CN113105558A - Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein and preparation method of standard substance thereof - Google Patents
Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein and preparation method of standard substance thereof Download PDFInfo
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- CN113105558A CN113105558A CN202110355044.6A CN202110355044A CN113105558A CN 113105558 A CN113105558 A CN 113105558A CN 202110355044 A CN202110355044 A CN 202110355044A CN 113105558 A CN113105558 A CN 113105558A
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a saccharomyces cerevisiae expression human rPDGF-HSA fusion protein, which comprises the following steps: (1) constructing a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rPDGF-HSA; (2) preparing and transforming rPDGF-HSA fusion protein engineering bacteria; (3) inducible expression and purification of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria. Simultaneously, a preparation method of the brewing yeast expressed human rPDGF-HSA fusion protein standard is also provided. The invention uses saccharomyces cerevisiae to express recombinant protein (rPDGF-HSA) formed by fusing human rPDGF and 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 brewing yeast expression human rPDGF-HSA fusion protein and a preparation method of a standard substance thereof.
Background
Platelet Derived Growth Factor (PDGF) is an important mitogenic factor and chemical inducer, can stimulate division and proliferation of connective tissue cells, and has close relation with growth and development of body tissues, wound healing, atherosclerosis and occurrence and development of tumors. The PDGF family is composed of 5 different dimeric subtypes including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD, which are formed by folding A, B, C, D four polypeptide chains with each other through disulfide bonds. They exert their biological functions by binding to PDGF alpha and beta receptors. PDGF-BB can bind to both alpha and beta receptors with high affinity, and its proliferation promoting effect is stronger than that of PDGF of other subtypes.
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.
PDGF is currently obtained mainly through two routes: firstly, extracting from the tissue cells of organisms; secondly, an expression vector is constructed by using a genetic engineering technology, and a host is used for expressing recombinant PDGF. The PDGF is naturally purified by complex process and high cost, and the popularization and application of the PDGF 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.
Disclosure of Invention
In order to overcome the technical problems in the background art, the invention utilizes saccharomyces cerevisiae to express recombinant protein (rPDGF-HSA) formed by fusing human PDGF and HSA, has simple production process, low cost and uniform product, establishes an economic, efficient and stably expressed long-acting recombinant human PDGF standard protein with high concentration and high activity, and provides a basis for establishing the quality standard of the human rPDGF protein.
A preparation method of saccharomyces cerevisiae expression human rPDGF-HSA fusion protein comprises the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rPDGF-HSA specifically comprises the following steps:
designing and obtaining rPDGF-HSA gene sequence and fusion protein rPDGF-HSA amino acid sequence;
designing and amplifying a target gene according to a rPDGF-HSA nucleotide sequence, recovering and double-enzyme-digesting a PCR product and pYES2/CT-MF alpha plasmid, connecting the PCR product and the pYES2/CT-MF alpha plasmid by using T4 DAN ligase, transferring the plasmid into E.coli DH5 alpha competent cells for culture, and obtaining a positive clone pYES2/CT-MF alpha-rPDGF-HSA;
(2) the preparation and transformation of rPDGF-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-rPDGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-rPDGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the inducible expression and purification of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSc1/pYES2/CT-MF alpha-rPDGF-HSA obtained in the step (2), and purifying by metal ion affinity chromatography and anion exchange chromatography in sequence to obtain the wine brewing yeast expression human rPDGF-HSA fusion protein.
Further, in the step (1), a gene sequence of rPDGF-HSA and an amino acid sequence of fusion protein rPDGF-HSA are designed and obtained, and the specific steps are as follows:
according to the property of pYES2/CT-MF alpha carrier (figure 1) and Saccharomyces cerevisiae host codon preference, the gene sequence of human rPDGF-HSA and the amino acid sequence of human rPDGF-HSA are designed, the gene sequence of human rPDGF-HSA is shown in a sequence table 1, and the amino acid sequence of human rPDGF-HSA is shown in a sequence table 2.
