Recombinant follicle-stimulating hormone and Fc fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a recombinant follicle-stimulating hormone and Fc fusion protein as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The fusion protein comprises two different peptide chains, the two peptide chains are folded into a stable heterodimer through non-covalent binding between a follicle stimulating hormone alpha subunit and a follicle stimulating hormone beta subunit and interaction between Fc-CH3 'A and Fc-CH3' B, and the Fc-CH3 'A chain and the Fc-CH3' B chain are mutant chains obtained after amino acid mutation is conducted on a wild type IgG1Fc chain. According to the invention, the CH3 region of the Fc chain is modified, so that the assembling efficiency and stability of the heteroduplex of the recombinant fusion protein are remarkably improved. The recombinant fusion protein provided by the invention is high in biological activity and long in half-life period, and can promote superovulation of animals, estrus synchronization of the animals and fetal birth. When the hCG is combined with the hCG, the effect of the natural PMSG can be achieved, and the hCG can be used as a substitute of the natural PMSG.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant follicle-stimulating hormone and Fc fusion protein, and a preparation method and application thereof. Background Art

[0002] Gonadotropins (Gn) are glycoprotein hormones that regulate gonadal development in vertebrates, promoting the production and secretion of sex hormones. They are widely used in assisted reproductive technologies (ART) in livestock to stimulate ovarian function. Pregnant mare serum gonadotropin (PMSG) is the most widely used hormone, promoting follicular development, oocyte maturation, and ovulation. In non-equine species, PMSG exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like biological activities. However, repeated use of PMSG may lead to ovarian hyperstimulation and induce a series of immune responses, potentially impacting the health of reproductive animals. PMSG is typically obtained from the blood of pregnant horses between 45 and 90 days of gestation, which raises various animal welfare concerns. The PMSG content in pregnant mare serum is extremely low, making extraction difficult and the purification process complex, resulting in poor batch-to-batch stability. Extraction of PMSG from pregnant mare serum generates large amounts of waste serum, contributing to environmental pollution. Consequently, there is a growing demand in livestock production for PMSG alternatives that can effectively improve livestock reproductive performance.

[0003] The combined use of follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) is a viable alternative to PMSG. However, due to the short half-life of natural FSH in the bloodstream (approximately 5 hours), multiple injections are required to stimulate follicle maturation and ovulation. However, repeated injections can cause severe stress in animals. Furthermore, animal-derived natural FSH carries the risk of disease transmission. Furthermore, the high cost of FSH preparation and purification increases livestock production costs, significantly limiting its widespread application in animal husbandry.

[0004] Recombinant in vitro expression of FSH is a technological alternative to natural extraction. Large-scale recombinant FSH expression requires addressing four key challenges: first, improving the efficiency of recombinant FSH expression and increasing expression levels; second, extending FSH's blood half-life, reducing the number of injections and, consequently, reducing animal stress; third, improving the assembly efficiency of FSH α and β chain heterodimers; and fourth, facilitating the separation and purification of the end product.

[0005] At present, people have studied a variety of strategies to extend the half-life of porcine follicle-stimulating hormone (FSH) derived from natural pituitary glands. For example, patent document CN201710833452.1 discloses directly or indirectly linking porcine FSH α chain and β chain to Fc fragments for fusion expression, thereby extending its blood half-life and improving its biological activity. The Fc fragment of immunoglobulin IgG1 is fused with FSH α chain or β chain for expression. Since the Fc fragment can interact with receptors such as FcRn in the body, FcRn can protect the Fc fusion protein from degradation. This type of receptor can bind to the Fc fragment in the fusion protein molecule, recover the fusion protein from the endosome and re-release it to the extracellular space, avoiding lysosomal degradation of the fusion protein and thus extending the half-life of FSH in the body. The chromatographic medium coupled with Protein A has a high affinity for the Fc fragment. Therefore, the Protein A chromatographic medium can be used to efficiently separate and purify FSH expressed by Fc fusion, which greatly simplifies the purification preparation process, improves product purification efficiency, and reduces production costs.

[0006] Natural FSH is a heterodimeric protein composed of an α chain and a β chain. During recombinant expression, co-expression of these two chains may form some heterodimers, but homodimers are much more numerous. Therefore, the correct assembly of FSH heterodimers is crucial. In achieving heterodimer assembly in bispecific antibodies, strategies such as knobs into holes, ionic interactions, electrostatic steering, coiled-coil, leucine zipper, and helix-turn-helix motifs within the antibody Fc fragment can significantly improve the pairing accuracy of the two Fc fragments, thereby increasing the assembly efficiency of heterodimer molecules.

[0007] In the design of the Knobs into Holes structure, site-directed mutations S354C and T366W were performed on the CH3 region of one Fc chain, and site-directed mutations Y349C, T366S, L368A, and Y407V were performed on the CH3 region of the other Fc chain. Except for the disulfide bond formed between S354C of the CH3 of one chain and Y349C of the CH3 of the other chain, the assembly efficiency of the two chains was improved by the "knobs-into-holes" structure between the remaining amino acids. However, the interaction forces between the amino acid groups (such as electrostatic effects, hydrophobic effects, etc.) were not considered, which affected the assembly efficiency of the target protein FSHα and β heterologous duplex.

