CN109851674B - Preparation and purification method of recombinant human serum albumin/growth hormone fusion protein for treating children's dwarf syndrome - Google Patents

Abstract

The invention discloses a recombinant human serum albumin/growth hormone fusion protein, a preparation and purification method of the recombinant fusion protein and application of the recombinant fusion protein in preparing a medicament for treating children short stature. The amino acid sequence of the recombinant human serum albumin/growth hormone fusion protein is SEQ ID NO.1, and the nucleic acid sequence is SEQ ID NO. 2. The preparation process of the invention obtains high-density expression recombinant fusion protein by constructing expression yeast engineering bacteria, and obtains the recombinant human serum albumin/growth hormone fusion protein which can be clinically used by a purification process. The recombinant human serum albumin/growth hormone fusion protein obtained by the preparation and purification method is an innovative medicine structure for treating the childhood dwarf syndrome, achieves the long-acting property of once administration for two weeks, is more suitable for the requirements of children on medication, and has better curative effect, less administration frequency and lower production cost.

Description

Preparation and purification method of recombinant human serum albumin/growth hormone fusion protein for treating children's dwarf syndrome

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant human serum albumin/growth hormone fusion protein for treating children's dwarf syndrome, a preparation and purification method thereof and application thereof in preparing a medicament for treating the children's dwarf syndrome.

Background

Human Serum Albumin (HSA) is a soluble monomeric protein that makes up half of the total amount of protein in the blood. Albumin, as a basic carrier, carries, inter alia, delivery fatty acids, steroids and hormone molecules, the stable inert nature of which is an important factor in maintaining blood pressure. Serum albumin is a globular, non-glycosylated, 65 kilodalton molecular weight, 585 amino acid serum protein. This protein (albumin precursor) is then secreted extracellularly by the process of conversion of the Golgi apparatus to remove the leader polypeptide. Serum albumin has 35 cysteines, and in blood albumin is a monomer with 17 disulfide bonds (see Brown JR, "albumin structure, function and use" Pergamon, new york, 1977). When the polypeptide is not secreted, the albumin product in the yeast cell is in a mismatched state, will lose 90% of its antigenicity (compared to the native state albumin in plasma), and form insoluble albumin aggregates, without the biological activity of albumin. Albumin is now extracted from human plasma for clinical use. The production of recombinant expression of albumin (rHSA) by microorganisms has been disclosed in patents EP330451, EP361991 and in China issued patent ZL2004010057313.7 in forestry, Rich rock.

Albumin is the main component in blood, and has a content of 40 g per liter of blood in human body, and a half-life of 14-20 days. Albumin is also commonly used as a stabilizer in pharmaceutical formulations, particularly in the manufacture of biopharmaceuticals and vaccines. In conclusion, by utilizing the genetic engineering technology, after the human serum albumin and the therapeutic protein are fused in yeast or vertebrate cells and expressed into recombinant fusion protein, the fusion protein has great advantages, so that the fusion protein can resist the enzymolysis in vivo, the service life of the therapeutic protein in vitro and in vivo can be greatly prolonged, the stability and the longer half-life of the therapeutic protein in serum can be increased during neutralization and storage, the long-acting effect of the protein medicament can be achieved, the administration frequency can be greatly reduced, and a better treatment effect can be obtained in the clinical treatment of major diseases.

Human growth hormone (hGH) is a protein hormone secreted by the anterior lobe of the pituitary, has the effect of promoting growth, is closely related to the anabolism of the body, and is an important medicament for treating dwarfism, burns, wounds, bone fractures and hemorrhagic ulcers. Because the half-life period in vivo is only about 0.5-2 hr, the medicine needs to be injected every day for a long time (for years, 5-10 years) to exert the curative effect, and the long-acting effect of the medicine is needed for the treatment period. For this reason, long-acting drugs, particularly those suitable for children, are urgently required in the market. The long-acting recombinant human serum albumin/growth hormone fusion protein of gene recombination has the same action as human endogenous growth hormone, stimulates the differentiation and proliferation of cartilage cells at epiphyseal end, stimulates the growth of cartilage matrix cells, stimulates the differentiation and proliferation of osteoblasts, and causes the acceleration of linear growth and the widening of bones. Promoting whole body protein synthesis, correcting negative nitrogen balance state after trauma such as operation, and correcting hypoproteinemia caused by severe infection and liver cirrhosis; stimulating the synthesis of immunoglobulin, stimulating the proliferation of lymphoid tissue, macrophage and lymphocyte, and enhancing the anti-infection capacity; stimulating collagen cells of the burn wound surface and the operation incision to synthesize fiber cells, dividing and proliferating macrophages, accelerating wound healing, promoting cardiac muscle protein synthesis, increasing cardiac muscle contraction force, reducing cardiac muscle oxygen consumption, regulating fat metabolism, and reducing the levels of serum cholesterol and low-density lipoprotein; supplementing deficiency or deficiency of growth hormone, and regulating fat metabolism, bone metabolism, and functions of heart and kidney of adult.

Can be clinically used for treating the following main indications.

1. For slow growth in children due to endogenous growth hormone deficiency; 2. For the treatment of severe burns; 3. The compound is used for growth hormone deficiency caused by a well-defined hypothalamus-pituitary disease and obvious deficiency of growth hormone confirmed by two different growth hormone stimulation tests.

In 1985, recombinant human growth hormone (rhGH) administered once a day was approved for sale on the market, replacing human pituitary-derived growth hormone that had been inactivated based on safety considerations, and became a human growth hormone resource that could be obtained indefinitely. Over the next 20 years, mainly with the support of rhGH producers, pediatric endocrinologists were first run a series of clinical trials in an attempt to demonstrate that rhGH treatment can improve growth rate and final height in children with short stature due to certain diseases other than Growth Hormone Deficiency (GHD), including Turner's Syndrome (TS), Chronic Renal Insufficiency (CRI), Small for Gestational Age (SGA), Prader Willi Syndrome (PWS), or Idiopathic Short Stature (ISS). Although the recombinant human growth hormone achieves the aim of clinical treatment, the administration frequency is once a day, the administration time of children is at least 1 to 3 years, the compliance of patients is extremely low, the treatment cost is huge, and the ordinary families are difficult to cooperate with treatment according to the requirements of medical advice, so that the drug-receiving population is greatly limited. Therefore, the PEG recombinant human growth hormone is available, and can be administrated once a week, but the cost is several times of that of the common recombinant human growth hormone, and the preparation also contains phenol as a protein preparation formula component. Therefore, the invention is an innovation, and an innovative medicine structure which can be used for the administration of the medicine once every two weeks for children patients, the production and preparation cost is 1/100 of PEG-metagrowth hormone, the compliance of the patients is greatly improved, and the treatment cost is greatly reduced is expected to be obtained. The present invention will facilitate more urgent needs of treatment and will limit the population to receive the necessary treatments at compliance and treatment costs, thus achieving unexpected technical advantages and creativity.

Disclosure of Invention

The invention aims to solve the technical problems and provides a preparation and purification method of recombinant human serum albumin/growth hormone fusion protein for treating children dwarfism.

The invention is realized according to the following technical scheme.

A recombinant human serum albumin/growth hormone fusion protein for treating children's dwarf syndrome has an amino acid sequence of SEQ ID NO. 1.

