CN103539861A - Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) - Google Patents
Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) Download PDFInfo
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Abstract
The invention discloses a long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (hFSH-Fc) and a preparation method thereof. The hFSH-Fc is a dimerization fusion protein. An amino acid sequence of the hFSH-Fc comprises an hFSHbeta subunit, CTP (carboxy-terminal peptide), an hFSHalpha subunit, a flexible peptide linker and a human IgG (immunoglobulin G) Fc variant from the N terminal to the C terminal in sequence. The hFSH-Fc has longer half-life in vivo and smaller side effects than existing hFSH. The invention also relates to an application of a recombinant hFSH-Fc composition to preparation of drugs for treating and/or preventing infertility.
Description
Technical field
The present invention relates to molecular biology and field of medicaments.More specifically, the present invention relates to long-acting recombinant human follicle stimulating hormone fusion rotein, its preparation method and application.This fusion rotein Half-life in vivo significant prolongation, its curative effect is better than existing Human Fallicle-Stimulating Hormone.
Background technology
At present, in world wide, Infertility rate, up to 15%, becomes outside continue cancer and cardiovascular and cerebrovascular diseases, has a strong impact on the third-largest disease of human health.China's Infertility number accounts for reproduction number of women more than 10%, and its sickness rate is obvious ascendant trend.Human follicle-stimulating element (Human follicle-stimulating hormone is called for short hFSH) is the common major ingredient that is used for the treatment of male and female infertility medicine of existing market, and China's existing market comprises that people urinates FSH medicine and the Recombinant FSH in source.
The developed countries such as urine source hFSH exists that virus is polluted, raw material sources are limited, collections is difficult, content is low and the problem such as purge process is complicated, American-European generally do not accept to urinate that product-derived goes on the market and sale.Comparatively speaking, restructuring hFSH has advantages of that at aspects such as its purity, antigenicity, security, virus-free infections urine source hFSH is incomparable.HFSH is a kind of glycosylated protein, and it is 43KD that SDS-PAGE detects its molecular weight.As a kind of medicine, guarantee its biological activity, necessary condition is to have correct three-dimensional structure and glycosylation modified.Have that to improve posttranslational modification function be the main cause that mammalian cell is selected as most of biological medicament protein expression hosts.Wherein, Chinese hamster ovary cell (Chinese Hamster Ovary Cell, CHO) be for the most successful host cell of eukaryote exogenous gene expression, existing increasing pharmaceutical protein has obtained high efficient expression therein, a lot of medicines are put on market, as EPO, G-CSF etc.Compare with other expression system, this system has many advantages, as have a complete translation post-treatment process, comprise glycosylation, hydroxylation, make the external source eukaryotic gene product of expressing can keep its natural structure and activity, and expression product is secreted into outside born of the same parents, is conducive to the separation and purification of foreign protein.
Although the existing reconstituted drug hFSH listing that utilizes Chinese hamster ovary celI to produce at present, but still there are the following problems: first, its transformation period is short, in order to reach effective result for the treatment of, needs frequent drug administration clinically, to patient, brings very big misery.For example, when when ovulating, patient must every day 1-2 intramuscular or subcutaneous injection hFSH, conventionally need to inject 8-12 days even for more time.This treatment plan is often with bad side effect, comprise nerve, internal secretion and immune stimulation and toxic side effect, and cause common complication as ovarian hyperstimulation syndrome, show as ovary increase, the increase of whole body capillary permeability and ascites and form, severe one can jeopardize patient's life.Secondly, the hFSH expression amount of recombinating is at present low, and preparation process is complicated and production cost is huge.In addition, hFSH is the glycosylated protein with two strands (α chain and β chain), and interchain connects with non covalent bond, the biological activity of the correct folding guarantee hFSH of two chains.The normal combination that how to keep two chains in protein expression and purge process is a challenge.Defect for existing restructuring hFSH and like product, the present invention utilizes the restructuring hFSH product of Protocols in Molecular Biology, cells produce and the purifying process development of new of optimization, with prolong half-life, keep its biological activity, improve restructuring hFSH protein expression level, thereby increase its drug effect, reduce side effect.
Summary of the invention
The present invention aims to provide Gonal-F's fusion rotein (Human follicle-stimulating hormone-Fc fusion protein, be called for short hFSH-Fc), its preparation method and application, to solve the problem that hFSH expression amount is low, purifying is difficult, Half-life in vivo is short of recombinating in prior art.
An object of the present invention is to provide restructuring hFSH-Fc fusion rotein, this fusion rotein is dimerization fusion rotein, its aminoacid sequence holds C end to comprise successively hFSH β subunit, CTP, hFSH α subunit, peptide linker (Linker from N, be called for short L) and human IgG Fc variant (vIgG4Fc, vIgG1Fc), as shown in SEQ ID NO:2,4 (hFSH β-CTP-hFSH α-L-vIgG4Fc, hFSH β-CTP-hFSH α-L-vIgG1Fc aminoacid sequence), above-mentioned fusion rotein is referred to as hFSH-Fc.
The aminoacid sequence of described hFSH β subunit has been removed the 1-18 amino acids residue in conventional hFSH β subunit, as shown in SEQ ID NO:7.
Described CTP (carboxy-terminal peptide, c-terminal peptides) aminoacid sequence is from 28-34 amino-acid residue of HCG β chain C-terminal, preferably CTP is 33 amino acid residue sequences from HCG β chain C-terminal, as shown in SEQ ID NO:6.
The amino acid residue sequence of described hFSH α subunit has been removed the 1-24 amino acids residue in conventional hFSH α subunit, as shown in SEQ ID NO:5.
Described peptide linker contains 2-20 amino-acid residue, and peptide linker contains the amino-acid residue that two or more are selected from glycine, Serine, L-Ala and Threonine, and preferred peptide linker aminoacid sequence is
GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。
Described human IgG Fc variant comprises: human IgG 4 hinge regions, CH2 and CH3 region that (1) contains Ser228Pro and Leu235Ala sudden change; (2) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.
Below specifically introduce human IgG Fc variant of the present invention, peptide linker and CTP function:
IgG Fc variant
The immunoglobulin (Ig) of IgG class is rich in protein in human blood, and their transformation period can be up to 21 days, and Fc fragment be in IgG holder compared with long half-lift major cause, there is the effect of stabilize proteins simultaneously.
