CN113151343A - Preparation method of saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein and standard thereof - Google Patents
Preparation method of saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein and standard thereof Download PDFInfo
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- CN113151343A CN113151343A CN202110354980.5A CN202110354980A CN113151343A CN 113151343 A CN113151343 A CN 113151343A CN 202110354980 A CN202110354980 A CN 202110354980A CN 113151343 A CN113151343 A CN 113151343A
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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 EGF-HSA fusion protein, which comprises the following steps: (1) constructing a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-hEGF-HSA; (2) preparing and transforming hEGF-HSA fusion protein engineering bacteria; (3) inducible expression and purification of INVSC1/pYES2/CT-MF alpha-hEGF-HSA engineering bacteria. Meanwhile, a preparation method of the saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein standard is also provided. The invention utilizes the recombinant protein (hEGF-HSA) formed by fusing the long-acting recombinant human EGF expressed by the saccharomyces cerevisiae and the 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 EGF-HSA fusion protein and a preparation method of a standard substance thereof.
Background
Since the discovery of human epidermal growth factor (hEGF) in the early 60 s, its wide application was determined by its special biological effects and potentially enormous economic benefits, although the history of research was not long, and its discovery gained the nobel prize for physiology and medicine in 1986. Numerous studies have shown that EGF has the functions of promoting cell proliferation and epithelial regeneration, and thus is widely used for promoting the healing of surgical wounds and other wound surfaces, treating various corneal-related diseases, promoting the healing of gastrointestinal mucosal ulcers, and treating tumors. Human epidermal growth factor (hEGF) was first purified from human urine in 1974, and has a structure consisting of 53 amino acids, a molecular weight of 6045 daltons, 6 intramolecular disulfide bonds consisting of cysteines, and EGF having no glycosyl sites, is very stable, heat-resistant, acid-resistant, widely present in body fluids and various glands, mainly synthesized by submandibular glands and duodenum, and has been found to be specifically elevated in milk, urine, and semen, but at lower concentrations in serum, in most body fluids of the human body.
hEGF is widely present in most body fluids of human bodies, but the content is not high, and the traditional extraction method for preparing EGF is high in cost and reaches the selling price of millions of dollars per gram, so that the preparation of the EGF in large quantity is difficult, and the clinical application is greatly limited. Since the 80 s, recombinant human epidermal growth factor (hEGF) prepared by genetic engineering techniques was introduced for clinical research and has been greatly developed. Many pharmaceutical companies have turned their products from the laboratory to the market, especially some pharmaceutical companies abroad. For example, EGF eye drops, developed by Zambon, Italy, are marketed in Switzerland under the Gentel trade name, and are used for the treatment of various corneal diseases and for promoting the healing of corneal epithelium. Companies such as Chiron, Amgen, Sumitomo, Wutian, Hitachi, etc., in the United states, developed EGF having ulcer treating effect, and entered later clinical trials in order. EGF is a multifunctional growth factor, which can induce growth and migration of various histiocytes in vivo and in vitro by combining with a receptor (EGFR) thereof, and promote expression of differentiation genes, thereby playing a role in maintaining normal metabolism of epithelial cells. Therefore, EGF marker detection is also more used for clinical diagnosis of laryngeal cancer (Sun Nengzhu et al, 1998), lung cancer (Maolu, 2010; Gong Cheng Xiang et al, 2013), innovative fracture healing (Wang Lei Junying, 2014), and the like.