[0008] Electrostatic steering is a way of regulating the formation of protein dimers by utilizing electrostatic interactions between oppositely charged amino acids and protein domains. In the design of electrostatic steering interaction structures, site-directed mutations are performed on the amino acids of the two chains, mainly considering that the amino acids with a close spatial distance on the two chains mutate into positively charged amino acids or negatively charged amino acids, and utilizing their electrostatic attraction to improve the assembly efficiency of heteroduplexes. For example, patent document CN115975043A discloses the use of a lock mode or an electrostatic steering mode for transformation to achieve efficient assembly of heterodimers of Fc and follicle-stimulating hormone α or β chain fusion proteins. However, since the CH3 region of the wild-type Fc fragment has a hydrophobic interaction region and two electrostatic interaction regions, if the transformation without full consideration of its wild-type structure is carried out, its interaction force may be increased, but it may also lead to a decrease in its stability, affecting the assembly efficiency of the target protein FSH α and β chains, and even causing a decrease in its activity.

[0009] Therefore, how to fully consider the two forces of hydrophobic interaction and electrostatic interaction to achieve the modification of the Fc CH3 region and thereby improve the assembly efficiency of the Fc-FSH fusion protein heteroduplex is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0010] The purpose of the present invention is to provide a recombinant follicle-stimulating hormone and Fc fusion protein, which improves the assembly efficiency of the Fc-FSH fusion protein heterologous duplex by modifying the Fc CH3 region, thereby improving its biological activity.

[0011] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a recombinant follicle-stimulating hormone and Fc fusion protein, comprising two different peptide chains, one of which is a peptide chain formed by the follicle-stimulating hormone α subunit directly or indirectly linked to the Fc-CH3'A chain or Fc-CH3'B chain via a linking element, and the other is a peptide chain formed by the follicle-stimulating hormone β subunit directly or indirectly linked to the Fc-CH3'B chain or Fc-CH3'A chain via a linking element; the two peptide chains fold into a stable heterodimer through non-covalent binding between the follicle-stimulating hormone α subunit and the β subunit and interaction between Fc-CH3'A and Fc-CH3'B; Compared to the wild-type IgG1 Fc chain, the Fc-CH3'A chain has, according to EU numbering, mutations of Q347E or Q347D, K360E or K360D, T366L or T366V, K370D or K370E, K392D or K392E, F405A or F405V, Y407V or Y407L, K409D or K409E; and the Fc-CH3'B chain has, according to EU numbering, mutations of Q347K or Q347R, E356K or E356R, E357K or E357R, T366L or T366V, D399K or D399R, F405A or F405V, Y407V or Y407L.

[0012] The Fc-CH3'A and Fc-CH3'B chains are mutant chains resulting from amino acid mutations in wild-type IgG1 Fc chains. The CH3 domains of the two chains of the wild-type IgG1 Fc fragment have three interaction regions: two electrostatic interaction regions and one hydrophobic interaction region. This invention, taking into account these three interactions, conducts site-directed mutagenesis of the two chains. By subjecting the amino acids in the two electrostatic interaction regions and their surrounding areas to site-directed mutagenesis, the two electrostatic interaction regions of one chain (CH3A) are negatively charged, while the two electrostatic interaction regions of the other chain (CH3B) are positively charged. This minimizes the formation of CH3A:CH3A and CH3B:CH3B homodimers and significantly improves the correct pairing rate of CH3A:CH3B heterodimers. Furthermore, site-directed mutagenesis of the amino acids in and around the hydrophobic interaction region enhances the hydrophobic interaction within the hydrophobic interaction region, further improving the assembly efficiency of the heterodimers.

[0013] Specifically, the mutation scheme for CH3A is as follows: in electrostatic interaction region 1, according to EU numbering, glutamine (Q) at position 347 is mutated to glutamic acid (E) or aspartic acid (D), and lysine (K) at position 370 is mutated to aspartic acid (D) or glutamic acid (E); in electrostatic interaction region 2, according to EU numbering, lysine (K) at positions 360, 392, and 409 are all mutated to glutamic acid (E) or aspartic acid (D); in the hydrophobic interaction region, according to EU numbering, threonine (T) at position 366 is mutated to leucine (L) or valine (V), phenylalanine (F) at position 405 is mutated to alanine (A) or valine (V), and tyrosine (Y) at position 407 is mutated to valine (V) or leucine (L). After mutation, the Fc-CH3'A chain is obtained.

[0014] Mutation scheme for CH3B: In electrostatic interaction region 1, according to EU numbering, glutamine (Q) at position 347 was mutated to lysine (K) or arginine (R), and aspartic acid (D) at position 399 was mutated to lysine (K) or arginine (R); in electrostatic interaction region 2, according to EU numbering, glutamic acid (E) at positions 356 and 357 were mutated to lysine (K) or arginine (R); in the hydrophobic interaction region, threonine (T) at position 366 was mutated to leucine (L) or valine (V), phenylalanine (F) at position 405 was mutated to alanine (A) or valine (V), and tyrosine (Y) at position 407 was mutated to valine (V) or leucine (L), according to EU numbering. These mutations resulted in the Fc-CH3'B chain.

[0015] Research in this paper demonstrates that the aforementioned amino acid mutations, through the synergistic effect of electrostatic steering and hydrophobic interactions, can significantly improve the assembly efficiency of the heteroduplex fusion protein, thereby enhancing its biological activity. The two peptide chains formed by the fusion of the Fc-CH3'A chain and Fc-CH3'B chain with the α and β subunits of follicle-stimulating hormone can form a properly assembled, stable heterodimer of the recombinant follicle-stimulating hormone and Fc fusion protein.

[0016] Preferably, compared to the wild-type IgG1 Fc chain, the Fc-CH3'A chain has mutations Q347E, K360D, T366V, K370D, K392E, F405A, Y407V, and K409E according to EU numbering; and the Fc-CH3'B chain has mutations Q347K, E356K, E357R, T366V, D399K, F405A, and Y407V according to EU numbering.

[0017] As a specific embodiment of the present invention, the amino acid sequence of the Fc-CH3'A chain is shown as SEQ ID NO.1, and the amino acid sequence of the Fc-CH3'B chain is shown as SEQ ID NO.2.

