CN108794634A

CN108794634A - The long-acting human growth hormone (HGH) fusion protein and its preparation and use of recombination

Info

Publication number: CN108794634A
Application number: CN201710305492.9A
Authority: CN
Inventors: 许必雄; 郭颀然
Original assignee: SYNDEGEN (SHANGHAI) BIOTECHNOLOGY CO Ltd
Current assignee: SYNDEGEN (SHANGHAI) BIOTECHNOLOGY CO Ltd
Priority date: 2017-05-03
Filing date: 2017-05-03
Publication date: 2018-11-13

Abstract

The present invention provides the long-acting human growth hormone (HGH) fusion proteins and its preparation and use of recombination.Specifically, the present invention provides a kind of fusion protein, and the fusion protein has structure shown in Formulas I：In formula, X is growth hormone element；Y1 and Y2 respectively stands alone as Fc elements；L is nothing or connection peptide；"-" is covalent bond (such as peptide bond)；" ... " indicates that the n interchain disulfide bonds between Y1 and Y2, wherein n are 1,2,3,4,5 or 6,

Description

The long-acting human growth hormone (HGH) fusion protein and its preparation and use of recombination

Technical field

The present invention relates to drug fields, more particularly, to the long-acting human growth hormone (HGH) fusion protein of recombination and its preparation And purposes.

Background technology

Growth hormone (HGH) is a kind of peptide hormone, it is to be synthesized, stored and divided by the somatotroph in hypophysis It secretes.Growth hormone molecule is made of 191 amino acid residues, 22124 dalton of relative molecular mass.Growth hormone is given birth to it Long hormone receptor (hGHR) combines in target cell surface, activates biological respinse.

Growth hormone can promote the development of animal and people and the proliferation of cell.One major physiological of growth hormone is made There is promotion synthesis with being tissue especially protein various to human body, bone articular cartilage and epiphyseal cartilage can be stimulated to grow, Can thus it increase.Human body results in growth retardation once lacking growth hormone.

At present clinically, growth hormone be used to treat the growth hormone deficiency of growthretardation and adult.In addition, close There has also been applications in terms of beauty and anti-aging for growth hormone over year, and body fat can actually be made by being verified by experiments growth hormone Again it secretes, the mucopolysaccharide in subcutaneous elastic fibrous tissue can be allowed to increase, skin is made to become solid flexible.

In clinical application, since growth hormone is very short in people's Half-life in vivo, about 2 hours half-life period, so treatment Medication needs that a daily needle, and dosage period length (such as may be up to about 6 months or more) is subcutaneously injected.Frequent injection growth Hormone can make troubles to patient, and drug cost is also more expensive.In order to solve this problem, the recombination life of long-actingization is developed Long hormone is very necessary.

Human growth hormone recombinant has multiple launch both at home and abroad at present, for example, strong person of outstanding talent is peaceful, Nutropin, NORDITROPIN, match increasing, peace Su Meng etc..However, these medications are short-acting product, patient needs injection daily.

In order to develop long-acting GH, have attempted to different structure through modification or modified GH, such as PEGylated GH Or GH fusion proteins.However, these performances through modification or modified GH are still unsatisfactory, it is apparent scarce that there are some Point：Such as long-term effect relatively low (be difficult to reach medication in 1 week primary or higher long-term effect) or activity it is relatively low.For example, (poly- with PEG Ethylene glycol) change growth hormone for, although growth hormone is made by that can significantly improve drug half-life after polyethyleneglycol modified Its long-actingization, but growth hormone often causes loss of activity in PEGylated production process so that and growth hormone is reduced than living. In addition, PEGylated growth hormone can also reduce affinity of the hormone to receptor.

In conclusion it is clinically used for a long time and improves for convenience curative effect, it is novel there is an urgent need in the art to develop Long-acting and high activity growth hormone product.

Invention content

It is an object of the invention to provide a kind of novel, long-acting and high activity growth hormone product and its preparation methods and use On the way.

In the first aspect of the present invention, a kind of fusion protein is provided, the fusion protein has to be tied shown in Formulas I Structure：

In formula,

X is growth hormone element；

Y1 and Y2 respectively stands alone as Fc elements；

L is nothing or connection peptide；

"-" is covalent bond (such as peptide bond)；

" ... " indicates that the n interchain disulfide bonds between Y1 and Y2, wherein n are 1,2,3,4,5 or 6.

In another preferred example, the L is the connection peptide that length is m amino acid, and wherein m is the positive integer of 1-80.

In another preferred example, m 2-50, preferably m be 3-30 (such as 3,4,5,6,7,8,9,10,11,12,13,14, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30).

In another preferred example, the connection peptide is selected from the group：GlyGlyGlyGlySerGlyGlyGlyGlySerGly GlyGlyGlySerGlyGlyGlyGlySer、GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer、 GlyGlyGlyGlySerGlyGlyGlyGlySer、GlyGlyGlyGlySer。

In another preferred example, X is the growth hormone polypeptides of people.

In another preferred example, X has SEQ ID NO.:Amino acid sequence shown in 1.

In another preferred example, X has SEQ ID NO.:Amino acid sequence shown in 3-193 of 1.

In another preferred example, Y1 and Y2 is each independently the Fc segments of immunoglobulin.

In another preferred example, the immunoglobulin is human immunoglobulin(HIg).

In another preferred example, the immunoglobulin is selected from the group：IgG, IgE, IgA, IgM, or combinations thereof.

In another preferred example, the immunoglobulin is selected from the group：IgG1, IgG2, IgG3, IgG4, or combinations thereof.

In another preferred example, the Y1 and Y2 is the Fc segments of wild-type immunoglobulin.

In another preferred example, the Y1 and Y2 is the Fc segments of saltant type immunoglobulin.

