CN103539861B - 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 is 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, be called for short hFSH) is the common major ingredient being used for the treatment of male and female infertility medicine of existing market, and China's existing market comprises FSH medicine and the Recombinant FSH that people urinates source.
The problems such as urine source hFSH exists virus contamination, raw material sources are limited, collection is difficult, content is low and purge process is complicated, the American-European developed country that waits generally does not accept the listing of urine product-derived and sale.Comparatively speaking, the hFSH that recombinates has the incomparable advantage of urine source hFSH in its purity, antigenicity, security, virus-free infection etc.HFSH is a kind of glycosylated protein, and it is 43KD that SDS-PAGE detects its molecular weight.As a kind of medicine, ensure its biological activity, necessary condition has 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 host.Wherein, Chinese hamster ovary cell (Chinese Hamster Ovary Cell, CHO) be for eukaryote exogenous gene expression the most successfully host cell, existing increasing pharmaceutical protein obtains high expression wherein, a lot of medicine is put on market, as EPO, G-CSF etc.Compared with other expression system, this system has many advantages, as having complete post translational processing process, comprise glycosylation, hydroxylation, the external source eukaryotic gene product of expression is enable to keep its natural structure and activity, and make expression product be secreted into outside born of the same parents, be conducive to the separation and purification of foreign protein.
Although the existing reconstituted drug hFSH utilizing Chinese hamster ovary celI to produce goes on the market at present, but still there are the following problems: first, its transformation period is short, clinically in order to reach effective result for the treatment of, needing frequent drug administration, bringing considerable distress to patient.Such as, when for ovulating, patient must every day 1-2 intramuscular or subcutaneous injection hFSH, usually need injection 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, whole body capillary permeability increases and ascites is formed, severe one can jeopardize patient vitals.Secondly, restructuring hFSH expression amount is low at present, 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 how keeping two chains in Protein expression and purification process is a challenge.For the defect of existing recombinate hFSH and like product, the present invention utilizes the restructuring hFSH product of the Protocols in Molecular Biology of optimization, cells produce and purifying process development of new, with prolong half-life, keep its biological activity, raising restructuring hFSH protein expression level, thus increase its drug effect, reduction 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 in prior art the problem that hFSH expression amount is low, purifying is difficult, Half-life in vivo is short of recombinating.
An object of the present invention is to provide restructuring hFSH-Fc fusion rotein, this fusion rotein is dimeric fusion protein, its aminoacid sequence holds C end to comprise hFSH β subunit, CTP, hFSH α subunit, peptide linker (Linker successively 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 eliminates 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 from 28-34 amino-acid residue of HCG β chain C-terminal, preferred 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 eliminates 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 region that (1) suddenlys change containing Ser228Pro and Leu235Ala, CH2 and CH3 region; (2) the human IgG1's hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.
Below human IgG Fc variant of the present invention, peptide linker and CTP function is specifically introduced:
IgG Fc variant
The immunoglobulin (Ig) of IgG class is most rich in protein in human blood, and their transformation period can 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 receptors (Fc γ Rs), by antibody dependent cellular cytotoxicity (ADCC) approach disease for digest substance, or the combination of the C1q part of (2) and the first complement component C1, cause cytotoxicity (CDC) approach depending on complement, thus cracking pathogenic agent.At four kinds of human IgG isotypes, (in (IgG1, IgG2, IgG3, IgG4), IgG1 can effectively in conjunction with a binding affinity order of magnitude lower than IgG1 of Fc γ Rs, IgG4 and Fc γ Rs.Human IgG1 can also effectively in conjunction with C1q, and activating complement cascade reaction, and the extreme ability lacking activating complement cascade of IgG4 performance.For medical use, the Fc region of restructuring dimerizing protein must can not mediate effector function and cracking or remove these cells.Therefore, the Fc region of hFSH-Fc must be non-cracking performance, namely in conjunction with Fc γ Rs and C1q in trigger effect subfunction, and Fc preferably non-activity.Obviously, a kind of natural IgG isotype is not had to be applicable to producing hFSH-L-Fc restructuring dimerizing protein.In order to obtain the Fc of non-cracking performance, some amino acid mutations in native Fc region must be made, to reduce its effector function.
