CN107286248B - High-glycosylation human growth hormone (HGH) fusion protein and preparation method thereof and purposes - Google Patents

High-glycosylation human growth hormone (HGH) fusion protein and preparation method thereof and purposes Download PDF

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CN107286248B
CN107286248B CN201710202564.7A CN201710202564A CN107286248B CN 107286248 B CN107286248 B CN 107286248B CN 201710202564 A CN201710202564 A CN 201710202564A CN 107286248 B CN107286248 B CN 107286248B
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fusion protein
hgh
ser
val
human
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CN107286248A (en
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***
陈思
郑云程
李子瑞
马心鲁
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Anyuan Pharmaceutical Technology (shanghai) Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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)
    • C—CHEMISTRY; METALLURGY
    • 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)
    • A—HUMAN NECESSITIES
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

The invention discloses a kind of high-glycosylation human growth hormone (HGH) fusion protein.Fusion protein described in the human growth hormone (HGH) fusion protein of the present invention contains human growth hormone (HGH) (hGH), flexible peptide linker (L), at least one human chorion gonadotrophic hormone beta subunit carboxyl terminal rigidity peptide (CTP) and human immunoglobulin(HIg) Fc fragments successively from N-terminal to C-terminal.The invention also discloses a kind of method for efficiently preparing this kind of fusion protein.The fusion protein that the present invention is built has drug effect inside more excellent than restructuring hGH, circulating half-life inside extension, and administration frequency substantially reduces and bioavilability improves；Its production process is also simpler, efficient simultaneously.

Description

High-glycosylation human growth hormone (HGH) fusion protein and preparation method thereof and purposes
Technical field
The present invention relates to fusion protein field, more particularly it relates to which a kind of high-glycosylation human growth hormone (HGH) merges Albumen and its production and use.
Background technology
Human growth hormone (HGH) (human growth hormone, hGH) is one secreted by anteriorpituitary acidophic cell Kind proteohormone, is made up of 191 amino acid, and its physiological function is chiefly to facilitate metabolism and grown.HGH passes through Somatomedin or IGF mediation increase bone length, so as to reach promotion human body in cartilaginous tissue Linear growth.Recent years, the more functions of hGH were found, including were promoted skeletal muscle and Myocyte growth, promoted albumen The effects such as matter synthesis, regulation function of immune system and enhancing immune defense ability.HGH larger scale clinical application is also from initial The prevention and treatment of the children short stature of growth hormone deficiency, it is extended to the neck such as burn, acute pancreatitis, the elderly's anti-aging Domain, its clinical indication is also in continuous extension.
Children and adult can be treated in the case of growth hormone deficiency by external source supplementation with growth hormones. It is about 0.3 hour that half-life period is removed inside natural hGH, and half-life period is removed inside current commercialized human growth hormone recombinant About 2-3 hours, will soon be removed after injection by liver, kidney, so need daily subcutaneous administrations, to patient with Carry out many pains.In addition, long term injections human growth hormone recombinant produces antibody in a few patients body and affected the treatment.Therefore, A kind of technology is needed to keep higher activity while hGH circulating half-life in vivo is extended.Build recombinant long-acting, height The human growth hormone (HGH) of activity, the immunogenicity reduced and higher bioavilability turns into the primary mesh of drug development of future generation Mark.
For stable protein, the scavenging action of enzymolysis and kidney is prevented, can be modified using such as polyethylene glycol (PEG) (Sada et al.,J.Ferment Bioeng 71:137-139,1991), it is glycosylation modified transformation (U.S. Patent number US 7, 217,689) method of (WO 93/15199) and with other protein is merged to promote body absorption, prevent from being degraded and kidney Remove.The albumen of PEG modifications can prevent from hydrolyzing, and will not cause serious side effects, but PEG belongs to non-with target protein coupling Specific covalent combines, this to be coupled the interaction between different degrees of obstruction target protein and its acceptor at random and cause Its activity in vivo reduces.Increase glycosylation can increase the molecular weight of target protein, and critical line is filtered especially for kidney is in Protein (~30KD), glycosylation can reduce sensitiveness of the protein to protease hydrolytic.Korean Patent Publication No KR10-2013-0029713 discloses adds N- glycosyls by introducing at least one mutation on α -1 antitrypsins, attempts to increase Add the method for Half-life in vivo.The glycosylation modified of protein has two classes, including O- sugar chains and N- sugar chains.However, sugar chain is attached And adding can cause physiologically active protein matter to inactivate, and can additionally adhere to the selection face in the site of the physiologically active protein matter of sugar chain Also it is very narrow, therefore do not widely used also to the glycosylation engineering technology of the extra glycosylation site of protein introducing.
It is to connect the gene of physiologically active protein to improve albumen pharmacokinetics with another method of internal stability On the expressing gene for the albumen that one has a high stability, by gene recombination technology to produce fusion protein.Fc merges egg Be in vain using the technologies such as genetic engineering by certain have biological activity functional protein molecule (soluble ligand, acceptor or its He needs to extend the bioactive substance of half-life period) with Fc segment compositions and caused novel recombinant protein.Such fusion protein Not only remain the biological activity of functional protein molecule, moreover it is possible to extend the half-life period of fusion protein and improve its stability, Fc The interchain disulfide bond of fragment is advantageous to fusion molecule and forms dimer, so as to strengthen ligand binding capacity and improve bioactivity； The introducing of Fc fragments also advantageously improves expression of the fusion molecule in mammalian cell.Therefore manufacture contains and people The connected fusion protein in the Fc regions of IgG albumen, it will help extend the circulating half-life of medicine and/or increase its biology and live Property.Patent CN102875683 discloses a kind of Fc fusion proteins of long-acting recombinant human growth hormone, becomes in hGH and human IgG Fc One section of flexible peptide linker is added in body to reduce space steric effect, it is contemplated that target is to make its extended serum half lives, biology Activity increase, so as to improve pharmacokinetics and drug effect.However, because hGH avtive spot is mainly in C-terminal, C-terminal fusion Other albumen leverage hGH activity and function, show the hGH-L-vFc's of dimerization in CN102875683 patents Molar specific activity is 1.2nM, and it is still not ideal enough that its activity compares restructuring hGH.Find that simple extension is soft according to present invention research Property peptide linker, increase Fc with the space length of hGH C-terminals can not solve problem, it is necessary to which other methods solve to merge part pair The problem of hGH activity influences.It is contemplated that find new fusion method, further improve fusion protein serum half-life and Activity in vivo.
CTP is the small peptide of one section of β-subunit carboxyl terminal from human chorionic gonadotrophin (hCG).Four kinds and reproduction Related peptide hormone, follicle-stimulating hormone (FSH) (FSH), lutropin (LH), thyroid-stimulating hormone (TSH) and chorionic gonadotrophin Hormone (hCG) contains identical α-subunit and each special β-subunit.Compared with other three kinds of hormones, hCG Half-life in vivo It is obviously prolonged, this is mainly derived from distinctive carboxy terminal peptide (CTP) (Fares FA et al.Proc Natl on its β-subunit Acad Sci USA.89:4304–4308,1992).CTP contains 37 amino acid residues, and it has 4 O- glycosylation sites, Terminal is sialic acid residues.CTP can increase the level of glycosylation of protein, improve the activity of target protein, while negatively charged, Highly sialylated CTP can resist scavenging action of the kidney to it, so as to extend the half-life period of albumen in vivo.The U.S. is special Profit 13,195,931 discloses a kind of method by connecting human chorionic gonadtropin carboxy terminal peptide (CTP) on hGH, makes Growth hormone circulating half-life only extends about 6 times, but does not refer to the size of several hGH/CTP chimeric proteins external activities； The chimeric protein of internal drug effect display 1 or 2 CTP molecule of fusion is poor compared with restructuring hGH effect, only CTP-hGH-CTP-CTP This chimeric protein drug effect for being fitted together to 3 CTP molecules slightly above recombinates hGH.The present inventor will creatively have multiple O- A part of the CTP polypeptides in glycosyl site as connection peptide, for connecting hGH and Fc fragments, not as fusion part hair The effect of waving, because of the native glycosylation sites that it has, the half-life period of fusion protein can not only be made further to extend, bioavilability Improve, while be also greatly reduced fusion part Fc to hGH steric effect, it is maintained higher biological activity.
