CN102875683A

CN102875683A - Fc fusion protein of long-acting recombinant human growth hormone

Info

Publication number: CN102875683A
Application number: CN2011101932103A
Authority: CN
Inventors: 金宜慧; 刘瑞贤; 周若芸; 严孝强; 王宇鹏; 李强; 孙乃超
Original assignee: Xuhua (shanghai) Biological Research & Technology Center Co Ltd
Current assignee: Xuhua (shanghai) Biological Research & Technology Center Co Ltd
Priority date: 2011-07-11
Filing date: 2011-07-11
Publication date: 2013-01-16
Anticipated expiration: 2031-07-11
Also published as: CN102875683B

Abstract

The invention discloses a Fc fusion protein of long-acting recombinant human growth hormone. The Fc fusion protein (hGH-L-vFc fusion protein) disclosed herein contains human growth hormone, flexible peptide linker of about 2-20 amino acids, and human IgG Fc mutant. The Fc mutant is not lytic and has tiny side effect of adverse Fc-mediator. The invention further discloses a method for preparing or generating the fusion protein with high expression level. The hGH-L-vFc fusion protein disclosed herein has prolonged serum half-life period and increased biological activity, so as to improve the pharmacokinetics and drug efficacy, and needs few times of injection required in treatment.

Description

The Fc fusion rotein of long-acting recombinant human tethelin

Technical field

The present invention relates to molecular biology and medical field.More specifically, the present invention relates to a kind of Fc fusion rotein and method for making and purposes of long-acting recombinant human tethelin.

Background technology

Human growth hormone (hGH) is to be produced and release by pituitary body, and 191 amino acid of total length, molecular weight are the peptide hormone of 22kDa.It has participated in most people's normal growth and the regulation and control of growth, is the major hormone that stimulates physical growth, and presents multiple biological effect, comprises linear growth, physique formation, lactation, macrophage activation and similar insulin action etc.In addition, tethelin can also stimulate the metabolism of bone, cartilage and muscle.

Tethelin growth hormone receptor special with it (hGHR) mutually combines on the target cell surface, mediates out biochemical cascade reaction, thereby activates biological effect.A kind of sequential mechanism is followed in the combination of tethelin and acceptor molecule usually; Be combined with first acceptor molecule by the binding site 1 in the tethelin first, be combined with second acceptor molecule by the binding site 2 of tethelin more thereafter.This combination by a tethelin molecule and two growth hormone receptor molecules is to intensify the necessary step of biological activity, growth regulation and growth.Particularly, binding site 2 has comprised 8 amino acid and other several amino acid from belonging in spiral-line 1 and the spiral-line 3 of tethelin molecule N end.1 of binding site comprises near the amino acid of the tethelin molecule amino acid within the spiral-line 1 and C-end.8 amino acid of binding site 1 have accounted in conjunction with 85% of total energy.In other words, these 8 amino acid with the process of receptors bind in played the part of conclusive role.If the position of these 8 amino-acid residues with other aminoacid replacement, will be changed rapidly its ability of being combined with growth hormone receptor (for example seeing Wells et al.Recent Prog.Horm.Res., 48:253-75,1993).This 8 seed amino acid is section (K41, L45, P61 between spiral-line 1 and spiral-line 2, R64) and the end of the carboxyl C in the spiral-line 4 half section (K172, T175, F176, and 4 amino-acid residues of its back segment just in time are positioned near the C-terminal of 191 amino acid whose tethelin of total length R178).Therefore near the amino acid the tethelin C end with the process of receptors bind in very important.

In other words, amino acid and its function of tethelin C end are closely related, hold so at present GH is carried out usually avoiding C when genetic engineering modified, also do not adopt the C-terminal of people GH and the mode that other albumen merges.

Children and adult are in the growth hormone deficiency situation, and replenishing recombinant human somatropin (rhGH) is desirable therapeutic modality.But its shortcoming is that recombinant human somatropin's drug effect in human body is very short, and its intravenous serum is removed about 20 minutes of transformation period.If with the subcutaneous injection recombinant human somatropin, its peak Plasma Concentration is wanted just can reach after several hours usually, and it eliminates the transformation period then is 3 to 8 hours.Therefore, recombinant human somatropin's treatment need to be injected weekly 3 times, or once a day, to keep suitable serum growth hormone level.Need to accept for a long time the patient of growth hormone therapy for those, often owing to can not inject on time, cause result for the treatment of to reduce.Long-acting, highly active recombinant human somatropin thereby the target that becomes medicament for this reason to improve.

Between 1999 to 2004, gene engineering (Genentech) company and A Erkaimosi (Alkermes) limited-liability company develop Nutropin Depot in market sale, this product is a kind of tethelin of sustained release form, the long-acting characteristic of tool, its frequency injection of the patient who receives treatment can reduce to for per 2 or 4 weeks once, and did not need injection every day.But since production cost too high, this product in 2004 by withdraw.

During this period, several human cytokines medicines are through carrying out that structural modification prolongs that half its phase and improving of declining tired in the body and the s-generation product that created these pharmaceutical grade proteins with some polymkeric substance such as polyoxyethylene glycol (PEG).The combination of protein drug and polyoxyethylene glycol covalency can increase the valid period of protein usually, and reduces its clearance rate in human body.The recombinant human somatropin is no exception.The document of some relevant polyoxyethylene glycol tethelin shows that several multi-form polyoxyethylene glycol tethelin have the transformation period longer than the restructuring human growth hormone really, but have often caused bioactive loss in the Pegylation process of protein.

The immunoglobulin (Ig) of IgG class is rich in protein in the human blood.Their transformation period can be up to 21 days.Existing report with the Fc zone of IgG and other oroteins (such as various cytokines and soluble receptors) in conjunction with and form fusion rotein (referring to, such as people such as Capon, Nature, 337:525-531,1989; The people such as Chamow, Trends Biotechnol., 14:52-60,1996; U.S. Patent No. 5,116,964 and 5,541,087).Typical fusion rotein is a heavy protein dimer, is to be connected with albumen by the cysteine residues in the IgG Fc hinge region, and forms similar IgG but lack the molecule of CH1 zone and light chain.Because structural homology, the external pharmacokinetic properties that the Fc fusion rotein shows and the human IgG of isotype are quite similar.Therefore make and contain the hGH fusion rotein that links to each other with the Fc zone of human IgG protein, the biological activity that will help to prolong the circulating half-life of hGH and/or increase it.

