CN114920819A - Recombinant human growth hormone mutant, polyethylene glycol derivative thereof, preparation method of polyethylene glycol derivative and pharmaceutical application of polyethylene glycol derivative - Google Patents
Recombinant human growth hormone mutant, polyethylene glycol derivative thereof, preparation method of polyethylene glycol derivative and pharmaceutical application of polyethylene glycol derivative Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH] (Somatotropin)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
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- C12N15/09—Recombinant DNA-technology
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention provides a recombinant human growth hormone mutant, a polyethylene glycol derivative thereof, a preparation method of the polyethylene glycol derivative and a pharmaceutical application thereof. The recombinant human growth hormone of the inventionThe amino acid sequence of the mutant is shown in SEQ ID NO.1, and the recombinant human growth hormone mutant is obtained by mutating leucine Leu at the 101 st position of the amino acid sequence of the human growth hormone shown in SEQ ID NO.3 into cysteine Cys. The invention adopts the gene engineering technology to integrate the recombinant human growth hormone mutant gene into escherichia coli (E.coli) In the engineering bacteria, a large amount of high-activity high-purity recombinant human growth hormone mutant protein is obtained through fermentation and purification. Meanwhile, the polyethylene glycol is used for site-directed modification of the 101-Cys amino acid of the recombinant human growth hormone mutant protein, the half-life period of a modified product is prolonged compared with that of an unmodified protein, the immunogenicity is reduced, the modified product is uniform, and compared with a prototype enzyme, the modified product has better stability and lower production cost.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to site-directed mutagenesis recombinant human growth hormone and polyethylene glycol site-directed modification thereof, wherein the site-directed mutant is a human growth hormone cysteine mutant and relates to site-directed covalent connection between a thiol reaction type polyethylene glycol reagent and the human growth hormone cysteine mutant. The invention further relates to the use of site-directed mutagenesis or modification of growth hormone, e.g. as a stable, long-acting growth hormone or the like.
Background
Growth hormone
Human Growth Hormone (hGH) is a single peptide chain protein Hormone secreted by anterior pituitary eosinophils, and comprises 191 amino acid residues, two pairs of intramolecular disulfide bonds (C54-C65, C182-C189), an isoelectric point of 4.9, a molecular weight of about 22 kDala, and no glycosylation in the molecule. The medicine has the physiological functions of regulating metabolism, stimulating protein synthesis, accelerating fat degradation and the like, can promote the growth and development of skeleton and muscle tissues, is a specific medicine for treating short stature of children caused by the deficiency or insufficiency of endogenous recombinant human growth hormone (rhGH), can promote the healing of wounds, and is also used for treating injuries and serious burns and in surgical operations.
Because the in vivo half-life of the human growth hormone is short and is only about 0.5-2 hours, the human growth hormone can achieve good treatment effect only by injecting once or twice every day. The result is that frequent and long-term injections both increase the cost of treatment and cause much pain and inconvenience to the patient, and severely reduce patient compliance, becoming a major obstacle to the market spread of growth hormone and its market growth. Therefore, it is necessary to improve the dosage form of growth hormone, develop long-acting preparation of growth hormone, reduce the administration cost and pain of patients, and improve the quality of life and compliance of patients.
Polyethylene glycol modification
Polyethylene glycol (PEG) is a hydrophilic polymer with neutral pH, no toxicity and high water solubility, and has repeating units of oxyethylene groups and end groups of two hydroxyl groups, and is in a linear or branched chain structure. The curative effect of the medicines can be improved or the application range of the medicines can be enlarged by coupling the activated polyethylene glycol to medicine carriers such as proteins, polypeptides, small molecular organic medicines, liposomes and the like by a chemical method.
It is generally accepted in the art that the following changes in properties occur in most proteins after modification with polyethylene glycol: reduced immunogenicity and antigenicity; the circulation half-life period is prolonged; increased solubility; resistance to protease hydrolysis; the bioavailability is improved; the toxicity is reduced; the thermal stability and the mechanical stability are increased; isoelectric point, electrophoretic behavior, kinetic properties, etc. In addition, it is important that the modification of the protein with polyethylene glycol causes the decrease of the cytological activity, mainly because the groups introduced in the final product, including PEG and the bond between PEG and the modified protein, are also related to the coupling conditions, the generated by-products, etc.
The polyethylene glycol modified protein technology is widely applied to the field of biological medicines, and amino modification is common in PEG protein medicines on the market at present. For example, PEG modified interferon alpha-2 a of Roche company and PEG modified interferon alpha-2 b of Schering-Plough company have slower clearance speed in serum and reduced medication times; PEG modified ADA, PEG modified G-CSF, new drug PEGylated urate oxidase for treating gout approved by FDA in 2010 and on the market, and the like. The modified medicines are proved to have prolonged half-life, weakened immunogenicity, reduced administration times, improved curative effect, greatly reduced immunogenicity and reduced administration frequency.
Pegylated growth hormone
In patent CN102989001B, the inventors disclose a method for modifying human growth hormone with polyethylene glycol, in which method polyethylene glycol is transiently linked to human growth hormone, and the method prolongs the in vivo half-life of human growth hormone. But still have a short half-life compared to the stably modified growth hormone product.
In patent CN102838671B, the invention discloses introducing unnatural amino acid site in growth hormone protein and using polyethylene glycol to site-specifically modify the site to obtain stable long-acting growth hormone protein conjugate. Since unnatural amino acids were introduced into growth hormone proteins, the safety impact could not be determined.
In patent CN1253472C, the inventors disclose a method for N-terminal specific modification of polyethylene glycol with growth hormone. In patent CN1477126A, the inventors disclose a method for modifying growth hormone with long-acting polyethylene glycol, which modifies amino group in growth hormone at a single point, and significantly prolongs the half-life of growth hormone. However, the polyethylene glycol modification of the growth hormone in the patent is mainly aimed at the amino modification in the growth hormone, and as the number of amino sites capable of being modified in the growth hormone is large, byproducts of different modification sites or products of multi-site modification can appear in the modification process, so that the uniformity of the modified product is reduced, the subsequent purification and clinical application of the product are not favorable, and the yield of the target protein is reduced.
In order to solve the problems existing in the prior modification technology, the preparation of a stable pegylated growth hormone product with more uniform structure has important clinical and economic significance.