[0018] In the present invention, the Fc-CH3'A chain and the Fc-CH3'B chain can be covalently linked to the amino terminus (N-terminus) or carboxyl terminus (C-terminus) of the follicle-stimulating hormone α subunit (FSHα chain) or the follicle-stimulating hormone β subunit (FSHβ chain).

[0019] Specifically, the two peptide chains of the fusion protein are: the N-terminus of the follicle-stimulating hormone α subunit is connected to the C-terminus of the Fc-CH3'A chain to form Fc-CH3'A-FSHα, and the N-terminus of the follicle-stimulating hormone β subunit is connected to the C-terminus of the Fc-CH3'B chain to form Fc-CH3'B-FSHβ; Alternatively, the N-terminus of the follicle-stimulating hormone β subunit is linked to the C-terminus of the Fc-CH3'A chain to form Fc-CH3'A-FSHβ and the N-terminus of the follicle-stimulating hormone α subunit is linked to the C-terminus of the Fc-CH3'B chain to form Fc-CH3'B-FSHα; Alternatively, the C-terminus of the follicle-stimulating hormone α subunit is linked to the N-terminus of the Fc-CH3'A chain to form FSHα-Fc-CH3'A and the C-terminus of the follicle-stimulating hormone β subunit is linked to the N-terminus of the Fc-CH3'B chain to form FSHβ-Fc-CH3'B; Alternatively, the C-terminus of the FSH β subunit is linked to the N-terminus of the Fc-CH3'A chain to form FSHβ-Fc-CH3'A and the C-terminus of the FSH α subunit is linked to the N-terminus of the Fc-CH3'B chain to form FSHα-Fc-CH3'B.

[0020] In the present invention, the Fc-CH3'A chain and the Fc-CH3'B chain can be directly connected to the FSH α and β chains, or can be indirectly connected through a linking element. The linking element can be, but is not limited to, a flexible polypeptide. Connection through a flexible polypeptide can reduce the impact on the FSH subunit structure. Preferably, the flexible polypeptide is (Gly-Gly-Gly-Gly-Ser) n , n is 1 to 10. More preferably, n is 4.

[0021] In the present invention, the follicle-stimulating hormone may be, but is not limited to, porcine follicle-stimulating hormone.

[0022] Specifically, the amino acid sequence of the porcine follicle-stimulating hormone α subunit is shown in SEQ ID NO.3, and the amino acid sequence of the porcine follicle-stimulating hormone β subunit is shown in SEQ ID NO.4.

[0023] As a specific embodiment of the present invention, the two peptide chains of the fusion protein are: Fc-CH3'A-FSHα with an amino acid sequence as shown in SEQ ID NO.5 and Fc-CH3'B-FSHβ with an amino acid sequence as shown in SEQ ID NO.6; or Fc-CH3'A-FSHβ with an amino acid sequence as shown in SEQ ID NO.7 and Fc-CH3'B-FSHα with an amino acid sequence as shown in SEQ ID NO.8; or FSHα-Fc-CH3'A with an amino acid sequence as shown in SEQ ID NO.9 and FSHβ-Fc-CH3'B with an amino acid sequence as shown in SEQ ID NO.10; or FSHβ-Fc-CH3'A with an amino acid sequence as shown in SEQ ID NO.11 and FSHα-Fc-CH3'B with an amino acid sequence as shown in SEQ ID NO.12.

[0024] The recombinant follicle-stimulating hormone and Fc fusion protein provided by the present invention has high activity and a long half-life. A single administration of a small dose can promote follicle development in mammals, promote induced fertilization and ripening of animals, and improve the efficiency of artificial reproduction.

[0025] Another object of the present invention is to provide a method for preparing the recombinant follicle-stimulating hormone and Fc fusion protein, the method comprising the following steps: (1) Using the pCDNA3.4-TOPO vector as the original vector, a DNA sequence containing a multiple cloning site, a bovine growth hormone polyA sequence, a CMV enhancer sequence, and a CMV promoter sequence was inserted into the TA cloning site. The nucleotide sequence of the DNA sequence is shown in SEQ ID NO. 13, thereby constructing a dual expression vector pCDNA3.4Double containing two CMV promoter sequences; (2) The coding sequences of the two peptide chains were respectively inserted into the multiple cloning sites downstream of the two CMV promoters of the dual expression vector to construct a heterodimer expression plasmid; (3) The heterodimer expression plasmid is transfected into CHO S cells, the culture fluid is collected after cell culture, and the recombinant follicle-stimulating hormone and Fc fusion protein is obtained by separation and purification.

[0026] The present invention constructs a dual expression vector containing two expression modules, inserts the coding sequences of the two peptide chains into the downstream of two CMV promoters respectively, and expresses two different peptide chains simultaneously through single vector dual expression, which helps to improve the recombination assembly efficiency.

[0027] Preferably, the nucleotide sequence encoding the Fc-CH3'A chain in the coding sequences of the two peptide chains is shown as SEQ ID NO.14; the nucleotide sequence encoding the Fc-CH3'B chain is shown as SEQ ID NO.15.

[0028] Preferably, the nucleotide sequence encoding the α subunit of follicle-stimulating hormone in the coding sequences of the two peptide chains is shown as SEQ ID NO.16; the nucleotide sequence encoding the β subunit of follicle-stimulating hormone is shown as SEQ ID NO.17.

[0029] Preferably, the nucleotide sequence encoding the linking element in the coding sequence of the two peptide chains is GGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGC.

[0030] Another object of the present invention is to provide the use of the recombinant follicle-stimulating hormone and Fc fusion protein in the preparation of drugs for promoting animal superovulation, animal estrus synchronization, and conception and birth.