By analyzing the aminoacid sequence of the IgG isotype of different genera, the Fc part finding near CH2 region N-terminal is presented in the combination of IgG Fc and Fc γ Rs and works, and is bonded to C1q and closes part and parcel and be positioned near the CH2 region carboxyl terminal of human IgG.In order to reduce the combination of Fc and Fc γ Rs and C1q and the effector function caused, the present invention uses following human IgG Fc variant (as Fig. 1):
(1) IgG4Fc variant (vIgG4Fc): human IgG 4 hinge region containing Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region;
(2) IgG1Fc variant (vIgG1Fc): the human IgG1's hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.
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 activity of length to restructuring dimerizing protein of connection peptides is extremely important.Existing people reports erythropoietin (EPO) derivative (as dipolymer), compared with EPO monomer, restructuring dimerizing protein containing 2 complete EPO regions (3 to 7 amino acid peptide joints of being separated by) shows the activity that weakens (see Qiu H etc., J Biol Chem, 273:11173-11176,1998).But 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 significantly improves (Sytkowski AJ etc., J Biol Chem, 274:24773-24778,1999; U.S. Patent No. 6,187,564).This possible explanation is the connection peptides increased between restructuring dimerizing protein two portions, makes two portions of this molecule can exercise its function (see Ashkenazi A etc., Curr Opin in Immunol, 9:195-200,1997) respectively.
The present inventor is through long-term and deep research, and the hinge region peptide linker devising a kind of uniqueness is first to reduce space steric effect, and can obtain the C end of hFSH α chain and the restructuring dimerizing protein of Fc variant coupling, there is soft peptide linker centre.This restructuring dimerizing protein not only can not cause the afunction of hFSH, can maintain, even improve the biological activity of hFSH-Fc restructuring dimerizing protein on the contrary.The amino acid residue sequence of preferred peptide joint is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
CTP
Glycosylation plays an important role for the activity of albumen and transformation period, and the glycosylation site on albumen has two classes, comprises O-glycopeptide chain and N-glycopeptide chain.CTP is one section of 28-34 amino-acid residue from HCG β key C-terminal, the transformation period having bibliographical information HCG relatively to grow compared with hFSH, is 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 dimeric fusion protein, and its aminoacid sequence holds C end to comprise hFSH β subunit, CTP, hFSH α subunit, peptide linker and human IgG Fc variant successively from N.Human IgG Fc variant has the effect extending Half-life in vivo and stabilize proteins, and Fc variant is non-cracking performance, can reduce and be combined with Fc γ Rs and C1q and the effector function that triggers.CTP can improve protein-active and extend Half-life in vivo, its non-immunogenicity, and the α chain and the β chain that connect 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.Carry out the C end of hFSH α chain and the coupling of Fc variant by the peptide linker of softness, can maintain, even improve the biological activity of restructuring hFSH-Fc fusion rotein.
CTP, peptide linker and human IgG Fc variant are connected in FSH and form novel restructuring hFSH-Fc fusion rotein by the present invention first in an orderly manner, in this fusion rotein, the positional alignment of human IgG Fc variant, CTP and peptide linker can maintain the correct sterie configuration of FSH, and do not affect its biological activity, and can the significant prolongation transformation period, can frequency of injection be reduced during clinical application, reduce the toxic side effect produced 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) expression vector of coding restructuring hFSH-Fc fusion rotein is built;
(2) stably express of restructuring hFSH-Fc fusion rotein in mammalian host cell;
(3) concentration cultivation restructuring hFSH-Fc fusion rotein;
(4) purification of restructuring hFSH-Fc fusion rotein.
Specifically, the expression vector step building 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 containing hFSH-Fc destination gene expression plasmid pCDNA3-hFSH-Fc (Fig. 5).The nucleotide sequence optimization of gene selects design based on the codon of mammalian host cell preference.
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 containing hFSH-Fc albumen is transfected into suitable mammalian host cell, and screening obtains the cell strain 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, tame DHFR (Dihydrofolate Reductase, Tetrahydrofolate dehydrogenase) the defective type Chinese hamster ovary celI adapting to suspension growth in serum free medium and (be called for short CHO-DHFR
-).
Described transfection method comprises calcium phosphate method, electroporation transfection method, liposome transfection, and preferred transfection method is electroporation transfection method.
