CN115960246A - Recombinant cyclic human growth hormone-Fc fusion protein and application thereof - Google Patents

Abstract

The invention discloses a recombinant cyclic human growth hormone-Fc fusion protein and application thereof. The fusion protein uses two connecting peptides to sequentially connect the Fc fragment, human growth hormone and the Fc fragment from N end to C end to form a molecule, and the two Fc fragments form 1 or more pairs of disulfide bonds or no disulfide bond after folding to form the cyclic Fc-hGH-Fc. The cyclic Fc-hGH-Fc fusion protein has higher renaturation ratio than the expression renaturation of the monomer Fc/Fc-hGH, and forms less Fc/Fc homodimer and dimer hGH-Fc/hGH-Fc, thereby providing convenience for later purification. The fusion protein fermentation preparation process is simple, convenient to purify, high in yield and low in cost. The fusion protein of the invention weakens the protein elimination effect caused by a growth hormone receptor by using the steric hindrance effect of Fc, further prolongs the half life period of the medicine and increases the medicine effect.

Description

Recombinant cyclic human growth hormone-Fc fusion protein and application thereof

Technical Field

The invention relates to the field of genetic engineering medicines, in particular to a recombinant cyclic human growth hormone-Fc fusion protein and application thereof.

Background

Human growth hormone (hGH) is a polypeptide hormone (SEQ ID NO: 1) that is synthesized, stored and secreted by growth hormone cells in the pituitary. The human growth hormone molecule is composed of 191 amino acid residues, and the relative molecular mass is 22124 daltons. Under physiological conditions, growth hormone binds to a growth hormone receptor (hGHR) on the surface of a target cell to activate corresponding downstream channels. Growth hormone promotes the development and cell proliferation of animals and humans, and has the main physiological effects of promoting the synthesis of various tissues of human body, especially protein, and stimulating the growth of bone joint cartilage and bone epiphyseal cartilage. Growth hormone can promote the increase of human body, and once the human body lacks growth hormone, the growth of human body is stopped.

Clinical studies have shown that wild-type growth hormone has a very short half-life in humans (half-life of about 2 hours), so that conventional therapeutic administration of recombinant human growth hormone requires one injection per day of subcutaneous injection, and the administration period is long (up to 6 months or more). Frequent injections of growth hormone can cause inconvenience to the patient, and mental stress caused by the injections also reduces the quality of life of the patient. In addition, compliance with the frequency of administration by patients becomes an important factor hindering the therapeutic effect, and the cost of administration is also expensive. In order to solve this problem, it is necessary to develop recombinant human growth hormone with long-lasting effect. At present, recombinant human growth hormone at home and abroad has been marketed in a plurality of products, for example, currently marketed recombinant growth hormone drugs include Pfizer's Genotropin, eli Lilly's Humatrope, genentech's nuttropin, novonortropin of NovoNordisk, jianhaoning, saizeng, and busu lovely, however, these drugs are all short-acting products, and patients need to inject daily, so the market is in urgent need of a long-acting and safe novel growth hormone drug.

Fc fusion proteins are a safe and effective technique for extending the plasma half-life of protein drugs, and up to 9 Fc fusion proteins are currently approved by the FDA. Since the Fc protein is derived from a conserved region of human immunoglobulin, it has advantages of high safety and low immunogenicity. Most fusion protein drugs exist in a homodimer-based form, while the hGH-Fc/hGH-Fc dimer form of the fusion protein shortens the half-life of the drug and increases the frequency of administration by increasing growth hormone receptor-mediated protein clearance compared to the monomeric form (i.e., fc/hGH-Fc). Monomeric Fc fusion proteins (Fc/hGH-Fc) containing a protein drug linked to only one Fc domain have improved half-life or biological activity compared to homodimers. In 2017, shanghai Changdian Biotechnology Limited disclosed an Fc fusion protein (Fc/hGH-Fc) fused to only one hGH in the patent application, which showed superior efficacy to the hGH-Fc/hGH-Fc dimer in the animal experiments. However, since Fc/hGH-Fc is a heterodimer expressed by mammalian cells, impurity molecules that inevitably produce homodimers during the production process, i.e., fc fusion protein Fc/Fc without growth hormone and hGH/hGH-Fc fusion protein containing two growth hormone molecules, are formed in the middle and late stages of the cell sap because of their close properties, which leads to hGH-Fc/hGH-Fc and Fc/Fc that are difficult to remove by purification, and in a large proportion, so that Fc/hGH-Fc has problems of process instability and difficulty in scale-up of production during the production process, resulting in reduced pharmaceutical potency. Meanwhile, although the drug efficacy and pharmacokinetics of Fc/hGH-Fc are greatly improved compared with hGH-Fc/hGH-Fc homodimer proteins, the half-life is still not long enough due to limitations on growth hormone receptor-mediated protein degradation and the like.

In order to develop more long-acting hGH, various research institutes pharmaceutical factories have also attempted to develop modified or engineered hGH of different structures. For example, PEG hGH fusion proteins CN 108794634A and CN 108136043B have aldehyde groups at both ends of PEG, and are coupled twice to form Fc/hGH-PEG-Fc in a monomer form or cell biplasmid expresses Fc and hGH-Fc to form Fc/hGH-Fc in a monomer form; or CN 108136043A forms hGH-fat acid modified protein by late chemical coupling. These modified or engineered hghs have properties that are still unsatisfactory and suffer from some significant disadvantages: for example, taking PEG (polyethylene glycol) modified growth hormone as an example, although the half-life of the growth hormone can be obviously improved and the growth hormone can be prolonged after being modified by polyethylene glycol, the growth hormone often causes activity loss in the PEG production process, so that the specific activity of the growth hormone is reduced. Or the later chemical modification method is complex in process production and has high requirements on factory implementation. Meanwhile, there are reports in the literature (J Clin Endocrinol Metab, june 2020, 105 (6): 1-13), and preclinical studies in primates have shown that autopsy revealed that PEG accumulates in the ependymal cells of the choroid plexus, thereby causing toxicity, which is also an important reason for limiting the widespread use of PEGylated modified hGH. In addition, PEG has been reported to cause problems such as generation of anti-drug antibodies and fat collapse at injection sites in long-term use, which limits the wide application of PEG-modified hGH.

In view of the above, in order to facilitate long-term clinical use and improve therapeutic effects, there is a need to develop a novel long-acting growth hormone product that is safe, effective, simple in process, low in cost, and high in yield.

