CN113151344A - Saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein and preparation method of standard substance thereof - Google Patents
Saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein and preparation method of standard substance thereof Download PDFInfo
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- CN113151344A CN113151344A CN202110354982.4A CN202110354982A CN113151344A CN 113151344 A CN113151344 A CN 113151344A CN 202110354982 A CN202110354982 A CN 202110354982A CN 113151344 A CN113151344 A CN 113151344A
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method of saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein, which comprises the following steps: (1) constructing a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-aFGF-HSA; (2) preparing and converting aFGF-HSA fusion protein engineering bacteria; (3) inducible expression and purification of INVSC1/pYES2/CT-MF alpha-aFGF-HSA engineering bacteria. Meanwhile, a preparation method of the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein standard is also provided. The invention utilizes the long-acting recombinant human aFGF expressed by saccharomyces cerevisiae and the recombinant protein (aFGF-HSA) formed by fusing HSA, and has simple production process, low cost and uniform product.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein and a preparation method of a standard substance thereof.
Background
The acidic fibroblast growth factor (aFGF) is obtained by separating Thomas from bovine brain, is a Fibroblast Growth Factor (FGFs) family member, has the same efficacy as bFGF, has better stability under weak acid solution, is an important growth factor for various cells, and mainly plays roles in increasing angiogenesis, nerve fiber regeneration and cell repair after cell injury.
In recent years, a great deal of scholars have generated great interest in the biological functions of aFGF, and have made extensive studies on its high-efficiency expression and clinical application. In 1974, Gospodalowicz et al isolated and purified a polypeptide from bovine pituitary gland, named fibroblast growth factor, which had a promoting effect on the proliferation of the fibroblast line, BALB/c 3T 3. The fibroblast growth factor is divided into two types, which are respectively found from different positions, one type is stable under an acidic condition, is called an acidic fibroblast growth factor, and has an isoelectric point of about 5.6; the other one performs its biological function under alkaline conditions and is called basic fibroblast growth factor, with an isoelectric point of 9.6.
The human aFGF gene is located on chromosome 4 and is a single copy gene consisting of 2 large introns and 3 exons. aFGF is distributed mainly in kidney and brain tissue, is a cytoplasmic protein that itself lacks an N-terminal signal peptide, acting mainly on peripheral organelles by two means, autocrine and paracrine. The human aFGF polypeptide consists of 154 amino acid residues, has the molecular weight of 16kD, and is a beta-sheet protein without disulfide bonds. The secondary structure of aFGF includes clover structure comprising 12 reverse parallel beta chains and 4 chains constituting the folded unit of substructure connected into stable integral body. The response process of aFGF receptors on cells is mediated by aFGF tyrosine kinase receptor (aFGFR). The aFGF-induced dimerization reaction is the main form for its activity and signal transduction, and heparin plays an important role in this process. Signaling cascades and nuclear translocation are the major means by which aFGF exerts its own effects. The biological properties of aFGF are closely related to the various functional regions in its polypeptide chain sequence, the major functional regions including the receptor binding region, the heparin binding region and the nuclear translocation region.
Human aFGF had 95% effect on acne, ochre, whitening, skin elasticity and skin damage repair. aFGF promotes the production of new cells to replace the original cells and reduce the content of melanin and colored cells in the skin. In addition, aFGF can act on the skin surface, can reduce the damaging effect of ultraviolet rays on the skin, can improve the skin's glossiness, can retain skin moisture, and can reduce skin wrinkles. The aFGF also has good injury repair effect, can promote the growth of granulation tissues of the wound surface, induces the formation and re-epithelization of capillary embryo, and has obvious promotion effect on the wound surface repair. The growth of endothelial cells in the blood vessel can be promoted by aFGF, and the growth effect of the blood vessel can be improved. Pandit and other researches find that aFGF and other growth factors have the effects of remarkably inducing angiogenesis and promoting the repair of body surface, ischemic viscera, spinal nerves, brain and the like. Ischemic heart disease requires increased angiogenesis and aFGF is an excellent therapeutic for this disease. The expression level of aF GF in the body of patients with unstable angina is greatly improved compared with that of various cardiovascular diseases. In coronary atherosclerotic heart disease, aFGF plays an important role in the growth of coronary artery collaterals. aFGF enables rapid growth of neurons in vitro by increasing their survival. The aFGF can act on the hippocampal part in the brain to increase the glucose in the brain and promote the memory function to be improved. The neurotrophic effect of aFGF is also shown in the repair of central and peripheral nerve damage and can promote the functional regeneration of spinal nerve roots.
Human aFGF is mainly distributed in brain tissues, retina, adrenal glands, bones and other parts, and plays a wide biological effect in the aspects of accelerating wound healing and tissue repair, promoting bone growth and nourishing nerves. The process for naturally purifying the aFGF is complex, the cost is high, and the product has large molecular weight, so that the popularization and the application of the product are limited. The expression of foreign genes by gene engineering technology has become one of the efficient ways to obtain target proteins, and is mainly divided into prokaryotic expression and eukaryotic expression according to the difference of expression vectors and host bacteria. Although the prokaryotic expression system has low cost, the system has the defects of easy formation of inclusion bodies, low biological activity of the obtained protein and the like, and the target protein with high activity can be obtained by utilizing the eukaryotic expression system to carry out exogenous expression.
