protein liquid is set
In the beaker equipped with 2L PBS solution (pH 7.4), through magnetic stirrer 4 hours in 4 DEG C of environment, complete for the first time
After desalination, then 2L PBS solution is re-replaced, continues stirring 4 hours, it can be to aFGF protein liquid desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by ultra-filtration centrifuge tube
Photometer measurement concentration, and its concentration is adjusted to 200 μ g/mL using PBS；
Step 5 prepares heparin protection agent solution；
Heparin protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, and mixed solution is by phosphoric acid
Salt buffer (pH 7.4) and heparin composition, and concentration of the heparin in the phosphate buffer is 2000 μ g/mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
Heparin protects agent solution, and 1:5 is mixed by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -50 DEG C of refrigerators and saves.
Comparative example 2
Using chondroitin sulfate as the protective agent of aFGF protein, concrete operation step are as follows:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, when bacterial density grow to 600nm absorbance be 0.6 when, under 18 DEG C of environment
Bacterium is collected by centrifugation by IPTG inducing expression 16h, through 4 DEG C, 8000 revs/min in aFGF protein；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution (pH 7.4)
In, and release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, removes big portion
Divide impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product is through nickel ion gel chromatography column point
From affording aFGF protein sterling liquid using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；
The specific preparation method of eluent 1 and eluent 2 and the preparation method phase of eluent 1 and eluent 2 in embodiment 1
Together；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: 20mL aFGF protein sterling liquid is fitted into bag filter, closes bag filter, then the bag filter equipped with aFGF protein liquid is set
In the beaker equipped with 2L PBS solution (pH 7.4), through magnetic stirrer 4 hours in 4 DEG C of environment, complete for the first time
After desalination, then 2L PBS solution is re-replaced, continues stirring 4 hours, it can be to aFGF protein liquid desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by protein concentration pipe
Photometer measurement concentration, and its concentration is adjusted to 200 μ g/mL using PBS；
Step 5 prepares chondroitin sulfate protection agent solution；
Chondroitin sulfate protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, mixed solution
It is made of phosphate buffer (pH 7.4) and chondroitin sulfate, and concentration of the chondroitin sulfate in the phosphate buffer
For 2000 μ g/mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
Chondroitin sulfate protects agent solution, and 1:5 is mixed by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -50 DEG C of refrigerators and saves.
Comparative example 3
Using λ type-carragheen as the protective agent of aFGF protein, concrete operation step are as follows:
The expression of step 1, aFGF protein: aFGF protein coded sequence is inserted into the pET-11b plasmid containing histidine tag
In after, imported into e. coli strain bl21, when bacterial density grow to 600nm absorbance be 0.6 when, under 18 DEG C of environment
Bacterium is collected by centrifugation by IPTG inducing expression 16h, through 4 DEG C, 8000 revs/min in aFGF protein；
The purifying of step 2, aFGF protein: the bacterium collected in step 1 is resuspended in phosphate buffer solution (pH 7.4)
In, and release aFGF protein through ultrasound cracking is broken, bacterial lysate is adsorbed through heparin gel chromatographic column, removes big portion
Divide impurity protein, and aFGF protein crude product is afforded by eluent 1；AFGF protein crude product is through nickel ion gel chromatography column point
From affording aFGF protein sterling liquid using eluent 2；
Wherein, eluent 1 is formulated by trishydroxymethylaminomethane hydrochloride buffer and NaCl, and trihydroxy methyl amino
Mass concentration of the methane in eluent 1 is 50mmol/L, and mass concentration of the NaCl in eluent 1 is 1.5mol/L, elution
The pH of liquid 1 is 7.2；
Eluent 2 is formulated by trishydroxymethylaminomethane hydrochloride buffer and imidazoles, and trishydroxymethylaminomethane
Mass concentration in eluent 2 is 50mmol/L, and mass concentration of the imidazoles in eluent 2 is 0.3mol/L, eluent 2
PH is 7.2；
The specific preparation method of eluent 1 and eluent 2 and the preparation method phase of eluent 1 and eluent 2 in embodiment 1
Together；
Step 3, desalination: typically containing the imidazoles and inorganic salts ingredients of high concentration in the aFGF protein eluent being purified into,
Need to bag filter dialyse by way of by aFGF protein eluent imidazoles and inorganic salts be replaced as PBS, concrete operation method
Are as follows: 20mL aFGF protein sterling liquid is fitted into bag filter, closes bag filter, then the bag filter equipped with aFGF protein liquid is set
In the beaker equipped with 2L PBS solution (pH 7.4), through magnetic stirrer 4 hours in 4 DEG C of environment, complete for the first time
After desalination, then 2L PBS solution is re-replaced, continues stirring 4 hours, it can be to aFGF protein liquid desalination；
Step 4, the adjustment of aFGF protein concentration: the aFGF protein liquid after desalination is used by micro light splitting by ultra-filtration centrifuge tube
Photometer measurement concentration, and its concentration is adjusted to 200 μ g/mL using PBS；
Step 5 prepares λ type-carragheen protection agent solution；
λ type-carragheen protection agent solution is made of by volume fraction 50% glycerol and 50% mixed solution, mixed solution
It is made of phosphate buffer (pH 7.4) and λ type-carragheen, and λ type-concentration of the carragheen in the phosphate buffer
For 2000 μ g/mL；
Step 6, aFGF protein and protective agent mixing: by after desalination in step 4 aFGF protein liquid and step 5 in prepare
1:5 is mixed λ type-carragheen protection agent solution by volume, obtains mixed liquor；
Step 7 dispenses the mixed liquor in step 6, is placed in -50 DEG C of refrigerators and saves.
