CN102875683B

CN102875683B - Fc fusion protein of long-acting recombinant human growth hormone

Info

Publication number: CN102875683B
Application number: CN201110193210.3A
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: 2014-06-11
Anticipated expiration: 2031-07-11
Also published as: CN102875683A

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 produced and discharged by pituitary body, the peptide hormone that 191 amino acid of total length, molecular weight are 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 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 conventionally; First be combined with first acceptor molecule by the binding site 1 in tethelin, 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 tethelin molecule N end 8 amino acid and other several amino acid from belonging in spiral-line 1 and spiral-line 3.1 of binding site comprises near amino acid amino acid and the C-end of tethelin molecule within spiral-line 1.8 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.For example, if the position of these 8 amino-acid residues, with other aminoacid replacement, will be changed rapidly to its ability of being combined with growth hormone receptor (seeing Wells et al.Recent Prog.Horm.Res., 48:253-75,1993).This 8 seed amino acid is section (K41 between spiral-line 1 and spiral-line 2, L45, P61, R64) the half section of (K172 of carboxyl C end and in spiral-line 4, T175, F176, R178), and 4 amino-acid residues of its back segment are just in time positioned near the C-terminal of 191 amino acid whose tethelin of total length.Therefore near amino acid 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 therefore at present GH is carried out to conventionally avoid 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 growth hormone deficiency situation, and supplementing 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, its intravenous serum is removed approximately 20 minutes transformation period.If with subcutaneous injection recombinant human somatropin, its peak Plasma Concentration is wanted just can reach after several hours conventionally, and it eliminates the transformation period 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.The patient that need to accept for a long time growth hormone therapy for those, often owing to can not inject on time, causes 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 every 2 or 4 weeks once, and does not need injection every day.But, because production cost is too high, this product was withdrawn from market in 2004.

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

The immunoglobulin (Ig) of IgG class is rich in protein in human blood.Their transformation period can be up to 21 days.Existing report by the Fc region of IgG and other oroteins (as various cytokines and soluble receptors) in conjunction with and formation fusion rotein (referring to, the people such as 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 IgG Fc hinge region, and forms similar IgG but lack the molecule of CH1 region and light chain.Due to structural homology, the external pharmacokinetic properties that Fc fusion rotein shows and the human IgG of isotype are quite similar.Therefore manufacture and contain the hGH fusion rotein being connected with the Fc region of human IgG protein, the biological activity that extends the circulating half-life of hGH and/or increase it contributing to.

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

Due to its difficulty in essence that is built with of hGH-L-vFc fusion rotein, still both do not there is 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

Object of the present invention is just to provide one and has highly bioactive hGH-L-vFc fusion rotein and its production and use.

In a first aspect of the present invention, a kind of restructuring hGH-L-vFc fusion rotein is provided, described fusion rotein holds 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) human IgG2's hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change;

(ii) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change;

(iii) human IgG l hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.

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

In another preference, the aminoacid sequence of described fusion rotein is as 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 having removed after the hGH leading peptide of-26 to-1 amino acids residue.

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

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

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

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

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

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

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

(b) protein that purification step (a) is expressed, wherein recombination fusion protein, on mole foundation, has the Bioactivity similar or higher with rhGH, 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 two or more and is selected from the amino acid of glycine, Serine, L-Ala and Threonine.

In another preference, described fusion rotein aminoacid sequence 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 of encoding fusion protein is introduced to Chinese hamster ovary celI, generate the derivative clone of CHO;

(b) cultivate the derivative clone of this CHO, thus expressed fusion protein; With

(c) fusion rotein that purification step (b) is expressed,

Wherein recombination fusion protein is hGH-L-vFc fusion rotein, it is characterized by and shows high Bioactivity,, on mole foundation, has the Bioactivity similar or higher with people GH and longer transformation period; Wherein between people GH and IgG Fc variant, exist containing 2-20 the amino acid whose flexible peptide linker of having an appointment; Contain 2 or multiple amino acid and be selected from the amino acid of glycine, Serine, L-Ala and Threonine with flexible peptide linker; The human IgG2 that wherein human IgG Fc variant contains the hinge region, CH2 and CH3 region: the Pro331Ser sudden change that are selected from following human IgG; 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 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 combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.