Human Serum Albumin (HSA) is a main protein in Human blood, is composed of 585 amino acids, is a soluble protein with the highest content in a Human circulatory system, and has the concentration of 34-54 g/L in blood. HSA is synthesized by the liver, and the serum half-life period is long and can reach 19 days. HSA plays an important role in regulating blood osmotic pressure, nourishing, promoting wound healing and the like, and is widely used for clinical treatment of ascites due to cirrhosis, burns, shock and the like. In addition, HSA has the characteristics of no immunogenicity, good human compatibility, wide tissue distribution, no enzyme activity and the like, so that HSA becomes an ideal recombinant protein fusion vector. The molecular weight of the recombinant protein can be increased by constructing a fusion protein technology, so that the half-life period is prolonged, and the stability of the recombinant protein is effectively improved.
For aFGF index detection, a standard substance is an essential component in production, and in biological product standardization, quality control and efficacy evaluation, the standard substance is a scale for medicine quality evaluation and plays a very important role, but the existing aFGF detection reagent in a laboratory has no standard substance.
Disclosure of Invention
In order to overcome the problems, the invention utilizes the recombinant protein (aFGF-HSA) formed by fusing the long-acting recombinant human aFGF expressed by saccharomyces cerevisiae and HSA, has simple production process, low cost and uniform product, establishes an economic, high-efficiency, stably-expressed, high-concentration and high-activity long-acting recombinant human aFGF standard protein, and provides a basis for establishing the quality standard of the long-acting recombinant human aFGF protein.
A preparation method of saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein comprises the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-aFGF-HSA specifically comprises the following steps:
artificially optimizing and obtaining human acidic fibroblast growth factor and human serum albumin factor to obtain plasmid pMD 19-T-aFGF-HSA;
carrying out double enzyme digestion on plasmid pYES2/CT-MF alpha and plasmid pMD19-T-aFGF-HSA, respectively cutting glue to recover aFGF-HSA gene fragments and pYES2/CT-MF alpha vectors, and then connecting by using T4 DNA ligase to obtain positive clone pYES2/CT-MF alpha-aFGF-HSA;
(2) the preparation and transformation of the aFGF-HSA fusion protein engineering bacteria specifically comprise the following steps:
preparing a common solution and a culture medium for a saccharomyces cerevisiae expression system;
transforming the pYES2/CT-MF alpha-aFGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-aFGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the induced expression and purification of INVSC1/pYES2/CT-MF alpha-aFGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSC1/pYES2/CT-MF alpha-aFGF-HSA obtained in the step (2), and purifying by metal ion affinity chromatography and anion exchange chromatography in sequence to obtain the saccharomyces cerevisiae expression recombinant human aFGF-HSA fusion protein.
Further, in the step (1), the human acidic fibroblast growth factor and the human serum albumin factor are artificially optimized and obtained to obtain the plasmid pMD19-T-aFGF-HSA, and the specific steps are as follows:
according to the property of pYES2/CT-MF alpha vector (figure 1) and Saccharomyces cerevisiae host codon preference, a recombinant human aFGF-HSA gene sequence and an amino acid sequence of the recombinant human aFGF-HSA are designed, wherein the recombinant human aFGF-HSA gene sequence is shown in a sequence table 1, and the amino acid sequence of the recombinant human aFGF-HSA is shown in a sequence table 2;
the sequence was inserted into pMD19-T Simple Vector in the order aFGF-HSA, which was terminated at the 5 'end with Not I cleavage site and at the 3' end with XbaI cleavage site, to give plasmid pMD 19-T-aFGF-HSA.
Further, in the step (1), plasmid pYES2/CT-MF alpha and plasmid pMD19-T-aFGF-HSA are subjected to double enzyme digestion, gel cutting is carried out to recover aFGF-HSA gene fragment and pYES2/CT-MF alpha vector respectively, then T4 DNA ligase is used for connection, and positive clone pYES2/CT-MF alpha-aFGF-HSA is obtained, and the specific steps are as follows:
carrying out double digestion on the plasmid pYES2/CT-MF alpha and the plasmid pMD19-T-aFGF-HSA by using endonuclease Not I and endonuclease Xba I respectively;
performing enzyme digestion reaction for 3 hours at 37 ℃ in a metal bath, detecting the enzyme digestion product by 2 percent agarose gel electrophoresis, respectively cutting gel, recovering aFGF-HSA gene fragments and pYES2/CT-MF alpha vectors, and then connecting by using T4 DNA ligase;
the recombinant plasmid is transformed into escherichia coli (DH5 alpha), a positive clone is selected on an LB plate culture medium containing ampicillin, and the positive clone is selected through bacterial liquid PCR (forward primer aFGF-HSA-F: 5'-GAAATTACCACGTTTACCGCTCTGA-3'; reverse primer aFGF-HSA-R: 5'-TAGATTAGTGATGGTGATGGTGATG-3') and double enzyme digestion (Not I and Xba I) identification, so that the positive clone pYES2/CT-MF alpha-aFGF-HSA is selected.
