Long-acting recombinant single-chain follicle-stimulating fusion protein, gene, preparation method therefor, and use thereof

Abstract

The present invention relates to the technical field of molecular biology and drugs, provides a long-acting recombinant single-chain follicle-stimulating fusion protein, a gene, a preparation method therefor, and a use thereof, further provides a gene sequence of a linker sequence for the fusion protein, and provides an expression vector of the gene of the fusion protein, a cell, and a drug. The linker sequence can link an FSHα subunit and an FSHβ subunit together at the gene level, thereby encoding a fusion protein having a single peptide chain. Moreover, the gene can be expressed in a long-acting and stable manner and has a relatively high expression level. The obtained long-acting recombinant single-chain follicle-stimulating fusion protein has high activity and can be used for a single injection to replace natural FSH requiring multiple injections.

Description

A long-acting recombinant single-chain follicle-stimulating hormone fusion protein, its gene, preparation method, and its applications Technical Field

[0001] This invention relates to the fields of molecular biology and pharmaceutical technology, specifically to a long-acting recombinant single-chain follicle-stimulating hormone fusion protein, its gene, preparation, and application. Background Technology

[0002] Gonadotropins in mammals, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are glycoprotein hormones secreted by gonadotropic cells in the anterior pituitary gland. They are regulated by hypothalamic gonadotropin-releasing hormone (GnRH, also known as luteinizing hormone-releasing hormone, LHRH), ovarian hormones, and inhibin. FSH stimulates ovarian growth in female animals, increases ovarian weight, and stimulates follicle growth and development; it also promotes the development of seminiferous epithelium and sperm formation in male animals. Different levels of FSH and LH in an animal's body, as well as their ratio (FSH / LH), have varying effects on reproductive capacity, particularly on the strength of estrus in females, the duration of estrus, the number of mature follicles, the timing of ovulation, oocyte quality, and related biological effects. Comparing the duration of estrus in animals such as cattle, horses, sheep, and pigs, cattle have the shortest duration, horses the longest, and sheep and pigs fall in between. In animal production and veterinary clinical practice, FSH is commonly used to induce estrus, ovulation, and superovulation in female animals, as well as to treat ovarian dysfunction and maintain the sexual function of breeding animals.

[0003] Both FSH and LH consist of two polypeptide chains (or subunits) connected non-covalently to form functional units, which can then bind to their respective receptors to exert their corresponding biological effects. FSH and LH from the same species have identical α-subunit amino acid sequences; the specificity of their hormone functions depends on their respective β-subunit sequences. Currently, veterinary FSH is mainly derived from animal pituitary tissue extracts, which not only contain varying amounts of LH, leading to unstable biological effects, but also pose a potential risk of carrying biotransmitted infectious agents. Due to the short half-life of natural FSH, multiple injections are necessary to achieve the desired effect, generally 1-2 times daily for 3-4 consecutive days. Frequent injections not only increase labor costs but also easily cause stress reactions in animals and affect drug efficacy. With the advancement and widespread use of genetic engineering technology, many target proteins have been obtained through gene recombination technology. To date, two main genetic engineering strategies for recombinant FSH have been reported. One strategy involves expressing and folding the α and β polypeptide chains separately, then binding them together non-covalently to form a heterodimer. Due to the limited efficiency of the two free subunits binding together, the functional expression level of this recombinant protein is low, meaning that most of the expressed polypeptide chain is in a non-functional single-subunit state. Therefore, many researchers have turned to exploring a second strategy: how to link the α and β polypeptide chains together at the gene level to encode a single-peptide fusion protein.

[0004] For example, Chinese invention patent (publication number: CN109970871A, publication date: 2019.07.05) discloses a recombinant human follicle-stimulating hormone (rFSH) and its genetically engineered strain, as well as a recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria for the gene, and the fermentation of the recombinant bacteria to obtain recombinant human follicle-stimulating hormone (rFSH); it also provides the encoding gene of the protein and an engineered bacterium (recombinant Pichia pastoris) containing the encoding gene. The recombinant human follicle-stimulating hormone (rFSH) produced by the method of this invention has the advantages of long-acting and stable effects, and is suitable for use as an ovulation-inducing hormone in humans and animals. Its recombination strategy adds three elements to the two subunits b and a, namely CTP, peptide linker and Fc sequence fragments. However, the excessive amount of exogenous sequences raises concerns about stimulating immune responses after repeated use.

