WO2019051954A1 - 长效重组猪fsh融合蛋白及其制备方法与应用 - Google Patents

长效重组猪fsh融合蛋白及其制备方法与应用 Download PDF

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WO2019051954A1
WO2019051954A1 PCT/CN2017/108854 CN2017108854W WO2019051954A1 WO 2019051954 A1 WO2019051954 A1 WO 2019051954A1 CN 2017108854 W CN2017108854 W CN 2017108854W WO 2019051954 A1 WO2019051954 A1 WO 2019051954A1
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pfsh
subunit
amino acid
fusion protein
acid sequence
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PCT/CN2017/108854
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English (en)
French (fr)
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罗昊澍
师磊
韩国
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北京伟杰信生物科技有限公司
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Priority to AU2017431494A priority Critical patent/AU2017431494B2/en
Priority to EP17925156.6A priority patent/EP3722323A4/en
Priority to US16/609,543 priority patent/US11299527B2/en
Priority to NZ760019A priority patent/NZ760019B2/en
Priority to RU2020102568A priority patent/RU2747291C1/ru
Priority to CA3065112A priority patent/CA3065112A1/en
Priority to BR112020001332-3A priority patent/BR112020001332A2/pt
Publication of WO2019051954A1 publication Critical patent/WO2019051954A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates to the field of biomedicine and animal breeding technology, in particular to a long-acting recombinant pig FSH fusion protein and a preparation method and application thereof.
  • Porcine Follicle-stimulating hormone is a gonadotropin secreted by the anterior pituitary of pigs. It is non-covalently linked by an alpha subunit and a beta subunit. The alpha subunit is highly conserved and porcine luteinizing The alpha subunits of Porcine Luteinizing Hormone (pLH), Porcine Chorionic Gonadotro-phin (pCG), and Porcine Thyroid Stimulating Hormone (pTSH) are identical; there are differences in ⁇ subunits. Determine the functional specificity of FSH.
  • pFSH can promote the growth and maturation of sow endometrium, ovary and follicles; promote the synthesis and secretion of estrogen; induce the development of boar seminiferous tubules and maintain spermatogenesis.
  • pFSH is commonly used in the field of animal reproduction for the simultaneous treatment of estrus, superovulation, embryo transfer, and ovarian diseases of female animals. Studies have shown that pig FSH is better than cattle and sheep in FSH, so pFSH is of great value to livestock production and economic animal reproduction.
  • FSH horse serum gonadotropin
  • PMSG pregnant horse serum gonadotropin
  • human FSH is not suitable for animal, pig pituitary FSH requires frequent administration, PMSG is prone to adverse effects, and animal breeding field, especially economic animals, requires long-acting animal source FSH.
  • the object of the present invention is to provide a novel long-acting recombinant pig FSH fusion protein and a preparation method and application thereof.
  • Fc fusion protein technology is one of the most widely used and stable technologies in the long-term development of protein drugs, and FSH is fused with Fc by genetic engineering technology to produce a novel recombinant protein FSH-Fc. Not only can the high biological activity of FSH be preserved, but also a longer half-life can be obtained. At the same time, the obtained recombinant protein has high purity, relatively uniform quality, no LH and high safety factor.
  • Use mammalian expression systems especially Chinese hamsters Ovarian cells (CHO) express recombinant proteins and obtain protein molecules that are closest to each other in terms of molecular structure, physical and chemical properties, and biological functions.
  • the long-acting recombinant porcine FSH fusion protein of the invention comprises the fusion proteins pFSH-Fc-1 and pFSH-Fc-2, wherein the alpha subunit of the porcine FSH fusion protein pFSH-Fc-1 is directly or Indirectly fused to the Fc fragment by a linker element, the beta subunit is bound to the alpha subunit by van der Waals force or a linker; the beta subunit of the porcine FSH fusion protein pFSH-Fc-2 is fused directly or indirectly to the Fc fragment by a linker element Above, the alpha subunit binds to the beta subunit via van der Waals forces or linking elements.
  • the porcine FSH fusion protein comprises two peptide chains, which conforms to the following equation: (pFSH ⁇ : pFSH ⁇ -L-Fc) 2 or (pFSH ⁇ : pFSH ⁇ -L-Fc) 2 , wherein pFSH ⁇ refers to the ⁇ -Asia of porcine FSH removal signal peptide
  • the colon represents the relationship between the porcine FSH ⁇ subunit and the ⁇ subunit in a van der Waals force
  • pFSH ⁇ refers to the ⁇ subunit of the porcine FSH removal signal peptide
  • L represents the pFSH ⁇ or pFSH ⁇ subunit and the Fc fragment
  • Fc refers to immunity An Fc fragment of globulin or a mutant thereof
  • parenthesis subscript 2 indicates that the porcine FSH fusion protein is a divalent homodimer.
  • amino acid sequence of pFSH ⁇ is represented by SEQ ID NO: 1, or a protein consisting of an amino acid sequence having 90% or more homology with SEQ ID NO: 1 and having an equivalent function.
  • the amino acid sequence of the pFSH ⁇ is represented by SEQ ID NO: 3, or a protein consisting of an amino acid sequence having 90% or more homology with SEQ ID NO: 3 and having an equivalent function.
  • the Fc comprises an immunoglobulin hinge region as well as CH2 and CH3 regions.
  • the immunoglobulin is from human, pig, cow, sheep, horse or dog. Immunoglobulins are classified into IgG, IgM, IgA, IgD, IgE, and each immunoglobulin includes various subtypes such as IgG1, IgG2, IgG3, IgG4.
  • the Fc mutant refers to an Fc variant comprising one or several mutated amino acid positions on the Fc fragment, such as a human IgG2 Fc variant comprising a human IgG2 hinge of the Pro331Ser mutation. Zone, CH2 and CH3 zones.
  • the Fc is derived from a porcine immunoglobulin, i.e., pFc, comprising the hinge region, CH2 and CH3 regions of porcine immunoglobulin.
  • the amino acid sequence of the pFc is represented by SEQ ID NO: 5, or a protein consisting of an amino acid sequence having 80% or more homology with SEQ ID NO: 5 and having an equivalent function.
  • the ligation relationship of the pFSH ⁇ or pFSH ⁇ subunit to the Fc is a direct splicing linkage or a linker linkage, preferably via a linker.
  • linker is a flexible polypeptide consisting of 2-20 flexible amino acids selected from at least one of Gly, Ser, Ala and Thr.
  • the linker is (Gly-Gly-Gly-Gly-Ser) n , wherein n is an integer between 2 and 5, more preferably n is 3.
  • the pFSH ⁇ -L-Fc is: i) a protein consisting of the amino acid sequence shown in SEQ ID No. 6;
  • Iii a protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID No. 6, and having an equivalent function.
  • the pFSH ⁇ -L-Fc is: iv) a protein consisting of the amino acid sequence shown in SEQ ID No. 8;
  • v a protein derived from iv) in which the amino acid sequence of SEQ ID No. 8 is substituted, deleted and/or added with one or several amino acids and functions equally;
  • a protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID No. 8 and having an equivalent function.
  • the invention also provides an expression cassette, expression vector, cloning vector, engineered or transgenic cell line comprising a nucleic acid comprising the fusion protein described above.
  • the long-acting recombinant porcine FSH fusion protein of the present invention can be prepared by artificially synthesizing genes encoding pFSH ⁇ , pFSH ⁇ , pFSH ⁇ -L-Fc and pFSH ⁇ -L-Fc, and Codon optimization, the optimized genes were cloned into the eukaryotic expression vector; the pFSH ⁇ recombinant vector and the pFSH ⁇ -L-Fc recombinant vector were simultaneously transformed into eukaryotic cells, and expressed in eukaryotic cells, and the target protein was isolated and purified; The pFSH ⁇ recombinant vector and the pFSH ⁇ -L-Fc recombinant vector simultaneously transform the eukaryotic cells and express them in eukaryotic cells to isolate and purify the target protein.