Further, in the step (1), a target gene is designed and amplified according to a nucleotide sequence of rPDGF-HSA, a PCR product and pYES2/CT-MF alpha plasmid are recovered and subjected to double enzyme digestion, the PCR product and the pYES2/CT-MF alpha plasmid are connected by using T4 DAN ligase, and are transferred into E.coli DH5 alpha competent cells for culture, so that a positive clone pYES2/CT-MF alpha-rPDGF-HSA is obtained, and the specific steps are as follows:
designing an amplification primer according to a PDGF-HSA nucleotide sequence, wherein:
a forward primer: 5'ATAAGAATGCGGCCGCAATGTCTCTAGGAA 3';
reverse primer: 5'CTAGTCTAGATTAGTGATGGTGATGGTG 3';
performing PCR amplification;
preparing 1% agarose gel, separating PCR amplification products by electrophoresis, quickly cutting a target strip under an ultraviolet lamp, and recovering the target gene PCR amplification products by using a DNA gel recovery kit;
extracting pYES2/CT-MF alpha plasmid from DH5 alpha/pYES 2/CT-MF alpha;
carrying out double enzyme digestion on the plasmid pYES2/CT-MF alpha and the recovered PCR product by Not I and Xba I respectively, and recovering the double enzyme digestion PCR product and the plasmid pYES2/CT-MF alpha by glue;
connecting the PCR amplification product recovered by double enzyme digestion with pYES2/CT-MF alpha plasmid by T4DNA ligase at 37 ℃ for about 30 min;
and (3) transforming the connection product into E.coli DH5 alpha competent cells, selecting a positive transformant after Amp resistance screening, and culturing to obtain a positive clone pYES2/CT-MF alpha-rPDGF-HSA.
Further, 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: 20g of peptone and 10g of yeast extract, adding purified water to reach the constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20 Xglucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
SC-U deficient medium: 6.70g of YNB (YNB) non-amino acid nitrogen source, 0.01% amino acid mixture I1g, 0.005% amino acid mixture II 0.5g, adding distilled water to a constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20% glucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
wherein the 0.01% amino acid mixture I is arginine, leucine, threonine, lysine, tryptophan, cysteine and adenine; the 0.005% amino acid mixture II is aspartic acid, serine, histidine, proline, isoleucine, phenylalanine, valine, tyrosine, and methionine;
SC-U induction medium: 20g of peptone and 10g of yeast extract, adding purified water to a constant volume of 700ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding 100ml of sterile 20% galactose into an ultra-clean bench, and adding 2.0% agar powder into a solid culture medium.
Further, in the step (2), the specific steps for obtaining the positive clone INVSC1/pYES2/CT-MF α -rPDGF-HSA include:
pYES2/CT-MF α -rPDGF-HSA was transformed into s.cerevisiae INVSC1 competent cells using the electrotransformation:
adding 10 μ l pYES2/CT-MF α -rPDGF-HSA plasmid into 80 μ l Saccharomyces cerevisiae INVScl competent cell, blowing and sucking to mix them uniformly, transferring into a precooled electric shock cup, ice-bathing for 5min, and wiping off 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;
selecting a transformant, inoculating the transformant into an SC-U liquid culture medium, and culturing at the constant temperature of 30 ℃ and 200 rpm;
PCR reaction is carried out by taking the cultured bacterial liquid as a template, and positive clones of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA are screened.
Further, in the step (3), the induced expression of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria comprises the following specific steps:
selecting single colony of INVSC1/pYES2/CT-MF alpha-rPDGF-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 OD600nmReaching 0.4, and the induction time is 36 h;
the supernatant of the induced expression was centrifuged and filtered through a 0.22 μm filter and the filtrate was collected.
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 (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 rPDGF-HSA protein stock solution after metal ion affinity chromatography.
Further, in the step (3), the anion exchange chromatography comprises the following steps:
and (2) replacing the collected protein stock solution after the metal ion affinity chromatography purification into a Tris buffer solution, then loading the sample, collecting rPDGF-HSA protein peak through a DEAE anion exchange chromatography column which is well balanced by the Tris buffer solution, eluting with a Tris-NaCl eluent, and washing off the impurity protein, thus obtaining the purified human rPDGF-HSA fusion protein.
A preparation method of a human rPDGF-HSA fusion protein standard comprises the following steps:
and (3) filtering the prepared rPDGF-HSA fusion protein solution by using a 0.22 mu m filter membrane for sterilization, diluting the solution by using 10mM phosphate buffer, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose freeze-drying protective agent into the diluted solution, and carrying out freeze-vacuum drying on the diluted solution to obtain the rPDGF-HSA fusion protein standard product.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes the recombinant protein (rPDGF-HSA) formed by fusing the platelet-derived 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, and the economic and efficient preparation method of the rPDGF-HSA standard substance established by the invention has positive significance in the standardization, quality control and efficacy evaluation of biological products.