Fc is from the Fc region of immunoglobulin (Ig), and Fc has vital role in the immune defense of eliminating pathogen.The effector function of IgG is mediated by Fc, by two kinds of main mechanisms: the combination of (1) and cell surface Fc acceptor (Fc γ Rs), by antibody dependent cellular cytotoxicity (ADCC) approach digestion pathogenic agent, or the combination of the C1q of (2) and the first complement component C1 part, cause cytotoxicity (CDC) approach that depends on complement, thus cracking pathogenic agent.Four kinds of human IgG isotypes (in (IgG1, IgG2, IgG3, IgG4), IgG1 can be effectively in conjunction with Fc γ Rs, IgG4 with the binding affinity of Fc γ Rs than the low order of magnitude of IgG1.Human IgG1 can also be effectively in conjunction with C1q, and activating complement cascade reaction, and the extreme ability that lacks activating complement cascade of IgG4 performance.For medical use, the Fc region of restructuring dimerization albumen must not can mediate effector function and cracking or remove these cells.Therefore, the Fc region of hFSH-Fc must be non-cracking performance, and in conjunction with Fc γ Rs and C1q and aspect trigger effect subfunction, Fc is non-activity preferably.Obviously, do not have a kind of natural IgG isotype to be applicable to producing hFSH-L-Fc restructuring dimerization albumen.In order to obtain the Fc of non-cracking performance, must make some amino acid mutations in natural Fc region, to reduce its effector function.
By analyzing the aminoacid sequence of the IgG isotype of different genera, find that near the Fc partial display N-terminal of CH2 region works in the combination of IgG Fc and Fc γ Rs, and be bonded to pass part and parcel with C1q, be positioned near the CH2 region carboxyl terminal of human IgG.The effector function causing in order to reduce the combination of Fc and Fc γ Rs and C1q, the present invention uses following human IgG Fc variant (as Fig. 1):
(1) IgG4Fc variant (vIgG4Fc): human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change;
(2) IgG1Fc variant (vIgG1Fc): human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.
Above-mentioned Fc variant shows much lower effector function than natural IgG4Fc and IgG1Fc, is more suitable for preparation restructuring hFSH-Fc fusion rotein.
Peptide linker
The length of connection peptides is extremely important to the activity of restructuring dimerization albumen.Existing people has reported erythropoietin (EPO) derivative (as dipolymer), compare with EPO monomer, the restructuring dimerization albumen that contains 2 complete EPO regions (3 to 7 the amino acid peptide joints of being separated by) shows the activity that weakens (referring to Qiu H etc., J Biol Chem, 273:11173-11176,1998).Yet when the length of the interregional peptide linker of these two EPO is 17 amino acid, the in vitro and in vivo biological activity of dimer EPO molecule obviously improves (Sytkowski AJ etc., J Biol Chem, 274:24773-24778,1999; U.S. Patent No. 6,187,564).The connection peptides of this possible explanation for increasing between restructuring dimerization albumen two portions, makes two portions of this molecule can exercise respectively its function (referring to Ashkenazi A etc., Curr Opin in Immunol, 9:195-200,1997).
The inventor is through long-term and deep research, and the hinge region peptide linker that has designed first a kind of uniqueness reduces space steric effect, can make the C end of hFSH α chain and the restructuring dimerization albumen of Fc variant coupling, and there is soft peptide linker centre.This restructuring dimerization albumen not only can not cause the afunction of hFSH, can maintain on the contrary, even improve the biological activity of hFSH-Fc restructuring dimerization albumen.The amino acid residue sequence of preferred peptide joint is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
CTP
Glycosylation played an important role for activity and the transformation period of albumen, and the glycosylation site on albumen has two classes, comprised O-glycopeptide chain and N-glycopeptide chain.CTP be one section from 28-34 amino-acid residue of HCG β key C-terminal, have the bibliographical information HCG transformation period relatively long compared with hFSH, be mainly derived from this CTP peptide section.It contains the glycosylation site that 4 O-connect, and can increase the level of glycosylation of albumen, improves the activity of albumen and extends the albumen transformation period in vivo.
Restructuring hFSH-Fc fusion rotein of the present invention has following characteristics: this recombination fusion protein is dimerization fusion rotein, and its aminoacid sequence holds C end to comprise successively hFSH β subunit, CTP, hFSH α subunit, peptide linker and human IgG Fc variant from N.Human IgG Fc variant has the effect that extends Half-life in vivo and stabilize proteins, and Fc variant is non-cracking performance, can reduce the effector function of being combined with Fc γ Rs and C1q and triggering.CTP can improve protein-active and extend Half-life in vivo, and its non-immunogenicity connects α chain and the β chain of hFSH by CTP, not only have no side effect, and can make there is certain steric hindrance between two chains, is conducive to it and correctly folds and do not affect function.By soft peptide linker, carry out the coupling of C end with the Fc variant of hFSH α chain, can maintain, even improve the biological activity of restructuring hFSH-Fc fusion rotein.
The present invention is connected to CTP, peptide linker and human IgG Fc variant in an orderly manner first in FSH and forms novel restructuring hFSH-Fc fusion rotein, in this fusion rotein, the positional alignment of human IgG Fc variant, CTP and peptide linker can maintain the sterie configuration that FSH is correct, and do not affect its biological activity, and can the significant prolongation transformation period, during clinical application, can reduce frequency of injection, reduce the toxic side effect producing in existing treatment plan.
Another object of the present invention is to provide the preparation method of restructuring hFSH-Fc fusion rotein, and this preparation method comprises:
(1) build the expression vector of coding restructuring hFSH-Fc fusion rotein;
(2) stably express of restructuring hFSH-Fc fusion rotein in mammalian host cell;
(3) high-density cells is cultivated restructuring hFSH-Fc fusion rotein;
(4) purification of restructuring hFSH-Fc fusion rotein.