  • the eukaryotic expression vector includes, but is not limited to, pcDNA3.1, and the eukaryotic cells include, but are not limited to, 293, CHO cells.
  • the present invention also provides the use of the above long-acting recombinant porcine FSH fusion protein for the preparation of a medicament for promoting animal reproduction (including simultaneous estrus, superovulation, etc.) and for treating reproductive related diseases in animals.
  • the animal includes, but is not limited to, pig, cow, sheep, horse or dog; preferably pig.
  • the present invention further provides a medicament for promoting animal reproduction (including simultaneous estrus, superovulation, etc.) and for treating a reproductive-related disease of an animal prepared from the above-described long-acting recombinant porcine FSH fusion protein.
  • the invention also provides the use of the above long-acting recombinant porcine FSH fusion protein in the field of animal breeding.
  • Modified proteins including the two fusion proteins pFSH-Fc-1, pFSH-Fc-2 or porcine FSH, are glycosylated, PEGylated, acetylated or bound to BSA, etc., all within the scope of the present invention.
  • the engineered protein including the fusion protein pFSH-Fc-1, pFSH-Fc-2 or porcine FSH protein, which is fused with porcine Fc or with other proteins, does not alter the activity of porcine FSH protein, and belongs to the present invention. protected range.
  • the present invention has the following advantages:
  • the long-acting recombinant porcine FSH fusion protein and/or porcine FSH protein provided by the present invention, and the derivative protein or modified protein thereof, have a half-life of about 60 h, which is higher than the pituitary FSH but lower than the PMSG. Does not require continuous injection, and does not cause adverse reactions to livestock due to long half-life; and there is no immunogenicity in sows, no drug resistance Sexual antibodies.
  • the long-acting recombinant porcine FSH fusion protein and/or porcine FSH protein provided by the present invention, and their derived proteins or modified proteins, have more than 90% effectiveness in promoting sow estrus, and are used for treating ill-fed sows.
  • the effectiveness is above 85%, the average number of ovulations in the head is about 27, and the average number of litters in the head is about 13, and the estrus and superovulation effect is better than PMSG, hFSH-hFc and pFSH-hFc.
  • the long-acting recombinant porcine FSH fusion protein and/or porcine FSH protein provided by the present invention, and the derived protein or modified protein thereof, can also improve the estrus rate of cattle and sheep and promote the super-discharge effect: cow The number of embryos per head is about 8.0, and the number of embryos available in the head is about 6.1, which are higher than PMSG, hFSH-hFc and pFSH-hFc. The efficiency of promoting estrus in ewes is over 90%, which is also better than PMSG and hFSH. -hFc and pFSH-hFc.
  • the long-acting recombinant porcine FSH fusion protein and/or porcine FSH protein provided by the present invention, and the derived protein or modified protein thereof, can effectively provide a safe and effective method for promoting the reproduction rate of animals, especially economic animals. drug.
  • the long-acting recombinant pig FSH fusion protein provided by the invention can effectively prolong the half-life of pig FSH, reduce the number of administrations, reduce the immunogenicity, and improve the estrus rate and the number of ovulation of pigs, cattle, sheep, etc., without It has an adverse reaction to animals and can replace the application of pig pituitary FSH and PMSG in animal breeding.
  • Figure 1 is a diagram showing the SDS-PAGE electrophoresis of pFSH-Fc-1 and pFSH-Fc-2 in Example 1 of the present invention.
  • a and C are denatured electropherograms of pFSH-Fc-1 and pFSH-Fc-2, respectively;
  • B and D are non-denaturing electropherograms of pFSH-Fc-1 and pFSH-Fc-2, respectively.
  • MK Protein Marker; 1: Clarified fermentation broth; 2: Protein A flow through solution; 3: Protein A collection solution; 4: Capto S collection solution; 5: Capto Q collection solution.
  • the structural formula of pFSH-Fc-1 is (pFSH ⁇ : pFSH ⁇ -L-Fc) 2
  • the structural formula of pFSH-Fc-2 is (pFSH ⁇ : pFSH ⁇ -L-Fc) 2 .
  • Figure 2 is a diagram showing the rat ovary of the pFSH-Fc-1, pFSH-Fc-2 and PMSG groups in Example 2 of the present invention.
  • 10 IU, 20 IU, and 40 IU showed that the rats in the pFSH-Fc-1, pFSH-Fc-2, and PMSG groups were injected with 10 IU, 20 IU, and 40 IU, respectively.
  • Figure 3 is a diagram showing the superovulation of superovulation of primiparous sows of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups in Example 4 of the present invention.
  • hFSH-hFc and pFSH-hFc described in the following examples are both ⁇ subunits linked to hFc, and the construction method is the same as that of pFSH-Fc-2, and the gene sequences of human FSH ⁇ , human FSH ⁇ , and human Fc are searched, and codon-optimized.
  • the hFSH ⁇ , hFSH ⁇ -L-hFc, pFSH ⁇ , pFSH ⁇ -L-hFc genes were artificially synthesized, and the hFSH ⁇ and hFSH ⁇ -L-hFc, pFSH ⁇ and pFSH ⁇ -L-hFc recombinant plasmids were transiently transformed into 293 cells for expression and purified to obtain hFSH. -hFc and pFSH-hFc.
  • the gene pool was searched for gene sequences of porcine FSH ⁇ (GenBank NM-214446.1), porcine FSH ⁇ (GenBank NM-213875.1) and porcine Fc (GenBank BAE20056). Codon optimization was performed: pFSH ⁇ nucleotide sequence as shown in SEQ ID NO: 2; pFSH ⁇ as shown in SEQ ID NO: 4; pFSH ⁇ -L-pFc sequence as shown in SEQ ID NO: 7; pFSH ⁇ -L- The pFc sequence is set forth in SEQ ID NO: 9.
  • the artificially synthesized pFSH ⁇ , pFSH ⁇ , pFSH ⁇ -L-pFc and pFSH ⁇ -L-pFc genes were cloned into the vector pcDNA3.1, respectively.
  • Recombinant vectors of pFSH ⁇ and pFSH ⁇ -L-pFc, pFSH ⁇ and pFSH ⁇ -L-pFc were electroporated into 293 cells, respectively.
  • pFSH-Fc-1 and pFSH-Fc-2 purified and verified for transiently expressed proteins.
  • the recombinant vectors of pFSH ⁇ and pFSH ⁇ -L-pFc, pFSH ⁇ and pFSH ⁇ -L-pFc were linearized and then electroporated into CHO cells to obtain stable cell lines of pFSH-Fc-1 and pFSH-Fc-2. .
  • the stable cells of pFSH-Fc-1 and pFSH-Fc-2 were fermented in a fermentor, and the fermentation broth was subjected to two-stage deep filtration membrane to remove cells and cell debris, and then filtered through a 0.22 ⁇ m filter to obtain clarification. Fermentation broth.
  • the fermentation broth was first purified by affinity chromatography ProteinA (MabSelect SuReTM, GE Healthcare): first equilibrated to the baseline with an equilibration solution (50 mM glycine, 0.15 M NaCl, pH 7.2) and then washed with an eluent (50 mM glycine, pH 3.0). Remove and collect the eluent.
  • the Protein A collection solution was further purified by cation exchange Capto S (GE Healthcare) column chromatography: the pH was adjusted to 6.5 with 1 M NaOH, the conductivity was adjusted to 4.5 to 5.0 ms/cm, and the equilibration solution (50 mM glycine pH 6.5) was used. ) Balance, load, collect the flow through the effluent.