Described screening method first utilizes selection markers to screen, and then can improve expression amount by amplification selected marker 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 marker gene known in the art, comprise GFP (Green Fluorescent Protein, green fluorescent protein), RFP (Red Fluorescent Protein, red fluorescent protein), preferred fluorescent marker gene is GFP.Amplification selected marker is DHFR sequence known in the art or GS (Glutaminesynthetase, glutamine synthetase) sequence, and preferred amplification selected gene is DHFR.Due to CHO-DHFR-cell self disappearance Tetrahydrofolate dehydrogenase (DHFR), cannot self tetrahydrobiopterin synthesis folic acid, so just can must survive in the nutrient solution that with the addition of 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 the substratum of above-mentioned additive also can grow, what is more important due to DHFR can by folacin MTX (Methotrexate, methotrexate) suppressed, under MTX concentration selective pressure, DHFR gene a lot of copy number that must increase could be survived, thus obtains anti-MTX clone; Again owing to tending to the goal gene of DHFR gene co-transfection the same area of being incorporated into together with it on cell chromosome, so the sequence fragment of encoding exogenous recombinant protein also increases along with the amplification of DHFR gene, the cell clone of mass expressing external albumen can be obtained.
The step of concentration cultivation restructuring hFSH-Fc fusion rotein is: the above-mentioned stable cell line screened 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.Cell culture processes of the present invention can realize the lifting of concentration cultivation, recombinant proteins and output, the degree of glycosylation of recombinant protein increases and sialic acid content improves.
The optimization of described cell culture condition comprises cooling culture method, specifically, when cell density reaches 1 × 10
7individual/mL time, temperature is down to 33 DEG C by 37 DEG C, at such a temperature cultivate until express output no longer increase.The method can improve the activity level of expressing protein and the cumulative withdrawal of recombinant protein.
The optimization method of described cell culture condition also comprises and adds special additive in the medium, preferably, adds 100 μMs of Cu at basic medium
2+, feeding substratum adds 2mM ManNAc (N-ethanoyl-D-epichitosamine).The method can make the degree of glycosylation of restructuring hFSH-Fc fusion rotein increase, and sialic acid content improves about 20%.
The purification step of restructuring hFSH-Fc fusion rotein is:
1) Protein A affinity chromatography: centrifugal, collects supernatant, according to the characteristic of albumen coupling Fc fragment of 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, the impurity further after the above-mentioned ProteinA purifying of removal in target protein.
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, the recombinant protein formed can obtain efficient purifying easily by ProteinA affinity chromatography.The fusion rotein purity obtained 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 avoid α-α dimer, the dimeric appearance of β-β, enormously simplify purification step, reduce production cost.
Also object of the present invention there is provided the pharmaceutical composition containing 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%; Preferably 0.1-50wt%; Better, 5-40wt%) recombinant long-acting hFSH-Fc fusion rotein of the present invention, and pharmaceutically acceptable carrier.Usually, the fusion rotein of the present invention of significant quantity can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and 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.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Described pharmaceutical composition should aseptically manufacture.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 (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio, metabolism, transformation period etc. of described fusion rotein; The severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.
Another object of the present invention is that restructuring hFSH-Fc fusion rotein is treating and/or preventing the application in people's infertility.
Restructuring hFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, thus improve pharmacokinetics and drug effect, compare with existing FSH, clinical application can reduce patient's frequency injection, 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 formed by CTP, peptide linker and human IgG Fc variant (vIgG4Fc or vIgG1Fc) and the orderly coupling of hFSH, maintain the correct sterie configuration of FSH, can the Half-life in vivo of 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 correctly fold with covalent linkage, avoid the formation of α-α dimer and β-β dimer, enormously simplify purifying process, reduce production cost.
3, restructuring hFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, its transformation period is existing restructuring more than 3 times of hFSH, and clinical application can reduce patient's frequency injection, reduces the side effect that existing treatment plan produces.
Accompanying drawing explanation
Fig. 1. show the comparison of human IgG1, IgG4 and the hinge region of their variants and the aminoacid sequence in CH2 region.Relatively 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.Numbering amino acid residues demarcates according to EU number system.
Fig. 2. show restructuring hFSH-Fc strand and dimerization domain schematic diagram.A) strand hFSH-Fc; B) hFSH-Fc of dimerization.