In another preferred example, the Fc segments of the saltant type immunoglobulin have amino acid sequence selected from the group below Row：M1-IgG4-Fc, M2-IgG4-Fc, M3-IgG4-Fc, M4-IgG4-Fc, M5-IgG4-Fc, M6-IgG4-Fc or its group It closes.

In another preferred example, there are 2,3 or 4 interchain disulfide bonds between Y1 and Y2.

In another preferred example, the fusion protein has high activity, the fusion protein activity A1 and wild type The ratio between active A0 of people GH (A1/A0) be 50-200%, preferably 70-150%, be more preferably 80-120%, most preferably for 90-110%.

In another preferred example, the Y1 has SEQ ID NO.:Amino acid sequence shown in 4.

In another preferred example, the Y1 is selected from the group：M1-IgG4-Fc,M2-IgG4-Fc,M3-IgG4-Fc,M4- IgG4-Fc, M5-IgG4-Fc, M6-IgG4-Fc or combinations thereof.

In another preferred example, the fusion protein has low immunogenicity or non-immunogenicity for people.

In another preferred example, the Y1 has SEQ ID NO.:Amino acid sequence shown in 3-229 of 4.

In another preferred example, the Y1 has SEQ ID NO.:Amino acid sequence shown in 5.

In another preferred example, the Y1 has SEQ ID NO.:Amino acid sequence shown in 3-229 of 5.

In another preferred example, the Y2 has SEQ ID NO.:Amino acid sequence shown in 5.

In another preferred example, the Y2 is selected from the group：M1-IgG4-Fc,M2-IgG4-Fc,M3-IgG4-Fc,M4- IgG4-Fc, M5-IgG4-Fc, M6-IgG4-Fc or combinations thereof.

In another preferred example, the Y2 has SEQ ID NO.:Amino acid sequence shown in 4.

In another preferred example, the Y2 has SEQ ID NO.:Amino acid sequence shown in 3-229 of 4.

In another preferred example, the Y2 has SEQ ID NO.:Amino acid sequence shown in 3-229 of 5.

In the second aspect of the present invention, a kind of growth hormone fusion protein product is provided, the product contains following Component：

Growth hormone fusion protein shown in Formulas I described in component (a) the first aspect of the present invention；

Fusion protein shown in component (b) Formula II；

In formula, X, L, Y1, "-" and " ... " are as defined above；

With fusion protein shown in component (c) formula III；

In formula, Y2 and " ... " are as defined above；

Also, the product meets formula Fa：

Qa/ (Qa+Qb+Qc) × 100% >=Qsa (Fa)

In formula,

Qa is the quantity of component (a)；

Qb is the quantity of component (b)；

Qc is the quantity of component (c)；

Qsa is 85%-99.9%, preferably 90-99.5%, more preferably 90-95%.

In another preferred example, the product meets formula Fb：

Qb/ (Qa+Qb+Qc) × 100%≤Qsb (Fb)

In formula,

Qa is the quantity of component (a)；

Qb is the quantity of component (b)；

Qc is the quantity of component (c)；

Qsb is 3%-10%, preferably 3%-5%.

In another preferred example, the product meets formula Fc：

Qc/ (Qa+Qb+Qc) × 100%≤Qsc (Fc)

In formula,

Qa is the quantity of component (a)；

Qb is the quantity of component (b)；

Qc is the quantity of component (c)；

Qsc is 3%-10%, preferably 3%-6%.

In another preferred example, the product is liquid formulation, semisolid preparations or solid formulation.

In another preferred example, the product is pharmaceutical solutions or lyophilized preparation.

In another preferred example, the product is injection.

In the third aspect of the present invention, a kind of nucleic acid molecules are provided, the nucleic acid molecules are used to encode the of the present invention The nucleic acid molecules of fusion protein shown in Formulas I described in one side, and the nucleic acid molecules include the first nucleic acid molecules and Second nucleic acid molecules；

Wherein, the first polypeptide shown in the first nucleic acid molecule encoding " X-L-Y1 "；And the second nucleic acid molecule encoding " Y2 " institute The second polypeptide shown.

In the fourth aspect of the present invention, a kind of expression vector is provided, the expression vector contains the third of the present invention Nucleic acid molecules described in aspect.

In the fifth aspect of the present invention, a kind of host cell is provided, the host cell contains the 4th of the present invention Expression vector described in aspect is integrated with core described in the third aspect of the present invention in the genome of the host cell Acid molecule.

In another preferred example, the host cell includes：Prokaryotic cell or eukaryocyte.

In another preferred example, the host cell is selected from the group：Escherichia coli, yeast, CHO, BHK, HEK-293.

In another preferred example, first nucleic acid molecules and the second nucleic acid molecules are all located on same expression vector.

In another preferred example, first nucleic acid molecules and the second nucleic acid molecules are all incorporated into the host cell In genome.

In the sixth aspect of the present invention, provides and merged shown in a kind of Formulas I produced described in the first aspect of the present invention The method of albumen, including step：

(a) under conditions suitable for the expression, the host cell described in the fifth aspect of the present invention is cultivated, contains table to obtain Up to the culture solution of albumen；

(b) fusion protein shown in the Formulas I is isolated from the culture solution.

In another preferred example, the method in step (a) and (b) between, further include step (b0)：It is being suitble to more than first Peptide " X-L-Y1 " and the second polypeptide " Y2 " are incubated under conditions of forming interchain disulfide bond, are merged shown in Formulas I to be formed Albumen.

In another preferred example, in step (b), the separation includes：Saltout, affinity chromatography, ion-exchange chromatography, Hydrophobic chromatography, molecular exclusion chromatography, reversed phase chromatography, high performance liquid chromatography.

In the seventh aspect of the present invention, a kind of pharmaceutical composition is provided, the pharmaceutical composition contains the present invention's Fusion protein described in first aspect and pharmaceutically acceptable carrier.