In addition, because tethelin molecule C terminal amino acid in bioactive high importance, therefore must consider how to overcome the consequence of bringing when connecting the Fc halfbody in advance when making up hGH-Fc fusion rotein (hGH-Fc).If directly huge IgG Fc halfbody is bound up on the C end, can causes space steric effect for the C end, and affect widely the affable degree of tethelin binding site to acceptor.At first, the combination of fusion rotein and first growth hormone receptor will be impaired because of the steric hindrance that the Fc halfbody brings, the second acceptor on its subsequent and cell surface follow-up in conjunction with may further weakening because of this steric effect.Therefore, according to the amino acid whose importance of above-mentioned tethelin C-terminal (K172, T175, F176, R178), the hGH-Fc fusion rotein will be damaged to the avidity of acceptor, cause its biological activity to reduce even forfeiture.Therefore present technology all is to avoid the Fc halfbody directly is bound up on the C-terminal, in order to avoid its activity wrecks.Most of recombinant proteins all are attached to the Fc halfbody N-terminal of tethelin molecule.But this substitute mode also has its defective, because the binding site of tethelin molecule and its acceptor is distributed in two terminals, no matter be that Fc is received N end or C end, the biological activity of fusion rotein all may go to pot because of the steric effect that Fc brings.

Owing to its difficulty in essence that is built with of hGH-L-vFc fusion rotein, still both do not had up to now the gratifying GH derivative of transformation period significant prolongation.Therefore, this area, high reactivity long-acting in the urgent need to developing, the hGH derivative that can produce with rational cost.

Summary of the invention

Purpose of the present invention just provides a kind of highly bioactive hGH-L-vFc fusion rotein and its production and use that has.

In a first aspect of the present invention, a kind of restructuring hGH-L-vFc fusion rotein is provided, described fusion rotein holds the C end to contain successively people GH, peptide linker and human IgG Fc variant from N,

And described human IgG Fc variant is selected from lower group:

(i) contain human IgG2's hinge region, CH2 and the CH3 zone that Pro331Ser suddenlys change;

(ii) contain human IgG 4 hinge regions, CH2 and the CH3 zone that Ser228Pro and Leu235Ala suddenly change;

(iii) contain human IgG l hinge region, CH2 and the CH3 zone that Leu234Val, Leu235Ala and Pro331Ser suddenly change.

In another preference, described peptide linker contains 2-20 amino acid, and described peptide linker is present between people GH and the human IgG Fc variant; And described peptide linker contains two or more amino acid that are selected from glycine, Serine, L-Ala and Threonine.

In another preference, the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:18,20 or 22.

In another preference, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:18,20 or 22 that has removed after the hGH leading peptide of-26 to-1 amino acids residue.

In another preference, described hGH-L-vFc fusion rotein has the Bioactivity similar or higher with rhGH, longer transformation period on mole foundation.

In a second aspect of the present invention, provide the described restructuring of a kind of first aspect present invention of the encoding hGH-L-vFc dna molecular of fusion rotein.

In a third aspect of the present invention, the strain of a kind of CHO derived cell is provided, described cell in its growth medium in per 24 hours, produce to surpass 1,000,000 cells of 10 μ g/ such as the described restructuring of first aspect present invention hGH-L-vFc fusion rotein.

In another preference, the strain of described CHO derived cell in per 24 hours, produces the described restructuring of the first aspect present invention hGH-L-vFc fusion rotein that surpasses 1,000,000 cells of 30 μ g/ in growth medium.

In another preference, the cell strain that described CHO derives contains the dna sequence dna of coding hGH-L-vFc fusion rotein, and described dna sequence dna has the nucleotide sequence shown in the SEQ ID NO:17,19 or 21.

In a fourth aspect of the present invention, a kind of method for preparing the described recombination fusion protein of first aspect present invention is provided, comprise step:

(a) fusion rotein in its growth medium during per 24 hours in, express to surpass 10 μ g/10 ⁶Under the condition of (1,000,000) individual cell, cultivate the described cell strain of a third aspect of the present invention; With

(b) protein of purification step (a) expression, wherein recombination fusion protein has the Bioactivity similar or higher with rhGH on mole foundation, the longer transformation period.

In another preference, described recombination fusion protein has 2-20 amino acid whose flexible peptide between people GH and human IgG Fc variant; And described flexible peptide linker contains the amino acid that two or more are selected from glycine, Serine, L-Ala and Threonine.

In another preference, described fusion rotein aminoacid sequence such as SEQ ID NO:18,20 or 22 not.

In a fifth aspect of the present invention, the method for the expression amount of the recombination fusion protein that a kind of raising contains people GH, flexible peptide linker and human IgG Fc variant is provided, described method comprises:

(a) DNA with encoding fusion protein introduces Chinese hamster ovary celI, generates the clone that CHO derives;

(b) cultivate the clone that this CHO derives, thus expressed fusion protein; With

(c) fusion rotein of purification step (b) expression,

Wherein recombination fusion protein is the hGH-L-vFc fusion rotein, it is characterized by to show high Bioactivity, namely on mole foundation, has and the similar or higher Bioactivity of people GH and longer transformation period; Wherein between people GH and IgG Fc variant, exist and contain 2-20 the amino acid whose flexible peptide linker of having an appointment; Contain the amino acid that 2 or a plurality of amino acid are selected from glycine, Serine, L-Ala and Threonine with flexible peptide linker; Wherein human IgG Fc variant contains hinge region, CH2 and the CH3 zone that is selected from following human IgG: the human IgG2 of Pro331Ser sudden change; The human IgG 4 of Ser228Pro and Leu235Ala sudden change; Human IgG1 with Leu234Val, Leu235Ala and Pro331Ser sudden change.

In another preference, the DNA of described encoding fusion protein has the nucleotide sequence shown in the SEQ ID NO:17,19 or 21.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.

Description of drawings

Fig. 1 has shown the comparison of the aminoacid sequence in the hinge region of human IgG1, IgG2, IgG4 and their variants and CH2 zone.Compare three parts: amino acid region 228,234-237 and 330-331.The amino acid mutation of these variants shows with bold Italic.The amino-acid residue numbering is to demarcate according to the EU number system.

Fig. 2 has shown the hGH-L-vFc of HindIII-EcoRI fragment in the pGFP expression vector _{γ 2}Nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 is to the leading peptide of the-1st, hGH.Maturation protein contains hGH (amino-acid residue 1 to 191), peptide linker (amino-acid residue 192 to 207) and Fc variant (amino-acid residue 208 to 430).In the Fc zone, the Nucleotide of runic and corresponding amino acid variant mark with underscore.

Fig. 3 has shown the hGH-L-vFc of HindIII-EcoRI fragment in the pGFP expression vector _{γ 4}Nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 is to the leading peptide of the-1st, hGH.Maturation protein contains hGH (amino-acid residue 1 to 191), peptide linker (amino-acid residue 192 to 207) and Fc variant (amino-acid residue 208 to 436).In the Fc zone, the Nucleotide of runic and corresponding amino acid variant mark with underscore.

Fig. 4 has shown the hGH-L-vFc of HindIII-EcoRI fragment in the pGFP expression vector _{γ 1}Nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 is to the leading peptide of the-1st, hGH.Maturation protein contains hGH (amino-acid residue 1 to 191), peptide linker (amino-acid residue 192 to 207) and Fc variant (amino-acid residue 208 to 434).In the Fc zone, the Nucleotide of runic and corresponding amino acid variant mark with underscore.