In summary content, many protein fusion methods are constructed to develop long acting protein class medicine at present, But so far still without activity and half-life period very gratifying long-acting hGH preparations.Therefore, there is an urgent need to develop length for this area Effect, high activity, purification step are simple, are easy to the long-acting hGH of industrialized production.
The content of the invention
The present invention designs to solve the problems, such as that restructuring hGH serum half-lifes are short and bioactivity is poor and is prepared for one kind The long-acting hGH fusion proteins of high-glycosylation.The present inventor's in-depth study by long-term, the peptide linker of uniqueness is devised first To reduce the steric hindrance between fusion molecule, the fusion protein formed is connected with Fc by hGH C-terminal, it is middle with flexible peptide and firm Property peptide connection.Unexpectedly, this fusion protein improves about than the external activity for the hGH-L-vFc that former the present inventor develops 1 times (referring to China Patent No. CN102875683).In addition, also achieve unexpected technique effect, i.e., described fusion egg White bioavilability also greatly improves.
In the first aspect of the present invention, there is provided one kind restructuring hGH fusion proteins, the fusion protein from N-terminal to C-terminal according to It is secondary to contain human growth hormone (HGH) (hGH), flexible peptide linker (L), at least one human chorion gonadotrophic hormone beta-subunit carboxyl terminal Rigid peptide (CTP) and human immunoglobulin(HIg) Fc fragments；Wherein, the preferred human IgG Fc variants (being expressed as vFc) of Fc fragments.
Wherein, the preferred non-immunogenic of the flexible peptide linker, and produce enough distances between hGH and Fc, Minimize mutual steric effect.It is preferred that connect using the flexible peptide containing following 2 or multiple Amino acid profiles Head：Gly (G), Ser (S), Ala (A) and Thr (T).Preferably, the flexible peptide linker includes G and S residues.Connect the length of peptide Degree is to the active extremely important of fusion protein.For the purpose of the present invention, it is preferable that the structure of the flexible peptide linker amino acid composition Formula is (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are greater than or equal to 0 integer, and a+b+c+d >=1.
In some embodiments of the present invention, the peptide linker is selected from following sequence：
(a)L1：GSGGGSGGGGSGGGGS；
(b)L2：GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(c)L3：GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(d)L4：GGGGSGGGGSGGGGSGGGGS.
Wherein, the human chorion gonadotrophic hormone beta-subunit carboxy terminal peptide rigidity peptide (CTP) is selected from by human chorionic Full length sequence that the amino acids of gonadotrophin beta subunit carboxyl terminal the 113rd to 145 are formed or its fragment are (hereinafter referred to as CTP), specifically, the CTP rigidity peptide includes SEQ ID NO：The sequence of 1 or its truncation.
Preferably, the CTP rigidity peptide includes at least two glycosylation site；For example, a preferred embodiment of the present invention In, the CTP includes 2 glycosylation sites, and exemplarily, the CTP includes SEQ ID NO：10 amino acid at 1N ends, i.e., SSSS*KAPPPS* (* represents glycosylation site)；Or the CTP includes SEQ ID NO：14 amino acid at 1C ends, i.e. S* RLPGPS*DTPILPQ；And for example, in another embodiment, the CTP includes 3 glycosylation sites, exemplarily, the CTP bags The NO of ID containing SEQ：16 amino acid at 1N ends, i.e. SSSS*KAPPPS*LPSPS*R；For another example, it is described in other embodiments CTP includes 4 glycosylation sites, and exemplarily, the CTP sequences include 28,29,30,31,32 or 33 amino acid and started The the 113rd, 114,115,116,117 or 118 in human chorion gonadotrophic hormone beta subunit, terminate at the 145th.Specifically, The CTP includes SEQ ID NO：28 amino acid at 1N ends, i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.It is every kind of Possibility all represents the standalone embodiment of the present invention.
In further embodiments, CTP rigid elements provided by the invention and natural CTP amino acid sequences at least 70% are same Source；In further embodiments, CTP rigid elements provided by the invention and natural CTP amino acid sequences at least 80% are homologous； In other embodiments, CTP rigid elements provided by the invention and natural CTP amino acid sequences at least 90% are homologous；Another In a little embodiments, CTP rigid elements provided by the invention and natural CTP amino acid sequences at least 95% are homologous.
Preferably, in embodiments of the invention, the CTP rigidity peptide preferably includes following sequence units：
(i)CTP1：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP2：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP3：SSSSKAPPPS；
(iv)CTP4：SRLPGPSDTPILPQ.
Fusion protein of the present invention can also include the above-mentioned CTP rigidity peptide sequence unit of 2 or more than 2, such as originally In one embodiment of invention, the CTP includes 2 CTP3Unit：SSSSKAPPPSSSSSKAPPPS(CTP3-CTP3, or represent For (CTP3)2)。
Wherein, extend half-life period part and preferably be selected from Immunoglobulin IgG, IgM, IgA Fc fragments；More preferably from human IgG1, IgG2, IgG3 or IgG4 and its variant Fc fragments；Further, the human IgG Fc variants, which include, is located at wild type human IgG At least one of Fc is amino acid modified, and variant have reduce effector function (ADCC and/or CDC effects) and/or with Neonatal receptor FcRn binding affinity enhancing.Further, human IgG Fc variants may be selected from the following group：
(a)vFcγ1：Containing Leu234Val, Leu235Ala and Pro331Ser mutation human IgG1's hinge region, CH2 and CH3 regions (such as SEQ ID NO：Amino acid sequence shown in 4)；
(b)vFcγ2-1：Human IgG2's hinge region, CH2 and CH3 regions (such as SEQ ID NO containing Pro331Ser mutation：5 Shown amino acid sequence)；
(c)vFcγ2-2：Containing Pro331Ser, Thr250Gln and Met428Leu mutation human IgG2's hinge region, CH2 and CH3 regions (such as SEQ ID NO：Amino acid sequence shown in 6)；
(d)vFcγ4：The hinge region of human IgG 4, CH2 and CH3 regions containing Ser228Pro and Leu235Ala mutation are (such as SEQ ID NO：Amino acid sequence shown in 7).
More preferably, the amino acid sequence of the hGH-L-CTP-vFc fusion proteins such as SEQ ID NO：Shown in 2.
In the second aspect of the present invention, there is provided recombinate hGH-L-CTP-vFc described in one kind coding first aspect present invention The DNA molecular of fusion protein, it is preferable that described DNA sequence dna has SEQ ID NO：Nucleotide sequence shown in 3.
According to the third aspect of the invention we, there is provided a kind of carrier, the carrier include the DNA sequences described in second aspect of the present invention Row.
According to the fourth aspect of the invention, there is provided a kind of host cell, the host cell include third aspect present invention institute Carrier is stated, or has transfected above-mentioned carrier.
In the embodiment of the present invention, host cell is CHO derived cell strain DXB-11.
Preferably, the host cell produces million thin more than 30 μ g/ in its growth medium in every 24 hours The restructuring hGH-L-CTP-vFc fusion proteins as described in the first aspect of the invention of born of the same parents.
In the fifth aspect of the present invention, there is provided prepared by one kind recombinates hGH-L-CTP-vFc described in first aspect present invention The method of fusion protein, methods described include：
(a) DNA of encoding fusion protein is introduced into Chinese hamster ovary celI, generation CHO derived cells system；
(b) in screening step (a) during every 24 hours, express more than 30 μ g/106(million) high yield of individual cell is thin Born of the same parents' strain；
(c) cell line that incubation step (b) screens, expressed fusion protein；
(d) zymotic fluid that step (c) obtains, purified fusion protein are harvested.
Preferably, the CHO derived cells system in step (a) is DXB-11.
In the sixth aspect of the present invention, the invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes pharmacy Upper acceptable carrier, excipient or diluent, and the hGH-L-CTP-vFc fusion proteins of effective dose.