Accompanying drawing explanation

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

Fig. 2 has shown the hGH-L-vFc of HindIII-EcoRI fragment in pGFP expression vector _{γ 2}nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 are 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 Fc region, 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 pGFP expression vector _{γ 4}nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 are 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 Fc region, 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 pGFP expression vector _{γ 1}nucleotide sequence and the aminoacid sequence of derivation.Amino-acid residue-26 are 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 Fc region, 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, through long-term and deep research, has designed first a kind of original hinge region peptide linker and has reduced space steric effect, and the C that can make hGH holds the fusion rotein being connected with Fc, and there is soft peptide linker centre.Surprisingly, this fusion rotein not only can not cause the afunction of GH, can maintain on the contrary, even improve the biological activity of tethelin-Fc fusion rotein.Complete on this basis the present invention.

Particularly, hGH-L-vFc fusion rotein of the present invention holds 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 region: the human IgG1 of (A) containing Leu234Val, Leu235Ala and Pro331Ser sudden change; (B) human IgG2 of containing Pro331Ser sudden change; (C) human IgG 4 that contains Ser228Pro and Leu235Ala sudden change.Preferably, use an about 2-20 amino acid length, contain the flexible peptide linker that following 2 kinds or multiple amino acids form: glycine, Serine, L-Ala and Threonine.IgG Fc variant is non-cracking performance, and contains amino acid mutation compared with natural IgG Fc.This type of hGH-L-vFc fusion rotein, on mole foundation, has the Bioactivity similar or higher with rhGH.And hGH-L-vFc _{γ 2}body in serum remove the transformation period have phenomenal growth than rhGH.

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

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

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

Fc element

Fc element is from the Fc region of immunoglobulin (Ig), and Fc is tool vital role in the immune defense of eliminating pathogen.The effector function of IgG is mediated by Fc and by two kinds of main mechanisms: 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 part of the first complement component Cl, cause cytotoxicity (CDC) approach that depends on complement, thereby dissolve pathogenic agent.In four kinds of human IgG isotypes, IgG1 and IgG3 can be effectively in conjunction with Fc γ R.IgG4 with the binding affinity of Fc γ R than a low order of magnitude of IgG1 or IgG3, and the combination of IgG2 and Fc γ R low be difficult to measure.Human IgG1 and IgG3 can also be effectively in conjunction with Clq, and activating complement cascade reaction.Human IgG2 is very weak to the fixed action of complement, and IgG4 seems quite lacking aspect the ability of activating complement cascade reaction.For being applied to people's treatment, in the time that hGH-Fc fusion rotein is incorporated into the lip-deep hGHR of target cell, must can not there be ill effect subfunction in the Fc region of fusion rotein, thereby can not dissolve or remove these target cell.Therefore, the necessary right and wrong in the Fc region of hGH-Fc are deliquescent, thereby to being incorporated into Fc γ Rs and Clq trigger effect subfunction aspect, Fc region must be non-activity.Obviously, do not have a kind of natural IgG isotype to be applicable to producing hGH-Fc fusion rotein.In order to obtain the Fc of non-solubility, must make some amino acid mutations in natural Fc region, to reduce its effector function.

See through the relatively demonstration of the aminoacid sequence of the IgG isotype of people and mouse, Fc fragment is near the tool vital role in the combination of IgG Fc and Fc γ Rs of the sequence N-terminal of CH2 region.Its 234 importance to 237 motifs in the antibody of genetically engineered construction prove (referring to, the people such as 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 EU number system described in 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 on 234 sites, Leu changes Phe into.Not with IgG2 that Fc γ Rs is combined, occur that replace in two sites and a site is deleted, thus formation Val234-Ala-Gly237 sequence (Fig. 1).In order to reduce combination and the ADCC activity of Fc and Fc γ R, the Leu235 in IgG4 can replace with Ala (referring to, the people such as such as Hutchins, Proc.Natl.Acad.Sci.USA, 92:11980-11984,1995).Glu233-Leu-Leu235 sequence in IgG1 antibody was once replaced by the Pro233-Val-Ala235 correlated series with in IgG2.This change makes IgG1 variant in mouse, lose the ability of removing target cell through Fc γ R-mediation.