  • the Capto S collection solution was finely purified by anion exchange Capto Q (GE Healthcare) column chromatography: the pH was adjusted to 8.0 with 1 M NaOH, equilibrated to baseline with an equilibration solution (50 mM glycine pH 8.0), followed by eluent (50 mM).
  • the activities of pFSH-Fc-1 and pFSH-Fc-2 were measured using a rat ovarian weight gain method (Steelman-Pohley method).
  • the product of the present invention is intended to be used in place of animal breeding in the field of animal breeding. Therefore, the activity of the sample is determined according to the 2015 edition of the Chinese Pharmacopoeia "Hemoglobin Bioassay", using PMSG as a standard.
  • the specific implementation is as follows: pFSH-Fc-1 (specific activity estimated 10000 U / mg), pFSH-Fc-2 (specific activity estimated 10000 U / mg) and PMSG are formulated into 40 IU, 20 IU and 10 IU high, medium and low doses .
  • Rats of 21-day-old, 40-55 g female SD (Sprague Dawley) were randomly divided into 9 groups, 6 in each group. Each rat was subcutaneously injected with 0.5 ml of the corresponding drug. After 6 days, the rats were sacrificed, weighed, dissected, the ovaries were removed, weighed, and converted into ovary weight per 100 g body weight (Fig. 2).
  • the specific activity of pFSH-Fc-1 was calculated to be about 9600 U/mg using the Pharmacopoeia Bioassay Statistics BS2000 software, and the specific activity of pFSH-Fc-2 was about 10700 U/mg.
  • Ten SD female rats of about 40 g were randomly divided into two groups: pFSH-Fc-1 group and pFSH-Fc-2 group.
  • the levels of pFSH-Fc-1 and pFSH-Fc-2 in the serum were measured using an FSH ELISA kit, and each blood sample was analyzed three times. Using Pksolver software to calculate pFSH-Fc-1 half-life was 57.2h, pFSH-Fc-2 half-life was 63.4h, higher than pig natural FSH, lower than PMSG.
  • the estrus sows were bred with the same boars three times, each time 12 h. After 36 hours of the first breeding, the donor pigs were surgically harvested and the number of ovulations was counted (Fig. 3).
  • the results are shown in Table 1.
  • the donor pigs in each group had good estrus, and the estrus rate of the primipara and sows in the pFSH-Fc-1 group were both 90% and 95%, which was higher than that of the PMSG group (60% and 65%).
  • the hFSH-hFc group (65% and 70%) and the pFSH-hFc group (80% and 85%) were significantly different from the PMSG group and the hFSH-hFc group (P ⁇ 0.05).
  • the pFSH-Fc-2 group had 100% estrus in both primiparous and sows, and the difference between PMSG group and hFSH-hFc group was very significant (P ⁇ 0.01).
  • the number of ovulation in each group of sows was higher than that in normal natural estrus sows (8-14 pieces/ Head), the average number of ovulations in the pFSH-Fc-1 group was 27.7 and 27.5, respectively, higher than the PMSG group (19.8 and 20.2) and the hFSH-hFc group (22.1 and 21.6).
  • the average number of ovulations in the pFSH-Fc-2 group was 29.9 and 29.1, respectively, which was higher than that of the PMSG group, the hFSH-hFc group and the pFSH-hFc group, and the PMSG group and hFSH-hFc.
  • the group ratio was significantly different (P ⁇ 0.05).
  • Group 5 pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups, each group of sows were injected with drugs according to the method of Example 4. After the sow is in estrus, the sows who choose to estrus are bred with the same boars 3 times, at intervals of 12 h. The litter size of each group of sows was recorded in detail.
  • the results are shown in Table 2.
  • the total litter size (123 and 125) in the pFSH-Fc-1 and pFSH-Fc-2 groups was higher than the PMSG group (65) and the hFSH-hFc group (68).
  • the pFSH-hFc group (99 heads) was significantly different from the PMSG and hFSH-hFc groups (P ⁇ 0.05).
  • the litter size (13.7 and 13.9) of the pFSH-Fc-1 and pFSH-Fc-2 groups was also higher than that of the PMSG group (10.1 heads), the hFSH-hFc group (10.5 heads), and the pFSH-hFc group (12.4). head).
  • Each group of donor cows was intramuscularly injected with 1000 IU of corresponding drugs (Day 5), and 0.5 mg of cloprostenol (PG) was injected at the same time.
  • the estrus was removed (Day 10), and the estrus was observed. The climb will prevail.
  • the first insemination was performed, and after 24 hours, the second insemination was performed.
  • the embryos were collected on Day16 non-surgical rinsing and the number of embryos was counted.
  • the donor bovine super-discharge effect was remarkable in each administration group (naturally, one cow produced only one embryo at a time), and the pFSH-Fc-1 group and the pFSH-Fc-2 group had a cow head.
  • the number of embryos (7.9 and 8.7) was higher than that of the PMSG group (5.7), the hFSH-hFc group (6.1), and the pFSH-hFc group (7.3), and the difference was significant between the PMSG group and the hFSH-hFc group (P). ⁇ 0.05).
  • the number of available embryos in the pFSH-Fc-1 and pFSH-Fc-2 groups were higher than those in the PMSG group (3.5), the hFSH-hFc group (3.9), and the pFSH-hFc group (5.7). (), and the difference between the PMSG group and the hFSH-hFc group was significant (P ⁇ 0.05).
  • the number of embryos that were not available in the pFSH-Fc-1 and pFSH-Fc-2 groups (1.8 and 1.3) were lower than those in the PMSG group (2.2) and the hFSH-hFc group (2.2), and the pFSH-Fc-2 group.
  • the pFSH-hFc group (1.6) was significantly different from the PMFS group and the hFSH-hFc group (P ⁇ 0.05).
  • the antigenic immunity of the pFSH-Fc-1 and pFSH-Fc-2 proteins was examined by detecting the drug-resistant antibody (ADA) of the drug in the serum of the sample by using the Bridging-ELISA method.
  • ADA drug-resistant antibody
  • Forty primiparous large white sows were selected and divided into 4 groups: pFSH-Fc-1 group, pFSH-Fc-2 group, hFSH-hFc and pFSH-hFc group.
  • Each group of donor pigs was intramuscularly injected with 1000 IU of the drug into the neck, administered once every three days for a total of 5 weeks (13 times of administration), and the first dose was recorded as Day0.
  • Blood was collected at the following times: first drug (Day-2), third drug (Day6), fifth drug (Day12), and the sixth to last drug (Day 15, 18, 21, 24, 27, 30, 33, 36), the third day after the last administration (Day 39).
  • the blood was centrifuged, serum was collected, and the OD 490 nm value of ADA in the serum was detected in the ELISA plate coated with pFSH-Fc-1, pFSH-Fc-2, hFSH-hFc or pFSH-hFc, respectively.
  • Positive control samples were prepared with anti-pFSH-Fc-1, anti-pFSH-Fc-2, anti-hFSH-hFc or anti-pFSH-hFc rabbit monoclonal antibody diluted with 100% mixed pig serum, respectively; negative control (N) samples were injected with the same A PBS buffer was prepared from the primiparous white sow serum.
  • SCP threshold
  • the results are shown in Table 6.
  • the SCPs of the pFSH-Fc-1 and pFSH-Fc-2 donor pig samples were all less than 1.15, and no ADA was detected, indicating that 1000 IU of pFSH-Fc-1 or pFSH-Fc-2 large white mother was injected. Pigs do not produce drug-resistant antibodies.
  • the SCP of Day27 and subsequent sows 1, 2, 4, 7, and 9 were greater than 1.15 (first detected in Day27, last detected in Day39), and other sows Day30 and later.