Furthermore, the nucleic acid sequence of the recombinant human serum albumin/growth hormone fusion protein is SEQ ID NO. 2.

Furthermore, the recombinant human serum albumin/growth hormone fusion protein gene is expressed by a recombinant engineering host cell; the recombinant engineering host cell is yeast, CHO cell, plant, insect, bacteria or animal cell; the recombinant human serum albumin/growth hormone fusion protein gene is transferred into plants and animals for expression through plasmid DNA vector expression, virus vector expression or transgenic technology.

Furthermore, the recombinant human serum albumin/growth hormone fusion protein gene is constructed by a gene engineering technology to express; the fusion protein is obtained by expressing the recombinant fusion protein by means of transformation and transfection or infection using a vector plasmid.

Furthermore, the recombinant human serum albumin/growth hormone fusion protein gene selects a yeast expression vector to transform pichia pastoris, and the fusion protein is secreted into a culture solution.

Furthermore, the saccharomyces host expressed by the recombinant human serum albumin/growth hormone fusion protein gene is a saccharomyces pasteurianus.

The high-density expression method of the recombinant human serum albumin/growth hormone fusion protein for treating the childhood dwarf comprises the following steps:

a. construction and expression of recombinant human serum albumin/growth hormone fusion protein carrier plasmid

Using total RNA extracted from human fetal liver as template, synthesizing and amplifying HSA gene by reverse transcription polymerase chain reaction; separating and purifying HSA DNA fragment by electrophoresis, cloning the DNA fragment to a yeast expression vector to obtain pYZ-HSA recombinant plasmid; artificially synthesizing GH nucleotide sequence by adopting a DNA full sequence synthesis method; using enzyme-digested target gene GH and pYZ-HSA recombinant plasmids, and connecting the two to obtain pYZ-HSA/GH recombinant plasmids;

b. transformation and preparation of pichia pastoris expression engineering bacteria

The pYZ-HSA/GH recombinant plasmid is subjected toPmeI, inoculating a pichia pastoris X33 competent cell after the treatment of the restriction enzyme, and inoculating the transformed yeast on a YPD plate culture medium containing Zeocin antibiotics for culture;

c. screening of recombinant yeast engineering bacteria

Culturing a plurality of yeast colonies containing genes to be expressed in a basic culture solution containing Zeocin antibiotics and having buffer capacity and glycerol respectively; after culturing for a period of time, centrifugally collecting thalli, and then suspending the thalli in a same basic culture solution without glycerol, wherein the content of methanol is 0.5%; adding 100% methanol every 24 hours until the final concentration is 0.5%, respectively collecting culture supernatant at different time points, and screening recombinant yeast engineering strains for expressing specific proteins;

d. large-scale high-density expression rHSA/GH fusion protein

Using 500L and 1000L biological fermentation tank systems, taking an engineering strain sub-library, starting small-scale shaking culture, then amplifying to a first-stage seed tank and a second-stage seed tank, and entering a production fermentation tank; after culturing until the glycerol in the tank is consumed, the dissolved oxygen is returned to the bottom limit and then is returned to the bottom limit, and the glycerol feeding is started; after the feeding is finished and the dissolved oxygen is increased again, starting methanol feeding, entering an induction and recombinant protein production stage, and maintaining the methanol feeding for 72 hours; the yield is not less than 3 g/L of fermentation supernatant.

The purification method of the recombinant human serum albumin/growth hormone fusion protein for treating the children short stature comprises the following purification steps of carrying out high-density expression on the obtained recombinant human serum albumin/growth hormone fusion protein; purity obtained is not less than 99%:

a. primary purification affinity chromatography column: Capto-MMC

And (3) an equilibrium buffer: 20-100 mM NaAc-HAc, 5-20 mM cysteine, 2-8% glycine, pH 4.0-6.0

Washing with a miscellaneous buffer solution: 20 to 100mM PB, 5 to 20mM cysteine, 2 to 8% glycine, pH6.0 to 6.5

Elution buffer: 20-100 mM PB, 5-20 mM cysteine, 2-8% glycine, 0.5-2.0M NH₄Cl or other high salt solution, pH 6.5-8.5

b. Hydrophobic medium chromatography column: phenyl Sepharose^TM High Performance

And (3) an equilibrium buffer: 20-100 mM Tris-HCl or PB, 5-20 mM cysteine, 2-8% glycine, 1-2M NH₄Cl or (NH)₄）₂SO₄，pH5.0～9.0

Elution buffer: 20 to 100mM Tris-HCl or PB, pH5.0 to 9.0

c. Ion purification and separation chromatographic column: q Sepharose^TM Fast Flow

And (3) an equilibrium buffer: 20-100 mM Tris-HCl or PB, 5-20 mM cysteine, 2-8% glycine, pH 6.0-9.0

Washing with a miscellaneous buffer solution: 20-100 mM PB, 180mM NaCl, 5-20 mM cysteine, 2-8% glycine, pH 6.0-9.0

Elution buffer: 20 to 100mM Tris-HCl or PB, 200 to 1000 mM NaCl or NH₄Cl, 5-20 mM cysteine, 2-8% glycine, pH 6.0-9.0

d. A chromatographic column: sephacryl S-200HR

And (3) an equilibrium buffer: 5-50 mM PB, 2-8% glycine, pH 5-8.

Further, a method for purifying recombinant human serum albumin/growth hormone fusion protein for treating children short stature, which comprises the following purification steps of:

a. primary purification affinity chromatography column: Capto-MMC

And (3) an equilibrium buffer: 30mM NaAc-HAc, 10mM cysteine, 5% glycine, pH5.5

Washing with a miscellaneous buffer solution: 20mM PB,10mM cysteine, 5% glycine, pH6.5

Elution buffer: 20mM PB,10mM cysteine, 5% glycine, 1.0M NH₄Cl ，pH7.5

b. Hydrophobic medium chromatography column: phenyl Sepharose^TM High Performance

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, 1.3M NH₄Cl，pH6.7

Elution buffer: 20mM PB, pH6.7

c. Ion purification and separation chromatographic column: q Sepharose^TM Fast Flow

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, pH7.2

Washing with a miscellaneous buffer solution: 20mM PB, 180mM NaCl, 10mM cysteine, 5% glycine, pH7.2

Elution buffer: 20mM PB, 800mM NaCl, 10mM cysteine, 5% glycine, pH7.2

d. A chromatographic column: sephacryl S-200HR

And (3) an equilibrium buffer: 20mM PB, 5% glycine, pH 7.2. Whether or not the above can give corresponding

An application of the recombinant human serum albumin/growth hormone fusion protein in preparing a medicament for treating the childhood dwarf syndrome. Less frequent dosing, which could be once every 2 weeks, was achieved for the first time.

The present invention obtains the following advantageous effects.

1. The invention directly carries out molecular fusion between the C-end of the human serum albumin gene and the N-end of the human growth hormone gene (No connecting peptide, No-Linker is arranged between the C-end of the human serum albumin gene and the N-end of the human growth hormone gene). Through research and repeated tests, the long-acting recombinant human serum albumin/growth hormone fusion protein expression pichia pastoris engineering bacteria are constructed. Establishes a new technology and a method for specifically separating and purifying the recombinant human serum albumin/growth hormone fusion protein. The fusion protein can be secreted, and can be combined with an antibody specifically recognizing anti-human serum albumin, and also can be combined with an antibody specifically recognizing human growth hormone.