Another further aspect, the invention provides the hGH-L-CTP-vFc fusion proteins for treating because of endogenous growth Dysplasia caused by hormone secretion deficiency, and build caused by Turner syndrome is short and small, chronic renal failure, The illnesss such as Prader-Willi syndromes or idiopathic build are short and small.
Another aspect, the invention provides the fusion protein for preparing the use in treating osteoporosis agents On the way, in particular for slow down because the age increases and the bone-loss that occurs.
To sum up, fusion protein structure of the present invention has the characteristics that：
1st, the fusion part human IgG Fc variants that fusion protein uses are non-cracking performances, are reduced and Fc γ Rs and Clq With reference to and trigger effector function；
2nd, no matter the fusion protein for preparing of the present invention is respectively provided with good steady during fermentation, purge process and storage It is qualitative；
3rd, its Half-life in vivo can not only further be extended by L-CTP connection hGH and Fc variants, CTP rigidity peptide；Simultaneously By the iris action of multiple glycosylation side chains, increase the space length between fusion molecule, promote each autofolding shape of hGH and Fc sections Into correct three-dimensional conformation and the respective bioactivity that is independent of each other, compared with hGH-L-vFc, hGH-L-CTP-vFc biology Activity increases substantially；
4th, fusion protein has the immunogenicity reduced, makes it induce risk caused by neutralizing antibody to reduce；
5th, compared with PEG-rhGH, hGH-L-CTP-vFc has the bioavilability improved, and blood concentration fluctuation is small, rises Effect faster, while has circulating half-life inside extension, can reduce frequency of injection, improves patient compliance；
6th, compared with recombinating hGH, hGH-L-CTP-vFc purification steps are simple, purification efficiency is high.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferable technical scheme.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention：
IgG Fc variants
Non- cracking performance Fc variants
Fc elements derive from the constant region fc fragment of Immunoglobulin IgG, and it rises in the immune defense of eliminating pathogen Important function.The IgG of Fc mediations effector function, which plays, passes through two kinds of mechanism：(1) with cell-surface Fc receptors (Fc γ Rs) With reference to, by phagocytosis or splitting action or killing cell pass through antibody-dependent cytotoxicity (ADCC) approach digest cause of disease Body, or (2) are combined with the C1q of the first complement component C1, trigger complement-dependent cytotoxicity (CDC) approach, so as to crack disease Substance.For the treatment applied to people, when hGH-L-CTP-vFc fusion proteins be incorporated into hGH on target cell surface by During body, the Fc regions of fusion protein do not have ill effect subfunction preferably, so as to dissolve or remove these target cells. Therefore, hGH-Fc Fc regions must be non-deliquescent, to being incorporated into Fc γ Rs and Clq so as to trigger effect subfunction side Face, Fc regions are preferably inactive.In four kinds of human IgG hypotypes, IgG1 and IgG3 can effectively combine Fc γ Rs, IgG4 and Fc γ Rs binding affinity is relatively low, and IgG2 and Fc γ Rs combination is low must be difficult to determine, so human IgG2 does not almost have ADCC Effect.In addition, human IgG1 and IgG3 can also effectively with reference to C1q activating complement cascade reaction.Human IgG2 is combined relatively with C1q It is weak, and IgG4 do not combined with C1q (Jefferis R etc., Immunol Rev, 1998,163:59-76), so human IgG2 CDC is imitated Should be also weaker.Obviously, it is especially suitable for producing hGH fusion proteins without a kind of natural IgG hypotypes.In order to not had effector work( The non-cracking performance Fc of energy, most effectual way are to complement, receptor binding domains mutation transformation in Fc fragments, adjust Fc and associated receptor Binding affinity, reduce or eliminate ADCC and CDC effects, only retain Fc long circulating half-life characteristics, without produce cell Toxicity.More non-cracking performance Fc variants include mutational site and may refer to Shields RL etc., J Biol Chem, and 2001, 276(9):6591-604 or Chinese invention patent CN 201280031137.2.
Pharmacokinetically improved Fc variants
IgG plasma half-time depends on the combination of it and FcRn, is typically combined in pH 6.0, in (the blood plasma of pH 7.4 Dissociated when pH).By the research to both binding sites, the site combined on IgG with FcRn is transformed, is allowed to tie in pH 6.0 The increase of conjunction ability.The verified mutation for combining some residues of the important people's Fc γ domains of FcRn can increase serum half Decline the phase.Especially, in people Fc γ 1, when three residues are substituted by other 19 conventional amino acids, some point mutation display increase FcRn binding affinities (Hinton etc., J Biol Chem, 279 (8):6213-6216,2004).Hinton etc. has found 2 mutant of T250Q and M428L make respectively and FcRn combination increases by 3 and 7 times.2 sites of simultaneous mutation, then combine increase 28 times.In rhesus monkeys, M428L or T250QM428L mutant shows that plasma half-time increases by 2 times of (Paul R.Hinton Deng, J Immunol, 2006,176:346-356).In addition, there is research to carry out T250Q/ to the Fc sections of five kinds of humanized antibodies M428L, which is mutated, not only improves Fc and FcRn interaction, and is found in pharmacokinetic trial with skin inside then Lower drug administration by injection, Fc mutant antibodies pharmacokinetic parameter compared with wild-type antibodies improve, internal exposed amount increase, clearance rate Reduce, subcutaneous bioavilability is improved (.MAbs.Taylor＆Francis such as Datta-Mannan A, 2012,4 (2): 267-273.)。
Carboxy terminal peptide (CTP)
CTP is the small peptide of one section of β-subunit carboxyl terminal from human chorionic gonadotrophin (hCG).Four kinds and reproduction Related peptide hormone follicle-stimulating hormone (FSH) (FSH), lutropin (LH), thyroid-stimulating hormone (TSH) and chorionic gonadotrophin Hormone (hCG) contains identical α-subunit and each special β-subunit.Compared with other three kinds of hormones, hCG Half-life in vivo It is obviously prolonged, this is mainly derived from distinctive carboxy terminal peptide (CTP) (Fares FA etc., Proc Natl on its β-subunit Acad Sci USA,1992,89:4304-4308).CTP contains 37 amino acid residues, and it has 4 O- glycosylation sites, Terminal is sialic acid residues.Negatively charged, highly sialylated CTP can resist scavenging action of the kidney to it, so as to extend The half-life period of albumen in vivo.Thus, inventor creatively increases at least one after the flexible peptide linker of suitable length CTP polypeptides so that the half-life period of fusion protein further extends, and bioavilability improves.
In addition, by increasing CTP peptides between hGH and Fc variants, equivalent to adding one section of peptide that is rigidly connected.This aspect It ensure that the hGH of N- ends fusion does not interfere with Fc variants and FcRn binding site, so as to influence half-life period；Other Fc's Protein A binding sites are critically important for purification step in preparation technology, and connection CTP ensures that the hGH of N- ends fusion also will not " covering " its binding site with Protein A.On the other hand, CTP addition also make it that the Fc fragments of about 25KD sizes will not The hGH of N- ends fusion correct folding is disturbed, so that the decline or forfeiture of its biological activity/function.With multiple glycosyls Side chain rigidity CTP polypeptides, relative to the random coil of this kind of flexible peptide linkers of (GGGGS) n, it can form stable stand Body conformation, this " barrier " effect promote hGH and Fc sections independently to fold to form correct three-dimensional conformation and be independent of each other respective Bioactivity.
Fusion protein and its production method
Fusion protein of the present invention is generally prepared by the method for biosynthesis.According to nucleotide sequence of the present invention, sheet The code nucleic acid of the present invention can be easily made in technical field personnel with various known methods.These methods are such as, but not limited to： PCR, DNA are artificial synthesized etc., and specific method can be found in J. Pehanorm Brookers,《Molecular Cloning:A Laboratory guide》.As the present invention A kind of embodiment, the method for Overlap extension PCR can be carried out again by subsection synthesis nucleotide sequence to build the present invention's Nucleic acid sequence encoding.