About antibody to Fc γ R be combined with Clq CH2 region that vital the second position is located in human IgG near near of carboxyl terminal (referring to, the people such as such as Duncan, Nature, 332:738-740,1988).In four kinds of human IgG isotypes, in this part, only there is a site to show and replace: the Ser330 in IgG4 and Ser331 have replaced Ala330 and the Pro331 (Fig. 1) in IgG1, IgG2 and IgG3.The existence of Ser330 does not affect the combination of Fc γ R and Clq.Substitute Pro331 with Ser and make IgG1 lose and the binding affinity of Clq, and with Pro substitute the complement fixation activity that Ser331 part retained IgG4 (referring to, the people such as 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 fusion rotein is variant (vFc), for building efficient hGH-L-vFc fusion rotein.Human IgG2 is in conjunction with Fc γ R, but demonstrates faint complement activity.There is the Fc of Pro331Ser sudden change _{γ 2}variant should be than natural Fc _{γ 2}variant activity lower, and be not still incorporated into Fc γ R.IgG4Fc is activating variant cascade reaction defectiveness, and the highest low approximately order of magnitude of isotype (IgG1) (ten times) of the binding affinity specific activity of it and Fc γ R.With natural Fc _{γ 4}compare, there is the Fc of Leu235Ala sudden change _{γ 4}variant should show

micro-effector function.There is the Fc of Leu234Val, Leu235Ala and Pro331Ser sudden change _{γ 1}also show than natural Fc _{γ 1}much lower effector function.These Fc variants are all more suitable for preparing 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 prepared by biosynthetic method conventionally.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, the method that can carry out again overlapping extension PCR by salvage nucleotide sequence builds nucleic acid sequence encoding of the present invention.

The present invention also provides a kind of expression vector, comprises the encode sequence of fusion rotein of the present invention and the connected expression regulation sequence of operability with it.Described " operability is connected " or " being operationally connected in " refer to so a kind of situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promotor control sequence, it is exactly to be operationally 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 encoding sequence of fusion rotein of the present invention is inserted to suitable restriction site, make 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.Preferably eukaryotic cell of described host cell, such as but not limited to CHO, 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 having good stability.

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

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

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

3) by 2) recombinant expression vector import suitable host cell;

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

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

Described encoding sequence importing host cell can be adopted to 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 residue in centrifugal removal suspension, collect clear liquid.Can identify by agarose gel electrophoresis technology.

The character that can be basic homogeneous by the above-mentioned fusion protein purification preparing, for example, be single band on SDS-PAGE electrophoresis.For example, in the time that recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane to separate described albumen, the such as company such as Millipore, Pellicon product, first will express supernatant and concentrate.Further purifying in addition of the method that concentrated solution can adopt gel chromatography, or adopt 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.Finally, also available hydroxyapatite adsorption chromatography, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and RPLC (RP-HPLC) are to 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 to carry out purifying to the fusion rotein of expressing.According to the characteristic of used affinity column, can utilize conventional method, 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 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 to carry out purifying.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical composition, and it contains significant quantity (as 0.000001-90wt%; Preferably 0.1-50wt%; Better, 5-40wt%) fusion rotein of the present invention, and pharmaceutically acceptable carrier.Conventionally, the fusion rotein of the present invention of significant quantity can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, 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 (as toxicity, stimulation and transformation reactions), 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.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into 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 manufactured under aseptic condition.The dosage of activeconstituents is 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 patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Conventionally,, when fusion rotein of the present invention gives with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) every day, can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, can 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. extend 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 contribute to the growth disappearance for the treatment of because of endogenous growth hormone hyposecretion significantly, and the build that Tener syndromes causes is short and small, chronic renal failure, Prader-Willi syndromes, the illness such as idiopathic build is short and small.