In another preferred example, the dosage form of the pharmaceutical composition is injection, freeze drying powder injection, the injection of preliminary filling needle Agent.

In the eighth aspect of the present invention, a kind of method treated GH and lack relevant disease, including step are provided：Giving needs The fusion protein described in object the first aspect of the present invention or the growth hormone fusion egg described in the second aspect of the present invention wanted Pharmaceutical composition described in white product or the seventh aspect of the present invention.

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, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows monomeric form fusion protein (hGH-L-Fc/Fc) and duplex form fusion protein (hGH-L-Fc)₂'s Structure.

Fig. 2 GHFc-3-1 and GHFc-1-3 schematic arrangements

Fig. 3, which is shown, to be gone in SD in hypophysis rat, and various forms of growth hormone fusion proteins all have internal growth promotion Long-acting.

Specific implementation mode

The present inventor after extensive and in-depth study, passes through the engineered or modified GH's to a variety of different structures Screening for the first time it was unexpectedly observed that a kind of GH fusion proteins of specific structure not only have excellent long-term effect, but also also retains High activity, to realize the primary target of medication in 1-2 weeks for the first time.In addition, the preparation letter of the GH fusion proteins of the present invention Just, purity is high, impurity is few, it is easier to meet the strict demand of clinical application.The present invention is completed on this basis.

Term

As used herein, term " fusion protein of the present invention ", " GH fusion proteins of the invention ", " fusion egg of the invention White compound ", " GH fusion proteins compound of the invention " are used interchangeably, and refer to having described in first aspect present invention The growth hormone of the modified long-acting high activity of Formulas I structure.Typically, GH fusion proteins of the invention are that the GH of monovalence is merged Albumen.It should be understood that the term includes predominantly or the polypeptide set that is constituted of the fusion protein of all Formulas I structures, such as (i) (((component c) is constituted to be mixed component a) fusion protein shown in Formulas I for component b) and/or the second polypeptide with (ii) first polypeptide Object.

Immunoglobulin Fc element

In the present invention, applicable immunoglobulin is not particularly limited, and can be from people or other mammals Immunoglobulin element or its mutation-ure and derivative.Be preferred from the immunoglobulin of people, for example, IgG, IgE, IgA, IgM, Or combinations thereof.

Immunoglobulin G includes four subclass：IgG1,IgG2,IgG3,IgG4.The protein structure of this four subclass has Prodigious similitude, all there are four regions：One variable region (VH), three constant regions (CH1, CH2, CH3).Fc segments are by two Constant region (CH2-CH3) is formed, wherein there are one disulfide bond in the regions CH2 so that two Fc segment monomer compositions are covalently tied The homodimer of conjunction.Under normal physiological conditions, the concentration of IgG is taken second place with IgG1 highests, IgG2 in human plasma, IgG3 and IgG4 concentration is relatively low.

A kind of preferred Fc elements are human IgG 4Fc segments or its mutation-ure, derivative.

In the present invention, with GH (such as hGH) by connecting the connected Fc segments of peptide from the constant of Immunoglobulin IgG Area, it plays an important role in the immune defense of eliminating pathogen.In four kinds of human IgG hypotypes, IgG1 and IgG2, IgG3, IgG4 can be combined with hGH or its Isoforms by connecting peptide.

Since wild type IgG4-Fc, various mutations are carried out, obtain Mi-IgG4-Fc (such as M1-IgG4-Fc, M2- IgG4-Fc、M3-IgG4-Fc、M4-IgG4-Fc、M5-IgG4-Fc、M6-IgG4-Fc)。

In the present invention, a kind of preferred Fc elements are the obtained Fc elements being mutated to wild type IgG-Fc, example Such as include the IgG4-Fc of the K of mutation S228P, F234A, L235A and removal C-terminal.

In the present invention, compared with wild type IgG4-Fc, currently preferred two Fc can have each independently to be selected from The one or more of the following group or the Membrane filter of whole：

(1) two amino acid of AE are increased in the front ends wild type IgG4-Fc；

(2)S228P.The mutation can further avoid IgG4-Fc and form intrachain disulfide bond, be unlikely to form single-stranded Fc；(such as By SEQ ID NO.:The 9th sports P from S in 2)；

(3) F234A and L235A.The mutation can further decrease cell mediated cytotoxicity (ADCC)；(such as by SEQ ID NO.:The 15th of 2 sports A from F, by SEQ ID NO.:The 16th of 2 sports A from L) and

(4) K for removing C-terminal, keeps C-terminal consistent.

A kind of particularly preferred IgG4-Fc is that have the above-mentioned IgG4-Fc being all mutated, such as SEQ ID NO.:3).

Second class is mutated：So that Fc forms the mutation of heterodimer

(1) Mut3=T366S, L368A, Y407V；(such as by SEQ ID NO.:The 147th of 2 sports S from T, by SEQ ID NO.:The 149th of 2 sports A from L, by SEQ ID NO.:The 188th of 2 sports V from Y)；

(2) Mut1=T366W is such as shown in SEQ ID NO.:The 147th of 2 sports W from T.

The sequence SEQ ID NO. of original shape (i.e. wild type) IgG4-Fc:2

M1-IgG4-Fc is original shape IgG4-Fc sequences plus (1), (2), (3), (4) in above-mentioned Membrane filter；SEQ ID NO.:3

M2-IgG4-Fc is original shape IgG4-Fc sequences plus (1), (2), (3), (4) and upper in above-mentioned Membrane filter State (1) in the mutation of the second class；SEQ ID NO.:4

M3-IgG4-Fc is original shape IgG4-Fc sequences plus (1), (2), (3), (4) and upper in above-mentioned Membrane filter State (2) in the mutation of the second class；SEQ ID NO.:5

M4-IgG4-Fc is original shape IgG4-Fc sequences plus (2), (3), (4) in above-mentioned Membrane filter；That is SEQ ID NO.:3-229 of 3.