[0031] The fusion protein provided by the present invention can be applied to animal superovulation, animal estrus synchronization, conception and birthing to promote animal reproduction, or to treat animal infertility, or to induce in vitro maturation and in vitro fertilization of oocytes.

[0032] The animal is a mammal, which may be, but is not limited to, a pig, a cow, a sheep, and the like.

[0033] Preferably, the drug further comprises a pharmaceutically acceptable carrier, which is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and having no toxic side effects on the host or subject.

[0034] The present invention has the following beneficial effects: (1) The present invention fully considers the two forces of hydrophobic interaction and electrostatic interaction to achieve the modification of the CH3 region of the IgG1 Fc chain. By performing site-directed mutagenesis on the hydrophobic interaction regions and the amino acids in the vicinity of the two Fc chains, the hydrophobic interaction force is enhanced; by performing site-directed mutagenesis on the amino acids in the two electrostatic interaction regions and the regions in the vicinity, the electrostatic attraction is enhanced, thereby improving the assembly efficiency of the heterologous double chain of the recombinant follicle-stimulating hormone and Fc fusion protein and the stability of the recombinant fusion protein.

[0035] (2) The present invention provides a dual expression vector pCDNA3.4Double containing two CMV promoter sequences, which can be used to simultaneously express two peptide chains for assembling recombinant follicle-stimulating hormone and Fc fusion protein, with high expression efficiency, which helps to improve the efficiency of recombinant assembly.

[0036] (3) The recombinant follicle-stimulating hormone (FSH)-Fc fusion protein provided by the present invention has high biological activity and a long half-life, and can promote superovulation, estrus synchronization, and conception and farrowing in animals. Combined with hCG, it can achieve the effects of natural PMSG and can be used as a substitute for natural PMSG for estrus synchronization and batch production management in sows. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the interaction between the CH3 regions of the two chains of the IgG1 Fc fragment.

[0038] Figure 2 It is expressed in a fusion format of Fc CH3 and FSHα / β chains.

[0039] Figure 3 Schematic diagram of the pcDNA3.4-TOPO vector map.

[0040] Figure 4 Schematic diagram of the pCDNA3.4Double vector map.

[0041] Figure 5 The SDS-PAGE results of Fc / FSH fusion protein before purification, wherein the first lane is the marker, the second lane is Fc' / FSHαβ, the third lane is Fc' / FSHβα, the fourth lane is FSHαβ / Fc', and the fifth lane is FSHβα / Fc'.

[0042] Figure 6 The SDS-PAGE results after purification of Fc / FSH fusion protein are shown in FIG1 , wherein lane 1 is Marker, lane 2 is Fc' / FSHαβ, lane 3 is Fc' / FSHβα, lane 4 is FSHαβ / Fc', and lane 5 is FSHβα / Fc'.

[0043] Figure 7 This figure shows the effect of Fc / FSH fusion protein on rat ovarian development. From left to right, the injections are Fc' / FSHαβ, Fc' / FSHβα, FSHαβ / Fc', and FSHβα / Fc'. From top to bottom, the injection doses are 20, 40, and 80 iu / mL. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0046] In the present invention, the amino acid sequence is from N-terminus to C-terminus from left to right; the nucleotide sequence is from 5′-terminus to 3′-terminus from left to right.

[0047] Example 1: Site-directed mutagenesis 1. Screening candidate amino acid mutation sites for the interaction between the two chains of the IgG1 Fc fragment Search the crystal structure of IgG1Fc from the PDB database, see PDB code 3AVE for details. Figure 1As can be seen in the figure, the CH3 domains of the two chains of the Fc fragment have three interaction regions: two electrostatic interaction regions and one hydrophobic interaction region. The first electrostatic interaction region is formed by D399, K370, and K439 on the first Fc chain (CH3A) and K409, E357, and D356 on the second chain (CH3B), respectively, forming electrostatic steering interactions. The hydrophobic interaction region is formed by L351, Y407, and T366 on CH3A and T366, Y407, and L351 on CH3B. The second electrostatic interaction region is formed by K409, K392, E356, and E357 on CH3A and D399, K439, and K370 on CH3B.

[0048] The present invention targets these three interaction regions and, while maintaining the original interaction properties, performs site-directed mutagenesis to mutate the electrostatic interaction region of CH3A into negatively charged amino acids and the electrostatic interaction region of CH3B into positively charged amino acids. By replacing amino acids, the hydrophobicity of the amino acids in the hydrophobic region is further enhanced, thereby enhancing the hydrophobic interaction force.

[0049] 2. Site-directed mutagenesis of the Fc CH3 domain Mutation scheme for CH3A (amino acid mutations according to EU numbering): Electrostatic interaction region 1: Q347E or Q347D, K370D or K370E; Electrostatic interaction region 2: K360E or K360D, K392D or K392E, K409D or K409E; Hydrophobic interaction region: T366L or T366V, F405A or F405V, Y407V or Y407L; Mutation scheme for CH3B (amino acid mutations according to EU numbering): Electrostatic interaction region 1: Q347K or Q347R, D399K or D399R; Electrostatic interaction region 2: E356K or E356R, E357K or E357R; Hydrophobic interaction region: T366L or T366V, F405A or F405V, Y407V or Y407L; Through site-specific modification of amino acids, the two electrostatic interaction regions of CH3A are negatively charged, and the two electrostatic interaction regions of CH3B are positively charged, which can minimize the formation of CH3A:CH3A and CH3B:CH3B homodimers; at the same time, the hydrophobic interaction of the hydrophobic interaction regions is enhanced.