[0005] There are two main forms of constructing single-peptide chain fusion proteins: indirect fusion with the addition of an external linker (linker sequence) and direct fusion with the ends joined together. Since the function of a protein molecule depends on the correctness of its spatial structure, the two subunits of native FSH must fold freely and fully before they can bind together via non-covalent bonds to achieve a functional spatial structure; otherwise, they cannot be recognized by their receptor. Therefore, indirect fusion is superior to direct fusion, as the former's structure provides adequate space for the correct folding of the α and β polypeptide subunits. The key to achieving indirect fusion lies in selecting the linker sequence between the two subunits. If the target protein is long-acting FSH, the linker sequence design must balance extending the half-life and improving the biological effect. Regarding the commonalities of indirect fusion, the factors influencing the fusion protein include whether the sequence length itself provides adequate space for the free folding of the two subunits, the physicochemical properties (rigidity / flexibility, hydrophilicity / hydrophobicity, charge polarity, etc.) of the amino acid residues in the sequence leading to mutual influence between amino acid residues, and whether the histidine tag used for downstream purification interferes with the correctness of folding. The three-dimensional relationship between the two subunits and the connecting sequence directly affects the biological activity of the fusion protein and its expression level. Experiments have shown that sometimes a difference of only one nucleotide in the gene sequence is enough to affect the expression level of the target protein; or a difference of only one amino acid can lead to significantly different biological effects. Regarding the characteristics of recombinant glycoproteins, in addition to selecting a suitable host, the design of the connecting sequence must consider whether glycosylation is necessary, and if so, how to determine the type, site, and number of glycosylations in the connecting sequence and their impact on the biological function of the target protein. Furthermore, the folding state of the protein is constrained by many factors during expression in host cells. The same gene sequence is subject to different constraints during expression in different host cells, resulting in varying degrees of glycosylation and expression levels of recombinant fusion glycoproteins. Although several recombinant single-chain FSHs have been reported to be prepared using different linker sequences via indirect fusion technology, and studies have demonstrated that recombinant fusion FSHs with glycosylated linkers exhibit increased glycosylation levels and in vivo half-life compared to wild-type FSH, their biological effects do not show a simple linear relationship with the increase in glycosylation sites. The optimal sequence for low-dose, high-efficacy FSH remains to be further explored. To date, there is no clear and unified theoretical guidance on how to design specific functional fusion proteins. Researchers can only design linker sequences through dry experiments using bioinformatics methods, and then verify their effectiveness through trial and error using wet molecular biology methods. Therefore, for recombinant single-chain follicle-stimulating hormone (FSH), there is currently no single, unchanging, universally applicable linker sequence. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a long-acting recombinant single-chain follicle-stimulating hormone (FSH) fusion protein, its gene, preparation method, and applications, particularly providing a linker sequence for fusing the two FSH subunits. This long-acting recombinant single-chain FSH fusion protein exhibits high expression and activity, and can be used for a single injection to replace multiple injections of natural FSH.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention uses the sheep follicle-stimulating hormone (FSH) gene as an example to design a series of long-acting recombinant single-chain FSHs containing different linker sequences, based on the rotational freedom, hydrophilicity / hydrophobicity, charge effect, and the length and rigidity of the linker sequences of amino acid monomers. The expression product is a single-chain fusion protein linked from the N-terminus to the C-terminus in the order of FSHβ-linker sequence-FSHα (FSHβ-Linker-FSHα). Their expression levels and biological effects are then compared to select the optimal sequence.

[0009] The expression level was measured using Chinese hamster ovary (CHO) cells as the host cell, and the amount and biological effects of recombinant proteins secreted by the host cells into their culture medium were detected.

[0010] The biological effects described were determined according to the follicle-stimulating hormone bioassay method in the Chinese Pharmacopoeia quality standards.

[0011] The research process for the long-acting recombinant single-stranded FSH includes the following steps:

[0012] Step 1: Based on the rotational freedom, hydrophilicity / hydrophobicity, charge effect, length, rigidity / flexibility, and glycosylation characteristics of the amino acid monomers that form the candidate linker sequences between the follicle-stimulating hormone (FSH) α and β peptide chains, a series of candidate long-acting recombinant single-chain FSHs with different linker sequences were designed. The order from the amino terminus to the carboxyl terminus was FSHβ-Linker-FSHα. The coding sequence of one of the fusion proteins was synthesized from the whole genome to provide a template for subsequent research.

[0013] Step 2: Long-acting recombinant single-stranded FSH coding sequences containing different linker sequences are obtained through sequential recombination via localized substitution PCR. These different linker sequences include control and experimental group sequences.

[0014] Step 3: The FSH gene sequences containing different linkers and double restriction sites (e.g., Nhe I and EcoRI, or XbaI and XmaI) obtained by PCR replacement and amplification (confirming that the coding sequence is located within the correct reading frame) are cloned into the target fragments obtained by double digestion with the same double restriction enzymes [e.g., pcDNA3.1(+)]. T4 ligase is then used to ligate the fragments, making FSHβ-Linker-FSHα-pcDNA3.1(+) a circular structure. This circular structure is then transformed into competent DH5α E. coli. The expressed product of this gene is a secretory target protein.

[0015] Step 4: Extract plasmids from the transformed E. coli, and after determining the sequences, transfect them into Chinese hamster ovary (CHO) cells to transiently express the target protein in parallel experiments.

[0016] Step 5: Analyze and compare the expression levels of the target gene using the supernatant from the transiently expressing cell culture. Select one clone with the highest expression from both the control and experimental groups for further study.

[0017] Step 6: The selected control group and experimental group FSHβ-Linker-FSHα-pcDNA3.1(+) were linearized by single enzyme digestion and then transfected into CHO cells.

[0018] Step 7: Select transfected CHO cells for further monoclonalization and compare expression levels.

[0019] Step 8: Take one stable, high-expressing clone from each of the control and experimental groups, also known as a cell line, for further study.

[0020] Step 9: Culture two cell lines in parallel, take equal amounts of the expression material from each, and compare their biological effects according to the methods specified in the pharmacopoeia to determine the optimal sequence.

[0021] The present invention obtains a long-acting recombinant single-chain follicle-stimulating hormone fusion protein through the above research process. The protein structure is FSHβ-Linker E2-FSHα from the N-terminus to the C-terminus, where FSHβ is the follicle-stimulating hormone β subunit, FSHα is the follicle-stimulating hormone α subunit, and Linker E2 is the linking sequence between the two subunits; the amino acid sequence of Linker E2 is shown in SEQ ID NO: 12.