  • the serum sample SCP was greater than 1.15 (first detected in Day 30 and last detected in Day 39), indicating that all sows in the hFSH-hFc group were detected with ADA, with a positive rate of 100%.
  • the SCP of the sows Day30 after 2, 3, 6, 7, and 9 and the subsequent SCP were greater than 1.15 (first detected in Day 30, last detected in Day 39)
  • sows 1, 5, and 8 Days 36 and subsequent serum samples had SCP greater than 1.15 (first detected on Day36, last detected on Day39)
  • SCPs for all samples 4 and 10 were less than 1.15, indicating that 8 sows were detected in the pFSH-hFc group.
  • ADA the positive rate was 80%.
  • the above results indicate that 1000 IU of hFSH-hFc or pFSH-hFc large white sows were injected to produce drug-resistant antibodies.
  • the present invention provides two long-acting recombinant porcine FSH fusion proteins, including pFSH-Fc-1 and pFSH-Fc-2, wherein the ⁇ subunit/ ⁇ subunit is directly or indirectly fused to the Fc fragment via a linker element, ⁇ subunit/ The alpha subunit binds to the alpha subunit/beta subunit via van der Waals forces or linking elements.
  • the porcine FSH fusion protein can be prepared based on genetic engineering techniques using a eukaryotic expression system.
  • the two porcine FSH fusion proteins provided by the invention have good pharmacological effect, longer half-life than the natural pig FSH; no adverse effects on the animal, no immunogenicity on the sow, no anti-drug antibody, and can replace the pregnant horse serum
  • PMSG gonadotropin

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Abstract

两种长效重组猪FSH融合蛋白，包括pFSH-Fc-1和pFSH-Fc-2，其α亚基/β亚基直接或间接通过连接元件融合至Fc片段上，β亚基/α亚基通过范德华力或连接元件与α亚基/β亚基结合。所述猪FSH融合蛋白可基于基因工程技术，利用真核表达系统制备得到。两种猪FSH融合蛋白药效好，比天然猪FSH半衰期长；又不会对动物产生不良效应，可替代孕马血清促性腺激素(PMSG)在动物繁殖生产中的使用。两种猪FSH融合蛋白可制备动物繁殖领域药物。

Description

长效重组猪FSH融合蛋白及其制备方法与应用 技术领域
本发明涉及生物医药和动物繁殖技术领域，具体地说，涉及一种长效重组猪FSH融合蛋白及其制备方法与应用。
背景技术
猪促卵泡素(Porcine Follicle-stimulating hormone，简称pFSH)是一种猪垂体前叶分泌的促性腺激素，由一个α亚基和一个β亚基非共价连接，其中α亚基高度保守，与猪促黄体素(Porcine LuteinizingHormone,pLH)、猪绒毛膜促性腺激素(Porcine Chorionic Gonadotro–phin,pCG)、猪促甲状腺素(Porcine Thyroid Stimulating Hormone,pTSH)的α亚基完全相同；β亚基存在差异，主要决定FSH的功能特异性。pFSH可以促进母猪子宫内膜、卵巢和卵泡的生长和成熟；促进雌激素的合成与分泌；诱导公猪曲细精管的发育和维持精子生成。pFSH在动物繁殖领域中常用于同期发情、超数排卵、胚胎移植、母畜卵巢疾病的治疗。有研究表明猪FSH用于牛、羊的超排效果要好于牛羊自身的FSH，因此pFSH对畜牧生产和经济动物繁殖具有重要价值。
目前已有相关专利和文章报道了FSH融合蛋白的表达，但大部分是关于人FSH的重组表达与应用，将人FSH用于动物繁殖领域，也获得了一些成功。然而尽管人与家畜同属于哺乳动物，但在蛋白序列上仍存在较大的种属差异(人和猪FSHα亚基氨基酸序列同源性为73％，β亚基同源性为93％，Fc片段同源性为73％)。如果长期将人重组FSH用于动物上，会被动物的免疫系统识别诱生抗体(包括中和抗体)，这些抗体特异性结合动物FSH的受体结合位点，从而阻断药物的生物学活性，导致人FSH在动物体内的生物利用度越来越低，限制了人FSH在动物，尤其是需要长期使用FSH的猪、牛、羊等经济动物上的应用，这也是目前市售的人重组FSH不用于动物繁殖领 域的原因之一。
目前市面上畜用FSH产品主要包括猪垂体提取的FSH和孕马血清促性腺激素(PMSG)。猪垂体FSH，如Folltropin-V(加拿大)，半衰期短(5h)，需要频繁给药，导致终端客户给养和管理成本高；并且从猪垂体组织中提纯时很难将猪FSH与LH分离、纯化难度大、产能低，所以在实际应用中受到很大限制。PMSG是一种马属动物胎盘尿囊绒毛膜细胞分泌的糖蛋白激素，兼具FSH(高)和LH(低)活性，具有促进动物卵巢和睾丸生殖机能的作用。在动物繁殖领域，常用于诱发雌性动物同期发情、超数排卵，治疗雌性动物繁殖障碍和卵巢功能不全；促进雄性动物曲细精管发育和精子生成。但因PMSG分子含有较高的己糖和唾液酸，在动物体内半衰期太长(120h)，用于动物超数排卵时，容易引发母畜的多种不良效应，如卵巢囊肿，妊娠初期胚胎的提前退化；并且商品化的PMSG从孕马血清中提取纯化，用于其它动物时会产生抗体，存在一定的免疫原性，不能长时间使用。此外，从怀孕母马采集血清制备PMSG，经常因采血过多导致母马流产、胎马死亡，不符合动物伦理。
因此基于人FSH不适用于动物、猪垂体FSH需要频繁给药、PMSG易产生不良效应，动物繁殖领域特别是经济动物需要长效的动物源FSH。
发明内容
本发明的目的是提供一种新型的长效重组猪FSH融合蛋白及其制备方法与应用。
本发明的构思如下：Fc融合蛋白技术是蛋白药物长效化研发中最广泛使用且最稳定的技术之一，利用基因工程技术将FSH与Fc融合产生新型重组蛋白FSH-Fc。不仅能保留FSH的高生物活性，还可以获得较长的半衰期；同时得到的重组蛋白纯度高，质量相对均一，不含有LH，安全系数高。使用哺乳动物表达体系，尤其是中国仓鼠 卵巢细胞(CHO)表达重组蛋白，能获得在分子结构、理化特性和生物学功能方面最接近天然的蛋白分子。
为了实现本发明目的，本发明的长效重组猪FSH融合蛋白，包括融合蛋白pFSH-Fc-1和pFSH-Fc-2，其中所述猪FSH融合蛋白pFSH-Fc-1的α亚基直接或间接通过连接元件融合至Fc片段上，β亚基通过范德华力或连接元件与α亚基结合；所述猪FSH融合蛋白pFSH-Fc-2的β亚基直接或间接通过连接元件融合至Fc片段上，α亚基通过范德华力或连接元件与β亚基结合。
所述猪FSH融合蛋白包括两条肽链，符合以下方程：(pFSHβ:pFSHα-L-Fc)2或(pFSHα:pFSHβ-L-Fc)2，其中pFSHβ是指猪FSH去除信号肽的β亚基；冒号代表猪FSHβ亚基和α亚基以范德华力连接的关系；pFSHα是指猪FSH去除信号肽的α亚基；L代表pFSHα或pFSHβ亚基与Fc片段的连接关系；Fc是指免疫球蛋白的Fc片段或其突变体；括号外下标2代表所述猪FSH融合蛋白为二价同二聚体。