4. reduce frequency of injection, make patient have better quality of the life.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people 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 builds coding hGH-L-vFc _{γ 2}the gene of fusion rotein coding

1. preparation is containing the gene order plasmid of people-tethelin

HGH-L-vFc _{γ 2}fusion rotein encoding sequence is combined by several DNA fragmentations.The preparation of the gene of the leading peptide that contains people GH and maturation protein coding, be according to NCBI reference number NM_000515.3 the gene order artificial synthesis of manned-tethelin prepare.Synthetic method is first to advance to 3 ' terminal from 5 ' terminal along the double chain DNA sequence of hGH gene, prepares the oligonucleotide fragment that four upper and lower chains replace, length is about 180 bases.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.Again by this four oligonucleotide fragments, be combined into the nucleotide fragments that length is about 650 bases with polymerase chain reaction technology (PCR) thereafter.For the ease of clone, Restriction Enzyme inscribe site will be contained in the two ends of synthesized gene.Table 1 has been listed the oligonucleotide sequence for cloning 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’

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

2 preparations are containing L-vFc _{γ 2}the plasmid of sequence

12 amino-acid residues (GluArgLysCysCysValGluCysProProCysPro) that comprise 4 halfcystines are contained in the hinge region of human IgG2's heavy chain.In these 4 cysteine residues, the 3rd and the 4th participates in the formation of two heavy interchain disulfide bonds; And the 1st of elimination and the 2nd cysteine residues can prevent that nonspecific disulfide linkage is bonding.Fc in the present invention _{γ 2}can to be prescinded be 7 amino-acid residues (ValGluCysProProCysPro) in hinge region.The RNA for preparing from human leukocyte and suitable 5 ' (SEQ ID NO:3) and 3 ' (SEQ ID NO:4) primer can be used in the Fc region of IgG2, make the Fc containing people by reverse transcription and PCR _{γ 2}the gene of encoding sequence.The Fc of hinge, CH2 and the CH3 region complete sequence that and then contains IgG2 with this _{γ 2}dNA fragmentation is as template, by 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 first to use natural Fc _{γ 2}as template, make primer containing the oligonucleotide (table 1) of mutant nucleotide sequence and produce two partly overlapping DNA fragmentations of tool, then with in 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; Finally use 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 Pro331Ser sudden change.The encoding sequence that SEQ ID NO:7 primer contains 16 amino acid peptide joints also comprises BamHI restriction endonuclease sites, and SEQ ID NO:4 contains EcoRI restriction endonuclease sites.This is made to DNA fragmentation that length is about 700bp with BamHI and EcoRI restriction endonuclease excision end, after agarose gel electrophoresis purifying, insert BamHI and the EcoRI site of accepting carrier (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: cut hGH fragment from phGH plasmid with HindIII and BamHI, then purify with agarose gel electrophoresis.On the other hand by pL-vFc ₂plasmid is with HindIII and BamHI cuts and purify with agarose gel electrophoresis.The hGH fragment of purifying inserted to 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 existing between hGH and Fc part, has increased the flexibility in hGH region, 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 using can contain 2 kinds or more multiselect from following amino acid: glycine, Serine, L-Ala and Threonine.One of common peptide linker example is the peptide linker that contains Gly-Ser peptide structure plate, as GlyGlyGlyGlySer.