2. The rHSA/GH nucleotides of the present invention can be introduced into host cells using recombinant cloning techniques to allow expression of the fusion protein. The engineered host cells can be cultured in media containing conventional nutrients and appropriately modified to facilitate the promoter. The culture conditions for selection of transformants or for amplification of the nucleotide chain encoding rHSA/GH, such as temperature, pH and selection of expression cells, are controlled in a suitable manner of operation.

The recombinant human serum albumin/growth hormone fusion protein gene (nucleotide sequence) expression of the invention needs to be expressed by recombinant engineering host cells. The recombinant engineering host cell can be yeast, CHO cell, plant, insect, bacteria or animal, can be directly expressed by plasmid DNA vector, can be expressed by virus vector, and can be transferred into plant and animal body by transgenic technology for expression.

The recombinant human serum albumin and human growth hormone fusion protein mentioned in the invention is preferably constructed by using genetic engineering technology for expression. The preferred method of obtaining the fusion protein is to use a vector plasmid to express the fusion protein by means of transformation and transfection or infection. In particular, it is preferable to transform Pichia pastoris using a transformable yeast expression vector and secrete the fusion protein into the culture broth. A preferred Saccharomyces host for expression of rHSA/GH may be a Pasteurella species of Pichia. The advantage of using yeast to express rHSA/GH is that the yeast system can produce high quality mature fusion protein and can be secreted into the culture solution for easy purification.

The development of genetic engineering of yeast allows foreign genes to be expressed in yeast and secrete protein products outside cells. The advantages of using yeast to express secreted proteins include, but are not limited to, high expression yields, protein solubility, correct folding, and ease of large-scale production and purification.

3. The present invention provides a method for producing rHSA/GH protein more efficiently and cost-effectively by using yeast. The invention also provides and discloses a ton-level large-scale fermentation process of pichia pastoris engineering bacteria expressing the high-yield non-heat source recombinant rHSA/GH protein and a method for separating and purifying fusion protein, wherein the expression amount of the protein in a 1000L fermentation tank of the pichia pastoris is not less than 1g/L, the purity after purification can reach 99 percent, and the endotoxin content is not more than 0.5 EU/mg.

The invention designs a combined novel weak cation exchange chromatography, a hydrophobic column chromatography, an ion exchange column chromatography and a gel column chromatography which are sequentially combined to remove impurities such as impure proteins, saccharides, lipids, host proteins and the like, and finally obtains the high-purity protein.

The invention uses pichia expression system to prepare recombinant human serum albumin/growth hormone fusion protein, the recombinant protein medicine prepared by the system is completely suitable for clinical application, and the residual amount of host protein in the innovative medicine stock solution researched and developed by the invention also meets the requirements of 2015 edition of Chinese pharmacopoeia three parts.

The invention also accomplishes a 25 ℃ rapid test of high dose protein pharmaceutical water injection to ensure that there is no problem with the stability of the new dosage form (a long term stability test at 25 ℃ for 6 months is equivalent to that the formulation can be stored stably for 3 years under the specified storage conditions of 2-8 ℃).

The invention ensures that the production cost of the long-acting recombinant human serum albumin/growth hormone fusion protein injection after scale production is between 5 yuan and 10 yuan. At present, recombinant human growth hormone products are sold in the market, the production cost is high, and the sale price is gradually reduced in competition. The long-acting human growth hormone of the invention which can be administrated for 1 time in 2 weeks is a new generation human growth hormone medicament, has low production cost, large productivity and less injection times, and can completely gradually replace the existing short-acting and long-acting human growth hormone products. The technology of the present invention has great and obvious advantages in market competition.

Drawings

FIG. 1 shows pYZ-HSA recombinant plasmids of the present inventionEcoRⅠ&XhoI, enzyme digestion identification chart;

FIG. 2 is a diagram showing the growth of a plate after electrotransformation of pYZ-HSA/GH recombinant yeast expression engineering bacteria of the present invention;

FIG. 3 is a SDS-PAGE protein electrophoresis chart of the large-scale high-density expression rHSA/GH protein of the present invention;

FIG. 4 is SDS-PAGE protein electrophoresis picture of rHSA/GH protein purified and secreted by QFF ion exchange column of the invention.

FIG. 5 is a reduced and non-reduced electrophoretogram of rHSA/GH protein after purification according to the present invention;

FIG. 6 is a liquid chromatography purity profile of purified rHSA/GH protein of the present invention;

FIG. 7 is a Western immunoblot of purified rHSA/GH fusion proteins of the present invention against anti-human HSA murine monoclonal antibody;

FIG. 8 is a Western immunoblot pattern of purified rHSA/GH fusion proteins of the present invention against monoclonal antibodies against human GH;

FIG. 9 is a graph of rHSA/GH fusion protein peptide mass fingerprinting of the present invention;

FIG. 10 is a graph showing the measurement of blood concentration of growth hormone in rats of 3 different structures after subcutaneous administration of the present invention.

Detailed Description

The invention is further described with reference to the following figures and examples.

Example 1: human Serum Albumin (HSA) gene expression and construction of vector plasmid

The HSA gene is synthesized and amplified by reverse transcription polymerase chain reaction with total RNA extracted from human fetal liver as template. 5 micrograms of total RNA were subjected to reverse transcriptase (from GIBCO/BRL) to synthesize the corresponding DNA strand with 18T + lN oligonucleotide primers (N is random nucleotides). The reaction conditions were 45 ℃ for 20 minutes, then the temperature was raised to 55 ℃ and the incubation continued for 40 minutes.

The oligonucleotide primer sequence is as follows: 5'-GAATTCATGAAGTGGGTAACCTTTATTTCC-3' and 5'-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3'

Two are providedEcoR I recognition sites are respectively added at two ends of an HSA gene and cloned into an expression vector. PCR reaction conditions were 94 ℃, 4 min, and DNA encoding HSA was further amplified for 35 cycles: 94 ℃, 30 seconds; 58 ℃, 30 seconds; 72' C, 2min 30 sec. At the end of the cycling reaction, a further prolonged reaction of 72 ℃ was given for 10 minutes. Gel electrophoresis shows that the PCR amplification product is about 1850 alkaliAnd (4) base pair. The PCR product was inserted into a PCRII vector (Invitrogen) by TA cloning. The resulting plasmid was designated PCR-HSA. The DNA sequencing result shows that the DNA sequence of the human serum albumin is the same as the published DNA sequence of the human serum albumin. By usingEcoRI was subjected to restriction enzyme digestion of the PCR product, and the HSA DNA fragment was purified by electrophoretic separation and cloned into a yeast expression vector (pYZ vector, available from Tianjin Ying Biotechnology Ltd.). The transformed DH5a competent cells were screened for positive monoclonals on low-salt LB-agar plates containing the antibiotic Zeocin, which was designated pYZ-HSA by the plasmid which was assayed for the insertion of the HSA gene (see FIG. 1, pYZ-HSA recombinant plasmidEcoRⅠ&XhoI, enzyme digestion identification, wherein the size of a target band is about 1850bp and is consistent with the expected size of the target band. Sequencing confirmed forward and reverse).