Specifically, the expression vector step that builds coding restructuring hFSH-Fc albumen is: adopt artificial synthesis to obtain coding restructuring hFSH-Fc antigen-4 fusion protein gene and carried out codon optimized nucleotide sequence (as shown in SEQ ID NO:1,3), be inserted into mammalian cell expression vector (as pCDNA3), obtain and contain hFSH-Fc destination gene expression plasmid pCDNA3-hFSH-Fc (Fig. 5).The nucleotide sequence optimization of gene is that the codon based on mammalian host cell preference is selected design.
Described mammalian cell expression vector can adopt commercially available but be not limited to, as: pCDNA3, pCMV/ZEO, pIRES, pDR, pBK, pSPORT etc. can be used for the carrier that eukaryotic cell system is expressed, preferably, and pCDNA3.
The stably express step of restructuring hFSH-Fc fusion rotein in mammalian host cell is: the expression plasmid that contains hFSH-Fc albumen is transfected into suitable mammalian host cell, and screening obtains the cell strain of stablizing high-expression target proteins.
Described mammalian host cell comprises CHO, HEK293, COS, BHK, NS0 and Sp2/0 cell.Preferably, Chinese hamster ovary celI; More preferably, tamed DHFR (Dihydrofolate Reductase, Tetrahydrofolate dehydrogenase) defective type Chinese hamster ovary celI (the abbreviation CHO-DHFR that adapts to suspension growth in serum free medium
-).
Described transfection method comprises calcium phosphate method, electroporation transfection method, liposome transfection, and preferred transfection method is electroporation transfection method.
Described screening method is first to utilize selection markers to screen, and then by amplification selected marker, can improve expression amount and obtain and stablize overexpression cell line.Selection markers is any one suitable selectivity resistance marker known in the art, as ZEO (Zeocin, bleomycin), G418 (aminoglycoside antibiotics), PUR (puromycin, tetracycline), HYP (Hygromycin, Totomycin), preferred resistant gene is ZEO; Selection markers also can be any one fluorescent mark gene known in the art, comprise GFP (Green Fluorescent Protein, green fluorescent protein), RFP (Red Fluorescent Protein, red fluorescent protein), preferred fluorescent mark gene is GFP.Amplification selected marker is DHFR sequence or GS (Glutaminesynthetase, glutamine synthetase) sequence known in the art, and the selected gene that preferably increases is DHFR.Due to CHO-DHFR-cell self disappearance Tetrahydrofolate dehydrogenase (DHFR), cannot self tetrahydrobiopterin synthesis folic acid, so must just can survive in the nutrient solution that has added xanthoglobulin (hypoxanthine), thymus pyrimidine (Thymidine) and glycine.And by goal gene and DHFR gene co-transfection, not only obtain not containing the cell clone that also can grow on the substratum of above-mentioned additive, what is more important is because DHFR can be by folacin MTX (Methotrexate, methotrexate) suppress, under MTX concentration selective pressure, DHFR gene a lot of copy number that must increase could be survived, thereby obtains anti-MTX clone; Again because the goal gene with DHFR gene co-transfection tends to be incorporated into the same area on cell chromosome together with it, so the sequence fragment of encoding exogenous recombinant protein also increases along with the amplification of DHFR gene, can obtain the cell clone of mass expressing external albumen.
The step that high-density cells is cultivated restructuring hFSH-Fc fusion rotein is: the above-mentioned stable cell line screening is proceeded to shaking flask or biological reactor carries out large scale culturing, particularly, the present invention, by the optimization to cell culture condition, obtains the cell culture fluid of high expression level restructuring hFSH-Fc fusion rotein.The degree of glycosylation that cell culture processes of the present invention can be realized high-density cells cultivation, recombinant proteins and output lifting, recombinant protein increases and sialic acid content raising.
The optimization of described cell culture condition comprises cooling culture method, specifically, and when cell density reaches 1 * 10
7individual/during mL, temperature to be down to 33 ℃ by 37 ℃, at this temperature, cultivate until express output and no longer increase.The method can improve the cumulative withdrawal of activity level and the recombinant protein of expressing protein.
The optimization method of described cell culture condition is also included in substratum and adds special additive, preferably, at basic medium, adds 100 μ M Cu
2+, feeding substratum adds 2mM ManNAc (N-ethanoyl-D-epichitosamine).The method can make to recombinate degree of glycosylation of hFSH-Fc fusion rotein increases, and sialic acid content improves approximately 20%.
The purification step of restructuring hFSH-Fc fusion rotein is:
1) Protein A affinity chromatography: centrifugal, collect supernatant, the characteristic of albumen coupling Fc fragment according to the present invention, utilizes affine Protein A column chromatography.
2) hydrophobic chromatography column purification: adopt hydrophobic chromatography post, different according to the hydrophobicity of restructuring hFSH-Fc fusion rotein, further remove the impurity in target protein after above-mentioned ProteinA purifying.
Described drainage column comprises Butyl Sepharose4Fast Flow, Octyl Sepharose4Fast Flow, Phenyl Sepharose6Fast Flow, Butyl-S Sepharose6Fast Flow, Butyl Sepharose4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B.Preferably, Phenyl Sepharose6Fast Flow.
The preparation method of restructuring hFSH-Fc fusion rotein disclosed in this invention, expression output is high and because of the coupling with IgG Fc variant, formed recombinant protein can obtain efficiently purifying easily by ProteinA affinity chromatography.The fusion rotein purity obtaining after hydrophobic chromatography is further purified reaches more than 98%.In addition, in the restructuring hFSH-Fc fusion rotein that the present invention announces, α chain and β chain can correctly fold, and have avoided α-α dimer, the dimeric appearance of β-β, have greatly simplified purification step, have reduced production cost.
An also object of the present invention has been to provide the pharmaceutical composition that contains restructuring hFSH-Fc fusion rotein, it is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring hFSH-Fc fusion rotein of the present invention of significant quantity.
Specifically, described pharmaceutical composition contains significant quantity (as 0.000001-90wt%; 0.1-50wt% preferably; Better, recombinant long-acting hFSH-Fc fusion rotein of the present invention 5-40wt%), 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.
Described pharmaceutically acceptable carrier comprises (but being not limited to): sucrose, N.F,USP MANNITOL, polysorbas20 (Tween20), methionine(Met), salt solution, damping fluid, glucose, water, glycerine and composition 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 the pattern of administration and the severity of 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.