4. build hGH-L-vFc fusion protein expression vector

For expressing hGH-L-vFc fusion rotein, the complete genome coding of hGH-L-vFc fusion rotein need be inserted to mammalian expression vector as pcDNA3 (Invitrogen, Carlsbad, CA).Method is by phGH-L-vFc _{γ 2}plasmid cuts hGH-L-vFc fragment from HindIII and EcoRI site, then purifies with agarose gel electrophoresis.On the other hand by expression vector with HindIII with EcoRI cuts and purify with agarose gel electrophoresis.The hGH-L-vFc fragment of purifying inserted to expression vector cut after, 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 transfected bacterium, and in transfected mammalian cell, gives G418 resistance.In addition, work as host cell, as hamster ovary cell (Chinese Hamster Ovary, CHO), Tetrahydrofolate dehydrogenase (Dihydrofolate reductase, DHFR) genetic expression defectiveness time, pGFP2 expression vector contains DHFR gene, thereby can under existing, methotrexate (Methotrexate, MTX) jointly increase transfected intracellular hGH-L-vF ₂fusion gene and DHFR gene (referring to, for example U.S. Patent No. 4,399,216), thus hGH-L-vF improved _{γ 2}the expression level of fusion rotein.

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

Embodiment 2 builds coding hGH-L-vFc _{γ 4}the gene of fusion rotein coding

Due to dissociating of heavy interchain disulfide bond in hinge region, the human IgG 4 of a part can dissociate and form the molecule that is considered to incomplete antibody.This situation conventionally can not occur in other three-type-person IgG isotype molecule.Document demonstration, 228 sites in IgG1 and IgG2 are Pro, and are Ser228 in this site of IgG4.The Ser228 residue of IgG4 is made to monamino acid with Pro and 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, by Fc _{γ 4}carry out Leu235Ala sudden change, can make this Fc _{γ 4}variant reduces the bonding force with Fc γ R.This kind of sudden change, adds aforementioned Ser228Pro sudden change, will make fusion rotein in the time of purifying, more can obtain even, complete prepared product.

Build containing hGH-L-vFc _{γ 4}the method of fusion rotein encoding gene is as follows:

Use the RNA for preparing from human leukocyte and suitable 5 ' primer (SEQ ID NO:8) and 3 ' primer (SEQ IDNO:9), obtain the Fc region (Fc containing human IgG 4 by reverse transcription and PCR _{γ 4}) coding gene fragment.Next by the Fc of the hinge that contains IgG4 obtaining, CH and CH3 region 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}), in this process, Ser228 and Leu235 will be substituted by Pro and Ala respectively.At this mutation process, the 5 ' primer (SEQ ID NO:10) of first using 3 ' primer (SEQ IDNO:9) and containing Leu235Ala sudden change is with pcr amplification CH2 and CH3 region.Secondly with SEQ ID NO:12 as 5 ' primer and SEQ ID NO:9 as 3 ' primer, in PCR, be 60 bases by the fragment of this amplification and synthetic length and couple together containing the oligonucleotide (SEQ ID NO:11) that Ser228Pro and Leu235Ala suddenly change.The encoding sequence that SEQ ID NO:12 primer contains 16 amino acid Gly-Ser peptide linkers (comprising BamHI site).The DNA fragmentation length that PCR obtains after connecting is about 700bp, and containing BamHI and EcoRI restriction enzyme site.After this DNA fragmentation is excised with BamHI and EcoRI restriction endonuclease and purifying with agarose gel electrophoresis, insert the carrier of accepting cutting with BamHI and EcoRI restriction endonuclease equally, can be cloned into containing 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}when fusion gene, cut hGH fragment from phGH plasmid with HindIII and BamHI, then purify with agarose gel electrophoresis.On the other hand by pL-vFc _{γ 4}plasmid is with HindIII and BamHI cuts and purify with agarose gel electrophoresis.The hGH fragment of purifying inserted to 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.Finally by phGH-L-vFc _{γ 4}plasmid cuts hGH-L-vFc fragment from HindIII and EcoRI site, then purifies with agarose gel electrophoresis.On the other hand by expression vector with HindIII with EcoRI cuts and purify with agarose gel electrophoresis.The hGH-L-vFc fragment of purifying inserted to expression vector cut after, obtain final expression vector plasmid, called after pGFP4 thereafter.