Present invention also offers a kind of expression vector, operates comprising the sequence for encoding fusion protein of the invention and therewith Property connected expression regulation sequence.Described " being operatively connected " or " being operably coupled to " refers to such a situation, i.e., linear Some parts of DNA sequence dna can adjust or control the activity of same linear DNA molecule other parts.If for example, promoter The transcription of control sequence, then it is exactly to be operably coupled to coded sequence.
Expression vector can use commercially available be such as, but not limited to：PcDNA3, pIRES, pDR, pUC18 etc. can be used for eucaryon thin The carrier of born of the same parents' system expression.Those skilled in the art can select suitable expression vector according to host cell.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can be conventionally by restricted Enzyme is sheared and splicing, and the coded sequence of the fusion protein of the present invention is inserted into suitable restriction site, the weight of the present invention is made Group expression vector.
Present invention also offers the host cell for expressing fusion protein of the present invention, wherein the fusion protein containing the present invention Coded sequence.Described host cell is preferably eukaryotic, such as, but not limited to CHO, COS cells, 293 cells, and RSF is thin Born of the same parents etc..As the preferred embodiment of the present invention, described cell is Chinese hamster ovary celI, and it can express the fusion protein of the present invention well, The fusion protein that binding activity is good, has good stability can be obtained.
The present invention also provides a kind of method that fusion protein of the present invention is prepared with recombinant DNA, and its step includes：
1) nucleotide sequence (such as SEQ ID NO of encoding fusion protein are provided：3 sequences)；
2) nucleotide sequence 1) is inserted into suitable expression vector, obtains recombinant expression carrier；
3) recombinant expression carrier 2) is imported into suitable host cell；
4) culture converts host cell under conditions suitable for the expression；
5) supernatant, and purified fusion protein product are collected.
The coded sequence is imported into host cell can use a variety of known technologies of this area, such as, but not limited to：Phosphorus Sour calcium precipitate, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkali metal from Sub- method.
Culture and expression about host cell can be found in Olander RM Dev Biol Stand 1996；86：338. Clear liquid can be collected by centrifuging the cell and residue that remove in suspension.It can be identified by agarose gel electrophoresis technology.
It can be substantially uniform property by the above-mentioned fusion protein purification prepared, such as be on SDS-PAGE electrophoresis Single band.For example, when recombinant protein is secreting, expressing, the albumen, example can be separated using the milipore filter of commercialization Such as Millipore, Pellicon Products, first will expression supernatant concentration.The method that concentrate can use gel chromatography Further purified, or purified using the method for ion-exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or sun from Sub- displacement chromatography.Gel-type vehicle can be the matrix that agarose, glucan, polyamide etc. are usually used in protein purification.Q- or SP- groups It is ideal ion-exchange group.Finally, also hydroxylapatite adsorption can use to chromatograph, metal chelate chromatography is hydrophobic mutual The methods of acting on chromatography and RPLC (RP-HPLC) is to the further polishing purification of above-mentioned purified product.Above-mentioned institute There is purification step to utilize different combinations, purity of protein is reached substantially uniform.
Using fusion of the affinity column of the specific antibody containing the fusion protein, acceptor or part to expression Albumen is purified.According to the characteristic of used affinity column, using the method for routine, such as high-salt buffer, change pH Amalgamation polypeptide of the method elution of bound on affinity column.Selectively, the aminoterminal of described fusion protein or c-terminus be also One or more polypeptide fragments can be contained, as protein tag.Any suitable label may be used to the present invention.For example, institute The label stated can be FLAG, HA, HAl, c-Myc, 6-His or 8-His etc..These labels can be used for pure to fusion protein progress Change.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, and it contains effective dose (such as 0.000001-90wt%；Preferably 0.1-50wt%；More preferably, 5-40wt%) fusion protein of the invention, and pharmaceutically acceptable carrier.Generally, may be used The fusion protein of the present invention of effective dose is formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, its Middle pH ordinarily be about 5-8, it is preferred that pH is about 6-8.Term " effective dose " or " effective dose " refer to can be to people and/or animal Produce function or amount that is active and being received by people and/or animal.The composition of " pharmaceutically acceptable " applies to people And/or mammal and without excessive bad side reaction (such as toxicity, stimulation and allergy), i.e., with rational benefit/wind The material of dangerous ratio.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and dilute Release agent.
Pharmaceutically acceptable carrier includes (but being not limited to)：Salt solution, buffer solution, glucose, water, glycerine, ethanol and It is combined.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the invention can be made into injection form, example The aqueous solution such as with physiological saline or containing glucose and other assistant agents is prepared by conventional method.Described drug regimen Thing preferably aseptically manufactures.The dosage of active component is therapeutically effective amount.The pharmaceutical preparation of the present invention may also be fabricated which slow Release formulation.
The effective dose of fusion protein of the present invention can be with the pattern of administration and the order of severity etc. of disease to be treated And change.The selection of preferable effective dose can be determined (such as to pass through by those of ordinary skill in the art according to various factors Clinical test).Described factor includes but is not limited to：The pharmacokinetic parameter of described fusion protein such as biological utilisation Degree, metabolism, half-life period etc.；The order of severity of the disease to be treated of patient, the body weight of patient, the immune state of patient, administration Approach etc..Generally, when the fusion protein of the present invention is (preferable with about 0.00001mg-50mg/kg the weight of animals daily 0.0001mg-10mg/kg the weight of animals) dosage give, gratifying effect can be obtained.For example, compeled by treatment situation Highly necessary ask, dosage separated several times can be given daily, or dosage is reduced pari passu.
Brief description of the drawings
Fig. 1, show in pAPG-3 expression vectors the nucleotide sequence of the fusion protein of Spe I-EcoR I fragments and The amino acid sequence of derivation.Peptide linker position in maturation protein is marked with lower stroke of dotted line.In Fc regions, the nucleotides of runic Marked with corresponding amino acid variant with underscore.
Fig. 2, show the ability that hGH fusion proteins stimulate Nb2-11 cells propagation.
Fig. 3, the growth curve for showing each group after the administration of hGH fusion proteins.
Fig. 4, show hGH fusion proteins after being administered to rat tibia pathologic examination result.Note：A：Model group； B：TL groups；C：TM groups；D：TH groups；E：SL groups；F：SM groups；G：SH groups.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part such as Sambrook et al., molecular cloning：Laboratory manual (New York：Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.
The expression plasmid of embodiment 1, structure coding hGH fusion proteins
Gene order and different length flexible peptide linker, the different length CTP for encoding hGH leader peptides and ripe hGH are firm The gene order of property peptide and different IgG Fc variants is all the Chinese hamster ovary celI preference codon of artificial optimization, through chemical synthesis process Obtain.For the ease of purpose fragment to be inserted to the specific site of expression vector, respectively there is a limit at synthesized fragment 5 ' and 3 ' end Enzyme site, respectively SpeI and EcoRI in property enzyme processed.Fusion SpeI and EcoRI digestions after sequence verification, are then inserted Enter between using PCDNA3.1 as template and improved expression plasmid PXY1A1 corresponding restriction enzyme site, obtain track fusion Plasmid pAPG.PXY1A1 plasmids are including but not limited to following important expression component:1) human cytomegalovirus early promoter and Enhancer needed for the high heterogenous expression of mammalian cell；2) Double Selection label, there is kalamycin resistance in bacterium, There is G418 resistances in mammalian cell；3) mouse dihydrofolate reductase (DHFR) gene expression frame, when host cell is During DHFR gene defection types, amethopterin (MTX) can coamplification fusion and DHFR genes (referring to United States Patent (USP) US 4, 399,216)。
As shown in table 1, the present invention constructs a series of hGH-Fc fusion proteins, and the flexible peptide that they have different length connects Head, CTP compositions and also position difference, and IgG Fc variants (vFc) element composition of several different subtypes.Wherein APG-3's Nucleotide sequence and the amino acid sequence of translation are as shown in Figure 1.