所述pFSHα的氨基酸序列如SEQ ID NO:1所示，或者与SEQ IDNO:1同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
所述pFSHβ的氨基酸序列如SEQ ID NO:3所示，或者与SEQ IDNO:3同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
所述Fc包含免疫球蛋白铰链区以及CH2和CH3区。
所述免疫球蛋白来自人、猪、牛、羊、马或狗。免疫球蛋白分为IgG、IgM、IgA、IgD、IgE，每种免疫球蛋白包括各种亚型，如IgG1、IgG2、IgG3、IgG4。
所述Fc突变体是指Fc片段上含有一个或几个突变氨基酸位点的Fc变体，例如人IgG2Fc变体，其含有Pro331Ser突变的人IgG2绞链 区、CH2和CH3区。
优选地，所述Fc来自猪的免疫球蛋白，即pFc，包含猪免疫球蛋白的铰链区、CH2和CH3区。所述pFc的氨基酸序列如SEQ ID NO:5所示，或者与SEQ ID NO:5同源性在80％以上的，且具有同等功能的氨基酸序列构成的蛋白。
所述pFSHα或pFSHβ亚基与Fc的连接关系为直接拼接连接或通过linker连接，优选通过linker连接。
其中，所述linker是由2-20个柔性氨基酸组成的柔性多肽，所述柔性氨基酸选自Gly、Ser、Ala和Thr中的至少一种。
优选地，所述linker为(Gly-Gly-Gly-Gly-Ser)n，其中n为2-5之间的整数，更优选n为3。
优选地，所述pFSHα-L-Fc为：i)由SEQ ID No.6所示的氨基酸序列构成的蛋白；或
ii)SEQ ID No.6所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白；或
iii)与SEQ ID No.6所示氨基酸序列同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
优选地，所述pFSHβ-L-Fc为：iv)由SEQ ID No.8所示的氨基酸序列构成的蛋白；或
v)SEQ ID No.8所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由iv)衍生的蛋白；或
vi)与SEQ ID No.8所示氨基酸序列同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
本发明还提供表达盒、表达载体、克隆载体、工程菌或转基因细胞系，其包括包含编码上述融合蛋白的核酸。
本发明的长效重组猪FSH融合蛋白可按如下方法制备得到：人工合成pFSHα、pFSHβ、pFSHα-L-Fc和pFSHβ-L-Fc的编码基因，并 进行密码子优化，优化后的基因分别克隆到真核表达载体中；将pFSHα重组载体和pFSHβ-L-Fc重组载体同时转化真核细胞，并在真核细胞中表达，分离纯化目标蛋白；将pFSHβ重组载体和pFSHα-L-Fc重组载体同时转化真核细胞，并在真核细胞中表达，分离纯化目标蛋白。
所述真核表达载体包括但不限于pcDNA3.1，所述真核细胞包括但不限于293、CHO细胞。
本发明还提供上述长效重组猪FSH融合蛋白在制备用于促进动物繁殖(包括同期发情、超数排卵等)以及治疗动物生殖相关疾病的药物中的应用。其中，所述动物包括但不限于猪、牛、羊、马或狗；优选猪。
本发明进一步提供由上述长效重组猪FSH融合蛋白制备的用于促进动物繁殖(包括同期发情、超数排卵等)以及治疗动物生殖相关疾病的药物。
本发明还提供上述长效重组猪FSH融合蛋白在动物繁殖领域中的应用。
经过修饰的蛋白，包括两种融合蛋白pFSH-Fc-1、pFSH-Fc-2或猪FSH经过糖基化、聚乙二醇化、乙酰化或与BSA结合等，均属于本发明的保护范围。
经过改造的蛋白，包括两种融合蛋白pFSH-Fc-1、pFSH-Fc-2或猪FSH蛋白与猪Fc或与其他蛋白融合构成的不改变猪FSH蛋白活性的融合蛋白，均属于本发明的保护范围。
与现有技术相比，本发明具有以下优点：
(一)本发明提供的长效重组猪FSH融合蛋白和/或猪FSH蛋白，以及它们的衍生蛋白或经过修饰的蛋白，半衰期约为60h，高于猪垂体FSH，但低于PMSG，使用时既不需要连续注射，又不会因半衰期过长对家畜产生不良反应；并且对母猪不存在免疫原性，不产生抗药 性抗体。
(二)本发明提供的长效重组猪FSH融合蛋白和/或猪FSH蛋白，以及它们的衍生蛋白或经过修饰的蛋白，促进母猪发情的有效性达90％以上，治疗乏情母猪的有效性在85％以上，头均排卵数约为27枚，头均产仔数约为13头，发情和超排效果优于PMSG、hFSH-hFc和pFSH-hFc。
(三)本发明提供的长效重组猪FSH融合蛋白和/或猪FSH蛋白，以及它们的衍生蛋白或经过修饰的蛋白，亦能提高牛、羊的发情率，促进超排效果：母牛的头均胚胎数约为8.0枚，头均可用胚胎数约为6.1枚，均高于PMSG、hFSH-hFc和pFSH-hFc；促进母羊发情的有效率在90％以上，亦优于PMSG、hFSH-hFc和pFSH-hFc。
(四)本发明提供的长效重组猪FSH融合蛋白和/或猪FSH蛋白，以及它们的衍生蛋白或经过修饰的蛋白，可以有效地为促进动物尤其经济动物的繁殖率，提供安全、有效的药物。
(五)应用于动物繁殖领域，与人FSH相比，免疫原性更低、生物活性更高；与猪垂体天然FSH相比纯度更高、半衰期更长；与PMSG相比，副作用更小、免疫原性更低。
(六)本发明提供的长效重组猪FSH融合蛋白，可有效延长猪FSH的半衰期，减少给药次数，降低免疫原性；提高猪、牛、羊等的发情率和排卵数，又不会对动物产生不良反应，可替代猪垂体FSH和PMSG在动物繁殖领域中的应用。
附图说明
图1为本发明实施例1中pFSH-Fc-1和pFSH-Fc-2的SDS-PAGE电泳图。其中，A和C分别是pFSH-Fc-1和pFSH-Fc-2的变性电泳图；B和D分别是pFSH-Fc-1和pFSH-Fc-2的非变性电泳图。MK：蛋白Marker；1：澄清发酵液；2：Protein A流穿液；3：Protein A收集液；4：Capto S收集液；5：Capto Q收集液。pFSH-Fc-1的结构式为 (pFSHβ:pFSHα-L-Fc)2，pFSH-Fc-2的结构式为(pFSHα:pFSHβ-L-Fc)2。
图2为本发明实施例2中pFSH-Fc-1、pFSH-Fc-2和PMSG组的大鼠卵巢图。其中，10 IU、20 IU和40 IU分别表示pFSH-Fc-1、pFSH-Fc-2和PMSG组大鼠分别注射10 IU、20 IU和40 IU量的相应药品。
图3为本发明实施例4中pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组1号初产母猪超排的卵巢图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a LaboratoryManual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中述及的hFSH-hFc和pFSH-hFc均是β亚基与hFc连接，构建方法与pFSH-Fc-2相同，检索人FSHα、人FSHβ、人Fc的基因序列，密码子优化后人工合成hFSHα、hFSHβ-L-hFc、pFSHα、pFSHβ-L-hFc基因，分别将hFSHα和hFSHβ-L-hFc、pFSHα和pFSHβ-L-hFc重组质粒瞬转转入293细胞中表达，纯化得到hFSH-hFc和pFSH-hFc。
实施例1 pFSH-Fc-1和pFSH-Fc-2蛋白的制备
在基因库中搜索猪FSHα(GenBank NM-214446.1)、猪FSHβ(GenBank NM-213875.1)和猪Fc(GenBank BAE20056)的基因序列。进行密码子优化：pFSHα核苷酸序列，如SEQ ID NO:2所示；pFSHβ如SEQ ID NO:4所示；pFSHα-L-pFc序列，如SEQ ID NO:7所示；pFSHβ-L-pFc序列，如SEQ ID NO:9所示。
将人工合成的pFSHα、pFSHβ、pFSHα-L-pFc和pFSHβ-L-pFc基因，分别克隆到载体pcDNA3.1中。分别将pFSHβ和pFSHα-L-pFc、pFSHα和pFSHβ-L-pFc的重组载体电转转入293细胞中表达 pFSH-Fc-1和pFSH-Fc-2，对瞬转表达的蛋白进行纯化验证活性。存在活性后分别将pFSHβ和pFSHα-L-pFc、pFSHα和pFSHβ-L-pFc的重组载体线性化后电转转入CHO细胞中获得pFSH-Fc-1和pFSH-Fc-2的稳转细胞系。