Fig. 3 has shown hGH-L-vFc in 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 are 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 (underscore marks) of variant.

Embodiment 3 builds 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 region of human IgG1's heavy chain.In these 3 cysteine residues, the 2nd and the 3rd participates in the formation of two heavy interchain disulfide bonds.The 1st cysteine residues participates in the disulfide-bonded with IgG light chain.Owing to there is no light chain in 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 by Fc _{γ 1}hinge region prescind, to eliminate the 1st cysteine residues (AspLysThrHisThrCysProProCysPro).Use the RNA for preparing from human leukocyte 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.By the Fc that contains obtaining _{γ 1}the DNA fragmentation of the hinge region of brachymemma and CH2 and CH3 complete sequence, as template, carries out the triple site mutations of Leu234Val, Leu235Ala and Pro331Ser to produce Fc _{γ 1}variant (vFc _{γ 1}).

Guide the method for these sudden changes as follows: first to prepare 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 to make as 3 ' primer as 5 ' primer and with SEQ ID NO:5 with SEQ ID NO:14.This 5 ' primer contains Leu234Val, Leu235Ala sudden change, and 3 ' primer contains 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, by 5 ' and 3 ' fragment get up covering the joint area that Pro331Ser suddenlys change.Finally, 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.The encoding sequence that SEQ ID NO:16 primer contains 16 amino acid Gly-Ser peptide linkers (comprising BamHI site).The DNA fragmentation that the length obtaining is about to 700bp through BamHI and EcoRI cuts and purify with agarose gel electrophoresis after, insert BamHI and the EcoRI site of accepting carrier, 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 be first with HindIII and BamHI after phGH plasmid cuts hGH fragment, purifying, then insert pL-vFc _{γ 1}5 ' the end (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}fusion gene fragment through HindIII and EcoRI cuts and purify with agarose gel electrophoresis after, HindIII and the EcoRI site that can insert mammalian expression vector.The vector plasmid called after pGFP1 of the final expression obtaining.

Fig. 4 has shown hGH-L-vFc in 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 are 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 (underscore marks) of variant.

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 to mammalian host cell strain, to express hGH-L-vFc fusion rotein.Stable in order to obtain, high-caliber expression, preferred host cell strain be tool DHFR enzyme defect Chinese hamster ovary celI (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.When transfection, first in cuvette, place 2-5 × 10 ⁷individual cell, add subsequently the linearizing plasmid DNA of 10 μ g BspCI, evenly mix, then be positioned over (Gene Pulser Electroporator in electroporation apparatus, Bio-Rad Laboratories, Hercules, CA), voltage is arranged on to 250V electric capacity and is arranged on 960 μ Fd enforcement electroporations.After 2 days, make substratum into growth medium containing 0.8mg/mlG418 in transfection.After transfection approximately 2 weeks, transfectant G418 medicine to resistance can grow up to macroscopic population of cells (clone).Now can secrete with anti-human IgG Fc enzyme-linked immunosorbent assay (ELISA) screening the transfectant of fusion rotein.In addition, also can do quantitative analysis with the fusion rotein that anti-hGH test is carried out expressing by ELISA.After selected fusion rotein is compared with the transfectant of high expression level, by 96 orifice plate limes superiors dilutions, these produce the population of cells of high-level Fc fusion rotein subclone.

Reach the expression of fusion rotein higher level in order to make to select and pass through the transfectant cell clone of subclone, suitable method is to use the gene dosage that increases fusion rotein with the program of DHFR gene coamplification.Conventionally, 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 and normal synthetic nucleosides by MTX medicine, 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 maintains normal growth to produce more enzyme by cell.The antigen-4 fusion protein gene of transfection is adjacent with DHFR gene, therefore can jointly increase with DHFR gene in the process of DHFR gene amplification.Through the Stepwise Screening of progressive concentration MTX, select the transfectant that can grow in up to 1 μ g/ml MTX substratum, again carry out subclone by Method of Limited Dilution method.By measuring secretion rate, the cell strain of subclone is further analyzed, finally select several secretion rate horizontal exceedings approximately 10 (preferably approximately 30) μ g/10 ⁶the cell strain of (1,000,000) individual cell/24 hour.The cell strain that these are selected uses serum-free growth medium to cultivate, and allows 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 hour _{γ 2}.HGH-LvFc in nutrient solution _{γ 2}the concentration of fusion rotein is measured with 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 the rotating and culturing bottle of 100 milliliters.