Disclosure of Invention

In order to solve the defects of the prior art, the invention mainly aims to provide a recombinant cyclic human growth hormone-Fc fusion protein. The cyclic molecule fusion protein related by the invention has higher renaturation ratio than monomer expression renaturation yield, does not form Fc/Fc homodimer and dimeric hGH-Fc/hGH-Fc, and provides convenience for later purification. In addition, the cyclic molecule of the present invention comprises only one polypeptide chain, which is advantageous for increasing production efficiency and reducing production costs, compared to the conventional Fc/hGH-Fc heterodimer form. The fusion protein of the invention has simple fermentation preparation process, convenient purification, high yield and low cost. The fusion protein of the invention reduces the protein clearance effect caused by growth hormone receptor by using the steric hindrance effect of Fc, thereby further prolonging the half-life period of the drug. Further animal experiments show that the cyclic growth hormone Fc-fusion protein of the present invention exhibits a longer half-life and superior efficacy in rat experiments than the conventional Fc/hGH-Fc heterodimer form.

Another object of the present invention is to provide a gene encoding the above recombinant cyclic human growth hormone-Fc fusion protein.

Still another object of the present invention is to provide the use of the above recombinant cyclic human growth hormone-Fc fusion protein.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a recombinant cyclic human growth hormone-Fc fusion protein, wherein the molecular linkage is Fc-hGH-Fc, the fusion protein is composed of human growth hormone, two linker peptides and two Fc fragments which may be the same or different, wherein the first Fc monomer fragment, human growth hormone and the second Fc monomer fragment are sequentially linked from N-terminus to C-terminus to form a molecule, wherein one linker peptide is present between the first Fc fragment and human growth hormone, the other linker peptide is present between human growth hormone and the other Fc fragment, the amino acid sequences of the two linker peptides are the same or different, and 1 or more pairs of disulfide bonds or no disulfide bonds are formed between the two Fc fragments after folding is completed, because the two Fc fragments may spontaneously form a dimer, and the whole molecule is then linked end to form the cyclic human growth hormone-Fc fusion protein (as shown in fig. 1).

Wherein the amino acid sequence of the growth hormone is shown as SEQ ID NO. 1.

In some embodiments, the Fc fragment is selected from the Fc fragment of human IgG, preferably human IgG4 or IgG 1; further preferably an Fc fragment of human IgG4.

In some embodiments, the sum of the number of amino acids of the two linking peptides is 5 or more, and the linking peptide is preferably a sequence with low immunogenicity and flexible structure, and is further preferably a GS repeat, a GGGGS repeat or a random sequence containing only Q, S, E, P, T, G and A7 amino acids.

In another preferred embodiment, the amino acid sequence of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc is shown in SEQ ID NO 2.

In a second aspect of the present invention, there is provided a DNA molecule encoding the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to the first aspect of the present invention.

In a third aspect of the invention there is provided an expression vector, preferably a eukaryotic or prokaryotic expression vector, comprising a DNA molecule according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a host cell comprising an expression vector according to the third aspect of the invention, said cell being selected from a eukaryotic cell or a prokaryotic cell; preferably E.coli.

In a fifth aspect of the present invention, there is provided a method for producing a recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to the first aspect of the present invention, comprising the steps of:

(a) Culturing a host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby obtaining a culture solution containing an Fc-hGH-Fc fusion protein expressing cyclic human growth hormone-Fc;

(b) And (3) separating the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc from the culture solution.

In another preferred embodiment, in step (b), the separating comprises: the thalli is crushed under high pressure, washed by PBS buffer solution twice, and the inclusion body is recovered; dissolving the inclusion body; renaturation process of fusion protein Fc-hGH-Fc; and (5) purifying the fusion protein Fc-hGH-Fc.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

In a seventh aspect of the present invention, there is provided a use of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to the first aspect of the present invention or the pharmaceutical composition according to the sixth aspect of the present invention for the manufacture of a medicament for the treatment of a GH deficiency related disease.

The beneficial effect that above technical scheme produced lies in:

(1) The fusion protein fermentation preparation process is simple, convenient to purify and high in yield, 7g of target protein can be obtained in one liter of fermentation strain, and the cost is low.

(2) The renaturation rate of the fusion protein is higher than that of monomer expression renaturation, the renaturation rate of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc is 65%, and the renaturation rate of monomer Fc/hGH-Fc is 25%; and the formation of Fc/Fc homodimers and hGH-Fc/hGH-Fc homodimers is avoided, so that convenience is brought to later purification.

(3) The fusion protein of the invention reduces the protein clearance effect caused by growth hormone receptor by using the steric hindrance effect of Fc, thereby further prolonging the half-life period of the drug.

(4) Compared with the PEG long-acting GH preparation on the current market, the invention has no risk of hepatotoxicity and nephrotoxicity caused by introduction of PEG. The risk of immunogenicity due to glycosylation is also reduced compared to that expressed in eukaryotic cells.

(5) The molecules of the invention have better efficacy in animal experiments than Fc/hGH-Fc.

Drawings

FIG. 1 is a schematic representation of the structure of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc;

FIG. 2 shows SDS-PAGE plots for expression of Fc and hGH-Fc fusion proteins and for expression of cyclic Fc-hGH-Fc fusion proteins during preparation of monomeric Fc/hGH-Fc fusion proteins;

a is an SDS-PAGE gel picture of 10% of the Fc-only fermentation during the preparation of the monomeric Fc/Fc-hGH fusion protein;

b is a 10% SDS-PAGE gel of the fermentation of hGH-Fc during the preparation of the monomeric Fc/Fc-hGH fusion protein;

c is a 10% SDS-PAGE gel picture of fermentation bacteria collection of the cyclic Fc-hGH-Fc fusion protein; d is a protein molecular weight marker;

FIG. 3 is a diagram showing the quantitative determination of cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc after the disruption;

FIG. 4 is a photograph of an inclusion body denatured and renatured SDS-PAGE gel of a cyclic Fc-hGH-Fc, monomeric Fc/hGH-Fc fusion protein;

a is a 4% -12% SDS-PAGE gradient gel plot after renaturation of the cyclic Fc-hGH-Fc fusion protein, the first lane is a reduction state plot after protein renaturation, the second lane is a protein marker, and the third lane is a non-reduction state plot after renaturation; b is an SDS-PAGE gel picture after the renaturation of the monomer Fc/hGH-Fc fusion protein, a first lane is a non-reduction state picture after the renaturation of the protein, a second lane is a protein marker, and a third lane is a reduction state picture after the renaturation of the protein; c is a protein molecular weight Marker;