[0022] Preferably, the nucleotide sequence of the gene encoding Linker E2 is shown in SEQ ID NO: 7.

[0023] Furthermore, the present invention also discloses a gene encoding the fusion protein of the claim, the nucleotide sequence of which is shown in SEQ ID NO: 13.

[0024] Furthermore, the present invention also discloses an expression vector containing a gene encoding the fusion protein.

[0025] Furthermore, the present invention also discloses a host cell containing the expression vector described above, or containing a gene encoding the fusion protein described above.

[0026] Furthermore, the present invention also discloses a method for preparing the fusion protein, which includes the following steps:

[0027] 1) The fully synthesized FSHβ-Linker E2-FSHα fragment was inserted into the expression vector pcDNA3.1(+) by double digestion with Nhe I and EcoRI to construct a recombinant expression plasmid;

[0028] 2) The recombinant expression plasmid was transformed into competent DH5α Escherichia coli, and the plasmid was extracted after culturing, screening and verification;

[0029] 3) Transfect the recombinant plasmid obtained in step 2) into host cells, such as CHO-K1 cells, to induce the expression of the recombinant FSHβ-LinkerE2-FSHα protein.

[0030] Furthermore, the present invention also discloses the use of the fusion protein or the gene in the preparation of a drug that stimulates the growth of female animal ovaries; or in the preparation of a drug that stimulates the growth of male sperm and sexual function.

[0031] Furthermore, the present invention also provides a pharmaceutical composition comprising the long-acting recombinant single-chain follicle-stimulating hormone fusion protein or the expression vector, as well as a pharmaceutically acceptable carrier and / or excipient.

[0032] Furthermore, the present invention also provides a pharmaceutical preparation comprising the aforementioned pharmaceutical composition.

[0033] Preferably, the pharmaceutical preparation is an injection.

[0034] In summary, this invention provides a novel gene sequence for linking recombinant long-acting single-chain FSH fusion proteins, capable of connecting the FSHα and FSHβ subunits at the gene level to encode a single-peptide fusion protein. Furthermore, this gene can be expressed stably and for a long period, exhibiting high expression levels. The resulting long-acting recombinant single-chain follicle-stimulating hormone fusion protein is highly active and can be used for a single injection to replace multiple injections of natural FSH. Attached Figure Description

[0035] Figure 1 shows the recombinant protein expression level characterized by Western blotting (1, untransfected cell culture supernatant; 2, empty vector pcDNA3.1(+) transfected cell culture supernatant; the ligation sequence numbers of 3-12 are C1, C2, C3, C4, E1, E2, E3, E4, E5 and E6 respectively).

[0036] Figure 2 shows the expression levels of recombinant FSH carrying different linker sequences.

[0037] Figure 3 is a dose-ovarian weight line graph comparing the bioactivity of follicle-stimulating hormone analogs using the follicle-stimulating hormone bioassay. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0039] Example 1: Expression of Long-Acting Recombinant Single-Chain Follicle-Stimulating Hormone Fusion Protein

[0040] 1. Design of candidate sequences for long-acting recombinant single-stranded follicle-stimulating hormone (FSH) genes. Taking the sheep FSH gene as an example, a series of long-acting recombinant single-stranded FSH genes containing different linker sequences were designed based on the rotational freedom, hydrophilicity / hydrophobicity, charge effect, and the length and rigidity of the linker sequences of the amino acid monomers. The gene sequence from the 5' end to the 3' end is FSHβ-Linker-FSHα. The corresponding sequences of FSHβ and FSHα are obtained from GenBank, with the numbers NM_001009798.1 and NM_001009464.1, respectively. The FSHβ subunit coding sequence retains its signal peptide and start codon coding sequences but deletes the stop codon, while the FSHα subunit coding sequence deletes the signal peptide and start codon coding sequences but retains its stop codon. Six linker sequences were designed, numbered E1 to E6 (SEQ ID NO: 6-11), based on their length, rigidity, hydrophilicity / hydrophobicity, and charge effects, and each sequence carried a histidine tag and different O-glycosylation and N-glycosylation sites. Four previously reported sequences were used as controls, numbered C1 to C4 (SEQ ID NO: 2-5). The long-acting recombinant single-stranded follicle-stimulating hormone gene FSHβ-Linker C1-FSHα, linked by linker sequence C1, was synthesized, and its sequence is SEQ ID NO: 1. Other linker sequences were synthesized using primers, purified by PAGE, and synthesized in 1 OD increments. The synthesized single-stranded DNA sequences were stored at -20°C for the preparation of other full-length sequences.

[0041] 2. Prepare the other 9 complete sequences.

[0042] 2.1 Using localized substitution PCR, Linker C1 in FSHβ-Linker C1-FSHα was sequentially replaced with different linker sequences. The corresponding primer names were F1, R1, F2C2-F2C4, F3C2-F3C4, R2C2-R2C4, R3C2-R3C4, F2E1-F2E6, F3E1-F3E6, R2E1-R2E6, and R3E1-R3E6, as shown in Table 1. F1 and R1 contain restriction endonucleases Nhe I and EcoRI at their 5' ends, respectively.