Further, in the step (2), the preparation of the common solution and culture medium of the saccharomyces cerevisiae expression system comprises the following specific steps:
YPD medium: dissolving peptone 20g, yeast extract 10g, and glucose 20g (20 g agar powder is added when preparing solid culture medium) in 800ml water, diluting to 1L, and autoclaving at 121 deg.C for 20 min;
SC-U selection Medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid (if SC-U selective plate culture medium is prepared, 20g of agar powder is additionally added), 900ml of deionized water is added, autoclave sterilization is carried out at 121 ℃ for 20min, 100ml of 20% glucose solution which is filtered and sterilized is added when the temperature is cooled to 50 ℃, and the mixture is uniformly mixed and stored at 4 ℃ for standby.
SC-U induction medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 800ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of filter-sterilized 20% glucose solution and 100ml of filter-sterilized 20% galactose solution, mixing uniformly, and storing at 4 ℃ for later use.
Further, in the step (2), the specific steps for obtaining the positive clone INVSC1/pYES2/CT-MF alpha-aFGF-HSA are as follows:
pYES2/CT-MF alpha-aFGF-HSA was transformed into Saccharomyces cerevisiae INVSC1 competent cells by an electrical transformation method:
adding 10 μ l pYES2/CT-MF α -aFGF-HSA plasmid into 80 μ l Saccharomyces cerevisiae INVScl competent cells, blowing and sucking to mix them uniformly, transferring into a precooled electric shock cup, performing ice bath for 5min, and wiping the outer wall of the electric shock cup;
adjusting a Bio-Rad electric converter to a fungus grade, selecting PIC, placing an electric shock cup on the Bio-Rad electric converter for electric shock, quickly adding 500 mu l of precooled 1M sorbitol solution into the electric shock cup, uniformly mixing, and coating an SC-U plate;
carrying out inversion culture at constant temperature of 30 ℃ until monoclonals grow out;
grown in SC-U selection medium (containing ampicillin) were Saccharomyces cerevisiae transformants containing pYES2/CT-MF α -aFGF-HSA, and the positive clones of INVSC1/pYES2/CT-MF α -aFGF-HSA were screened by PCR (forward primer: aFGF-HSA-F; reverse primer: aFGF-HSA-R) from the culture broth.
Further, in the step (3), the induced expression of the engineering bacteria containing INVSC1/pYES2/CT-MF alpha-aFGF-HSA specifically comprises the following steps:
single colonies of INVSC1/pYES2/CT-MF α -aFGF-HSA were picked and inoculated into 20ml SC-U selection cultureCulturing the culture medium at 30 deg.C overnight with shaking at 220rpm, and determining its OD600nmLight absorption value, transferring the calculated bacterial liquid with corresponding volume into 100ml SC-U induction culture medium to ensure that the initial OD600nmUp to 0.4;
centrifuging at 4 ℃ and 1500g for 5min, collecting thalli, suspending the thalli by using 1-2 ml of SC-U induction culture medium, re-inoculating 100ml of SC-U induction culture medium, placing in shaking culture at 30 ℃ for 96h, centrifuging at 4 ℃ and 15000g for 5min, collecting thalli and supernatant, centrifuging the supernatant subjected to induced expression, filtering through a 0.22 mu m filter membrane, and collecting filtrate.
Further, in the step (3), the metal ion affinity chromatography comprises the following steps:
the filtrate after centrifugation and filtration through a 0.22 μm membrane filter was subjected to self-column packing using a filler for Nickel ion chelate affinity chromatography of chemical Sepharose (TM) Fast Flow from GE Healthcare, and Ni was washed with 3 column volumes of purified water2+Chelating affinity chromatography column, balancing 2-3 column volumes with PBS;
detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value of 280nm wavelength are both stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min;
further passing through a chromatography column with PBS, and washing away the foreign proteins not bound to the chromatography column until OD280nmAnd (4) stabilizing. And then passing the protein through a chromatographic column by using PBS buffer solution containing 500mM of imidazole, eluting and collecting the protein corresponding to an elution peak to obtain the recombinant human aFGF-HSA protein stock solution after metal ion affinity chromatography.
Further, in the step (3), the anion exchange chromatography comprises the following steps:
replacing a protein stock solution collected after metal ion affinity chromatography purification into Binding Buffer II (50mM tris (hydroxymethyl) aminomethane, pH8.5) by DEAE anion exchange chromatography, loading the sample through a DEAE anion exchange chromatography column well balanced by the Binding Buffer II, and collecting an aFGF-HSA fusion protein peak;
eluting with Elution Buffer II (50mM tris, 1M NaCl, pH8.5), washing off foreign proteins, and collecting proteins corresponding to Elution peaks, namely the purified recombinant human aFGF-HSA fusion protein obtained by the invention.
A preparation method of a recombinant human aFGF-HSA fusion protein standard comprises the following steps:
and (2) filtering and sterilizing the recombinant human aFGF-HS A fusion protein solution purified by metal ion affinity chromatography and anion exchange chromatography by using a 0.22 mu m filter membrane, diluting the solution by using 10mmol/L phosphate buffer solution, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose freeze-drying protective agent to perform freeze vacuum drying, and drying to obtain the recombinant human aFGF-HSA fusion protein standard product.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes the recombinant protein (aFGF-HSA) formed by fusing the long-acting recombinant human acidic fibroblast growth factor secreted and expressed by saccharomyces cerevisiae and HSA, and improves the stability of the recombinant protein. The saccharomyces cerevisiae secretion expression system is a eukaryotic expression system, can express protein at a high level and secrete the protein into a culture medium, and has the advantages of simple production process, low cost, uniform product and no immunogenicity.