Disclosure of Invention
The invention aims to provide a human growth hormone mutant and a preparation method thereof, the method mutates leucine (Leu) at the 101-position of human growth hormone into cysteine (Cys), integrates mutant genes into escherichia coli engineering bacteria, and obtains a large amount of high-activity high-purity human growth hormone mutant protein through fermentation and purification.
The second purpose of the invention is to provide a human growth hormone mutant polyethylene glycol modified compound, a preparation method and a pharmaceutical application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in the first aspect of the invention, the amino acid sequence of the recombinant human growth hormone mutant is shown in SEQ ID NO.1, and the recombinant human growth hormone mutant is obtained by mutating leucine Leu at the 101-th site of the amino acid sequence of the human growth hormone shown in SEQ ID NO.3 into cysteine Cys.
In a second aspect of the present invention, there is provided a nucleic acid encoding the recombinant human hormone mutant according to the first aspect, wherein the nucleotide sequence is shown in SEQ ID No. 2.
In a third aspect of the present invention, there is provided a method for preparing a recombinant human growth hormone mutant as described in the first aspect, the method comprising the steps of:
1) constructing an expression vector and transforming escherichia coli engineering bacteria:
inserting the mutated human growth hormone gene fragment into a restriction enzyme cutting site of an expression vector, identifying a recombinant expression plasmid, and transforming the recombinant plasmid into an expression host escherichia coli (E.coil) to obtain an engineering bacterium, wherein the expression vector is a pET series vector;
2) screening, culturing and induced expression of positive clones:
screening out positive clones, performing fermentation culture in a culture medium, and inducing to express a target protein;
3) and (3) separation and purification of an expression product:
collecting Escherichia coli thallus, and purifying by bacteria breaking, ion exchange chromatography, molecular sieve chromatography, and hydrophobic chromatography to obtain human growth hormone mutant protein.
The cDNA sequence of the human growth hormone mutant is shown in SEQ ID NO.2, and a protein with the amino acid sequence shown in SEQ ID NO.1 is coded; the coding sequence of the human growth hormone is obtained by designing a primer and performing PCR amplification after base mutation of a cDNA sequence of a gene library.
Preferably, the pET series vector in step 1) is one or more of pET24, pET28, pET30 or pET 39.
Preferably, the restriction site of the expression vector in step 1) is XbaI/XhoI.
Preferably, the engineering bacteria in the step 1) are E.coli BL21(DE3), E.coli DH5 alpha or E.coli Rosetta.
Preferably, kanamycin is used as a resistance screen in the positive clone screening in the step 2).
Preferably, the culture temperature in the step 2) is controlled to be 25-40 ℃, and the culture temperature is controlled to be 28-33 ℃.
Preferably, the final concentration of IPTG in the step 2) is 0.1-1.5 mmol/L as an inducer, and the final concentration of IPTG is 0.5-1 mmol/L.
Preferably, the induced expression temperature of the step 2) is controlled to be 25-40 ℃, and the induced expression temperature is controlled to be 28-33 ℃.
Preferably, the chromatography in step 3) is one or more of ion exchange chromatography, molecular sieve chromatography and hydrophobic chromatography.
In a fourth aspect of the present invention, there is provided a polyethylene glycol derivative of the recombinant human growth hormone mutant according to the first aspect, wherein the polyethylene glycol derivative is formed by linking a polyethylene glycol modifier to the Cys site of cysteine 101 of the recombinant human growth hormone mutant. The thiol group of the 101-bit mutation Cys amino acid of the polyethylene glycol and the recombinant human growth hormone mutant specifically reacts, each recombinant human growth hormone mutant molecule specifically combines 1 polyethylene glycol molecule, and the modification site is the 101-bit mutation Cys amino acid.
Preferably, the polyethylene glycol modifier molecule is any one of mPEG-maleimide (mPEG-MAL), mPEG-orthopyridyl disulfide, mPEG-vinylsulfone, mPEG-iodoacetamide and mPEG-orthopyridyl disulfide; most preferably, the polyethylene glycol modifier molecule is mPEG-maleimide (mPEG-MAL).
Preferably, the average molecular weight of the polyethylene glycol modifier is 4kDa to 40 kDa; the average molecular weight of the preferably selected polyethylene glycol modifier is 20kDa to 40 kDa; most preferably, the polyethylene glycol modifier has an average molecular weight of 40 kDa.
Preferably, the polyethylene glycol modifier is linear chain or branched chain polyethylene glycol containing a single active functional group, and the most preferred polyethylene glycol modifier is branched chain polyethylene glycol containing a single active functional group, and the structural formula is as follows:
wherein j is a positive integer of 1-12, m and n can be the same or different integers, and m + n ranges from 880-940; r is the active functional group of polyethylene glycol, such as maleimide, disulfide, vinyl sulfone, iodoacetamide, orthopyridyl disulfide and the like.
In a fifth aspect of the present invention, there is provided a method for preparing a polyethylene glycol derivative of the recombinant human growth hormone mutant as described in the fourth aspect.
1) The method adopts recombinant human growth hormone mutant protein with the purity of more than 95 percent, and polyethylene glycol modifier molecules react with free cysteine (Cys) in the recombinant human growth hormone mutant in neutral or alkaline aqueous medium.
2) Isolating the product of the polyethylene glycol mono-modified recombinant human growth hormone mutant from the modification reaction mixture.
Preferably, in the step 1), the polyethylene glycol modifier is one of mPEG-maleimide (mPEG-MAL), mPEG-o-pyridyl disulfide, mPEG-vinyl sulfone, mPEG-iodoacetamide and mPEG-n-pyridyl disulfide; preferably mPEG-maleimide (mPEG-MAL).
Preferably, in the step 1), the average molecular weight of the polyethylene glycol modifier is 4kDa to 40 kDa; the average molecular weight of the preferably selected polyethylene glycol modifier is 20kDa to 40 kDa; most preferably, the polyethylene glycol modifier has an average molecular weight of 40 kDa.
Preferably, in the step 1), the polyethylene glycol modifier is linear chain or branched chain polyethylene glycol containing a single active functional group; preferably branched polyethylene glycol.