FIG. 5 is a graph showing the detection of various indices of the purified cyclic Fc-hGH-Fc fusion protein;

a is SEC-HPLC detection result after purification of the cyclic Fc-hGH-Fc fusion protein, and the purity is 97.28%;

b is a UPLC detection result after the purification of the cyclic Fc-hGH-Fc fusion protein, and the purity is 95%;

c is an LC-Mass detection result after the purification of the cyclic Fc-hGH-Fc fusion protein, the LC-Mass detection result is 77654.5Da, and the theoretical molecular weight is 77653.8658Da;

d is the 4% -12% Nu-PAGE gel graph detection result after the purification of the cyclic Fc-hGH-Fc fusion protein, and the sample loading amount is 5 mug/hole;

FIG. 6 is a graph showing the detection of various indices of the purified monomeric Fc/hGH-Fc fusion protein;

a is a SEC-HPLC detection result after the purification of the monomer Fc/hGH-Fc fusion protein, and the purity is 97.89%;

b is a UPLC detection result after the monomer Fc/hGH-Fc fusion protein is purified, and the purity is 94.5%;

c is the LC-Mass detection result of the purified monomer Fc/hGH-Fc fusion protein of 73252.00Da, and the theoretical molecular weight is 73251.67Da;

d is the SDS-PAGE gel pattern detection result of 8% -15% after the purification of the monomer Fc/hGH-Fc fusion protein, the sample loading is 5 mug/hole;

FIG. 7 somatotrophic potency assay of monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc on a pituitary-extirpated rat model- -comparison between AUC groups for weight gain in rats in each group;

a is the comparison between AUC groups of the weight increase of rats, B is a histogram of the weight increase in A;

FIG. 8 test of the growth promoting efficacy of monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc on pituitary-extirpated rat models- -comparison between the body length growth AUC groups of rats in each group;

a is the comparison between the AUC groups of the body length increment of the rats, and B is a histogram of the body length increment in the A;

FIG. 9 test of the growth promoting efficacy of monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc on pituitary-extirpated rat models- -comparison between groups of AUC groups of tail length growth of rats;

a is the comparison between the AUC groups of tail length increment of rats, and B is a histogram of tail length increment in A;

FIG. 10 growth-promoting efficacy of monomeric hGH-Fc/Fc and cyclic Fc-hGH-Fc on pituitary-extirpated rat models- -comparison between groups of serum IGF-1 content in rats of each high-dose group tested;

a is the comparison between the rat serum IGF-1 content groups, B is the bar chart of the increase of the tail length in A;

Detailed Description

Definition of terms

As used herein, the terms "fusion protein of the present invention", "cyclic Fc-hGH-Fc fusion protein", "recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc", "cyclic Fc-hGH-Fc", "Fc-hGH-Fc", and "Fc-hGH-Fc" are used interchangeably and refer to a fusion protein as described in the first aspect of the present invention, which consists of human growth hormone, two linking peptides and two identical or different Fc fragments, i.e., two linking peptides are used to link the Fc fragment, human growth hormone and Fc fragment in sequence from N-terminus to C-terminus to form one molecule, wherein one linking peptide is present between the Fc fragment and human growth hormone, the other linking peptide is present between human growth hormone and the other Fc fragment, the amino acid sequences of the two linking peptides are identical or different, and 1 or two pairs of disulfide bonds are formed after the two Fc fragments are folded.

As used herein, the terms "Fc/hGH-Fc", "Fc/Fc-hGH", "hGH-Fc/Fc", "monomeric Fc/hGH-Fc", "monomeric hGH-Fc/Fc", "Fc/hGH-Fc heterodimer protein", "hGH-Fc/Fc heterodimer protein" are used interchangeably (SEQ ID NO:3 and SEQ ID NO: 4). The protein refers to a heterodimeric protein Fc/hGH-Fc formed by a monomer Fc and an hGH-Fc fusion protein, wherein 1 pair or more disulfide bonds exist between two Fc molecules, and the two monomer Fc molecules in the heterodimeric protein can be identical or not.

In the present invention, the immunoglobulin to be used is not particularly limited, and may be an immunoglobulin element derived from a human or other mammal, or a mutant or derivative thereof. Preferably an immunoglobulin of human origin, such as IgG, igE, igA, igM, or combinations thereof.

Human immunoglobulin G includes four subclasses: igG1, igG2, igG3, igG4. The protein structures of these four subclasses have great similarity, all having four regions: one variable domain (VH), three constant domains (CH 1, CH2, CH 3). The Fc fragment consists of two constant regions (CH 2-CH 3) with one or more pairs of disulfide bonds in the CH2 region, such that the two Fc fragment monomers constitute a covalently bound homodimer. Under normal physiological conditions, igG concentrations in human plasma are highest at IgG1, second at IgG2, and lower at IgG3 and IgG4.

A preferred Fc element is a human IgG4-Fc fragment, or a mutant, derivative thereof.

In the present invention, the Fc fragment to which GH (e.g., hGH) is linked via a linker peptide is derived from the constant region of immunoglobulin IgG. Of the four human IgG subtypes, igG1 and IgG2, igG3 and IgG4 can be bound to hGH or its allosteric effectors via a linker peptide.

From a wild-type IgG4-Fc (the amino acid sequence of which is shown in SEQ ID NO: 1), various mutations were performed to obtain IgG4-Fc containing FL234-235AK, N297E, N315Q, N384Q mutations and K removed from the C-terminus.

The invention provides a fusion protein, which optionally contains a connecting peptide (also called linker protein, linker peptide). The size and complexity of the linker peptide may affect the activity of the protein. In general, the linker peptide should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. Meanwhile, the influence of alpha helix or beta folding and the like formed in the connecting peptide on the stability of the fusion protein is avoided. Meanwhile, the connecting peptide should be selected from amino acids with low immunogenicity as much as possible.

The length of the linker peptide is generally 1 to 80 amino acids, preferably 2 to 50 amino acids, and more preferably 5 to 40 amino acids.

In the present invention, the preferred linker peptide has a length of 30 to 40 amino acids,

the preferred flexible linker peptide is SEPATSGSETPGTSESATPESGPGTSTEPS EG which contains 32 amino acids.

