CN103554269B - Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc) - Google Patents
Recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc) Download PDFInfo
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Abstract
The invention discloses a recombinant porcine follicle-stimulating hormone-Fc fusion protein (pFSH-Fc) and a preparation method thereof. The fusion protein is a dimerized fusion protein, wherein the amino acid sequence of the fusion protein sequentially comprises pFSH beta subunits, carboxy-terminal peptide (CTP), pFSH alpha subunits, peptide connectors and human IgG Fc variants from a terminal N to a terminal C. Compared with existing pFSH, the pFSH-Fc has a longer in-vivo half-life period and better efficacies. The invention further relates to application of the pFSH-Fc to preparation of medicaments in the animal breeding fields.
Description
Technical field
The present invention relates to molecular biology and herding field.More specifically, the present invention relates to Recombinant Swine follicle stimulating hormone fusion rotein (pFSH-Fc), its preparation method and the application in herding reproduction thereof.The follicle stimulating hormone that this fusion rotein is originated with existing pig pituitary compares, and has longer transformation period and better drug effect.
Background technology
Pig follitropin (Porcine Follicle-stimulating hormone, be called for short pFSH) be the medicine main component that animal reproduction field is commonly used, existing listing pFSH is the glycoprotein hormones extracted from pig pituitary frontal lobe, and it is 43KD that SDS-PAGE detects its molecular weight.PFSH can promote the growth of ovarian follicle in sow ovary, comprises a ripe ovum in each ovarian follicle.During these follicle maturitys, can secrete oestrogenic hormon, the latter can stimulate sow to show typical quiet vertical estrus behavior.In addition, pFSH can also stimulate the growth of male animal seminiferous tubule epithelium and secondary spermatocyte, promotes spermiogenesis tail.
The pFSH that pig pituitary extracts collects and separation and purification difficulty, also there is the defects such as virus contamination, raw material sources are limited, purity is low, production cost is high, and its transformation period short-range missile causes and needs frequent drug administration, uses limited simultaneously.
Comparatively speaking, the pFSH that recombinates has the incomparable advantage of biochemical FSH product in its purity, antigenicity, security, virus-free infection etc.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 that existing increasing pharmaceutical protein obtains high expression wherein, and a lot of people is gone on the market with recombinant protein medicine for eukaryote exogenous gene expression the most successfully host cell.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.
PFSH 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 pFSH of two chains.The normal combination how keeping two chains in Protein expression and purification process is a challenge.Up to now, restructuring pFSH product is not also had for field of veterinary.
Summary of the invention
The present invention aims to provide Recombinant Swine follicle stimulating hormone fusion rotein (Porcine follicle-stimulating hormone-Fc fusion protein, be called for short pFSH-Fc), its preparation method and application, this restructuring pFSH-Fc fusion rotein is applied to animal reproduction field, similar with existing pig pituitary pFSH activity, and there is the features such as purity is high, transformation period significant prolongation.
An object of the present invention is to provide a kind of restructuring pFSH-Fc fusion rotein, this fusion rotein is dimeric fusion protein, its aminoacid sequence holds C end to comprise pFSH β subunit successively from N, CTP, pFSH α subunit, peptide linker (Linker, be called for short L) and human IgG Fc variant, as SEQ ID NO:2, 4, (pFSH β-CTP-pFSH α-L-vIgG2Fc shown in 6, pFSH β-CTP-pFSH α-L-vIgG4Fc or pFSH β-CTP-pFSH α-L-vIgG1Fc aminoacid sequence), preferred aminoacid sequence is as SEQ ID NO:2 (pFSH β-CTP-pFSH α-L-vIgG2Fc), above-mentioned fusion rotein is referred to as pFSH-Fc.
The aminoacid sequence of described pFSH β subunit eliminates the 1-18 amino acids residue in conventional pFSH β subunit, as shown in SEQ ID NO:8.
The aminoacid sequence of described CTP (carboxy-terminal peptide) is from 28-34 amino-acid residue of people 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:9.
The amino acid residue sequence of described pFSH α subunit eliminates the 1-24 amino acids residue in conventional pFSH α subunit, as shown in SEQ ID NO:7.