将pFSH-Fc-1和pFSH-Fc-2的稳转细胞在发酵罐中进行发酵培养，发酵液先经过两级深层过滤膜包去除细胞和细胞碎片，之后用0.22μm滤膜过滤，获得澄清的发酵液。发酵液首先用亲和层析ProteinA(MabSelect SuReTM,GE Healthcare)纯化：先用平衡液(50mM甘氨酸，0.15M NaCl，pH 7.2)平衡至基线，然后用洗脱液(50mM甘氨酸，pH 3.0)洗脱、收集洗脱液。将Protein A收集液用阳离子交换Capto S(GE Healthcare)柱层析进一步纯化：收集液用1M NaOH调节pH值至6.5，加水调节电导为4.5～5.0ms/cm，用平衡液(50mM甘氨酸pH 6.5)平衡，上样，收集流穿流出液。将Capto S收集液用阴离子交换Capto Q(GE Healthcare)柱层析精细纯化：收集液用1MNaOH调节pH值至8.0，用平衡液(50mM甘氨酸pH 8.0)平衡至基线，之后用洗脱液(50mM甘氨酸，1M KCl，pH 8.0)洗脱，收集得到纯化蛋白。对纯化的目的蛋白进行SDS-PAGE凝胶电泳(图1)。实施例2 pFSH-Fc-1和pFSH-Fc-2蛋白的活性检测
采用大鼠卵巢增重法(Steelman-Pohley法)测定pFSH-Fc-1和pFSH-Fc-2的活性。本发明产品旨在代替PMSG在动物繁殖领域使用，因此根据2015年版《中国药典》“血促性素生物测定法”测定样品活性，以PMSG作为标准品。具体实施如下：将pFSH-Fc-1(比活估计10000U/mg)、pFSH-Fc-2(比活估计10000U/mg)和PMSG均配制成40IU、20IU和10IU高、中、低三个剂量。选取日龄21-23天、体重40-55g雌性SD(Sprague Dawley)大鼠随机分成9组，每组6只。每只大鼠皮下注射0.5ml相应药品，6日后，将大鼠处死，称体重，解剖，摘出卵巢，称重，换算成每100g体重的卵巢重(图2)。 使用中检所《药典生物检定统计BS2000》软件计算pFSH-Fc-1的比活约为9600U/mg，pFSH-Fc-2的比活约为10700U/mg。
实施例3 pFSH-Fc-1和pFSH-Fc-2蛋白的药代动力学研究
选取10只40g左右的SD雌性大鼠，随机分成两组：pFSH-Fc-1组和pFSH-Fc-2组。皮下注射20IU/kg体重的相应药品，分别在给药0、1、2、4、8、12、24、48、72、96、120、144h后取血100μl，3000rpm离心取血清-80℃冻存。使用FSH ELISA试剂盒检测血清中pFSH-Fc-1和pFSH-Fc-2的含量，每个血样重复分析三次。使用Pksolver软件计算pFSH-Fc-1半衰期为57.2h，pFSH-Fc-2半衰期为63.4h，高于猪天然FSH、低于PMSG。
实施例4 pFSH-Fc-1和pFSH-Fc-2蛋白在促进初产和经产母猪同期发情和超数排卵中的应用
分别选取100头初产大白母猪、断奶2周后没有发情的经产大白母猪，体重85-100kg，品种相同，体征接近。随机分为5组：pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组；各组组内再分为初产母猪组和经产母猪组。分别在各组供体猪耳后颈部肌肉注射1000IU相应药品，间隔72h后注射500IU HCG。5天后观察各组母猪的发情情况。除了将初产母猪组发情的1号大白母猪进行屠宰取卵巢拍照外，其他组的发情母猪均与同系公猪配种3次，每次间隔12h。在第一次配种36h后，对供体猪手术采卵，计算排卵数(图3)。
结果如表1所示，各组供体猪发情良好，pFSH-Fc-1组初产和经产母猪发情率均为90％和95％，高于PMSG组(60％和65％)、hFSH-hFc组(65％和70％)和pFSH-hFc组(80％和85％)，与PMSG组和hFSH-hFc组比差异显著(P<0.05)。pFSH-Fc-2组初产和经产母猪均100％发情，与PMSG组和hFSH-hFc组比差异非常显著(P<0.01)。
各组母猪的排卵数均高于正常自然发情母猪的排卵数(8-14枚/ 头)，pFSH-Fc-1组初产和经产母猪的头均排卵数分别为27.7枚和27.5枚，高于PMSG组(19.8枚和20.2枚)、hFSH-hFc组(22.1枚和21.6枚)和pFSH-hFc组(25.7枚和26.2枚)，且与PMSG组和hFSH-hFc组比差异显著(P<0.05)。pFSH-Fc-2组初产和经产母猪的头均排卵数分别为29.9枚和29.1枚，亦高于PMSG组、hFSH-hFc组和pFSH-hFc组，且与PMSG组和hFSH-hFc组比差异显著(P<0.05)。
Figure PCTCN2017108854-appb-000001
实施例5 pFSH-Fc-1和pFSH-Fc-2蛋白在提高母猪产仔数的应用
选择50头初产大白母猪，体重85-100kg，品种相同，体征接近。分5组：pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组，依实施例4中方法对各组母猪注射药品。母猪发情后，选择发情的母猪与同系公猪配种3次，每次间隔12h。详细记录各组母猪的产仔数。
结果如表2所示pFSH-Fc-1组和pFSH-Fc-2组母猪总产仔数(123头和125头)高于PMSG组(65头)、hFSH-hFc组(68头)和pFSH-hFc组(99头)，且与PMSG和hFSH-hFc组比差异显著(P<0.05)。pFSH-Fc-1和pFSH-Fc-2组的胎均产仔数(13.7头和13.9头)亦高于PMSG组(10.1头)、hFSH-hFc组(10.5头)和pFSH-hFc组(12.4头)。
表2 pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc
对初产母猪产仔数的比较
Figure PCTCN2017108854-appb-000002
注：同列系列肩标不同大小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
实施例6 pFSH-Fc-1和pFSH-Fc-2蛋白在治疗乏情母猪中的应用
选择100头断奶后21天以上未发情的乏情大白母猪，随机分成5组pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组。分别在各组供体猪耳后颈部肌肉注射1000IU相应药品，间隔72h后注射500IU HCG。观察母猪的发情特征：如阴户红肿、有粘液；压背时出现静立反应。依实施例4和5将发情后的母猪与公猪进行配种，记 录受胎情况。
结果如表3所示，乏情母猪对药品反应敏感，pFSH-Fc-1组和pFSH-Fc-2组母猪的发情率(85％和90％)均高于PMSG组(55％)、hFSH-hFc组(65％)和pFSH-hFc组(75％)，且与PMSG组比差异显著(P<0.05)。pFSH-Fc-1组和pFSH-Fc-2组母猪的受孕率(88.2％和88.9％)亦高于PMSG组(63.6％)、hFSH-hFc组(61.5％)和pFSH-hFc组(73.3％)，差异均不显著(P>0.05)。
表3 pFSH-FC-1、pFSH-FC-2、PMSG、hFSH-hFC和pFSH-hFC
对诱导乏情母猪的发情比较
Figure PCTCN2017108854-appb-000003
注：同列系列肩标不同大小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
实施例7 pFSH-Fc-1和pFSH-Fc-2蛋白在促进母牛超数排卵中的应用
选择50头3-6岁龄体质健康、无疾病的荷斯坦母牛，随机分为pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组。各组母牛在原有饲喂基础上加喂1kg精料，同时肌肉注射VA、VD和VE。每头供体牛埋植孕酮阴道栓CIDR(含黄体酮1.56g/支)。放栓当天记为Day0，各组供体牛分别肌肉注射1000IU相应药品(Day5)，同时再注射0.5mg氯前列烯醇(PG)，撤栓(Day10)，观察发情，以供体牛接受公牛爬跨为准。站立发情12h后第一次输精，24h后第二次输精。于Day16非手术冲洗采集胚胎，统计胚胎数。