HSA gene DNA sequence (GeneBank accession No.: AY 728024):

ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATAA

human Serum Albumin (HSA) mature peptide amino acid sequence (GeneBank accession number: AY 728024):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

example 2: expression of recombinant human serum albumin/growth hormone fusion gene (rHSA/GH) and construction of vector plasmid

The nucleotide sequence of GH mature peptide (GeneBank accession No.: NP-000506) was artificially synthesized by DNA full sequence synthesis (synthesized by Biotechnology Ltd., New England of Beijing Ongji science). Respectively at its N terminal withBsu36I cleavage site, to which C terminal is addedXhoI, enzyme cutting site, and using the two enzymes to respectively cut the synthesized target gene GH and pYZ-HSA recombinant plasmids; connection of a target gene GH and a pYZ-HSA recombinant plasmid: the method comprises the following specific steps: mixing the enzyme-cut target gene GH and pYZ-HSA recombinant plasmid according to a certain proportion, adding a connection buffer and a ligase, and connecting at 22 DEG C1 hour; transforming DH5 alpha competent cells by the ligation product, and comprises the following specific steps: the ligation product was added to DH 5. alpha. competent cells, ice-washed for 30 minutes, heat-shocked at 42 ℃ for 90 seconds, ice-washed for 2 minutes, added to the medium and centrifuged at 150rpm, 45 minutes and 12000rpm for 1 minute in a shaker, and plated. Sequencing and verifying, comprising the following specific steps: selecting a monoclonal, carrying out amplification culture, carrying out small-quantity plasmid extraction, carrying out enzyme digestion identification, selecting a plasmid with the size consistent with that of a target fragment obtained by enzyme digestion, carrying out sequencing confirmation, wherein sequencing primers are P5 and P2, and finally, the plasmid subjected to sequencing confirmation is named pYZ-HSA/GH.

P5：5'-AAGGAGACCTGCTTTGCCG -3

P2：5'-CAAATGGCATTCTGACATCC-3

rHSA/GH nucleotide DNA sequence (Seq ID number 2):

ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATTCCCAACCATTC CCTTATCCCGTCTGTTCGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAG TTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGA GTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGC TCATCCAGTCGTGGCTGGAGCCCGTGCAGTTTCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGAC AGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCC CCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGA ACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCT GTGGAGGGCAGCTGTGGCTTCTAGTGA

the nucleotide sequence of the mature peptide of human growth hormone is underlined.

rHSA/GH mature peptide amino acid sequence (Seq ID number 1):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLFPTIPLSRLFDNAMLRAHRLHQL AFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANS LVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFL RIVQCRSVEGSCGF

underlined is the amino acid sequence of the mature peptide of human growth hormone

Example 3: transformation and preparation of pichia pastoris expression engineering bacteria

The colonies of Pichia pastoris strain X33 were inoculated into 50ml centrifuge tubes containing 5ml YPD medium and incubated overnight at 30 ℃ at 250 rpm. The next day, 0.2ml of overnight culture was transferred into 500ml YPD medium and placed in a 2-liter Erlenmeyer flask. Rotary culturing at 30 deg.C for 2-3 hr to make cell density reach OD₆₀₀And (5) = 1.3-1.5. The yeast was collected by centrifugation and resuspended in 500ml of ice-chilled sterile water and washed twice. The yeast was then washed once suspended in 20ml of ice-chilled 1M Sortbitol solution.

pYZ-HSA/GH plasmid DNA constructed in example 2 was subjected toPmeI after restriction enzyme treatment, a linear plasmid molecule is formed. Mu.g of linearized plasmid DNA was mixed with 80. mu.l of the treated yeast and placed in a 0.2 cm thick electrode cup on an electrotransfer apparatus. The electric pulse condition is a voltage of 7500V/CM, and the electrode interval time is 5-10 (ms). Immediately after the shock treatment, 1ml of ice-chilled 1M Sorbitol solution was added to the yeast and then transferred to a 15ml tube. The transformed yeast was placed in an incubator at 30 ℃ for 2 hours, and then inoculated on YPD plate medium containing Zeocin antibiotic. The clones grown by resistance selection are characterized by the insertion of their genes by molecular biology methods. The expression and secretion of the protein were examined by SDS-PAGE or Western blotting with specific antibodies (see FIG. 2).

Example 4: screening of recombinant yeast engineering bacteria

Several yeast colonies containing the gene to be expressed were cultured in basic culture medium containing Zeocin antibiotic, buffer capacity and glycerol, respectively. Culturing at 300 rpm until the thallus density reaches OD₆₀₀And (2-6). Centrifuging the culture at 1500 rpm for 15min to collect thallus, and re-suspending in the same basic culture liquid without adding any other additiveContains glycerol and 0.5% methanol, and has cell density up to OD₆₀₀=1.0, the culture was continued. The yeast is induced by methanol, and the foreign protein starts to express under the action of a promoter. Thereafter, 100% methanol was added every 24 hours to a final concentration of 0.5%. Culture supernatants were collected separately at different time points. The expression of the recombinant human serum albumin/growth hormone fusion protein is preliminarily determined by SDS-PAGE denaturing polyacrylamide gel electrophoresis, and the recombinant yeast engineering strain expressing the target specific protein is screened.

Example 5 Large Scale high Density expression of rHSA/GH fusion proteins

The invention uses a 1000L (ton grade) biological fermentation tank system to develop ton grade large-scale production and fermentation process of the rHSA/GH fusion protein and preparation process and technical establishment of kilogram grade rHSA/GH protein. The recombinant yeast engineering bacteria are prepared into an engineering bacteria seed bank according to a developed process program. Inoculating an engineering bacteria seed bank into a triangular flask, starting from small-scale shake culture, then amplifying to a first-stage seed tank and a second-stage seed tank, and entering a production fermentation tank (1 ton volume). After the glycerol in the tank is completely consumed by culture, the dissolved oxygen is returned to the bottom limit and then is returned to the bottom limit, and the glycerol feeding is started. After the feeding is finished and the dissolved oxygen is raised again, the methanol feeding is started, the induction and recombinant protein production stage is carried out, and the methanol feeding is maintained for 72 hours. Samples can be taken at different culture time points to test for expression of the recombinant protein. The intracellular and culture medium contents of the secreted protein were analyzed by SDS-PAGE and the expression level and purity were monitored at each step. The results showed that the expression level of rHSA/GH protein in the fermentation broth was around 3-10g/L (see FIG. 3, lane M: protein Standard molecular weight (from top to bottom: 97.4kD, 66.2kD, 43.0kD, 31.0kD, 20.1kD, 14.4 kD), lane 1: reduced rHSA/GH with a molecular weight of 88.6kD, which was induced to express 52 hours, lane 2: reduced rHSA/GH with a molecular weight of 88.6kD, which was induced to express 72 hours, lane 3: non-reduced rHSA/GH with a molecular weight of 88.6kD, lane 4: non-reduced rHSA/GH with a molecular weight of 72 hours, molecular weight of 88.6kD, and lane 5: self-made MK (molecular weight from top to bottom: 83kD, 66 kD)). SDS-PAGE is divided into non-reducing SDS-PAGE and reducing SDS-PAGE, which are electrophoresed under denaturing conditions. The difference between reduced and non-reduced SDS-PAGE is the addition or absence of reducing agents (e.g., DTT or 2-mercaptoethanol, etc.) during sample processing.

