Novel long-acting human FSH and LH fusion protein as well as preparation method and application thereof

Abstract

The FSH and LH fusion protein is composed of a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide. Wherein the first polypeptide comprises a beta subunit of FSH, a beta subunit of LH, a hinge, a CH2 structural domain and a CH3 structural domain, or the beta subunit of LH, the beta subunit of FSH, the hinge, the CH2 structural domain and the CH3 structural domain from an N terminal to a C terminal; the second polypeptide comprises a hinge, a CH2 structural domain and a CH3 structural domain from the N terminal to the C terminal; the third polypeptide and the fourth polypeptide are both composed of alpha subunits of FSH. The FSH and LH fusion protein has the functions of FSH and LH at the same time; meanwhile, compared with recombinant or natural FSH and LH, the heterodimer has a longer half-life period, can complement gonadotropin in a long-acting, mild and continuous manner, and is suitable for various FSH and LH deficiency related diseases such as male hypogonadotropin gonad hypofunction, female pituitary insufficiency, hypogonadotropin amenorrhea or hypogonadotropin infertility. The medicine economy is better, and the patient compliance is better.

Description

Novel long-acting human FSH and LH fusion protein, its preparation method and application Technical Field The present invention relates to the field of biopharmaceuticals, in particular to a novel long-acting human FSH and LH fusion protein, its preparation method and application. Background Art Human gonadotropins are a family of glycoprotein hormones secreted by the anterior pituitary gland, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH). FSH and LH play important roles in the regulation of reproductive function and sexual characteristics. These hormones are composed of an α subunit and a β subunit bound by non-covalent bonds. Among them, the β subunit of FSH consists of 111 amino acids, the β subunit of LH consists of 121 amino acids, and the α subunits of FSH, LH, CG and TSH are exactly the same, consisting of 92 amino acids. Due to the differences in the sequences and structures of the β subunits of different glycoprotein hormones, different biological functions of different hormones are brought about. In women, FSH promotes the development and maturation of follicles, induces ovulation, inhibits ovarian atresia, and promotes the expression of LH receptors. LH produces androgen during the process of stimulating theca cells of follicles, and also provides a basis for estrogen synthesis. FSH can activate aromatase in granulosa cells to convert androgen into estrogen. Therefore, FSH and LH jointly promote the production of estrogen. In men, FSH promotes the development of spermatogenic epithelium, and synergistically with LH and androgen promotes the occurrence and maturation of sperm. LH mainly acts on Leydig cells in the testis in men, promoting Leydig cells in the testis to synthesize and release testosterone. FSH and LH play crucial roles in reproductive-related physiological processes. Therefore, FSH can be used alone or in combination with LH for the treatment of infertility. FSH and LH can be extracted from the urine of postmenopausal women, or can be produced by DNA recombinant technology. For example, the marketed recombinant FSH drug Gonalf and recombinant LH drug Luveris produced by Merck & Co., Inc. FSH and LH from urine sources have defects such as limited sources, uneven activities of crude extracts from different sources, and potential risks of infectious pathogen contamination. Recombinant FSH and LH have stable sources, better purity and uniform product quality characterization. Menotropins hMG is extracted from urine and contains follicle-stimulating hormone FSH and luteinizing hormone LH in a ratio of 1:1. As an ovulation-inducing drug, it also has the effect of treating female amenorrhea, irregular menstruation, and low quality of male germ cells. The process of ovulation induction treatment requires long-term treatment, with continuous administration for 8 - 10 days, and at least one subcutaneous injection of FSH per day. In the mid-late stage of follicular development, LH and FSH can produce a synergistic effect to promote follicular development. When the follicle is only under the action of FSH and lacks LH protection, it can lead to structural abnormalities and oocyte degeneration. Some studies have pointed out that in the superovulation cycle using the ultra-long protocol, the rate of high-quality embryos is lower when using FSH alone compared with the combined use of recombinant FSH + recombinant LH or recombinant FSH + hMG. Comparing the clinical effects of the combined use of recombinant FSH + recombinant LH or recombinant FSH + hMG in the long protocol of follicular phase ovulation induction, it was found that: the endometrial thickness, estradiol, and LH levels after treatment in both groups were increased compared with those before treatment, but there was no statistically significant difference between the two treatments (Wu Runxiang, et al., Comparison of the clinical effects of recombinant human follicle-stimulating hormone combined with recombinant human luteinizing hormone and menotropins in the long protocol of follicular phase ovulation induction [J], China Contemporary Medicine, Vol. 28, No. 23, August 2021). Therefore, the combined use of FSH and LH in clinical practice already has a relatively deep theoretical basis and application experience. However, whether it is FSH or LH extracted from urine or produced recombinantly, it has the limitation of a short half-life. The half-life of FSH is only 12 hours. Therefore, in clinical practice, it is necessary to administer by intramuscular or subcutaneous injection every day, which brings local reactions and discomfort to patients. Therefore, the development of long-acting FSH and LH drugs has practical clinical value. In the prior art, Merck of Germany launched elonva (Patents US5338835 and US5585345), which achieved the purpose of extending the half-life by fusing the CTP group of hCG to the C-terminal of the β subunit of FSH, and the half-life in humans reached 69 hours. Patent US0186662 published the constructed FSH-Fc fusion protein, and the half-life in rats was increased to 60 hours. Patent CN103509121B published the constructed FSH-Fc / Fc heterodimeric protein, in which the α subunit was free and combined with the β subunit through non-covalent bonds, having a more flexible receptor-binding region, and the half-life in rats reached 30 - 40 hours, while maintaining good FSH activity. The above findings, on the one hand, solve the problem of optimizing the efficacy of long-term ovulation induction treatment in clinical practice by combining recombinant LH or hMG, and on the other hand, solve the problem of short half-life by the fusion methods of CTP and Fc. Currently, there is no recombinant ovulation induction drug that simultaneously has the functions of FSH and LH and is long-acting. Summary of the Invention The present invention provides a fusion protein that simultaneously has the functions of FSH and LH and is long-acting. The fusion protein is composed of a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide; wherein, The first polypeptide, from the N-terminus to the C-terminus, comprises: the β subunit of FSH, the β subunit of LH, a hinge, a CH2 domain, and a CH3 domain, wherein the β subunit of FSH is directly or indirectly connected to the β subunit of LH through a linker peptide; the β subunit of LH is directly or indirectly connected to the hinge through a linker peptide; or the first polypeptide, from the N-terminus to the C-terminus, comprises: the β subunit of LH, the