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 biological products, biological methods are mostly adopted for the effectiveness index of quality evaluation, and the methods have large variability. Therefore, the standard substance is an essential component in production, and plays a very important role in standardization of biological products, quality control and efficacy evaluation, and is a scale for quality evaluation of medicines. While natural EGF is widely present in most body fluids of the human body, it is not present in high amounts. The traditional extraction method for preparing EGF is high in cost, and reaches the selling price of millions of dollars per gram, so that the EGF is difficult to prepare in large quantities, and the clinical application is greatly limited. At present, engineering bacteria for expressing exogenous EGF by using a gene recombination technology comprise escherichia coli, lactobacillus, bacillus, saccharomyces cerevisiae and pichia pastoris. Oka et al secretly expressed EGF in E.coli, and the EGF concentration in the supernatant was only 1.026 mg/L; the expression amount of the EGF expressed in the Brevibacillus brevis by Yamagata and the like is about 260 mg/L; wanyang et al expressed pEGF in Pichia pastoris by multi-copy screening, the pEGF concentration in supernatant was 82 mg/L; and D.N.Lee and the like express pEGF in pichia pastoris, and the expression quantity of the pEGF reaches 870 mg/L; pEGF is expressed in Lactobacillus by Q.C.K.Cheung, etc., and the expression level of pEGF in the supernatant is about 500. mu.g/L. Pascalal et al expressed a small amount of the fusion protein of pEGF in saccharomyces cerevisiae and the biological activity of the fusion protein was not further studied. In addition, although EGF can be efficiently induced and expressed in pichia pastoris, methanol induction is required for the induction and expression of pichia pastoris, and further processing and detoxification treatment are required in production application; and the Q.C.K.Cheung and the like induce and express pEGF in lactobacillus which is beneficial bacterium, but the expression amount is too low (500 mug/L), and the significance is not great for large-scale fermentation production. At present, there is no report on the secretory expression of EGF gene in beneficial bacteria Saccharomyces cerevisiae.
Disclosure of Invention
In order to overcome the technical problems in the background art, the invention utilizes the recombinant protein (hEGF-HSA) formed by fusing the long-acting recombinant human EGF and HSA expressed by saccharomyces cerevisiae, has simple production process, low cost and uniform product, establishes an economic, high-efficiency and stably-expressed long-acting recombinant human EGF standard protein with high concentration and high activity, and provides a basis for establishing the quality standard of the long-acting recombinant human EGF protein.
A preparation method of saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein comprises the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-hEGF-HSA specifically comprises the following steps:
artificially optimizing and obtaining human epidermal growth factor and human serum albumin factor to obtain plasmid pMD 19-T-hEGF-HSA;
carrying out double enzyme digestion on plasmid pYES2/CT-MF alpha and plasmid pMD19-T-hEGF-HSA, respectively cutting gel to recover a hEGF-HSA gene fragment and a pYES2/CT-MF alpha vector, and then connecting by using T4 DNA ligase to obtain positive clone pYES2/CT-MF alpha-hEGF-HSA;
(2) the preparation and transformation of the hEGF-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-hEGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-hEGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the induced expression and purification of the INVSC1/pYES2/CT-MF alpha-hEGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSc1/pYES2/CT-MF alpha-hEGF-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 EGF-HSA fusion protein.
Further, in the step (1), the plasmid pMD19-T-hEGF-HSA is obtained by artificially optimizing and obtaining human epidermal growth factor and human serum albumin factor, 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, designing recombinant human EGF-HSA gene sequence and recombinant human EGF-HSA amino acid sequence, wherein the recombinant human EGF-HSA gene sequence is shown in sequence table 1, and the recombinant human EGF-HSA amino acid sequence is shown in sequence table 2;
the sequence was inserted into pMD19-T Simple Vector in the order hEGF-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-hEGF-HSA.
Further, in the step (1), double enzyme digestion is carried out on plasmid pYES2/CT-MF alpha and plasmid pMD19-T-hEGF-HSA, and the hEGF-HSA gene fragment and pYES2/CT-MF alpha vector are recovered by cutting gel respectively, and then T4 DNA ligase is used for connection to obtain positive clone pYES2/CT-MF alpha-hEGF-HSA, and the specific steps are as follows:
carrying out double digestion on the plasmid pYES2/CT-MF alpha and the plasmid pMD19-T-hEGF-HSA by using endonuclease Not I and endonuclease Xba I respectively;
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 to recover an hEGF-HSA gene fragment and a pYES2/CT-MF alpha vector, 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 bacterium liquid PCR (forward primer hEGF-HSA-F: 5'-GAAATTACCACGTTTACCGCTCTGA-3'; reverse primer hEGF-HSA-R: 5'-TAGATTAGTGATGGTGATGGTGATG-3') and double enzyme digestion (Not I and Xba I) identification, so that the positive clone pYES2/CT-MF alpha-hEGF-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 (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 standby 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.