Preferably, in the step 1), the mass ratio of the polyethylene glycol molecules to the recombinant human growth hormone mutant protein in the modification reaction is 3: 1-8: 1;
preferably, in the step 1), the pH value of the modification reaction system is controlled to be 7-9.5; the pH value is preferably 8.0-9.2.
Preferably, in the step 2), the unmodified recombinant human growth hormone mutant protein and free polyethylene glycol are removed by using an ion exchange column Capto Q, a PB buffer solution with pH8.0 and 20mmol/L is used as a loading buffer solution, the flow rate is 10ml/min, a PB solution with 20mmol/L and a NaCl solution with 120mmol/L and a PB solution with 20mmol/L and a NaCl solution with 500mmol/L are respectively used as eluent, the target protein is eluted at 120mmol/L NaCl, and the elution peak is the product of the polyethylene glycol single-modified recombinant human growth hormone mutant.
In the sixth aspect of the invention, the application of the polyethylene glycol derivative of the recombinant human growth hormone mutant as described in the fourth aspect in the preparation of a medicament for treating slow growth of children caused by pathological factors such as endogenous growth hormone deficiency is provided. According to the six examples of in vivo activity detection of recombinant human growth hormone mutant protein and derivatives thereof, the polyethylene glycol single-modified recombinant human growth hormone mutant has the same effect as growth hormone protein in animals, so that the polyethylene glycol single-modified recombinant human growth hormone mutant can be applied to the preparation of medicines for treating slow growth of children caused by pathological factors such as endogenous growth hormone deficiency.
The invention has the following technical effects:
1) the invention adopts the gene engineering technology to integrate the recombinant human growth hormone mutant gene into an escherichia coli (E.coli) engineering bacterium, and obtains a large amount of high-activity high-purity recombinant human growth hormone mutant protein through fermentation and purification. Meanwhile, the polyethylene glycol is used for site-specific modification of the Cys amino acid at the 101 th site of the recombinant human growth hormone mutant protein, the half-life period of a modified product is prolonged compared with that of an unmodified protein, the immunogenicity is reduced, the modified product is uniform, and compared with a prototype enzyme, the modified product has better stability and lower production cost. Therefore, the polyethylene glycol modified recombinant human growth hormone mutant protein realizes the balance of aspects of high specific activity, long half-life period, low immunogenicity, uniform and stable quality, low cost and the like, and has industrial value.
2) It can be seen from the eighth and tenth embodiments of the present invention that only one single-point modification product of polyethylene glycol is present in the modification products of the polyethylene glycol derivatives of the recombinant human growth hormone mutant of the present invention, and the modification site is the mutated 101-position Cys, which indicates that the post-mutation modification product has good homogeneity and no modification products at other sites.
3) As can be seen from the third and fourth examples of the embodiment of the present invention, the polyethylene glycol derivative of the recombinant human growth hormone mutant of the present invention adopts thiol single-site-directed modification, and thus, no by-product is produced after modification, the protein recovery rate is above 90%, and the protein recovery rate is high.
Drawings
FIG. 1 shows the results of protein fermentation expression of recombinant human growth hormone mutants:
lane 1: a standard protein molecular weight;
lane 3: inducing expression of a preprotein;
lane 4: inducing the expression of the protein for 1 h;
lane 5: inducing the expression of the protein for 2 h;
lane 6: inducing the expression of the protein for 3 hours;
lane 7: and (4) inducing protein expression for 4 h.
FIG. 2 shows the result of protein purification of recombinant human growth hormone mutant:
lane 1: a standard protein molecular weight;
lane 3: purifying recombinant human growth hormone mutant protein Q;
lane 5: hydrophobic purification of recombinant human growth hormone mutant protein;
lane 6: desalting and concentrating recombinant human growth hormone mutant protein.
FIG. 3 shows the result of purity of RP-HPLC of recombinant human growth hormone mutant protein.
FIG. 4 shows the results of Y-mPEG-MAL modification of recombinant human growth hormone mutant protein and purification:
lane 2: a standard protein molecular weight;
lane 3: before the recombinant human growth hormone mutant protein is modified;
lane 4: modifying the mass ratio of the Y-mPEG-MAL to the recombinant human growth hormone mutant protein of 3: 1;
lane 5: modifying the Y-mPEG-MAL with the recombinant human growth hormone mutant protein in a mass ratio of 5: 1;
lane 6: modifying the mass ratio of the Y-mPEG-MAL to the recombinant human growth hormone mutant protein of 8: 1;
lane 7: purifying the Y-mPEG-MAL modified recombinant human growth hormone mutant protein.
FIG. 5 shows the molecular sieve purity results of purified samples of Y-mPEG-MAL modified recombinant human growth hormone mutant protein.
FIG. 6 shows the result of MALDI-TOF MS detection of recombinant human growth hormone mutant protein (GHcys 101).
FIG. 7 shows the result of MALDI-TOF MS detection of a protein modified sample (Y-mPEG (40kDa) -GHcys101) of a recombinant human growth hormone mutant.
FIG. 8 shows the results of mPEG-MAL modification of recombinant human growth hormone mutant protein with different molecular weights and purification:
lane 1: a standard protein molecular weight;
lane 2: mPEG-MAL (5kDa) modified recombinant human growth hormone mutant protein;
lane 3: mPEG-MAL (10kDa) modified recombinant human growth hormone mutant protein;
lane 4: mPEG-MAL (20kDa) modified recombinant human growth hormone mutant protein;
lane 5: mPEG-MAL (40kDa) modified recombinant human growth hormone mutant protein;
lane 6: before the recombinant human growth hormone mutant protein is modified;
lane 7: purifying mPEG-MAL (5kDa) modified recombinant human growth hormone mutant protein;
lane 8: purifying mPEG-MAL (10kDa) modified recombinant human growth hormone mutant protein;
lane 9: purifying mPEG-MAL (20kDa) modified recombinant human growth hormone mutant protein;
lane 10: purifying mPEG-MAL (40kDa) modified recombinant human growth hormone mutant protein.
FIG. 9 shows the results of the PEG modified recombinant human growth hormone mutant protein with different activating groups:
lane 2: a standard protein molecular weight;
lane 3: mPEG-o-pyridyldisulfide modified recombinant human growth hormone mutant protein;
lane 4: mPEG-vinylsulfone modified recombinant human growth hormone mutant protein;
lane 5: mPEG-iodoacetamide modified recombinant human growth hormone mutant protein;
lane 6: mPEG-orthopyridyl disulfide modified recombinant human growth hormone mutant protein;
lane 7: before the recombinant human growth hormone mutant protein is modified.