[0050] The specific plan is as follows: According to the amino acid sequence of human IgG1 (hIgG1) with accession number P01857 in the UniProt database, the wild-type IgG1 Fc region amino acid sequence was obtained: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0051] The IgG1 Fc was modified using negatively charged amino acids to obtain the negatively charged chain CH3'A(-), the sequence of which is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP E VYTLPPSREEMT D NQVSLTCLV D GFYPSDIAVEWESNGQPENNY E TTPPVLDSDGSFFLYS E LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Compared to the wild-type IgG1 Fc region, the negatively charged chain has Q347E, K360D, K370D, K392E, and K409E mutations according to EU numbering.

[0052] The IgG1 Fc was modified using positively charged amino acids to obtain a positively charged chain CH3'B(+), the sequence of which is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP K VYTLPPSR KR MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Compared to the wild-type IgG1 Fc region, the positive chain has mutations Q347K, E356K, E357R, and D399K according to EU numbering.

[0053] The IgG1 Fc was modified to obtain a chain CH3'F carrying hydrophobic amino acids, the sequence of which is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF A L V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Compared to the wild-type IgG1 Fc region, the hydrophobic region has T366V, F405A, and Y407V mutations according to EU numbering.

[0054] The IgG1 Fc was modified using negatively charged amino acids to obtain a negatively charged CH3'A chain with enhanced hydrophobic region interaction. The sequence is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP E VYTLPPSREEMT D NQVSL V CLV D GFYPSDIAVEWESNGQPENNY E TTPPVLDSDGSF A L V S ELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Compared to the wild-type IgG1 Fc region, the mutant chain has the mutations Q347E, K360D, T366V, K370D, K392E, F405A, Y407V, and K409E according to EU numbering.

[0055] The IgG1 Fc was modified using positively charged amino acids to obtain a chain CH3'B that carries a positive charge and has enhanced hydrophobic region interaction. The sequence is as follows: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP K VYTLPPSR KR MTKNQVSL V CLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSF A L V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Compared to the wild-type IgG1 Fc region, the mutant chain has the mutations Q347K, E356K, E357R, T366V, D399K, F405A, and Y407V according to EU numbering.

[0056] Example 2: Fusion Expression of Fc CH3 with FSH α and β Chains 1. Integration solution The chain after CH3A mutation is called CH3'A, and the chain after CH3B mutation is called CH3'B.

[0057] CH3'A and CH3'B are connected to FSHα and β chains through flexible polypeptides to form fusion protein molecules. Flexible polypeptides are composed of flexible amino acids. The typical flexible polypeptide sequence is (Gly-Gly-Gly-Gly-Ser)n, abbreviated as (G4S). n , where "n" is generally between 1 and 10, especially 3 or 4.

[0058] According to the amino acid sequence of the porcine FSH (pFSH) α subunit with accession number P01219 in the UniProt database, the amino acid sequence of the mature peptide of the pFSH α subunit was obtained: FPDGEFTMQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKS (SEQ ID NO. 3).

[0059] According to the amino acid sequence of the porcine FSH (pFSH) β subunit with accession number P01228 in the UniProt database, the amino acid sequence of the pFSH β subunit mature peptide was obtained: CELTNITITVEKEECNFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFSEMKE (SEQ ID NO. 4).

[0060] CH3'A and CH3'B can be covalently linked to the amino or carboxyl termini of FSHα and β chains via a flexible polypeptide (G4S)4, such as Figure 2 .

[0061] The specific plan is as follows: The sequence of the fusion protein CH3'A / FSHα is shown in SEQ ID NO. 5; the sequence of the fusion protein CH3'B / FSHβ is shown in SEQ ID NO. 6.

[0062] The sequence of the fusion protein CH3'A / FSHβ is shown in SEQ ID NO.7; the sequence of the fusion protein CH3'B / FSHα is shown in SEQ ID NO.8.

[0063] The sequence of the fusion protein FSHα / CH3'A is shown in SEQ ID NO.9; the sequence of the fusion protein FSHβ / CH3'B is shown in SEQ ID NO.10.

[0064] The sequence of the fusion protein FSHβ / CH3'A is shown in SEQ ID NO.11; the sequence of the fusion protein FSHα / CH3'B is shown in SEQ ID NO.12.

[0065] 2. Construction of heterodimer expression plasmid The pCDNA3.4-TOPO vector was used as the original vector (see vector spectrum for details). Figure 3), and a 1944 bp sequence was inserted into the TA cloning site of the original vector, as shown in SEQ ID NO.13. This sequence contains a multiple cloning site (MCS1), the sequence of which is shown at positions 1 to 40 of the sequence shown in SEQ ID NO.13; a bovine growth hormone (bGH) polyA sequence, the sequence of which is shown at positions 50 to 274 of the sequence shown in SEQ ID NO.13; a CMV enhancer sequence, the sequence of which is shown at positions 320 to 699 of the sequence shown in SEQ ID NO.13; and a CMV promoter sequence, the sequence of which is shown at positions 700 to 903 of the sequence shown in SEQ ID NO.13. By inserting the above sequences, a dual expression vector pCDNA3.4Double containing two CMV promoter sequences was constructed (see vector map for details). Figure 4 ).

[0066] Using the above vector, the coding sequences for the fusion protein CH3'A / FSHα and the fusion protein CH3'B / FSHβ were inserted downstream of CMV1 and CMV2, respectively, to construct a heterodimer expression plasmid. This vector enables dual expression from a single vector. This vector simultaneously expresses both the α and β chain fusion proteins, achieving high expression and recombinant assembly efficiencies.