Example 6: purification and characterization of secreted rHSA/GH fusion proteins

The rHSA/GH fusion protein is directly secreted into a supernatant through fermentation culture of yeast engineering bacteria. After separating the supernatant containing the secreted serum albumin-growth hormone fusion protein from the thalli by continuous flow centrifugation, the column chromatography purification firstly adopts a composite novel weak cation exchange chromatography-Capto-MMC chromatography of the American GE company, because the expressed supernatant has complex components and certain concentration of salt, the Capto-MMC chromatography can bear higher flow velocity and high combination capacity, and the requirement on the concentration of the salt in the fermented supernatant is not high, so that the method is very suitable for the first-step column chromatography collection of the fermented liquid.

The Capto-MMC chromatography can be performed by using 20-100 mM NaAc-HAC buffer solution with pH of 4.0-6.0, and the elution buffer solution can be 20-100 mM phosphate buffer solution containing more than 0.5M NH₄Cl or other high salt solutions, with a pH range of 6.5-8.5, and organic solvents of various concentrations may also be used. Glycine or cysteine may also be added to the buffer in an amount sufficient to maintain protein stability.

Through a large number of screening and exploratory experiments, the test flow is as follows:

primary purification affinity chromatography column: Capto-MMC

And (3) an equilibrium buffer: 30mM NaAc-HAc, 10mM cysteine, 5% glycine, pH5.5

Washing with a miscellaneous buffer solution: 20mM PB,10mM cysteine, 5% glycine, pH6.5

Elution buffer: 20mM PB,10mM cysteine, 5% glycine, 1.0M NH₄Cl，pH7.5。

The primary pure component containing the target protein peak through Capto-MMC is specially explored for the convenience and effectiveness of the production process, and the process steps which can directly enter the hydrophobic medium filler for separation and purification are specially explored. For this purpose, the hydrophobic fillers in butyl-, phenyl-or octyl groups are compared and screened, and 20-100 mM Tris-HCl or phosphorus are respectively selectedAcid buffer solution with pH value of 5.0-9.0, and adding 1-2M NH₄Cl or (NH) of 1-2M₄）₂SO₄The buffer (2) is an equilibrium buffer containing no NH₄Cl or (NH)₄）₂SO₄The buffer solution of (2) is an elution buffer solution, and a certain amount of glycine or cysteine can be added into the buffer solution to maintain the stability of the protein. The process comprises eluting the peak of Capto-MMC column with 1-2M NH at pH 5.0-9.0₄Cl or (NH) of 1-2M₄）₂SO₄Diluting with 20-100 mM Tris-HCl or phosphate buffer solution for several times, or directly adding high-concentration NH into the elution peak of a Capto-MMC chromatographic column₄Cl or (NH)₄）₂SO₄The solution is adjusted to NH with the concentration of 1-2M₄Cl or (NH) of 1-2M₄）₂SO₄Loading on a hydrophobic column, washing the column with an equilibration buffer, eluting with an elution buffer, and collecting an elution peak. The hydrophobic column can further remove impurities such as polymer, carbohydrate, lipid, degraded protein, pigment and the like which may exist, and further improve the purity of the target protein.

Through a large number of screening and exploratory experiments, the test flow is as follows:

hydrophobic medium chromatography column: phenyl Sepharose^TM High Performance

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, 1.3M NH₄Cl，pH6.7

Elution buffer: 20mM PB, pH 6.7.

The sample purified by hydrophobic chromatography is further directly re-purified from an anion exchange column such as Q or DEAE in view of the production process. Ion exchange chromatography allows further purification of the target protein by a separation mechanism different from that of hydrophobic chromatography. Tris-HCl or phosphate buffer solution with the pH value of 20-100 mM and the pH value of 6.0-9.0 can be selected as the equilibrium buffer solution, and a certain amount of glycine or cysteine can be added into the buffer solution to keep the stability of the protein. NaCl or NH with a concentration of 200-1000 mM can be added into the equilibrium buffer solution₄Cl was used as elution buffer. Passing through the vaginaThe sample for ion exchange chromatography can usually reach a purity of more than 95%.

Through a large number of screening experiments, the test procedure is as follows:

ion purification and separation chromatographic column: q Sepharose^TM Fast Flow

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, pH7.2

Washing with a miscellaneous buffer solution: 20mM PB, 180mM NaCl, 10mM cysteine, 5% glycine, pH7.2

Elution buffer: 20mM PB, 800mM NaCl, 10mM cysteine, 5% glycine, pH7.2

The elution peak collected after ion exchange chromatography can be directly loaded on gel chromatography columns such as Sephacryl, Superdex or Sephadex, etc., for desalting and liquid changing. The gel chromatography uses 5-50 mM Phosphate Buffer (PB) as equilibrium buffer, and glycine or cysteine with certain amount can be added into the buffer to maintain the stability of the protein. The buffer solution system of the target protein rHSA-GH obtained under the anion exchange chromatography condition can be changed into 5-10 mM phosphate buffer solution which can be directly used for preparation, and other means such as dialysis or ultrafiltration are not needed, so that possible pollution, protein denaturation, protein degradation, aggregation and other losses in the treatment step are reduced.

Through a large number of screening experiments, the test procedure is as follows:

a chromatographic column: sephacryl S-200HR

And (3) an equilibrium buffer: 20mM PB, 5% glycine, pH7.2

In the whole purification process, supernatant samples of the fermentation liquor after centrifugation can be directly collected by using a Capto MMC column, collected target proteins can be added with ammonium sulfate to directly go to a hydrophobic column, the elution conductivity of the hydrophobic column is low, the samples are highly concentrated, the requirements of ion exchange chromatography can be met through simple dilution treatment or Sephadex G-25 desalination, the sample concentration after the ion exchange chromatography is high, the volume is small, and the method is just suitable for gel column chromatography to carry out liquid change treatment. The whole purification process is reasonable in sequence, simple and convenient to operate and easy for industrial scale-up production, the single component of the final product can reach more than 95%, and the residual amount of the host protein is less than 0.05% when the polymer, the dimer and the related protein of the target protein are calculated as the target protein. The separation and purification production process of the recombinant human serum albumin/growth hormone fusion protein formed by the invention can be used for large-scale production, and the final purity reaches an unexpected high purification effect of more than 99.95% (see fig. 4 and fig. 5, MK used in fig. 4 is a self-made marker, and the molecular weight of MK is 83KD and 66KD from top to bottom).

Example 7: purity determination of rHSA/GH fusion protein by liquid chromatography

Using shimadzu LC-10Avp Plus high performance liquid chromatograph, column: TSK-GEL G2000SWXL 7.8X 300mm column from TOSOH. Using 50mM PB-0.1M NaCl buffer solution, pH7.0 as mobile phase, flow rate: 0.8ml/min, detection wavelength: 280 nm.

Through the optimized separation and purification process established by the above procedure, rHSA/GH fusion protein with the purity of 97.059% is obtained (see figure 6), and the content of the related protein of the target protein (polymer and degradation product of the target protein) is about 3%.

Example 8: detection of recombinant human serum albumin/growth hormone fusion protein yeast host protein residue

And detecting by using a double-antibody sandwich enzyme-linked immunosorbent assay. The host protein residue of the recombinant human serum albumin/growth hormone fusion protein (rHSA/GH) should be not more than 0.01% of the total protein.