Fig. 3. show the nucleotide sequence of hFSH-Fc (hFSH β-CTP-hFSH α-L-vIgG4Fc) and the aminoacid sequence of derivation of HindIII-EcoRI fragment in pCDNA3 expression vector.The nucleotide sequence of hFSH-Fc comprises coding 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. show the nucleotide sequence of hFSH-Fc (hFSH β-CTP-hFSH α-L-vIgG1Fc) and the aminoacid sequence of derivation of HindIII-EcoRI fragment in pCDNA3 expression vector.The nucleotide sequence of hFSH-Fc comprises coding 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. show the eukaryon expression plasmid collection of illustrative plates of constructed coding hFSH-Fc fusion rotein.This expression plasmid total length 9063bp, containing 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. show the accumulation tendency graphic representation of hFSH-Fc cell strain expression-secretion hFSH-Fc albumen of recombinating in 7L bio-reactor.
Fig. 7 .Westem bloting result shows 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 (about 140kDa) of the present invention; Lane3: restructuring hFSH-vIgG4Fc fusion rotein (about 140kDa) of the present invention.
Fig. 8. show strand and the 10%SDS-PAGE electrophoretogram of dimerization hFSH-Fc under reductive condition and non reducing conditions.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. show restructuring hFSH-Fc fusion rotein of the present invention, restructuring hFSH and people and urinate the metabolic chart of hFSH in rat body.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1. builds the expression vector of coding restructuring hFSH-Fc fusion rotein
Gene order design is optimized based on Chinese hamster ovary celI preference codon, adopt the method synthesis of synthetic through the fusion gene of the signal peptide containing coding hFSH albumen β chain optimized and mature peptide section thereof, CTP and hFSH α chain mature peptide section, synthesized 756bp DNA fragmentation is inserted transfer vector as between the EcoRV restriction enzyme site in pUC57, obtain hFSH plasmid (phFSH), use the exactness of DNA sequencing method validation insertion sequence.
Synthetic contains the coding flexible peptide linker (Linker is called for short " L ") of BamHI (5 ' end) and EcoRI (3 ' end) restriction enzyme site and fusion gene L-vIgG4Fc and L-vIgG1Fc of human IgG Fc variant (vIgG4Fc and vIgG1Fc) fragment respectively.The fusion gene fragment of acquisition is inserted into respectively transfer vector as between BamHI and the EcoRI site of PUC19, obtains the plasmid containing two kinds of variants, be respectively pL-vIgG4Fc and pL-vIgG1Fc.By the gene order of DNA sequencing checking 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 fusion gene fragment of the signal peptide of coding hFSH albumen β chain and mature peptide section thereof, CTP and hFSH α chain mature peptide section, purified said gene fragment is inserted into 5 '-end of peptide linker in pL-vIgG4Fc and pL-vIgGIFc plasmid respectively, and T4 ligase enzyme connects and composes phFSH-L-vIgG4Fc and phFSH-L-vIgGIFc plasmid.Constructed fusion gene is made up of hFSH β, CTP, hFSH α, peptide linker and Fc variant gene, and as shown in Figure 2 a, dimerization domain 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 for purifying hFSH-L-Fc fragments are inserted into mammalian cell expression plasmid as between the corresponding restriction enzyme site of pCDNA3 (Invitrogen), final acquisition is 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 promotor CMV needed for mammal cell with high efficient expression alien gene albumen; This plasmid also containing two kinds of selected markers, thus 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, Tetrahydrofolate dehydrogenase (DHFR) gene of the mouse contained by PCDNA3 expression vector, makes it when methotrexate (MTX) exists, can coamplification pFSH-Fc fusion gene and DHFR gene.