FIG. 10 shows the comparison results of amino-modified recombinant human growth hormone mutant protein and sulfhydryl-modified human growth hormone mutant protein:
lane 1: a standard protein molecular weight;
lane 2: Y-mPEG-NHS modified recombinant human growth hormone mutant protein (amino modified);
lane 3: purifying the Y-mPEG-NHS modified recombinant human growth hormone mutant protein (amino modification);
lane 4: Y-mPEG-MAL modifies the mutant protein of the recombinant human growth hormone (sulfhydryl modification);
lane 5: purifying the Y-mPEG-MAL modified recombinant human growth hormone mutant protein (sulfhydryl modification);
lane 6: before the recombinant human growth hormone mutant protein is modified.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example one construction of recombinant human growth hormone mutant protein Gene and screening of Positive clones
According to a human growth hormone base sequence (SEQ ID NO.4) published by pubmed, rare codons in a template gene are synonymously replaced by preferred codons of E.coli through base optimization, and bases for expressing 101-bit Leu amino acid are replaced by bases for expressing Cys amino acid, so that a primer is designed by taking the bases as a template.
Upstream primer (L1): SEQ ID NO.5
5’-GACAGCCCAGATCTGGGTACCGACGACGACGACAAGTTCCC-3’；
Downstream primer (L2): SEQ ID NO.6
5’-GTGGTGGTGGTGGTGCTCGAGTTAGAAACCACAAGAACCTTCAACA-3’；
Restriction enzyme cutting sites Xba I and Xho I are respectively introduced into the upstream primer and the downstream primer. The primers are used for amplifying recombinant human growth hormone mutant protein genes, the amplified recombinant human growth hormone mutant protein gene fragments are subjected to Xba I and Xho I double enzyme digestion, are connected to an expression vector with a corresponding cut (the vector pET28a is subjected to Xba I and Xho I double enzyme digestion), are transformed into E.coli DH5 alpha (purchased from Beijing Solebao science and technology Limited), and are identified as recombinant expression plasmids pET28a-GH on the premise of ensuring correct reading frames, and the recombinant sequences are correct after DNA sequencing comparison. The recombinant plasmid was transformed into expression host e.coli BL21(DE3) (purchased from beijing solibao technologies ltd), kanamycin was used as resistance screening to obtain engineering bacteria, and the recombinant clones were verified to be correct by PCR.
EXAMPLE II expression and purification of recombinant human growth hormone mutant protein
The recombinant human growth hormone mutant protein engineering bacteria obtained in the first embodiment are subjected to fermentation culture, the culture temperature is 30 ℃, 1mmol/L IPTG (Beijing Ding Guosheng Biotech, Ltd.) is used as an inducer, induction is carried out for 3 hours at 30 ℃, thalli are collected by centrifugation, the protein is expressed in a soluble form, and the expression result is shown in figure 1.
Coarse purification: dissolving the thallus collected by fermentation with TE, breaking the thallus by a high-pressure homogenizer, and centrifuging at 4 ℃ to collect supernatant.
And (3) fine purification: the three-step purification process of Q anion exchange chromatography, hydrophobic chromatography and molecular sieve desalination is adopted in sequence. The method comprises the following specific steps:
the supernatant is separated and purified by ion exchange chromatography, and diluted with 20mmol/LPB (national drug group chemical reagent Co., Ltd.) buffer solution with pH of 8.0 to make its conductivity less than 5 mS/cm. The Capto Q (GE) column was equilibrated with the same buffer, and after loading, the column was washed with the equilibration buffer to a baseline, and eluted with NaCl (national pharmaceutical group chemical Co., Ltd.) solutions at concentrations of 0.02mol/L, 0.10mol/L, and 0.15mol/L, respectively, and the target protein was eluted at 0.10mol/L NaCl, and the target protein peak was collected.
Target protein supplementation (NH) 4 ) 2 SO 4 (national chemical group, Ltd.) was added to the mixture so that the final concentration was 1.0mol/L and 1.2mol/L (NH) was added to the sample 4 ) 2 SO 4 (national chemical group chemical Co., Ltd.), 20mmol/L PB (national chemical group chemical Co., Ltd.), Phenyl-Sepharose Fast Flow (GE Co.) column equilibrated with a buffer solution having a pH of 6.0, using (NH) at concentrations of 0.5mol/L, 0.1mol/L and 0.02mol/L, respectively 4 ) 2 SO 4 (national pharmaceutical group chemical Co., Ltd.) solution elution of the target protein at 0.1mol/L of (NH) 4 ) 2 SO 4 Eluting the protein, and collecting the target protein peak.
The obtained hydrophobic chromatographic peak was desalted by Sephadex G-25(GE Co.), concentrated by a membrane pack (MILLIPORE) having a molecular weight cut-off of 5kDa, and the equilibrium solution was 20mmol/L PB (national drug group chemical Co., Ltd.), and pH was 7.6. The sample is desalted and concentrated to obtain the recombinant human growth hormone mutant protein, the purity of the product is analyzed by SDS-PAGE, the result is shown in figure 2, the purity of the sample is more than 95% by RP-HPLC purity detection, and the result is shown in figure 3.
EXAMPLE III PEG conjugation of recombinant human growth hormone mutant proteins
The recombinant human growth hormone mutant protein of the present invention can be conjugated with PEG with various molecular weights, and in a preferred embodiment, PEG is branched mPEG-MAL (Xiamen Sainuo Pong Biotech, Inc.) with an average molecular weight of about 40 kDa.
1) Recombinant human growth hormone mutant protein concentrate
The recombinant human growth hormone mutant protein can be concentrated by using an ultrafiltration membrane package. The specific method comprises the following steps: the protein sample purified in example two was concentrated using a membrane package with a molecular weight cut-off of 5kDa (MILLIPORE) to about 10 mg/ml.