β subunit of FSH, a hinge, a CH2 domain, and a CH3 domain, wherein the β subunit of LH is directly or indirectly connected to the β subunit of FSH through a linker peptide, and the β subunit of FSH is directly or indirectly connected to the hinge through a linker peptide. The second polypeptide, from the N-terminus to the C-terminus, comprises: a hinge, a CH2 domain, and a CH3 domain; The third polypeptide consists of the α subunit of FSH; The fourth polypeptide consists of the α subunit of LH; There is a non-covalent interaction between the first polypeptide and the third polypeptide; there is a non-covalent interaction between the first polypeptide and the fourth polypeptide; the first polypeptide and the second polypeptide are linked by a disulfide bond. Preferably, there is a non-covalent interaction between the β subunit of FSH in the first polypeptide and the third polypeptide; there is a non-covalent interaction between the β subunit of LH in the first polypeptide and the fourth polypeptide; the first polypeptide and the second polypeptide are linked by a disulfide bond in the hinge region. Preferably, the β subunit of FSH is indirectly connected to the β subunit of LH through a linker peptide. The linker peptide is used to connect the two subunits and maintain a certain spatial distance. Commonly used linker peptides include flexible linker peptides (GGGGS) n , where n represents an integer from 1 to 4, and rigid linker peptides (EAAAK) n, where n represents an integer from 2 to 5 (Yu Jian, Sarra Setrerrahmane, Xu Hanmei, Selection and Application of Linker Peptides in Fusion Protein Design, Pharmaceutical Biotechnology, 2016, 23(3): 260-263). Those skilled in the art can obtain the linker short peptide suitable for the FSH-LH fusion protein of the present application through a limited number of experiments. Preferably, the amino acid sequence of the linker short peptide is as shown in SEQ ID NO: 2. Preferably, the hinge, CH2 domain, and CH3 domain of the first polypeptide and the second polypeptide are from IgG1, IgG2, or IgG4 antibodies. The Fc region composed of CH2-CH3 of the antibody can bind to the cell surface FcRn receptor, and the recycling mechanism mediated by FcRn enables the Fc regions of IgG1, 2, and 4 antibodies to have the effect of extending the half-life of recombinant proteins (Roland E. Kontermann. Half-life extended biotherapeutics [J] Expert Opinion on Biological Therapy. 2016 Jul; 16(7): 903-15). In a specific embodiment, the hinge, CH2 domain, and CH3 domain are from IgG4. This embodiment is only for illustration and not for limiting the sources of the hinge, CH2 domain, and CH3 domain of the present application. Those skilled in the art know that using the hinge, CH2 domain, and CH3 domain of other different types of antibodies in the present application can also extend the half-life of recombinant proteins. The positions and sequences of the CH2 domain and CH3 domain of different types of antibodies can be determined by the publicly available prior art. For example, the positions (determined according to the Kabat numbering system) and sequences of the CH2 domain or CH3 domain of IgG are as follows in the table: Preferably, the hinge in the first polypeptide and the second polypeptide contains modifications based on the natural hinge. Taking the hinge of IgG4 as an example, according to the Eu numbering rule, it contains the S228P mutation to enhance the stability of IgG4 antibodies and reduce the formation of half-antibodies. It is also possible to design the L235E or D265A mutation to further reduce the Fc effector function of IgG4 (WO9428027, US7332581), design M252Y, S253T, T256E to extend the half-life (US7083784), design S228P / L235E / R409K to reduce the aggregation problem of IgG4 at low pH (WO2006033386), design to delete G446 and K447 to reduce the C-terminal heterogeneity of IgG4 (WO2009041613), etc. When using IgG1 or IgG2, various modifications disclosed in the art can also be adopted. Preferably, the CH3 domain contains modifications that facilitate the formation of heterodimers of the first polypeptide and the second polypeptide. For example, amino acids with different side chain sizes are replaced in the Fc domain to create a "hole" in one chain and a "knob" in the paired chain, promoting the formation of heavy chain heterodimers through the interaction of KIH pairing (i.e., the knob into hole technique), such as the modifications listed in Table 5 of CN104011221A. Another example is to change the charge effect of amino acid residues in the Fc domain to create a "positive charge" in one chain and a "negative charge" in the paired chain, promoting the formation of heavy chain heterodimers through the repulsion of like charges and the attraction of opposite charges, such as the modifications listed in Table 6 of CN104011221A. In the examples of this application, the CH3 domain in the first polypeptide contains S375C and T389W mutations according to the Kabat numbering rule for forming a knob; the CH3 domain in the second polypeptide contains Y370C, T389S, L391A, and Y438V mutations for forming a hole; vice versa. This example is only for illustration and not for limiting the modification methods for promoting the heterodimerization of the first polypeptide and the second polypeptide in this application. The C-terminal deletion of the Fc domain is the main cause of C-terminal heterogeneity. During the synthesis and secretion of antibodies in mammalian cells, endogenous carboxypeptidase in the cells will excise lysine at the C-terminus of the antibody heavy chain. Incomplete excision will result in 0, 1, or 2 C-terminal lysine residues remaining in one antibody molecule. Therefore, the Fc fusion proteins in the examples of this application will have varying degrees of lysine deletion, resulting in C-terminal heterogeneity. The CH2 domain or the CH3 domain may also contain some amino acid substitutions that have been disclosed in the prior art. For example, the CH2 domain contains an N314X substitution (the position determined according to the Kabat numbering system) to remove the glycosylation site of the CH2 domain, and X is any amino acid other than Q. The above-mentioned β-subunit, LH β-subunit, and α-subunit sequences of FSH can be the same as those of natural human FSH β-subunit, LH β-subunit, and α-subunit, or can have appropriate amino acid additions, deletions, and substitutions based on the natural human FSH β-subunit, LH β-subunit, and α-subunit. In a specific embodiment, the amino acid sequence of the β subunit of FSH is as shown in SEQ ID NO: 1; the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 2; the amino acid sequence of the β subunit of LH is as shown in SEQ ID NO: 3; the α subunit of FSH is the same as the α subunit of LH, and the amino acid sequence is as shown in SEQ ID NO: 4; the amino acid sequence of IgG4 hinge-CH2-CH3 (containing S228P, S375C, T389W) is as shown in SEQ ID NO: 5; the amino acid sequence of IgG4 hinge-CH2-CH3 (containing S228P, Y370C, T389S, L391A, Y438V) is as shown in SEQ ID NO: 6. The method for preparing the above fusion protein comprises the following steps: (I). Vector construction Insert the coding gene of the first polypeptide into the AvrII and BstZ17I sites of pZHK1.3 to obtain the pZHK1.3-FSHβ-LHβ-Fc expression vector; Insert the coding gene of the third polypeptide / fourth polypeptide into the EcoRV and PacI sites of pZHK5.18, and insert the coding gene of the second polypeptide into the AvrII and BstZ17I sites to obtain the pZHK5.18-FSHα / Fc expression vector; (II). Expression in CHO-S cells Electroporate the expression vector into CHO-S cells, and screen to obtain a high-expression stable cell line by the pressure method of MTX + puromycin + hygromycin. Amplify