Connect α chain and the β chain of hFSH by CTP peptide section, two chains can be convenient to and correctly fold.The coupling 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 about 20 or less (but can not be less than 2) amino acid whose peptide linkers; certainly by the peptide linker of 1 Amino acid profile also in protection scope of the present invention, should use containing or be selected from by 2 or more the peptide linker of following Amino acid profile: glycine, Serine, L-Ala and Threonine.The peptide linker of the embodiment of the present invention contains Gly-Ser peptide component, and its aminoacid sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
Embodiment 2. is recombinated the stably express of hFSH-Fc fusion rotein in mammalian cell
Expression plasmid pCDNA3-hFSH-L-Fc embodiment 1 built is transfected into DHFR deficient CHO host cell (CHO-DHFR
-), Fig. 2 b shows the schematic diagram of restructuring dimerization hFSH-Fc fusion rotein.Adopt electric robin to 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
7the linearizing plasmid DNA of 10 μ g PvuI is added in individual cell.In transfection two days later, substratum is made into the 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.DHFR amplification selected marker is utilized to improve the expression level of restructuring dimerizing protein, for this reason in the growth medium containing progressive concentration MTX, with the restructuring dimerizing protein gene of DHFR gene coamplification transfection.With the transfectant that limiting dilution method subclone can grow in up to 10 μMs/mL MTX substratum.Do to analyze further to the secretion rate of subclonal cell line.Screening secretion level exceedes about 5 (preferably about 20) μ g/10
6the cell strain of (namely 1,000,000) individual cell/24 hour, obtains and stablize high expression level and recombinates the cell strain of hFSH-Fc fusion rotein.
Embodiment 3. is recombinated the production of hFSH-Fc fusion rotein and purifying
The high yield cell strain that embodiment 2 is obtained, first in culture dish, carry out serum-free domestication cultivate, then transfer in shaking flask and carry out suspension domestication cultivation, carry out the screening of substratum in domestication process simultaneously, add growth conditions, the growth tendency of 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 μMs of Cu
2+, feeding substratum adds 2mM ManNAc (N-ethanoyl-D-epichitosamine), and the method can make the degree of glycosylation of restructuring hFSH-Fc fusion rotein increase, and sialic acid content improves about 20%.Tame successfully, carry out cell amplification, amplification, to q.s, is carried out the monitoring of 7L bio-reactor and is cultivated, at cell density more than 1 × 10
7individual/mL time start to be cooled to 33 DEG C of cultivations, batch growth cycle is 20 days.With 1ml ProteinA column chromatography, preliminary purification is carried out to recombinant protein, measure the expression amount of two kinds of restructuring hFSH-Fc fusion roteins, result shows, and 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, collects supernatant, according to the characteristic of albumen coupling Fc fragment of the present invention, utilizes affinity chromatography method, supernatant liquor is loaded onto the Protein A post that phosphate buffer saline (PBS) balances; After fusion rotein to be reorganized is incorporated into ProteinA, this post is washed with PBS, until OD280 value is lower than 0.01, then be the Recombinant FSH-Fc fusion rotein of sodium-acetate buffer elution of bound of 4.0 with 20mM pH, finally collect liquid with the 1M Tris-HCl damping fluid Neutralization effect of pH10.0.The hFSH-Fc purity of protein of purifying can reach more than 95%.
2) hydrophobic chromatography: with hyperfiltration process, above-mentioned protein A activity being collected fluid exchange is 20mM Tris-HCl-1.5M NaCl (pH8.0) damping fluid, by this sample pipetting volume to the phenyl-6Fast Flow post using 20mM Tris-HCl-1.5M NaCl (pH8.0) equilibrated, first with identical balance liquid drip washing, use 20mM Tris-HCl-1.35M NaCl (pH8.0) drip washing again, finally use 20mM Tris-HCl-0.5M NaCl (pH8.0) buffer solution elution.
Western bloting result shows the successful expression of the present invention two kinds restructuring hFSH-Fc fusion rotein in Chinese hamster ovary celI, as shown in Figure 7, SDS-PAGE gel electrophoresis collection of illustrative plates under non reducing conditions, people urinates the hFSH (commerical prod) in source and restructuring hFSH-Fc fusion rotein of the present invention shows corresponding target protein hybridising band at 43kDa and 140kDa respectively, demonstrates in the restructuring hFSH fusion rotein that the present invention obtains containing hFSH albumen.Fig. 8 is the SDS-PAGE gel electrophoresis collection of illustrative plates of two kinds of hFSH-Fc fusion roteins under reduction and non reducing conditions of purifying.Result shows, and the hFSH-Fc purity of protein of purifying of the present invention can reach more than 98%, hFSH-Fc molecular weight is under the reducing conditions molecular weight under non reducing conditions 50%.