Description of the drawings:
FIG. 1 plasmid pYES2/CT-MF α map;
FIG. 2SDS-PAGE identification of purified human rPDGF-HSA protein
Description of sequence listing:
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-rPDGF-HSA
1.1 Artificial optimization and acquisition of platelet-derived 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 carrier (figure 1) and the codon preference of Saccharomyces cerevisiae host, the gene sequence of human rPDGF-HSA and the amino acid sequence of human rPDGF-HSA are designed, the gene sequence of human rPDGF-HSA is shown in a sequence table 1, and the amino acid sequence of human rPDGF-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 PDGF and HSA sequences; CATCACCATCACCATCAC is a 6 × His tag sequence.
1.2 construction of pYES2/CT-MF α -rPDGF-HSA expression vector
Designing an amplification primer according to a rPDGF-HSA nucleotide sequence, wherein:
a forward primer: 5'ATAAGAATGCGGCCGCAATGTCTCTAGGAA 3';
reverse primer: 5'CTAGTCTAGATTAGTGATGGTGATGGTG 3'.
PCR conditions were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 2.5min for 29 cycles; final extension at 72 ℃ for 10 min.
Preparing 1% agarose gel, separating PCR amplification products by electrophoresis, quickly cutting a target strip under an ultraviolet lamp, and recovering the target gene PCR amplification products by using a DNA gel recovery kit.
The enzyme was cleaved in a metal bath at 37 ℃ for 3 h. After the digestion, electrophoresis was performed on 2% agarose gel, and the PCR product and plasmid were recovered by double digestion.
The PCR amplification product recovered by double digestion was ligated with pYES2/CT-MF α plasmid using T4DNA ligase at 37 ℃ for about 30 min.
Ligation system (10 μ l): the PCR product was recovered in 5. mu.l by double digestion, the pYES2/CT-MF α fragment was recovered in 3. mu.l by double digestion, and each of T4DNA ligase and 10 Xligase buffer was 1. mu.l.
The ligation product is transformed into E.coli DH5 alpha competent cells, and positive transformants are picked for culture after Amp resistance selection.
The PCR of bacterial liquid is used to identify the successful introduction of gene into the vector, and the pYES2/CT-MF alpha-rPDGF-HSA vector is obtained by sequencing.
Example 2: preparation and transformation of rPDGF-HSA fusion protein engineering bacteria
2.1 preparation of common solution and culture Medium for Saccharomyces cerevisiae expression System
YPD medium: 20g of peptone and 10g of yeast extract, adding purified water to reach the constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20 Xglucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
SC-U deficient medium: 6.70g of YNB (YNB) non-amino acid nitrogen source, 0.01% amino acid mixture I1g, 0.005% amino acid mixture II 0.5g, adding distilled water to a constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20% glucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
wherein the 0.01% amino acid mixture I is arginine, leucine, threonine, lysine, tryptophan, cysteine and adenine; the 0.005% amino acid mixture II is aspartic acid, serine, histidine, proline, isoleucine, phenylalanine, valine, tyrosine, and methionine;
SC-U induction medium: 20g of peptone and 10g of yeast extract, adding purified water to a constant volume of 700ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding 100ml of sterile 20% galactose into an ultra-clean bench, and adding 2.0% agar powder into a solid culture medium.
2.2 pYES2/CT-MF alpha-rPDGF-HSA transformed Saccharomyces cerevisiae
pYES2/CT-MF α -rPDGF-HSA was transformed into s.cerevisiae INVSC1 competent cells by electrotransformation.
Mu.l of pYES2/CT-MF α -rPDGF-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 is adjusted to the fungus grade, and the electric shock cup is placed on the Bio-Rad electric converter for electric shock. Quickly adding 500. mu.l of precooled 1M sorbitol solution into an electric shock cup, uniformly mixing, coating an SC-U solid plate, and carrying out inversion culture at constant temperature of 30 ℃ until monoclonals grow out.
The transformants were picked and inoculated into SC-U liquid medium, and cultured at 30 ℃ at a constant temperature of 200 rpm. PCR reaction is carried out by taking the bacterial liquid as a template, and positive clones are identified and screened, so that the engineering bacteria INVSC1/pYES2/CT-MF alpha-rPDGF-HSA are obtained.