A further object of the present invention is the application of restructuring hFSH-Fc fusion rotein in treating and/or preventing people's infertility.
Restructuring hFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, thus pharmacokinetics and drug effect improved, with existing FSH comparison, in clinical application, can reduce patient infusion number of times, reduce side effect, alleviate patient suffering and economical load.
The advantage of restructuring hFSH-Fc fusion rotein of the present invention and preparation method thereof is summarized as follows:
1, restructuring hFSH-Fc fusion rotein of the present invention is the new fusion protein being formed by CTP, peptide linker and human IgG Fc variant (vIgG4Fc or vIgG1Fc) and the orderly coupling of hFSH, maintained the correct sterie configuration of FSH, Half-life in vivo that can significant prolongation albumen, greatly improve the expression amount of hFSH in Chinese hamster ovary celI, and its in vitro and in vivo is active similar with existing natural hFSH.
2, the α chain of dimerization strand hFSH-Fc fusion rotein and β chain are correctly folding with covalent linkage, avoid forming α-α dimer and β-β dimer, have greatly simplified purifying process, reduce production costs.
3, restructuring hFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, its transformation period is existing restructuring the more than 3 times of hFSH, in clinical application, can reduce patient infusion number of times, reduces the side effect that existing treatment plan produces.
Accompanying drawing explanation
Fig. 1. shown the comparison of the hinge region of human IgG1, IgG4 and their variants and the aminoacid sequence in CH2 region.Compare this three partial amino-acid series: 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. shown restructuring hFSH-Fc strand and dimerization structural representation.A) strand hFSH-Fc; B) hFSH-Fc of dimerization.
Fig. 3. shown the nucleotide sequence of hFSH-Fc (hFSH β-CTP-hFSH α-L-vIgG4Fc) of HindIII-EcoRI fragment in pCDNA3 expression vector and the aminoacid sequence of derivation.The nucleotide sequence of hFSH-Fc comprises that coding is containing leading peptide (1-18), hFSH β chain, CTP, ripe hFSH α chain, peptide linker, IgG4Fc variant (vIgG4Fc).Ripe restructuring hFSH-Fc fusion rotein contains ripe hFSH β chain (19-129), CTP (130-162), ripe α chain (163-254), peptide linker (255-270) and IgG4Fc variant (vIgG4Fc) (271-499).
Fig. 4. shown the nucleotide sequence of hFSH-Fc (hFSH β-CTP-hFSH α-L-vIgG1Fc) of HindIII-EcoRI fragment in pCDNA3 expression vector and the aminoacid sequence of derivation.The nucleotide sequence of hFSH-Fc comprises that coding is containing leading peptide (1-18), hFSH β chain, CTP, ripe hFSH α chain, peptide linker, IgG1Fc variant (vIgG1Fc).Ripe restructuring hFSH-Fc fusion rotein contains ripe hFSH β chain (19-129), CTP (130-162), ripe α chain (163-254), peptide linker (255-270) and IgG1Fc variant (vIgGIFc) (271-497).
Fig. 5. shown the eukaryon expression plasmid collection of illustrative plates of constructed coding hFSH-Fc fusion rotein.This expression plasmid total length 9063bp, contains 10 oligogene segments, comprises 1.CMV promotor; 2. target gene hFSH-Fc; 3.IRES; 4.Zeocin resistance screening gene; 5.BGH terminator; 6.SV40 promotor; 7.DHFR amplification gene; 8.SV40 terminator; 9. ampicillin resistance gene (Ampicillin); 10.ColE1 replication orgin (Ori).
Fig. 6. shown the accumulation tendency graphic representation of the hFSH-Fc cell strain expression-secretion hFSH-Fc albumen of recombinating in 7L bio-reactor.
Fig. 7 .Westem bloting result has shown the successful expression of restructuring hFSH-Fc fusion rotein in Chinese hamster ovary celI.Non-reduced glue, Lane1: the hFSH (about 43kDa) in urine source; Lane2: restructuring hFSH-vIgG1Fc fusion rotein of the present invention (about 140kDa); Lane3: restructuring hFSH-vIgG4Fc fusion rotein of the present invention (about 140kDa).
Fig. 8. shown strand and the dimerization hFSH-Fc 10%SDS-PAGE electrophoretogram under reductive condition and non-reduced condition.Lane1: non-reduced glue, restructuring dimerization hFSH-vIgG1Fc fusion rotein (about 140kDa); Lane2: non-reduced glue, restructuring dimerization hFSH-vIgG4Fc fusion rotein (about 140kDa); Lane3: go back virgin rubber, recombinant single chain hFSH-vIgGIFc fusion rotein (about 70kDa); Lane4: go back virgin rubber, recombinant single chain hFSH-vIgG4Fc fusion rotein (about 70kDa).
Fig. 9. shown that restructuring hFSH-Fc fusion rotein of the present invention, restructuring hFSH and people urinate the metabolic chart of hFSH in rat body.
Embodiment
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, according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Gene order design is to be optimized based on Chinese hamster ovary celI preference codon, adopt synthetic the encode signal peptide of hFSH albumen β chain and the fusion gene of mature peptide section, CTP and hFSH α chain mature peptide section thereof of containing through optimizing of method of synthetic, the 756bp DNA fragmentation of synthesized is inserted to transfer vector as between the EcoRV restriction enzyme site in pUC57, obtain hFSH plasmid (phFSH), used the exactness of DNA sequencing method validation insertion sequence.
Synthetic contains BamHI (5 ' end) and the flexible peptide linker of coding (Linker is called for short " L ") of EcoRI (3 ' end) restriction enzyme site and fusion gene L-vIgG4Fc and the L-vIgG1Fc of human IgG Fc variant (vIgG4Fc and vIgG1Fc) fragment respectively.The fusion gene fragment of acquisition is inserted into respectively to transfer vector as between the BamHI of PUC19 and EcoRI site, obtains the plasmid containing two kinds of variants, be respectively pL-vIgG4Fc and pL-vIgG1Fc.By DNA sequencing, verify the gene order of L-vIgG4Fc and L-vIgG1Fc.For preparing two kinds of hFSH-L-Fc fusion genes, with restriction enzyme SpeI and BamHI double digestion phFSH plasmid, after gel electrophoresis, glue reclaims the signal peptide of coding hFSH albumen β chain and the fusion gene fragment of mature peptide section, CTP and hFSH α chain mature peptide section thereof, purified said gene fragment is inserted into respectively 5 '-end of peptide linker in pL-vIgG4Fc and pL-vIgGIFc plasmid, and T4 ligase enzyme connects and composes phFSH-L-vIgG4Fc and phFSH-L-vIgGIFc plasmid.Constructed fusion gene is comprised of hFSH β, CTP, hFSH α, peptide linker and Fc variant gene, and as shown in Figure 2 a, dimerization structure as shown in Figure 2 b for its single-stranded structure.