Further, in the step (2), the specific steps for obtaining the positive clone INVSC1/pYES2/CT-MF alpha-hEGF-HSA include:
pYES2/CT-MF alpha-hEGF-HSA was transformed into Saccharomyces cerevisiae INVSC1 competent cells by an electrical transformation method:
adding 10 μ l pYES2/CT-MF alpha-hEGF-HSA plasmid into 80 μ l Saccharomyces cerevisiae INVScl competent cells, blowing and sucking to mix them uniformly, then transferring 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), Saccharomyces cerevisiae transformant containing pYES2/CT-MF alpha-hEGF-HSA was subjected to PCR (forward primer: hEGF-HSA-F; reverse primer: hEGF-HSA-R) screening of positive clones of INVSC1/pYES2/CT-MF alpha-hEGF-HSA.
Further, in the step (3),
the induced expression of the engineering bacteria containing INVSC1/pYES2/CT-MF alpha-hEGF-HSA comprises the following specific steps:
selecting single colony of INVSC1/pYES2/CT-MF alpha-hEGF-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 into 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, centrifuging the supernatant subjected to induction 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 filter was subjected to self-column packing using a packing for Chelating affinity chromatography of nickel ions by chemical Sepharose (TM) Fast Flow of 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 solution through a chromatographic column by using PBS buffer solution containing 500mM imidazole, eluting and collecting protein corresponding to an elution peak to obtain the recombinant human EGF-HSA protein stock solution after metal ion affinity chromatography.
Further, in the step (3), the anion exchange chromatography comprises the following steps:
adopting DEAE anion exchange chromatography, replacing a protein stock solution collected after metal ion affinity chromatography purification into Binding Buffer II (50mM tris (hydroxymethyl) aminomethane, pH8.5), loading the sample through a DEAE anion exchange chromatography column well balanced by the Binding Buffer II, and collecting a hEGF-HSA fusion protein peak;
eluting with Elution Buffer II (50mM tris, 1M NaCl, pH8.5), washing off foreign protein, and collecting protein corresponding to an Elution peak, namely the purified recombinant human EGF-HSA fusion protein obtained by the invention.
A preparation method of a recombinant human EGF-HSA fusion protein standard comprises the following steps:
and (2) filtering and sterilizing the recombinant human EGF-HSA 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 EGF-HSA fusion protein standard product.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes the recombinant protein (hEGF-HSA) formed by fusing the long-acting recombinant human epidermal growth factor secreted and expressed by the saccharomyces cerevisiae and the 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 hEGF 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 EGF-HSA protein
FIG. 3Western Blot to identify purified recombinant human EGF-HSA protein
Description of sequence listing:
Sequence table 2 amino acid sequence of recombinant human EGF-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-hEGF-HSA
1.1 artificial optimization and acquisition of human epidermal growth factor and human serum albumin factor:
the following related genes and amino acid sequences are obtained through GenBank inquiry, the gene sequence is optimized by using the amino acid sequence expressed by the target gene in the experiment, and then two segments of genes are artificially synthesized to construct an expression vector. According to the property of pYES2/CT-MF alpha vector (figure 1) and Saccharomyces cerevisiae host codon preference, the recombinant human EGF-HSA gene sequence and the amino acid sequence of the recombinant human EGF-HSA are designed, the recombinant human EGF-HSA gene sequence is shown in a sequence table 1, and the amino acid sequence of the recombinant human EGF-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 hEGF and HSA sequences; CATCACCATCACCATCAC is a 6 × His tag sequence.
The sequence was inserted into pMD19-T Simple Vector in the order hEGF-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-hEGF-HSA.
1.2 construction of pYES2/CT-MF alpha-hEGF-HSA expression vector
Plasmid pYES2/CT-MF α and plasmid pMD19-T-hEGF-HSA were digested simultaneously with endonuclease NotI 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 hEGF-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-hEGF-HSA is selected by bacterial liquid PCR (forward primer hEGF-HSA-F: 5'-GGCCGCAATGAACTCTGATTCTGAA-3'; reverse primer hEGF-HSA-R: 5'-GGTGATGGTGATGTAACCCTAAAGC-3') and double enzyme digestion (NotI and Xba I) identification.
Example 2: preparation and transformation of hEGF-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 and 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 for 20min at 121 ℃, 100ml of 20% glucose solution for filtration and 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.