[0067] The methods for constructing expression plasmids for the fusion proteins CH3'A / FSHβ and CH3'B / FSHα, the fusion proteins FSHα / CH3'A and FSHβ / CH3'B, and the fusion proteins FSHβ / CH3'A and FSHα / CH3'B were the same as above. Heterodimer expression plasmids without mutation, with electrostatic reorientation mutation alone, or with hydrophobic mutation alone were also constructed as controls.

[0068] The whole gene synthesis in the above method was commissioned to GenScript Biotech Co., Ltd. The nucleotide sequence encoding the CH3'A chain in the fusion protein coding sequence is shown in SEQ ID NO.14; the nucleotide sequence encoding the CH3'B chain is shown in SEQ ID NO.15; the nucleotide sequence encoding the FSH α chain is shown in SEQ ID NO.16; the nucleotide sequence encoding the FSH β chain is shown in SEQ ID NO.17; and the nucleotide sequence encoding the flexible polypeptide (G4S)4 is GGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGC.

[0069] 3. Heterodimeric protein expression CHO S cells were subcultured and proliferated until the cell density reached approximately 4 × 10 6 ~6×10 6Live cells / mL. One day before transfection, CHOS cells were split into vials to a density of 3×10 6 ~4×10 6 Viable cells / mL. Determine the viable cell density and cell viability. The cell density should reach approximately 7×10 6 ~10×10 6 Dilute the cells to 6 × 10 viable cells / mL using fresh expression medium pre-warmed to 37°C. 6 viable cells / mL. Prepare ExpiFectamine™ CHO / plasmid DNA complex. TM Incubate the CHO / plasmid DNA complex at room temperature for 1-5 minutes, then slowly transfer the mixture to a shake flask. Incubate the transfected cells in a 37°C, 8% CO2 incubator with shaking. One day after transfection, add a volume of feed medium to the shake flask, mixing gently while adding. Return the shake flask to a 37°C, 5% CO2 incubator and incubate with shaking. Five days after transfection, add feed medium a second time and immediately return the shake flask to a 32°C incubator and incubate with shaking. 12-14 days after transfection, collect the culture medium by centrifugation for purification of the recombinant protein.

[0070] 4. Isolation and purification of heterodimeric proteins CHO S cells and cell debris were removed by centrifugation, and the supernatant was loaded onto a Pierce Protein A column equilibrated with phosphate buffer. The column was washed with 10 column volumes of phosphate buffer to remove specifically bound proteins and other components. The target protein was eluted with 100 mM glycine-HCl buffer (pH 3.5). The target protein solution was neutralized with 1 M Tris-HCl buffer (pH 9.0). The neutralized target protein solution was loaded onto a CaptoMMC column equilibrated with 20 mM phosphate buffer (pH 7.0). The column was first washed with the phosphate buffer and then eluted with a gradient of 20 mM PBS-0.5 M NaCl (pH 6.0) to collect the purified protein. A 10 μg sample was mixed with 6× loading buffer (6:1) and subjected to SDS-PAGE electrophoresis on an 8-16% precast gel.

[0071] The results are as follows Figure 5 and Figure 6As shown, the first lane is a marker, the second lane is Fc' / FSHαβ (CH3'A-(G4S)4-FSHα:CH3'B-(G4S)4-FSHβ), the third lane is Fc' / FSHβα (CH3'A-(G4S)4-FSHβ:CH3'B-(G4S)4-FSHα), the fourth lane is FSHαβ / Fc' (FSHα-(G4S)4-CH3'A:FSHβ-(G4S)4-CH3'B), and the fifth lane is FSHβα / Fc' (FSHβ-(G4S)4-CH3'A:FSHα-(G4S)4-CH3'B).

[0072] Example 3: Biological activity of Fc / FSH fusion protein The biological activity of the FSH fusion protein was determined using the rat ovarian weight gain method, referring to the hemoglobin bioassay method in the "Chinese Veterinary Drug Quality Standards." The test samples prepared in Example 2 were prepared, along with hemoglobin standards, at three concentration gradients: 80, 40, and 20 iu / mL, based on the different estimated values ​​obtained from the preliminary activity test.

[0073] Female rats, 20 to 21 days old and weighing approximately 50 g, with a variance of no more than 10 g, from the same source were divided according to body weight into 12 groups of 8 rats each. 0.5 mL of standard or test solution at various concentrations was injected subcutaneously. Six days after injection, the rats were sacrificed, weighed, and dissected. The ovaries were removed, the attached tissues and oviducts were stripped, and the surface fluid was aspirated and weighed directly. The ovary weight was converted to ovary weight per 100 g body weight for calculation of biological activity. The results are shown in Table 1.

[0074] Table 1. Biological activities of Fc / FSH fusion proteins Note: Fc(E) indicates that Fc is subjected to electrostatic shift mutation only; Fc(Hi) indicates that Fc is subjected to hydrophobic mutation only. The mutation method is shown in Example 1.

[0075] As shown in Table 1, compared with the unmutated wild-type Fc chain fusion protein, all mutations, whether electrostatic redirection, hydrophobicity, or a combination of both, significantly increased FSH activity. Compared with the control group using either electrostatic redirection or hydrophobicity mutation alone, the combined electrostatic redirection and hydrophobicity mutation significantly enhanced porcine follicle-stimulating hormone (FSH) activity. Electrostatic redirection mutations in the Fc chain, denoted by Fc(E), involve mutating specific amino acids in the two Fc chains to positively or negatively charged amino acids, respectively. These mutations utilize the attractive forces between positive and negative charges to enhance the assembly efficiency of the heteroduplex. The data in the table show that regardless of whether Fc(E) is located upstream or downstream of the fusion protein, the recombinant FSH activity is lower than that achieved with the combined mutation. Hydrophobicity mutations in the Fc chain, denoted by Fc(Hi), involve mutating specific amino acids in the two Fc chains to hydrophobic amino acids. These mutations utilize hydrophobic interactions between hydrophobic amino acids to enhance the assembly efficiency of the heteroduplex. The data in the table show that regardless of whether Fc(Hi) is located upstream or downstream of the fusion protein, the activity of the recombinant FSH is lower than that of the combined mutation group. This suggests that electrostatic steering and hydrophobic interactions work synergistically to improve the assembly efficiency of the FSH heteroduplex, thereby enhancing biological activity.