Preparation of fusion proteins

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

As used herein, "isolated recombinant fusion protein" means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.

The full-length nucleotide sequence or its fragment of the fusion protein of the present invention or its elements (e.g., GH, fc) can be obtained by PCR amplification, recombinant methods, or synthetic methods. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells encoded with the vector or fusion protein coding sequences of the invention, and methods for producing the proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, the following steps are provided:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying protein from culture medium or cell.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a protein of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, NS0, COS7, or 293 cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells, which are capable of DNA uptake, can be harvested after exponential growth phase and subsequently treated with CaCl ₂ Methods, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

As used herein, "inclusion bodies" refer to high density, insoluble protein particles formed when a foreign gene is expressed in prokaryotic cells, particularly when expressed efficiently in e.

As used herein, "denaturation of inclusion bodies" refers to the process of re-solubilizing the inclusion bodies in aqueous solution using detergents such as high concentrations of urea, guanidine hydrochloride, or other conditions.

As used herein, "inclusion body refolding" refers to the process of refolding the above-described resolubilized inclusion bodies into a functional protein under suitable conditions.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Pharmaceutical compositions and methods of administration

The invention also provides a composition comprising an effective amount of the fusion protein of the invention, and a pharmaceutically acceptable carrier. Typically, the fusion proteins of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to about 8, preferably about 6 to about 8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in a human and/or animal and is acceptable to the human and/or animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the techniques realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, fourth edition), or according to the manufacturer's recommendations.

In the following examples, the Fc of the cyclic Fc-hGH-Fc fusion protein, the Fc of the monomeric Fc/hGH-Fc was site-mutated starting from the Fc fragment wild-type of IgG4 (amino acid sequence shown in SEQ ID NO: 5) described below, and hGH was prepared using the wild-type sequence of the library (amino acid sequence shown in SEQ ID NO: 1). Wherein the amino acid sequence of the cyclic Fc-hGH-Fc fusion protein is shown in SEQ ID NO. 2; the amino acid sequence of Fc in the monomer Fc/hGH-Fc is shown in SEQ ID NO. 4, and the amino acid sequence of hGH-Fc in the monomer Fc/hGH-Fc is shown in SEQ ID NO. 5.

Example 1: design, synthesis and plasmid construction of cyclic Fc-hGH-Fc fusion protein

1. Expression plasmid construction

Designing a primer, constructing a new plasmid pET41a-Fc-hGh-Fc, converting a BL21 (DE 3) strain for expression test, wherein the amino acid sequence of the expressed fusion protein is shown as SEQ ID NO.2, the structural schematic diagram of the circular Fc-hGH-Fc fusion protein is shown as figure 1, and the basic process is as follows: designing a primer, carrying out PCR amplification, carrying out vector enzyme digestion, recombining, sequencing, extracting a plasmid, transforming BL21 (DE 3), carrying out small-amount expression and carrying out large-amount expression.

The primers are synthesized by Beijing optisco New Biotechnology Co., ltd, and according to the operation method mentioned in molecular cloning, overlapping PCR is carried out to obtain the target fragment, and then recombinant connection, transformation, sequencing and bacteria protection of the fragment and the universal vector are carried out.

2. Plasmid extraction

Extracted according to the operating methods of Qiagen Mini-prep Kit and Qiagen Endofree Maxi-prep Kit. And finally determining and obtaining a correct target gene sequence through sequencing.

Example 2 expression of Cyclic Fc-hGH-Fc and monomeric Fc/hGH-Fc fusion proteins and preparation of Inclusion bodies

The cyclic Fc-hGH-Fc fusion protein can be effectively expressed in a prokaryotic system, and has the advantages of high expression quantity, short time and low cost, the whole fermentation expression process is only three days, and the implementation scheme is as follows:

1. preparation of E.coli expressing circular Fc-hGH-Fc

The plasmid obtained in example 1 was transformed according to a molecular biological methodDissolving into BL21 (DE 3) competent cells, adding a bacterial liquid containing target protein into a 250mL triangular flask containing a Kan LB liquid culture medium, and performing shaking culture at 30 ℃ and 220rpm overnight; the next day, the overnight-cultured bacterial suspension was transferred into Kan-resistant 500mL TB flasks (flask volume: 2L) at an initial OD of 1 ₆₀₀ About 0.1, 37 ℃ at 220rpm, cultured to OD ₆₀₀ =1.5 (about 2.5 h); cooling to 30 ℃; adding IPTG to a final concentration of 0.5 mM; culturing overnight at 30 ℃ and 220 rpm; the supernatant was then removed and the pellet retained.

2. Expression of Cyclic Fc-hGH-Fc fusion proteins

Inoculating the strain obtained in the step 1 into a 50mL LB culture medium according to the inoculation amount of 1% to be cultured for 4-5h to serve as a seed, adding the seed into a 2.8L fermentation tank containing FDM0 culture medium to start fermentation, and setting the fermentation conditions as follows: stirring speed/200 rpm, culture temperature/37 ℃; ventilation volume/3L/min, pH value/7.0, pressure/0 Bar, dissolved oxygen 100%, during the culture process, maintaining temperature at 37 deg.C, pH 7.0, and tank pressure at 0Bar. Gradually increasing stirring speed and ventilation to maintain a certain dissolved oxygen concentration, and feeding when the dissolved oxygen rises. When the cell density had grown to an OD600 value of 53, induction was carried out by adding IPTG at a final concentration of 0.5mM and setting the temperature at 37 ℃. Sampling from 0h of induction without induction to the end of fermentation every 1h, analyzing the sample by SDS-PAGE, stopping fermentation after the induction time reaches 6 h, centrifuging, discarding the supernatant, and retaining the thallus. The expression of the cyclic Fc-hGH-Fc fusion protein generates insoluble inclusion bodies, the thalli are crushed under high pressure, washed by PBS buffer solution with the pH value of 7.4 twice, and the inclusion bodies are recovered, in the process, the expression and the crushing of the fusion protein only need to be completed once to obtain the inclusion bodies with high yield, 467g of wet thalli and 215g of the inclusion bodies are obtained from 2.8L of fermentation liquor.

Expression of the cyclic Fc-hGH-Fc fusion protein and disrupted washing of the inclusion bodies, the entire process taking 3 days.