2.2 pYES2/CT-MF alpha-hFLG-HSA transformed Saccharomyces cerevisiae
pYES2/CT-MF alpha-hEGF-HSA was transformed into s.cerevisiae INVSC1 competent cells by electrical transformation.
Add 10. mu.l pYES2/CT-MF α -hEGF-HSA plasmid to 80. mu.l Saccharomyces cerevisiae INVSc competent cells, mix them well by aspiration, and then transfer to a pre-chilled 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), Saccharomyces cerevisiae transformant containing pYES2/CT-MF alpha-hEGF-HSA was subjected to PCR (forward primer: hEGF-HSA-F; reverse primer: hEGF-HSA-R) screening of positive clones of INVSC1/pYES2/CT-MF alpha-hEGF-HSA.
Example 3: inducible expression, detection and purification of INVSC1/pYES2/CT-MF alpha-hEGF-HSA engineering bacteria
3.1 inducible expression and detection of engineering bacteria
Selecting single colony of INVSC1/pYES2/CT-MF alpha-hEGF-HSA, inoculating to 20ml SC-U selection medium, shaking at 30 deg.C and 220rpmThe culture was carried out overnight. 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 72.6kDa 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), and the specific bands at about 72.6kDa can be observed by using rabbit Anti-hEGF polyclonal antibody (Anti-FGF1 antibody, ab207321) to identify the induction expression supernatant of the saccharomyces cerevisiae strain containing pYES2/CT-MF alpha-hEGF-HSA plasmid by Western Blot (figure 3, a Lane M: Marker, a Lane 1: the unloaded plasmid saccharomyces cerevisiae strain product, a Lane 2: GF hEGF-HSA protein), so that the eukaryotic expression saccharomyces cerevisiae engineering bacteria containing the human epidermal growth factor and carrying pYES2/CT-MF alpha-hEGF-HSA can generate recombinant proteins at about 72.6kDa by galactose induction of the fusion proteins of the human epidermal growth factor and serum albumin .
3.2 purification of recombinant human EGF-HAS fusion protein
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. Passing through a chromatographic column by using PBS buffer solution containing 500mM imidazole, eluting and collecting protein corresponding to an elution peak to obtain the metal ion affinityAnd the hEGF-HSA protein stock solution after chromatographic elution.
3.2.2 anion exchange chromatography
DEAE anion exchange chromatography: the hEGF-HSA protein stock solution eluted by the metal ion affinity chromatography is replaced into a Binding Buffer II (50mM tris (hydroxymethyl) aminomethane, pH8.5), and then the hEGF-HSA fusion protein peak is collected by loading the sample through a DEAE anion exchange chromatography column well balanced by the Binding Buffer II. 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 EGF-HSA fusion protein.
Example 4: hEGF-HSA cell growth promotion effect detection
4.1 reagent preparation
4.1.1 complete broth: measuring calf serum 100ml, adding culture solution to constant volume to 1L;
4.1.2 maintenance broth: measuring 4ml of calf serum, and adding the culture solution to a constant volume of 1L;
4.1.3 digestive juice: weighing 0.2g of disodium ethylene diamine tetraacetate, 8g of sodium chloride, 0.2g of potassium chloride, 1.152g of disodium hydrogen phosphate, 0.2g of monopotassium phosphate and 2.5g of trypsin, adding purified water to dissolve, fixing the volume to 1L, and filtering and sterilizing by a 0.22 mu m filter membrane.
4.1.4 thiazole blue (MTT) solution: 0.1g of MTT powder was weighed, dissolved in 20ml of PBS, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4 ℃ in the dark.
4.2 preparation of test articles
Mu.l of the sample was diluted 10-fold in 900. mu.l of complete medium and the procedure was repeated until the dilution was 1000-fold. Taking 1000-fold diluent to perform 4-fold incremental gradient dilution in sequence from 1:4 in a 96-well cell culture plate (the specific operation steps are firstly adding 150 mu l of complete culture solution in the 96-well cell culture plate, adding 50 mu l of 1000-fold diluted sample solution in the 1 st column of the 96-well plate, performing blow-on dilution, after full dilution, extracting 50 mu l in the 2 nd column of the 1 st column of the well, performing blow-on dilution, extracting 50 mu l in the 3 rd column of the well after full dilution, performing blow-on dilution, repeating the operation till the 10 th column, performing 10 dilutions (1-10 wells) in total, and simultaneously setting one repeat well for each dilution.