结果如表4所示，各给药组供体牛超排效果显著(自然情况下，一头母牛一次只产生一枚胚胎)，pFSH-Fc-1组和pFSH-Fc-2组母牛头均胚胎数(7.9枚和8.7枚)高于PMSG组(5.7枚)、hFSH-hFc组(6.1枚)和pFSH-hFc组(7.3枚)，且与PMSG组和hFSH-hFc组比差异显著(P<0.05)。pFSH-Fc-1和pFSH-Fc-2组母牛头均可用胚胎数(6.1枚和7.4枚)均高于PMSG组(3.5枚)、hFSH-hFc组(3.9枚)和pFSH-hFc组(5.7枚)，且与PMSG组和hFSH-hFc组比差异显著(P<0.05)。pFSH-Fc-1和pFSH-Fc-2组母牛头均不可用胚胎数(1.8枚和1.3枚)低于PMSG组(2.2枚)和hFSH-hFc组(2.2枚)，pFSH-Fc-2组低于pFSH-hFc组(1.6枚)，且pFSH-Fc-2组与PMSG组和hFSH-hFc组比差异显著(P<0.05)。
表4 pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc
对荷斯坦母牛超数排卵的比较
Figure PCTCN2017108854-appb-000004
注：同列系列肩标不同大小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
实施例8 pFSH-Fc-1和pFSH-Fc-2蛋白在促进母山羊同期发情中的应用
选择75只1.5-3岁，体重30-45kg、体质健康、无疾病的母山羊，随机分为pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc组。于发情周期任一天给供体羊放置孕酮阴道栓并记为Day0，各供体羊分别肌肉注射300IU相应药品(Day10)，撤栓(Day12)。观察 母山羊的发情表现，并用试情公羊进行试情。以母羊外阴发红、流黏液并接受爬跨视为发情，计算发情率。
结果如表5所示，各组母羊发情明显，pFSH-Fc-1组和pFSH-Fc-2组母羊发情率均为93.3％，高于PMSG组(60％)、hFSH-hFc组(73.3％)和pFSH-hFc组(80.0％)，且与PMSG组比差异显著(P<0.05)。
表5 pFSH-Fc-1、pFSH-Fc-2、PMSG、hFSH-hFc和pFSH-hFc
对母山羊的发情比较
Figure PCTCN2017108854-appb-000005
注：同列系列肩标不同大小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
实施例9 pFSH-Fc-1和pFSH-Fc-2蛋白的抗原免疫性检测
通过使用Bridging-ELISA法检测样品血清中药品的抗药性抗体(ADA)以检测pFSH-Fc-1和pFSH-Fc-2蛋白的抗原免疫性。分别选取40头初产大白母猪，分为4组：pFSH-Fc-1组、pFSH-Fc-2组、hFSH-hFc和pFSH-hFc组。分别在每组供体猪耳后颈部肌肉注射1000IU药品，每三天给药一次，共给药5周(给药13次)，第一次给药记为Day0。分别在以下时间采集血液：首次药前(Day-2)，第三次药前(Day6)，第五次药前(Day12)，第六次至末次每次药前(Day15,18,21,24,27,30,33,36)，末次给药后第三天(Day39)。离心血液，收集血清，分别相应在pFSH-Fc-1、pFSH-Fc-2、hFSH-hFc或pFSH-hFc包被的酶标板中检测血清中ADA的OD490nm值。阳性对照样品分别用100％混合猪血清稀释的抗pFSH-Fc-1、抗pFSH-Fc-2、抗 hFSH-hFc或抗pFSH-hFc的兔单抗配制；阴性对照(N)样品用注射同量PBS缓冲液的初产大白母猪血清配制。用阈值(SCP)作为区分检测样品为阳性或阴性的判定数值，通过运用JMP
Figure PCTCN2017108854-appb-000006
统计分析计算SCP为1.15，即SCP≥1.15判定为阳性，样品含有ADA；否则为阴性，样品不含ADA。
结果如表6所示，pFSH-Fc-1组和pFSH-Fc-2组供体猪样品SCP均小于1.15，未检测到ADA，说明注射1000IU pFSH-Fc-1或pFSH-Fc-2大白母猪不会产生抗药性抗体。hFSH-hFc组中，1、2、4、7、9号母猪Day27及其后的血清样品SCP大于1.15(首次检出在Day27，末次检出在Day39)，其他母猪Day30及其后的血清样品SCP大于1.15(首次检出在Day30，末次检出在Day39)，说明hFSH-hFc组所有母猪均检测到ADA，阳性率为100％。pFSH-hFc组中，2、3、6、7、9号母猪Day30及其后的血清样品SCP大于1.15(首次检出在Day30，末次检出在Day39)，1、5、8号母猪Day36及其后的血清样品SCP大于1.15(首次检出在Day36，末次检出在Day39)，4号和10号母猪的所有样品SCP小于1.15，说明pFSH-hFc组有8头母猪检测到ADA，阳性率为80％。以上结果表明注射1000IU hFSH-hFc或pFSH-hFc大白母猪产生抗药性抗体。
表6 pFSH-Fc-1、pFSH-Fc-2、hFSH-hFc和pFSH-hFc
对大白母猪的抗原免疫性检测
Figure PCTCN2017108854-appb-000007
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本 领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供两种长效重组猪FSH融合蛋白，包括pFSH-Fc-1和pFSH-Fc-2，其α亚基/β亚基直接或间接通过连接元件融合至Fc片段上，β亚基/α亚基通过范德华力或连接元件与α亚基/β亚基结合。所述猪FSH融合蛋白可基于基因工程技术，利用真核表达系统制备得到。本发明提供的两种猪FSH融合蛋白药效好，比天然猪FSH半衰期长；又不会对动物产生不良效应，对母猪不存在免疫原性，不产生抗药性抗体，可替代孕马血清促性腺激素(PMSG)在动物繁殖生产中的使用，可以有效地促进动物尤其经济动物的繁殖率，具有较好的经济价值和应用前景。

Claims (10)

  1. 长效重组猪FSH融合蛋白，其特征在于，包括融合蛋白pFSH-Fc-1和pFSH-Fc-2；
    所述猪FSH融合蛋白pFSH-Fc-1的α亚基直接或间接通过连接元件融合至Fc片段上，β亚基通过范德华力或连接元件与α亚基结合；
    所述猪FSH融合蛋白pFSH-Fc-2的β亚基直接或间接通过连接元件融合至Fc片段上，α亚基通过范德华力或连接元件与β亚基结合。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述猪FSH融合蛋白包括两条肽链，符合以下方程：(pFSHβ:pFSHα-L-Fc)2或(pFSHα:pFSHβ-L-Fc)2，其中pFSHβ是指猪FSH去除信号肽的β亚基；冒号代表猪FSHβ亚基和α亚基以范德华力连接的关系；pFSHα是指猪FSH去除信号肽的α亚基；L代表pFSHα或pFSHβ亚基与Fc片段的连接关系；Fc是指免疫球蛋白的Fc片段或其突变体；括号外下标2代表所述猪FSH融合蛋白为二价同二聚体。
  3. 根据权利要求2所述的融合蛋白，其特征在于，所述pFSHα的氨基酸序列如SEQ ID NO:1所示，或者与SEQ ID NO:1同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白；所述pFSHβ的氨基酸序列如SEQ ID NO:3所示，或者与SEQ ID NO:3同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
  4. 根据权利要求2所述的融合蛋白，其特征在于，所述Fc包含免疫球蛋白铰链区以及CH2和CH3区；
    所述免疫球蛋白来自人、猪、牛、羊、马或狗；
    所述Fc突变体是指Fc片段上含有一个或几个突变氨基酸位点的Fc变体，包括人IgG2Fc变体，其含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述Fc来自猪的免疫球蛋白，即pFc，所述pFc的氨基酸序列如SEQ ID NO:5所示，或者 与SEQ ID NO:5同源性在80％以上的，且具有同等功能的氨基酸序列构成的蛋白。
  6. 根据权利要求2所述的融合蛋白，其特征在于，所述pFSHα或pFSHβ亚基与Fc的连接关系为直接拼接连接或通过linker连接，优选通过linker连接；其中所述linker是由2-20个柔性氨基酸组成的柔性多肽，所述柔性氨基酸选自Gly、Ser、Ala和Thr中的至少一种；
    优选地，所述linker为(Gly-Gly-Gly-Gly-Ser)n，其中n为2-5之间的整数，更优选n为3。