Fig. 5 shows growth and the secretion hGH-LvFc thereof of rotating and culturing bottle inner cell strain _{γ 2}The concentration trend curve figure of fusion rotein.

Fig. 6 has shown hGH-L-vFc _{γ 2}Purifying protein stimulates the ability of Nb2 cell proliferation.

Embodiment

The inventor has designed first a kind of original hinge region peptide linker and has reduced space steric effect through long-term and deep research, and the C that can make hGH holds the fusion rotein that is connected with Fc, and there is soft peptide linker the centre.Surprisingly, this fusion rotein not only can not cause the afunction of GH, can keep even improve on the contrary the biological activity of tethelin-Fc fusion rotein.Finished on this basis the present invention.

Particularly, hGH-L-vFc fusion rotein of the present invention holds the C end to contain successively people GH, peptide linker and human IgG Fc variant from N.Preferably, wherein people Ig Fc variant contains and is selected from following variant hinge region, CH2 and CH3 zone: the human IgG1 of (A) containing Leu234Val, Leu235Ala and Pro331Ser sudden change; (B) contain the human IgG2 that Pro331Ser suddenlys change; (C) contain the human IgG 4 that Ser228Pro and Leu235Ala suddenly change.Preferably, use flexible peptide linker about 2-20 amino acid length, that contain following 2 kinds or multiple amino acids formation: glycine, Serine, L-Ala and Threonine.IgG Fc variant is non-cracking performance, and compares with natural IgG Fc and to contain amino acid mutation.This type of hGH-L-vFc fusion rotein has the Bioactivity similar or higher with rhGH on mole foundation.And hGH-L-vFc _{γ 2}Body in serum remove the transformation period and than rhGH phenomenal growth arranged.

Another embodiment of the present invention behaviour Ig Fc variant contains hinge region, CH2 and CH3 zone.Amino acid mutation is contained 228,234,235 and 331 (by the definite positions of EU number system) in its CH2 zone, thereby reduces the effector function of Fc.

In another embodiment of the present invention, a kind of clone preparation of deriving from mammal cell line such as CHO or the method for producing this recombination fusion protein are disclosed.Cultivate the clone of transfection so that recombination fusion protein in its growth medium during 24 hours to surpass 10 (preferably as 30) μ g/10 ⁶Express under the level of (1,000,000) individual cell.These hGH-L-vFc fusion roteins demonstrate the Bioactivity of height and the interior serum half-life of longer body and without bad side effect, thereby have improved pharmacokinetics and drug effect, and then have reduced dosage and the frequency injection of realizing that similar drug effect is required.

In addition, the inventor also finds, the peptide linker that adds between hGH and human IgG Fc variant improves the Bioactivity of hGH-L-Fc in two ways: (1) makes the Fc zone away from the hGHR binding site on the hGH, (2) make a hGH away from another hGH structural domain, thus make two hGH zones can be respectively with target cell on the hGHR reaction.And people's IgG Fc variant contains amino acid mutation in the CH2 zone in 228,234,235,331 sites, thereby reduces the effector function of Fc.

The Fc element

The Fc element is from the Fc zone of immunoglobulin (Ig), and Fc is the tool vital role in the immune defense of eliminating pathogen.The effector function of IgG passes through two kinds of main mechanisms by the Fc mediation: the combination of (1) and cell surface Fc acceptor (Fc γ Rs), cause by antibody-dependent cellular cytotoxicity (ADCC) approach, by killer cell by phagolysis or splitting action and Pharynx gulps down pathogenic agent; (2) with the combination of the Clq of the first complement component Cl part, cause cytotoxicity (CDC) approach that depends on complement, thus the dissolving pathogenic agent.In four kinds of human IgG isotypes, IgG1 and IgG3 can be effectively in conjunction with Fc γ R.The binding affinity of IgG4 and Fc γ R is than low order of magnitude of IgG1 or IgG3, and IgG2 and Fc γ R in conjunction with low the mensuration that is difficult to.Human IgG1 and IgG3 can also be effectively in conjunction with Clq, and the activating complement cascade reaction.As if the human IgG2 is very weak to the fixed action of complement, and IgG4 is quite lacking aspect the ability of activating complement cascade reaction.For being applied to people's treatment, when the hGH-Fc fusion rotein was incorporated into the lip-deep hGHR of target cell, must can not there be the ill effect subfunction in the Fc of fusion rotein zone, thereby can not dissolve or remove these target cell.Therefore, the necessary right and wrong in the Fc of hGH-Fc zone are deliquescent, thereby to being incorporated into Fc γ Rs and Clq trigger effect subfunction aspect, the Fc zone must be non-activity.Obviously, there is not a kind of natural IgG isotype to be fit to produce the hGH-Fc fusion rotein.In order to obtain the Fc of non-solubility, must make some amino acid mutations in the natural Fc zone, to reduce its effector function.

See through the relatively demonstration of aminoacid sequence of the IgG isotype of people and mouse, near sequence in the combination of IgG Fc and Fc γ Rs the tool vital role of the Fc fragment N-terminal of CH2 zone.Its 234 to the importance of 237 motifs in the antibody of genetically engineered construction proof (referring to, such as people such as Duncan, Nature, 332:563-564,1988).Amino-acid residue that the present invention carries numbering is to demarcate (" SEQUENCES of PROTEINS of IMMUNOLOGICAL INTEREST " according to the described EU number system of the people such as Kabat, the 5th edition, United States Department of Health and Human Services, 1991).In four kinds of human IgG isotypes, the bonding force of IgG1 and IgG3 and Fc γ Rs is the highest, and both have identical Leu234-Leu-Gly-Gly237 sequence (Fig. 1).IgG4 is very low with the avidity that Fc γ Rs is combined, and sees its sequence and shows amino acid and replaced, and namely Leu changes Phe on 234 sites.Not in IgG2 that Fc γ Rs is combined, occur then that replace in two sites and a site is deleted, thus formation Val234-Ala-Gly237 sequence (Fig. 1).For the combination that reduces Fc and Fc γ R and ADCC active, the Leu235 among the IgG4 can replace with Ala (referring to, such as people such as Hutchins, Proc.Natl.Acad.Sci.USA, 92:11980-11984,1995).Glu233-Leu-Leu235 sequence in the IgG1 antibody was once replaced with the Pro233-Val-Ala235 correlated series among the IgG2.This change has lost the IgG1 variant and sees through the ability that target cell is removed in Fc γ R-mediation in mouse.

About antibody to Fc γ R be combined with Clq CH2 zone that vital the second position is located in human IgG near carboxyl terminal neighbouring (referring to, such as people such as Duncan, Nature, 332:738-740,1988).In four kinds of human IgG isotypes, only have a site to show in this part and replace: the Ser330 among the IgG4 and Ser331 have replaced Ala330 and the Pro331 (Fig. 1) among IgG1, IgG2 and the IgG3.The existence of Ser330 does not affect the combination of Fc γ R and Clq.Substitute Pro331 with Ser and then make IgG1 lose binding affinity with Clq, and with Pro substitute Ser331 partly kept the complement fixation of IgG4 active (referring to, such as people such as Tao, J.Exp.Med., 178:661-667,1993; The people such as Xu, J.Biol.Chem., 269:3469-3474,1994).