Several hGH fusion proteins composition of table 1, structure
Embodiment 2, transient expression difference fusion protein and active determination in vitro
A series of expression plasmids obtained in embodiment 1, DNAFect LT reagents are used in 30ml shaking flask (ATGCell) 3 × 10 are transfected7Chinese hamster ovary celI, the cell through transfection grows 5 days in serum free growth medium, according to corresponding Fusion protein concentration in ELISA method measure supernatant, and determine its extracorporeal biology with the method described in embodiment 5 and live Property.ELISA results show that the transient expression amount of several plasmids under this condition is more or less the same, but their activity is but shown Larger difference (table 2).
Table 2, transient expression various fusion proteins external activity EC50Value compares
Wherein, APG-1 molar specific activity is defined as 100% by us.APG-2 activity is the 80.9% of APG-1, table The bright activity that fusion protein is not only can effectively improve by extending peptide linker, i.e., mutual space steric effect can not be with peptide The extension of joint and weaken.Even, long peptide linker can not only improve the activity of fusion protein, can make albumen mistake on the contrary Fold, secreted with inactive multimeric forms.It is presumed that reason, long flexible peptide linker makes the flexibility ratio of fusion protein It is higher, can freely it rotate, this may make hGH stereochemical structure closer to Fc areas, and add CTP between, On the one hand one end rigidity peptide linker quite being added, made away from each other, it is often more important that CTP contains multiple sugar side chains, relative to The random coil form of flexible peptide linker, CTP can form fixed space conformation, can effectively separate fusion protein Difference in functionality area, this is more beneficial for two parts and is independently folded into correct three-dimensional conformation, maintains higher activity.Pass through APG- 1st, APG-2 and APG-3 expression activitiy demonstrates the correctness of this supposition.And CTP is placed in the APG-4 of Fc C-terminals activity only The activity that the APG-3 of Fc N-terminals 61.3%, APG-4 is placed in for CTP is similar to the APG-1 without CTP.And APG-3 activity Higher than APG-4, APG-5, APG-6 and APG-7 activity will also be far above other fusion proteins.Result above, it was demonstrated that CTP is to melting The activity of hop protein is extremely crucial, and CTP is placed in Fc N-terminal and is more beneficial for improving the activity of fusion protein.
The expression of embodiment 3, fusion protein in transfectional cell series
The expression plasmid of restructuring is transfected into mammalian host cell line, to express hGH-L-CTP-vFc fusion proteins. In order to stablize high-caliber expression, preferable host cell line be DHFR deficient CHO- cells (referring to United States Patent (USP) US 4, 818,679).A kind of preferable transfection method is electroporation, and other methods, including calcium phosphate co-sedimentation, fat can also be used to turn Dye and Protoplast fusion.In electroporation, with the Gene Pulser for being arranged to 300V electric fields and 1050 μ Fd electric capacity Electroporator (Bio-Rad Laboratories, Hercules, CA), 5 × 10 are placed in cuvette7Individual cell, Then add 50 μ g plasmids and carry out electroporation.In transfection two days later, culture medium is made to the grown cultures of the G418 containing 0.6mg/mL into Base.After transfection about 2 weeks, the transfectant resistant to G418 medicines can grow up to macroscopic population of cells.Use anti-human igg Fc elisa assay method, screen the transfectant resistant to selection medication.Also anti-hGH ELISA can use to carry out fusion egg The quantitative analysis of white expression quantity.By the well culture plate of Method of Limited Dilution 96, subclone produces the hole of high-level Fc fusion proteins.
In order to realize the expression of fusion protein higher level, preferably coamplification is carried out with the DHFR genes by MTX Drug inhibitions. In the growth medium containing progressive concentration MTX, with the antigen-4 fusion protein gene of DHFR genes coamplification transfection.Use Method of Limited Dilution The transfectant that can be grown in up to 6 μM of MTX culture mediums of subclone.Determine secretion rate the cell line of subclone is entered to advance The analysis of one step.Secretion rate level is more than about 30 (being preferably about 50) μ g/106The cell line of (i.e. million) individual cell/24 hour Adapt to the suspension culture using serum free growth medium.Then conditioned medium purified fusion protein is used again.
Embodiment 4, fusion protein purifying with it is qualitative
The conditioned medium containing fusion protein is titrated to pH 7~8 with 1N NaOH, then with 0.45 micron of nitric acid Cellulose filter filters.Filtrate is loaded onto on the Protein A posts of phosphate buffer saline (PBS) balance.It is to be fused Protein binding discards the component of outflow after Protein A posts.The post is washed with PBS, the OD values at 280nm are less than 0.01.Then with the fusion protein for the citrate buffer solution elution of bound that 0.1M pH are 3.75.The 1M of 0.4 volume of eluent K2HPO4Neutralize, merge the component containing purifying protein, and use PBS.Then with 0.22 micron of nitrocellulose filter Filtering, and it is stored in 4 DEG C.Under non reducing conditions, protein product is identified and analyzed by SDS-PAGE.By the use of BSA as Standard, pass through the quantitative fusion protein of BCA protein analyses.
The Bioactivity analysis of embodiment 5, fusion protein
Bred using Nb2-11 cells to detect the In vitro biological activity of restructuring hGH-L-CTP-Fc fusion proteins.With containing The Fischer's culture mediums for having 10% hyclone normally cultivate mouse lymphoma cell Nb2-11 (U.S.'s ATCC cell banks). Using serum free medium, 1000ng/ml originates 3 times of gradient dilution fusion proteins, obtains the sample of 8 various concentrations, per hole 100 μ l, added to 96 orifice plates, culture medium is negative control.The cell in logarithmic phase growth period is taken, is washed with serum free medium Wash cell once, adjustment density is every milliliter 3 × 106Individual cell, it is added to per the μ l of hole 100 in above-mentioned 96 orifice plate.37 DEG C, 5%CO2Cultivated 48 hours in incubator, use CCK-8 kits (Cell Counting Kit, purchased from the holy biological section of Shanghai assist Skill Co., Ltd) detection cell proliferative conditions.The absorbance at 450nm is determined with ELIASA, by OD readings relative to fusion The concentration mapping of albumen, gained dose-effect curve calculate the bioactivity of fusion protein.
Fig. 2 shows the ability that hGH fusion proteins stimulate Nb2 cells propagation.Table 3 is effective for the half of different fusion proteins Concentration value (half maximal effective concentration, EC50).Due to the amino acid of growth hormone C-terminal and its Function is closely related, and Fc is directly connected with hGH C sections can influence its bioactivity.After connection peptide is added in hGH and Fc, hGH The activity raising of fusion protein.It can be seen from the results that APG-3 activity, compared with APG-1, activity improves will by about one time.This Be probably because on the one hand CTP plays the function of itself, on the other hand as the peptide that is rigidly connected between Fc and hGH, CTP with it is soft Property peptide coupling, this structure makes fusion protein be folded into more favourable three-dimensional structure, ensure that hGH bioactivity.
The EC of table 3, hGH fusion proteins50Value
Determination of biological activity inside embodiment 6, fusion protein
Long-acting somatotrophic pharmacodynamic study in vivo is carried out to fusion protein.Choose 4 week old SPF level male SD rats, body Weight 60-80g, is provided by National Institute for Food and Drugs Control's Experimental Animal Center.2 weeks clean conditions menisectomies are extractd before experiment Hypophysis, it is convalescence to remove hypophysis Post operation 2 weeks, and the change of selection rat body weight is qualified less than operation consent body weight ± 10% before being administered Healthy animal, hypophysis rat is gone to be divided into 7 groups, every group 7 by body weight is uniformly random.Administering mode is subcutaneously injected for neck, middle dose Amount is scaled 1.26IU/kg/d with clinical dosage, and setting agent is away from 1:3, PEG-rhGH (Changchun Jinsai Medicine Co., Ltd) High, medium and low dosage group is respectively 27IU/kg/7d (SH), 9IU/kg/7d (SM) and 3IU/kg/7d (SL), and APG-3 is high, medium and low Dosage group is respectively 27IU/kg/7d (TH), 9IU/kg/7d (TM), 3IU/kg/7d (TL).Various dose APG-3 fusion proteins With PEG-rhGH weekly administrations once, i.e. d1, d7, d14, d21, d28, continuous 5 weeks, it is administered 5 times altogether, model group gives solvent.