Restriction enzyme SpeI/EcoRI is double digestion phFSH-L-vIgG4Fc and phFSH-L-vIgG1Fc plasmid respectively, and DNA gel-purified obtains hFSH-L-vIgG4Fc and hFSH-L-vIgG1Fc fragment.Three kinds of good hFSH-L-Fc fragments of purifying are inserted into mammalian cell expression plasmid as between the corresponding restriction enzyme site of pCDNA3 (Invitrogen), final acquisition containing fusion gene expression plasmid pCDNA3-hFSH-L-vIgG1Fc and pCDNA3-hFSH-L-vIgG4Fc, be referred to as pCDNA3-hFSH-Fc plasmid, as shown in Figure 5.This plasmid is containing the required promotor CMV of mammal cell with high efficient expression alien gene albumen; This plasmid also contains two kinds of selected markers, thereby can have amicillin resistance in bacterium, can have bleomycin (zeocin) resistance in mammalian cell.In addition, when host cell is DHFR genetic expression defective type, the Tetrahydrofolate dehydrogenase of the mouse that PCDNA3 expression vector is contained (DHFR) gene, makes it when methotrexate (MTX) exists, can coamplification pFSH-Fc fusion gene and DHFR gene.
The α chain and the β chain that by CTP peptide section, connect hFSH, can be convenient to two chains correctly folding.The coupling of carrying out hFSH and Fc fragment by peptide linker (preferably for flexible joint) can improve the biological activity of albumen; for the purpose of the present invention; preferably length is approximately 20 or (but can not be less than 2) amino acid whose peptide linker still less; certainly by the peptide linker of 1 Amino acid profile also in protection scope of the present invention, should use and contain or by 2 or the peptide linker of oneself following Amino acid profile of multiselect more: glycine, Serine, L-Ala and Threonine.The peptide linker of the embodiment of the present invention contains Gly-Ser peptide member, and its aminoacid sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
The stably express of embodiment 2. restructuring hFSH-Fc fusion roteins in mammalian cell
The expression plasmid pCDNA3-hFSH-L-Fc that embodiment 1 is built is transfected into DHFR deficient CHO host cell (CHO-DHFR
-), Fig. 2 b has shown the schematic diagram of restructuring dimerization hFSH-Fc fusion rotein.Adopt electricity to turn method and carry out transfection, use the Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) of 960 μ Fd electric capacity, its electric field is set to 250V, in cuvette 2～5 * 10
7in individual cell, add the linearizing plasmid DNA of PvuI for 10 μ g.In transfection two days later, make substratum into growth medium containing 100 μ g/mL Zeocin resistant maker genes, obtain the transfectant through resistance primary dcreening operation.Adopt the method for westernblotting, with the expression of anti-hFSH antibody test hFSH-Fc.Utilize DHFR amplification selected marker to improve the expression level of restructuring dimerization albumen, in the growth medium that contains progressive concentration MTX, use the restructuring dimerization protein gene of DHFR gene coamplification transfection for this reason.The transfectant that can grow in up to 10 μ M/mL MTX substratum with Method of Limited Dilution method subclone.The secretion rate of subclonal cell line is done further to analyze.Screening secretion level surpasses approximately 5 (preferably approximately 20) μ g/10
6the cell strain of (1,000,000) individual cell/24 hour, obtains and stablize the recombinate cell strain of hFSH-Fc fusion rotein of high expression level.
Production and the purifying of embodiment 3. restructuring hFSH-Fc fusion roteins
The high yield cell strain that embodiment 2 is obtained, first in culture dish, carrying out serum-free domestication cultivates, then transfer in shaking flask and suspend and tame cultivation, in domestication process, carry out the screening of substratum simultaneously, the growth conditions, the growth tendency that add different composition observation of cell, and the biochemical indicator such as the activity of expression product and sialic acid, preferred cell culture condition is: basic medium adds 100 μ M Cu
2+, feeding substratum adds 2mM ManNAc (N-ethanoyl-D-epichitosamine), and the method can make to recombinate degree of glycosylation of hFSH-Fc fusion rotein increases, and sialic acid content improves approximately 20%.After taming successfully, carry out cell amplification, amplification, to q.s, is carried out the monitoring of 7L bio-reactor and is cultivated, and at cell density, surpasses 1 * 10
7individual/to start to be cooled to 33 ℃ of cultivations during mL, batch growth cycle is 20 days.With 1ml ProteinA column chromatography, recombinant protein is carried out to preliminary purification, measure the expression amount of two kinds of restructuring hFSH-Fc fusion roteins, result demonstration, the cumulative withdrawal that restructuring hFSH-L-vIgG4Fc and restructuring hFSH-L-vIgG1Fc cell strain are expressed is respectively 0.82g/L, 0.73g/L (Fig. 6).
The purifying of restructuring hFSH fusion rotein comprises the following steps:
1) Protein A affinity chromatography: centrifugal, collect supernatant, according to the present invention, the characteristic of albumen coupling Fc fragment, utilizes affinity chromatography method, supernatant liquor is loaded onto to the Protein A post of phosphate buffer saline (PBS) balance; Fusion rotein to be reorganized is incorporated into after ProteinA, with PBS, wash this post, until OD280 value is lower than 0.01, then Recombinant FSH-Fc fusion rotein of the sodium-acetate buffer elution of bound that is 4.0 with 20mM pH, finally with in the 1M Tris-HCl damping fluid of pH10.0 and the active liquid of collecting.The hFSH-Fc purity of protein of purifying can reach more than 95%.