Fusion rotein and production method thereof

The invention provides the novel effective hGH-L-Fc fusion rotein of a class, the Fc element in the fusion rotein is variant (vFc), to be used for making up efficient hGH-L-vFc fusion rotein.Human IgG2's debond Fc γ R, but demonstrate faint complement activity.Fc with Pro331Ser sudden change _{γ 2}Variant should be than natural Fc _{γ 2}The variant activity lower, and still debond in Fc γ R.IgG4Fc is activating variant cascade reaction defectiveness, and it hangs down about order of magnitude (ten times) with the highest isotype (IgG1) of the binding affinity specific activity of Fc γ R.With natural Fc _{γ 4}Compare, have the Fc of Leu235Ala sudden change _{γ 4}Variant should show

Little effector function.Fc with Leu234Val, Leu235Ala and Pro331Ser sudden change _{γ 1}Also show than natural Fc _{γ 1}Much lower effector function.These Fc variants all are more suitable for preparing the hGH fusion rotein than naturally occurring human IgG Fc.In the preparation of non-dissolving Fc, also can introduce other and replace, and not jeopardize circulating half-life or cause bad conformational change.

Fusion rotein of the present invention is usually by biosynthetic method preparation.According to nucleotide sequence of the present invention, the art personnel can make coding nucleic acid of the present invention with various currently known methodss easily.These methods are such as but not limited to PCR, DNA synthetic etc., and concrete method can be referring to J. Pehanorm Brooker, " molecular cloning experiment guide ".As one embodiment of the present invention, can make up nucleic acid sequence encoding of the present invention by the method that the salvage nucleotide sequence carries out overlapping extension PCR again.

The present invention also provides a kind of expression vector, comprises the encode sequence of fusion rotein of the present invention and the expression regulation sequence that links to each other of operability with it.Described " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and namely the activity of same linear DNA sequence other parts can be regulated or control to some part of linear DNA sequence.For example, if the transcribing of promotor control sequence, it is exactly operationally to be connected in encoding sequence so.

Expression vector can adopt commercially available such as but not limited to: pcDNA3, pIRES, pDR, pUC18 etc. can be used for the carrier that eukaryotic cell system is expressed.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 shear and splicing by Restriction Enzyme according to ordinary method, and the suitable restriction site of encoding sequence insertion with fusion rotein of the present invention makes recombinant expression vector of the present invention.

The present invention also provides the host cell of expressing fusion rotein of the present invention, wherein contains the encoding sequence of fusion rotein of the present invention.Described host cell is eukaryotic cell preferably, such as but not limited to CHO, and COS cell, 293 cells, RSF cell etc.As optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can express fusion rotein of the present invention well, can obtain in conjunction with active good the fusion rotein that has good stability.

The present invention also provides a kind of and prepares the method for fusion rotein of the present invention with recombinant DNA, and its step comprises:

1) provides the nucleotide sequence (such as SEQ ID NO:17 sequence) of encoding fusion protein;

2) with 1) nucleotide sequence be inserted into suitable expression vector, obtain recombinant expression vector;

3) with 2) recombinant expression vector import the appropriate host cell;

4) cultivate under conditions suitable for the expression transformed host cell;

5) collect supernatant liquor, and the purified fusion protein product.

Described encoding sequence is imported host cell can adopt the multiple known technology of this area, such as but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkalimetal ion method.

About cultivation and the expression of host cell can be referring to Olander RM Dev Biol Stand 1996; 86:338.Can by cell and the residue in the centrifugal removal suspension, collect clear liquid.Can identify by agarose gel electrophoresis technology.

The character that can be basic homogeneous with the above-mentioned fusion protein purification for preparing for example is single band at the SDS-PAGE electrophoresis.For example, when recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane to separate described albumen, the product such as the company such as Millipore, Pellicon at first will be expressed supernatant and be concentrated.The method that concentrated solution can adopt gel chromatography is purifying in addition further, or adopts the method purifying of ion exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or cation-exchange chromatography.Gel matrix can be the matrix that agarose, dextran, polymeric amide etc. are usually used in protein purification.Q-or SP-group are comparatively desirable ion-exchange groups.At last, available hydroxyapatite adsorption chromatography also, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and RPLC (RP-HPLC) are to the further refining purifying of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.

Can utilize the affinity column of the specific antibody, acceptor or the part that contain described fusion rotein that the fusion rotein of expressing is carried out purifying.According to the characteristic of employed affinity column, can utilize conventional method, such as the amalgamation polypeptide of method elution of bound on affinity column such as high-salt buffer, change pH.Selectively, the aminoterminal of described fusion rotein or carboxyl terminal also can contain one or more polypeptide fragments, as the albumen label.Any suitable label may be used to the present invention.For example, described label can be FLAG, HA, HAl, c-Myc, 6-His or 8-His etc.These labels can be used for fusion rotein is carried out purifying.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical composition, and it contains significant quantity (such as 0.000001-90wt%; Better 0.1-50wt%; Better, fusion rotein of the present invention 5-40wt%), and pharmaceutically acceptable carrier.Usually, the fusion rotein of the present invention of significant quantity can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably, pH is about 6-8.Term " significant quantity " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.The composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (such as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.

Pharmaceutically acceptable carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Usually pharmaceutical preparation should be complementary with administering mode, and pharmaceutical composition of the present invention can be made into the injection form, for example with physiological saline or contain glucose and the aqueous solution of other assistant agents is prepared by ordinary method.Described pharmaceutical composition should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity.Pharmaceutical preparation of the present invention also can be made into sustained release preparation.

The significant quantity of fusion rotein of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (for example by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of described fusion rotein, metabolism, transformation period etc.; The severity of the disease that the patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Usually, the dosage when fusion rotein of the present invention every day with about 0.00001mg-50mg/kg the weight of animals (better 0.0001mg-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.

The advantage of fusion rotein of the present invention is as follows:

1. prolong the circulating half-life of GH and/or increase biological activity.

2. the minimizing of serum Chinese traditional medicine fluctuation of concentration, security improves, the improvement of tolerance.

3. the hGH-L-vFc fusion rotein that contains non-dissolving Fc variant can help to treat the growth disappearance because of the endogenous growth hormone hyposecretion significantly, and the build that the Tener syndromes causes is short and small, chronic renal failure, Prader-Willi syndromes, the illness such as the idiopathic build is short and small.