3. Expression of monomeric Fc/Fc-hGH fusion proteins and disruption and washing of Inclusion bodies

And constructing plasmids for the monomer Fc/hGH-Fc in the same way, wherein the method for expressing the monomer Fc/hGH-Fc in a prokaryotic system is the same as that for expressing the cyclic Fc-hGH-Fc fusion protein, but in the expression process, the expression is carried out in two tanks, wherein one tank is only Fc, the other tank is hGH-Fc, after the two tanks are expressed in batches, the bacteria are respectively broken and washed, and the method is the same as the conditions of the buffer solution for breaking the bacteria and washing the cyclic Fc-hGH-Fc to respectively obtain the inclusion bodies only containing Fc or Fc-hGH.

4. Protein expression and inclusion body crushing and washing detection

As shown in FIG. 2, both the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc/hGH-Fc fusion protein were expressed predominantly in inclusion bodies during fermentation.

5. Detection of concentration of Cyclic Fc-hGH-Fc fusion protein

The expression level of the cyclic Fc-hGH-Fc fusion protein was determined by UPLC quantification. In the method of performing UPLC quantitative analysis, the flow rate is 50 ℃;0.4ml/min; for a total of 16min, UV was detected at 215nm. Mobile phase a was an aqueous solution containing 0.1% trifluoroacetic acid and mobile phase B was an acetonitrile solution containing 0.085% trifluoroacetic acid. First, balance was carried out at a ratio of 95% A/5% B for 1 minute. At 1.5 min, the ratio of A and B was raised to 70%/30% of the initial protein elution ratio, followed by a linear gradient elution for 10 min, with the ratio of B increasing gradually to 60%. The column was then washed for 1.5 minutes at 90% b and finally re-equilibrated by switching to the initial equilibrium ratio. The inclusion body standard substance selects early purified protein as the standard substance, and a multi-point method is used for preparing a standard curve for quantification. And determining the concentration of the sample to be detected according to the peak area (controlling the concentration of the sample to be detected within the concentration range detected by the standard curve). The results are shown in FIG. 3: after the bacteria are broken, the cyclic Fc-hGH-Fc is detected, and about 7g of target protein can be obtained in 1L of zymocyte.

6. Conclusion

The expression of the annular Fc-hGH-Fc fusion protein and the preparation process of the inclusion body are simpler and easier to operate than the preparation process of the monomer Fc/Fc-hGH fusion protein, only one fermentation and one bacteria breaking and washing are needed, so that the time and the cost are saved. Meanwhile, most of protein is precipitated, and the recovery rate of the protein in the inclusion body is very high, thereby laying a foundation for the renaturation in the later period and the improvement of the yield.

Example 3 renaturation of a Cyclic Fc-hGH-Fc fusion protein with a control monomeric Fc/Fc-hGH fusion protein

1. Renaturation of cyclic Fc-hGH-Fc fusion proteins

After the bacteria of the cyclic Fc-hGH-Fc fusion protein are broken, the operation of renaturation can be carried out. 50g of inclusion bodies (approx. 0.65L of fermentation broth) were solubilized with 750ml of 15 volumes of a lysis solution containing: 4-8M Urea, 1-10mM DTT (final concentration) was added at pH 8.5. And (3) dissolving at normal temperature for 2-3 hours for quantification, wherein 4400mg of target protein is obtained, and determining the concentration of the cyclic Fc-hGH-Fc fusion protein in the dissolved solution to prepare for subsequent renaturation. After 2-3 hours of solubilization, the solution was diluted to a concentration of 2-4mg/ml of cyclic Fc-hGH-Fc fusion protein, 1.5-3M urea, pH8, while adding 2-5mM cysteine, and stirred at room temperature overnight. The next day UPLC tested its recovery rate. 2800mg of target protein with good renaturation can be obtained, and the renaturation rate is 65%.

2. Renaturation process of monomeric Fc/hGH-Fc fusion protein

The method of the monomer Fc/hGH-Fc in the renaturation process is basically consistent with that of the cyclic Fc-hGH-Fc fusion protein, and because the expression and the preparation of the inclusion bodies are carried out separately, the dissolution and the quantification are carried out according to the proportion of 15w/v respectively in the dissolution process of the inclusion bodies. According to the quantitative results, the molar concentration is 1:1, 10g of Fc and 20g of hGH-Fc, about 6.2g of protein in the renaturation solution after mixing, the subsequent operation after mixing being identical to the renaturation operation of the cyclic Fc-hGH-Fc fusion protein. After overnight renaturation, 1.54g of monomeric Fc/hGH-Fc protein was obtained, with a renaturation of 25%. In this process, the quantification is required twice, and the time and error in the quantification process easily cause the mixture ratio to be uneven in the mixing and renaturation process at the later stage, so that Fc, hGH-Fc, fc/Fc, hGH-Fc/hGH-Fc are easily generated. Because the components have similar properties and structural characteristics with the monomer Fc/hGH-Fc fusion protein, the components are difficult to distinguish in the later purification process, and the difficulty of the purification process is increased.

3. Renaturation result detection of cyclic Fc-hGH-Fc and monomeric Fc/hGH-Fc proteins

FIG. 3 is a diagram showing the quantitative detection of the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc fusion protein after the disruption of the bacteria, and FIG. 4 is a SDS-PAGE gel before and after the renaturation of the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc/hGH-Fc fusion protein. As can be seen from the SDS-PAGE gel images, after renaturation treatment, the desired target protein, cyclic Fc-hGH-Fc fusion protein and monomeric Fc/Fc-hGH, are formed.

4. Conclusion

In the whole operation process of renaturation, the operation of the annular Fc-hGH-Fc fusion protein is simple, the renaturation efficiency is high, impurities are few, 7g of target protein can be expressed by 1L of fermentation liquor of escherichia coli, and 4.3g of target protein can be obtained from expression to bacterium breaking to final renaturation. The expression renaturation yield is much higher than that of monomer Fc/hGH-Fc, and the renaturation solution has less Fc/Fc and hGH-Fc/hGH-Fc impurities, and the purification at the later stage is easy. Also compared with the monomer form of the Fc/hGH-Fc fusion protein (CN 108794634A) GH-Linker-Fc/Fc expressed in a eukaryotic system, the expression amount is only 3.2g/L, and after the monomer form of the Fc/hGH-Fc fusion protein is purified by the Mab-selectisure affinity medium of GE company, 6% of hGH-Fc/hGH-Fc and Fc/Fc and 1.2% of other impurities still exist, so that the cyclic Fc-hGH-Fc fusion protein implemented by the scheme has obvious industrial value advantages.