[0043] Table 1. Primer List

[0044]

[0045]

[0046] 2.2 The following nine candidate sequences encoding single-stranded FSH were obtained by localized substitution PCR:

[0047] 2.2.1

[0048] FSHβ-Linker C2: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2C2, and R3C2 as primers.

[0049] Linker C2-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2C2, F3C2 and R1 as primers.

[0050] FSHβ-Linker C2-FSHα: PCR amplification product using FSHβ-Linker C2 and Linker C2-FSHα as a combined template and F1 and R1 as primers.

[0051] 2.2.2

[0052] FSHβ-Linker C3: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2C3, and R3C3 as primers.

[0053] Linker C3-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2C3, F3C3 and R1 as primers.

[0054] FSHβ-Linker C3-FSHα: PCR amplification product using FSHβ-Linker C3 and Linker C3-FSHα as a combined template and F1 and R1 as primers.

[0055] 2.2.3.

[0056] FSHβ-Linker C4: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2C4, and R3C4 as primers.

[0057] Linker C4-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2C4, F3C4, and R1 as primers.

[0058] FSHβ-Linker C4-FSHα: PCR amplification product using FSHβ-Linker C4 and Linker C4-FSHα as a combined template and F1 and R1 as primers.

[0059] 2.2.4.

[0060] FSHβ-Linker E1: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E1, and R3E1 as primers.

[0061] Linker E1-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E1, F3E1 and R1 as primers.

[0062] FSHβ-Linker E1-FSHα: PCR amplification product using FSHβ-Linker E1 and Linker E1-FSHα as a combined template and F1 and R1 as primers.

[0063] 2.2.5.

[0064] FSHβ-Linker E2: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E2, and R3E2 as primers.

[0065] Linker E2-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E2, F3E2 and R1 as primers.

[0066] FSHβ-Linker E2-FSHα: PCR amplification product using FSHβ-Linker E2 and Linker E2-FSHα as a combined template and F1 and R1 as primers.

[0067] 2.2.6.

[0068] FSHβ-Linker E3: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E3, and R3E3 as primers.

[0069] Linker E3-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E3, F3E3 and R1 as primers.

[0070] FSHβ-Linker E3-FSHα: PCR amplification product using FSHβ-Linker E3 and Linker E3-FSHα as a combined template and F1 and R1 as primers.

[0071] 2.2.7.

[0072] FSHβ-Linker E4: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E4, and R3E4 as primers.

[0073] Linker E4-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E4, F3E4 and R1 as primers.

[0074] FSHβ-Linker E4-FSHα: PCR amplification product using FSHβ-Linker E4 and Linker E4-FSHα as a combined template and F1 and R1 as primers.

[0075] 2.2.8.

[0076] FSHβ-Linker E5: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E5, and R3E5 as primers.

[0077] Linker E5-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E5, F3E5 and R1 as primers.

[0078] FSHβ-Linker E5-FSHα: PCR amplification product using FSHβ-Linker E5 and Linker E5-FSHα as a combined template and F1 and R1 as primers.

[0079] 2.2.9.

[0080] FSHβ-Linker E6: PCR amplification product using SEQ ID NO: 1 as a template and F1, R2E6, and R3E6 as primers.

[0081] Linker E6-FSHα: PCR amplification product using SEQ ID NO: 1 as a template and F2E6, F3E6, and R1 as primers.

[0082] FSHβ-Linker E6-FSHα: PCR amplification product using FSHβ-Linker E6 and Linker E6-FSHα as a combined template and F1 and R1 as primers.

[0083] 2.3 The above localization substitution involves preparing a single-stranded FSH candidate sequence containing a unique ligation sequence through three rounds of PCR (PCR system shown in Table 2-4):

[0084] Table 2. PCR system for the first round

[0085]

[0086] R2*: Depending on the primer name of the amplification target, the corresponding primers are R2C2, R2C3, R2C4, R2E1, R2E2, R2E3, R2E4, R2E5, or R2E6.

[0087] R3**: Depending on the primer name of the amplification target, the corresponding primers are R3C2, R3C3, R3C4, R3E1, R3E2, R3E3, R3E4, R3E5, or R3E6.

[0088] The parameters for the first round of PCR reaction were as follows: 95℃ for 1 min, then cycling at 95℃ for 20 s, #℃ for 20 s, 72℃ for 1 min, for 30 cycles, followed by a final hold at 72℃ for 10 min. The resulting PCR products were separated by 1% agarose gel electrophoresis, extracted, and stored at 4℃ for later use. Where # represents the annealing temperature: C2: 58-72℃, preferably 68℃; C3: 58-72℃, preferably 70℃; C4: 58-70℃, preferably 68℃; E1: 58-72℃, preferably 70℃; E2: 58-72℃, preferably 72℃; E3: 58-72℃, preferably 72℃; E4: 58-72℃, preferably 72℃; E5: 65-72℃, preferably 72℃; E6: 60-72℃, preferably 72℃.

[0089] The annealing temperatures used in this experiment were all optimized temperatures.

[0090] Table 3. Second round PCR system

[0091]

[0092] F2*: Depending on the primer name of the amplification target, the corresponding primers are F2C2, F2C3, F2C4, F2E1, F2E2, F2E3, F2E4, F2E5, or F2E6.

[0093] F3**: Depending on the primer name of the amplification target, the corresponding primers are F3C2, F3C3, F3C4, F3E1, F3E2, F3E3, F3E4, F3E5, or F3E6.