  7. 根据权利要求2所述的融合蛋白，其特征在于，所述pFSHα-L-Fc为：i)由SEQ ID No.6所示的氨基酸序列构成的蛋白；或
    ii)SEQ ID No.6所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白；或
    iii)与SEQ ID No.6所示氨基酸序列同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白；
    所述pFSHβ-L-Fc为：iv)由SEQ ID No.8所示的氨基酸序列构成的蛋白；或
    v)SEQ ID No.8所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由iv)衍生的蛋白；或
    vi)与SEQ ID No.8所示氨基酸序列同源性在90％以上的，且具有同等功能的氨基酸序列构成的蛋白。
  8. 表达盒、表达载体、克隆载体、工程菌或转基因细胞系，其特征在于，其包括包含编码如权利要求1-7任一项所述融合蛋白的核酸。
  9. 权利要求1-7任一项所述融合蛋白的制备方法，其特征在于，人工合成pFSHα、pFSHβ、pFSHα-L-Fc和pFSHβ-L-Fc的编码基因，并进行密码子优化，优化后的基因分别克隆到真核表达载体中；将pFSHα重组载体和pFSHβ-L-Fc重组载体同时转化真核细胞，并在真核细胞中表达，分离纯化目标蛋白；将pFSHβ重组载体和pFSHα-L-Fc重组载体同时转化 真核细胞，并在真核细胞中表达，分离纯化目标蛋白；
    优选地，所述真核表达载体为pcDNA3.1，所述真核细胞包括293、CHO细胞。
  10. 权利要求1-7任一项所述融合蛋白在制备用于促进动物繁殖，包括同期发情、超数排卵，以及治疗动物生殖相关疾病的药物中的应用；其中，所述动物包括猪、牛、羊、马或狗；优选猪。
PCT/CN2017/108854 2017-09-15 2017-11-01 长效重组猪fsh融合蛋白及其制备方法与应用 WO2019051954A1 (zh)

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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108531508B (zh) * 2018-03-20 2021-07-16 西北民族大学 一种绵羊促卵泡激素基因重组表达制备方法
CN108676096B (zh) * 2018-05-22 2022-03-29 北京伟杰信生物科技有限公司 重组猪fsh-ctp融合蛋白及其制备方法与应用
CN108503705A (zh) * 2018-07-10 2018-09-07 北京伟杰信生物科技有限公司 一种重组绒促性素（rhCG）的纯化方法
CN109336981A (zh) * 2018-09-19 2019-02-15 北京伟杰信生物科技有限公司 一种重组马绒毛膜促性腺激素融合蛋白及其制备方法和其应用
CN109942717A (zh) * 2019-04-24 2019-06-28 上海延立药业有限公司 一种长效重组人促卵泡激素及其制备方法和应用
CN111848814B (zh) * 2020-06-21 2022-12-06 北京伟杰信生物科技有限公司 一种重组猪il-29融合蛋白及其制备方法与应用
CN114957442B (zh) * 2022-04-05 2023-07-14 中国水产科学研究院黄海水产研究所 一种具有促进大菱鲆卵巢发育功能的重组蛋白

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050186662A1 (en) * 2004-01-28 2005-08-25 Syntonix Pharmaceuticals, Inc. Heterodimeric follicle stimulating hormone-Fc (FSH-Fc) fusion proteins for the treatment of infertility
CN103509121A (zh) * 2012-11-22 2014-01-15 苏州康宁杰瑞生物科技有限公司 一种fsh融合蛋白及其制备方法和用途
CN103539860A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组人促卵泡激素融合蛋白
CN103539862A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组促卵泡激素及其应用
CN103554268A (zh) * 2013-11-01 2014-02-05 广州诺新生物技术有限公司 长效重组促卵泡激素及其应用
CN106496331A (zh) * 2016-11-08 2017-03-15 北京启康兴业生物医药科技有限公司 一种新型FSH‑Fc融合蛋白及其制备方法和用途

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030166525A1 (en) * 1998-07-23 2003-09-04 Hoffmann James Arthur FSH Formulation
CN2924049Y (zh) * 2006-04-06 2007-07-18 重庆宗申技术开发研究有限公司 发动机火花塞异步点火装置
JP5563572B2 (ja) * 2008-07-23 2014-07-30 ハンミ サイエンス カンパニー リミテッド 三末端官能基を有する非ペプチド性重合体を用いた生理活性ポリペプチド薬物結合体
US8784834B2 (en) * 2012-07-24 2014-07-22 Sbc Virbac Biotech Co., Ltd. Recombinant fusion interferon for animals
US8956623B2 (en) * 2012-07-24 2015-02-17 Sbc Virbac Limited Recombinant fusion interferon for animals
AU2014296215A1 (en) * 2013-07-31 2016-02-11 Amgen Inc. Stabilization of Fc-containing polypeptides
CN103554269B (zh) * 2013-11-01 2015-02-11 广州联康生物科技有限公司 重组猪促卵泡激素融合蛋白

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050186662A1 (en) * 2004-01-28 2005-08-25 Syntonix Pharmaceuticals, Inc. Heterodimeric follicle stimulating hormone-Fc (FSH-Fc) fusion proteins for the treatment of infertility
CN103509121A (zh) * 2012-11-22 2014-01-15 苏州康宁杰瑞生物科技有限公司 一种fsh融合蛋白及其制备方法和用途
CN103539860A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组人促卵泡激素融合蛋白
CN103539862A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组促卵泡激素及其应用
CN103554268A (zh) * 2013-11-01 2014-02-05 广州诺新生物技术有限公司 长效重组促卵泡激素及其应用
CN106496331A (zh) * 2016-11-08 2017-03-15 北京启康兴业生物医药科技有限公司 一种新型FSH‑Fc融合蛋白及其制备方法和用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NM-213875.1
"Pharmacopoeia of China", article "Serum Gonadotropin Bioassay"
SAMBROOK JRUSSELL DW, MOLECULAR CLONING: A LABORATORY MANUAL, 2001

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