the obtained high-expression stable mammalian cell line and inoculate it into Dynamis medium at an inoculation density of 0.5×10 6 cell / ml, and perform fed-batch culture at 37°C, 8% CO2, and 130 rpm. The feeding medium is Efficient Feed C + supplement. After 12-17 days of expression, collect the expression supernatant, filter and clarify it with 0.22 μm, and then purify it to obtain the above fusion protein. The present invention also relates to a pharmaceutical composition comprising the above fusion protein, which further comprises a pharmaceutically acceptable excipient. The present invention also relates to the use of the above fusion protein in stimulating follicular development and assisted reproduction. The fusion protein of the present invention can provide the dual hormonal functions of FSH and LH simultaneously through a single molecule, can provide support in multiple links such as follicle growth, maturation and ovulation, and the long-acting molecular design significantly prolongs the drug half-life, making the blood drug concentration more stable after entering the body, enabling the release of FSH and LH activities to be more uniform and continuous, the signal transduction after binding to the receptor to be milder, providing a more stable hormonal support environment for follicle development, and helping to reduce the adverse reactions caused by fluctuating or excessive hormone levels. During the natural development of follicles in the female body, the coordinated action of multiple hormones is required for regulation in each cycle, including FSH, LH, and downstream estrogen and progesterone. Follicle recruitment and growth mainly require FSH. Although the role of LH is weak in the early development of follicles in the natural cycle, it is also indispensable. LH increases the sensitivity of granulosa cells to FSH and promotes follicle recruitment. By measuring the expression of LH receptor (LHR) and FSH receptor (FSHR) in granulosa cells and cumulus cells of normal human follicles with diameters of 3 - 20 mm, it is found that LHR already exists in the early stage of follicle development, and the LHR level is relatively stable during follicle development, while FSHR gradually decreases as the follicle develops and enlarges. LH also has a negative selection effect on non-dominant follicles. During the development of dominant follicles, the dependence on FSH gradually decreases, and the dependence on LH gradually increases. In dominant follicles with a diameter of ≥14 mm, FSH, under the synergistic action of estrogen, induces a sudden increase in the expression of LHR in granulosa cells, ensuring that only one dominant follicle develops, matures and ovulates in each cycle. It can be seen that physiological levels of LH are obviously very important for follicle development. If the LH level is abnormal, it will lead to abnormal follicle development. In the field of assisted reproductive technology, the addition of LH drugs during ovulation induction was initially based on the two-cell two-gonadotropin theory, that is, theca cells secrete androgens under the stimulation of LH to promote follicle development. On the other hand, androgens stimulate the expression of FSHR on the surface of granulosa cells, and cooperate with IGF1 to increase follicle recruitment. LH also binds to LHR on granulosa cells to promote and maintain follicle development and estrogen secretion in the mid-follicular phase, etc. [1,2]. Low levels of LH in the body can reduce endometrial receptivity, thereby reducing the embryo implantation rate. However, in current clinical ovulation induction protocols, the main drug used is FSH. There are numerous discussions regarding the necessity of adding LH, the treatment window for addition, and the dosage of addition, but there is no unified conclusion at home and abroad. Some scholars believe that adding LH has no obvious positive significance, as endogenous LH can meet the demand. Other scholars believe that the serum LH level is not proportional to its biological activity. For patients with diminished ovarian reserve (DOR), advanced age, or slow response, the sensitivity of their LH receptors decreases. However, in the studies from which these conclusions are derived, the drug used is HMG, which contains the component hCG, and LH and hCG share receptors [3] , and it cannot be excluded that the insufficient biological activity of hMG is related to this [4] . Clinical studies have shown that the combined use of rFSH and rLH may benefit patients over 35 years old more. The results of a large RCT experiment with a ratio of FSH:LH = 2:1 showed that the addition of rLH significantly increased the implantation rate of women aged 36 - 39, and the continuous pregnancy rate also increased to a certain extent, but young patients did not benefit significantly [5] . The possible reason for the benefit in older patients may be that, on the one hand, the endocrine changes in ovarian aging are first reflected in the increased serum concentration of FSH in the early follicular phase, but this is not accompanied by an increase in LH [6] , but is accompanied by a progressive decrease in the basal androgen level [7-8] . Furthermore, the ability of follicles to synthesize androstenedione after FSH injection significantly decreases in older patients, but the ability to secrete estrogen is compensated by aromatase [9] . The added LH increases the synthesis of androgen and then the action of aromatase to synthesize estrogen

[0010] , promoting the recovery of egg quality, the improvement of embryo quality, and the implantation rate. In young patients, because the quality of their own eggs is relatively high, it may mask the supplementary effect of LH, which is also consistent with the result that the apoptosis rate of cumulus granulosa cells decreases in the LH-supplemented group,

[0011] indicating better egg quality. Based on the above basic research and clinical research background, the fusion protein of the present application, as a long-acting recombinant hormone drug with dual targets of FSH and LH, is particularly suitable for: female infertility caused by hypogonadotropic or normogonadotropic ovarian insufficiency to stimulate follicle growth; controlled ovarian hyperstimulation for inducing multiple follicle development in ART such as IVF-ICSI; controlled ovarian hyperstimulation for women with decreased ovarian reserve or premature ovarian failure in IVF-ICSI. In addition, patients with polycystic ovary syndrome (PCOS) often present with chronic anovulation, and follicular development is arrested at the small and medium follicle stages. In the ovulation induction treatment of such patients, due to the increased sensitivity to exogenous hormone stimulation, ovarian hyperstimulation syndrome is more likely to occur. The fusion protein of the present application has characteristics such as long-acting and gentle release, can provide a relatively balanced ovarian stimulation, make follicular development and maturation more gentle, reduce the reaction of patients to excessive hormone sensitivity, and help reduce the risk of ovarian hyperstimulation and reduce the inhibition of follicular development by abnormal LH peaks. Therefore, the fusion protein of the present application also has application prospects for the ovulation induction treatment of related infertility in this part of patients. The present invention also relates to the application of the above-mentioned fusion protein in diseases associated with FSH and LH deficiency. The deficiency of FSH and LH is caused by reduced production or action of gonadotropins due to internal and external factors, and these conditions are usually characterized by low sex hormones and low / normal gonadotropins (Yasmin et al., 2013; Gordon et al., 2017). Bosch et al. pointed out that in clinical practice, it was found that both congenital and acquired multi-factors could lead to the deficiency of gonadotropins LH and FSH (Bosch et al., Hum Reprod. 