2) Coupling of recombinant human growth hormone mutant protein and mPEG-MAL
The recombinant human growth hormone mutant protein is coupled with mPEG-MAL, and the single PEG derivative is written as follows: Y-mPEG (40kDa) -GHcys 101. The coupling reaction steps are as follows: an appropriate volume of the concentrated solution obtained in step 1 of this example was taken, the protein concentration was adjusted to 5mg/ml, the pH was adjusted to 8.0, mPEG-MAL (Xiamen Nippon Biotech Co., Ltd.) and the purified protein were mixed in the mass ratios of 3:1, 5:1 and 8:1, respectively, and the mixture was dissolved with stirring and reacted at 4 ℃ for 24 hours, and the degree of coupling of the reaction was detected by SDS-PAGE (shown in FIG. 4).
EXAMPLE four purification of recombinant human growth hormone mutant protein derivatives
The specific conditions for purifying the desalted sample by ion exchange chromatography using Capto Q (GE) were as follows: 20mmol/L PB (national medicine group chemical reagent Co., Ltd.) has pH of 8.0, the buffer system is loaded, the flow rate of the buffer solution is 10ml/min, 20mmol/L PB (national medicine group chemical reagent Co., Ltd.), 120mmol/L NaCl (national medicine group chemical reagent Co., Ltd.), 20mmol/L PB (national medicine group chemical reagent Co., Ltd.) and 500mmol/L NaCl (national medicine group chemical reagent Co., Ltd.) are respectively used as eluent, the target protein is eluted at 120mmol/L NaCl, and the elution peak is the product of the polyethylene glycol mono-modified recombinant human growth hormone mutant. The purity of the single-point modified product of the finally obtained recombinant human growth hormone mutant protein is over 95 percent, the protein yield is calculated, and the single-point modified protein yield is more than 90 percent (an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map is shown in figure 4, and the purity of a molecular sieve is shown in figure 5).
Example five in vitro Activity assay of recombinant human growth hormone mutant proteins and derivatives thereof
The recombinant human growth hormone mutant protein and the derivative thereof are subjected to in vitro activity detection by referring to a growth hormone in United states pharmacopoeia in vitro activity detection method, and the specific method is as follows.
In vitro biological assays were performed using rat lymphoma Nb2-11 cells (CTCC). Nb2-11 cells were cultured in 1640 medium containing 10% fetal bovine serum, 10% horse serum, and 1 Xpenicillin streptomycin at 37 ℃ under 5% CO 2 And culturing in a saturated humidity incubator.
Taking the normal cultured cell strain in logarithmic phase, centrifuging at 218g for 7min, counting under a counting plate, and adjusting the cell concentration to 3 x 10 5 One/ml, spread on 1 96 black-bottom well plate, 50 μ L of cell suspension per well except in the first column, medium B (1640 medium with 1% HS) equivalent in the first column.
Preparing a recombinant human growth hormone standard substance (China institute for testing biological products of drugs), a recombinant human growth hormone mutant protein (GHcys101) and a recombinant human growth hormone mutant protein modified sample Y-mPEG (40kDa) -GHcys101 into a 2ng/ml solution by using a culture medium B. And (3) diluting each sample by 2-time gradient of 10 gradients, transferring 50 mu L of each sample to a corresponding position of a 96-well plate with a black bottom, slightly mixing the samples for 30s after the transfer is finished, and putting the 96-well plate in an incubator for incubation for 28 h.
After 28h incubation, the plate was removed and 100. mu.L of substrate luminescence (CellTiter-
Cell viability assay kit), shaking table incubation for 15min at room temperature (in the dark), standing incubation for 15min at room temperature (in the dark), and detecting luminescence on a fluorescence detector. And (3) carrying out statistical analysis on the detection data, and calculating the in-vitro activity of the recombinant human growth hormone mutant protein (GHcys101) and the recombinant human growth hormone mutant protein modified sample Y-mPEG (40kDa) -GHcys101 according to the activity of the growth hormone protein standard product. The specific results are shown in the following table:
TABLE 1 in vitro Activity of recombinant human growth hormone mutant proteins and derivatives thereof
| Sample name | Cytological specific Activity (IU/mg) |
| Recombinant human growth hormone |
3 |
| Recombinant human growth hormone mutant protein (GHcys101) | 3.15 |
| Y-mPEG(40kDa)-GHcys101 | 0.36 |
The result shows that the in vitro biological activity of the polypeptide is not influenced after the 101-position Leu is mutated into Cys, but the EC50 is increased by a plurality of times after the modification of the polyethylene glycol, but the proliferation effect of the polypeptide on cells is not changed, and the polypeptide still has higher activity.
EXAMPLE VI in vivo Activity assay of recombinant human growth hormone mutant proteins and derivatives thereof
The in vivo activity of the recombinant human growth hormone mutant protein and the derivative thereof is determined according to the growth hormone bioassay method of the Chinese pharmacopoeia 2020 edition 1219, and the titer and the test error are calculated according to the quantitative reaction parallel line assay method in the bioassay statistical method of the general rule 1431.
The hypophysectomized rat is used as an animal model, the national standard rhGH (China institute for drug and biological products) is used as a positive control, and the solvent is used as a negative control to carry out in-vivo protein activity detection. Qualified hypophysectomized rats were screened and randomly grouped according to body weight. Each group had 10 rats; positive control group rats with low dose (105. mu.g/kg/d) and high dose (420. mu.g/kg/d); the GHcys101 group rats have low dose (105 mu g/kg/d), medium dose (420 mu g/kg/d) and high dose (1.68 mg/kg/d); Y-mPEG (40kDa) -GHcys101 group rats low dose (157.5mg/kg/d), medium dose (630. mu.g/kg/d), high dose (2.52 mg/kg/d). The administration route of each group of animals is neck subcutaneous injection, and the administration volume is 0.5 ml. Wherein the negative control group is administered (vehicle) 1 time per day for 6 days; the positive control group and the GHcys101 group were administered 1 time a day for 6 days; the Y-mPEG (40kDa) -GHcys101 group rats were dosed once on the same day as the positive control group. Rats were sacrificed 24h after the last administration of the positive control group, the body weight and tibial epiphyseal plate width of the rats were measured, and the activity of the samples was calculated according to the pharmacopoeia method. The specific results are shown in the following table:
TABLE 2 in vivo Activity of recombinant human growth hormone mutant proteins and derivatives thereof
| Sample name | In vivo Activity (IU/mg) |
| Recombinant human growth hormone |
3 |
| Recombinant human growth hormone mutant protein (GHcys101) | 3.23 |
| Y-mPEG(40kDa)-GHcys101 | 5.87 |
The result shows that the biological activity of the growth hormone after 101-site Leu is mutated into Cys is equivalent to that before mutation, and the biological activity of the growth hormone after polyethylene glycol modification is about 2 times that of the common growth hormone. The Y-mPEG (40kDa) -GHcys101 has higher biological activity for promoting the growth of animal bodies and long-acting pharmacological action when being administrated once than growth hormone which needs to be administrated by injection every day.