[0076] Figure 4 The figure shows the weight gain of mouse ovaries after injection of 20, 40, and 80 iu / mL of Fc'-FSHAB, Fc'-FSHBA, FSHAB-Fc', and FSHBA-Fc', respectively. As can be seen from the figure, with increasing doses, the weight of mouse ovaries increased significantly, suggesting that it has obvious activity in promoting ovarian maturation.

[0077] Example 4: Pharmacokinetic study of Fc / FSH fusion protein Male rats weighing approximately 200 g were given a single subcutaneous injection of the fusion protein at a dose of 400 iu / kg body weight. Blood (0.5 mL) was collected from the orbital cavity at 1, 2, 4, 8, 12, 24, 2, 3, 4, 5, 7, 9, 11, and 14 days after the injection. Serum was collected and FSH concentrations at each blood sampling point were measured simultaneously using an FSH ELISA kit. Each sample was analyzed in triplicate. The half-life of the fusion protein was calculated using WinNonlin software. The results are shown in Table 2.

[0078] Table 2. Half-life of Fc / FSH fusion proteins As shown in the table above, the half-life of recombinant proteins with Fc located at the N-terminus of the FSH α / β chains is significantly longer than that with Fc located at the C-terminus, suggesting that fusion proteins with Fc located at the N-terminus are more stable. Compared with unmutated Fc fusion proteins, both electrostatic shifts and hydrophobicity mutations can extend half-life. Compared with amino acid mutations based solely on electrostatic shifts or hydrophobicity, Fc-FSH fusion proteins with combined Fc mutations exhibit significantly longer half-life. This suggests that simultaneous electrostatic shifts and hydrophobicity mutations can extend the half-life of recombinant porcine FSH.

[0079] Example 5: Fc:FSH fusion protein promotes superovulation in mice Thirty-six ICR female mice, approximately 5 weeks old and of similar weight, were divided equally into 12 groups and injected with 10 iu of Fc:FSH and 2 iu of hCG. Sixteen hours later, the mice were sacrificed by cervical dislocation. The oviducts were transferred to a watch dish containing PBS, the adipose tissue removed, and the mice rinsed thoroughly. The oviducts were then placed on the stage of a stereomicroscope. The ampulla of the oviduct was located under a 20x or 40x magnification lens, the tube was fixed, and the ampulla was opened with a needle. The oocyte clusters were transferred to a solution containing hyaluronidase. Once the oocytes were dispersed into single cells, they were pipetted into an oocyte retrieval cup, washed three times with PBS, and the number of oocytes was counted.

[0080] Table 3. Effects of Fc:FSH fusion protein on superovulation in mice As can be seen from the table above, compared with simple electrostatic redirection or hydrophobic interaction mutations, recombinant FSH in the Fc-FSHBA and FSHBA-Fc schemes can increase the number of mouse ovulations. This suggests that combined electrostatic redirection and hydrophobic interaction mutations in the Fc fragment can help improve the in vivo activity of recombinant FSH and promote mouse ovulation.

[0081] Example 6: Effect of Fc:FSH fusion protein on reproductive performance of gilts Based on the previous mouse ovarian weight gain method and mouse ovulation induction experiments, it can be seen that the synergistic mutation of amino acids based on electrostatic steering and hydrophobic interactions can improve the biological activity and blood half-life of recombinant FSH; considering the scale of sow farming, we selected four combined mutation FSH fusion proteins for sow production experiments.

[0082] Two hundred gilts of similar age and weighing over 110 kg were randomly divided into five groups. They were fed 5 mL (20 mg) of allylprogesterone continuously for 18 days and then moved to stables. Forty-two hours after stopping allylprogesterone, 1000 IU of Fc:FSH fusion protein plus 100 IU of hCG were injected intramuscularly. GnRH was injected 80 hours later, and the first scheduled insemination was performed 24 hours after GnRH injection, and the second scheduled insemination was performed 40 hours after GnRH injection. One month later, sows were examined for pregnancy using ultrasound. After farrowing, the litter size was counted. The results are shown in Table 4.

[0083] Table 4. Effects of Fc:FSH fusion protein on reproductive performance of gilts The results showed that, compared to injections of natural pregnant mare serum gonadotropin (PMSG), all four recombinant FSHs demonstrated superior efficacy in promoting estrus and ovulation in sows. Production performance indicators, such as pregnancy rate and litter size, were also similar to those achieved with natural PMSG. This suggests that the four recombinant porcine follicle-stimulating hormones designed in this invention, when used in combination with hCG for ovulation in sows, can replicate the efficacy of natural PMSG and serve as an alternative to natural PMSG for estrus synchronization and batch production management in sows.