Example 4 purification of Cyclic human growth hormone-Fc fusion protein (Fc-hGH-Fc) and control monomeric Fc/hGH-Fc fusion proteins

1. Process for purifying cyclic Fc-hGH-Fc fusion protein

The fusion protein is purified mainly by chromatography, for example, by anion exchange chromatography or cation exchange chromatography or gel filtration chromatography, or by hydrophobic chromatography, reverse phase chromatography, all of which can be combined to achieve substantial homogeneity of protein purity. The expressed fusion protein may also be purified using affinity columns containing antibodies, receptors or ligands specific for the fusion protein, such as recombinant rproteinA and rproteinG. Since no homodimer is produced during renaturation, the late purification is easy. After purification, the yield of cyclic Fc-hGH-Fc fusion protein from the renaturation solution to the final product was 14%: the UPLC purity of the final product is 94.2%, and the SEC-HPLC purity is as follows: 97.28%, as shown in FIG. 5.

In the method of performing UPLC quantitative analysis, the flow rate is 50 ℃;0.4ml/min; for a total of 16min, UV was detected at 215nm. Mobile phase a was an aqueous solution containing 0.1% trifluoroacetic acid and mobile phase B was an acetonitrile solution containing 0.085% trifluoroacetic acid. First, balance was carried out at a ratio of 95% A/5% B for 1 minute. At 1.5 min, the proportion of B was raised to 30% of the initial proportion of protein elution, followed by a linear gradient elution for 10 min, with the proportion of B increasing gradually to 60%. The column was then washed for 1.5 minutes at 90% b and finally re-equilibrated by switching to the initial equilibrium ratio. The inclusion body standard substance selects early purified protein as the standard substance, and a multi-point method is used for preparing a standard curve for quantification. And determining the concentration of the sample to be detected according to the peak area.

SEC-HPLC can monitor the aggregation, concentration and purity of the protein. The instrument is Agilent 1100. Detection was performed with TSK gel G2000SWxl,7.8mm 300mm column. The method comprises the following steps: the flow rate was 0.4ml/min, the column temperature was set to room temperature, UV280. The mobile phase is as follows: 2xPBS,5% IPA/aqueous solution, pH3.0. Run time was 45 minutes. The protein maker (Gel Filtration Standard, cat #: 151-1901) of BIO-RAD was used as a reference for molecular weight.

After purification, the sample was analyzed for composition by LC-Mass. The liquid phase was selected from the aqueous phase of an ACQUITY UPLC Protein BEH C4,300A,1.7um,2.1X 100mm column from Waters (P/N: 186004496), and in the method for quantitative analysis, the flow rate was 0.4ml/min, the column temperature was 70 ℃ and the detection UV was 215/280nm. Mobile phase a was an aqueous solution containing 0.1% fa, and mobile phase B was an acetonitrile solution containing 0.1% fa. First, balance was carried out at a ratio of 95% A/5% B for 1 minute. At 1.5 min, the proportion of B was raised to 20% of the initial proportion of protein elution, followed by a linear gradient elution for 10 min, with the proportion of B increasing gradually to 60%. The column was then washed for 1 minute at 95% b and finally re-equilibrated by switching to the initial equilibrium ratio. The mass spectrum was selected for the Full Scan mode, cationic polarity, cone voltage 70V, mass range High (400-7000 m/z), scan rate 1Hz, capillary voltage 1.5KV, and desorption temperature 550 ℃.

2. Human growth hormone-Fc fusion protein Fc/hGH-Fc purification process

Since a mixture of various fusion products including (hGH-Fc/hGH-Fc), homodimer type Fc fusion protein (Fc/Fc) and monomer type human growth hormone-Fc protein (hGH-Fc) is generated in the Fc/Fc-hGH fusion protein in the renaturation solution, monomer type Fc protein, the generation of a large amount of impurities results in low yield, low purity, and instability. Meanwhile, due to the generated impurities and the physical and biochemical properties of the target protein, the impurities and the target protein are very similar, and the impurities and the target protein are difficult to be removed completely through an affinity column, an ion column, a hydrophobic column and mixed packing during purification, and finally, a SEC column has to be used for removing residual hGH-Fc, fc/Fc, fc and hGH-Fc so as to achieve high-purity monomeric Fc/hGH-Fc fusion protein. After 4 rounds of purification including the last step of SEC, the yield of the monomeric Fc/hGH-Fc fusion protein from the renaturation solution to the final product was 14%, the purity of UPLC as the final product was 94.5%, the purity of SEC-HPLC was 97.89%, and endotoxin was 0.17EU/mg for 222mg of the target protein (10 g of Fc inclusion body in monomeric form and 20g of human growth hormone-Fc protein (hGH-Fc) inclusion body in monomeric form, which were combined to be 6.2g of protein dissolved in the renaturation solution, after mixing, the subsequent renaturation was identical to that of the cyclic Fc-hGH-Fc fusion protein.

The results of detecting various indexes of the protein after the monomer Fc/hGH-Fc fusion protein is purified are shown in Table 1 and FIG. 6.

TABLE 1 detection results of various indexes of the protein during the preparation process of the cyclic Fc-hGH-Fc fusion protein and the control group monomer Fc/hGH-Fc fusion protein and the process

3. And (4) conclusion:

by comparing the processes from fermentation to production of the cyclic Fc-hGH-Fc fusion protein and the monomeric Fc/hGH-Fc fusion protein (Table 1), it can be seen that the cyclic Fc-hGH-Fc fusion protein has a simple molecular structure and high stability, so that the previous renaturation process avoids the generation of a mixture with similar properties, and the gel filtration chromatography is avoided to remove residual hGH-Fc/hGH-Fc, fc/Fc, human growth hormone-Fc protein in monomeric form (hGH-Fc) and Fc protein in monomeric form during the use of a purification medium, so that the cyclic Fc-hGH-Fc fusion protein with high purity can be obtained. Compared with a monomer Fc/hGH-Fc fusion protein (CN 108794634A) GH-Linker-Fc/Fc expressed by a eukaryotic system, the hGH-Fc/hGH-Fc, fc/Fc and 1.2% of impurities are still remained after the purification by a Magselectsure affinity medium of GE company because a certain component of mixture with similar properties exists in cell fluid. Therefore, the cyclic Fc-hGH-Fc fusion protein implemented by the scheme also shows more advantages of industrial value in the aspect of purification.