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, preferred peptide linker aminoacid sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
Described human IgG Fc variant comprises: human IgG2's hinge region that (1) suddenlys change containing Pro331Ser, CH2 and CH3 region; (2) human IgG 4 hinge region containing Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region; (3) the human IgG1's hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.Preferred human IgG Fc variant is, the human IgG2's hinge region containing 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.In four kinds of human IgG isotypes, IgG1 and IgG3 can effectively in conjunction with Fc γ Rs.The binding affinity of IgG4 and Fc γ Rs than a low order of magnitude of IgG1 and IgG3, and IgG2 and Fc γ Rs in conjunction with so low that to be difficult to measure.Human IgG1 and IgG3 can also effectively in conjunction with C1q, and activating complement cascade reaction.Human IgG2 is very weak to fixing of complement, 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 pFSH-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 pFSH-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.For this reason, the present invention uses following human IgG Fc variant:
(1) IgG2Fc variant (vIgG2Fc): the human IgG2's hinge region containing Pro331Ser sudden change, CH2 and CH3 region;
(2) IgG4Fc variant (vIgG4Fc): human IgG 4 hinge region containing Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region;
(3) IgG1Fc variant (vIgG1Fc): the human IgG1's hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.Preferred human IgG Fc variant is, the human IgG2's hinge region containing Pro331Ser sudden change, CH2 and CH3 region.
Peptide linker
The activity of length to restructuring dimerizing protein of connection peptides is extremely important.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 pFSH 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 pFSH, can maintain, even improve the biological activity of pFSH-Fc restructuring dimerizing protein on the contrary.The amino acid residue sequence of preferred peptide joint is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
In addition, the present invention also finds, the peptide linker added between pFSH α and human IgG Fc variant improves the Bioactivity of pFSH-Fc in two ways: (1) makes Fc region away from the structural domain on pFSH, (2) make a pFSH away from the structural domain of another pFSH, thus reduce space steric effect.And the IgG Fc variant of people contains amino acid mutation in CH2 region in 228,234,235,331 sites, thus reduce the effector function of Fc.
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 people HCG β key C-terminal, the transformation period having bibliographical information HCG relatively to grow compared with FSH, is mainly derived from HCG β key C-terminal 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 pFSH-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 pFSH β subunit, CTP, pFSH 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 Fc γ Rs and C1q and combine and the effector function of triggering.CTP can improve protein-active and extend Half-life in vivo, connects α chain and the β chain of pFSH, can make there is certain steric hindrance between two chains, thus facilitate it correctly to fold with CTP.Carry out the C end of pFSH α chain and the coupling of Fc variant by the peptide linker of softness, can maintain, even improve the biological activity of restructuring pFSH-Fc fusion rotein.
Another object of the present invention is to provide the preparation method of restructuring pFSH-Fc fusion rotein, and this preparation method comprises:
(1) expression vector of coding restructuring pFSH-Fc fusion rotein is built;
(2) stably express of restructuring pFSH-Fc fusion rotein in mammalian host cell;
(3) concentration cultivation restructuring pFSH-Fc fusion rotein;
(4) purification of restructuring pFSH-Fc fusion rotein.
Specifically, the expression vector step building coding restructuring pFSH-Fc albumen is: adopt artificial synthesis to obtain the nucleotide sequence (as shown in SEQ ID NO:1,3,5) of coding restructuring pFSH-Fc antigen-4 fusion protein gene, the expression vector (as pCMV-DHFR) being inserted into mammalian cell expression vector (as pCBAEA3) or improving, obtains containing pFSH-Fc destination gene expression plasmid pCBAEA3-pFSH-Fc (Fig. 6).Preferred nucleotide sequence is as SEQ ID NO:1.
Described mammalian cell expression vector can adopt commercially available but be not limited to, as: pCDNA3, pCBAEA3, pCMV/ZEO, pIRES, pDR, pBK, pSPORT etc. can be used for the carrier that eukaryotic cell system is expressed, preferably, and pCBAEA3.
The stably express step of restructuring pFSH-Fc fusion rotein in mammalian host cell is: the expression plasmid containing pFSH-Fc albumen is transfected into suitable mammalian host cell, utilizes selection markers screening and amplification selected marker to obtain 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, DHFR (Dihydrofolate Reductase, Tetrahydrofolate dehydrogenase) the defective type Chinese hamster ovary celI (being called for short CHO-DHFR-) adapting to suspension growth in serum free medium has been tamed.
Described transfection method comprises calcium phosphate method, electric robin, liposome transfection, and preferred transfection method is electric robin.