The invention also prepares a detection standard substance, takes the aFGF international standard substance as a standard for cooperative calibration, meets the regulation through appearance, sterility and moisture detection, and keeps the biological activity stable for 24 months at the temperature of-20, 4, 25 and 37 ℃.
Description of the drawings:
FIG. 1 plasmid pYES2/CT-MF α map;
FIG. 2SDS-PAGE identification of purified recombinant human aFGF-HSA protein
FIG. 3Western Blot to identify purified recombinant human aFGF-HSA protein
Description of sequence listing:
sequence table 1 nucleotide sequence of recombinant human aFGF-HSA of the invention
Sequence table 2 amino acid sequence of recombinant human aFGF-HSA of the invention
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
The specific implementation mode is as follows:
example 1: construction of Saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-aFGF-HSA
1.1 artificial optimization and acquisition of human acidic fibroblast growth factor and human serum albumin factor:
the following related genes and amino acid sequences are obtained through GenBank inquiry, and the gene sequence is optimized by using the amino acid sequence expressed by the target gene in the experiment, and then the artificial synthesis is carried out to construct an expression vector. According to the property of pYES2/CT-MF alpha vector (figure 1) and Saccharomyces cerevisiae host codon preference, an aFGF-HSA gene sequence and an amino acid sequence of recombinant human aFGF-HSA are designed, wherein the recombinant human aFGF-HSA gene sequence is shown in a sequence table 1, and the amino acid sequence of recombinant human aFGF-HSA is shown in a sequence table 2.
In the sequence table 1, GCGGCCGC is Not I enzyme cutting site, and TCTAGA is Xba I enzyme cutting site; ATG is initiation codon, TAA is termination codon; GCAGAGGCGGCGGCTAAGGAAGCTGCAGCCAAAGCC is a base sequence corresponding to Linker connecting aFGF and HSA sequences; CATCACCATCACCATCAC is a 6 × His tag sequence.
The sequence was inserted into pMD19-T Simple Vector in the order aFGF-HSA, which was terminated at the 5 'end with Not I cleavage site and at the 3' end with XbaI cleavage site, to give plasmid pMD 19-T-aFGF-HSA.
1.2 construction of pYES2/CT-MF α -aFGF-HSA expression vector
Plasmid pYES2/CT-MF α and plasmid pMD19-T-aFGF-HSA were digested simultaneously with endonuclease Not I and endonuclease Xba I, respectively.
The enzyme digestion reaction system is as follows:
performing enzyme digestion reaction for 3h at 37 ℃ in a metal bath, detecting the enzyme digestion product by 2 percent agarose gel electrophoresis, respectively cutting gel, recovering aFGF-HSA gene fragment and pYES2/CT-MF alpha vector, and then connecting by using T4 DNA ligase.
The connection reaction system is as follows:
the reaction conditions are 16 ℃ and 14h, the recombinant plasmid is transformed into escherichia coli (DH5 alpha) by a conventional method (calcium chloride method), a positive clone is selected on an LB plate culture medium containing ampicillin, and the positive clone pYES2/CT-MF alpha-aFGF-HSA is selected by bacterial liquid PCR (forward primer aFGF-HSA-F: 5'-GAAATTACCACGTTTACCGCTCTGA-3'; reverse primer aFGF-HSA-R: 5'-TAGATTAGTGATGGTGATGGTGATG-3') and double enzyme digestion (Not I and Xba I) identification.
Example 2: preparation and transformation of aFGF-HSA fusion protein engineering bacteria
2.1 preparation of common solution and culture Medium for Saccharomyces cerevisiae expression System
YPD medium: dissolving peptone 20g, yeast extract 10g, and glucose 20g (20 g agar powder is added when preparing solid culture medium) in 800ml water, diluting to 1L, and autoclaving at 121 deg.C for 20 min;
SC-U medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid (if preparing SC-U selective plate culture medium, 20g of agar powder is additionally added), 900ml of deionized water is added, autoclave sterilization is carried out at 121 ℃ for 20min, 100ml of 20% glucose solution for filtration sterilization is added when the temperature is cooled to 50 ℃, and the mixture is uniformly mixed and stored at 4 ℃ for standby;
SC-U induction medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 800ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of filter-sterilized 20% glucose solution and 100ml of filter-sterilized 20% galactose solution, mixing uniformly, and storing at 4 ℃ for later use.
2.2pYES2/CT-MF alpha-aFGF-HSA transformed Saccharomyces cerevisiae
pYES2/CT-MF alpha-aFGF-HSA was transformed into Saccharomyces cerevisiae INVSC1 competent cells by electrotransformation.
Mu.l of pYES2/CT-MF α -aFGF-HSA plasmid was added to 80. mu.l of Saccharomyces cerevisiae INVScl competent cells, mixed well by aspiration, and then transferred to a pre-cooled cuvette. Ice-bath for 5min, and wiping the outer wall of the electric shock cup. The Bio-Rad electric converter was adjusted to the fungi range, PIC option, and the cuvette was placed on the Bio-Rad electric converter for electric shock. Add 500. mu.l of pre-chilled 1M sorbitol solution quickly to the cuvette, mix well and coat the SC-U plate. And (4) carrying out inverted culture at the constant temperature of 30 ℃ until a monoclonal antibody grows out. Grown in SC-U selection medium (containing ampicillin) were Saccharomyces cerevisiae transformants containing pYES2/CT-MF α -aFGF-HSA, and the positive clones of INVSC1/pYES2/CT-MF α -aFGF-HSA were screened by PCR (forward primer: aFGF-HSA-F; reverse primer: aFGF-HSA-R) from the culture broth.