EXAMPLE VII in vivo half-Life detection of recombinant human growth hormone mutant proteins and derivatives thereof
Adult male rats are randomly grouped into a positive group (a national standard rhGH (China pharmaceutical and biological product institute)), a recombinant human growth hormone mutant protein (GHcys101) group and a recombinant human growth hormone mutant protein modified sample Y-mPEG (40kDa) -GHcys101 group, wherein 6 of the groups are used, each group of the proteins are injected subcutaneously according to the dose of 1mg/kg, blood is taken at different time points, the content of hGH in serum is determined, the half-life period of each sample in vivo is calculated, and the result is shown in the following table:
TABLE 3 in vivo half-life of recombinant human growth hormone mutant proteins and derivatives thereof
| Sample name | Half-life in vivo t1/2(h) |
| Recombinant human growth hormone standard substance | 0.73±0.18 |
| Recombinant human growth hormone mutant protein (GHcys101) | 0.78±0.11 |
| Y-mPEG(40kDa)-GHcys101 | 18.37±0.56 |
The result shows that the half-life of the recombinant human growth hormone mutant protein (GHcys101) is equivalent to that of the national standard rhGH, and the half-life is prolonged by more than 20 times after the PEG site-directed modification, which indicates that the modified sample Y-mPEG (40kDa) -GHcys101 of the recombinant human growth hormone mutant protein has long-acting pharmacological action.
EXAMPLE VIII, Structure detection of recombinant human growth hormone mutant protein and its derivatives
1) Molecular weight detection
MALDI-TOF MS is adopted to detect the molecular weight of the recombinant human growth hormone mutant protein and the derivatives thereof, and the detection result is shown in figures 6-7. The results show that the molecular weight of the recombinant human growth hormone mutant protein (GHcys101) is about 22kDa, which is consistent with the theoretical molecular weight. The molecular weight of the purified recombinant human growth hormone mutant protein modification sample Y-mPEG (40kDa) -GHcys101 is about 61.6kDa, and the molecular weight is within 66.2 +/-10% of the theoretical molecular weight, which indicates that the recombinant human growth hormone mutant protein modification sample Y-mPEG (40kDa) -GHcys101 is a single-point modification product.
2) Modification site detection
Taking recombinant human growth hormone mutant protein (GHcys101) and a recombinant human growth hormone mutant protein modified sample Y-mPEG (40kDa) -GHcys101 (both solutions are not less than 1mg/ml), and fully dialyzing with 1% ammonium bicarbonate (national drug group chemical reagent, Inc.) solution respectively. Adding a trypsin (sigma company) solution (taking a trypsin mother solution, diluting the trypsin mother solution to be 0.1mg/ml by using a 1% ammonium bicarbonate (national medicine group chemical reagent, Inc.) solution) according to a ratio of 1:50(mg/mg) of trypsin to protein, carrying out enzyme digestion in a constant-temperature water bath at 37 ℃ for 24 hours, adding a 50% acetic acid (Shanghai Lingfeng chemical reagent, Inc.) solution according to a ratio of 1:10 to terminate the reaction, centrifuging the solution for 5min at a speed of 10000r/min per minute, and taking a supernatant liquid for separation for later use.
Detecting a peptide map of a sample subjected to enzyme digestion by using a liquid phase, wherein the specific parameters are as follows: chromatography column (advanced bio Peptide Map 2.1 × 150mm 2.7um, Agilent), detection wavelength 214 nm; flow rate: 0.5 ml/min; column temperature: 30 ℃; sample introduction amount: 100ul, mobile phase A (0.1% trifluoroacetic acid in water (sigma)), mobile phase B (0.1% trifluoroacetic acid in acetonitrile (sigma)), gradient elution was performed according to the following table:
TABLE 4 Peptiogram elution gradient
And (4) comparing the peptide diagram results before and after modification, finding out the PEG modified peptide fragment peak, loading for multiple times, and collecting the PEG modified peptide fragment peak.
And (3) carrying out N-terminal sequencing on the collected PEG modified peptide fragment peak, comparing the sequence with a peptide map enzyme digestion theoretical sequence, finding out a modified peptide fragment, and determining a PEG modified site.
The result shows that only one polyethylene glycol single-point modified product exists in the modified product, and the modified site is the mutated 101-position Cys, which shows that the modified product after mutation has good uniformity and no other site modified product.
EXAMPLE nine recombinant human growth hormone mutant proteins modified by polyethylene glycols of different molecular weights
1) Preparation of modified products
The recombinant human growth hormone mutant protein was concentrated using a membrane module with a molecular weight cut-off of 5kDa (MILLIPORE) and the sample was concentrated to about 10 mg/ml.
The concentrated recombinant human growth hormone mutant protein is subjected to coupling reaction with mPEG-MAL (5kDa, 10kDa, 20kDa and 40kDa) (Xiamen Sainuo Pong Biotech Co., Ltd.) with different molecular weights, and the coupling reaction steps are as follows: taking a proper volume of concentrated solution, adjusting the protein concentration to 5mg/ml, adjusting the pH to 8.0, mixing mPEG-MAL (Xiamen Sonopong Biotech Co., Ltd.) with different molecular weights and purified protein according to the mass ratio of 5:1, stirring and dissolving, reacting for 24h at 4 ℃, and detecting the modification efficiency by SDS-PAGE (the result is shown in figure 8).