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 pFSH-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 pFSH-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 pFSH-Fc fusion rotein increase, and sialic acid content improves about 20%.
The purification step of restructuring pFSH-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 ProteinA column chromatography.
2) hydrophobic chromatography column purification: adopt hydrophobic chromatography post, different according to the hydrophobicity of restructuring pFSH-Fc fusion rotein, the impurity further after the above-mentioned Protein A 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 pFSH-Fc fusion rotein disclosed in this invention, this preparation method can obtain and express output height restructuring pFSH-Fc fusion rotein.Because of the coupling with human IgG Fc variant, the recombinant protein formed can obtain efficient purifying easily by Protein A affinity chromatography.The fusion rotein purity obtained after hydrophobic chromatography is further purified reaches more than 98%.In addition, in the restructuring pFSH-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 pFSH-Fc fusion rotein, it is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring pFSH-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 pFSH-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; Animal want severity, the body weight of animal, the immune state, route of administration etc. of animal of disease therapy.
Another object of the present invention is the application of restructuring pFSH-Fc fusion rotein in animal reproduction field.
Restructuring pFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, thus improve pharmacokinetics and drug effect, compare with existing pFSH, can frequency injection be greatly reduced, use more convenient.
The advantage of restructuring pFSH-Fc fusion rotein of the present invention and preparation method thereof is summarized as follows:
1. restructuring pFSH-Fc fusion rotein of the present invention is the new fusion protein formed by the orderly coupling of Fc and CTP and pFSH, maintains the correct sterie configuration of pFSH, can the Half-life in vivo of significant prolongation albumen, greatly improves the expression amount of pFSH in Chinese hamster ovary celI.
2. the α chain of dimerization strand pFSH-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 pFSH-Fc fusion rotein Half-life in vivo significant prolongation of the present invention, its transformation period is more than 10 times of existing pig pituitary FSH, can reduce frequency injection, and its drug effect is more excellent.
Accompanying drawing explanation
Fig. 1. show the comparison of human IgG1, IgG2, IgG4 and the hinge region of their variants and the aminoacid sequence in CH2 region.
Fig. 2 shows pFSH-Fc strand and dimerization domain schematic diagram.A) the strand pFSH-Fc before non-dimerization; B) pFSH-Fc of dimerization.
Fig. 3. show the nucleotide sequence of pFSH-Fc (pFSH β-CTP-pFSH α-L-vIgG2Fc) and the aminoacid sequence of derivation of HindIII-EcoRI fragment in pCBAEA3 expression vector.The nucleotide sequence of pFSH-Fc comprises coding containing leading peptide, pFSH β chain, CTP, ripe pFSH α chain, peptide linker, human IgG2 Fc variant (vIgG2Fc).Ripe restructuring pFSH-Fc fusion rotein contains ripe pFSH β chain, CTP, ripe α chain, peptide linker and human IgG2 Fc variant (vIgG2Fc).
Fig. 4. show the nucleotide sequence of pFSH-Fc (pFSH β-CTP-pFSH α-L-vIgG4Fc) and the aminoacid sequence of derivation of HindIII-EcoRI fragment in PCBAEA3 expression vector.The nucleotide sequence of pFSH-Fc comprises coding containing leading peptide, pFSH β chain, CTP, ripe pFSH α chain, peptide linker, human IgG 4Fc variant (vIgG4Fc).Ripe restructuring pFSH-Fc fusion rotein contains ripe pFSH β chain, CTP, ripe α chain, peptide linker and IgG4Fc variant (vIgG4Fc).
Fig. 5. show the nucleotide sequence of pFSH-Fc (pFSH β-CTP-pFSH α-L-vIgG1Fc) and the aminoacid sequence of derivation of HindIII-EcoRI fragment in PCBAEA3 expression vector.The nucleotide sequence of pFSH-Fc comprises coding containing leading peptide, pFSH β chain, CTP, ripe pFSH α chain, peptide linker, human IgG1 Fc variant (vIgG1Fc).Ripe restructuring pFSH-Fc fusion rotein contains ripe pFSH β chain, CTP, ripe α chain, peptide linker and human IgG1 Fc variant (vIgG1Fc).