Claims

1. A recombinant follicle-stimulating hormone and Fc fusion protein, characterized in that: The invention comprises two different peptide chains, one of which is a peptide chain formed by the follicle-stimulating hormone α subunit directly or indirectly linked to the Fc-CH3'A chain or Fc-CH3'B chain through a linker element, and the other is a peptide chain formed by the follicle-stimulating hormone β subunit directly or indirectly linked to the Fc-CH3'B chain or Fc-CH3'A chain through a linker element; the two peptide chains fold into a stable heterodimer through non-covalent binding between the follicle-stimulating hormone α subunit and the β subunit and the interaction between Fc-CH3'A and Fc-CH3'B; Compared to the wild-type IgG1 Fc chain, the Fc-CH3'A chain has, according to EU numbering, mutations of Q347E or Q347D, K360E or K360D, T366L or T366V, K370D or K370E, K392D or K392E, F405A or F405V, Y407V or Y407L, K409D or K409E; and the Fc-CH3'B chain has, according to EU numbering, mutations of Q347K or Q347R, E356K or E356R, E357K or E357R, T366L or T366V, D399K or D399R, F405A or F405V, Y407V or Y407L.

2. The recombinant follicle-stimulating hormone and Fc fusion protein according to claim 1, wherein Compared to the wild-type IgG1 Fc chain, the Fc-CH3'A chain has mutations Q347E, K360D, T366V, K370D, K392E, F405A, Y407V, and K409E according to EU numbering; and the Fc-CH3'B chain has mutations Q347K, E356K, E357R, T366V, D399K, F405A, and Y407V according to EU numbering.

3. The recombinant follicle-stimulating hormone and Fc fusion protein according to claim 2, wherein: The amino acid sequence of the Fc-CH3'A chain is shown in SEQ ID NO. 1, and the amino acid sequence of the Fc-CH3'B chain is shown in SEQ ID NO.

2.

4. The recombinant follicle-stimulating hormone and Fc fusion protein according to claim 1, wherein The two peptide chains are: the N-terminus of the follicle-stimulating hormone α subunit is connected to the C-terminus of the Fc-CH3'A chain to form Fc-CH3'A-FSHα and the N-terminus of the follicle-stimulating hormone β subunit is connected to the C-terminus of the Fc-CH3'B chain to form Fc-CH3'B-FSHβ; Alternatively, the N-terminus of the follicle-stimulating hormone β subunit is linked to the C-terminus of the Fc-CH3'A chain to form Fc-CH3'A-FSHβ and the N-terminus of the follicle-stimulating hormone α subunit is linked to the C-terminus of the Fc-CH3'B chain to form Fc-CH3'B-FSHα; Alternatively, the C-terminus of the follicle-stimulating hormone α subunit is linked to the N-terminus of the Fc-CH3'A chain to form FSHα-Fc-CH3'A and the C-terminus of the follicle-stimulating hormone β subunit is linked to the N-terminus of the Fc-CH3'B chain to form FSHβ-Fc-CH3'B; Alternatively, the C-terminus of the FSH β subunit is linked to the N-terminus of the Fc-CH3'A chain to form FSHβ-Fc-CH3'A and the C-terminus of the FSH α subunit is linked to the N-terminus of the Fc-CH3'B chain to form FSHα-Fc-CH3'B.

5. The recombinant follicle-stimulating hormone and Fc fusion protein according to claim 1, wherein: The follicle-stimulating hormone is porcine follicle-stimulating hormone, the amino acid sequence of the porcine follicle-stimulating hormone α subunit is shown in SEQ ID NO.3, and the amino acid sequence of the porcine follicle-stimulating hormone β subunit is shown in SEQ ID NO.

4.

6. The recombinant follicle-stimulating hormone and Fc fusion protein according to claim 5, wherein: The two peptide chains are: Fc-CH3'A-FSHα with an amino acid sequence as shown in SEQ ID NO.5 and Fc-CH3'B-FSHβ with an amino acid sequence as shown in SEQ ID NO.6; or Fc-CH3'A-FSHβ with an amino acid sequence as shown in SEQ ID NO.7 and Fc-CH3'B-FSHα with an amino acid sequence as shown in SEQ ID NO.8; or FSHα-Fc-CH3'A with an amino acid sequence as shown in SEQ ID NO.9 and FSHβ-Fc-CH3'B with an amino acid sequence as shown in SEQ ID NO.10; or FSHβ-Fc-CH3'A with an amino acid sequence as shown in SEQ ID NO.11 and FSHα-Fc-CH3'B with an amino acid sequence as shown in SEQ ID NO.

12.

7. The method for preparing the recombinant follicle-stimulating hormone and Fc fusion protein according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Using the pCDNA3.4-TOPO vector as the original vector, a DNA sequence containing a multiple cloning site, a bovine growth hormone polyA sequence, a CMV enhancer sequence, and a CMV promoter sequence was inserted into the TA cloning site. The nucleotide sequence of the DNA sequence is shown in SEQ ID NO. 13, thereby constructing a dual expression vector pCDNA3.4Double containing two CMV promoter sequences; (2) The coding sequences of the two peptide chains were respectively inserted into the multiple cloning sites downstream of the two CMV promoters of the dual expression vector to construct a heterodimer expression plasmid; (3) The heterodimer expression plasmid is transfected into CHO S cells, the culture fluid is collected after cell culture, and the recombinant follicle-stimulating hormone and Fc fusion protein is obtained by separation and purification.

8. The preparation method according to claim 7, wherein The nucleotide sequence encoding the Fc-CH3'A chain in the coding sequences of the two peptide chains is shown in SEQ ID NO.14; the nucleotide sequence encoding the Fc-CH3'B chain is shown in SEQ ID NO.15; the nucleotide sequence encoding the follicle-stimulating hormone α subunit is shown in SEQ ID NO.16; and the nucleotide sequence encoding the follicle-stimulating hormone β subunit is shown in SEQ ID NO.

17.

9. Use of the recombinant follicle-stimulating hormone and Fc fusion protein according to any one of claims 1 to 6 in the preparation of a drug for promoting superovulation, estrus synchronization, and conception and farrowing in animals.

10. The use according to claim 9, characterized in that The drug further includes a pharmaceutically acceptable carrier.

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