The instrument comprises the following steps: enzyme-linked immunosorbent assay (e.g., BIO-RAD, Model 680)

Reagent: (1) detection Kit immunoenzymetic Assay Kit for the Measurement of picha PASTORIS Host Cell Proteins (Cygnus, usa), cat #: cat # F140, detection wavelength 450/650 nm;

(2) preparation of a standard solution: 0ng, 1ng, 4ng, 20ng, 75ng and 250ng of CHO cell protein per 1ml using standard solutions provided in the kit;

(3) test solution: when the protein content is 5mg/ml, 60-80 times dilution (dilution with water for injection) is adopted; a 70-fold dilution is recommended.

(4) A recovery rate measuring well (double well) was provided.

Assay methods (subject to the instructions provided by the kit manufacturer):

(the standard substance and the sample to be tested can be tested by providing multiple holes to improve the accuracy of the test)

(1) Sample dilution: the sample was diluted to 1mg/ml with water for injection.

(2) Preparation of recovery sample: mix well 15 μ l of each diluted sample solution with 15 μ l of 250 ng/ml standard for use.

(3) And (3) sucking 25 mu l of the standard substance, the sample and the recovery rate sample, adding into a 96-well plate, and adding 100 mu l of anti-P.

(4) The membrane is sealed and the 96-well plate is covered, and then put into a shaker for shaking at 24 ℃ and 180rpm for 3 hours.

(5) The solution was decanted, 350. mu.l of diluted wash solution "was added to each well (the stock solution in the kit was diluted 20 times with water for injection), and the solution was decanted and washed 4 times in total.

(6) Add 100. mu.l of "TMB Substrate", cover, and let stand at room temperature for 30 minutes.

(7) Add 100. mu.l stop solution ", set the microplate reader reading at 450/650nm, draw a standard curve with Excel and calculate the host protein content and recovery of the sample.

0 was adjusted using 0ng/ml standard wells as a blank and the OD was read from each well. Computer analysis software was used for reading and data analysis.

(8) And (4) taking the concentration corresponding to the absorbance of the standard solution as a standard curve, and obtaining the corresponding host protein content on the standard curve by the absorbance of the test solution. Calculated according to the following formula:

wherein c is the content of host protein in the test solution, ng/ml;

d is the dilution multiple of the test sample;

t is the protein content of the test sample, mg/ml.

And (3) calculating and judging a result:

percent host protein = host protein concentration/test protein concentration

Recovery rate = [ recovery rate determination pore concentration- (test sample concentration/2) ]/100

The detection results of the host proteins of 3 batches of stock solutions 9075Y20160801, 9075Y20170501 and 9075Y20180301 of rHSA/GH prepared by using the method are shown in the following table:

and (4) judging a result: the residual amount of host protein in the recombinant human serum albumin/growth hormone fusion protein stock solution should be not more than 0.01% of the total protein.

The host protein of the yeast is protein secreted by the yeast generated in the accessory process of the yeast, enzyme and commercial value protein produced by degraded yeast. The host protein residue can enter blood after the drug is administrated by subcutaneous injection to stimulate the organism to generate immune response, or the host protein residue is characterized by high fever, shivering and the like, and the smaller the host protein residue is, the better the host protein residue is. The detection result of the high-purity injectable recombinant human serum albumin/growth hormone fusion protein product produced by the production and preparation process shows that the residual amount of host protein is below 0.01 percent. Namely, the purity of the target protein reaches 99.99 percent.

Example 9: immunoblot analysis of recombinant human serum albumin/growth hormone fusion proteins

The rHSA/GH fusion protein obtained by purification is subjected to SDS-PAGE electrophoresis, the protein is transferred to a cellulose acetate membrane by an electrophoresis apparatus, and then a specific antibody (a first antibody) is used for identifying the corresponding protein. And then a secondary antibody with horseradish peroxidase is used for recognizing and binding to the primary antibody, and the mark is left on the cellulose acetate membrane after DAB color development.

The yeast expressed and secreted rHSA/GH fusion protein was detected with an anti-human HSA mouse monoclonal antibody (Sigma Cat #: A6684), and it was confirmed that the protein sequence contained human serum albumin. Similarly, after the electrophoresis gel transfer of the same sample, the Western blotting experiment with Anti-GH antibody (cat #: BA 14243) of Anti-GH monoclonal antibody from Abcam company of America shows that the fusion protein has the same antigenicity as GH. For this purpose, it was confirmed that the expressed rHSA/GH fusion protein had antigenicity of both human serum albumin and human growth hormone (see FIGS. 7 and 8).

Example 10: mass spectrum molecular weight determination of recombinant human serum albumin/growth hormone fusion protein

1. Experimental methods

(1) Sample pretreatment method

Complete molecular weight determination: analyzing the sample solution by using 5mg/ml stock solution and 4 mul (sample loading amount is 20 mug) of sample injection; and diluting the adaptive sample of the system to 2mg/ml by using purified water, uniformly mixing, and carrying out analysis by sampling 5 mu l (sample loading amount is 10 mu g).

(2) UPLC method parameters

Mobile phase a was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. The chromatographic Column is Nano-Micro UniPS3-300 Column, 3 mu m, 2.1mm X50 mm, [ Part Nr.UPS03300 ]. The temperature of the column incubator is set to 80 ℃, the temperature of the sample chamber is set to 4 ℃, and the flow rate is set to 0.2 mL/min.

(3) Parameters of mass spectrometry method

Samples were desalted through chromatography columns and then subjected to detection scanning mass spectrometry using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Analysis duration: 6min, detection mode: positive ion mode, scan range: 800-: 7500

(4) Data analysis parameters

LCMS results mass spectrum deconvolution analysis was performed on Biopharma Finder (version 3.0) software.

2. Conclusion of the experiment

The theoretical molecular weight of the system adaptation sample Avastin is: 149197.20, the measured value is: 149198.41, the error from the theoretical value is: 8.1ppm, and the system adaptability result meets the detection requirement. The mass spectrometry data of the recombinant human serum albumin/growth hormone fusion protein injection measured under the condition are deconvoluted, and the average molecular weight is as follows: 88542.21 Da. This is very close to the theoretical molecular weight of 88583.45Da for the rHSA/GH mature peptide amino acid sequence.

Example 11 recombinant human serum Albumin/growth hormone fusion protein peptide Mass fingerprinting (peptide matching coverage) assay

The sample is reduced and denatured in 6M Guanidine hydrochloride solution containing DTT, IAA is added for alkylation, buffer solution is replaced into enzymolysis buffer solution, Trypsin, Glu-C and Chymotrypsin are added for enzymolysis, and LCMS is used for analysis. The specific operation process is as follows: adding a sample of 75 micrograms into 248 microliters of Solution F and 4 microliters of Solution C for 30min at 37 ℃; adding 9.6 muL Solution D, keeping out of the sun for 30min at room temperature, and adding muL Solution C; buffer exchange: taking a new ultrafiltration tube (20 kD), adding 350 mu L Solution H, centrifuging for 12000 rpmx for 15min, and discarding the upper and lower layer liquid. Adding a sample into an ultrafiltration tube, adding a proper amount of Solution H, centrifuging for 12000 rpmx for 15min, discarding the lower-layer waste liquid, and repeating for 4 times; transferring the sample to a centrifuge tube, adding a proper amount of Solution H to adjust the concentration of the Solution to be 0.7 ug/uL; dividing into 3 parts, adding Glu-C (1: 10), Trypsin (1: 15) and Chrymotrypsin (1: 20) for enzymolysis, and keeping the temperature at 37 ℃ for 18 h; formic acid was added to inactivate the enzyme. The enzymatic products were centrifuged and analyzed on LCMS.