2) hydrophobic chromatography: be 20mM Tris-HCl-1.5M NaCl (pH8.0) damping fluid by the active collection of above-mentioned protein A fluid exchange with hyperfiltration process, by this sample pipetting volume to the phenyl-6Fast Flow post of crossing by 20mM Tris-HCl-1.5M NaCl (pH8.0) balance, first use identical balance liquid drip washing, use again 20mM Tris-HCl-1.35M NaCl (pH8.0) drip washing, finally use 20mM Tris-HCl-0.5M NaCl (pH8.0) buffer solution elution.
Western bloting result has shown the successful expression of two kinds of restructuring hFSH-Fc fusion roteins of the present invention in Chinese hamster ovary celI, as shown in Figure 7, SDS-PAGE gel electrophoresis collection of illustrative plates under non-reduced condition, hFSH (commerical prod) and restructuring of the present invention hFSH-Fc fusion rotein that people urinates source show corresponding target protein hybridization band at 43kDa and 140kDa respectively, have verified in the restructuring hFSH fusion rotein that the present invention obtains and have contained hFSH albumen.Fig. 8 be purifying two kinds of hFSH-Fc fusion roteins reduction and non-reduced condition under SDS-PAGE gel electrophoresis collection of illustrative plates.Result shows, the hFSH-Fc purity of protein of purifying of the present invention can reach more than 98%, the molecular weight of hFSH-Fc under reductive condition be under non-reduced condition molecular weight 50%.
The inside and outside determination of activity of embodiment 4. restructuring hFSH-Fc fusion roteins
The external activity (immunogen activity) of restructuring hFSH-Fc fusion rotein of the present invention adopts the FSH enzyme immunoassay detection kit that BIOCHECK (U.S.) company produces to detect, method reference reagent box specification sheets.Activity in vivo adopts the ovary weightening finish method in 2010 editions < < British Pharmacopoeia > > to detect.Protein content determination is used LOWRY quantitative method.Get HCG preparation, add 0.1% albumin phosphate buffered saline buffer (pH7.2 ± 0.2) solution, make the trial-product diluent that contains 70IU/mlHCG.According to standard substance labelled amount, restructuring hFSH, people, urinate hFSH and restructuring hFSH-Fc fusion rotein is estimated to tire, with trial-product diluent (pH7.2 ± 0.2), standard substance, restructuring hFSH, people are urinated to hFSH, restructuring hFSH-L-vIgG4Fc and the hFSH-L-vIgG1Fc that recombinates is mixed with the solution containing FSH1.67IU/ml dosage.Select 19～28 ages in days, the age must not differ and surpasses 3, and body weight must not differ and surpassed the female mouse of Wistar of 10 grams, is divided into standard substance, restructuring hFSH group, people and urinates hFSH group, restructuring hFSH-L-vIgG4Fc and restructuring hFSH-L-vIgG1Fc group, every group of 6 animals.Subcutaneous injection after rat neck, injects twice every day, and per injection volume is 0.2ml, injects continuously three days, and every day is in same time administration.Last drug administration by injection, after 24 hours, adopts dislocation method to put to death animal according to order of administration, gets ovary, peels off after blotting surface-moisture and weighs, and records organ weight.According to standard substance ovary, weightening finish adopts parallel line analysis method to calculate restructuring hFSH, people urinates hFSH and the activity of the hFSH-Fc that recombinates.Record external activity that restructuring hFSH-L-vIgG4Fc, restructuring hFSH-L-vIgG1Fc, restructuring hFSH and people urinate hFSH and be respectively 10005,10020,9928 and 9321IU/ml, its activity in vivo is respectively 10120,10012,8190 and 9051U/ml, result shows, two kinds of restructuring hFSH-Fc fusion roteins of the present invention have inside and outside biologic activity.
The pharmacokinetics of embodiment 5. restructuring hFSH-Fc fusion roteins is measured
Be divided into restructuring hFSH-Fc (comprising restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc), people urinates hFSH and restructuring hFSH administration group, 5 of every group of male wistar rats of selecting body weight 200-250g, give subcutaneous injection by 15IU/kg respectively.Restructuring hFSH group and people urinate hFSH group after administration 1,2,3,4,6,8,12,36,56h gets blood, restructuring hFSH-Fc group respectively after administration 1,2,4,8,12,24,56,120,176,200,264,340h gets blood, after the centrifugal 5min of 3000rpm, draw serum ,-20 ℃ of preservations.Adopt ELISA test kit (BIOCHECK, the U.S.) to measure FSH immunocompetence in each time point blood plasma.Use PKSolver2.0 software, by statistics moments method, calculate and respectively organize main pharmacokinetic parameter.Each organizes pharmacokinetic curve as shown in Figure 9, and the transformation period, result was as table 1.Result shows, restructuring hFSH and people urinates the elimination transformation period of hFSH in rat body and is about respectively 11.35 ± 1.0h and 12.7 ± 2.8h, and wait dosage the present invention greatly to extend recombinate elimination transformation period of hFSH-Fc fusion rotein, the elimination transformation period of restructuring hFSH-vIgG4Fc, restructuring hFSH-vIgG1Fc fusion rotein is respectively 40.64 ± 10.28h and 38.34 ± 12.35h, is that restructuring hFSH and people urinate the more than 3 times of FSH.
Table 1 the present invention recombinates transformation period of hFSH-Fc
The short super Effect of Ovulation of embodiment 6. restructuring hFSH-Fc fusion roteins to female rats
75 of Healthy female SD rats, body weight 200-250g normally feeds and supports quarantine observation more than 7 days under experimental situation.Be divided at random 5 groups: negative control group, restructuring hFSH-vIgG4Fc administration group, restructuring hFSH-vIgG1Fc administration group, restructuring hFSH control group, people urinate hFSH control group, every group of 15 rats, each is organized rat body weight and distributes similar.First by vaginal smear method, observe animal oestrus cycle one-period.Afterwards, restructuring hFSH-vIgG4Fc administration group, restructuring hFSH-vIgG1Fc administration group are pressed 45IU/kg dosage in dioestrus first day subcutaneous injection administration 1 time; Restructuring hFSH group and people urinate hFSH group and in dioestrus first day, start successive administration 3 days (15IU/kg/ days) by total dose 45IU/kg, and negative control group is to equivalent physiological saline.All groups were in (proestrum) subcutaneous injection HCG (10IU/ only) on the 4th.(rutting sedson) on the 5th carries out ovary separation, and weighs after rat is anaesthetized, liquid-solid fixed with BouinShi to separated ovary, paraffin embedding, does serial section to fixing ovary every 6 μ m, under microscope, carry out histological observation, and record follicle number.