4. reduction frequency of injection makes the patient that better quality of the life be arranged.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1 makes up coding hGH-L-vFc _{γ 2}The gene of fusion rotein coding

1. preparation contains the gene order plasmid of people-tethelin

HGH-L-vFc _{γ 2}The fusion rotein encoding sequence is combined by several dna fragmentations.Contain the preparation of the gene of the leading peptide of people GH and maturation protein coding, be according to NCBI reference number NM_000515.3 the gene order of manned-tethelin prepare with artificial synthesis.Synthetic method is to advance to 3 ' terminal from 5 ' terminal along the double chain DNA sequence of hGH gene first, prepares four oligonucleotide fragments that chain replaces, length is about 180 bases up and down.The terminal of each fragment contains respectively the complementary overlap of 20 bases of having an appointment with the terminal of next complementary strand fragment.Thereafter again with these four oligonucleotide fragments, (PCR) is combined into the nucleotide fragments that length is about 650 bases with polymerase chain reaction technology.For the ease of the clone, Restriction Enzyme inscribe site will be contained in the two ends of institute's synthetic gene.Table 1 has been listed the oligonucleotide sequence that is used for clone hGH-L-vFc fusion rotein.

Table 1

SEQ ID NO	Primer sequence
			1	5’-cccaagcttggcgcggagatggctacaggctcccgga-3’
2	5’-cggatccgaagccacagctgccctcca-3’
		3	5’-gtcgagtgcccaccgtgccca-3’
4	5’-ggaattctcatttacccggagacaggga-3’
		5	5’-tggttttctcgatggaggctgggaggcct-3’
6	5’-aggcctcccagcctccatcgagaaaacca-3’
		7	5’-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagtcgagtgcccaccgtgccca-3’

8	5’-gagtccaaatatggtccccca-3’
		9	5’-ggaattctcatttacccagagacaggga-3’
10	5’-cctgagttcgcggggggacca-3’
		11	5’-gagtccaaatatggtcccccatgcccaccatgcccagcacctgagttcgcggggggacca-3’
12	5’-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagagtccaaatatggtccccca-3’
		13	5’-gacaaaactcacacatgccca-3’
14	5’-acctgaagtcgcggggggaccgt-3’
		15	5’-gacaaaactcacacatgcccaccgtgcccagcacctgaagtcgcggggggaccgt-3’
16	5’-cggatccggtggcggttccggtggaggcggaagcggcggtggaggatcagacaaaactcacacatgccca-3’

When the PCR composite reaction, introduced the restriction enzyme site (SEQ ID NO:1) of HindIII as the sequence of 5 ' Oligonucleolide primers; 3 ' primer has then been introduced BamHI site (SEQ ID NO:2).The hGH dna fragmentation that the length that obtains is about 650bp is terminal with HindIII and the excision of BamHI restriction endonuclease, behind the agarose gel electrophoresis purifying, be inserted into HindIII and the BamHI site of accepting carrier, namely the clone obtains containing the plasmid of the gene order of people-tethelin, called after phGH.The sequence of its contained hGH gene can be verified by dna sequencing.

2 preparations contain L-vFc _{γ 2}The plasmid of sequence

12 amino-acid residues (GluArgLysCysCysValGluCysProProCysPro) that comprise 4 halfcystines are contained in the hinge zone of human IgG2's heavy chain.In these 4 cysteine residues, the 3rd and the 4th formation that participates in disulfide linkage between two heavy chains; Can prevent that nonspecific disulfide linkage is bonding and eliminate the 1st and the 2nd cysteine residues.Fc in the present invention _{γ 2}It can be prescinded be 7 amino-acid residues (ValGluCysProProCysPro) in the hinge zone.The Fc zone of IgG2 is available from the RNA of human leukocyte preparation and suitable 5 ' (SEQ ID NO:3) and 3 ' (SEQ ID NO:4) primer, makes by reverse transcription and PCR to contain people Fc _{γ 2}The gene of encoding sequence.And then the Fc that contains hinge, CH2 and the CH3 zone complete sequence of IgG2 with this _{γ 2}Dna fragmentation is as template, with Fc _{γ 2}In the Ser in 331 sites replace to Pro, produce Fc _{γ 2}Pro331Ser (vFc _{γ 2}) variant.The introducing of this Substitution is to use first natural Fc _{γ 2}Make primer as template, the oligonucleotide (table 1) that contains mutant nucleotide sequence and produce two partly overlapping dna fragmentations of tool, then with among the overlapping PCR they being assembled: SEQ ID NO:3 as 5 ' primer and with SEQ ID NO:5 as 3 ' primer generation, 5 ' fragment; Generate 3 ' fragment as 5 ' primer and with SEQ ID NO:4 as 3 ' primer with SEQ ID NO:6; Use at last SEQ ID NO:7 as 5 ' primer and SEQ ID NO:4 as 3 ' primer, these two fragments are got up at the joint area that covers the Pro331Ser sudden change.SEQ ID NO:7 primer contains the encoding sequence of 16 amino acid peptide joints and comprises the BamHI restriction endonuclease sites, and SEQ ID NO:4 then contains the EcoRI restriction endonuclease sites.It is terminal with BamHI and the excision of EcoRI restriction endonuclease that this is made the dna fragmentation that length is about 700bp, behind the agarose gel electrophoresis purifying, inserts BamHI and the EcoRI site of accepting carrier (such as pUC19), can clone and obtain containing L-vFc _{γ 2}The plasmid of sequence, pL-vFc _{γ 2}Its contained L-vFc _{γ 2}The sequence of gene can be verified with dna sequencing.

3 preparation hGH-L-vFc _{γ 2}Fusion gene

HGH-L-vFc _{γ 2}The preparation method of fusion gene is as follows: downcut the hGH fragment with HindIII and BamHI from the phGH plasmid, then use the agarose gel electrophoresis purifying.On the other hand with pL-vFc ₂Plasmid cuts and uses the agarose gel electrophoresis purifying with HindIII and BamHI.The hGH fragment of purifying inserted pL-vFc thereafter _{γ 2}5 ' end of plasmid peptide linker obtains phGH-L-vFc _{γ 2}Plasmid.This fusion gene contains complete hGH, Gly-Ser peptide linker and Fc _{γ 2}Variant gene.

The peptide linker that exists between hGH and Fc part has increased the flexibility in hGH zone, thus improved its biological activity (referring to, for example U.S. Patent No. 6,797,493 and 6,900,292).For the purpose of the present invention, preferably peptide linker is that length contains have an appointment 20 or less amino acid; And the amino acid that uses can contain 2 kinds or more multiselect from following amino acid: glycine, Serine, L-Ala and Threonine.One of common peptide linker example is to contain the peptide linker that the Gly-Ser peptide makes up plate, such as GlyGlyGlyGlySer.