Example 3: inducible expression, detection and purification of INVSC1/pYES2/CT-MF alpha-aFGF-HSA engineering bacteria
3.1 inducible expression and detection of engineering bacteria
Single colonies of INVSC1/pYES2/CT-MF α -aFGF-HSA were picked, inoculated into 20ml of SC-U selection medium, and shake-cultured overnight at 220rpm at 30 ℃. Measuring the OD thereof600nmLight absorption value, transferring the calculated bacterial liquid with corresponding volume into 100ml SC-U induction culture medium to ensure that the initial OD600nmUp to 0.4.
Centrifuging at 4 ℃ and 1500g for 5min, collecting thalli, suspending the thalli by using 1-2 ml of SC-U induction culture medium, re-inoculating 100ml of SC-U induction culture medium, placing in 30 ℃ for shake culture for 96h, centrifuging at 4 ℃ and 15000g for 5min, collecting thalli and supernatant, and filtering the supernatant subjected to induced expression by a 0.22 mu m filter membrane.
The supernatant protein liquid obtained by induction expression can be observed to have obvious specific bands at about 86kDa by SDS-PAGE electrophoresis (figure 2, wherein, a Lane M: Marker, a Lane 1: the induction supernatant containing the unloaded plasmid Saccharomyces cerevisiae strain, a Lane 2, the induction supernatant), the induction expression supernatant of the Saccharomyces cerevisiae strain containing pYES-FGF 2/CT-MF alpha-aFGF-HSA plasmid is identified by rabbit Anti-aFGF polyclonal antibody (Anti-FGF1 antibody, ab207321) through Western blot, the specific bands at about 86kDa can be observed (figure 3, wherein, a Lane M: Marker, a Lane 1: the unloaded plasmid Saccharomyces cerevisiae strain product, and a Lane 2: aFGF-HSA protein), therefore, the eukaryotic expression pYES2/CT-MF alpha-FGF-HSA engineering bacteria containing the human acidic fibroblast growth factor prepared by the invention can generate the recombinant human acidic fibroblast growth factor and the recombinant human acidic fibroblast growth factor at about 86kDa through galactose induction Fusion protein of serum albumin.
3.2 purification of recombinant human aFGF-HAS fusion proteins
3.2.1 Metal ion affinity chromatography
The culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter for loading. The column was self-packed using the GE Healthcare company chemical Sepharose TM Fast Flow Nickel ion chelate affinity chromatography packing, and the Ni was washed with 3 column volumes of purified water2+Chelating affinity chromatography column, then using PBS to balance 2-3 column volumes. And (3) detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value are both stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min. Further passing through a chromatography column with PBS, and washing away the foreign proteins not bound to the chromatography column until OD280nmAnd (4) stabilizing. And then passing the protein through a chromatographic column by using PBS buffer solution containing 500mM of imidazole, eluting and collecting the protein corresponding to an elution peak to obtain aFGF-HSA protein stock solution eluted by metal ion affinity chromatography.
3.2.2 anion exchange chromatography
DEAE anion exchange chromatography, the aFGF-HSA protein stock solution eluted by metal ion affinity chromatography is replaced into Binding Buffer II (50mM tris (hydroxymethyl) aminomethane, pH8.5), and then the sample is loaded through a DEAE anion exchange chromatography column well balanced by the Binding Buffer II, and an aFGF-HSA fusion protein peak is collected. Eluting with Elution Buffer II (50mM tris, 1M NaCl, pH8.5), washing off foreign protein, and collecting protein corresponding to an Elution peak to obtain the saccharomyces cerevisiae expression recombinant human aFGF-HSA fusion protein.
Example 4: preparation and detection of aFGF-HSA standard substance
4.1 preparation of aFGF-HSA Standard
Filtering and sterilizing the protein solution after the purification of the recombinant aFGF-HSA by using a 0.22 mu m filter membrane, diluting the protein solution by using 10mmol/L phosphate buffer solution, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose freeze-drying protective agent into the diluted protein solution, and performing freeze-vacuum drying.
4.2 detection of aFGF-HSA Standard
Protein content determination: the protein concentration of the purified aFGF-HSA standard substance was measured by the Bradford method, and the protein content was 2.0 mg/ml.
Purity identification by SDS-PAGE: the purity of aFGF-HSA standard substance is determined by SDS-PAGE, the purity is 95%, and the relative molecular weight is 86 kD.
And (4) HPLC purity identification: aFGF-HSA standards were analyzed on a μ RPC 18 ST 4.6/100 reverse phase column. The main peak area accounts for 99.23 percent of the total area by calculating the peak area.