Fig. 6 shows the eukaryon expression plasmid collection of illustrative plates of constructed coding pFSH-Fc fusion rotein.This expression plasmid contains 10 oligogene segments, comprises 1.CMV promotor; 2. target gene pFSH-Fc; 3.EMCV IRES; 4.DHFR amplification gene; 5.IRES; 6. Enhanced green fluorescent protein gene EGFP; 7.BGH terminator; 8.SV40 promotor; 9. neomycin resistance gene (Neomycin); 10.SV40 terminator; 11.PUC replication origin (ori); 12. penicillin resistance screening-genes (Ampcillin).
Fig. 7. show the activity trend graphic representation of cell strain expression-secretion pFSH-Fc albumen in 7L bio-reactor.
Fig. 8 .Western bloting result shows the successful expression of restructuring pFSH-Fc fusion rotein in Chinese hamster ovary celI.Non-reduced glue, Lane1: restructuring pFSH-vIgG1Fc fusion rotein (about 140kDa) of the present invention; Lane2: restructuring pFSH-vIgG2Fc fusion rotein (about 140kDa) of the present invention; Lane3: restructuring pFSH-vIgG4Fc fusion rotein (about 140kDa) of the present invention; Lane4: pig pituitary FSH (about 43kDa).
Fig. 9. show the present invention and to recombinate the 10%SDS-PAGE electrophoretogram of pFSH-Fc under non reducing conditions.Lane1: restructuring pFSH-vIgG1Fc fusion rotein (about 140kDa) of the present invention; Lane2: restructuring pFSH-vIgG2Fc fusion rotein (about 140kDa) of the present invention; Lane3: restructuring pFSH-vIgG4Fc fusion rotein (about 140kDa) of the present invention
Figure 10. show restructuring pFSH-Fc fusion rotein and the metabolic chart of pig pituitary FSH in rat body of purifying.
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 (New York: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 pFSH-Fc fusion rotein
Adopt the fusion gene of the signal peptide of the method synthesis of synthetic containing coding pFSH albumen β chain and mature peptide section thereof, CTP and pFSH α chain mature peptide section, synthesized 756bp DNA fragmentation is inserted transfer vector as between the EcoRV restriction enzyme site in pUC57, obtain pFSH plasmid (ppFSH), use the exactness of DNA sequencing method validation insertion sequence.
Synthetic contains the coding flexible peptide linker (Linker detects " L ") of BamHI (5 ' end) and EcoRI (3 ' end) restriction enzyme site and fusion gene L-vIgG2Fc, L-vIgG4Fc and L-vIgG1Fc of Fc variant (vIgG2Fc, 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 three kinds of variants, be respectively pL-vIgG2Fc, pL-vIgG4Fc and pL-vIgG1Fc.The gene order of L-vIgG2Fc, L-vIgG4Fc and L-vIgG1Fc is verified by DNA sequencing.For preparation pFSH-L-Fc fusion gene, with restriction enzyme SpeI and BamHI double digestion pFSH plasmid, after gel electrophoresis, glue reclaims the fusion gene fragment of the signal peptide of coding pFSH albumen β chain and mature peptide section thereof, CTP and pFSH α chain mature peptide section, purified said gene fragment is inserted into 5 '-end of peptide linker in pL-vIgG2Fc, pL-vIgG4Fc and pL-vIgG1Fc plasmid respectively, and T4 ligase enzyme connects and composes pFSH-L-vIgG2Fc, pFSH-L-vIgG4Fc and pFSH-L-vIgG1Fc plasmid.Constructed fusion gene is made up of pFSH β, CTP, pFSH 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 ppFSH-L-vIgG2Fc, ppFSH-L-vIgG4Fc and ppFSH-L-vIgG1Fc plasmid respectively, and DNA gel purifying obtains pFSH-L-vIgG2Fc, pFSH-L-vIgG4Fc and pFSH-L-vIgG1Fc fragment.Three kinds of good for purifying pFSH-L-Fc fragments are inserted into mammalian cell expression plasmid as between the corresponding restriction enzyme site of pCBAEA3, final acquisition is containing fusion gene expression plasmid pCBAEA3-pFSH-L-vIgG2Fc, pCBAEA3-pFSH-L-vIgG4Fc and pCBAEA3-pFSH-L-vIgG1Fc, be referred to as pCBAEA3-pFSH-Fc plasmid, as shown in Figure 6.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 Liu Suanyan NEOMYCIN SULPHATE (Neomycin) resistance in mammalian cell.In addition, when host cell is DHFR genetic expression defective type, Tetrahydrofolate dehydrogenase (DHFR) gene of the mouse contained by PCBAEA3 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 pFSH by CTP peptide section, two chains can be convenient to and correctly fold.The coupling carrying out pFSH 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 pFSH-Fc fusion rotein in mammalian cell