Embodiment 4. recombinate hFSH-Fc fusion rotein In vitro and in vivo activity measure
The FSH enzyme immunoassay detection kit that the external activity (immunogen activity) of restructuring hFSH-Fc fusion rotein of the present invention adopts BIOCHECK (U.S.) company to produce detects, method reference reagent box specification sheets.Activity in vivo adopts the ovarian weight augmentation method in 2010 editions " British Pharmacopoeia " to detect.Protein content determination uses LOWRY quantitative method.Get HCG preparation, add 0.1% albumin phosphate buffered saline buffer (pH7.2 ± 0.2) solution, make the trial-product diluent containing 70IU/mlHCG.According to standard substance labelled amount, restructuring hFSH, people urinates hFSH and restructuring hFSH-Fc fusion rotein is estimated to tire, and standard substance, restructuring hFSH, people is urinated hFSH, restructuring hFSH-L-vIgG4Fc and restructuring hFSH-L-vIgG1Fc be mixed with solution containing FSH1.67IU/ml dosage with trial-product diluent (pH7.2 ± 0.2).Select 19 ~ 28 ages in days, the age must not differ more than 3 days, the female mouse of Wistar that body weight must not differ more than 10 grams, is divided into standard substance, restructuring hFSH group, people urinates hFSH group, restructuring hFSH-L-vIgG4Fc and restructuring hFSH-L-vIgG1Fc group, often organizes 6 animals.Subcutaneous injection after rat neck, every day injects twice, and per injection volume is 0.2ml, continuously injection 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, record organ weight.According to the activity that standard substance ovarian weight augmentation adopts parallel line analysis method calculating restructuring hFSH, people urinates hFSH and restructuring hFSH-Fc.Record the 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.
Embodiment 5. recombinate hFSH-Fc fusion rotein pharmacokinetics measure
Be divided into restructuring hFSH-Fc (comprising restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc), people urinates hFSH and restructuring hFSH administration group, often group selects the Male Wistar Rats 5 of body weight 200-250g, gives subcutaneous injection respectively by 15IU/kg.Restructuring hFSH group and people urinate hFSH group upon administration 1,2,3,4,6,8,12,36,56h gets blood, restructuring hFSH-Fc group respectively upon administration 1,2,4,8,12,24,56,120,176,200,264,340h gets blood, after the centrifugal 5min of 3000rpm, draw serum ,-20 DEG C of preservations.ELISA kit (BIOCHECK, the U.S.) is adopted to measure FSH immunocompetence in each time point blood plasma.Use PKSolver2.0 software, calculate each group of main pharmacokinetic parameter by statistics moments method.As shown in Figure 9, the transformation period, result was as table 1 for each group of pharmacokinetic curve.Result shows, restructuring hFSH and people urinate the elimination transformation period of hFSH in rat body and are about 11.35 ± 1.0h and 12.7 ± 2.8h respectively, 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 more than 3 times that restructuring hFSH and people urinate FSH.
Table 1 the present invention recombinates transformation period of hFSH-Fc
Embodiment 6. is recombinated the Tropic hyper ovulation effect of hFSH-Fc fusion rotein to female rats
Healthy female SD rat 75, body weight 200-250g, normally feeds and supports quarantine observation more than 7 days under experimental situation.Be divided into 5 groups at random: negative control group, restructuring hFSH-vIgG4Fc administration group, restructuring hFSH-vIgG1Fc administration group, restructuring hFSH control group, people urinate hFSH control group, and often organize 15 rats, the distribution of each group rat body weight is similar.First animals into heat cycle one-period is observed by vaginal smear method.Afterwards, hFSH-vIgG4Fc administration group, the restructuring hFSH-vIgG1Fc administration group of recombinating presses 45IU/kg dosage in dioestrus first day subcutaneous administrations 1 time; Restructuring hFSH group and people urinate hFSH group and start successive administration 3 days (15IU/kg/ days) by total dose 45IU/kg in dioestrus first day, and negative control group is to normal 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 anaesthetizing rat, liquid-solid fixed to the ovary BouinShi be separated, paraffin embedding, does serial section to fixing ovary every 6 μm, carry out histological observation under microscope, and record follicle number.