And (3) carrying out chromatographic separation on the sample enzymolysis product by using UPLC. Mobile phase a was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. The chromatographic Column is Waters ACQUITY UPLC Peptide BEH C18 Column, 130A, 1.7 μm, 2.1mm X150 mm, [ Part Nr.186003556 ]. The temperature of a column incubator is set to 60 ℃, the temperature of a sample chamber is set to 4 ℃, the flow rate is set to 0.3 mL/min, the detection wavelength is 215nm, and the sample loading amount is 15 mug.

Samples were desalted and separated by UPLC and then subjected to detection scan mass spectrometry using an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Analysis duration: 90min, detection mode: positive ion mode, scan range: 300-: 30000/7500.

The LC-MS/MS result of the enzymolysis product searches a target protein sequence on a Biophama Finder (version 3.0), and after the results of three enzymolysis products of Trypsin and Glu-C, Chymotrypsin are integrated, the coverage rate of the primary structure of the obtained recombinant human serum albumin/growth hormone fusion protein injection is as follows: 100% (see FIG. 9, the upper panel in FIG. 9 shows the results of Trypsin enzymatic hydrolysate, the middle panel shows the results of Glu-C enzymatic hydrolysate, and the lower panel shows the results of Chymotrysin enzymatic hydrolysate).

Example 12: rat in vivo bioactivity assay for rHSA/GH fusion proteins

1. Method for removing weight of hypophysis rat

On the day of the test, recombinant PEG-human growth hormone was taken as a reference substance (purchased from Catharanthus roseus), and the reference substance and the sample were prepared into a solution with a concentration of 450 mug/kg by using a 0.9% sodium chloride solution containing 0.1% bovine serum albumin.

The determination method comprises the steps of taking a rat with the same source and strain, birth for 26-28 days, weight of 60-80 g, the same sex and health, removing hypophysis by an operation 2-3 weeks before the test, and feeding in a barrier environment after the operation to recover the rat. Rats with weight change less than +/-10% of that before the operation 2-3 weeks after the pituitary removal operation are divided into 4 groups according to the weight at random, at least 5 rats in each group are numbered, and the weight is recorded. 0.4ml of control solution or test solution is injected subcutaneously into the neck, the first day is administered for 1 time, the rat is sacrificed on day 7, the weight is weighed, necropsy can be performed after the experiment is finished, the sphenoid saddle area is cut open, the rat with residual pituitary is removed by visual inspection. The response was determined as grams of body weight gain after each animal was dosed. The results and experimental errors were calculated according to the quantitative response parallel assays in the bioassay statistics (general rule 1431).

Test data:

this calculation resulted in a 9.0g body weight gain comparable to 450 μ g PEG-rhGH only when 675 μ g rHSA/GH administration was used.

2. The method is a method for determining the potency of a test sample by comparing the degree of increase of the growth hormone standard (S) and the test sample (T) in the width of a tibial epiphyseal plate of a hypophysectomized rat.

The standard solution and the test solution are prepared by the same weight method.

Assay this method can be performed simultaneously with the hypophysectomized rat body weight method. After the weight method experiment is finished, the shines of two legs are taken down, placed in 10% formaldehyde solution for preservation, cut along the sagittal plane from the middle of the top of the proximal end of the shin, placed in 10% formaldehyde solution for preservation, washed for 10 minutes, placed in acetone solution for 10 minutes, washed for 3 minutes, placed in 2% silver nitrate solution for dyeing for 2 minutes, washed, placed in water and irradiated by strong light until becoming brown and black, fixed in 10% sodium thiosulfate solution for 30 seconds, and placed in 80% ethanol solution for measurement. During measurement, left and right slices of lmm are cut along the plane, and the width of the tibial epiphyseal plate is measured under a microscope to serve as a reaction value. Titers and experimental errors were calculated as a quantitative response parallel assay in the bioassay statistics (general rule 1431).

Test data:

the results of the body weight show that the biological activity of 675 μ g recombinant human serum albumin/growth hormone fusion protein is equivalent to 450 μ g of PEG-rhGH, and thus the in vivo biological specific activity value of rHSA/GH in rats is calculated to be about 4 IU/mg (this is because the human serum albumin is not considered to be necessarily a xenogeneic body in rats, and a binding antibody is generated, and the action efficacy of GH in the drug is reduced by the binding antibody). However, the bioactivity (drug effect) of key human growth hormone in vivo rat is measured by the increase of the width of the epiphyseal plate of the rat, and then the bioactivity of the rHSA/GH is particularly and obviously superior to that of the PEG-rhGH. The recombinant human serum albumin/growth hormone fusion protein obtained by the technology of the invention has unexpected proliferation effect.

According to the Chinese pharmacopoeia second part (2015 edition) and the drug specifications of the commercial recombinant human growth hormone (rhGH) and the pegylated recombinant human growth hormone (PEG-rhGH), the biological specific activity values of the two are about 3 IU/mg and 6 IU/mg respectively. 1mg of PEG-rhGH corresponds to 2mg of rhGH; theoretically, on a molar basis, 4 mg of rHSA/GH (88.5 kD, 1 GH contained therein, molecular weight about 22kD, 1/4 accounted for) should theoretically correspond to 1mg of rhGH; an rHSA/GH concentration of 8mg should theoretically correspond to 1mg of PEG-rhGH (about 60kD, 2 GH and 19kD polyethylene glycol molecules, but protein content detection techniques determine that the sugar content is not included in mg weight). The result of the actual rat measurement is that the biological specific activity of rHSA/GH is 5.3 times higher than that of rhGH; the specific biological activity value of the recombinant human serum albumin/growth hormone fusion protein is 10 times higher than that of PEG-rhGH, so that the recombinant human serum albumin/growth hormone fusion protein prepared by the production process has unexpectedly high biological activity in rats. This result was obtained because the metabolic process and elimination pattern of the fusion protein in rats was completely different from that of rhGH and the chemically modified long-acting PEG-rhGH in vivo. The result shows the special clinical application value and scientific result of the invention.

Example 13: determination of in vivo half-life of recombinant human serum albumin/growth hormone fusion protein rat

Sprague-Dawley rats (SD, SPF grade, hermaphrodite half) were used. The body weights of the rats were measured one day (D-1) before the administration of the drug, wherein the body weight of the male rat was about 276-372g, and the body weight of the female rat was about 221-280 g. The positive control drug 1 is recombinant human growth hormone (rhGH) for common injection (purchased from Catharanthus roseus company), and the specification is 3 mg/ml/count; the positive control drug 2 is long-acting PEG recombinant human growth hormone injection (PEG-rhGH) (purchased from Catharanthus roseus company) with the specification of 9 mg/ml/count. The test reagent is the recombinant human serum albumin/growth hormone fusion protein injection of the invention, and the specification is as follows: 5mg/0.5 ml/piece (protein concentration 10 mg/ml). When the test drug is administrated to rats, the administration dose is 200 mug/kg according to the rhGH content of only 1/4 in each mg of protein in the fusion protein. Each administration volume was 300 μ l. Blood was taken 1h before administration, 2min, 0.5h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 168h and 240h after administration. After inhalation anesthesia with isoflurane, the orbital venous plexus was bled, approximately 0.2mL per blood sample. Blood samples were collected, transferred into EP tubes containing EDTA, shaken well, placed in crushed ice (not more than 2h of storage in crushed ice), and then centrifuged at 4000 rpm for 10 min at low temperature (about 4 ℃) to separate plasma for storage below-20 ℃ for analysis.