N-terminal amino acid sequence determination:
(a) SDS-PAGE electrophoresis: performing SDS-PAGE electrophoresis on an aFGF-HSA protein sample, loading the prepared polyacrylamide gel on a vertical electrophoresis apparatus before loading, and performing empty run for 30min at a constant voltage of 50V;
(b) film transfer: electrotransfering the protein to a PVDF (polyvinylidene fluoride) membrane, wherein the electrotransfer buffer solution is CAPS buffer solution;
(c) ponceau red staining: putting the PVDF membrane into a ponceau dye solution for dyeing for 30min, and washing away the background color by water;
(d) cutting the target band, placing the target band in an Eppendorf tube, preserving at-20 ℃, and performing N-terminal amino acid sequencing by an Edman degradation method;
(e) the N-terminal twenty amino acid sequences are EAEAYVEFPRAAAMAEGEIT, which indicates that the purified target protein is aFGF-HSA.
In conclusion, the invention utilizes the recombinant protein (aFGF-HSA) formed by fusing the long-acting recombinant human aFGF expressed by the saccharomyces cerevisiae and the HSA, overcomes the defects that in the prior art, an inclusion body is easily formed by adopting an expression system of escherichia coli, lactobacillus, brevibacillus and pichia pastoris, and the obtained protein has lower biological activity, and has simple production process, low cost and uniform product; meanwhile, an important recombinant human aFGF-HSA standard substance is provided, and plays an important role in biological product standardization, quality control and efficacy evaluation. The long-acting recombinant human aFGF-HSA fusion protein prepared by the invention has better uniformity, stability and recovery rate, and can obviously reduce the production cost.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> research institute of biological product industry, Inc. of Utafel, Utaki
<120> preparation methods of saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein and standard substance thereof
<160> 2
<170> SIPOSequenceListing 1.0
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gcggccgcaa tggcggaagg cgaaattacc acgtttaccg ctctgaccga aaagttcaac 60
ctgccgccgg gcaactacaa aaaaccaaaa ctgctgtact gttctaacgg cggccacttc 120
ctgcgtatcc tgccggacgg tacggtagat ggtactcgtg atcgtagcga tcagcacatc 180
cagctgcagc tgtccgctga atctgttggt gaggtttaca tcaagtccac tgagactggt 240
cagtatctgg ctatggatac ggacggcctg ctgtacggta gccaaactcc gaacgaggag 300
tgcctgtttc tggagcgcct ggaagaaaac cactataaca cctacatttc taaaaagcac 360
gcagaaaaaa actggttcgt tggtctgaaa aaaaacggtt cctgcaaacg tggtccgcgc 420
acgcactacg gtcagaaagc tatcctgttc ctgccgctgc cggttagctc cgacgcagag 480
gcggcggcta aggaagctgc agccaaagcc atgaagtggg ttacgtttat ctccctatta 540
tttctgttct catccgccta ctccagaggt gttttcagga gagatgctca caaatctgag 600
gttgctcata gattcaagga tttgggtgaa gaaaacttta aggccttagt gttaatagct 660
ttcgcacaat acctgcaaca gtgtcctttt gaagaccatg tcaaattagt taatgaagtc 720
accgaatttg ctaagacgtg cgttgctgat gagtctgccg aaaattgtga caaatcactg 780
catacattgt tcggtgataa gctatgtacc gttgcaactc ttagagaaac gtacggagag 840
atggcggact gttgtgctaa acaagaacct gaaagaaatg aatgtttttt gcaacacaaa 900
gatgataatc caaacttgcc aagattggta agaccagaag ttgacgttat gtgtaccgct 960
tttcatgata atgaagaaac atttttgaaa aagtatcttt atgaaatagc aaggaggcat 1020
ccttacttct acgctccaga gttattattt tttgcaaaaa gatataaggc agcttttact 1080
gaatgttgtc aggctgcgga taaagccgca tgtctgttac ccaaattgga tgaattgaga 1140
gacgagggca aagctagtag tgccaaacaa agattaaaat gcgcttcatt acaaaaattt 1200
ggagaaagag cgtttaaggc ttgggccgta gcaagattgt ctcagagatt cccgaaagcc 1260
gaatttgcag aagtgagtaa actggtcaca gatttgacga aagttcacac agaatgttgt 1320
cacggagatt tattggaatg cgctgacgat agggctgact tagctaaata catatgcgag 1380
aatcaagatt ccatatcatc aaaattgaaa gaatgttgtg agaaaccatt attagaaaaa 1440
tcccactgta tagctgaagt tgagaacgat gaaatgcccg cggatttacc ctcccttgcg 1500
gctgacttcg ttgagtcaaa ggatgtttgc aagaattacg cggaggccaa ggatgttttt 1560
cttggcatgt ttttatatga gtatgccaga cgtcatccgg attattctgt agttctactg 1620
ttaaggcttg ccaagacata cgaaactacc ttagaaaaat gttgtgcggc tgccgatcca 1680
catgaatgtt acgcaaaagt ttttgatgaa ttaccgccgc ttgtcgagga gccacaaaat 1740
ttaattaaac aaaactgtga attatttgaa caattaggtg aatataaatt ccaaaacgca 1800
ttattggtca gatatacaaa aaaagtacct caggtttcca caccaacttt agtggaagtg 1860
tcacgtaacc taggcaaggt tggtagtaag tgctgtaaac acccagaagc taagagaatg 1920
ccatgcgctg aagattatct atcagtcgta cttaatcaac tgtgtgtcct acacgagaag 1980
actcctgtca gtgacagagt gacaaaatgt tgcaccgaga gcttagttaa tagaagaccg 2040
tgtttttcag cgctggaagt tgatgaaacc tatgttccaa aggagttcaa tgcagaaaca 2100
ttcaccttcc atgctgatat atgtactctt agtgaaaaag aaaggcagat caaaaaacaa 2160
actgccctgg tcgaattagt caaacataaa cctaaagcaa cgaaggaaca gttgaaggcc 2220
gtaatggatg atttcgcagc tttcgttgaa aaatgttgca aggctgatga caaagagaca 2280
tgttttgctg aagagggaaa aaaattggtg gcagcttctc aagccgcttt agggttacat 2340
caccatcacc atcactaatc taga 2364
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Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe
1 5 10 15
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser
20 25 30
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly
35 40 45
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu
50 55 60
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
65 70 75 80
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu
85 90 95
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr
100 105 110
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
115 120 125
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala
130 135 140
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Ala Glu Ala Ala Ala
145 150 155 160
Lys Glu Ala Ala Ala Lys Ala Met Lys Trp Val Thr Phe Ile Ser Leu
165 170 175
Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Asp
180 185 190
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu
195 200 205
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln
210 215 220
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe
225 230 235 240
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser
245 250 255
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg
260 265 270
Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu
275 280 285
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro
290 295 300
Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp
305 310 315 320
Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg
325 330 335
His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr
340 345 350
Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys
355 360 365
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser
370 375 380
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg
385 390 395 400
Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys
405 410 415
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val
420 425 430
His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg
435 440 445
Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser
450 455 460
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys
465 470 475 480
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu
485 490 495
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu
500 505 510
Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg
515 520 525
His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr
530 535 540
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys
545 550 555 560
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln
565 570 575
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr
580 585 590
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln
595 600 605
Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val
610 615 620
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala
625 630 635 640
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu
645 650 655
Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu
660 665 670
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr
675 680 685
Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile
690 695 700
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu
705 710 715 720
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys
725 730 735
Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala
740 745 750
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala
755 760 765
Ala Ser Gln Ala Ala Leu Gly Leu His His His His His His
770 775 780
Claims (9)
1. A preparation method of a saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein is characterized by comprising the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-aFGF-HSA specifically comprises the following steps:
artificially optimizing and obtaining human acidic fibroblast growth factor and human serum albumin factor to obtain plasmid pMD 19-T-aFGF-HSA;
carrying out double enzyme digestion on plasmid pYES2/CT-MF alpha and plasmid pMD19-T-aFGF-HSA, respectively cutting gel to recover aFGF-HSA gene fragment and pYES2/CT-MF alpha vector fragment, then connecting with T4 DNA ligase, and screening by an LB plate culture medium containing ampicillin to obtain a positive clone pYES2/CT-MF alpha-aFGF-HSA;
(2) the preparation and transformation of the aFGF-HSA fusion protein engineering bacteria specifically comprise the following steps:
preparing a common solution and a culture medium for a saccharomyces cerevisiae expression system;
electrically converting pYES2/CT-MF alpha-aFGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-aFGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the induced expression and purification of INVSC1/pYES2/CT-MF alpha-aFGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSC1/pYES2/CT-MF alpha-aFGF-HSA obtained in the step (2), and purifying by metal ion affinity chromatography and anion exchange chromatography in sequence to obtain the saccharomyces cerevisiae expression recombinant human aFGF-HSA fusion protein.
2. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein in the step (1), the human acidic fibroblast growth factor and the human serum albumin factor are artificially optimized and obtained to obtain the plasmid pMD19-T-aFGF-HSA, and the specific steps are as follows:
according to the property of pYES2/CT-MF alpha vector and the preference of saccharomyces cerevisiae host codon, a recombinant human aFGF-HSA gene sequence and an amino acid sequence of the recombinant human aFGF-HSA are designed, wherein the recombinant human aFGF-HSA gene sequence is shown in a sequence table 1, and the amino acid sequence of the recombinant human aFGF-HSA is shown in a sequence table 2;
the sequence was inserted into pMD19-T Simple Vector in the order aFGF-HSA, which was terminated at the 5 'end with Not I cleavage site and at the 3' end with XbaI cleavage site, to give plasmid pMD 19-T-aFGF-HSA.
3. The method of claim 1, wherein in the step (1), plasmid pYES2/CT-MF α and plasmid pMD19-T-aFGF-HSA are subjected to double digestion, and then aFGF-HSA gene fragment and pYES2/CT-MF α vector are recovered by cutting gel, and then connected with T4 DNA ligase to obtain positive clone pYES2/CT-MF α -aFGF-HSA, and the specific steps are as follows:
carrying out double digestion on the plasmid pYES2/CT-MF alpha and the plasmid pMD19-T-aFGF-HSA by using endonuclease Not I and endonuclease Xba I respectively;
performing enzyme digestion reaction for 3 hours at 37 ℃ in a metal bath, detecting the enzyme digestion product by 2 percent agarose gel electrophoresis, respectively cutting gel, recovering aFGF-HSA gene fragments and pYES2/CT-MF alpha vectors, and then connecting by using T4 DNA ligase;
transforming the recombinant plasmid into escherichia coli (DH5 alpha), selecting a positive clone on an LB plate culture medium containing ampicillin, and selecting a positive clone pYES2/CT-MF alpha-aFGF-HSA through bacterial liquid PCR and double enzyme digestion identification.
4. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein in the step (2), a solution and a culture medium commonly used for a saccharomyces cerevisiae expression system are prepared, and the specific method comprises the following steps:
YPD medium: dissolving peptone 20g, yeast extract 10g, and glucose 20g (20 g agar powder is added when preparing solid culture medium) in 800ml water, diluting to 1L, and autoclaving at 121 deg.C for 20 min;
SC-U selection Medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid (20 g of agar powder is additionally added when preparing SC-U selective plate culture medium), 900ml of deionized water is added, autoclave sterilization is carried out for 20min at 121 ℃, 100ml of 20% glucose solution for filtration sterilization is added when the temperature is cooled to 50 ℃, and the mixture is uniformly mixed and stored for later use at 4 ℃;
SC-U induction medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 800ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of filter-sterilized 20% glucose solution and 100ml of filter-sterilized 20% galactose solution, mixing uniformly, and storing at 4 ℃ for later use.
5. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein the specific steps for obtaining the positive clone INVSC1/pYES2/CT-MF α -aFGF-HSA in the step (2) are as follows:
pYES2/CT-MF α -aFGF-HSA was transformed into Saccharomyces cerevisiae INVSC1 competent cells using an electrical transformation method:
adding 10 μ l of pYES2/CT-MF α -aFGF-HSA plasmid into 80 μ l of Saccharomyces cerevisiae INVScl competent cells, blowing and sucking to mix the cells uniformly, transferring the cells into a precooled electric shock cup, carrying out ice bath for 5min, and wiping the outer wall of the electric shock cup;
adjusting a Bio-Rad electric converter to a fungus grade, selecting PIC, placing an electric shock cup on the Bio-Rad electric converter for electric shock, quickly adding 500 mu l of precooled 1M sorbitol solution into the electric shock cup, uniformly mixing, and coating an SC-U plate;
carrying out inversion culture at constant temperature of 30 ℃ until monoclonals grow out;
grown in SC-U selection medium (containing ampicillin) were Saccharomyces cerevisiae transformants containing pYES2/CT-MF α -aFGF-HSA, and the positive clones of INVSC1/pYES2/CT-MF α -aFGF-HSA were screened by PCR (forward primer: aFGF-HSA-F; reverse primer: aFGF-HSA-R) from the culture broth.
6. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein in the step (3), the inducible expression of INVSC1/pYES2/CT-MF α -aFGF-HSA engineering bacteria comprises the following specific steps:
selecting single colony of INVSC1/pYES2/CT-MF alpha-aFGF-HSA, inoculating to 20ml SC-U selection medium, shake culturing at 30 deg.C and 220rpm overnight, and determining its OD600nmLight absorption value, transferring the calculated bacterial liquid with corresponding volume into 100ml SC-U induction culture medium to ensure that the initial OD600nmUp to 0.4;
centrifuging at 4 ℃ and 1500g for 5min, collecting thalli, suspending the thalli by using 1-2 ml of SC-U induction culture medium, re-inoculating 100ml of SC-U induction culture medium, placing in shaking culture at 30 ℃ for 96h, centrifuging at 4 ℃ and 15000g for 5min, collecting thalli and supernatant, centrifuging the supernatant subjected to induced expression, filtering through a 0.22 mu m filter membrane, and collecting filtrate.
7. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein in the step (3), the metal ion affinity chromatography comprises the following steps:
the filtrate after centrifugation and filtration through a 0.22 μm membrane filter was subjected to self-column packing using a filler for Nickel ion chelate affinity chromatography of chemical Sepharose (TM) Fast Flow from GE Healthcare, and Ni was washed with 3 column volumes of purified water2+Chelating affinity chromatography column, balancing 2-3 column volumes with PBS;
detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value of 280nm wavelength are both stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min;
further passing through a chromatography column with PBS, and washing away the foreign proteins not bound to the chromatography column until OD280nmAnd (3) stabilizing, passing through a chromatographic column by using PBS buffer solution containing 500mM of imidazole, eluting and collecting protein corresponding to an elution peak to obtain the recombinant human aFGF-HSA protein stock solution after metal ion affinity chromatography.
8. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human aFGF-HSA fusion protein of claim 1, wherein in the step (3), the anion exchange chromatography comprises the following steps:
replacing a protein stock solution collected after metal ion affinity chromatography purification into a Binding Buffer II by DEAE anion exchange chromatography, loading the sample through a DEAE anion exchange chromatography column well balanced by the Binding Buffer II, and collecting an aFGF-HSA fusion protein peak;
eluting with Elution Buffer II, washing off foreign protein, and collecting protein corresponding to the Elution peak, namely the purified recombinant human aFGF-HSA fusion protein obtained by the invention.
9. A preparation method of a recombinant human aFGF-HSA fusion protein standard is characterized by comprising the following steps:
taking the recombinant human aFGF-HSA fusion protein solution prepared by the preparation method of any one of claims 1-8, filtering and sterilizing with a 0.22 μm filter membrane, diluting with 10mmol/L phosphate buffer, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose lyophilization protectant, and freeze-drying under vacuum to obtain the recombinant human aFGF-HSA fusion protein standard.
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