4. make up the hGH-L-vFc fusion protein expression vector

For expressing the hGH-L-vFc fusion rotein, need the complete genome coding of hGH-L-vFc fusion rotein is inserted mammalian expression vector such as pcDNA3 (Invitrogen, Carlsbad, CA).Method is with phGH-L-vFc _{γ 2}Plasmid downcuts the hGH-L-vFc fragment from HindIII and EcoRI site, then uses the agarose gel electrophoresis purifying.On the other hand expression vector is cut and uses the agarose gel electrophoresis purifying with HindIII and EcoRI.The hGH-L-vFc fragment of purifying inserted the expression vector after cutting, obtain final expression vector plasmid (being called pGFP2) thereafter.This pGFP2 contains required cytomegalovirus early gene promoter and the enhanser of high level expression in mammalian cell.This plasmid also contains alternative marker gene, thereby can give amicillin resistance in the transfected bacterium, and gives the G418 resistance in transfected mammalian cell.In addition, work as host cell, such as hamster ovary cell (Chinese Hamster Ovary, CHO), Tetrahydrofolate dehydrogenase (Dihydrofolate reductase, DHFR) genetic expression defectiveness the time, the pGFP2 expression vector contains the DHFR gene, thereby can in the presence of methotrexate (Methotrexate, MTX), jointly increase transfected intracellular hGH-L-vF ₂Fusion gene and DHFR gene (referring to, for example U.S. Patent No. 4,399, and 216), thereby improve hGH-L-vF _{γ 2}The expression level of fusion rotein.

Fig. 2 has shown hGH-L-vFc in the pGFP expression vector _{γ 2}Fusion gene (SEQ ID NO:17) and the aminoacid sequence (SEQ ID NO:18) of deriving, it contains sequence (amino-acid residue 1 to 191), 16 amino acid whose peptide linkers (GlySerGlyGlyGlySerGlyGlyGlyGlySerGLyGlyGlyGlySer) (amino-acid residue 192 to 207) and the Fc of leading peptide (26 to-1 amino acids), hGH _{γ 2}The sequence of Pro331Ser variant (underscore marks).

Embodiment 2 makes up coding hGH-L-vFc _{γ 4}The gene of fusion rotein coding

Owing to dissociating of disulfide linkage between heavy chain in the hinge zone, the human IgG 4 of a part can dissociate and form the molecule that is considered to incomplete antibody.This situation can not occur in other three-type-person IgG isotype molecule usually.Document shows that 228 sites in IgG1 and IgG2 are Pro, and is Ser228 in this site of IgG4.The Ser228 residue of IgG4 is made monamino acid with Pro replaces, can make IgG4 keep complete antibody molecule (referring to Angal etc., Molec.Immunol., 30:105-108,1993; Owens etc., Immunotechnology, 3:107-116,1997; U.S. Patent No. 6,204,007).In addition, with Fc _{γ 4}Carry out the Leu235Ala sudden change, can make this Fc _{γ 4}Variant reduces the bonding force with Fc γ R.This kind sudden change adds aforementioned Ser228Pro sudden change, will be so that fusion rotein more can obtain even, complete prepared product when purifying.

Structure contains hGH-L-vFc _{γ 4}The method of fusion rotein encoding gene is as follows:

With from the RNA of human leukocyte preparation and suitable 5 ' primer (SEQ ID NO:8) and 3 ' primer (SEQ IDNO:9), obtain containing the Fc zone (Fc of human IgG 4 by reverse transcription and PCR _{γ 4}) coding gene fragment.The Fc of the hinge that contains IgG4 that next will obtain, CH and CH3 zone complete sequence _{γ 4}Dna fragmentation as template, carry out Ser228Pro and Leu235Ala sudden change program to produce Fc _{γ 4}Variant (vFc _{γ 4}), Ser228 and Leu235 will be substituted by Pro and Ala respectively in this process.At this mutation process, the 5 ' primer (SEQ ID NO:10) of at first using 3 ' primer (SEQ IDNO:9) and containing Leu235Ala sudden change is with pcr amplification CH2 and CH3 zone.Secondly with SEQ ID NO:12 as 5 ' primer and SEQ ID NO:9 as 3 ' primer, in PCR be 60 bases with the fragment of this amplification and synthetic length and contain Ser228Pro and the oligonucleotide (SEQ ID NO:11) of Leu235Ala sudden change couples together.SEQ ID NO:12 primer contains the encoding sequence of 16 amino acid Gly-Ser peptide linkers (comprising the BamHI site).The dna fragmentation length that PCR obtains after connecting is about 700bp, and contains BamHI and EcoRI restriction enzyme site.This dna fragmentation with the excision of BamHI and EcoRI restriction endonuclease and with behind the agarose gel electrophoresis purifying, is inserted equally with the carrier of accepting of BamHI and the incision of EcoRI restriction endonuclease, can be cloned into and contain L-vFc _{γ 4}Plasmid, pL-vFc _{γ 4}L-vFc in this plasmid _{γ 4}The sequence of gene can be verified with dna sequencing.

Secondly, at preparation hGH-L-vFc _{γ 4}During fusion gene, downcut the hGH fragment with HindIII and BamHI from the phGH plasmid, then use the agarose gel electrophoresis purifying.On the other hand with pL-vFc _{γ 4}Plasmid cuts and uses the agarose gel electrophoresis purifying with HindIII and BamHI.The hGH fragment of purifying inserted pL-vFc thereafter _{γ 4}5 ' end of plasmid peptide linker obtains phGH-L-vFc _{γ 4}Plasmid.This fusion gene contains complete hGH, 16 amino acid whose Gly-Ser peptide linkers and Fc _{γ 4}Variant gene.At last with phGH-L-vFc _{γ 4}Plasmid downcuts the hGH-L-vFc fragment from HindIII and EcoRI site, then uses the agarose gel electrophoresis purifying.On the other hand expression vector is cut and uses the agarose gel electrophoresis purifying with HindIII and EcoRI.The hGH-L-vFc fragment of purifying inserted the expression vector after cutting, obtain final expression vector plasmid, called after pGFP4 thereafter.

Fig. 3 has shown hGH-L-vFc in the pGFP expression vector _{γ 4}Fusion gene (SEQ ID NO:19) and the fusion rotein sequence (SEQ ID NO:20) of deriving, it contains leading peptide (amino-acid residue-26 is to-1), hGH sequence (amino-acid residue 1 to 191), 16 amino acid whose peptide linkers (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) (amino-acid residue 192 to 207) and has Ser228Pro and the Fc of Leu235Ala sudden change _{γ 4}The fusion gene of variant (underscore marks).

Embodiment 3 makes up coding hGH-L-vFc _{γ 1}The gene of fusion rotein coding

15 amino-acid residues (GluProLysSerCysAspLysThrHisThrCysProProCysPro) that comprise 3 halfcystines are contained in the hinge zone of human IgG1's heavy chain.In these 3 cysteine residues, the 2nd and the 3rd formation that participates in disulfide linkage between two heavy chains.The 1st cysteine residues participates in the disulfide-bonded with the IgG light chain.Owing to do not have light chain in the Fc fusion protein molecule, this cysteine residues may match with other cysteine residues, and causes non-specific disulfide-bonded.Prevent this non-specific disulfide-bonded, can be with Fc _{γ 1}The hinge zone prescind, to eliminate the 1st cysteine residues (AspLysThrHisThrCysProProCysPro).With from the RNA of human leukocyte preparation and suitable 5 ' primer (SEQ ID NO:13) and 3 ' primer (SEQ ID NO:4), obtain containing Fc by reverse transcription and PCR _{γ 1}The gene of regional code.With the Fc that contains that obtains _{γ 1}The hinge zone of brachymemma and the dna fragmentation of CH2 and CH3 complete sequence carry out the triple site mutations of Leu234Val, Leu235Ala and Pro331Ser to produce Fc as template _{γ 1}Variant (vFc _{γ 1}).