M5-IgG4-Fc is original shape IgG4-Fc sequences plus (2), (3), (4) and above-mentioned the in above-mentioned Membrane filter (1) in the mutation of two classes；That is SEQ ID NO.:3-229 of 4.

M6-IgG4-Fc is original shape IgG4-Fc sequences plus (2), (3), (4) and above-mentioned the in above-mentioned Membrane filter (2) in the mutation of two classes；That is SEQ ID NO.:3-229 of 5.

Connect peptide

The present invention provides a kind of fusion proteins, it optionally contains connection peptide.It connects peptide size and complexity may It can influence the activity of albumen.In general, connection peptide should have enough length and flexibility, to ensure that two albumen of connection exist Spatially there are enough degree of freedom to play its function.Avoid being formed α spirals or β-pleated sheet etc. simultaneously in connection peptide to fusion protein Stability influence.

The length of connection peptide is generally 1-80 amino acid, and preferably 2-50 amino acid, is more preferably 3-30 amino Acid.

In the present invention, the preferred length for connecting peptide is 2-21 amino acid, and predominantly 2 kinds to 4 kinds include glycine, silk Propylhomoserin, threonine, alanine are combined with each other.Such as the connection peptide of 5 amino acid is GlyGlyGlyGlySer, 10 amino acid Connection peptide be GlyGlyGlyGlySerGlyGlyGlyGlySer, the connection peptide of 15 amino acid is The connection peptide of GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer, 20 amino acid is GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer；Preferred flexible peptide linker For the GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer containing 15 amino acid.

In the present invention, connection peptide moiety is represented with " L " in the expression of growth hormone Fc fusion proteins, i.e., " hGH-L-Fc ".

The composition of fusion protein

In the present invention, GH molecules include change the GH molecules of structure can be by the Fc segments of different connection peptide and separate sources Connection, such as IgG1, IgG2, IgG3, IgG4 and the equal connections for changing structure form fusion protein, these fusion proteins have biology work Property and long-term effect.

In the present invention, a kind of amino acid sequence such as SEQ ID NO. of preferred GH molecules:Shown in 1：

AEFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREET QQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYS KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

In the present invention, a kind of preferred GH molecules are hGH, amino acid sequence such as SEQ ID NO.:3-193 of 1 It is shown.

In the present invention, a kind of its amino acid sequence of preferred Fc is selected from the group：SEQ ID NO.:3,SEQ IDNO.:4 and/ Or SEQ ID NO.:5.

In the present invention, a kind of preferred connection peptide is the connection peptide (GS of 15 amino acid₄)₃。

The growth hormone Fc fusion proteins (GH-L-Fc) that two kinds of various configurations have been prepared in the present invention, are monomer respectively Form is expressed as hGH-L-Fc/Fc, and length is 435 amino acid；Duplex form is expressed as (hGH-L-Fc)₂, length is 664 Amino acid, as shown in Figure 1.

The preparation of fusion protein

As used herein, " separation " refers to that substance is separated from its primal environment (if it is crude, original Beginning environment is natural surroundings).If the polynucleotides and polypeptides under the native state in active somatic cell do not isolate and purify, But same polynucleotides or polypeptide are such as separated from other substances with existing in native state, then are isolated and purified.

As used herein, " recombination fusion protein of separation " refers to recombination fusion protein substantially free of natural associated therewith Other albumen, lipid, carbohydrate or other materials.Those skilled in the art can be purified heavy with the purified technology of protein of standard Group fusion protein.Substantially pure albumen can generate single master tape in non-reducing polyacrylamide gel.

The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the egg of identical amino acid sequence with the present invention White matter segment, analogs and derivatives.The variant of this polynucleotides can be the allelic variant or non-natural naturally occurred The variant of generation.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this field institute Know, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, missing or It is inserted into, but not the function of polypeptide is encoded from it is substantially changed.

As used herein, term " primer " refers to matching with template, can be with it under the action of archaeal dna polymerase Point carries out the general name of synthesis and the oligonucleotide acid of the DNA chain of template complementation.Primer can be natural RNA, DNA, can also It is any type of natural nucleotide.Primer can even is that non-natural nucleotide such as LNA or ZNA etc..Primer " generally " (or " substantially ") and the special sequence of one on a chain in template are complementary.Primer must be abundant with a chain in template Complementation could start to extend, but the sequence of primer need not be with the sequence complete complementary of template.For example, mutual at an end 3' and template The ends 5' of the primer of benefit add the preceding paragraph and the not complementary sequence of template, such primer still generally complementary with template.As long as having Sufficiently long primer can adequately be combined with template, and non-fully it is compound can also to form primer-template with template for complementary primer Object, to be expanded.

The nucleotide full length sequence or its segment of fusion protein of the present invention or its element (such as GH, Fc) can usually use PCR Amplification, recombination method or artificial synthesized method obtain.For PCR amplification method, can according to published related nucleotide sequence, Especially open reading frame sequence carrys out design primer, the commercially available libraries cDNA is used in combination or by routine side well known by persons skilled in the art The libraries cDNA prepared by method expand as template and obtain related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly Then the segment that each time amplifies is stitched together by PCR amplification by proper order again.

Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.

It is optimized for obtaining the gene of the present invention using the method for round pcr DNA amplification/RNA.Primer for PCR It can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA segments of amplification are such as detached and purified by gel electrophoresis.

The present invention also relates to the carriers of the polynucleotides comprising the present invention, and are compiled with the carrier or fusion protein of the present invention The code genetically engineered host cell of sequence, and the method that generates protein of the present invention through recombinant technique.