The count results of each group of rat follicle number is in table 2, result shows, compare with negative control, the present invention two kinds restructuring hFSH-Fc fusion rotein, Recombinant FSH and people urinate hFSH all has remarkable Tropic hyper ovulation effect (P < 0.01) to female rats, the present invention two kinds restructuring hFSH-Fc fusion rotein is only administered once, and recombinate hFSH and people urinate hFSH and need be administered three times and just can reach equal curative effect.
Table 2 is recombinated the ovulation effect of hFSH-Fc fusion rotein to female rats
Note: t checks, and compares with negative control group,
ap < 0.01
Embodiment 7. hFSH-Fc fusion rotein of recombinating causes the therapeutic action of female rats of not ovulating to male sex hormone
Get the female infant rats of 9 age in days SD 90 of being born on the same day, wherein 15 is Normal group: by the neutral tea oil 0.05ml of the disposable subcutaneous injection in nape portion; Remain 75 for modeling animal groups: every only by nape portion disposable subcutaneous injection testosterone propionate 1.25mg.70th age in days vaginal opening, continuous vaginal smear two oestrus cycles (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), 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, in persistence angling, prompting anovulatory rats Modelling success, rejects unsuccessful person.After 81st age in days, get rats in normal control group 13, distilled water is given by subcutaneous injection in 10ml/kg/ days, by above-mentioned anovulatory rats animal pattern, be divided into 5 groups at random by 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, administration 1 time in every 3 days, within continuous 15 days, are total to administration 5 times; Restructuring hFSH group and people urinate hFSH group and give subcutaneous injection by 7.5IU/kg/ time, administration every day 2 times, within continuous 15 days, are total to administration 30 times.After administration terminates, each group rat two oestrus cycles of vaginal smear all continuously, to judge Rat Ovulation situation.After vaginal smear examination ovulation, each group of rat is before the oestrus cycle, abdominal injection urethane of first weighing again is anaesthetized, ovary and the uterine cancer cell sample of choosing left side are put 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 then inner film thickness.
Table 3 shows each group of Rats During The Estrous Cycle situation result, table 4 shows each group of rat ovary and uterus pathology morphological outcomes, result shows to compare with negative control group, the present invention two kinds restructuring hFSH-Fc fusion rotein, restructuring hFSH and and people urinate hFSH and all have female rats of not ovulating and significantly actuate feelings restitution, large follicle number and corpus luteum number be more obvious than negative control group higher (P < 0.01) also, but the present invention two kinds restructuring hFSH-Fc fusion rotein administration 5 times, and recombinate hFSH and and people urinate hFSH and need administration just can reach equal drug effect 30 times.Result explanation, the present invention two kinds restructuring hFSH-Fc fusion rotein, restructuring hFSH and and people urinate hFSH and female rats Infertility of not ovulating is caused to male sex hormone all have remarkable therapeutic action, but the administration number of times of the present invention two kinds restructuring hFSH-Fc fusion rotein is obviously less than restructuring hFSH and people urinates hFSH.
Table 3 hFSH-Fc fusion rotein of recombinating actuates feelings recovery situation to female rats of not ovulating
Note: X
2inspection, compares with model negative control group,
ap < 0.01
Table 4 recombinates hFSH-Fc fusion rotein to rat ovary and the morphologic impact of uterus pathology of not ovulating
Note: t checks, and compares with model negative control group,
ap < 0.01
Embodiment 8. is recombinated the impact that hFSH-Fc fusion rotein stimulates 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 to urinate hFSH control group and restructuring hFSH control group, often organize 10.The rat that restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc administration group, people urinate hFSH and restructuring hFSH administration group plays every only according to 10IU injected sc relative medicine continuous 4 days at 22 ages in days, and the equal subcutaneous injection HCG30IU/ of 26 age in days all administrations group 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-cervical approach puts to death rat, cuts abdominal cavity and observes with or without 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 0.05ml0.1M NaOH solution again, with the speed of 3000r/min at room temperature centrifugal 10min, with spectrophotometer in the row colorimetric estimation of wavelength 600nm place, record OD value.Finally take off both sides ovary and use scales/electronic balance weighing immediately.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 point: without ascites; 2 points: a small amount of ascites exists; 3 points: middle amount ascites exists; 4 points: ascites obviously exists; 5 points: 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.