Guide the method for these sudden changes as follows: to prepare first two dna fragmentations that contain above-mentioned site mutation, then use natural Fc _{γ 1}They are assembled in overlapping PCR as template.First fragment, 5 ' fragment is also to make as 3 ' primer with SEQ ID NO:5 as 5 ' primer with SEQ ID NO:14.This 5 ' primer contains Leu234Val, Leu235Ala sudden change, and 3 ' primer contains the Pro331Ser sudden change.Second fragment, 3 ' fragment, available SEQ ID NO:6 also makes as 3 ' primer with SEQ ID NO:4 as 5 ' primer.Secondly, with SEQ ID NO:14 as 5 ' primer and SEQ ID NO:4 as 3 ' primer, 5 ' and 3 ' fragment is got up covering the joint area that Pro331Ser suddenlys change.At last, with SEQ ID NO:16 as 5 ' primer, SEQ ID NO:4 as 3 ' primer, by PCR the oligonucleotide (SEQ ID NO:15) (containing Leu234Val and Leu235Ala) of the fragment of the about 650bp of length of this amplification and synthetic 55 bases is coupled together.SEQ ID NO:16 primer contains the encoding sequence of 16 amino acid Gly-Ser peptide linkers (comprising the BamHI site).The dna fragmentation that the length that obtains is about 700bp through BamHI and EcoRI cuts and with the agarose gel electrophoresis purifying after, BamHI and the EcoRI site of carrier accepted in insertion, can be cloned into pL-vFc _{γ 1}Plasmid.The sequence of this gene can be verified with dna sequencing.

Prepare hGH-L-vFc _{γ 1}Fusion gene can use first HindIII and BamHI behind phGH plasmid cutting-out hGH fragment, purifying, inserts pL-vFc again _{γ 1}5 ' terminal (cutting and purifying with HindIII and BamHI equally) of plasmid peptide linker obtains phGH-L-vFc _{γ 1}Plasmid.This fusion gene contains hGH, 16 amino acid Gly-Ser peptide linkers and Fc _{γ 1}Variant gene.This hGH-L-vFc _{γ 1}The fusion gene fragment through HindIII and EcoRI cut and with the agarose gel electrophoresis purifying after, HindIII and the EcoRI site that can insert mammalian expression vector.The vector plasmid called after pGFP1 of resulting final expression.

Fig. 4 has shown hGH-L-vFc in the pGFP expression vector _{γ 1}Fusion gene sequence (SEQ ID NO:21) and the fusion rotein sequence (SEQ ID NO:22) of deriving, it contains leading peptide (amino-acid residue-26 is to-1), hGH sequence (amino-acid residue 1 to 191), 16 amino acid peptide joints (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) (amino-acid residue 192 to 207) and has Ser234Val, Leu235Ala and the Fc of Pro331Ser sudden change _{γ 1}The fusion gene of variant (underscore marks).

The expression of embodiment 4 fusion roteins in transfection cell strain

For realizing the expression of fusion rotein, restructuring pGFP1, pGFP2 or pGFP4 expression vector plasmid transfection can be entered the mammalian host cell strain, to express the hGH-L-vFc fusion rotein.For obtain to stablize, high-caliber expression, the Chinese hamster ovary celI that preferred host cell strain is tool DHFR enzyme defect (referring to, for example U.S. Patent No. 4,818,679).Preferred transfection method is electroporation.Other method comprises that coprecipitation of calcium phosphate, fat transfection and Protoplast fusion also can use.In cuvette, place first 2-5 * 10 during transfection ⁷Individual cell, add subsequently the 10 μ g linearizing plasmid DNA of BspCI, evenly mix, then be positioned over (Gene Pulser Electroporator in the electroporation apparatus, Bio-Rad Laboratories, Hercules, CA), voltage is arranged on 250V electric capacity is arranged on 960 μ Fd enforcement electroporation.After 2 days, substratum is contained instead the growth medium of 0.8mg/mlG418 in transfection.After about 2 weeks of transfection, the transfectant that the G418 medicine is had resistance can grow up to macroscopic population of cells (clone).The transfectant of available anti-human IgG Fc enzyme-linked immunosorbent assay this moment (ELISA) screening secretion fusion rotein.In addition, undertaken the fusion rotein of expressing is done quantitative analysis by ELISA also available anti-hGH test.Behind the selected transfectant of fusion rotein than high expression level, by diluting in 96 orifice plate limes superiors, these produce the population of cells of high-level Fc fusion rotein subclone.

In order to make transfectant cell clone selected and the process subclone reach the expression of fusion rotein higher level, suitable method is to use the gene dosage that increases fusion rotein with the program of DHFR gene coamplification.Usually, gene dosage increases, and the expression amount of fusion rotein can increase thereupon.In the growth medium that contains progressive concentration MTX, the activity of DHFR enzyme is suppressed by the MTX medicine and normal synthetic nucleosides, the normal growth of cell thereby be affected.Cell is that must the increase gene number of DHFR enzyme of the inhibition that overcomes this MTX medicine is kept normal growth to produce more enzyme by cell.The antigen-4 fusion protein gene of transfection is adjacent with the DHFR gene, and therefore the process in DHFR gene amplification can increase jointly with the DHFR gene.Through the Stepwise Screening of progressive concentration MTX, select the transfectant that can in up to 1 μ g/ml MTX substratum, grow, again carry out subclone by the Method of Limited Dilution method.By measuring secretion rate, the cell strain of subclone is further analyzed, select at last several secretion rate horizontal exceedings about 10 (preferably about 30) μ g/10 ⁶The cell strain of (1,000,000) individual cell/24 hours.Use the serum-free growth medium to cultivate these cell strains of selecting, allow it be adapted to gradually suspension culture.These cells can be secreted fusion rotein in substratum in process of growth, available purified fusion protein.

One of them cell strain, fixed number 9-10-39 can 20 μ g/10 ⁶The speed secretion hGH-LvFc of (1,000,000) individual cell/24 hours _{γ 2}HGH-LvFc in the nutrient solution _{γ 2}The concentration of fusion rotein is measured with the ELISA quantitative analysis, with the hGH-LvFc of purifying _{γ 2}Fusion rotein is made standardized solution.Cell strain 9-10-39 is progressively adapted to into suspension cell culture at 100 milliliters rotating and culturing bottle.