2021 May 17; 36(6):1469-1480). Congenital factors are mainly genetic or idiopathic causes, which are divided into two major categories according to the presence or absence of anosmia: those with olfactory impairment are called Kallmann syndrome (KS), and those with normal olfaction are called normosmic idiopathic hypogonadotropic hypogonadism (nlHH); acquired factors include inhibition of the HPG axis caused by strenuous exercise and eating disorders, abnormal secretion and action of gonadotropins caused by poorly controlled diabetes and thyroid diseases, pituitary dysfunction or pituitary dysfunction caused by compression of the tissues around the pituitary gland, etc. All these conditions meet the definition of hypogonadotropic hypogonadism (HH) by the International Committee for Monitoring Assisted Reproductive Technologies (ICMART). Regarding the treatment of hypogonadotropic hypogonadism, domestic and foreign guidelines or expert consensuses have all described the application of gonadotropins (FSH, LH). For example, in the "Expert Consensus on the Diagnosis and Treatment of Idiopathic Hypogonadotropic Hypogonadism" in China, the treatment options for male idiopathic hypogonadotropic hypogonadism are: testosterone replacement therapy, HCG / HMG combined spermatogenesis therapy, pulsatile GnRH spermatogenesis therapy; the treatment options for female idiopathic hypogonadotropic hypogonadism are: estrogen and progesterone replacement therapy, ovulation induction therapy. In the "EAU Guidelines on Male Hypogonadism (2018)" of the European Association of Urology, for male secondary hypogonadism caused by pituitary or hypothalamic dysfunction, the treatment principle for patients with fertility requirements is to supplement the deficiency of gonadotropins through FSH and LH analogs.In the "Society for endocrinology Guidelines for understanding diagnosing and treating fe male hypogonadism(2024)" of the British Society for Endocrinology, for the treatment of female hypogonadotropic or central hypogonadism, it is recommended to induce ovulation through gonadotropin (FSH and LH) treatment, and this treatment is also applicable to hypothalamic amenorrhea and the like. In addition, studies have shown that the replacement therapy of FSH and LH can play roles in inducing pubertal development / maturation, inducing fertility, etc. for congenital or idiopathic hypogonadotropic hypogonadism (Young, et al. Clinical management of Congenital hypogonadotropic hypogonadism. Endocr Rev. 2019). The fusion protein of the present invention simultaneously supplements two hormones, FSH and LH, and can supplement gonadotropin in a long-acting and mild and continuous manner, and is applicable to various diseases related to FSH and LH deficiencies exemplified above. For example, for patients with FSH and LH deficiencies such as male hypogonadotropic hypogonadism, it can respectively stimulate Sertoli cells and Leydig cells in the testis, promote the recovery of male spermatogenesis and testosterone secretion, and the characteristics of long-acting, mild release and activation of receptors help to avoid excessive testosterone or imbalance of Sertoli cells, and reduce the risk of negative feedback inhibition caused by excessive testosterone secretion, and the safety is more guaranteed. For patients with FSH and LH deficiencies such as female pituitary insufficiency, hypogonadotropic amenorrhea or hypogonadotropic infertility, the fusion protein of the present invention simultaneously provides exogenous FSH and LH supplementation, which helps to maintain normal follicular dynamics, achieve oocyte maturation and ovulation, reduce the possible low estrogen or hormonal disorders caused by simple FSH stimulation, and the characteristics of long-acting, slow release and promotion of target cell proliferation make the hormone stimulation more mild, and the follicular development process is more natural. The stable LH level helps to reduce the risk of overstimulation caused by the LH peak. For congenital hypogonadotropic hypogonadism, it can stably and long-acting provide LH and FSH activities closer to physiological stimulation in vivo, improve the deficiency of endogenous gonadotropin, help male testosterone secretion and spermatogenesis or female follicular maturation and estrogen synthesis, induce pubertal development, and improve fertility, etc. In addition, during perimenopause (menopause), due to the sharp decline in ovarian reserve, the estrogen synthesis ability is significantly reduced, which in turn leads to a series of physiological and psychological symptoms, namely menopausal syndrome. At the same time, the responsiveness of the ovaries of women with menopausal syndrome to FSH and LH stimulation also decreases, further exacerbating estrogen deficiency. For patients in perimenopause or menopause with remaining follicle reserves, exogenous supplementation of FSH and LH is theoretically expected to promote the synthesis of estrogen by residual follicles and improve some menopausal symptoms. The present invention provides a novel long-acting FSH-LH-Fc / Fc heterodimer or LH-FSH-Fc / Fc heterodimer, in which the α subunit combines with the FSHβ and LHβ subunits respectively through non-covalent interactions, and can simultaneously possess the functions of FSH and LH, with better activity. At the same time, the heterodimer has a longer half-life compared to recombinant or natural FSH and LH, and only one administration is required in clinical treatment to achieve the therapeutic effect of multiple administrations of combined FSH and LH or hMG, with better drug economy and better patient compliance. Brief Description of the Drawings Figure 1: Schematic diagram of the fusion protein structure Figure 2: Non-reducing SDS-PAGE of the purified FSH-LH monomer sample Figure 3: Non-reducing SDS-PAGE of the purified LH-FSH monomer sample Figure 4: Non-reducing SDS-PAGE of the one-step purified FSH-Fc / LH-Fc heterodimer sample Figure 5: Non-reducing SDS-PAGE of the one-step purified FSH-LH-Fc / Fc heterodimer sample Figure 6: SDS-PAGE pattern of the FSH-LH-Fc / Fc heterodimer. Among them, 1 is the reduced gel sample after boiling; 2 is the non-reduced gel sample after boiling; 3 is the non-reduced gel sample without boiling; M is the marker. Figure 7: In vitro activity of the FSH-LH-Fc / Fc heterodimer (FSH single reporter gene method) Figure 8: In vitro activity of the FSH-Fc / LH-Fc heterodimer (LH single reporter gene method) Figure 9: Among them, Figures 9a and 9b are the plasma concentration-time curves of the FSH-LH-Fc / Fc heterodimer and Gonal-F in rats respectively Figure 10: Downstream gene expression after KGN cells are treated with FSH-LH-Fc / Fc Figure 11: HE staining results of Example 10. Among them, Figures 11a, 11b, and 11c are the HE staining results of the solvent group, Gonal-F (6.4 nM) group, and FSH-LH-Fc (6.4 nM) group respectively Figure 12: Ovarian weight per 10 g body weight of each group of animals in Example 10 Figure 13: HE staining results of Example 11, where Figures 13a, 13b, and 13c are the HE staining results of the solvent group, FSH-LH-Fc (51.2 nM) group, and Luvelle group, respectively Figure 14: Seminal vesicle weight per 10 g body weight of animals in each group of Example 11 Figure 15. HE / IHC staining of ovaries of SD rats in Example 12 Figure 16. Statistics of the number of ovarian follicles and the thickness of cell layers in SD rats in Example 12 Figure 17. cAMP detection in CHO cells of Example 13 Figure 18. Bulging part of the ovarian ampulla of ICR mice in Example 14 Figure 19. Blastocysts obtained by in vitro culture of ICR mice in Example 15 Figure 20. Summary of hormone change levels during superovulation in cynomolgus monkeys in Example 17 Figure 21. Oocyte retrieval results in cynomolgus monkeys in Example 17 Detailed implementation manners Technical terms Naturally occurring IgG antibodies are tetramers that contain at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of four domains CH1, hinge region, CH2, and CH3, and the Fc fragment corresponds to CH2 and CH3 of the heavy chain. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain variable region (VH) and the light chain variable region (VL) are responsible for antigen recognition, especially the complementarity-determining regions (CDRs) therein, which are usually specific for different epitopes of the antigen. The constant region is mainly responsible for effector functions. IgG is further divided into four subclasses, IgG1, IgG2, IgG3, and IgG4. Their main structural differences are as follows: ① The hinge regions are different. The hinge regions of IgG1, IgG2, IgG3, and IgG4 contain 15, 12, 62, and 12 amino acids, respectively; ② The number and position of disulfide bonds are different. The Fc regions of different subclasses of IgG can all bind to the FcRn receptor on the cell surface, and the recycling mechanism mediated by FcRn makes the Fc region have the effect of prolonging the half-life. "Non-covalent interaction" refers to an interaction that maintains a certain spatial structure between or within molecules by means of a force with a dispersed change. Different from covalent bonds, electrons are not shared during non-covalent interactions. Non-covalent interactions can be further divided into: electrostatic interaction, Π-conjugation effect, van der Waals force, and hydrophobic effect. Pierce, J.G. and Parsons, T.F. Glycoprotein hormones: structure and function. Annu. Rev. Biochem., 1981, 50, 465-495, page 466, first paragraph states that "Pituitary gonadotropins include FSH, LH... Each hormone consists of two peptide chains or subunits, named α and β respectively... Hormone activity is only generated after a strong and specific non-covalent interaction between the α and β subunits", the last paragraph on page 478 states that "Hormone activity is only generated after the formation of the α-β complex", and "The receptor-binding domain can only act on the two subunits after the formation of the α-β complex". The recombinant FSH-LH-Fc / Fc or LH-FSH-Fc / Fc heterodimers of the present application show good FSH activity and LH activity both in vivo and in vitro (such as in Examples 5, 6, 7, 10, 11, etc.). Combining with the characterization data (such as in Examples 3, 4, etc.), it can be proved that in the recombinant FSH-LH-Fc / Fc or LH-FSH-Fc / Fc heterodimers of the present application, there is a non-covalent interaction between the FSHβ subunit and the α subunit of chain 1 (i.e., the first polypeptide), and there is a non-covalent interaction between the LHβ subunit and the α subunit. In addition, in the present application, FSHβ-LHβ-Fc (or LHβ-FSHβ-Fc) and FSHα (or LHα) are respectively expressed and translated into different peptide chains through different expression elements, and they are in a free state by themselves, and no covalent connection forms such as disulfide bonds will be generated between the chains naturally. The necessary condition for the expression product of the present application to produce active FSH or LH is that the β and α subunits of FSH and LH have completed spontaneous assembly to form a complete structure. Example 1 Construction of expression vectors for recombinant FSH-LH, LH-FSH, FSH-Fc / LH-Fc heterodimers, and FSH-LH-Fc / Fc heterodimers Modification of expression vector: The commercial vector pCHO1.0 (Thermo A13696-01) was purchased and amplified in our company, named pZHK1.3. There is an expression cassette 1 between the AvrII (5764bp) and BstZ17I (5775bp) sites, and an expression cassette 2 between the EcoRV (7833bp) and PacI (7845bp) sites in the vector. The vector contains a puromycin resistance gene (at positions 8860-9459bp) between AscI (8699bp) and KpnI (9824bp). The puromycin resistance gene between AscI and KpnI was replaced with a hygromycin resistance gene. The new gene fragment was synthesized by an outsourcing company. The puromycin resistance gene in the pZHK1.3 vector was replaced with a hygromycin resistance gene by restriction enzyme digestion and ligation with T4 ligase. The modified vector was named pZHK5.18. According to the search in UniProtKB, the amino acid sequences of the FSHα subunit (P01215), FSHβ subunit (P01225), LHβ subunit (P01229), and the hinge-CH2-CH3 sequence of the IgG4 heavy chain constant region (P01861) were obtained. In the following fusion proteins with different structures, the amino acid sequence of the β subunit of FSH is as shown in SEQ ID NO: 1; the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 2; the amino acid sequence of the β subunit of LH is as shown in SEQ ID NO: 3; the α subunits of FSH and LH are the same, and the amino acid sequence is as shown in SEQ ID NO: 4; the amino acid sequence of IgG4 hinge-CH2-CH3 (containing S228P, S375C, T389W) is as shown in SEQ ID NO: 5; the amino acid sequence of IgG4 hinge-CH2-CH3 (containing S228P, Y370C, T389S, L391A, Y438V) is as shown in SEQ ID NO: 6. 1) Design of FSH-LH monomer (the structural schematic diagram is shown in Figure 1A): The first chain: From the N-terminus to the C-terminus, it sequentially contains: FSHβ subunit - linker peptide - LHβ subunit. The amino acid sequence of the first chain is as shown in SEQ ID NO: 7. The second chain: FSHα subunit. After adding signal peptides to the above sequences, they were respectively synthesized by total gene synthesis. The second chain was inserted into the EcoRV and PacI sites of pZHK1.3 by restriction enzyme digestion and ligation, and then the first chain was inserted into the AvrII and BstZ17I sites of pZHK1.3 to obtain the pZHK1.3-FSH-LH expression vector. The predicted molecular weight of the target protein is about 50KD. 2) Design of LH-FSH monomer (the structural schematic diagram is shown in Figure 1B): The first chain: sequentially contains from the N-terminus to the C-terminus: LHβ subunit - linker peptide - FSHβ subunit. The amino acid sequence of the first chain is as shown in SEQ ID NO: 8. The second chain: FSHα subunit. After adding signal peptides to the N-terminus of the above sequences, they were respectively synthesized by total gene synthesis. The second chain was inserted into the EcoRV and PacI sites of pZHK1.3 by enzymatic digestion and ligation, and then the first chain was inserted into the AvrII and BstZ17I sites of pZHK1.3 to obtain the pZHK1.3-LH-FSH expression vector. The predicted molecular weight of the target protein is about 50 KD. 3) Design of FSH-Fc / LH-Fc heterodimer (the structural schematic diagram is shown in Figure 1C): The first chain: sequentially contains from the N-terminus to the C-terminus: FSHβ subunit - linker peptide - IgG4 hinge - CH2 - CH3. According to the Eu numbering rule, the hinge region contains S228P, which is used to enhance the stability of IgG4 antibody and reduce the formation of half antibodies (Rodolfo Bianchini et al. The Role of IgG4 in the Fine Tuning of Tolerance in IgE-Mediated Allergy and Cancer [J]. Int J Mol Sci. 2020, 16; 21(14):5017); according to the Kabat numbering rule, the CH3 domain contains S375C and T389W mutations, which are used to form the knob configuration (A. Margaret Merchant et al. An efficient route to human bispecific IgG [J]. Nature biotechnology (1998), 16(7):677 - 681). The amino acid sequence of the first