By the recombinant dna technology of routine, it can be used to express or produce recombination using the polynucleotide sequence of the present invention Albumen.In general there are following steps：

(1) with the polynucleotides (or variant) of the coding albumen of the present invention of the present invention, or with containing the polynucleotide Recombinant expression carrier converts or suitable host cell of transduceing；

(2) host cell cultivated in suitable culture medium；

(3) it is separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used to build DNA sequences encoding containing albumen of the present invention and suitable The expression vector of transcription/translation control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination Technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression Carrier further includes the ribosome bind site and transcription terminator of translation initiation.

In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg (GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.

The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable When host cell, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, the bacterial cell of streptomyces；Fungal cell is such as Yeast；Plant cell；The insect cell of drosophila S2 or Sf9；The zooblast etc. of CHO, NS0, COS7 or 293 cells.

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Protein in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.If It needs, can be separated by various separation methods using its physics, chemical and other characteristics and purifying protein.These methods are It is well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine uses albumen precipitation Agent handle (salting-out method), centrifugation, infiltration break bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, from The combination of sub- displacement chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Pharmaceutical composition and method of administration

The present invention also provides a kind of compositions, it contains the fusion protein of a effective amount of present invention, and pharmaceutically may be used The carrier of receiving.In general, can nontoxic, inert and pharmaceutically acceptable aqueous load be formulated in the fusion protein of the present invention In body medium, wherein pH ordinarily is about 5-8, preferably, pH is about 6-8.

As used herein, term " effective quantity " or " effective dose " refer to that can generate function or activity to people and/or animal And the amount that can be received by people and/or animal, such as 0.001-99wt%；Preferable 0.01-95wt%；More preferably, 0.1- 90wt%.

As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad Side reaction (such as toxicity, stimulation and allergy), i.e., with rational benefit/risk than substance.Term " can pharmaceutically connect The carrier received " refers to the carrier for Therapeutic Administration, including various excipient and diluent.

The pharmaceutical composition of the present invention contains the fusion protein and pharmaceutically acceptable of the present invention of safe and effective amount Carrier.This kind of carrier includes (but being not limited to)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Usual medicine Object preparation should match with administering mode, and pharmaceutical composition of the invention can be made into injection form, such as use physiological saline Or the aqueous solution containing glucose and other adjuvants is prepared by conventional method.The pharmaceutical composition is preferably in sterile item It is manufactured under part.The dosage of active constituent is therapeutically effective amount.The pharmaceutical preparation of the present invention may also be fabricated which sustained release preparation.

The effective quantity of fusion protein of the present invention can change with the pattern of administration and the severity of disease to be treated etc.. Preferred a effective amount of selection can depending on various factors be determined by those of ordinary skill in the art (such as to be tried by clinic It tests).The factor includes but not limited to：The pharmacokinetic parameter of fusion protein of the present invention for example bioavailability, metabolism, Half-life period etc.；Patient the severity of disease to be treated, the weight of patient, the immune state of patient, administration approach etc..

Main advantages of the present invention are：

(1) fusion protein preparation process of the present invention is simple, and purifying is convenient；

(2) fusion protein of the present invention is higher than work, has apparent long-term effect；

(3) fusion protein stability of the present invention is good, and it is primary to may be implemented medication in 1-2 weeks, substantially increases patient doctor from property And reduce drug cost；

(4) present invention is true protein drug, compared with current similar best PEGylated long-acting GH formulations, is not introduced The risk of the PEGylated hepatotoxicity wind agitation brought and renal toxicity.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and Number is calculated by weight.Involved experiment material can obtain unless otherwise specified from commercially available channel in the present invention.

Method

The expression of 1 hGH-L-Fc fusion proteins

In the present invention, hGH-L-Fc fusion proteins are expressed in CHO-K1 and CHO-S cells.

The expression of fusion protein is carried out using glutamine synthelase (GS) expression vector.First by the purpose base of hGH-L-Fc Because in DNA clone to first plasmid containing GS expressed sequences, the target gene of hGH-L-Fc is driven by CMV promoter, under Trip has SV40-polyA sequences.By in Fc-only target gene DNA clone to second expression vector, the purpose base of Fc-only Reason CMV promoter drives, and there are SV40-polyA sequences in downstream.The Fc-only expression units of second expression vector are used again Purification of target DNA segment after Not-Sal digestions is cloned between the sites Not-Sal of first expression vector, is formed most Whole expression plasmid.

The sterile final expression plasmid of purifying is subjected to large scale purification, the method that electricity consumption turns is thin to CHO-K1 or CHO-S Born of the same parents transfect, and are as follows：It is inoculated into complete medium, cultivates 2-5 days after transfection, wait for that cell state restores Afterwards, liquid is changed in centrifugation, and continuing culture using Selective agar medium, (Selective agar medium is to remove the complete medium of glutamine, and add Add 20-50uM MSX), it cultivates 2-5 days, when Cell viability is begun to ramp up, is inoculated in 96 orifice plates, is made with 1-0.33/hole With Selective agar medium culture 2-4 weeks, well-grown cell strain is picked out from 96 orifice plates, the detection for carrying out expression filters out Satisfactory cell strain.

The cell strain of expression hGH-L-Fc fusion proteins monomer and hGH-L-Fc fusion protein binarys can adapt in no blood It suspends in clear culture medium after cultivating, with 2 × 10⁵Density is passaged in 1L shaking flasks, 37 DEG C of temperature, 5% CO₂In environment, 120RPM Shaking table culture to density reaches 4 × 10⁶, temperature is reduced to 32 DEG C, the nutriment such as glucose, amino acid are supplemented daily, after 5-6 days Cell viability is reduced to 80-85% to stop culture.After cell is taken out in cell liquid 2000RCF centrifugations, then in 5000RCF centrifuging and takings It is clear to carry out protein purification.