Fig. 5 is presented in 100 milliliters the rotating and culturing bottle, the growth curve of this cell strain and secretion hGH-LvFc thereof _{γ 2}The concentration of fusion rotein.After 12 days, the concentration of the fusion rotein in the nutrient solution can be accumulate to about 1 grams per liter (g/L).

The purifying of embodiment 5 fusion roteins and qualitative

The conditioned medium that will contain the hGH-L-vFc fusion rotein with 1N NaOH is titrated to pH 7 to 8, and then the nitrocellulose strainer with 0.45 μ m filters.Filtrate is loaded onto on the Prosep A post of phosphate buffered saline (PBS) (PBS) balance.After fusion rotein is incorporated into Prosep A, discards stream and wear component.Wash this post with PBS, until the OD value at 280nm place is lower than 0.01.Then use the fusion rotein of the citrate buffer solution elution of bound of 0.1M pH3.75.1M K with 0.4 volume ₂HPO ₄Neutralization merges the component that contains purifying protein, and dialyses with PBS.Then solution filters with the nitrocellulose strainer of 0.22 μ m, and is stored in 4 ℃.Under non-reduced condition, the molecular weight ranges that records the hGH-L-vFc protein of purifying by SDS-PAGE is 90 to 100kDa.Under reductive condition, the albumen of purifying migrates to about 50kDa.With BSA as standard, by quantitative this fusion rotein of BCA protein analysis.

Embodiment 6 Bioactivity analyses

Bioactivity can be measured with the multiplication capacity method of transfectant or protein purification stimulation in rats lymphoma cell Nb2 cell.Although the propagation of Nb2 cell is to be subject to tethelin the lactation receptor for stimulating on the cell is reacted, but the test of Nb2 cell-proliferation activity can be used for estimating tethelin biological activity (referring to, such as people such as interior fields, J.Mol.Endocrinol., 23:347-353,1999).

Before test began about 48 hours, (RPMI 1640 substratum contained the beta-mercaptoethanol of 1% horse serum and 50 μ mol to slow down cell proliferation rate in the pre-detection substratum with cell transfer first.After insulation is cultivated, collecting cell and with every milliliter 4 * 10 ⁵The concentration of individual cell is suspended in substratum.Every part of 50 μ l cell samples are added in each hole of 96 hole tissue cultivating plates.These cells of analysis culture medium culturing that contain the various concentration hGH-L-vFc fusion roteins of 0.01-100nM or rhGH contrast with 50 μ l.At 37 ℃, 5%CO ₂Cultivate this flat board 48 hours in the humidified incubator, then in each hole, add 20 μ l Thiazolyl blues (being mixed with 2.5mg/ml with PBS).After five hours, in every hole, add the lysate that 100 μ l contain 10%SDS, spend the night in the rearmounted 37 ℃ of thermostat containers of sealed membrane sealing, with dissolved cell and formed first a ceremonial jade-ladle, used in libation.Then at 570nm this flat board is carried out optical density readings, wherein reference beam is made as 630nm.With the concentration mapping of OD reading with respect to the hGH-L-vFc fusion rotein.Can be measured the biological activity of hGH-L-vFc by the gained dose response curve.

Fig. 6 has shown hGH-L-vFc _{γ 2}Purifying protein stimulates the ability of Nb2 cell proliferation.Figure reads the weight break point of sigmoid curve thus, can calculate half numerical value (half maximal effective concentration, the EC of maximum effective concentration ₅₀) be 1.2nM.By mol, the Bioactivity and the rhGH that show of recombination fusion protein is similar.

The preparation of embodiment 7 pharmaceutical compositions

HGH-L-vFc fusion rotein 50 μ g

Physiological saline 1ml

Regulate pH to 6.7-7.2

Obtain containing the pharmaceutical composition of hGH-L-vFc fusion rotein.

All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. a restructuring hGH-L-vFc fusion rotein is characterized in that, described fusion rotein holds the C end to contain successively people GH, peptide linker and human IgG Fc variant from N,

And described human IgG Fc variant is selected from lower group:

(iii) contain human IgG1's hinge region, CH2 and the CH3 zone that Leu234Val, Leu235Ala and Pro331Ser suddenly change.

2. restructuring hGH-L-vFc fusion rotein as claimed in claim 1 is characterized in that described peptide linker contains 2-20 amino acid, and described peptide linker is present between people GH and the human IgG Fc variant; And described peptide linker contains two or more amino acid that are selected from glycine, Serine, L-Ala and Threonine.

3. restructuring hGH-L-vFc fusion rotein as claimed in claim 1 is characterized in that the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:18,20 or 22.

4. restructuring hGH-L-vFc fusion rotein as claimed in claim 1 is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:18,20 or 22 that has removed after the hGH leading peptide of-26 to-1 amino acids residue.

5. restructuring hGH-L-vFc fusion rotein as claimed in claim 1 is characterized in that, described hGH-L-vFc fusion rotein has the Bioactivity similar or higher with rhGH, longer transformation period on mole foundation.

6. the dna molecular of a coding restructuring claimed in claim 1 hGH-L-vFc fusion rotein.

7. CHO derived cell strain is characterized in that, described cell in its growth medium in per 24 hours, produce to surpass 1,000,000 cells of 10 μ g/ such as the arbitrary described restructuring hGH-L-vFc fusion rotein of claim 1-5.

8. CHO derived cell as claimed in claim 7 strain is characterized in that, in growth medium, in per 24 hours, produce surpass 1,000,000 cells of 30 μ g/ such as the arbitrary described restructuring hGH-L-vFc fusion rotein of claim 1-5.

9. the CHO as claimed in claim 7 cell strain of deriving is characterized in that, it contains the dna sequence dna of coding hGH-L-vFc fusion rotein, and described dna sequence dna has the nucleotide sequence shown in the SEQ ID NO:17,19 or 21.

10. a method for preparing the described recombination fusion protein of claim 1 is characterized in that, comprises step:

(a) fusion rotein in its growth medium during per 24 hours in, express to surpass 10 μ g/10 ⁶Under the condition of (1,000,000) individual cell, cultivate cell strain claimed in claim 7; With

11. method as claimed in claim 10 is characterized in that, is to have between people GH and the human IgG Fc variant 2-20 amino acid whose flexible peptide; And described flexible peptide linker contains the amino acid that two or more are selected from glycine, Serine, L-Ala and Threonine.

12. method as claimed in claim 10 is characterized in that, the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:18,20 or 22.

13. method as claimed in claim 10 is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:18,20 or 22 that has removed after the hGH leading peptide of-26 to-1 amino acids residue.

14. a raising contains the method for expression amount of the recombination fusion protein of people GH, flexible peptide linker and human IgG Fc variant, it is characterized in that described method comprises:

(c) fusion rotein of purification step (b) expression,

15. method as claimed in claim 14 is characterized in that, the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:18,20 or 22.

16. method as claimed in claim 14 is characterized in that, the DNA of described encoding fusion protein has the nucleotide sequence shown in the SEQ ID NO:17,19 or 21.