Continuous 4 days high dosage injection 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 hFSH-vIgG1Fc group of recombinating and ascites are marked and control group compares without difference, ovary weight is only had to increase, restructuring hFSH-vIgG4Fc and restructuring hFSH-vIgG1Fc does 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.Result is pointed out, the restructuring hFSH-vIgG4Fc of same injection higher dosage urinates hFSH with restructuring hFSH-vIgG1Fc, restructuring hFSH and people, it is less that restructuring hFSH-vIgG4Fc and the restructuring 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 in ascites scoring, peritoneal fluid and is compared
Note: t checks, and compares with Normal group,
ap < 0.01; Compared with restructuring hFSH-vIgG2Fc group;
bp < 0.01,
cp < 0.05.
Claims (9)
1. restructuring hFSH-Fc fusion rotein, it is characterized in that, described fusion rotein is dimeric fusion protein, monomer whose aminoacid sequence is held C to hold from N and is followed successively by hFSH β subunit, CTP, hFSH α subunit, peptide linker and human IgG Fc variant, and the aminoacid sequence of described monomer is as shown in SEQ ID NO:2 or 4.
2. the nucleic acid of coding restructuring according to claim 1 hFSH-Fc fusion rotein, is characterised in that its sequence is as shown in SEQ ID NO:1 or 3.
3. a preparation method for the restructuring hFSH-Fc fusion rotein of claim 1, its step comprises:
1) expression vector of coding restructuring hFSH-Fc fusion rotein is built:
Adopt artificial synthesis to obtain the gene of coding hFSH-Fc fusion rotein, be inserted into mammalian cell expression vector, obtain the expression vector containing hFSH-Fc antigen-4 fusion protein gene;
2) stably express of restructuring hFSH-Fc fusion rotein in mammalian host cell:
Expression vector containing hFSH-Fc antigen-4 fusion protein gene is transfected into mammalian host cell, the cell strain of screening stably express hFSH-Fc fusion rotein;
3) concentration cultivation restructuring hFSH-Fc fusion rotein:
The above-mentioned stable cell line screened is proceeded to shaking flask or cell response tank carries out large scale culturing, when cell density reaches 1 × 10
7individual/mL time, cool the temperature to 33 DEG C, at such a temperature cultivate until express output no longer increase;
4) purification of restructuring hFSH-Fc fusion rotein:
A) Protein A affinity chromatography: centrifugal, collects supernatant, according to the characteristic of albumen coupling Fc fragment, utilizes affine Protein A column chromatography;
B) hydrophobic chromatography column purification: remove the impurity after above-mentioned Protein A purifying in target protein further through hydrophobic chromatography purifying;
Described drainage column is Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow, Butyl-S Sepharose 6 Fast Flow, Butyl Sepharose 4B, Octyl Sepharose CL-4B or Phenyl Sepharose CL-4B.
4. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 3, the drainage column wherein described in step 4) is Phenyl Sepharose 6 Fast Flow.
5. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 4, the expression vector wherein described in step 1) is pCDNA3, pCMV/ZEO, pIRES or pCMV-DHFR; Wherein step 2) described in transfection use method be electroporation transfection method, calcium phosphate transfection, liposome transfection or Protoplast fusion; Described mammalian host cell is CHO, HEK293, BHK, NS0 or Sp2/0 cell.
6. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 5, the expression vector wherein described in step 1) is pCDNA3; Wherein step 2) described in transfection use method be electroporation transfection method; Described mammalian host cell is Chinese hamster ovary celI.
7. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 6, wherein said mammalian host cell is DHFR deficient CHO suspension cell.
8. pharmaceutical composition, is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring hFSH-Fc fusion rotein according to claim 1 of significant quantity.
9. the application of restructuring hFSH-Fc fusion rotein in the infertile disease drug of preparation treatment of claim 1.
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| US11123438B2 (en) | 2016-08-19 | 2021-09-21 | Ampsource Biopharma Shanghai Inc. | Linker peptide for constructing fusion protein |
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Effective date of registration: 20161108 Address after: 510703, No. 9, Tung Shing Street, Guangzhou economic and Technological Development Zone, Guangdong, China Patentee after: Guangzhou best Biotechnology Co., Ltd. Address before: 510663 Guangdong city of Guangzhou province Luogang District Science City on Guangzhou Road No. 3 international business incubator F Building 9 floor Patentee before: Guangzhou Youliankang Pharmaceutical Technology Co., Ltd. |