2 hGH-L-Fc fusion proteins monomers and binary isolate and purify

The purifying of fusion protein is mainly handed over using the method purifying of chromatography, such as using anion-exchange chromatography or cation It changes chromatography or uses gel permeation chromatography, or use hydrophobic chromatography, reversed phase chromatography.Hydroxylapatite adsorption layer can also be used Analysis, metal chelate chromatography etc., above-mentioned all purification steps can utilize different combinations, purity of protein finally made to reach substantially One.Also using the affinity column of the specific antibody containing the fusion protein, receptor or ligand such as recombination The mabSelectSure of rProteinA, rProteinG natural nProteinA, nProteinG or improved alkali resistant is to table The fusion protein reached is purified.

Cell culture fluid (hGH-l-Fc expression quantity about 3mg/ml) 2L containing hGH-l-Fc fusion proteins, 0.22 micron of nitre Acid cellulose film in-depth filtration, GE companies MabSelectSure affinity chromatography column purifications, 50mmol citrate buffer solutions pH3.3 Elution is often collected 10ml eluents addition 1ml 1M Tris pH8.5 and is neutralized.It collects eluent and passes through GE companies Phenyl hp Purifying, obtains final sample and carries out analysis detection.

3 hGH-L-Fc albumen Concentration Testings

The protein quantification of Fc fusion proteins is completed with two methods, is mutually compareed, i.e. lowry methods and uv-spectrophotometric inspection Survey method, method are well known General Experimental Procedures, can refer to Molecular Cloning:A Laboratory guide.

4 hGH-L-Fc SDS-Page electrophoresis detections

The SDS-Page running gels for preparing 15v/v% acrylamide concentrations, the sample buffer concentrated with 5 times dilute hGH- (5 times of concentration SDS sample buffer ingredients are 60mM Tris-HCl pH6.8,25% glycerine, 2%SDS, 14.4mM to L-Fc samples 2 mercapto ethanol, 0.1% bromophenol blue, aqueous solution；5 times concentrating sample buffer solutions and 10 microlitre samples of the electrophoresis Sample using 40 microlitres Product are sufficiently mixed), sample is boiled 5 minutes in boiling water, is centrifuged after cooling.Take supernatant loading (10-30 microlitres of loading) after centrifuging, control Constant pressure 150V processed terminates electrophoresis in about 80 minutes, and coomassie brilliant blue staining is observed after terminating electrophoresis.

Embodiment 1 investigates ratio of the two kinds of molecules of GHFc-1-3 and GHFc-3-1 in cell culture shared by monomer molecule

In order to make GHFc molecules that * 2 forms of monomeric form i.e. GH- (Fc) be more readily formed in the cell, the present invention The partial amino-acid of the parts IgG4-Fc is mutated, to which * 2 forms of GH- (Fc) be more readily formed.And the Fc of different mutation It is also the target of the invention investigated to be connect with GH, devises two kinds of molecules (see Fig. 2)：

One of which molecule be GH sequences using connection peptide with containing (1), (2), (3), (4) and upper in Membrane filter IgG4-Fc (i.e. M2-IgG4-Fc, the abbreviation mut3) connections for stating (1) in the mutation of the second class, and contain above-mentioned Membrane filter In (1), (2), (3), (4) and above-mentioned second class mutation in (2) IgG4-Fc (i.e. M3-IgG4-Fc, abbreviation mut 1) tie Close the GH-Fc fusion proteins formed, number GHFc-3-1.

Another molecule be GH sequences using connection peptide with containing in above-mentioned Membrane filter (1), (2), (3), (4) and IgG4-Fc (i.e. M3-IgG4-Fc, the abbreviation mut1) connections of (2) in the mutation of above-mentioned second class, and containing in Membrane filter (1), (2), (3), (4) and above-mentioned second class mutation in (1) IgG4-Fc (i.e. M2-IgG4-Fc, abbreviation mut3) combination The GH-Fc fusion proteins of formation, number GHFc-1-3.Remaining IgG4-Fc mutation are identical as design early period (to contain only above-mentioned first Class is mutated), mut3 and mut1 here are increased behind the mutational site designed in addition to early period to contribute to form heterodimeric The mutation (mutation of the second class) of body.

By two kinds of molecules, serum-free is expressed in same Chinese hamster ovary celI, and affinity purification obtains protein sample, utilizes SEC- HPLC analyzes expression ratio of the GHFc molecules of three kinds of different structures that may be present in cell.

GHFc-1-3 and GHFc-3-1 are expressed respectively using Chinese hamster ovary celI, with 2*10⁵Density is passaged in 1L shaking flasks, temperature 37 DEG C, 5% CO₂In environment, 120RPM shaking table cultures to density reach 4*10⁶, temperature is reduced to 32 DEG C, supplements Portugal daily The nutriment such as grape sugar, amino acid, Cell viability is reduced to 80-85% to stop culture after 5-6 days.Detect GHFc-3-1 tables in culture solution It is 3.2mg/ml up to amount, GHFc-1-3 expression quantity is 3.0mg/ml, without apparent differential expression.Utilize GE companies The culture solution purification process of two kinds of molecules of mabselectsure affinity medias pair.By GHFc-1-3 and GHFc-3-1 after purification It is analyzed using SEC-HPLC, specific chromatographic condition see the table below 1.

The chromatographic condition of table 1 SEC-HPLC analyses

As a result

Chromatogram is integrated, GHFc-3-1 expression ingredients is obtained using peak area percent method and GHFc-1-3 expresses ingredient Result see the table below 2.

2 two kinds of expression ingredient SEC-HPLC peak area percents of table

Wherein A is GH-Fc monomeric forms, that is, GH- (Fc) * 2；B is that GH-Fc duplex forms are (GH-Fc) * 2.

C is that the FcFc that Fc independent fragments are formed is (Fcmut3) * 2 or (Fcmut1) * 2, other to be expressed in cell for molecule The aggressiveness and catabolite formed in the process.