chain is as shown in SEQ ID NO: 9. The second chain: sequentially contains from the N-terminus to the C-terminus: LHβ subunit - linker peptide - IgG4 hinge - CH2 - CH3. According to the Eu numbering rule, the hinge region contains S228P; according to the Kabat numbering rule, the CH3 domain contains Y370C, T389S, L391A, and Y438V mutations, which are used to form the hole configuration and can form a knob in hole structure with the knob configuration. The amino acid sequence of the second chain is as shown in SEQ ID NO: 10. The third chain: FSHα subunit. The above sequences were respectively subjected to total gene synthesis. The third strand was inserted into the EcoRV and PacI sites of pZHK1.3 by restriction enzyme digestion and ligation, and then the first strand was inserted into the AvrII and BstZ17I sites of pZHK1.3 to obtain the pZHK1.3-FSH-Fc expression vector. The third strand was inserted into the EcoRV and PacI sites of pZHK5.18, and then the second strand was inserted into the AvrII and BstZ17I sites to obtain the pZHK5.18-LH-Fc expression vector. The predicted molecular weight of the target protein was approximately 100 KD. 4) Design of FSH-LH-Fc / Fc heterodimer (the structural schematic diagram is shown in Figure 1D): The first strand: sequentially contains from the N-terminus to the C-terminus: FSHβ subunit - linker peptide - LHβ subunit - IgG4 hinge - CH2 - CH3 (the hinge region contains S228P according to the Eu numbering rule, and the CH3 domain contains S375C and T389W mutations according to the Kabat numbering rule). The amino acid sequence of the first strand is as shown in SEQ ID NO: 11. The second strand: IgG4 hinge - CH2 - CH3 (the hinge region contains S228P according to the Eu numbering rule, and the CH3 domain contains Y370C, T389S, L391A, and Y438V mutations according to the Kabat numbering rule). The third strand: FSHα subunit. The above sequences were respectively subjected to total gene synthesis. The first strand was inserted into the AvrII and BstZ17I sites of pZHK1.3 by restriction enzyme digestion and ligation to obtain the pZHK1.3-FSHβ-LHβ-Fc expression vector. The third strand was inserted into the EcoRV and PacI sites of pZHK5.18, and then the second strand was inserted into the AvrII and BstZ17I sites to obtain the pZHK5.18-FSHα / Fc expression vector. The predicted molecular weight of the target protein was approximately 100 KD. In addition, the inventor also expressed the LH-FSH-Fc / Fc heterodimer. Its construction, preparation, purification, etc. can refer to the FSH-LH-Fc / Fc heterodimer. The difference between it and the above FSH-LH-Fc / Fc heterodimer is only that the fusion order of FSH and LH in the first strand is reversed, that is, it sequentially contains from the N-terminus to the C-terminus: LHβ subunit - linker peptide - FSHβ subunit - IgG4 hinge - CH2 - CH3. Process research shows that this design form has process feasibility in terms of purity and yield, and its effects in terms of in vivo and in vitro activities and long-acting properties are similar to those of FSH-LH-Fc / Fc. In the specific examples of this application, the FSH-LH-Fc / Fc heterodimer is taken as an example for description. Example 2 Expression of Recombinant FSH-LH, LH-FSH, FSH-Fc / LH-Fc Heterodimer, and FSH-LH-Fc / Fc Heterodimer The expression vector was electrotransfected into CHO-S cells, and high-expression stable cell lines were obtained by screening with different pressure methods. The vector electrotransfection combinations and pressure methods are shown in the following table: The obtained high-expression stable mammalian cell lines were amplified and then inoculated into Dynamis medium (purchased from Thermo Fisher, A2617501) at an inoculation density of 0.5×10 6 cell / ml, and batch feeding perfusion culture was carried out at 37°C, 8% CO2, and 130 rpm. The feeding medium was Efficient Feed C+supplement (purchased from Thermo Fisher, A250310). After 12-17 days of expression, the expression supernatant was collected, filtered and clarified with 0.22 μm, and then purified. Example 3 Purification of Recombinant FSH-LH, LH-FSH, FSH-Fc / LH-Fc Heterodimer, and FSH-LH-Fc / Fc Heterodimer 1) Purification of Recombinant FSH-LH and LH-FSH First, the CaptureSelect FSH affinity chromatography column (Thermo) was equilibrated with 20 mM Tris-HCl, pH 7.4. The feed solution was loaded onto the chromatography column, and then the column was washed with 20 mM Tris-HCl, pH 7.4. Finally, elution was carried out with 20 mM Tris-HCl, 2M MgCl2, pH 7.4, and the elution peak was collected. The non-reducing SDS-PAGE results of the purified FSH-LH monomer sample (Figure 2) and the non-reducing SDS-PAGE results of the purified LH-FSH monomer sample (Figure 3) showed that the size of the target band was correct, but the yield (mg of purified sample output ÷ fermentation scale L) was low, only 2-3 mg / L, and the purity was low, with many impurity proteins. It was difficult to effectively purify the sample through subsequent ion exchange chromatography (AEX, CEX) process exploration and difficult to scale up. 2) Purification of Recombinant FSH-Fc / LH-Fc Heterodimer: First, equilibrate the MabSelect Prism chromatography column (Cytiva) with 50 mM Tris-HCl, pH 7.4. Load the feed solution onto the column, then wash the column with 50 mM sodium acetate, 1 M NaCl, pH 5.5, and finally elute the protein with 50 mM sodium acetate, pH 3.5. After one-step purification, the non-reducing SDS-PAGE results are shown in Figure 4. It can be seen that there are multiple bands around 100 - 130 KD and an obvious presence of semi-products around 50 KD. It is judged that either FSH-Fc or LH-Fc may have overexpressed or underexpressed, resulting in the inability to correctly pair and form heterodimers, thus generating a large amount of semi-products and homodimers (the activity analysis results of Example 5 show that the semi-products generated are FSH-Fc monomers and the homodimers are FSH-Fc homodimers). Since the molecular weights of FSH-Fc and LH-Fc are relatively close, it is difficult to distinguish them from the electrophoresis results. This sample could not be effectively separated into heterodimers and homodimers after subsequent exploration of hydrophobic chromatography (HIC), ion exchange chromatography (AEX, CEX), or size exclusion chromatography processes. 3) Purification of recombinant FSH-LH-Fc / Fc heterodimer: First, equilibrate the Mabselect Prism chromatography column (Cytiva) with 50 mM Tris-HCl, pH 7.4. Load the feed solution onto the column, then wash the column with 50 mM sodium acetate, 1 M NaCl, pH 5.5, and finally elute the protein with 50 mM sodium acetate, pH 3.5. After one-step purification, the non-reducing SDS-PAGE results are shown in Figure 5. It can be seen that the sample shows three bands under non-reducing conditions, namely FSH-LH-Fc homodimer, FSH-LH-Fc / Fc heterodimer, and Fc dimer. Due to the large difference in molecular weights, these three bands can be easily judged from the electrophoresis results. The pH of the sample after affinity chromatography was adjusted to 8.0, and (NH4)2SO4 was added to the sample to a final concentration of 1.0 M. The sample was clarified by filtration through a 0.22 μm filter for use. A Phenyl HP hydrophobic chromatography column (Cytiva) was equilibrated with 20 mM Tris-HCl, 1.0 M (NH4)2SO4, pH 8.0. The sample was loaded onto the chromatography column and eluted with 20 mM Tris-HCl, pH 8.0, and the elution peak was collected. The non-reduced result of the sample after two-step purification is shown in lane 3 of Figure 6. It can be seen that the purified target FSH-LH-Fc / Fc heterodimer band is single, about 100 KD in size, and has a high purity. The reduced SDS-PAGE result of the sample after