Fig. 5 is presented in the rotating and culturing bottle of 100 milliliters, 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 nutrient solution can be accumulate to approximately 1 grams per liter (g/L).

The purifying of embodiment 5 fusion roteins and qualitative

The conditioned medium that contains hGH-L-vFc fusion rotein is titrated to pH 7 to 8 with 1N NaOH, then filters with the nitrocellulose strainer of 0.45 μ m.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.With the 1M K of 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 quantitatively this fusion rotein of BCA protein analysis.

Embodiment 6 Bioactivity analyses

Bioactivity can be measured by 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 cell is reacted, but the test of Nb2 cell-proliferation activity can be used for evaluating tethelin biological activity (referring to, the people such as such as interior field, J.Mol.Endocrinol., 23:347-353,1999).

Before test starts approximately 48 hours, first cell is transferred to in pre-detection substratum, (RPMI 1640 substratum, the beta-mercaptoethanol that contains 1% horse serum and 50 μ mol is to slow down cell proliferation rate.After insulation is cultivated, collecting cell 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 ₂in humidified incubator, cultivate this flat board 48 hours, then in each hole, add 20 μ l Thiazolyl blues (being mixed with 2.5mg/ml with PBS).After five hours, the lysate that adds 100 μ l to contain 10%SDS in every hole, spends the night in the rearmounted 37 ℃ of thermostat containers of sealed membrane sealing, with dissolved cell and the first a ceremonial jade-ladle, used in libation being formed.Then at 570nm, this flat board is carried out to optical density readings, wherein reference beam is made as 630nm.Concentration mapping by OD reading with respect to hGH-L-vFc fusion rotein.Can be measured the biological activity of hGH-L-vFc by 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, recombination fusion protein shows Bioactivity and rhGH are similar.

Embodiment 7 pharmaceutical composition preparations

HGH-L-vFc fusion rotein 50 μ g

Physiological saline 1ml

Regulate pH to 6.7-7.2

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

All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each piece of document.In addition should be understood that 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 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) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change;

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

2. restructuring hGH-L-vFc fusion rotein as claimed in claim 1, is characterized in that, the sequence of described peptide linker is as shown in SEQ ID NO.:18 the 192nd to 207.

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

4. restructuring hGH-L-vFc fusion rotein as claimed in claim 1, it is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence as shown in 27-460 position in 27-462 position or SEQ ID NO.:22 in 27-456 position, SEQ ID NO.:20 in SEQ ID NO:18.

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

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

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

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

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

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

(a), in growth medium, in described recombination fusion protein is during every 24 hours, expresses and exceed 10 μ g/10 ⁶under the condition of individual cell, cultivate cell strain claimed in claim 7; With

(b) the described recombination fusion protein that purification step (a) is expressed, wherein said recombination fusion protein, on mole foundation, has the Bioactivity similar or higher with rhGH, the longer transformation period.

11. methods as claimed in claim 10, is characterized in that, the sequence of described peptide linker is as shown in SEQ ID NO.:18 the 192nd to 207.

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

13. methods as claimed in claim 10, it is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence as shown in 27-460 position in 27-462 position or SEQ ID NO.:22 in 27-456 position, SEQ ID NO.:20 in SEQ ID NO:18.

The method of the expression amount of the recombination fusion protein that 14. 1 kinds of raisings contain people GH, flexible peptide linker and human IgG Fc variant, is characterized in that, described method comprises:

(c) fusion rotein that purification step (b) is expressed,

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

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

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