Conclusion

By analysis of experimental results, expression quantity of two kinds of molecules in cell culture fluid is almost the same, can reach The ratio of 3mg/ml, but by purifying post analysis, A, that is, monomer molecule in GHFc-3-1 molecules needed for us is more than 90% Monomer molecule in 92.8%, GHFc-1-3 molecule needed for us is 87.6%.In view of the product that we finally need is A, Remaining impurity is removed in isolating and purifying, such GHFc-3-1 molecules are more superior compared to GHFc-1-3 molecules.

2 hGH-L-Fc Bioactivities of embodiment are analyzed

Growth hormone Determination of biological activity is a kind of receptor potency increase measuring method measurement i.e. BAF methods based on cell.

BAF cell lines have reaction with dosage relative growth pattern to GH, therefore can be used for evaluating GH in proliferation assay Effect, BAF cell strains are incubated in DMEM high glucose mediums, rear to add 10% horse serum culture, and incubation time 16 hours is adjusted Whole cell density is 3*10^5/ml, takes 100 microlitres to drip in 96 orifice plates after mixing.Dilution standard GH solution is to final concentration of 0.01-1000.00ug/L takes 10ul standard GH solution to be added drop-wise in 96 orifice plates, cultivates 22 hours.22ul reactions are added per hole Liquid continues culture 3 hours, and light absorption value is measured at 490nm.The standard for drawing light absorption value and GH bioactivity equivalent relations is bent Line.It takes different growth hormone compound 10ul to be added drop-wise in 96 orifice plates to cultivate 22 hours, 22ul reaction solutions is added per hole, continue Culture 3 hours is measured light absorption value at 490nm, the active equivalents of different growth hormone compounds is calculated using standard curve.Mark Directrix curve takes the wild type GH and GHFc-3-1 of equimolar GH to carry out active comparison, the molecule of the GHFc-3-1 detected after generating Activity is suitable with the GH activity of wild type human, and ratio is 90%-110% (being the about 90-110% of wild type human GH).

3 hGH-L-Fc of embodiment pharmacodynamic experiments in Sprague Dawley (SD) rat

3.1 rat pituitary resections

The preceding 2 weeks clean conditions menisectomy Hypophysectomy of experiment, first with 25% urethane solution (0.3ml/100g mouse weight) Shallow fiber crops big white mouse, makes big white mouse lie low on rat frame, fixes 4 limbs and mouth, along the nearly lower jaw of cervical midline, one is cut with scissors It is about the notch of 1cm, both sides separate to the left and right by salivary gland, after left side crust is clamped with hemostat, pull open outward, you can clear Chu sees the sternothyroid for being covered in tracheae, the nearly pharyngeal place on the left of this flesh, with the layers-separated muscle of the curved tweezer of ophthalmology until touching Bone plate.Then the cleared muscle being attached on bone plate of cotton pellet is lived with small sharp spoon or with the curved tweezer of ophthalmology, on pharyngeal underface, a left side Between right two otic capsules, " ┬ " shape protrusion can be contacted, all dried the muscle of near " ┬ " shape and protruding part with cotton pellet Only, you can it is clearly seen from " ┬ " shape protrusion, there is a navy blue ugliness and ferociousness line (spheno-occipital cartilage joint) on pleuron, it is horizontal herein A small shrinkage pool is pierced with probe on the vertical same straight line of dashing forward of " ┬ " shape protrusion at 1mm above line, it is recessed that hand drill is then fixed on this At hole, hand drill is gently rotated, avoids overexertion and pushing, when sense pore has drilled through on hand, i.e., with examination in a small needle access aperture It visits, drilling should be tangent with horizontal line.When carrying out hypophysis absorption, one end is directed at drilling with the hypophysis suction pipe that suction bottle is connected, is opened Dynamic aspiration pump, draws hypophysis, after hypophysis suction, dries blood with cotton balls, immediately sutures skin incision, and is compiled to animal Number, weigh.Experimental animal pituitectomy is completed in 2 days.In inadvertent closure, if there is asphyxia phenomenon in animal, Can it be inserted into animal throat with a glass blowpipe immediately, it is rhythmical to tracheal gas insufflation, it is rescued.After operation rat feeding in In barrier environment, postoperative preceding 3 days rats drink normal water after 5% glucose water makes it restore.

The qualified animal of 3.2 screenings and animal packet

The 1st week is convalescence after going hypophysis to perform the operation, and weighs and records from the 2nd week, and weighed every 1 day 1 time up to Before medicine experiment, big white mouse of the changes of weight more than the 2nd week first day weight ± 10% after operation is weeded out in time, i.e. selection is given Rat body weight changes the qualified healthy animal less than the 2nd week first day weight ± 10% after operation before medicine experiment, equal by weight Even random grouping, goes hypophysis rat for totally 5 groups by 7/group.

3.3 different human growth hormone recombinant's fusion protein administrations

HGH treatment puberty short patients' clinical dosages country is generally：0.15~0.2IU/kg/d (ratify by U.S. FDA 0.3IU/kg/d), because rat effective dose is 6.3 times of people's clinical dosage (being calculated by body surface area), therefore it is converted to rat dosage For：0.2IU/kg/d × 6.3=1.26IU/kg/d goes hypophysis rat to be calculated by average 80g, experimental period 14d, 1.26IU/ Kg/d × 14d=17.6IU/kg, that is, 1.4IU/80g mouse/14d.That is administration hGH needs 5.8mg/kg (hGH standard items for two weeks altogether 1mg=3.0IU).According to the dosage in hGH, the PEG-hGH of different molecular weight, hGH-1-Fc/Fc and (hGH-1-Fc)₂Give The identical hGH amounts, that is, 5.8mgGH/kg of medicine compares the long lasting growth hormone of identical GH contents to going hypophysis mouse body weight increase It influences.