Example 3: inducible expression, detection and purification of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria
3.1 inducible expression and detection of engineering bacteria
Selecting single colony of INVSC1/pYES2/CT-MF alpha-rPDGF-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 OD600nmReaches 0.4, and the induction time is 36h。
The specific band of about 81kDa was observed on SDS-PAGE from the supernatant of INDSC 1/pYES2/CT-MF α -rPDGF-HSA induced expression, whereas the specific band was absent from the supernatant of Saccharomyces cerevisiae strain induced expression containing pYES2/CT-MF α empty plasmid (FIG. 2, wherein M, Marker; 1, concentrated induced supernatant; 2, induced supernatant of Saccharomyces cerevisiae strain containing empty plasmid).
3.2 purification of recombinant human PDGF-HAS fusion proteins
3.2.1 reagent preparation
(1) PBS buffer:
weighing the above components, dissolving in purified water, adjusting pH to 8.0, and diluting to 1L.
(2) PBS eluent:
weighing the above components, dissolving in purified water, adjusting pH to 8.0, and diluting to 1L.
(3) Tris buffer:
weighing the above components, dissolving in purified water, adjusting pH to 8.5, and diluting to 1L.
(4) Tris-NaCl eluent:
weighing the above components, dissolving in purified water, adjusting pH to 8.5, and diluting to 1L.
3.2.2 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 buffer solution 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 column with PBS buffer, and washing away the foreign proteins not bound to the column until OD280nmAnd (4) stabilizing. And then, passing the PBS eluent through a chromatographic column, eluting and collecting the protein corresponding to an elution peak to obtain the rPDGF-HSA protein stock solution.
3.2.2 anion exchange chromatography
And (3) displacing the protein stock solution subjected to metal ion affinity chromatography into a Tris buffer solution, then loading the sample, and collecting the rPDGF-HSA protein peak through a DEAE anion exchange chromatography column well balanced by the Tris buffer solution. Eluting with Tris-NaCl eluent, and washing to remove impurity protein to obtain the wine brewing yeast expression human rPDGF-HSA fusion protein.
Example 4: preparation and detection of rPDGF-HSA standard substance
4.1 preparation of rPDGF-HSA Standard
The purified rPDGF-HSA protein solution was sterilized by filtration through a 0.22 μm filter, diluted with 10mM phosphate buffer, and added with 10% glycerol, 0.12g/ml mannitol, and 0.025g/ml sucrose cryoprotectant, followed by lyophilization.
4.2 detection of rPDGF-HSA Standard
4.2.1 protein content determination
The protein concentration of the purified rPDGF-HSA standard substance is detected by a Lowry method, and the protein content is 2.0 mg/ml.
4.2.2 SDS-PAGE for purity characterization
The rPDGF-HSA standard is subjected to SDS-PAGE to identify the purity, the purity is 98%, and the relative molecular weight is 81 kDa.
4.2.3 HPLC purity characterization
The rPDGF-HSA standard substance is analyzed by a mu RPC 18 ST 4.6/100 reverse phase chromatographic column to obtain only one main absorption peak, and 2 peaks are mixed peaks.
The main peak area accounts for 99.28 percent of the total area by calculating the peak area.
4.2.4N-terminal amino acid sequence determination
And (3) carrying out SDS-PAGE electrophoresis on a PDGF-HSA protein sample, loading the prepared polyacrylamide gel on a vertical electrophoresis apparatus before loading, and carrying out air-assisted running for 30min at a constant voltage of 50V. The protein was electroporated onto PVDF (polyvinylidene fluoride) membrane, and the electroporation buffer was CAPS buffer. The PVDF membrane is placed in ponceau dye liquor for dyeing for 30min, and the background color is washed away by water. The band of interest was cut off and placed in an EP tube and stored at-20 ℃ and N-terminal amino acid sequencing was performed by Edman degradation.
The N-terminal amino acid sequence is EAEAYVEFPRAAAMSLGSLT, which indicates that the purified target protein is rPDGF-HSA.