Below for the method that various embodiments of the present invention and each comparative example provide, detection dextran sulfate is steady to aFGF protein
Qualitative and active protective effect.
One, protective effect of the molecule experiments detection dextran sulfate to aFGF protein thermal stability
1, test method
The experiment of aFGF protein thermal stability is predominantly detected in concentration range in the heparin of 2.5-2000 μ g/mL, λ type-card
The variation of aFGF protein melting temperature in drawing glue, dextran sulfate and chondroitin sulfate.By real-time PCR to aFGF egg
It is white to be heated, melting curve of the aFGF protein in four kinds of solution is detected, and evaluate the variation of aFGF protein melting temperature.
2, experimental result
The result is shown in Figure 1.
As shown in Figure 1, after dextran sulfate is added, the thermal stability of aFGF protein is significantly improved.Gradient concentration it is more
After sugar is mixed with aFGF protein, with the thermal stability of fluorescent quantitation technology detection albumen.Wherein, 40 μ g/mL heparin, 200 μ g/
ML λ type-carragheen, 10 μ g/mL dextran sulfates can improve albumen thermal stability significantly in conjunction with aFGF protein.Work as polysaccharide
When concentration is more than 5 μ g/mL, the polysaccharide of same concentrations is that dextran sulfate is better than heparin, heparin to the protective capability of aFGF protein
Better than λ type-carragheen.When polysaccharide concentration reaches 2000 μ g/mL, these three polysaccharide aFGF protein thermal stability can be improved to
60 DEG C or more, and dextran sulfate is most significant to the raising degree of aFGF protein thermal stability.However, low sulphated sulfuric acid is soft
Ossein is not obvious the protective effect of aFGF protein thermal stability.
Two, protective effect of the cell experiment detection dextran sulfate to aFGF protein thermal stability
1, method
By the method that each comparative example provides respectively by heparin, λ type-carragheen, chondroitin sulfate and the aFGF protein of protection
It is uniformly mixed and is placed on 37 DEG C that carry out 2 days and 1 day in cell incubator incubations, the method provided by embodiment 3 is (due to implementing
The method that example 1-3 is provided is substantially parallel to the protective effect of aFGF protein, so the method for only embodiment 3 being selected to provide in experiment
Detected) dextran sulfate is uniformly mixed to be placed on carries out 37 DEG C of 2 days and 1 day in cell incubator and be incubated for, and pass through
The bioactivity of albumen after cell detection is incubated for.
2, result
As a result as shown in Figure 2.
As shown in Figure 2, after 24 hours and 48 hours are incubated for, aFGF protein activity serious loss.Heparin, λ type-carragheen
There is greater activity with the aFGF of dextran sulfate protection, and chondroitin sulfate is to the active without obvious protective function of aFGF.Cause
This, dextran sulfate can be used as the protective agent of aFGF protein, the long-term preservation for aFGF protein.
It should be noted that involved in claims of the present invention when numberical range, it is thus understood that each numberical range
Any one numerical value can be selected between two endpoints and two endpoints, due to step method and the Examples 1 to 3 phase of use
Together, it repeats in order to prevent, the present invention describes preferred embodiment, and once a person skilled in the art knows basic wounds
The property made concept, then additional changes and modifications may be made to these embodiments.So the following claims are intended to be interpreted as includes
Preferred embodiment and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then 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.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987001728A1 (en) * | 1985-09-12 | 1987-03-26 | Biotechnology Research Partners, Ltd. | Recombinant fibroblast growth factors |
| CN1038765A (en) * | 1988-06-06 | 1990-01-17 | 武田药品工业株式会社 | The F G F composition of stabilisation and production thereof |
| CA2005154A1 (en) * | 1988-12-12 | 1990-06-12 | Takeda Chemical Industries, Ltd. | Method for purifying fibroblast growth factor protein |
| US5348941A (en) * | 1992-04-01 | 1994-09-20 | Merck & Co., Inc. | Stabilizers for fibroblast growth factors |
| CN102344930A (en) * | 2011-09-16 | 2012-02-08 | 江苏普罗赛生物技术有限公司 | Simple and convenient chemical industrial technology for acidic fibroblast growth factor (aFGF) |
-
2019
- 2019-05-29 CN CN201910458468.8A patent/CN110151977B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987001728A1 (en) * | 1985-09-12 | 1987-03-26 | Biotechnology Research Partners, Ltd. | Recombinant fibroblast growth factors |
| CN1038765A (en) * | 1988-06-06 | 1990-01-17 | 武田药品工业株式会社 | The F G F composition of stabilisation and production thereof |
| CA2005154A1 (en) * | 1988-12-12 | 1990-06-12 | Takeda Chemical Industries, Ltd. | Method for purifying fibroblast growth factor protein |
| US5348941A (en) * | 1992-04-01 | 1994-09-20 | Merck & Co., Inc. | Stabilizers for fibroblast growth factors |
| CN102344930A (en) * | 2011-09-16 | 2012-02-08 | 江苏普罗赛生物技术有限公司 | Simple and convenient chemical industrial technology for acidic fibroblast growth factor (aFGF) |
Non-Patent Citations (3)
| Title |
|---|
| CHANGYE SUN ET AL.: ""Sulfated polysaccharides interact with fibroblast growth factors and protect from denaturation"", 《FEBS OPEN BIO》 * |
| 刘青等: ""人酸性成纤维细胞生长因子在大肠杆菌的高效表达"", 《吉林大学学报（医学版）》 * |
| 周立社: "《分子生物检验技术》", 31 March 2012, 人民军医出版社 * |
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