Expression plasmid pCBAEA3-pFSH-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 pFSH-Fc fusion rotein.Adopt electroporation method 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 western blotting, with the expression of anti-pFSH antibody test pFSH-Fc, as Fig. 8.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/mLMTX substratum.Do to analyze further to the secretion rate of subclonal cell line.Screening secretion level exceedes about 10 (preferably about 20) μ g/10
6the cell strain of (namely 1,000,000) individual cell/24 hour, obtains the cell strain of three kinds of stable high expression levels restructuring pFSH-Fc fusion roteins, is respectively restructuring pFSH-L-vIgG2Fc, restructuring pFSH-L-vIgG4Fc and restructuring pFSH-L-vIgG1Fc.。
Embodiment 3. is recombinated the production of pFSH-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 pFSH-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 restructuring hFSH-Fc fusion rotein, result shows, and the restructuring dimerizing protein cumulative withdrawal that restructuring pFSH-L-vIgG2Fc, restructuring pFSH-L-vIgG4Fc and restructuring pFSH-L-vIgG1Fc cell strain are expressed is respectively 1.62g/L, 0.82g/L, 0.78g/L (Fig. 7).
The purifying of restructuring pFSH-Fc fusion rotein comprises the following steps:
1) ProteinA 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 ProteinA 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 restructuring pFSH-Fc fusion rotein of sodium-acetate buffer elution of bound of 4.0 with 20mM pH, finally collect liquid with the 1MTris-HCl damping fluid Neutralization effect of pH10.0.The pFSH-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-1.5MNaCl (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 restructuring pFSH-Fc fusion rotein in Chinese hamster ovary celI, as shown in Figure 8, SDS-PAGE gel electrophoresis collection of illustrative plates under non reducing conditions, pig pituitary FSH (commerical prod) and restructuring pFSH-Fc fusion rotein of the present invention show corresponding target protein hybridising band at 43kDa and 140kDa respectively, demonstrate in the restructuring pFSH fusion rotein that the present invention obtains containing FSH albumen.Fig. 9 is the SDS-PAGE gel electrophoresis collection of illustrative plates of pFSH-Fc fusion rotein under non reducing conditions of purifying.Result shows, and the pFSH-Fc purity of protein of purifying can reach more than 98%.
Embodiment 4. recombinate pFSH-Fc fusion rotein In vitro and in vivo activity measure
The FSH enzyme immunoassay detection kit that the external activity (immunogen activity) of restructuring pFSH-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.Estimate to tire according to standard substance labelled amount, pig pituitary FSH and restructuring pFSH-Fc fusion rotein, with trial-product diluent (pH7.2 ± 0.2), standard substance, pig pituitary FSH and restructuring pFSH-Fc fusion rotein are mixed with the working fluid closing high, normal, basic three dosage of FSH3.33IU/ml, 1.67IU/ml, 0.83IU/ml.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.Standard substance, pig pituitary FSH group and restructuring pFSH-Fc fusion rotein group are divided into high, normal, basic three dosage, often organize 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.Parallel line analysis method is adopted to calculate the activity of pig pituitary FSH and restructuring pFSH-Fc fusion rotein according to standard substance ovarian weight augmentation.The external activity recording restructuring pFSH-L-vIgG2Fc, pFSH-L-vIgG4Fc, pFSH-L-vIgG1Fc and pig pituitary FSH is respectively 9022,9020,8956 and 8321IU/ml, and its activity in vivo is respectively 8990,8975,8310 and 7523IU/ml.Result shows, restructuring pFSH-Fc fusion rotein of the present invention has inside and outside biologic activity.