In conclusion, the invention utilizes saccharomyces cerevisiae to express recombinant protein (rPDGF-HSA) formed by fusing human PDGF and HSA, overcomes the defects that in the prior art, an inclusion body is easily formed by adopting an escherichia coli, lactobacillus, brevibacillus and pichia pastoris expression system, and the obtained protein has lower biological activity, and has simple production process, low cost and uniform product; meanwhile, an important human rPDGF-HSA standard is provided, and plays an important role in biological product standardization, quality control and efficacy evaluation. The human rPDGF-HSA fusion protein prepared by the invention has better uniformity and stability and better 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, U
<120> preparation methods of saccharomyces cerevisiae expression human rPDGF-HSA fusion protein and standard substance thereof
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ttcttggttt ggcctccttg tgtagaagtc caaaggtgta gtggttgctg caacaacagg 180
aatgtccagt gtcgtcctac tcaagttcaa ttaagaccag tccaagttag aaaaatcgag 240
attgttagga aaaagcccat ttttaaaaaa gccaccgtta cactagaaga tcatttagct 300
tgtaagtgtg aaacagttgc tgcagctcgt cctgtcaccg cagaggcggc ggctaaggaa 360
gctgcagcca aagccatgaa gtgggttacg tttatctccc tattatttct gttctcatcc 420
gcctactcca gaggtgtttt caggagagat gctcacaaat ctgaggttgc tcatagattc 480
aaggatttgg gtgaagaaaa ctttaaggcc ttagtgttaa tagctttcgc acaatacctg 540
caacagtgtc cttttgaaga ccatgtcaaa ttagttaatg aagtcaccga atttgctaag 600
acgtgcgttg ctgatgagtc tgccgaaaat tgtgacaaat cactgcatac attgttcggt 660
gataagctat gtaccgttgc aactcttaga gaaacgtacg gagagatggc ggactgttgt 720
gctaaacaag aacctgaaag aaatgaatgt tttttgcaac acaaagatga taatccaaac 780
ttgccaagat tggtaagacc agaagttgac gttatgtgta ccgcttttca tgataatgaa 840
gaaacatttt tgaaaaagta tctttatgaa atagcaagga ggcatcctta cttctacgct 900
ccagagttat tattttttgc aaaaagatat aaggcagctt ttactgaatg ttgtcaggct 960
gcggataaag ccgcatgtct gttacccaaa ttggatgaat tgagagacga gggcaaagct 1020
agtagtgcca aacaaagatt aaaatgcgct tcattacaaa aatttggaga aagagcgttt 1080
aaggcttggg ccgtagcaag attgtctcag agattcccga aagccgaatt tgcagaagtg 1140
agtaaactgg tcacagattt gacgaaagtt cacacagaat gttgtcacgg agatttattg 1200
gaatgcgctg acgatagggc tgacttagct aaatacatat gcgagaatca agattccata 1260
tcatcaaaat tgaaagaatg ttgtgagaaa ccattattag aaaaatccca ctgtatagct 1320
gaagttgaga acgatgaaat gcccgcggat ttaccctccc ttgcggctga cttcgttgag 1380
tcaaaggatg tttgcaagaa ttacgcggag gccaaggatg tttttcttgg catgttttta 1440
tatgagtatg ccagacgtca tccggattat tctgtagttc tactgttaag gcttgccaag 1500
acatacgaaa ctaccttaga aaaatgttgt gcggctgccg atccacatga atgttacgca 1560
aaagtttttg atgaattcaa gccgcttgtc gaggagccac aaaatttaat taaacaaaac 1620
tgtgaattat ttgaacaatt aggtgaatat aaattccaaa acgcattatt ggtcagatat 1680
acaaaaaaag tacctcaggt ttccacacca actttagtgg aagtgtcacg taacctaggc 1740
aaggttggta gtaagtgctg taaacaccca gaagctaaga gaatgccatg cgctgaagat 1800
tatctatcag tcgtacttaa tcaactgtgt gtcctacacg agaagactcc tgtcagtgac 1860
agagtgacaa aatgttgcac cgagagctta gttaatagaa gaccgtgttt ttcagcgctg 1920
gaagttgatg aaacctatgt tccaaaggag ttcaatgcag aaacattcac cttccatgct 1980
gatatatgta ctcttagtga aaaagaaagg cagatcaaaa aacaaactgc cctggtcgaa 2040
ttagtcaaac ataaacctaa agcaacgaag gaacagttga aggccgtaat ggatgatttc 2100
gcagctttcg ttgaaaaatg ttgcaaggct gatgacaaag agacatgttt tgctgaagag 2160
ggaaaaaaat tggtggcagc ttctcaagcc gctttagggt tacatcacca tcaccatcac 2220
taatctaga 2229
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Met Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu
1 5 10 15
Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp
20 25 30
Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln
35 40 45
Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr
50 55 60
Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg
65 70 75 80
Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu
85 90 95
Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Ala Glu
100 105 110
Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Met Lys Trp Val Thr Phe
115 120 125
Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe
130 135 140
Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu
145 150 155 160
Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr
165 170 175
Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
180 185 190
Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys
195 200 205
Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
210 215 220
Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
225 230 235 240
Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
245 250 255
Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala
260 265 270
Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
275 280 285
Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
290 295 300
Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
305 310 315 320
Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
325 330 335
Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
340 345 350
Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
355 360 365
Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
370 375 380
Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala
385 390 395 400
Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