boiling is shown in lane 1 of Figure 6. It can be seen that three bands appear after the sample is boiled and reduced. The size of FSHβ-LHβ-Fc is about 63 KD, the Fc monomer is about 27 KD, and the FSHα subunit is about 20 KD. In the non-reduced and boiled state of the FSH-LH-Fc / Fc heterodimer, the non-covalent bond between the α subunit and the β subunit is opened, and the detached α subunit can be seen, and the result is shown in lane 2 of Figure 6. Therefore, the construction of the FSH-LH-Fc / Fc heterodimer is easier to express and correctly assemble than the FSH-Fc / LH-Fc heterodimer, and during the purification process, the content of different by-products can be conveniently judged from the non-reduced electrophoresis of SDS-PAGE by the molecular weight difference, and it can guide the optimization of the purification process. After two-step purification of the sample, a high-purity FSH-LH-Fc / Fc heterodimer protein can be obtained. The yield of the small-scale process reaches about 10 mg / L. The dosage of the long-acting FSH-LH preparation in clinical practice is extremely low. Therefore, after subsequent process optimization, this molecule has the feasibility of further scale-up production. Example 4 Structural Characterization of Recombinant FSH-LH-Fc / Fc Heterodimer I. Reducing and Deglycosylating Molecular Weight 1. Analytical method: (1) Buffer replacement treatment: Take 200 μg of the sample into an activated 10 kDa ultrafiltration tube and perform buffer replacement treatment, repeating 3 times. (2) A280 absorbance measurement: Use Nanodrop to measure the A280 absorbance of the sample after buffer replacement, with 100 mM Tris-HCl (pH 7.5) buffer as the blank control. The A280 absorbance of the sample is 1.533. (3) Denaturation treatment: Take about 10 μg of the sample (6 μl), place it in a 1.5 ml centrifuge tube, add 44 μl of 100 mM Tris-HCl (pH 7.5) buffer and 5 μl of RapiGest denaturing agent, vortex and mix well, then place it in a dry bath thermostat and incubate at 90.0 °C for 20 min. (4) Reduction and deglycosylation treatment: Add 0.6 μl of 1 M DTT solution and 0.5 μl of PNGaseF deglycosylase to the denatured sample. After vortexing and mixing, place it in a dry bath incubator and incubate at 37.0 °C for 2 h. (5) Acidification treatment: After the reduction and deglycosylation are completed, add 1 μl of FA to the reaction system to make its final concentration about 2%, and terminate the deglycosylation reaction. (6) Mass spectrometry detection: Inject the supernatant after centrifuging the acidified sample at 12000 rpm for 5 min for analysis. High performance liquid chromatography detection parameters: Mass spectrometer detection parameters: thermo Q Exactive mass spectrometer, ESI ion source, positive ion mode, Full MS - SIM detection mode. 2. Analysis results: The measured value of 10206.68 Da is consistent with the theoretical molecular weight of the 3rd chain; 25466.12 Da is consistent with the theoretical molecular weight corresponding to the loss of K at the C - terminus of the 2nd chain; the measured value of 52344.14 Da is consistent with the theoretical molecular weight corresponding to the loss of K at the C - terminus of the 1st chain. Summary of molecular weight detection results II. Amino acid sequence coverage 1. Analysis method: (1) Sample pretreatment Replacement and concentration: Take 400 μl of the sample, ultrafilter and replace it 3 times with 100 mM Tris - HCl, pH 7.5 buffer solution. Use Nanodrop to measure the A280 absorbance value of the sample after replacing the buffer solution, and use 100 mM Tris - HCl (pH 7.5) buffer solution as the blank control. Denaturation: Take 12.5 μg of protein into a 1.5 ml centrifuge tube, fix the volume to 50 μl, add 5 μl of 10% RepiGest SF denaturant, and denature at 90 °C for 20 min; Enzymatic digestion treatment: Take the denatured sample solution, add Trypsin or Chymotrypsin protease and PNGaseF, vortex and mix, then place it in a dry bath incubator and incubate at 37.0 °C for 16 h. Reduction treatment: After enzymatic digestion is completed, take 25 μl of each enzymatic digestion solution and place it in a 0.5 ml centrifuge tube. Add 0.3 μl of 1 M DTT solution to make its final concentration not less than 10 mM. After vortexing and mixing, place it in a dry bath incubator and incubate at 56.0 °C for 30 min. Alkylation treatment: Add 0.7 μl of 1 M IAM solution to the reduced sample to make its final concentration not less than 20 mM. After vortexing and mixing, place it in the dark at room temperature for 45 min. (2) Mass spectrometry detection: a. Non-reduced sample: Take 25 μl of the enzymatically digested sample into an injection vial, with an injection volume of 18 μl; b. Reduced sample: Transfer the alkylated sample to an injection vial and prepare for injection. The injection volume is 18 μl. High-performance liquid chromatography detection parameters: Mass spectrometer detection parameters: Thermo Q Exactive mass spectrometer, ESI ion source, positive ion mode, Full MS-dd-MS2 / dd-SIM detection mode. 2. Analysis results: Under the trypsin digestion method, the amino acid sequence coverage rate of the first chain is 99.4%, the second chain is 98.7%, and the third chain is 100%. III. K-loss modification 1. Analysis method: The same as amino acid coverage 2. Analysis results: The K-loss modification ratio of the first chain / second chain is 100%. Detection results of sample K-loss modification IV. Disulfide bonds 1. Analysis method: The same as amino acid coverage 2. Analysis results: 3 pairs of inter-chain disulfide bonds between the first chain and the second chain were identified, meeting expectations. Disulfide bond identification results Example 5 In vitro activity study of recombinant FSH-Fc / LH-Fc heterodimer and FSH-LH-Fc / Fc heterodimer (reporter gene method) (I) CHO cells recombinantly expressing FSH receptor (FSHR) and LH receptor (LHR) respectively: FSHR-CRE-Luc-CHO-K1 and LHR-CRE-Luc-CHO-K1 (constructed by the company itself), to detect the relative binding activity of the sample to the cell surface receptor FSHR or LHR. The cultured cells were prepared according to 5×10 4The cells / well cell density was spread on a 96-well plate (white plate), and the standard solution (commercial reagent) and the test solution (FSH-Fc / LH-Fc heterodimer and FSH-LH-Fc / Fc heterodimer of Example 1) were diluted to a starting concentration of 500 ng / mL with an appropriate amount of test culture medium; then a 3-fold gradient dilution was performed, with a total of 9 dilutions; 100 μL of the standard solution or the test solution after the gradient dilution was added to the 96-well plate cells, and cultured at 37.0°C and 5.0% CO2 for 4-6 hours; the cell culture plate to be tested was taken out and placed at room temperature (25°C±3°C) for 15 minutes to balance the culture plate temperature to room temperature, and the Bio-Lite luciferase reagent was balanced to room temperature at the same time; the cell supernatant was discarded, and 100 μL of the Bio-Lite luciferase reagent was added to each well; chemiluminescence detection was performed using a multifunctional microplate reader (25°C±3°C), and the luminescence intensity value was recorded. The logarithm of the molar concentration of the test sample and the standard was used as the horizontal axis, and the average fluorescence response was used as the vertical axis to draw a logistic four-parameter curve to calculate the half effective concentration (EC 50 ). The results in Figures 7 and 8 and the table below show that the recombinant FSH-LH-Fc / Fc heterodimer has equivalent biological activity to recombinant FSH (GONA-F) and recombinant LH (LERI) in vitro, respectively. 