[0094] The parameters for the second round of PCR reaction were as follows: 95℃ for 1 min, then cycling at 95℃ for 20 s, #℃ for 20 s, 72℃ for 1 min, for 30 cycles, followed by a final hold at 72℃ for 10 min. The resulting PCR products were separated by 1% agarose gel electrophoresis, extracted, and stored at 4℃ for later use. Where # represents the annealing temperature: C2: 58-70℃, preferably 68℃; C3: 59-71℃, preferably 70℃; C4: 59-71℃, preferably 70℃; E1: 55-71℃, preferably 70℃; E2: 55-72℃, preferably 71℃; E3: 55-72℃, 72℃; E4: 58-72℃, preferably 70℃; E5: 56-72℃, preferably 72℃; E6: 58-72℃, preferably 71℃.

[0095] The annealing temperatures used in this experiment were all optimized temperatures.

[0096] Table 4. PCR system for the third round

[0097]

[0098] The parameters for the third round of PCR reaction were as follows: 95℃ for 1 min, then cycling at 95℃ for 20 s, followed by 58-72℃ (preferably 72℃ for 20 s), then 72℃ for 1 min, for 25 cycles. Finally, the reaction was held at 72℃ for 10 min. The resulting PCR products were separated by 1% agarose gel electrophoresis, extracted, and stored at 4℃ for later use.

[0099] The annealing temperatures used in this experiment were all optimized temperatures.

[0100] The extraction and purification of the above PCR products were achieved by 1% agarose gel electrophoresis, which is well known to professionals in this industry. The target band was cut from the gel and purified using a column DNA purification kit (Shanghai Sangon Biotech). The concentration was then measured using a micro-volume UV spectrophotometer (Thermo Fisher Nanodrop).

[0101] 3. Assemble the expression plasmid.

[0102] 3.1. The 10 candidate single-stranded FSH sequences containing different ligation sequences and the expression plasmid pcDNA3.1(+) were digested with restriction endonucleases NheI and EcoRI (Shanghai Sangon Biotech). The digestion system is shown in Table 5:

[0103] Table 5. Double enzyme digestion system

[0104]

[0105] After mixing the above enzyme digestion system, incubate at 37°C for 4 hours. Separate the enzyme digestion products by 1% agarose gel electrophoresis. After electrophoresis, perform gel excision and column purification using techniques familiar to those skilled in the art.

[0106] 3.2 The candidate sequences containing different ligation sequences were digested with Nhe I and EcoRI to obtain the target fragments, which were then cloned into pcDNA3.1(+) plasmids that had been digested with the same double enzymes. Ligation was performed using ligase to form a circular structure of FSHβ-Linker-FSHα-pcDNA3.1(+). The ligation reaction system is shown in Table 6.

[0107] Table 6. Connection Reaction System

[0108]

[0109] The ligation products, namely FSH recombinant expression vectors containing different linkers, were transformed into competent DH5α *E. coli* using the calcium chloride (CaCl2) method, which is well-known to those skilled in the art. After plate culture, single colonies were picked, expanded, and the bacterial strain was preserved and plasmids extracted. The cloned gene expression product was confirmed by sequencing after verification using the double enzyme digestion method described in section 3.2 above. The cloned gene expression product is a secretory target protein.

[0110] 4. Transient expression of recombinant FSH in CHO-K1 cells.

[0111] Following techniques known to industry professionals, a cryopreserved CHO-K1 cell line was thawed, cultured, and expanded. 24 hours before transfection, a 10 cm cell culture dish containing newly confluent CHO-K1 cells was taken, the old culture medium was aspirated, the cells were washed three times with PBS, trypsinized for 1 minute, and then thoroughly mixed with 5 mL of F12 nutrient medium containing 10% serum. Cell counts were then performed, and the cell density was adjusted to 1 x 102. 6Cells / mL of culture medium were used to seed 6-well plates at a density of 0.5 mL / well. The constructed FSH expression vectors containing different linkers were dissolved in 25 μL of Ham's F12 serum-free nutrient medium at a concentration of 1 μg DNA; correspondingly, 1.5 μL of lipofectamine 2000 was dissolved in 25 μL of Ham's F12 serum-free nutrient medium. The solutions were mixed thoroughly and incubated at room temperature for 20 min. The mixture was then added to the cell-coated 6-well plates, gently pipetted to mix, and incubated at 37℃ in a 5% CO₂ incubator. After 6 h, the medium was replaced with fresh serum-free nutrient medium. After 6 days of cell culture, the supernatant was used for Western blotting to detect the expressed FSH protein: 4% polyacrylamide stacking gel and 12.5% ​​separating gel were prepared, and 15 μL of supernatant from each was used as a sample. After electrophoresis, the sample was electrotransferred onto a nitrocellulose membrane. Using rabbit anti-FSHα polyclonal antibody (1:400 dilution) as the primary antibody, the sample was incubated at 37°C for 1 h and washed three times with buffer. Then, goat anti-rabbit IgG-horseradish peroxidase-labeled secondary antibody (1:5000 dilution) was added and the sample was incubated at 37°C for 1 h. The sample was then developed and photographed using chemiluminescence, as shown in Figure 1.