405 410 415
Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
420 425 430
Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
435 440 445
Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
450 455 460
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr
465 470 475 480
Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
485 490 495
Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
500 505 510
His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
515 520 525
Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
530 535 540
Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys
545 550 555 560
Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
565 570 575
Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
580 585 590
Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val
595 600 605
Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
610 615 620
Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
625 630 635 640
Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
645 650 655
Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
660 665 670
Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
675 680 685
Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
690 695 700
Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys
705 710 715 720
Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu His His His His His
725 730 735
His
Claims (9)
1. A preparation method of saccharomyces cerevisiae expression human rPDGF-HSA fusion protein is characterized by comprising the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rPDGF-HSA specifically comprises the following steps:
designing and obtaining rPDGF-HSA gene sequence and fusion protein rPDGF-HSA amino acid sequence;
designing and amplifying a target gene according to a rPDGF-HSA nucleotide sequence, recovering and double-enzyme-digesting a PCR product and pYES2/CT-MF alpha plasmid, connecting the PCR product and the pYES2/CT-MF alpha plasmid by using T4 DAN ligase, transferring the plasmid into E.coli DH5 alpha competent cells for culture, and obtaining a positive clone pYES2/CT-MF alpha-rPDGF-HSA;
(2) the preparation and transformation of PDGF-HSA fusion protein engineering bacteria specifically comprise:
preparing a common solution and a culture medium for a saccharomyces cerevisiae expression system;
electrically transforming pYES2/CT-MF alpha-rPDGF-HSA obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clones INVSC1/pYES2/CT-MF alpha-rPDGF-HSA through culture of a culture medium, PCR amplification and screening;
(3) the inducible expression and purification of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria specifically comprise:
culturing and performing induced expression on the positive clone INVSc1/pYES2/CT-MF alpha-rPDGF-HSA obtained in the step (2), and purifying by metal ion affinity chromatography and anion exchange chromatography in sequence to obtain the wine brewing yeast expression human rPDGF-HSA fusion protein.
2. The method for preparing the saccharomyces cerevisiae expression human rPDGF-HSA fusion protein is characterized in that in the step (1), a gene sequence of rPDGF-HSA and an amino acid sequence of the fusion protein rPDGF-HSA are designed and obtained, 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 human rPDGF-HSA and the amino acid sequence of human rPDGF-HSA are designed, the gene sequence of human rPDGF-HSA is shown in the sequence table 1, and the amino acid sequence of human rPDGF-HSA is shown in the sequence table 2.
3. The method for preparing saccharomyces cerevisiae expression human rPDGF-HSA fusion protein according to claim 1, wherein in the step (1), the target gene is designed and amplified according to the rPDGF-HSA nucleotide sequence, the PCR product and pYES2/CT-MF α plasmid are recovered and subjected to double enzyme digestion, the PCR product and the pYES2/CT-MF α plasmid are connected by T4 DAN ligase, and the obtained product is transferred into E.coli DH5 α competent cells for culture, so as to obtain positive clone pYES2/CT-MF α -rPDGF-HSA, and the specific steps are as follows:
designing an amplification primer according to a nucleotide sequence of human rPDGF-HSA, wherein:
a forward primer: 5'ATAAGAATGCGGCCGCAATGTCTCTAGGAA 3';
reverse primer: 5'CTAGTCTAGATTAGTGATGGTGATGGTG 3';
performing PCR amplification;
preparing 1% agarose gel, separating PCR amplification products by electrophoresis, quickly cutting a target strip under an ultraviolet lamp, and recovering the target gene PCR amplification products by using a DNA gel recovery kit;
extracting pYES2/CT-MF alpha plasmid from DH5 alpha/pYES 2/CT-MF alpha;
carrying out double enzyme digestion on the plasmid pYES2/CT-MF alpha and the recovered PCR product by Not I and Xba I respectively, and recovering the double enzyme digestion PCR product and the plasmid pYES2/CT-MF alpha by glue;
connecting the PCR amplification product recovered by double enzyme digestion with pYES2/CT-MF alpha plasmid by using T4DNA ligase at 37 ℃ for about 30 min;
and (3) transforming the connecting product into an E.coliDH5 alpha competent cell, selecting a positive transformant after Amp resistance screening, and culturing to obtain a positive clone pYES2/CT-MF alpha-rPDGF-HSA.