Embodiment 5. recombinate pFSH-Fc fusion rotein pharmacokinetics measure
The present invention is divided into recombinate pFSH-Fc fusion rotein (comprise restructuring pFSH-vIgG1Fc, pFSH-vIgG2Fc and pFSH-vIgG4Fc) administration group and pig pituitary FSH administration group, often group selects the Male Wistar Rats 5 of body weight 200-250g, gives intramuscular injection respectively by 20IU/kg.Pig pituitary FSH group upon administration 1,2,3,4,6,8,12,36,60h gets blood, restructuring pFSH-Fc fusion rotein respectively upon administration 1,2,4,8,12,24,56,120,176,200,264,340h gets blood, after the centrifugal 5min of 3000rpm, get 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 kinetica4.4 software, calculate each group of main pharmacokinetic parameter by statistics apart from method.As shown in Figure 10, the transformation period, result was as table 1 for each group of pharmacokinetic curve.Result shows, the elimination transformation period of pig pituitary FSH in rat body is about 3.07h, and wait dosage the present invention recombinate pFSH-vIgG2Fc fusion rotein administration group, restructuring pFSH-vIgG4Fc fusion rotein administration group, restructuring pFSH-vIgG1Fc fusion rotein the elimination transformation period be about respectively 48.84h, 38.24h, 35.22h, be more than 10 times of pig pituitary FSH.
Table 1 the present invention recombinates transformation period of pFSH-Fc fusion rotein
Embodiment 6. pFSH-Fc fusion rotein of recombinating promotes the effect that young gilt oestruses in advance
Choose backup gilt (6 monthly ages puberty, 90-100kg), be divided into five groups at random: restructuring pFSH-vIgG1Fc administration group (100IU/ head), restructuring pFSH-vIgG2Fc administration group (100IU/ head), restructuring pFSH-vIgG4Fc administration group (100IU/ head), pig pituitary FSH control group (100IU/ head), negative control group (physiological saline), administration group all with the use of 400IU/ head HCG, can work in coordination with promotion follicle maturity.Intramuscular injection is carried out respectively, observed and recorded oestrus of sow situation, statistics Oestrus rate and estrus synchronization situation by group.The results are shown in Table 2, data show, recombinate pFSH-Fc fusion rotein and pig pituitary FSH of the present invention can both promote oestrusing in advance of young gilt, relative negative control group has significant difference (P<0.01), and the present invention's three kinds of recombination fusion protein Oestrus rate reach 90%, compare better efficacy with pig pituitary FSH, have significant difference (P<0.05), wherein effect optimum is restructuring pFSH-vIgG2Fc fusion rotein group.
Table 2 is recombinated the effect that pFSH-Fc fusion rotein is oestrused in advance to young gilt
Note: X
2inspection, compares with negative control group,
*p<0.01; Compare with pig pituitary FSH control group,
△for p<0.05.
Embodiment 7. is recombinated the therapeutic action of pFSH-Fc fusion rotein to weary feelings replacement gilt
Choose more than 10 monthly ages, body weight is at the weary feelings replacement gilt of more than 140kg, injection Cloprostenol injection liquid 1ml is with after getting rid of the case out of heat that causes because producing permanent corpus luteum, be divided into five groups at random: restructuring pFSH-vIgG1Fc fusion rotein administration group (100IU/ head), restructuring pFSH-vIgG2Fc fusion rotein administration group (100IU/ head), restructuring pFSH-vIgG4Fc fusion rotein administration group (100IU/ head), pig pituitary FSH control group (100IU/ head), negative control group (physiological saline), administration group is all with the use of 400IU/ head HCG, promotion follicle maturity can be worked in coordination with.Intramuscular injection is carried out respectively, the situation of oestrusing and become pregnant of observed and recorded sow by group.The results are shown in Table 3, compare with negative control group, the present invention three kinds restructuring FHS-Fc fusion rotein and pig pituitary FSH can significantly improve the Oestrus rate of weary feelings replacement gilt, and the present invention's three kinds of fusion rotein effects better than pig pituitary FSH (P<0.05).In addition, the non-return rate of restructuring pFSH-Fc fusion rotein group, apparently higher than negative control group and pig pituitary FSH control group, has significant difference (P<0.05).PFSH-vIgG2Fc fusion rotein curative effect of wherein recombinating is best.
Table 3 is recombinated the therapeutic action of pFSH-Fc fusion rotein to weary feelings replacement gilt
Note: X
2inspection, compares with negative control group,
*p<0.01,
*p<0.05; Compare with pig pituitary FSH control group,
△for p<0.05.