[0112] Each experiment was repeated three times. The expression levels of FSH recombined with different linker sequences were compared by analyzing the gray values ​​of the Western blot bands using ImageJ software, as shown in Figure 2. The results showed that the expression level of the recombinant protein was not linearly related to the linker length or its glycosylation state. The FSHβ-Linker E2-FSHα, which had the highest expression level, was selected for further investigation. The amino acid sequence of Linker E2 is shown in SEQ ID NO: 12. The FSHβ-Linker C4-FSHα, which had the highest expression level in the control group, was used as a control.

[0113] Example 2: Detection of expression level of long-acting recombinant single-chain follicle-stimulating hormone fusion protein

[0114] 1. Stable expression of recombinant single-stranded FSH in CHO-K1 cells

[0115] 1.1 Selection of G418 Concentration for Cell Screening

[0116] CHO-K1 cells (1 x 10⁻⁶) were seeded in 24-well plates. 5(1 cell per well). When the cells reached 50% confluence, the medium was replaced with G418, resulting in final G418 concentrations of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, and 700 μg / mL, with three replicates for each concentration. The medium was changed every three days. After 10 days, test results showed that no viable cells were present in the wells with G418 concentrations greater than 200 μg / mL. Therefore, the G418 concentration for cell selection medium was chosen to be 250 μg / mL.

[0117] 1.2 Linearization of Expression Vectors

[0118] The empty vector pcDNA3.1(+), expression vectors pcDNA3.1(+)-FSHβ-Linker E2-FSHα, and pcDNA3.1(+)-FSHβ-Linker C4-FSHα were linearized using the restriction endonuclease AcLI. The linearization systems of the expression vectors are shown in Table 7.

[0119] Table 7. Linearization and restriction enzyme digestion system for expression plasmids

[0120]

[0121] After mixing the above system, incubate it in a water bath at 37°C for 4 hours for enzyme digestion.

[0122] After separation of the enzyme digestion products by 1% agarose gel electrophoresis, gel bands containing the linearized expression vector and the empty vector were excised, purified, and recovered. The DNA concentration of the recovered products was then determined. The DNA was then ready for use.

[0123] 1.3 Stable transfection of CHO-K1 cells

[0124] Following techniques known to industry professionals, 24 hours before transfection, seed CHO cells in 24-well plates and incubate them at 37°C with 5% CO2 for 18-24 hours, ensuring a cell confluence of 50-70%, preferably 70%. Before transfection, replace the medium with serum-free Ham's F12K (Thermo Fisher Scientific). Dissolve 1 μg of DNA in 25 μL of Ham's F12K serum-free nutrient medium per well to obtain solution A; correspondingly, dissolve 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) in 25 μL of Ham's F12 serum-free nutrient medium to obtain solution B. Mix the two solutions (A and B) to obtain solution C, and incubate solution C at room temperature for 25-30 min, ideally 20 min. Add solution C to the cell culture wells of the 24-well plate and gently agitate to mix thoroughly. Cells were cultured at 37°C in a 5% CO₂ incubator for 7 h. The old culture medium was discarded, and 0.5 mL of fresh culture medium was added to each well. After 24 hours of culture, the medium was replaced with selection medium containing 250 μg / mL G418. The medium was changed every two days (selection medium containing G418). On day 8, no viable cells were observed in the plasmid-free transfection group. The cell density of the remaining groups was adjusted to 1 x 10⁻⁶ cells / well using Ham's F12 nutrient medium. 6 Cells / mL, 1 mL / well was transferred to a 6-well plate. Transfected cells were cultured for 7 days using selection medium. 15 μL of cell culture supernatant was collected for Western blotting. Results showed that the empty vector group was negative, while both the pcDNA3.1(+)-FSHβ-Linker C4-FSHα and pcDNA3.1(+)-FSHβ-Linker E2-FSHα expression vector groups were positive.

[0125] 1.4 Monoclonalization of Engineered Cells: After successful transfection of the two groups of cells containing the expression vector, accurate cell counts were performed. Each cell was serially diluted with culture medium to a concentration of 10 cells per mL, and then 100 μl / well was added to a 96-well plate. The plates were incubated in a 5% CO2 incubator. After 6 days, the number of monoclonal cells in each well was counted. 15 μl of cell culture medium from each well was collected, and recombinant FSH expression was monitored using the Western blot (WB) method described above. Clones with the highest expression levels from both the pcDNA3.1(+)-FSHβ-Linker C4-FSHα and pcDNA3.1(+)-FSHβ-Linker E2-FSHα groups were selected and monocloned again using the same method.

[0126] After three consecutive monoclonalizations, three clones of the cells with the highest expression levels were retained from each of the two cell lines: control group clones C41, C43, and C46, ​​and experimental group clones E24, E26, and E27 (the letters and the first number are the linker sequence numbers, and the second number is the clone number). These were further cultured and expanded. Both monoclonal cell lines were seeded at a density of 2 x 10^6 / mL and cultured in parallel. Seven days later, the cell culture supernatant was collected in sterile tubes, centrifuged for 20 minutes (2000-3000 rpm), and the supernatant was carefully collected.