The specific conditions for purifying the desalted sample by ion exchange chromatography using Capto Q (GE corporation) were as follows: 20mmol/L PB (national medicine group chemical reagent Co., Ltd.) has pH of 8.0, the buffer system is loaded, the flow rate of the buffer solution is 10ml/min, 20mmol/L PB (national medicine group chemical reagent Co., Ltd.), 120mmol/L NaCl (national medicine group chemical reagent Co., Ltd.), 20mmol/L PB (national medicine group chemical reagent Co., Ltd.) and 500mmol/L NaCl (national medicine group chemical reagent Co., Ltd.) are respectively used as eluent, the target protein is eluted at 120mmol/L NaCl, and the elution peak is the product of the polyethylene glycol mono-modified recombinant human growth hormone mutant. The purity of the finally obtained recombinant human growth hormone mutant protein single-point modification products modified by different molecular weights is over 95 percent (the SDS-PAGE detection result is shown in figure 8).
2) Detecting the in vivo half-life period of the PEG modified recombinant human growth hormone mutant protein with different molecular weights: according to the method of the seventh embodiment, the in vivo half-life period of the PEG modified recombinant human growth hormone mutant protein modified by PEG with different molecular weights is detected, and the detection results are shown in the following table.
TABLE 5 in vivo half-life of PEG modified recombinant human growth hormone mutant protein with different molecular weights
| Sample name | Half-life in vivo t1/2(h) |
| Recombinant human growth hormone standard substance | 0.71±0.13 |
| mPEG(5kDa)-GHcys101 | 4.37±0.51 |
| mPEG(10kDa)-GHcys101 | 10.46±0.98 |
| mPEG(20kDa)-GHcys101 | 12.57±0.33 |
| mPEG(40kDa)-GHcys101 | 16.88±0.72 |
| Y-mPEG(40kDa)-GHcys101 | 18.37±0.56 |
The result shows that the in vivo half-life of the modified sample of the recombinant human growth hormone mutant protein is longer along with the increase of the molecular weight of the modified polyethylene glycol, and the in vivo half-life of the modified product of the Y-type polyethylene glycol is longer than that of the linear polyethylene glycol, so that the modified sample of the recombinant human growth hormone mutant protein Y-mPEG (40kDa) -GHcys101 has long-acting pharmacological action.
EXAMPLE ten modification of recombinant human growth hormone mutant proteins with different active groups by polyethylene glycol
1) mPEG-o-pyridyl disulfide modified recombinant human growth hormone mutant protein
The concentration of the recombinant human growth hormone mutant protein is 5mg/ml, the pH is adjusted to 8.0, the mPEG-orthopyridyl disulfide (Xiamen Nippon Biotech Co., Ltd.) with the molecular weight of 40kDa and the purified protein are mixed according to the mass ratio of 5:1, stirred and dissolved, and the reaction is carried out for 24 hours at 4 ℃. The sample is taken for SDS-PAGE electrophoretic analysis, the result is shown in figure 9, and the sample modification has good uniformity.
2) mPEG-vinylsulfone modified recombinant human growth hormone mutant protein
The concentration of the recombinant human growth hormone mutant protein is 5mg/ml, the pH value is adjusted to 8.0, the mPEG-vinylsulfone with the molecular weight of 40kDa (Xiamen Sainuo Banggu Biotech Co., Ltd.) and the purified protein are mixed according to the mass ratio of 5:1, stirred and dissolved, and reacted for 24 hours at 4 ℃. The sample is taken for SDS-PAGE electrophoretic analysis, the result is shown in figure 9, and the sample modification has good uniformity.
3) mPEG-iodoacetamide modified recombinant human growth hormone mutant protein
The concentration of the recombinant human growth hormone mutant protein is 5mg/ml, the pH is adjusted to 8.0, the mPEG-iodoacetamide with the molecular weight of 40kDa (Xiamen Sainuo banger Biotech Co., Ltd.) and the purified protein are mixed according to the mass ratio of 5:1, stirred and dissolved, and the reaction is carried out for 24 hours at 4 ℃. The sample was analyzed by SDS-PAGE, and the results are shown in FIG. 9, and the sample modification was uniform.
4) mPEG-orthopyridyl disulfide modified recombinant human growth hormone mutant protein
The concentration of the recombinant human growth hormone mutant protein is 5mg/ml, the pH is adjusted to 8.0, the mPEG-orthopyridyl disulfide (Xiamen Nippon Biotech Co., Ltd.) with the molecular weight of 40kDa and the purified protein are mixed according to the mass ratio of 5:1, stirred and dissolved, and the reaction is carried out for 24 hours at 4 ℃. The sample is taken for SDS-PAGE electrophoretic analysis, the result is shown in figure 9, and the sample modification has good uniformity.
Comparative example I amino modified recombinant human growth hormone mutant protein
1) Preparation of modified products
The recombinant human growth hormone mutant protein was concentrated using a membrane module with a molecular weight cut-off of 5kDa (MILLIPORE) and the sample was concentrated to about 10 mg/ml.
The concentrated recombinant human growth hormone mutant protein is subjected to coupling reaction with Y-mPEG-NHS with the molecular weight of 40kDa, and the coupling reaction steps are as follows: taking a proper volume of concentrated solution, adjusting the protein concentration to 5mg/ml, adjusting the pH to 8.0, mixing 40kDa Y-mPEG-NHS (Xiamen Nuo bang Biotech Co., Ltd.) and purified protein according to the mass ratio of 5:1, stirring for dissolving, and reacting for 24h at 4 ℃ (the SDS-PAGE detection result is shown in figure 10).
The specific conditions for purifying the desalted sample by ion exchange chromatography using Capto Q (GE corporation) were as follows: 20mmol/L PB (national chemical reagent, Inc.) has pH of 8.0, a buffer system is loaded, the flow rate of the buffer solution is 10ml/min, 20mmol/L PB (national chemical reagent, Inc.), 20mmol/L NaCl (national chemical reagent, Inc.), 20mmol/L PB (national chemical reagent, Inc.), 120mmol/L NaCl (national chemical reagent, Inc.) solution, 20mmol/L PB (national chemical reagent, Inc.), 500mmol/L NaCl (national chemical reagent, Inc.) solution are respectively used as eluent, the target protein is eluted at 120mmol/L NaCl, and the elution peak is the product of the polyethylene glycol single-modified recombinant human growth hormone mutant. Finally, the purity of the single-point amino modified product of the recombinant human growth hormone mutant protein is more than 95%, the protein yield is calculated, and the single-point modified protein yield is about 50% (the SDS-PAGE detection result is shown in figure 10).