Embodiment 8. pFSH-Fc fusion rotein of recombinating to exceed the time limit therapeutic action out of heat to Suprapubic arch sling
Choose the Suprapubic arch sling that wean was not oestrused after 2 weeks, injection Cloprostenol injection liquid 1ml is with after getting rid of the case out of heat that causes because producing permanent corpus luteum, be divided into five groups at random: restructuring pFSH-vIgG1Fc fusion rotein administration group (100IU/ head), restructuring pFSH-vIgG2Fc fusion rotein administration group (100IU/ head), restructuring pFSH-vIgG4Fc fusion rotein administration group (100IU/ head), pig pituitary FSH control group (100IU/ head), negative control group (physiological saline), administration group is all with the use of 400IU/ head HCG, promotion follicle maturity can be worked in coordination with.Intramuscular injection is carried out respectively, the situation of oestrusing and become pregnant of observed and recorded sow by group.The results are shown in Table 4, compare with negative control group, the present invention three kinds restructuring FHS-Fc fusion rotein and pig pituitary FSH can significantly improve the Oestrus rate improving wean Suprapubic arch sling out of heat after 2 weeks, and the present invention's three kinds of fusion roteins are than pig pituitary FSH better effects if (P<0.05).In addition, the non-return rate of restructuring pFSH-Fc fusion rotein group, apparently higher than negative control group and pig pituitary FSH control group, has significant difference (P<0.05).PFSH-vIgG2Fc fusion rotein curative effect of wherein recombinating is optimum.
Table 4 pFSH-Fc fusion rotein of recombinating to exceed the time limit therapeutic action out of heat to Suprapubic arch sling pig
Note: X
2inspection, compares with negative control group,
*p<0.01,
*p<0.05; Compare with pig pituitary FSH control group,
△for p<0.05.
Claims (11)
1. restructuring pFSH-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 pFSH β subunit, CTP, pFSH α subunit, peptide linker and human IgG Fc variant, and the aminoacid sequence of described monomer is as shown in SEQ ID NO:2,4 or 6.
2. restructuring pFSH-Fc fusion rotein according to claim 1, monomer whose aminoacid sequence is as shown in SEQ ID NO:2.
3. the nucleic acid of coding restructuring according to claim 1 pFSH-Fc fusion rotein, its nucleotide sequence is as shown in SEQ ID NO:1,3 or 5.
4. the nucleic acid of coding restructuring according to claim 2 pFSH-Fc fusion rotein, its nucleotide sequence is as shown in SEQ ID NO:1.
5. a preparation method for the restructuring pFSH-Fc fusion rotein of claim 1, its step comprises:
1) expression vector of coding restructuring pFSH-Fc fusion rotein is built:
Adopt artificial synthesis to obtain the gene of encoding human pFSH-Fc fusion rotein, be inserted into mammalian cell expression vector, obtain the expression vector containing pFSH-Fc antigen-4 fusion protein gene;
2) stably express of restructuring pFSH-Fc fusion rotein in mammalian host cell:
Expression vector containing pFSH-Fc antigen-4 fusion protein gene is transfected into mammalian host cell, the cell strain of screening stably express pFSH-Fc fusion rotein;
3) concentration cultivation restructuring pFSH-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 pFSH-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.
6. the preparation method of restructuring pFSH-Fc fusion rotein according to claim 5, the drainage column wherein described in step 4) is Phenyl Sepharose 6 Fast Flow.
7. the preparation method of restructuring pFSH-Fc fusion rotein according to claim 6, the expression vector wherein described in step 1) is pCDNA3, pCBAEA3, 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.
8. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 7, 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.
9. the preparation method of restructuring hFSH-Fc fusion rotein according to claim 8, wherein said mammalian host cell is DHFR deficient CHO suspension cell.
10. pharmaceutical composition, is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring pFSH-Fc fusion rotein described in significant quantity claim 1 or 2.
The restructuring pFSH-Fc fusion rotein of 11. claims 1 promotes animals into heat in preparation, promotes the application of farrowing or treating in the infertile disease drug of animal of being impregnated.
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| CN107540748B (en) * | 2017-09-15 | 2020-07-14 | 北京伟杰信生物科技有限公司 | Long-acting recombinant porcine FSH fusion protein and preparation method and application thereof |
| CN108676096B (en) * | 2018-05-22 | 2022-03-29 | 北京伟杰信生物科技有限公司 | Recombinant porcine FSH-CTP fusion protein and preparation method and application thereof |
| CN110041434A (en) * | 2019-04-28 | 2019-07-23 | 广州威生医药科技有限公司 | Long-acting reorganization FSH fusion protein, preparation method and its application in sow timing semen deposition |
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