[0127] 1.5 Expression Level Detection: The concentration of recombinant FSH in the supernatant samples of two cell line culture media was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Yunclone). Two parallel standard series were set up, each with five wells. Standards were serially diluted using the standard diluent provided with the kit, with each well containing 100 μL of the diluted solution at concentrations of 600, 400, 200, 100, and 50 µg / L. A substrate blank well was also included, containing 100 μL of standard diluent. For the two series of test samples, three wells each contained 5-fold and 10-fold diluted test samples, with 50 μL of sample and standard solution (sample) added to each well. 50 μL of working solution A (prepared immediately before use) was added, and the plates were sealed and incubated at 37°C for 1 h. The liquid in the wells was discarded, and each well was washed with 350 μL of washing buffer for 1-2 min, then dried. The washing process was repeated three times. After the final wash and spin-drying, add 100 μL of working solution B (prepared immediately before use) to each well, seal the plate, and incubate at 37°C for 30 minutes. Discard the liquid in the wells, spin-dry, and wash the plate 5 times. Add 90 μL of TMB substrate solution to each well, seal the plate, and incubate at 37°C in the dark for color development (reaction time controlled at 10-20 minutes). Add 50 μL of stop solution to each well. Immediately use a multi-mode microplate reader to read the absorbance (OD value) at a wavelength of 450 nm, and measure the absorbance of each well sequentially. Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis, and obtain the linear regression equation y = 325.92x + 23.608, R² = 0.9987. Substitute the OD value of the sample into the equation to calculate the sample concentration, and then multiply by the dilution factor to obtain the actual concentration of the sample (Table 8). The t-test showed that, under the same cell density and culture conditions, the expression level of recombinant single-stranded FSH containing the linker sequence E2 was significantly higher than that of the control group (P<0.01). The highest concentration in the control group (pcDNA3.1(+)-FSHβ-LinkerC4-FSHα) was 1543 µg / L (clone number C41); the highest concentration in the experimental group (pcDNA3.1(+)-FSHβ-LinkerE2-FSHα) was 1927 µg / L (clone number E27). Based on µg / L, the amount of secreted target protein contained in 1.0 mL of cell culture medium from the C41 transfection group was approximately equal to the amount contained in 0.8 mL of cell culture medium from the E27 transfection group.

[0128] Table 8. Sample concentrations at different dilutions determined by ELISA and the calculated mean actual concentrations (µg / L)

[0129]

[0130] Example 3: Bioactivity assay of long-acting recombinant single-chain follicle-stimulating hormone fusion protein

[0131] 1. Detection of biological activity: One sample with the highest expression level of recombinant single-chain FSH was selected from each of the two engineered cell series with ligation sequences C4 and E2, i.e., clone numbers C41 and E27, respectively. The biological activity of recombinant single-chain FSH was further evaluated by measuring and evaluating the follicle-stimulating hormone bioassay method in the Chinese Pharmacopoeia quality standard.

[0132] 1.1. Solvent preparation: On the day of the experiment, weigh an appropriate amount of bovine serum albumin, add 0.9% sodium chloride solution to dissolve it, and prepare a 1 mg / mL solution. After complete dissolution, adjust the pH value to 7.2 with 1 mol / L sodium hydroxide solution.

[0133] 12. Preparation of Standard Solutions (S): On the day of the test, dissolve the injectable standard (GONAL-f, Merck Serono) thoroughly in the above solution according to its labeled potency to prepare three standard solutions of high, medium, and low concentrations (dS3, dS2, dS1: 12 IU / mL, 6 IU / mL, 3 IU / mL). The potency ratio (r) of adjacent solutions should be equal at 1:0.5. Store the standard solutions at 4℃ and use within 3 days.

[0134] 1.3. Preparation of two sets of test solutions: The two recombinant FSH proteins C41 and E27 prepared in Example 2 were used as test samples, and their titers (A) were estimated. T Each was 12 IU / µg. Calculations in Example 2 showed that the volume ratio of cell culture medium (supernatant) containing equal amounts of secretory target protein in C41 and E27 was 1:0.8. Supernatants from C41 and E27 were measured according to this ratio, and solutions of high, medium, and low concentrations were prepared for both series of test samples according to the standard solution preparation method [dC3 (1 µg / mL), dC2 (0.5 µg / mL), dC1 (0.25 µg / mL) and dE3 (1 µg / mL), dE2 (0.5 µg / mL), dE1 (0.25 µg / mL).].

[0135] 1.4. Results and Analysis: Healthy female Sprague Dawley rats aged 19-22 days (45±5 g) from the same source were randomly divided into 9 groups of 8 rats each on the day of the experiment. The rats were subcutaneously injected with either the standard or the test product. The standard solution was injected at 0.5 mL once daily for 3 consecutive days. The test products C41 and E27 series were injected only at 0.5 mL on day 1. Eighteen hours after the last administration, the animals were euthanized, weighed, dissected, and the ovaries were removed. The surrounding tissue was peeled off, the oviducts were removed, and the surrounding fluid was absorbed with filter paper. The ovaries were weighed directly and converted to ovarian weight per 10 g of body weight (Table 9). The results showed that the standard and the two test products each exhibited a dose-response relationship, and all three showed a linear and parallel relationship within the dose range (Figure 3). This indicates that the follicle-stimulating hormone analogue highly expressed in this invention has similar biological activity to the standard.

[0136] Table 9. Effects of each test sample on ovarian weight gain in rats* (ovarian weight mg / body weight 10g)

[0137]

[0138] * 8 animals per group. SL: Low dose of standard, SM: Medium dose of standard, SH: High dose of standard; CL: Low dose of control, CM: Medium dose of control, CH: High dose of control; EL: Low dose of test sample, EM: Medium dose of test sample, EH: High dose of test sample. ∑y(k): Sum within each dose group; ∑y(m): Sum between each block.