2) Modification site detection
Taking the recombinant human growth hormone mutant protein (GHcys101) and the amino modified sample (both solutions are not less than 1mg/ml), and respectively dialyzing with 1% ammonium bicarbonate (national group chemical reagent, Inc.) solution. Adding a trypsin (sigma company) solution (taking a trypsin mother solution, diluting the trypsin mother solution to be 0.1mg/ml by using a 1% ammonium bicarbonate (national medicine group chemical reagent limited)) solution according to a trypsin/protein ratio of 1:50(mg/mg), carrying out enzyme digestion in a constant-temperature water bath at 37 ℃ for 24 hours, adding a 50% acetic acid (Shanghai Lingfeng chemical reagent limited) solution according to a ratio of 1:10 to terminate the reaction, centrifuging the solution for 5min at 10000r/min per minute, and taking a supernatant liquid phase for separation for later use.
Detecting a peptide map of the sample subjected to enzyme digestion by using a liquid phase, wherein the specific parameters are as follows: chromatography column (advanced bio Peptide Map 2.1 x 150mm 2.7um, Agilent), detection wavelength 214 nm; flow rate: 0.5 ml/min; column temperature: 30 ℃; sample introduction amount: 100ul, mobile phase A (0.1% trifluoroacetic acid in water (sigma)), mobile phase B (0.1% trifluoroacetic acid in acetonitrile (sigma)), gradient elution was performed according to the following table:
TABLE 6 Peptiogram elution gradient
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 20 | 80 | 20 |
| 45 | 75 | 25 |
| 70 | 50 | 50 |
| 75 | 20 | 80 |
| 76 | 100 | 0 |
| 91 | 100 | 0 |
And (4) comparing the peptide diagram results before and after modification, finding out the PEG modified peptide fragment peak, loading for multiple times, and collecting the PEG modified peptide fragment peak.
And (3) carrying out N-terminal sequencing on the collected PEG modified peptide fragment peak, comparing with a peptide diagram enzyme digestion theoretical sequence, finding out a modified peptide fragment, and determining a PEG modified site.
The result shows that only 5 possible modified sites in the modified product are respectively N terminal, Lys38, Lys140, Lys145 and Lys 158; compared with a mutant recombinant human growth hormone protein modified sample, the modified product of the mutant sample is more uniform, the modification efficiency is higher in the modification process, and the yield of the modified product is higher, so that the modification of the recombinant human growth hormone mutant protein has higher economic value and product quality.
The above description is only an example of the present invention and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the specification or other related fields can be directly or indirectly applied to the present invention, and the same shall be included in the scope of the present invention.
Sequence listing
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Claims (16)
1. The recombinant human growth hormone mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO.1, and the recombinant human growth hormone mutant is obtained by mutating leucine Leu at the 101 th site of the amino acid sequence of the human growth hormone shown as SEQ ID NO.3 into cysteine Cys.
2. A nucleic acid encoding the recombinant human growth hormone mutant of claim 1, having the nucleotide sequence set forth in SEQ ID No. 2.
3. The polyethylene glycol derivative of the recombinant human growth hormone mutant as claimed in claim 1, wherein the polyethylene glycol derivative is formed by connecting a polyethylene glycol modifier to the Cys site of the 101 th cysteine of the recombinant human growth hormone mutant.
4. The polyethylene glycol derivative of the recombinant human growth hormone mutant according to claim 3, wherein: the polyethylene glycol modifier is mPEG-maleimide, mPEG-o-pyridyl disulfide, mPEG-vinyl sulfone, mPEG-iodoacetamide or mPEG-n-pyridyl disulfide.
5. The polyethylene glycol derivative of the recombinant human growth hormone mutant according to claim 3, wherein: the average molecular weight of the polyethylene glycol modifier is 4 kDa-40 kDa.
6. The polyethylene glycol derivative of the recombinant human growth hormone mutant according to claim 3, wherein: the polyethylene glycol modifier is straight chain or branched polyethylene glycol containing single active functional groups.
7. The polyethylene glycol derivative of the recombinant human growth hormone mutant according to claim 3, wherein: the polyethylene glycol modifier molecule is preferably mPEG-maleimide; the preferred average molecular weight of the polyethylene glycol modifier is 40 kDa; the polyethylene glycol modifier is preferably branched polyethylene glycol containing a single active functional group.
8. A method for preparing a polyethylene glycol derivative of a recombinant human growth hormone mutant as claimed in any one of claims 3 to 7, comprising the steps of:
1) reacting a polyethylene glycol modifier molecule with free cysteine Cys in the recombinant human growth hormone mutant in a neutral or alkaline aqueous medium;
2) separating the product of the recombinant human growth hormone mutant containing the polyethylene glycol modifier from the reaction mixture.
9. The method of claim 8, wherein: the polyethylene glycol modifier is any one of mPEG-maleimide, mPEG-ortho-pyridyl disulfide, mPEG-vinyl sulfone, mPEG-iodoacetamide and mPEG-ortho-pyridyl disulfide.
10. The method of claim 8, wherein: the molecular weight of the polyethylene glycol modifier is between 4kDa and 40 kDa.
11. The method of claim 8, wherein: the polyethylene glycol modifier is straight-chain or branched polyethylene glycol containing a single active functional group.
12. The method of claim 8, wherein: the mass ratio of polyethylene glycol modifier molecules to recombinant human growth hormone mutant protein in the modification reaction is 3: 1-8: 1.
13. The method of claim 8, wherein: the pH value of the modification reaction system is controlled to be 7-9.5.
14. The method of claim 8, wherein: said separating step comprises ion exchange column Capto Q chromatography.
15. The method according to any one of claims 9 to 14, wherein: the polyethylene glycol modifier molecule is preferably mPEG-maleimide; the preferred average molecular weight of the polyethylene glycol modifier is 40 kDa; the polyethylene glycol modifier is preferably branched polyethylene glycol containing a single active functional group; the pH value of the modification reaction system is preferably 8.0-9.2.
16. The use of the polyethylene glycol derivative of the recombinant human growth hormone mutant according to any one of claims 3 to 7 in the preparation of a medicament for treating slow growth of children caused by pathological factors such as endogenous growth hormone deficiency.
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