Example 5 detection of Cyclic Fc-hGH-Fc fusion protein Using Biacore SPR-8K with control monomeric Fc/hGH-Fc fusion protein Activity detection

The specific operation is as follows:

1) Sample dilution:

the running buffer was 20mM Hepes buffer, pH7.4,0.05% Tween-20.

Ligand hGH Receptor extracellular soluble region expression was constructed for this company (GHR, genBank: AAA 52555.1) using a concentration of 10. Mu.g/ml immobilized on CM5 chips in acetate buffer pH4.0 in an amount of 1000RU.

The analyte cyclic Fc-hGH-Fc, monomeric Fc/hGH-Fc concentration gradient was: 0nM,12.5nM,25nM,50nM, 100nM.

2) The SPR run was: the flow rate was 30. Mu.l/min, the binding time was 200 seconds and the dissociation time was 1000 seconds. The final result is fitted by SPR data analysis software to obtain a dissociation constant K _D The values are given below (table 2).

TABLE 2 dissociation constant K of the extracellular soluble domain of cyclic Fc-hGH-Fc fusion protein and control monomeric Fc/hGH-Fc fusion protein and ligand hGH Receptor _D

Molecular design	Dissociation constant K D
		Fc-GH-Fc	18.4nM
Fc/hGH-Fc	7.2nM

And (3) SPR detection data result analysis:

the data of the results of the binding experiment of the protein and the hGH-Receptor are shown in table 2, in the test, the affinity of the cyclic Fc-hGH-Fc to the Receptor GHR is weaker than that of the monomer Fc/hGH-Fc to the Receptor GHR by about 2 to 3 times in the binding experiment of the cyclic Fc-hGH-Fc fusion protein (KD =18.4 nM) and the control monomer Fc/hGH-Fc fusion protein (KD =7.2 nM) to the Receptor in vitro, which shows that in the Fc-hGH-Fc molecule, since the N-terminal and the C-terminal of hGH are both connected with Fc protein, fc causes a certain steric hindrance to the hGH binding Receptor GHR, reduces the binding to the Receptor GHR, thereby reducing the degradation mediated by the Receptor and prolonging the half-life of the hGH in vivo.

Example 6 pharmacodynamic experiment of Cyclic human growth hormone-Fc fusion protein (Fc-hGH-Fc) in rats

1. Experimental design and method

The hypophysis is removed by ear canal surgery of young female SD rats, ear canal puncture hypophysis surgery is carried out after all rats are induced and anesthetized by isoflurane, a three-dimensional positioning hypophysis instrument is adopted for the surgery, pink pituitary tissue is sucked out for suction and examination. After the hypophysis is removed in an operation, weighing is carried out at 2 and 3 weeks after the operation, and rats with the weight growth rate of 2-3 weeks within +/-10 percent and healthy appearance are selected as successful models for modeling. According to the weight gain rate, the selection of 42 models is preferred, and the weight gain rate (2-3 weeks after operation) is 1.8 +/-1.6%. And divided into 7 groups of model control group (0 nmol/kg), monomer Fc-hGH-Fc low (2.6 nmol/kg), high (24.0 nmol/kg) dose group, cyclic Fc-hGH-Fc low (2.6 nmol/kg), high (24.0 nmol/kg) dose group, and Jinsai low (2.6 nmol/kg), high (24.0 nmol/kg) dose group, with 6 animals per group, at random and balanced weight and weight gain rates. Wherein the mosaic is PEG modified hGH and is a positive control of the experiment.

The medicine is administered by subcutaneous injection after grouping for 1 time/week for 5 weeks, and is administered for 5 times in total, and the administration volume is 2.0mL/kg. After administration, body weight was weighed 1 time per day, body length and tail length were measured 1 time per week, and area AUC under gram-fold line of body weight gain was calculated _{(weight gain Ke Di)} AUC of area under body length growth broken line _{(body length growth amount)} And area AUC under tail length increase broken line _{(tail length growth amount)} . And blood was collected from the orbital venous plexus of D2, D4, D6, D8 and serum was isolated from the high dose group rats before the first administration, and the insulin-like growth factor 1 (IGF-1) content was determined by the Elisa method.

2. Data processing and statistical analysis

Data were logged and statistically analyzed using Excel 2013 and SPSS 22.0. The area AUC (weight gain gram) under the weight gain gram broken line, the area AUC (body length gain) under the body length gain broken line, and the area AUC (tail length gain) under the tail length gain broken line were calculated by the trapezoidal area method. The metrology data was tested for homogeneity of variance using the Leven's test, if the variance among groups is uniform (p)>0.01 Directly quoting results of single-factor analysis of variance (LSD) test to carry out pairwise comparison among groups; if the variance among the groups is not uniform (p)<0.01 Two-by-two comparisons between groups were performed using the Mann-Whitney U nonparametric test or the two independent sample t tests. Data are all provided with

(standard deviation) is shown. p is a radical of<0.05 Indicating that the difference is statistically significant.

3. Growth promoting efficacy experimental data of protein on hypophysis extirpation rat model

The comparison between AUC groups of body weight gain of rats is shown in FIG. 7. AUC after administration for each dose group of test sample monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc and for each dose group of Juzel, as compared with model control group _{(weight gain gram number)} Are all significantly larger than the model control group (p)<0.05). Ratio between different samples at the same doseIn comparison, AUC in the low dose groups of monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc _{(weight gain gram number)} Are all smaller than the mosaic increasing and decreasing dose group (p)<0.05，p＝0.05)。

The AUC group comparison of body length growth in rats is shown in FIG. 8. AUC after administration for each dose group of test sample monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc and for each dose group of Juzel, as compared with model control group _{(body length growth amount)} Are all significantly larger than the model control group (p)<0.05). Comparison between different samples at the same dose, AUC in monomeric Fc/hGH-Fc Low dose group _{(body length growth amount)} Significantly less than the mosaic increasing and decreasing dose group (p)<0.05). Whereas the Cyclic Fc-hGH-Fc low dose group AUC _{(body length growth amount)} Is significantly larger than the monomer Fc/hGH-Fc low dose group (p)<0.05)。

The comparison between the rat tail length increase AUC groups is shown in fig. 9; the test sample monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc dose groups and the AUC of the increase-of-Jinsai dose groups were compared with the model control group _{(tail length growth amount)} Are all significantly larger than the model control group (p)<0.05). Compared among different samples at the same dose, cyclic Fc-hGH-Fc increased more than monomeric Fc/hGH-Fc, and both increased more than the kinoform-decreased dose group. Monomeric Fc/hGH-Fc, cyclic Fc-hGH-Fc, and mosaic increasing dose group AUC _{(tail length growth amount)} There was no significant difference between the two comparisons.