4.3 measurement of cell growth promoting Activity
The method comprises the following steps:
(1) culturing mouse embryo fibroblast (BALB/C3T 3) with whole culture solution at 37 deg.C and 5% carbon dioxide, and controlling cell concentration to 1.0 × 10 per 1ml5～5.0×105The cells are used for biological activity determination 24-36h after passage;
(2) discarding the culture solution from the culture flask of step (1), digesting and collecting cells, preparing the whole culture solution to contain 5.0X 10 per 1ml4～8.0×104Inoculating the cell suspension of each cell into a 96-well plate, shaking up continuously in the inoculation process, keeping the same inoculation number of each well, keeping each well at 100 mu l, and culturing at 37 ℃ under the condition of 5% carbon dioxide;
(3) after the complete culture solution is cultured for 24 hours in the step (2), replacing the complete culture solution with a maintenance culture solution, and culturing for 24 hours at 37 ℃ by 5% carbon dioxide;
(4) after culturing for 24h in the step (3), removing the maintenance liquid from the prepared cell culture plate, adding 100 mu l of standard substance solution and test substance solution into each hole, and culturing for 64-72h at 37 ℃ and 5% carbon dioxide;
(5) and (3) detecting results by adopting an MTT colorimetric method: mu.l of MTT solution was added to each well, and the mixture was incubated at 37 ℃ with 5% carbon dioxide for 5 hours.
All the operations are carried out under aseptic conditions. After discarding the liquid in the culture plate, adding 100 μ l DMSO into each well, mixing well, measuring absorbance on a microplate reader with 630nm as reference wavelength and 570nm as test wavelength, and recording the measurement result.
4.4 test results
MTT method is adopted to detect the cell growth promoting effect of hEGF-HSA on BALB/C3T 3 cells, and the biological activity is 5.7 x 105IU/ml。
Example 5: preparation and detection of hEGF-HSA standard substance
5.1 preparation of hEGF-HSA standard substance
The purified protein solution of the recombinant hEGF-HSA is filtered and sterilized by a 0.22 mu m filter membrane, diluted by 10mm ol/L phosphate buffer, added with 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose freeze-drying protective agent, and frozen and dried in vacuum.
5.2 detection of hEGF-HSA Standard
Protein content determination: the protein concentration of the hEGF-HSA standard product after purification was measured by the Bradford method, and the protein content was 2.0 mg/ml.
Purity identification by SDS-PAGE: the purity of the hEGF-HSA standard product is determined by SDS-PAGE, the purity is 98 percent, and the relative molecular weight is 72.6 kD.
And (4) HPLC purity identification: the hEGF-HSA standard substance is analyzed by a mu RPC 18 ST 4.6/100 reverse phase chromatographic column, and the main peak area accounts for 99.00 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 hEGF-HSA protein sample, loading the prepared polyacrylamide gel on a vertical electrophoresis apparatus before loading, and performing idle running 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 E A E A Y V E F P R A A A M N S D S E C, which indicates that the purified target protein is hEGF-HSA.