4. The method for preparing saccharomyces cerevisiae expression human rPDGF-HSA fusion protein according to claim 1, wherein in the step (2), the preparation of the solution and culture medium commonly used in the saccharomyces cerevisiae expression system comprises the following steps:
YPD medium: 20g of peptone and 10g of yeast extract, adding purified water to reach the constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20 Xglucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
SC-U deficient medium: 6.70g of YNB (YNB) non-amino acid nitrogen source, 0.01% amino acid mixture I1g, 0.005% amino acid mixture II 0.5g, adding distilled water to a constant volume of 900ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding sterile 100ml of 20% glucose into an ultra-clean bench, and adding agar powder 2.0% into a solid culture medium;
wherein the 0.01% amino acid mixture I is arginine, leucine, threonine, lysine, tryptophan, cysteine and adenine; the 0.005% amino acid mixture II is aspartic acid, serine, histidine, proline, isoleucine, phenylalanine, valine, tyrosine, and methionine;
SC-U induction medium: 20g of peptone and 10g of yeast extract, adding purified water to a constant volume of 700ml, carrying out autoclaving at 121 ℃ for 20min, cooling to below 60 ℃, adding 100ml of sterile 20% galactose into an ultra-clean bench, and adding 2.0% agar powder into a solid culture medium.
5. The method for preparing Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein according to claim 1, wherein the specific steps of obtaining positive clone INVSc1/pYES2/CT-MF α -rPDGF-HSA in step (2) are:
pYES2/CT-MF α -rPDGF-HSA was transformed into s.cerevisiae INVSC1 competent cells using the electrotransformation:
adding 10 μ l pYES2/CT-MF α -rPDGF-HSA plasmid into 80 μ l Saccharomyces cerevisiae INVScl competent cell, blowing and sucking to mix them uniformly, transferring into a precooled electric shock cup, ice-bathing for 5min, and wiping off 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;
selecting a transformant, inoculating the transformant into an SC-U liquid culture medium, and culturing at the constant temperature of 30 ℃ and 200 rpm;
PCR reaction is carried out by taking the cultured bacterial liquid as a template, and positive clones of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA are screened.
6. The method for preparing Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein of claim 1, wherein in the step (3),
the inducible expression of INVSC1/pYES2/CT-MF alpha-rPDGF-HSA engineering bacteria comprises the following specific steps:
selecting single colony of INVSC1/pYES2/CT-MF alpha-rPDGF-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 OD600nmReaching 0.4, and the induction time is 36 h;
the supernatant of the induced expression was centrifuged and filtered through a 0.22 μm filter and the filtrate was collected.
7. The method for preparing Saccharomyces cerevisiae expression human rPDGF-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 was collected and self-purified by using a filler for Nickel ion chelate affinity chromatography (Sepharose TM Fast Flow) of the GE Healthcare companyLoading the column, washing Ni 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 rPDGF-HSA protein stock solution after metal ion affinity chromatography.
8. The method for preparing Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein of claim 1, wherein in the step (3), the anion exchange chromatography comprises the following steps:
and (2) replacing the collected protein stock solution after the metal ion affinity chromatography purification into a Tris buffer solution, then loading the sample, collecting rPDGF-HSA protein peak through a DEAE anion exchange chromatography column which is well balanced by the Tris buffer solution, eluting with a Tris-NaCl eluent, and washing off the impurity protein, thus obtaining the purified human rPDGF-HSA fusion protein.
9. A preparation method of a human rPDGF-HSA fusion protein standard is characterized by comprising the following steps:
taking the human rPDGF-HSA fusion protein solution prepared by the preparation method of any one of claims 1-8, filtering and sterilizing the solution by using a 0.22-micron filter membrane, diluting the solution by using 10mM phosphate buffer, adding 10% glycerol, 0.12g/ml mannitol and 0.025g/ml sucrose freeze-drying protective agent into the diluted solution, and carrying out freeze vacuum drying on the diluted solution to obtain the human rPDGF-HSA fusion protein standard product.
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