The variation in serum IGF-1 levels in the high dose groups of rats is shown in FIG. 10. Before administration, there was no significant difference between the groups with monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc and the increased dose of mosaic rat serum IGF-1 content. In D2, the serum IGF-1 content of rats was not significantly different in the monomeric Fc/hGH-Fc high-dose group compared with that in the Higashi dose group, and the cyclic Fc-hGH-Fc high-dose group was lower than that in the Higashi dose group. However, from D4 until the end of the experiment, the serum IGF-1 content in rats was significantly higher in the increased cyclic Fc-hGH-Fc group than in the high monomeric Fc/hGH-Fc group. There was no significant difference between the cyclic Fc-hGH-Fc, mosaic-increased dose groups. The monomeric Fc/hGH-Fc and cyclic Fc-hGH-Fc high-dose groups showed slightly lower serum IGF-1 levels than the mosaic-increased dose group.

4. Results of biological Activity assay

From FIGS. 7 to 10, it can be seen that the modified cyclic Fc-hGH-Fc fusion protein of the present invention is significantly superior to the monomeric Fc/hGH-Fc in both the in vitro binding to GH-receptor and the body weight gain, tail length growth and IGF-1 content in blood in animals, and the activity is equal to or even superior to the efficacy of the long-acting Jinsaing in the Chinese market. Meanwhile, the cyclic Fc-hGH-Fc fusion protein also shows excellent long-acting growth promoting effect.

Summary and discussion

In conclusion of the data, after the invention is modified, the unique cyclic Fc-hGH-Fc structural form can realize the high expression of prokaryotes in production, and ensure that the impurity Fc fusion protein in a dimer form is not generated in prokaryotic expression, so the renaturation and purification process is simpler and higher in yield compared with the monomer Fc/hGH-Fc expressed in eukaryotic cells or pronucleus respectively, the production cost of the long-acting growth hormone is greatly reduced, and the working hours are shortened. The auxin produced by the design and the process still keeps high activity and has good long-term effect, and meanwhile, the auxin does not introduce the risk of hepatotoxicity and nephrotoxicity caused by foreign substances (such as PEG) and reduces the risk of immunogenicity caused by glycosylation compared with the auxin expressed by eukaryotic cells.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Gln Gly Gln Pro Glu

145 150 155 160

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

165 170 175

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

180 185 190

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

195 200 205

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215

<210> 4

<211> 432

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ser Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met

1 5 10 15

Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu

20 25 30

Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln

35 40 45

Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg

85 90 95

Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val

100 105 110

Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly

115 120 125

Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr

130 135 140

Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys

145 150 155 160

Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu

165 170 175

Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly

180 185 190

Phe Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro Gln Gly Ala Pro

195 200 205

Gln Gly Ala Pro Gln Ser Cys Ala Pro Lys Ala Glu Gly Gly Pro Ser

210 215 220

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

225 230 235 240

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

245 250 255

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

260 265 270

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

275 280 285

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

290 295 300

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

305 310 315 320

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

325 330 335

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

385 390 395 400

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

420 425 430

<210> 5

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Cys Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

1 5 10 15

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

20 25 30

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

35 40 45

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

50 55 60

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

65 70 75 80

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

85 90 95

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

115 120 125

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

130 135 140

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

145 150 155 160

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

165 170 175

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

180 185 190

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

195 200 205

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215

Claims (10)

1. A recombinant human growth hormone-Fc fusion protein Fc-hGH-Fc is characterized in that the fusion protein consists of human growth hormone, two connecting peptides and two identical or different Fc fragments, namely the first Fc fragment, the human growth hormone and the second Fc fragment are sequentially connected to form a molecule by using the two connecting peptides from N end to C end, wherein one connecting peptide exists between the first Fc fragment and the human growth hormone, the other connecting peptide exists between the human growth hormone and the second Fc fragment, the amino acid sequences of the two connecting peptides are identical or different, and a dimer with 1 or more pairs of disulfide bonds or no disulfide bonds is formed after the two Fc fragments are folded to form the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc.

2. The fusion protein Fc-hGH-Fc of claim 1, wherein the Fc-fragment is selected from the group consisting of an Fc-fragment of human IgG, preferably of human IgG4 or IgG 1; further preferably an Fc fragment of human IgG4.

3. Fusion protein Fc-hGH-Fc according to claim 1, wherein the sum of the number of amino acids of said two linking peptides is at least 5, and wherein said linking peptide is a flexible polypeptide of amino acids, preferably of the amino acid type Asp, glu, gly, pro, ala, ser, thr, asn, gln, his.

4. The fusion protein Fc-hGH-Fc according to claim 1, wherein the amino acid sequence of the recombinant human growth hormone-Fc fusion protein Fc-hGH-Fc is shown in SEQ ID NO 2.

5. A DNA molecule of the recombinant human growth hormone-Fc fusion protein Fc-hGH-Fc according to any one of claims 1 to 4.

6. An expression vector comprising the DNA molecule of claim 5, wherein said vector is a eukaryotic or prokaryotic expression vector.

7. A host cell comprising the expression vector of claim 6, wherein said cell is selected from the group consisting of a eukaryotic cell or a prokaryotic cell; preferably E.coli.

8. A method for producing the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to any one of claims 1 to 4, comprising the steps of:

(a) Culturing the host cell of claim 7 under conditions suitable for expression to obtain a culture solution containing Fc-hGH-Fc that expresses cyclic human growth hormone-Fc fusion protein;

(b) Separating the cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc from the culture medium;

preferably, in step (b), said separating comprises: the thalli is crushed under high pressure, washed by PBS buffer solution twice, and the inclusion body is recovered; crushing and eluting the inclusion body to ensure that target protein does not exist in supernatant; renaturation process of fusion protein Fc-hGH-Fc; and (5) purifying the fusion protein Fc-hGH-Fc.

9. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

10. Use of the recombinant cyclic human growth hormone-Fc fusion protein Fc-hGH-Fc according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment of a disease associated with growth hormone deficiency.

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