First, rat body weight Changeement
Every rat daily weigh by the same time after administration, calculates daily rat body weight increase, the 35th day (d35) terminates real Test, rat weight.Certain day every the weight of animals (bw after administrationi) with administration before body weight (bw1) difference be that day increased body weight gram (if necessary, experiment can perform an autopsy on sb. number Δ bw (g) after terminating, and cut sella region, and visual inspection whether there is hypophysis residual, and rejecting has The animal of hypophysis remaining).Increased weight calculation formula：Δ bw=bwi–bw0, wherein：Δbw：Weight gain；bw0：Before administration (d0) body weight, bwi：Certain day body weight after administration.Measurement data is with mean ± standard deviationRepresent, multigroup calculating data are equal Number otherness compares through F variance test homogeneous, compares each group t two-by-two and examines progress statistical analysis.Fig. 3 shows administration The growth curve of each group afterwards.
Go hypophysis rat to start 7 week old during administration, be adult rat, after weekly administration 1 time, 5 weeks, rat 12 Week old, it is adult rat, has crossed growth animated period, growth is soon stagnated.As can be seen from Table 4, model group (is only given molten Matchmaker) body weight has almost no change, not increased, and each dosage group body weight highly significants of APG-3 and positive control drug PEG-rhGH Increase (P<0.01), and it is in dose-effect relationship, shows that there is APG-3 increase body growth, increase albumen to close as PEG-rhGH Into effect.After the 3rd administration, the Δ bw of each dosage group shows significant difference；And the 35th day upon administration, APG-3 is to big The body weight increase of mouse has the facilitation of highly significant, high dose APG-3 induction Hypophysectomized rats increased weights (Δ bw Value) it is about 1.5 times of PEG-rhGH high dose groups；And the increased weight of high dose PEG-rhGH inductions is slightly better than middle dosage APG- 3, and the ascendant trend of growth curve tends to be steady from d26.This be probably due to APG-3 compared to PEG-rhGH should have it is longer Inside active half-life, therefore after repeat administration, this accumulating effect in vivo causes APG-3 groups to be grown after the 3rd administration The ascendant trend of curve is more precipitous.
Table 4, administration before and after rat body weight change (N=7)
Note：With model group ratio,##P<0.01。
2nd, Cognition Function in Rats is studied
Rat is carried out dark avoidance task by (d35) after terminating experiment.Rat keeps away dark instrument i.e. use and is separated into bright, dark two Room Plastic box, there is an arch wicket to communicate between light and shade case, two bottoms are copper grid, and darkroom bottom there are copper grid and can be powered, voltage 36V.1 day before experiment, first rat training experiment is placed in the bright room of strong illumination, allows it to be found after voluntarily groping bright, dark Passage between room simultaneously enters darkroom, and repetition training rat treats rat entrance until it can find in 60s and enter darkroom The passage closed behind darkroom between bright, darkroom, it is powered in darkroom and gives the electro photoluminescence 1 time of 36V, 50Hz alternating current, duration About 5s, allow after rat rest 30s and repeat said process until it dare not enter the time in darkroom more than 300s in bright room, greatly Mouse repetition training in dark instrument reaction chamber is kept away, makes rat be run away to bright room by electric shock.Experimental day formally starts first to place rat In camera-lucida, 5min is adapted to, opens wicket, the time that rat enters camera bellows from camera-lucida is to keep away dark incubation period, and rat enters dark Case, camera bellows bottom pass to exchange electro photoluminescence, and rat escapes from darkroom immediately, and rat is worn into the number that camera bellows is shocked by electricity within 5min Shuttle number.Keep away dark instrument and record the number that rat in 5min enters darkroom automatically, as shuttle number, and first enter into darkroom when Between, as keep away dark incubation period, and carry out statistical analysis (being determined by pharmacological room, instrument is Beijing pharmacological room of medicine inspecting institute).
Dark avoidance task is the common method for screening nootropic.It is using animal good dark lucifuge (light and shade shuttle), The fear of aversive stimulus (such as foot electric shock) is set up with remembering, is the side of currently used measure muroid Memory result One of method.Criterion is that animal keeps away that dark latent time is longer, shuttle number is fewer, and animal learning memory capability is stronger.From table 5 In as can be seen that model group keeps away that dark latent time is most short, shuttle number is most, ability of learning and memory is worst, compared with model group, Each dosage groups of APG-3 keep away dark latent time and significantly extend (extend 290%~1220%), and APG-3 is high, middle dose group and PEG- RhGH middle dose groups and its difference all has significant (P compared with model group<0.05) (SH groups due in group gap compared with Greatly, in the absence of statistical significance)；The shuttle number of each dosage groups of APG-3 significantly reduces simultaneously, and its difference has compared with model group Significant (P<0.05).As a result show that APG-3 is respectively provided with certain raising as positive drug PEG-rhGH and goes hypophysis big Mouse cognitive function and the effect of intelligence development.
Table 5, rat dark avoidance task statistics (N=7)
Note：Compared with model group,#P<0.05,##P<0.01。
3rd, the determination study of rat insulin like factor -1
IGF-1 is growth factor important on growth axis, and it has different physiological roles, right in addition to body growth is adjusted Numerous cell lines, which have, promotes mitotic effect, including promotes osteoblastic proliferation, differentiation, improves the activity of Gegenbaur's cell And quantity, prevent TNF-a Induced Apoptosis in Osteoblasts.Growth hormone is the adjustment effect that growth is played by IGF-1 mediation, more at present Research thinks that the horizontal changes of serum I GF-1 are indexs relatively reliable, sensitive the effect of judging GH treatments.
Above-mentioned experiment terminates rear rat vena ophthalmica clump and takes blood, and every about 0.5mL, standing is after 30 minutes, 6000rpm at 4 DEG C 10min is centrifuged, only, -20 DEG C save backup the honest and upright and thrifty 0.2mL/ of separation rat serum.Melt when facing survey, use radioimmunological kit (HY- 082, RIA KIT, Beijing China English biotechnology research institute) method measure serum in Insulin-like growth factor II -1 (IGF-1) content. Measurement data is with mean ± standard deviationRepresenting, multigroup calculating data mean otherness compares through F variance test homogeneous, Compare each group t two-by-two and examine progress statistical analysis, the results are shown in Table 6.
After being administered 5 weeks, compared with model group, PEG-rhGH high dose group, middle dose group and each dosage group serum of APG-3 IGF-1 significantly raises (P<0.05 or P<0.01), PEG-rhGH low dose groups IGF-1 contents compared with model group do not become significantly Change.And the IGF-1 contents of each dosage groups of APG-3 are generally higher than PEG-rhGH.It can be seen from the results that APG-3 growth promotion is made With and stimulate IGF-1 synthesize ability be better than PEG-rhGH.
Table 6, administration after serum I GF-1 statistics (N=7)
Note：Compared with model group,##P<0.01。
4th, rat tail length, liver weight and the research of epiphyseal plate width
After d0 and experiment terminate d35 carbon dioxide asphyxia execution rat before administration, respectively with the every big rat-tail of tape measuring Root to tail point length is tail length (cm), and tail length increases calculation formula：Δ L=L35–L0, wherein：ΔL：Tail length increase；L0：Administration Preceding d0 tail lengths；L35：D35 tail lengths after administration.The rat of execution digs liver, removes adhering tissue, and physiological saline is cleaned, and filter paper is inhaled Dry surrounding watery blood weighs (g).
The rat of execution digs rear left tibias, peels off attachment muscle and connective tissue, and neutral 10% formaldehyde preserves, from Median sagittal plane incised tissue sample materials 3mm is thick at the top of shin bone proximal part, is cut into slices after FFPE.Haematoxylin Yihong After (hematoxylin eosin, HE) dyeing processing, resinene glue sealing, om observation simultaneously microphotograph.Quantitative measurment shin Bone epiphyseal plate (growth plate zona cartilaginea) width, cut into slices per mouse one, each section is not measuring in overlapped view, with unified mark Standard, the width of 20 epiphyseal plates is chosen in section, quantitatively calculated the average value of every mouse epiphyseal plate width, finally Go out the average value entirely organized.Measurement data is with mean ± standard deviationRepresent, multigroup calculating data mean otherness compares Through F variance test homogeneous, compare each group t two-by-two and examine progress statistical analysis, the results are shown in Table 7.