Each count results of organizing rat follicle number is in Table 2, result shows, with negative control comparison, the present invention two kinds of restructuring hFSH-Fc fusion roteins, Recombinant FSH and people urinate hFSH all has significantly short super Effect of Ovulation (P < 0.01) to female rats, two kinds of the present invention restructuring hFSH-Fc fusion roteins are only administered once, and restructuring hFSH and people urinate hFSH and need be administered three times and just can reach equal curative effect.
The ovulation effect of table 2 restructuring hFSH-Fc fusion rotein to female rats
Note: t check, with negative control group comparison,
ap < 0.01
90 of female infant rats of 9 age in days SD getting on the same day birth, wherein 15 be Normal group: by the disposable subcutaneous injection neutrality tea oil 0.05ml of nape portion; Remain 75 for modeling animal groups: every by the disposable subcutaneous injection testosterone propionate 1.25mg of nape portion.The 70th age in days vaginal opening, continuously two oestrus cycles of vaginal smear (oestrus cycle is 5 days, totally 10 days).The continuous vaginal smear of normal control treated animal should present typical case's oestrus cycle (proestrus, oestrus, metaoestrus, dioestrus), and the oestrus cycle person of not being true to type is rejected; And the vaginal epithelial cell of modeling animal groups animal should change without the oestrus cycle, be persistence angling, prompting anovulatory rats Modelling success, rejects unsuccessful person.After the 81st age in days, get 13 of rats in normal control group, by subcutaneous injection in 10ml/kg/ days, give distilled water, by above-mentioned anovulatory rats animal pattern, by being divided at random 5 groups after body weight layering: model negative control group, restructuring hFSH-vlgG4Fc, restructuring hFSH-vlgG1Fc administration group, Recombinant FSH control group and people urinate hFSH control group.Model negative control group rat gives distilled water by subcutaneous injection in 10ml/kg/ days; Restructuring hFSH-vlgG4Fc and restructuring hFSH-vlgG1Fc administration group give subcutaneous injection by 45IU/kg/ time, and within every 3 days, administration is 1 time, within continuous 15 days, is total to administration 5 times; Restructuring hFSH group and people urinate hFSH group and give subcutaneous injection by 7.5IU/kg/ time, and administration every day 2 times is total to administration 30 times for continuous 15 days.After administration finishes, each organizes equal continuous two oestrus cycles of vaginal smear of rat, to judge Rat Ovulation situation.After vaginal smear examination ovulation, each organized rat before the oestrus cycle, the abdominal injection urethane of first weighing again anesthesia, choosing ovary and the uterine cancer cell sample in left side puts in 10% neutral formalin fixing, carry out ovary and uterus PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM, the quantitative observation index of ovary is Follicles sum, corpus luteum number; Uterus observation index is inner film thickness.
Table 3 has shown respectively organizes Rats During The Estrous Cycle situation result, table 4 has shown respectively organizes rat ovary and uterus pathomorphism result, result shows and negative control group comparison, two kinds of restructuring hFSH-Fc fusion roteins of the present invention, restructuring hFSH and and people urinate hFSH the female rats of not ovulating all had and significantly actuates feelings restitution, large follicle number and corpus luteum number are also than negative control group obvious higher (P < 0.01), but two kinds of restructuring hFSH-Fc fusion roteins of the present invention administration 5 times, and restructuring hFSH and and people urinate hFSH and need administration just can reach equal drug effect 30 times.Presentation of results, two kinds of the present invention restructuring hFSH-Fc fusion roteins, restructuring hFSH and and people urinate hFSH and male sex hormone is caused to the female rats Infertility of not ovulating all have remarkable therapeutic action, but the administration number of times of two kinds of restructuring hFSH-Fc fusion roteins of the present invention is obviously less than restructuring hFSH and people urinates hFSH.
Table 3 restructuring hFSH-Fc fusion rotein is actuated feelings recovery situation to the female rats of not ovulating
Note: X
2check, with the comparison of model negative control group,
ap < 0.01
Table 4 restructuring hFSH-Fc fusion rotein is on rat ovary and the morphologic impact of uterus pathology of not ovulating
Note: t check, with the comparison of model negative control group,
ap < 0.01
The impact that embodiment 8. restructuring hFSH-Fc fusion roteins stimulate rat ovary
40 22 age in days female Wistar rats are divided at random and are divided into Normal group, restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc administration group, people and urinate hFSH control group and restructuring hFSH control group, 10 every group.The rat that restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc administration group, people urinate hFSH and restructuring hFSH administration group plays every according to continuous 4 days of 10IU dosage subcutaneous injection relative medicine at 22 ages in days, and the equal subcutaneous injection HCG30IU/ of all administration groups of 26 age in days only; Normal group is from 22-26 age in days subcutaneous injection every day equal-volume physiological saline.All groups all at the 28th age in days through tail vein injection 1%Evans Blue (EB, Sigma) dyestuff 0.1ml, after 30min, de-neck method is put to death rat, cuts abdominal cavity and observes and have or not ascites.Without ascites or a small amount of ascites person to intraperitoneal perfusion 5ml physiological saline, perfusion liquid is collected in 10ml test tube, all be diluted to 10ml, add again 0.05ml0.1M NaOH solution, with the speed of 3000r/min centrifugal 10min at room temperature, with spectrophotometer, in the row colorimetric estimation of wavelength 600nm place, record OD value.Finally take off both sides ovary and use immediately scales/electronic balance weighing.Observation index is: 1, abdominal cavity capillary permeability: according to normal concentration and OD value drawing standard curve thereof, calculate peritoneal fluid EB content.2, ascites scoring: 1 minute: without ascites; 2 minutes: a small amount of ascites exists; 3 minutes: middle amount ascites exists; 4 minutes: ascites obviously exists; 5 minutes: a large amount of ascites exists or ascites is overflowed from abdominal incision.3, ovary weight.According to the ovarian hyperstimulation syndrome Case definition of Golan, Ovarian Volume increases, abdominal cavity capillary permeability increases and ascites all occurs being diagnosed as ovarian hyperstimulation syndrome.