The detection of blood samples was carried out using the Quantikine ELISA Human Growth Hormone Immunoassay kit manufactured by R & D Systems, USA. The results showed that the blood half-life of rhGH was 1.5-2.5h, that of PEG-rhGH was 18-24h, and that of the recombinant human serum albumin/growth hormone (rHSA/GH) of the present invention was 58-80h (see FIG. 10). Just as rHSA/GH undergoes a significant and unexpected long metabolic cycle in animals, i.e., a long blood half-life, resulting in slow release of GH from the fusion protein, this half-life study also explains well why rhhsa/GH has been observed to have significantly higher biological specific activity than that of theoretical rats than rhGH and PEG-rhGH.

This result shows that the present invention has an unexpected advantage of long-acting drugs over all existing commercial products or growth hormone drugs under investigation. This result also fully supports the special requirement of the recombinant human serum albumin/growth hormone fusion protein prepared by the production process of the present invention as a drug that can be expected to achieve clinical treatment with administration frequency of once every 2 weeks, or once every 3 weeks, or even once every 4 weeks.

Sequence listing

<110> Tianjin Linda Biotechnology Ltd

TIANJIN SINOBIOTECH Ltd.

<120> preparation and purification method of recombinant human serum albumin/growth hormone fusion protein for treating childhood dwarf

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 776

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu Phe Pro Thr Ile Pro Leu Ser

580 585 590

Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu

595 600 605

Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu

610 615 620

Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser

625 630 635 640

Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser

645 650 655

Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu

660 665 670

Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr

675 680 685

Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu

690 695 700

Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr

705 710 715 720

Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His

725 730 735

Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg

740 745 750

Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg

755 760 765

Ser Val Glu Gly Ser Cys Gly Phe

770 775

<210> 2

<211> 2406

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60

gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120

gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180

gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240

gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300

gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360

gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420

agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480

aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540

tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600

tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660

agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720

gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780

gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840

agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900

gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960

gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020

aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080

aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140

ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200

tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260

cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320

caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380

tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440

ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500

tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560

tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620

tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt gaaacacaag 1680

cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740

aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800

gctgcaagtc aagctgcctt aggcttattc ccaaccattc ccttatcccg tctgttcgac 1860

aacgctatgc tccgcgccca tcgtctgcac cagctggcct ttgacaccta ccaggagttt 1920

gaagaagcct atatcccaaa ggaacagaag tattcattcc tgcagaaccc ccagacctcc 1980

ctctgtttct cagagtctat tccgacaccc tccaacaggg aggaaacaca acagaaatcc 2040

aacctagagc tgctccgcat ctccctgctg ctcatccagt cgtggctgga gcccgtgcag 2100

tttctcagga gtgtcttcgc caacagcctg gtgtacggcg cctctgacag caacgtctat 2160

gacctcctaa aggacctaga ggaaggcatc caaacgctga tggggaggct ggaagatggc 2220

agcccccgga ctgggcagat cttcaagcag acctacagca agttcgacac aaactcacac 2280

aacgatgacg cactactcaa gaactacggg ctgctctact gcttcaggaa ggacatggac 2340

aaggtcgaga cattcctgcg catcgtgcag tgccgctctg tggagggcag ctgtggcttc 2400

tagtga 2406

Claims (1)

1. A high-density expression and purification method of recombinant human serum albumin/growth hormone fusion protein for treating children's dwarf syndrome is characterized in that,

the high-density expression method comprises the following steps:

a. construction and expression of recombinant human serum albumin/growth hormone fusion protein vector plasmid:

using total RNA extracted from human fetal liver as template, synthesizing and amplifying HSA gene by reverse transcription polymerase chain reaction; separating and purifying HSA DNA fragment by electrophoresis, cloning the DNA fragment to a yeast expression vector to obtain pYZ-HSA recombinant plasmid; artificially synthesizing GH nucleotide sequence by adopting a DNA full sequence synthesis method; digesting the synthesized target gene GH and pYZ-HSA recombinant plasmids, and connecting the synthesized target gene GH and pYZ-HSA recombinant plasmids to obtain pYZ-HSA/GH recombinant plasmids;

wherein, the amino acid sequence of the recombinant human serum albumin/growth hormone fusion protein is shown as SEQ ID NO.1, and the coding nucleotide sequence is shown as SEQ ID NO. 2;

b. transformation and preparation of pichia pastoris expression engineering bacteria

The pYZ-HSA/GH recombinant plasmid is treated by Pme I restriction endonuclease and then is electrically transferred into Pichia pastoris X33 competent cells, and the transformed yeast is inoculated on a YPD plate culture medium containing Zeocin antibiotic for culture;

c. screening of recombinant yeast engineering bacteria

Culturing a plurality of yeast colonies containing genes to be expressed in a basic culture solution containing Zeocin antibiotics and having buffer capacity and glycerol respectively; after culturing for a period of time, centrifugally collecting thalli, and then suspending the thalli in a same basic culture solution without glycerol, wherein the content of methanol is 0.5%; adding 100% methanol every 24 hours until the final concentration is 0.5%, respectively collecting culture supernatant at different time points, and screening recombinant yeast engineering strains for expressing specific proteins;

d. large-scale high-density expression rHSA/GH fusion protein

Using a 500L or 1000L biological fermentation tank system, taking an engineering strain sub-library, starting small-scale shaking culture, then amplifying to a first-stage seed tank and a second-stage seed tank, and entering a production fermentation tank; after culturing until the glycerol in the tank is consumed, the dissolved oxygen is returned to the bottom limit and then is returned to the bottom limit, and the glycerol feeding is started; after the feeding is finished and the dissolved oxygen is increased again, starting methanol feeding, entering an induction and recombinant protein production stage, and maintaining the methanol feeding for 72 hours;

the purification of the recombinant human serum albumin/growth hormone fusion protein obtained by high-density expression comprises the following steps:

a. primary purification affinity chromatography column: Capto-MMC

And (3) an equilibrium buffer: 30mM NaAc-HAc, 10mM cysteine, 5% glycine, pH5.5 Wash buffer: 20mM PB,10mM cysteine, 5% glycine, pH6.5

Elution buffer: 20mM PB,10mM cysteine, 5% glycine, 1.0M NH4ClpH7.5;

b. hydrophobic medium chromatography column: phenyl Sepharose^TM High Performance

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, 1.3M NH4Cl, pH6.7

Elution buffer: 20mM PB, pH 6.7;

c. ion purification and separation chromatographic column: q Sepharose^TM Fast Flow

And (3) an equilibrium buffer: 20mM PB,10mM cysteine, 5% glycine, pH7.2

Washing with a miscellaneous buffer solution: 20mM PB, 180mM NaCl, 10mM cysteine, 5% glycine, pH7.2

Elution buffer: 20mM PB, 800mM NaCl, 10mM cysteine, 5% glycine, pH 7.2;

d. a chromatographic column: sephacryl S-200HR

And (3) an equilibrium buffer: 20mM PB, 5% glycine, pH 7.2.

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