As can be seen from Table 7, because model group rats do not grow, therefore model group rats tail before and after result display administration Length is substantially unchanged, and liver weight is also most light, and its shin bone epiphyseal plate width is also minimum；Compared with model group, APG-3 and PEG- Equally each dosage group rat tail lengths of rhGH and liver weight dramatically increase (P<0.01), shin bone epiphyseal plate width significantly broadening (P< 0.01 or P<0.05).As a result show that APG-3 and PEG-rhGH have the pharmacological action for promoting body growth.
Table 7, administration after tail length increase, liver weight, epiphyseal plate width statistics (N=7)
Note：Compared with model group,##P<0.01。
5th, rat bone density is studied
Right side femur, removes attachment muscle and connective tissue, gets rid of femoral head after every rat peels off after putting to death, per personal share Bone is placed in the test tube equipped with physiological saline 4 DEG C of preservations, takes out to dry during measure and puts HOLOGIC Dual-energy X-rays absorptionmetries (Hologic companies, the U.S.), with toy special-purpose software measure rat femur bone density (bone mineral density, BMD) it is corrected, surveys using model appended by instrument before with bone mineral content (bone mineral content, BMC), measure Every group 7 only measures together during amount, respectively the center-side of sweep measuring femur and BMD the and BMC values of distal end.Measurement data is with mean ± standard deviationRepresent, multigroup calculating data mean otherness compares through F variance test homogeneous, and each group is compared two-by-two Examined compared with t and carry out statistical analysis, the results are shown in Table 8.
As can be seen from Table 8, model group femur center-side and BMD the and BMC values of distal end are minimum, compared with model group, APG-3 and PEG-rhGH each dosage group center-side and the BMD and BMC of distal end significantly raise (P<0.01 or P<0.05).Bone Density is mineral quality contained in unit bone area in body bone tissue, is one of an important factor for influenceing bone strength, is to comment Valency bone strength common counter.This experimental studies results shows that APG-3 is respectively provided with increase bone mineral content as PEG-rhGH Effect, bone calcification can be promoted, increase bone density, promote the propagation of osteocyte, increased its activity, added the mineralization process of bone By force, bone amount increase；Therefore the clinically expected treatment available for osteoporosis, occur in particular for slowing down because the age increases Bone-loss.
Rat femur bone density and bone mineral content statistics after table 8, administration (N=7)
Note：Compared with model group,#P<0.05,##P<0.01。
6th, histopathologic examination
After rat is put to death, left side lower limb shin bone is dug, muscle and connective tissue is stripped clean, is placed in neutral formalin Preserve.Shin bone sample is made the histotomy of 4 μ m-thicks, takes vertical section, make HE dyeing through conventional decalcification, embedding.Section carries out disease Morphological observation and photomicrograph (× 2) are managed, Bone histomor-phometry metering is made using Image pro insight8.0 softwares.Every group 7 The section of rat, each section take middle part perimetry, calculate bone tissue area (Tissue Area, T.Ar)；Bone trabecula face Product (Trabecular Bone Area, Tb.Ar), bone trabecula girth (Trabecular Perimeter, Tb.Pm), bone trabecula Thickness (Trabecular thickness, Tb.Th), bone trabecula quantity (Trabecular number, Tb.N), bone trabecula point From degree (Trabecular separation, Tb.Sp).Measurement data is with mean ± standard deviationRepresent, multigroup calculating Data mean otherness compares through F variance test homogeneous, compares each group t two-by-two and examines progress statistical analysis, as a result sees Table 9.
Table 9, administration after rats shin bone bone trabecula measuration result statistics (N=7)
Note：Compared with model group,##P<0.01,#P<0.05。
Continued 9, administration after rats shin bone bone trabecula measuration result statistics (N=7)
Note：Compared with model group,##P<0.01,#P<0.05。
Morphologic observation, photomicrograph and data metering analysis are carried out to the pathological section of rats with left shin bone, it was therefore concluded that It is as follows：For model group in addition to bone trabecula separating degree is maximum, all other index parameters are minimum；Pathomorphologic observation is model group Bone trabecula is very thin and coloring is light, and morphosis integrality is poor, and with distortion and fracture, bone trabecula is short and growing state is bad, marrow Chamber is not of uniform size, and intracavitary hematopoietic cell is reduced.Each dosage group bone trabecula thickness increased, wherein the increasing of APG-3 middle dose groups Add (P<0.01)；Each dosage groups of all other index parameters APG-3 substantially increase (P<0.01 or P<0.05).As shown in figure 4, Pathomorphologic observation is that TH groups, TM groups and SH group bone trabecula are relative sturdy, and full and coloring is deeper, and bone trabecula morphosis is complete It is whole, there are not distortion or fracture, growing state is good, and ossis is relatively small, and intracavitary makes that erythrocyte number is more, and TL group bones are small Girder construction is better than model group.Bone trabecula separating degree is bigger, i.e., distance is bigger between bone trabecula, and the structure of bone is more bad, prompts bone Absorb increase, it may occur however that osteoporosis.Compared with model group, APG-3 and each dosage group bone trabecula separating degrees of PEG-rhGH are bright Aobvious to reduce, wherein APG-3 is high, the reduction of middle dose group and PEG-rhGH high dose group bone trabecula separating degrees has statistical significance (P<0.01 or P<0.05), only from the point of view of bone trabecula separating degree：Model group>SL>TL>SM>TM>SH>TH, prompt APG-3 and PEG-rhGH can improve osteoporosis.
Embodiment 7, the pharmacokinetics of fusion protein measure
The SPF levels male SD rat that selection 3-4 week old hypophysectomizes is (by National Institute for Food and Drugs Control experimental animal Center provides), 3 groups are randomly divided into, every group 3,27IU/kg PEG-rhGH (the limited duties of Changchun Kinsey medicine company are administered in single SC Ren companies), 27IU/kg (high dose group) and 9IU/kg (low dose group) APG-3 fusion proteins, investigate blood concentration and time Changing rule.Taken a blood sample upon administration through eye socket within 0,1,2,4,8,24,48,96 and 168 hour respectively, blood is placed in room temperature 5000r/min centrifuges 10min after 30min, isolates serum, -20 DEG C of preservations.It is each with being determined for ELISA method special hGH HGH contents in time point serum.By software PKSOLVER, each group main pharmacokinetic parameter is calculated.Each group medicine is for power Learn parametric results such as table 10.
The pharmacokinetic parameter of table 10, hGH fusion proteins in hypophysis rat is gone
As can be seen from the results, elimination phase half-life period of the PEG-rhGH medicines on SD rats pituitary are cut off is 9.89h, peak time are about 24h.The high dose group of fusion protein and the T of middle dose group1/2Respectively 11h and 12h, does not have substantially Have with the change of dosage and change.Compared to the rhGH of PEGylation, the half-life period of APG-3 fusion proteins is longer, the Fc on the one hand combined Variant effectively extends half-life period, on the other hand introduces CTP rigid structures, negatively charged, highly sialylated CTP can Resist scavenging action of the kidney to it so that the half-life period of fusion protein further extends.The peak time of fusion protein medicine About at 8 hours, peak is reached within 24 hours relative to PEG-rhGH, APG-3 fusion proteins work faster.As can be seen here, compared to PEG- RhGH, APG-3 show more excellent performance in biological activity, bioavilability and pharmacokinetics etc..
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.