Administration route：Neck is subcutaneously injected

Administered volume：Per 80g mouse per injection 0.5ml, dosage is adjusted with weight gain, such as 100g mouse per injections 0.625ml。

Administration time：Test solution and positive drug are each per mouse neck subcutaneous administrations d1 and d8 with reference to product PEG-hGH It is administered once and (is administered once within one week), rhGH same times daily, 1 time/d, continuous 14d.

3.4 Testing index (weight gain)

Every rat is weighed daily after administration, and d15 carbon dioxide asphyxia puts to death whole rats, weighs.Every animal i-th day (di) weight (bw_i) and preceding i.e. the 0th day (d0) weight (bw of administration₀) difference, that is, increased body weight in grams be that weight gain is (necessary When, it can perform an autopsy on sb. after experiment, cut sella region, whether there is or not hypophysis residual, rejecting has the dynamic of hypophysis remaining for visual inspection Object).Weight gain calculation formula：Δ bw=bw_i–bw₀Wherein：Δbw：Weight gain；bw₀：D0 weight, bw before administration_i：Administration I-th day weight afterwards.

Testing result such as the following table 3：

(d8) and (d15) weight gain after 2 weeks after 3 hGH-Fc fusion proteins of table are administered 1 week (N=5)

It can significantly find out from Fig. 3, be gone in hypophysis rat in SD, various forms of growth hormone fusion proteins have There is internal somatotrophic effect.

It is surprising that animal experiment shows in the case where being administered once within 1 week, at the same time under identical GH dosage, this Invention fusion protein hGH-L-Fc/Fc (monomer of asymmetric modification) shows more excellent, long-acting growth promoting function.? At the 15th day, the growth promoting function of fusion protein of the present invention is not only above current commodity PEG-hGH (improving about 40%), more much Higher than (the symmetrical GH-Fc dimers of similar modification) (29.5/8.5=347%), increase rate reaches about 250%.

In addition, fusion protein hGH-L-Fc/Fc of the present invention also unexpectedly have the characteristics that it is superior quick-acting.The 7th It when, the growth promoting function of fusion protein of the present invention is not only above current commodity PEG-hGH (improving about 69%), farther to be far above (hGH-L-Fc)₂(the symmetrical GH-Fc dimers of similar modification) (14.2/2.8=507%), increase rate reaches about 410%.

It discusses

The present invention has prepared the monomer and binary of growth hormone fusion protein, i.e., the growth hormone fusion protein of monovalence and The growth hormone fusion protein of divalent, due to that can have a large amount of binary and Fc simultaneously when growth hormone Fc fusion protein monomers are expressed Monomer exist, isolate and purify it is extremely difficult, separation yield it is relatively low；And the fusion protein of hGH-Fc duplex forms can be with them target Molecule such as receptor forms steric hindrance, and the Fc fusion protein activity of this duplex form is caused to decline.

The present invention is in order to improve the ratio of the growth hormone Fc fusion proteins of monomeric form in cell expression so that purifying Yield improves and Simple Purification step, and having carried out part to fusion protein structure is transformed, and has obtained growth hormone Fc fusion proteins Monomer purity is up to 93% monomer product, and zoopery evaluating drug effect shows that such monomer product activity merges egg than dimer The PEGylated fusion protein of bletilla is all significantly improved, and possesses good long-term effect.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

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Claims

1. a kind of fusion protein, which is characterized in that the fusion protein has structure shown in Formulas I：

In formula,

X is growth hormone element；

Y1 and Y2 respectively stands alone as Fc elements；

L is nothing or connection peptide；

"-" is covalent bond (such as peptide bond)；

2. fusion protein as described in claim 1, which is characterized in that the Y1 has SEQ ID NO.:Amino shown in 4 Acid sequence.

3. fusion protein as described in claim 1, which is characterized in that the Y2 has SEQ ID NO.:Amino shown in 5 Acid sequence.

4. a kind of growth hormone fusion protein product, which is characterized in that the product contains following components：

Growth hormone fusion protein shown in component (a) Formulas I described in claim 1；

Fusion protein shown in component (b) Formula II；

In formula, X, L, Y1, "-" and " ... " are as defined above；

With fusion protein shown in component (c) formula III；

In formula, Y2 and " ... " are as defined above；

Also, the product meets formula Fa：

Qa/ (Qa+Qb+Qc) × 100% >=Qsa (Fa)

In formula,

Qa is the quantity of component (a)；

Qb is the quantity of component (b)；

Qc is the quantity of component (c)；

Qsa is 85%-99.9%, preferably 90-99.5%, more preferably 90-95%.

5. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules melt for encoding shown in Formulas I described in claim 1 The nucleic acid molecules of hop protein, and the nucleic acid molecules include the first nucleic acid molecules and the second nucleic acid molecules；

Wherein, the first polypeptide shown in the first nucleic acid molecule encoding " X-L-Y1 "；And shown in the second nucleic acid molecule encoding " Y2 " Second polypeptide.

6. a kind of expression vector, which is characterized in that the expression vector contains the nucleic acid molecules described in claim 5.

7. a kind of host cell, which is characterized in that the host cell contains the expression vector described in claim 6, or The nucleic acid molecules described in claim 5 are integrated in the genome of the host cell.

8. a kind of method producing fusion protein shown in Formulas I described in claim 1, which is characterized in that including step：

(a) under conditions suitable for the expression, the host cell described in claim 7 is cultivated, to obtain the training of the albumen containing expression Nutrient solution；

9. a kind of pharmaceutical composition, which is characterized in that the pharmaceutical composition contains any described in claim 1-3 melt Hop protein and pharmaceutically acceptable carrier.

10. a kind of method treated GH and lack relevant disease, which is characterized in that including step：The object entitlement for giving needs is wanted Ask the growth hormone fusion protein product in 1-3 described in any fusion protein or claim 4 or claim 9 institute The pharmaceutical composition stated.