[0139] 2. Quantitative reaction parallel line determination: The experimental data were subjected to variance analysis and reliability test according to the statistical method of bioassay in Chinese Pharmacopoeia, and the potency of recombinant FSH was calculated.

[0140] 2.1. Analysis of variance and reliability test: The test results showed that under the given probability level, the regression terms between the standard and the control group (Table 10), the standard group and the experimental group (Table 11), and the control group and the experimental group (Table 12) were all highly significant (P<0.01), while the deviations from parallel, quadratic curve, and directional quadratic curve were not significant (P>0.05), proving that the experimental design was reasonable and the data were reliable.

[0141] Table 10. Analysis of variance and reliability test of standard and control group (SC)*

[0142]

[0143] * P<0.05 indicates a greater than 95% reliability in rejecting the null hypothesis, meaning the variation is significant; P<0.01 indicates highly significant variation; P>0.05 indicates no significant variation.

[0144] Table 11. Analysis of variance and reliability tests for standard and experimental (SE) samples*

[0145] *P<0.05 indicates a greater than 95% reliability in rejecting the null hypothesis, meaning the variation is significant; P<0.01 indicates highly significant variation; P>0.05 indicates no significant variation.

[0146] Table 12. Analysis of variance and reliability test of the control group and experimental group (CE)*

[0147]

[0148] * P<0.05 indicates a greater than 95% reliability in rejecting the null hypothesis, meaning the variation is significant; P<0.01 indicates highly significant variation; P>0.05 indicates no significant variation.

[0149] 2.2. Recombinant FSH Potency and Confidence Limit: Calculations showed that the potency of the control group (C) and the experimental sample (E) relative to the standard (S) at 12 IU was 11.65 IU and 12.66 IU, respectively. Importantly, the volume of cell supernatant used in the control group was greater than that used in the experimental sample at the same concentration, with a supernatant volume ratio of 1.25:1; however, the potency per unit mass (IU / µg) of the control group was lower than that of the experimental sample, with a potency ratio of 0.92:1 (Table 13). The confidence limit (FL%) determined according to the bioassay statistical method was 2.34% (Table 14), which meets the requirements of the Chinese Pharmacopoeia, indicating that the experimental results are reliable. This demonstrates that the potency of long-acting recombinant single-stranded FSH containing the E2 linker sequence is higher than that of an equal amount of recombinant FSH containing the C4 linker sequence, meaning that the FSH analogue containing the E2 linker sequence is superior to the FSH analogue containing the C4 linker sequence in terms of biological activity (Tables 13 and 14).

[0150] Table 13. Comparison of sample dosage and potency between control group (C) and experimental sample (E)

[0151]

[0152] Table 14. Results of confidence limit (FL%) determination of pairwise valence between blocks according to method (3.3)*

[0153]

[0154] * SC: Between the standard and the control group containing the C4 linker sequence; SE: Between the standard and the experimental group containing the E2 linker sequence; CE: Confidence limit of potency between the control and experimental groups. The Chinese Pharmacopoeia stipulates that the confidence limit must be less than 45%.

[0155] Based on the length, rigidity, hydrophobicity, and charge effect of the linking sequence between α and β peptide chains, this invention designs a set of different linking sequences to obtain a new FSH analog, namely a long-acting recombinant single-chain FSH with high expression and high activity, which can be used for a single injection to replace multiple injections of natural FSH.

[0156] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A long-acting recombinant single-chain follicle stimulating hormone fusion protein, characterized in that, The protein structure formula from N-terminal to C-terminal is FSHβ-Linker E2-FSHα, wherein FSHβ is follicle stimulating hormone β subunit, FSHα is follicle stimulating hormone α subunit, and Linker E2 is a connecting sequence between the two subunits; the amino acid sequence of the Linker E2 is shown as SEQ ID NO:

12.

2. The fusion protein of claim 1, wherein, The nucleotide sequence of the gene encoding the Linker E2 is shown as SEQ ID NO:

7.

3. A gene encoding the fusion protein of claim 1 or 2, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:

13.

4. An expression vector, characterized by, The expression vector contains a gene encoding the fusion protein of claim 1 or 2.

5. A host cell, characterized in that, The host cell contains the expression vector of claim 4, or contains a gene encoding the fusion protein of claim 1 or 2.

6. A method of producing the fusion protein of claim 1, characterized by, The preparation method comprises the following steps: 1) inserting the FSHβ-Linker E2-FSHα fragment into the expression vector pcDNA3.1(+) by double enzyme digestion of Nhe I and EcoR I to construct a recombinant expression plasmid; 2) transforming the competent DH5α E. coli with the recombinant expression plasmid, extracting the plasmid after culture, screening and verification; 3) transfecting the host cell with the recombinant plasmid obtained in step 2) to induce expression of the FSHβ-Linker E2-FSHα recombinant protein.

7. Use of the fusion protein of claim 1 or 2 or the gene of claim 3 in the preparation of a medicament for stimulating the growth of ovaries of female animals; or in the preparation of a medicament for stimulating the growth of sperm and sexual ability of male animals.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition contains the fusion protein of claim 1 or 2 or the expression vector of claim 4, and a pharmaceutically acceptable carrier and / or excipient.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the pharmaceutical composition of claim 8.

10. A pharmaceutical preparation according to claim 9, characterised in that The pharmaceutical preparation is an injection. The pharmaceutical preparation is an injection.

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