SEQUENCE LISTING
<110>Anyuan medical sci-tech（Shanghai）Co., Ltd
<120>High-glycosylation human growth hormone (HGH) fusion protein and preparation method thereof and purposes
<130> 2016
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> PRT
<213>HCG β CTP full length amino acid sequences
<400> 1
Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu
1 5 10 15
Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
20 25 30
Gln
<210> 2
<211> 469
<212> PRT
<213>APG-3 fusion protein amino acid sequences
<400> 2
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
1 5 10 15
Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro
35 40 45
Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg
50 55 60
Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu
65 70 75 80
Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95
Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp
100 105 110
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140
Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr
145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175
Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly
180 185 190
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
195 200 205
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Ser Ser Lys Ala
210 215 220
Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp
225 230 235 240
Thr Pro Ile Leu Pro Gln Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
245 250 255
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
305 310 315 320
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
340 345 350
Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 3
<211> 1467
<212> DNA
<213>APG-3 fusion protein nucleotide sequences
<400> 3
atgcgttcac tgggggctct gctgctgctg ctgtccgctt gtctggccgt gtccgccttc 60
ccaacaatcc ccctgtcccg actgtttgac aacgcaatgc tgagggcaca ccgactgcat 120
cagctggcat tcgatactta ccaggagttt gaggaggctt atatccctaa ggaacagaaa 180
tacagcttcc tgcagaatcc acagacctct ctgtgttttt ccgagagcat tcccacacct 240
agtaaccggg aggaaactca gcagaagtcc aatctggagc tgctgagaat ctccctgctg 300
ctgattcaga gctggctgga acccgtgcag ttcctgagaa gtgtgtttgc taactcactg 360
gtgtatggag catctgacag taatgtgtac gacctgctga aggatctgga ggaaggcatc 420
cagaccctga tggggcgact ggaggacggt tcccctagga caggccagat tttcaagcag 480
acttactcta agttcgatac caacagtcac aatgacgatg ccctgctgaa aaactacggc 540
ctgctgtatt gcttcaggaa ggacatggat aaagtggaga cctttctgag aattgtgcag 600
tgccgcagcg tggaaggctc ttgtggcttt ggatccggtg gcggtggctc cggtggaggc 660
ggaagcggcg gtggaggatc aggcggtgga ggtagcggcg gaggcggtag ctccagctct 720
agtaaagctc cccctccttc cctgccctca ccctcaagac tgcctggacc ttccgacact 780
cccatcctgc cacaggtgga gtgccctcca tgtccagcac cccctgtcgc aggtccatct 840
gtgttcctgt ttccacccaa gcctaaagac cagctgatga tctcccgcac cccagaagtc 900
acctgtgtgg tcgtggatgt gagccatgaa gaccccgagg tccagttcaa ttggtacgtg 960
gatggcgtcg aggtgcacaa cgctaagaca aaacctagag aagagcagtt caactctacc 1020
tttcgcgtcg tgagtgtgct gacagtcgtg caccaggact ggctgaatgg caaggagtat 1080
aagtgcaaag tgagcaacaa aggactgcct gcctcaatcg aaaagactat ttccaagacc 1140
aaaggacagc caagagagcc ccaggtgtac accctgcctc caagccgcga agagatgact 1200
aaaaatcagg tctctctgac ctgtctggtg aaggggtttt atcctagtga tatcgccgtg 1260
gaatgggagt caaacggtca gccagagaac aattacaaga ccacaccccc tatgctggac 1320
agcgatgggt ctttctttct gtatagcaaa ctgacagtgg acaagtctcg gtggcagcag 1380
ggtaacgtct tctcttgcag tgtgctgcac gaagcactgc acaatcatta cacccagaag 1440
tcactgtcac tgagcccagg aaaatga 1467
<210> 4
<211> 227
<212> PRT
<213>The amino acid sequences of vFc γ 1
<400> 4
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 5
<211> 223
<212> PRT
<213>VFc γ 2-1 amino acid sequences
<400> 5
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
1 5 10 15
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
20 25 30
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
65 70 75 80
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile
100 105 110
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
195 200 205
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 6
<211> 223
<212> PRT
<213>VFc γ 2-2 amino acid sequences
<400> 6
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
1 5 10 15
Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr
20 25 30
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
65 70 75 80
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile
100 105 110
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu
195 200 205
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 7
<211> 229
<212> PRT
<213>The amino acid sequences of vFc γ 4
<400> 7
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225

Claims (16)

1. a kind of human growth hormone recombinant's fusion protein, it is characterised in that the fusion protein contains someone successively from N-terminal to C-terminal Ball is immunized in growth hormone, flexible peptide linker, the carboxyl terminal rigidity peptide of at least one human chorion gonadotrophic hormone beta subunit and people Albumen Fc fragments, wherein,
The general structure of the amino acid composition of the flexible peptide linker is (GS) a (GGS) b (GGGS) c (GGGGS) d, wherein a, b, C and d is greater than or equal to 0 integer, and a+b+c+d >=1；
Moreover, the carboxyl terminal rigidity peptide of the human chorion gonadotrophic hormone beta subunit is selected from following sequence units：
(i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)SSSSKAPPPS；
(iv)SRLPGPSDTPILPQ。
2. fusion protein as claimed in claim 1, it is characterised in that the human immunoglobulin(HIg) fragment becomes for human IgG Fc Body.
3. fusion protein as claimed in claim 2, it is characterised in that the human IgG Fc variants, which include, is located at wild type human At least one of IgG Fc are amino acid modified, and variant have reduce effector function and/or with neonatal receptor FcRn Binding affinity enhancing.
4. fusion protein as claimed in claim 3, it is characterised in that the human IgG Fc is selected from the group：
(a) human IgG1's hinge region, CH2 and CH3 regions containing Leu234Val, Leu235Ala and Pro331Ser mutation；
(b) human IgG2's hinge region, CH2 and CH3 regions containing Pro331Ser mutation；
(c) human IgG2's hinge region, CH2 and CH3 regions containing Pro331Ser, Thr250Gln and Met428Leu mutation；
(d) hinge region of human IgG 4, CH2 and CH3 regions containing Ser228Pro and Leu235Ala mutation.
5. the fusion protein as described in claim 1 or 4, it is characterised in that the amino acid of the flexible peptide linker is selected from as follows Sequence：
(a)GSGGGSGGGGSGGGGS；
(b)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(c)GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(d)GGGGSGGGGSGGGGSGGGGS。
6. fusion protein as claimed in claim 1, it is characterised in that the fusion protein includes 1,2,3,4 or 5 human chorionic The carboxy terminal peptide rigid element of film gonadotrophin beta subunit.
7. fusion protein as claimed in claim 1, it is characterised in that the amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 2.
A kind of 8. DNA molecular for encoding the fusion protein described in claim any one of 1-7.
9. DNA molecular as claimed in claim 8, it is characterised in that described DNA molecular has SEQID NO：Core shown in 3 Nucleotide sequence.
10. a kind of carrier, it is characterised in that include the DNA molecular as described in claim 8 or 9.
11. a kind of host cell, it is characterised in that comprising carrier as claimed in claim 10, or transfected claim Carrier described in 10.
A kind of 12. method for preparing the recombination fusion protein as described in claim any one of 1-7, it is characterised in that including as follows Step：
(a) DNA of encoding fusion protein is introduced into Chinese hamster ovary celI, generation CHO derived cells system；
(b) in screening step (a) during every 24 hours, express more than 30 μ g/106The high yield cell line of individual cell；
(c) cell line that incubation step (b) screens, expressed fusion protein；
(d) zymotic fluid that step (c) obtains, purified fusion protein are harvested.
13. the method for Prepare restructuring fusion protein as claimed in claim 12, it is characterised in that the CHO in step (a) derives Cell line is DXB-11.
14. a kind of pharmaceutical composition, it is characterised in that the composition is containing any one of with good grounds claim 1-7 claim Described fusion protein and pharmaceutically acceptable carrier.
15. the fusion protein as described in claim any one of 1-7 is used to prepare treatment because of endogenous growth hormone hyposecretion Purposes in caused disease medicament, the disease are short and small, chronic including build caused by dysplasia, Turner syndrome Renal failure, Prader-Willi syndromes or the short and small illness of idiopathic build.
16. the fusion protein as described in claim any one of 1-7 is used to prepare the purposes in treatment osteoporosis agents.
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