Within continuous 4 days, high dosage is injected restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc, restructuring hFSH and people urinate hFSH, the result of table 5 shows, restructuring hFSH-vIgG4Fc, the rat abdominal cavity EB content of restructuring hFSH-vIgG1Fc group and ascites scoring and control group are relatively without difference, only have ovary weight to increase, restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc do not cause rat ovary hyperstimulation syndrome, and people urinates the rat ascites scoring of hFSH group and restructuring hFSH group, abdominal cavity EB content and ovary weight are all apparently higher than control group, restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc group, people urinates hFSH and restructuring hFSH has caused rat ovary hyperstimulation syndrome.Results suggest, the restructuring hFSH-vIgG4Fc of same injection higher dosage and restructuring hFSH-vIgG1Fc, restructuring hFSH and people urinate hFSH, it is less that restructuring hFSH-vIgG4Fc and the recombinate relative hFSH of restructuring of hFSH-vIgG1Fc and people urinate FSH side effect, and clinical application is safer.
Table 5 is respectively organized EB content and ovary weight comparison in ascites scoring, peritoneal fluid
Note: t check, with Normal group comparison,
ap < 0.01; Compare with restructuring hFSH-vIgG2Fc group;
bp < 0.01,
cp < 0.05.
Claims (10)
1. restructuring hFSH-Fc fusion rotein, is characterized in that, described fusion rotein is dimerization fusion rotein, and its aminoacid sequence holds C end to comprise successively hFSH β subunit, CTP, hFSH α subunit, peptide linker and human IgG Fc variant from N.
2. restructuring hFSH-Fc fusion rotein according to claim 1, the aminoacid sequence of wherein said hFSH β subunit is the aminoacid sequence shown in the hFSH β SEQ ID NO:7 after the 1-18 amino acids residue of having removed in conventional hFSH β subunit; The aminoacid sequence of wherein said CTP is 28-34 the amino-acid residue from HCG β chain C-terminal, and preferably CTP is 33 amino acid residue sequences from HCG β chain C-terminal, as shown in SEQ ID NO:6; The amino acid residue sequence of wherein said hFSH α subunit is the aminoacid sequence shown in the hFSH α SEQ ID NO:5 after the 1-24 amino acids residue of having removed in conventional hFSH α subunit; Wherein said peptide linker contains 2-20 amino acid, described peptide linker is present between hFSH α subunit and human IgG Fc variant, and peptide linker contains two or more amino acid that are selected from glycine, Serine, L-Ala and Threonine, preferred sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
3. according to the restructuring hFSH-Fc fusion rotein of claim 1, wherein said human IgG Fc variant is selected from lower group:
(i) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change;
(ii) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.
4. restructuring hFSH-Fc fusion rotein according to claim 1, is characterized in that, the aminoacid sequence of described fusion rotein is as shown in SEQ ID NO:2 and 4.
5. coding, according to the nucleotide sequence of the restructuring hFSH-Fc fusion rotein of claim 1, is characterised in that its sequence is as shown in SEQ ID NO:1 and 3.
6. a preparation method for the restructuring hFSH-Fc fusion rotein of claim 1, its step comprises:
1) build the expression vector of coding restructuring hFSH-Fc fusion rotein:
Adopt artificial synthesis to obtain the gene of coding hFSH-Fc fusion rotein, be inserted into mammalian cell expression vector, obtain the expression plasmid that contains hFSH-Fc antigen-4 fusion protein gene;
2) stably express of restructuring hFSH-Fc fusion rotein in mammalian host cell:
The expression vector that contains hFSH-Fc fusion rotein is transfected into mammalian host cell, the cell strain of screening stably express hFSH-Fc fusion rotein;
3) high-density cells is cultivated restructuring hFSH-Fc fusion rotein:
The above-mentioned stable cell line screening is proceeded to shaking flask or cell response tank carries out large scale culturing, when cell density reaches 1 * 10
7individual/during mL, temperature to be down to 33 ℃ by 37 ℃, at this temperature, cultivate until express output and no longer increase;
4) purification of restructuring hFSH-Fc fusion rotein:
A) Protein A affinity chromatography: centrifugal, collect supernatant, the characteristic of albumen coupling Fc fragment according to the present invention, utilizes affine Protein A column chromatography;
B) hydrophobic chromatography column purification: further remove the impurity in target protein after above-mentioned Protein A purifying through hydrophobic chromatography purifying;
Described drainage column comprises Butyl Sepharose4Fast Flow, Octyl Sepharose4Fast Flow, Phenyl Sepharose6Fast Flow, Butyl-S Sepharose6Fast Flow, Butyl Sepharose4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B.Preferably, Phenyl Sepharose6Fast Flow.
7. the preparation method of restructuring according to claim 6 hFSH-Fc fusion rotein, wherein step 1) described in expression vector be pCDNA3, pCMV/ZEO, pIRES, pDR, pBK, pSPORT or pCMV-DHFR, preferably, pCDNA3; Step 2 wherein) the cell transfecting method described in comprises electroporation transfection method, calcium phosphate transfection, liposome transfection and Protoplast fusion, preferably, and electroporation transfection method; Described mammalian host cell comprises CHO, HEK293, BHK, NS0 and Sp2/0 cell, preferably, Chinese hamster ovary celI, more preferably, DHFR deficient CHO suspension cell (CHO DHFR-).
8. the preparation method of restructuring according to claim 6 hFSH-Fc fusion rotein, wherein step 3) in be also included in substratum and add additive, preferably, at basic medium, add 100 μ M Cu
2+, feeding substratum adds 2mM ManNAc (N-ethanoyl-D-epichitosamine).
9. pharmaceutical composition, is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring hFSH-Fc fusion rotein described in the claim 1-8 any one claim of significant quantity.
10. the application of the restructuring hFSH-Fc fusion rotein of claim 1 in the infertile disease drug of preparation treatment.
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