In conclusion, the invention utilizes the recombinant protein (hEGF-HSA) formed by fusing the long-acting recombinant human EGF 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 EGF-HSA standard substance is provided, and plays an important role in biological product standardization, quality control and efficacy evaluation. The long-acting recombinant human EGF-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 EGF-HSA fusion protein and standard substance thereof
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tacattggtg aaagatgtca atacagagat ttgaagtggt gggagttgag acatcaccat 180
caccatcacg cagaggcgga ggctaaggaa gctgcagcca aagccatgaa gtgggttacg 240
tttatctccc tattatttct gttctcatcc gcctactcca gaggtgtttt caggagagat 300
gctcacaaat ctgaggttgc tcatagattc aaggatttgg gtgaagaaaa ctttaaggcc 360
ttagtgttaa tagctttcgc acaatacctg caacagtgtc cttttgaaga ccatgtcaaa 420
ttagttaatg aagtcaccga atttgctaag acgtgcgttg ctgatgagtc tgccgaaaat 480
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tttttgcaac acaaagatga taatccaaac ttgccaagat tggtaagacc agaagttgac 660
gttatgtgta ccgcttttca tgataatgaa gaaacatttt tgaaaaagta tctttatgaa 720
atagcaagga ggcatcctta cttctacgct ccagagttat tattttttgc aaaaagatat 780
aaggcagctt ttactgaatg ttgtcaggct gcggataaag ccgcatgtct gttacccaaa 840
ttggatgaat tgagagacga gggcaaagct agtagtgcca aacaaagatt aaaatgcgct 900
tcattacaaa aatttggaga aagagcgttt aaggcttggg ccgtagcaag attgtctcag 960
agattcccga aagccgaatt tgcagaagtg agtaaactgg tcacagattt gacgaaagtt 1020
cacacagaat gttgtcacgg agatttattg gaatgcgctg acgatagggc tgacttagct 1080
aaatacatat gcgagaatca agattccata tcatcaaaat tgaaagaatg ttgtgagaaa 1140
ccattattag aaaaatccca ctgtatagct gaagttgaga acgtagaaat gcccgcggat 1200
ttaccctccc ttgcggctga cttcgttgag tcaaaggatg tttgcaagaa ttacgcggag 1260
gccaaggatg tttttcttgg catgttttta tatgagtatg ccagacgtca tccggattat 1320
tctgtagttc tactgttaag gcttgccaag acatacgaaa ctaccttaga aaaatgttgt 1380
gcggctgccg atccacatga atgttacgca aaagtttttg atgaattcaa gccgcttgtc 1440
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aaattccaaa acgcattatt ggtcagatat acaaaaaaag tacctcaggt ttccacacca 1560
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gaagctaaga gaatgccatg cgctgaagat tatctatcag tcgtacttaa tcaactgtgt 1680
gtcctacacg agaagactcc tgtcagtgac agagtgacaa aatgttgcac cgagagctta 1740
gttaatagaa gaccgtgttt ttcagcgctg gaagttgatg aaacctatgt tccaaaggag 1800
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Met Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu
1 5 10 15
His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys
20 25 30
Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu
35 40 45
Lys Trp Trp Glu Leu Arg His His His His His His Ala Glu Ala Ala
50 55 60
Ala Lys Glu Ala Ala Ala Lys Ala Met Lys Trp Val Thr Phe Ile Ser
65 70 75 80
Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg
85 90 95
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
100 105 110
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
115 120 125
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
130 135 140
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
145 150 155 160
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
165 170 175
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
180 185 190
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
195 200 205
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
210 215 220
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
225 230 235 240
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
245 250 255
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
260 265 270
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
275 280 285
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
290 295 300
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
305 310 315 320
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
325 330 335
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
340 345 350
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
355 360 365
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
370 375 380
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
385 390 395 400
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
405 410 415
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
420 425 430
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
435 440 445
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
450 455 460
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
465 470 475 480
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
485 490 495
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
500 505 510
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
515 520 525
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
530 535 540
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
545 550 555 560
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
565 570 575
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
580 585 590
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
595 600 605
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
610 615 620
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
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Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
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Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
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Ala Ala Ser Gln Ala Ala Leu Gly Leu His His His His His His
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Claims (9)
1. A preparation method of saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein is characterized by comprising the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-hEGF-HSA specifically comprises the following steps:
artificially optimizing and obtaining human epidermal growth factor and human serum albumin factor to obtain plasmid pMD 19-T-hEGF-HSA;
carrying out double enzyme digestion on plasmid pYES2/CT-MF alpha and plasmid pMD19-T-hEGF-HSA, respectively cutting gel to recover a hEGF-HSA gene fragment and a pYES2/CT-MF alpha vector fragment, then connecting by using T4 DNA ligase, and screening by an LB plate culture medium containing ampicillin to obtain a positive clone pYES2/CT-MF alpha-hEGF-HSA;
(2) the preparation and transformation of the hEGF-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 transforming pYES2/CT-MF alpha-hEGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-hEGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the induced expression and purification of the INVSC1/pYES2/CT-MF alpha-hEGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSc1/pYES2/CT-MF alpha-hEGF-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 EGF-HSA fusion protein required by the invention.
2. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein is characterized in that in the step (1), the human epidermal growth factor and the human serum albumin factor are artificially optimized and obtained to obtain the plasmid pMD19-T-hEGF-HSA, and the specific steps are as follows:
according to the property of pYES2/CT-MF alpha carrier and the codon preference of saccharomyces cerevisiae host, the gene sequence of recombinant human EGF-HSA and the amino acid sequence of recombinant human EGF-HSA are designed, the gene sequence of recombinant human EGF-HSA is shown in a sequence table 1, and the amino acid sequence of recombinant human EGF-HSA is shown in a sequence table 2;
the sequence was inserted into pMD19-T Simple Vector in the order hEGF-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-hEGF-HSA.
3. The method of claim 1, wherein in step (1), plasmid pYES2/CT-MF α and plasmid pMD19-T-hEGF-HSA are subjected to double digestion, and hEGF-HSA gene fragment and pYES2/CT-MF α vector are recovered by cutting gel, and then connected by T4 DNA ligase to obtain positive clone pYES2/CT-MF α -hEGF-HSA, and the specific steps are as follows:
carrying out double digestion on the plasmid pYES2/CT-MF alpha and the plasmid pMD19-T-hEGF-HSA by using endonuclease Not I and endonuclease Xba I respectively;
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 to recover an hEGF-HSA gene fragment and a pYES2/CT-MF alpha vector, 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-hEGF-HSA through bacterial liquid PCR and double enzyme digestion identification.
4. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein of claim 1, wherein in the step (2), a common solution and a culture medium 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 standby 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 EGF-HSA fusion protein, according to claim 1, wherein the specific steps for obtaining the positive clone INVSC1/pYES2/CT-MF α -hEGF-HSA in the step (2) are as follows:
pYES2/CT-MF α -hEGF-HSA was transformed into s.cerevisiae INVSC1 competent cells using an electrical transformation method:
adding 10 μ l pYES2/CT-MF alpha-hEGF-HSA plasmid into 80 μ l Saccharomyces cerevisiae INVScl competent cells, blowing and sucking to mix them uniformly, then transferring 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;
the Saccharomyces cerevisiae transformant containing pYES2/CT-MF alpha-hEGF-HSA was grown in SC-U selection medium (containing ampicillin), and the bacterial liquid was PCR-screened for positive clones of INVSC1/pYES2/CT-MF alpha-hEGF-HSA.
6. The method for preparing Saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein of claim 1, wherein in the step (3),
the inducible expression of INVSC1/pYES2/CT-MF alpha-hEGF-HSA engineering bacteria comprises the following specific steps:
selecting single colony of INVSC1/pYES2/CT-MF alpha-hEGF-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 the thalli into 100ml of SC-U induction culture medium, placing the thalli in a 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 EGF-HSA fusion protein as claimed in 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 filter was subjected to self-column packing using a packing for Chelating affinity chromatography of nickel ions by chemical Sepharose (TM) Fast Flow of 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 imidazole, eluting and collecting protein corresponding to an elution peak to obtain the recombinant human EGF-HSA protein stock solution after metal ion affinity chromatography.
8. The method for preparing the saccharomyces cerevisiae expression long-acting recombinant human EGF-HSA fusion protein as claimed in claim 1, wherein in the step (3), the anion exchange chromatography comprises the following steps:
after the protein stock solution collected after the metal ion affinity chromatography purification is replaced into a Binding Buffer II, the sample is loaded through a DEAE anion exchange chromatographic column balanced by the Binding Buffer II, and the hEGF-HSA fusion protein peak is collected;
eluting with Elution Buffer II, washing off foreign protein and collecting protein corresponding to the Elution peak, namely the purified recombinant human EGF-HSA fusion protein obtained by the invention.
9. A preparation method of a recombinant human EGF-HSA fusion protein standard is characterized by comprising the following steps:
taking the recombinant human EGF-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 solution, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose lyophilization protectant, and freeze-drying under vacuum to obtain the recombinant human EGF-HSA fusion protein standard product.
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| CN114044830B (en) * | 2021-12-01 | 2024-07-05 | 清华大学 | Recombinant protein, protein sponge product containing recombinant protein and application |
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