50 Close. Recombinant FSH-Fc / LH-Fc heterodimers have poor activity. This indicates that the recombinant FSH-LH-Fc / Fc heterodimer design is an ideal design, which has the biological functions of both FSH and LH. Example 6 Effect of FSH-LH-Fc / Fc on Progesterone Production in KGN Cell Line 1. Grouping and Experimental Design Human ovarian granulosa cells KGN express FSH receptors. Under the action of FSH, KGN can secrete the steroid hormone progesterone. Based on the KGN cell line, under the condition of cell adherence culture, the test sample and blank group were given in 1% fetal bovine serum respectively. After 48 hours of culture, the progesterone production in the cell supernatant was detected using an ELISA kit. The experiment was divided into 2 groups. KGN were subcultured into 6-well plates, with 5*10 4 After 24 hours of attachment, FSH-LH-Fc / Fc was added to the test group at final concentrations of 1.16 nM and 0 nM. After 48 hours of treatment, the cell supernatant was collected and tested by ELISA. ELISA detection method (according to the kit instructions): Sample addition: Set up standard wells and sample wells, 50uL per standard well, 40uL per sample well, and 10uL per sample well; Add enzyme: Add 100 uL of enzyme-labeled reagent to each well. Seal the plate with a sealing film and incubate at 37 °C for 60 minutes; Prepare the washing solution and wash 5 times with the washing solution; Color development: First add 50 uL of chromogenic agent A to each well, then add 50 uL of chromogenic agent B, and develop color at 37 °C in the dark for 15 minutes; Termination: Add 50 uL of termination solution to each well; Reading: Read with an enzyme-labeled instrument at wavelengths of 450 nm and 630 nm, record and save the data for analysis; Use OriginPro software for non-linear fitting of the standard curve and back-calculation of concentration. II. Results The progesterone concentration in the supernatant of KGN cells is shown in the following table: III. Conclusions The above results indicate that FSH-LH-Fc / Fc has FSH activity in vitro and can promote the secretion of the steroid hormone progesterone by KGN cells expressing the FSH receptor. Example 7 FSH-LH-Fc / Fc Promotes the Expression of Genes Downstream of FSH and LH Receptors in the KGN Cell Line I. Grouping and Experimental Design Human ovarian granulosa cells KGN have the natural property of expressing FSH receptors and LH receptors and can be used to detect the activation of FSH receptors, LH receptors, and their downstream signaling pathways by FSH-LH-Fc / Fc. Based on the KGN cell line, under adherent cell culture conditions, high concentration (4.8 nM), low concentration (0.6 nM) of FSH-LH-Fc / Fc, and a blank group were given in 1% fetal bovine serum. After treatment for 48 hours, relative quantitative detection of the expression of specific genes in the qPCR transcriptome was performed. II. Results After treatment of KGN cells with the FSH-LH-Fc / Fc recombinant protein, the expression of FSH receptors, LH receptors, and their downstream signaling pathways is shown in Figure 10. The FSH-LH-Fc recombinant protein has FSH and LH activities in vitro and can promote the activation of downstream gene expression of FSH and LH by KGN cells expressing FSH receptors and LH receptors. Some genes did not show a dose-dependent effect, which may be due to an over-inhibitory effect. Example 8 The Drug Metabolism of FSH-LH-Fc / Fc Heterodimer in Rats Has Long-Term Efficacy I. Grouping and Experimental Design Based on adult female and male SD rats, the control or test article was administered subcutaneously, and the drug metabolism in the plasma of animals within 144 hours after the last dose was observed. A total of 2 groups were designed for the experiment, with 4 animals in each group, 2 males and 2 females. The groups were the Gonal-F group and the FSH-LH-Fc / Fc heterodimer group. Solvent: 1 mg / ml BSA, 0.9% sodium chloride. Gonal-F group: initial concentration of 44 μg / ml (600 IU / ml), diluted with the solvent to 3.6 μg / ml (50 IU / ml, 160 nM) as the control group. Prepare 160 nM FSH-LH-Fc / Fc heterodimer test article as the test article group. Both groups were administered once at a dose of 0.64 nmol / kg. Blood was collected from the orbital cavity at time points of 0 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, and 144 h after dosing. After coagulation, plasma was collected and stored at -20°C for ELISA detection at a later date. ELISA detection method: 1. Enzyme-linked immunosorbent assay (ELISA) plate coating: Anti-FSH antibody (14) 2 μg / ml + anti-LH antibody 2 μg / ml (1X PB, pH = 7.4) was used for detecting the plasma drug concentration of the FSH-LH-Fc / Fc heterodimer group, and anti-FSH antibody (06) 4 μg / ml (1XPB, pH = 7.4) was used for detecting the plasma drug concentration of the Gonal-F group, incubated overnight at 4°C. 2. Blocking: The incubated ELISA plate was washed once with the washing solution, the microplate was spun dry with a dehydrator, and 200 μl / well of blocking solution (2% BSA in PBST) was added and incubated at 37°C for 2 h for blocking. 3. Sample addition: After discarding the blocking solution in the wells of the blocked ELISA plate, 100 μl / well of the prepared samples were added to the ELISA plate, including the standard curve (concentration gradients were all 32 ng / ml, 16 ng / ml, 8 ng / ml, 4 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0 ng / ml, prepared with SD rat plasma and diluted 10-fold with the blocking solution), and serum samples (diluted 10-fold with the blocking solution), incubated at 37°C at 300 rpm for 2 h. 4. Detection of the analyte: The ELISA plate after incubating the samples was washed 3 times with the washing solution, and 100 μl / well of the prepared analyte was added to the ELISA plate for incubation, incubated at 37°C at 300 rpm for 1 h. The analyte for the Gonal-F group was anti-FSH (14) antibody-HRP, prepared at 1:200 in the blocking solution, and the analyte for the FSH-LH-Fc / Fc heterodimer group was abcam mouse anti-human IgG4pFc HRP, prepared at 1:30000 in the blocking solution. 5. Color development and termination of the reaction: The ELISA plate after incubation was washed 3 times with the washing solution, and 100 μl / well of TMB color development solution equilibrated at room temperature was added to the ELISA plate for light-shielded incubation at 25°C for 10 min. After color development was completed, 50 μl / well of the termination solution was used for termination. 6. Reading: Use an enzyme-linked immunosorbent assay (ELISA) reader to read at wavelengths of 450 nm and 630 nm, record and save the data for analysis. 7. Use OriginPro software for non-linear fitting of the standard curve and back-calculation of concentration, and use GraphPad Prism5 for plotting the curve graph and data analysis. II. Results The plasma drug concentrations of the FSH-LH-Fc / Fc heterodimer group and the Gonal-F group are shown in Figure 9 and the following table: III. Conclusion The above results show that the drug metabolism cycle of the FSH-LH-Fc / Fc heterodimer of this application is longer than that of the control drug after a single administration in adult rats. Its drug half-life is about 40 h, while the control drug Gonal-F is only 10 h. The fusion protein of this application has long-acting properties, and the drug release is more gentle and continuous. Example 9 The drug metabolism of FSH-LH-Fc / Fc heterodimer in cynomolgus monkeys has long-acting properties I. Grouping and experimental design Based on adult female cynomolgus monkeys, inject the control product or the test product subcutaneously, and observe the drug metabolism in the plasma of the animals within 336 hours after the last administration. The experiment was designed into 3 groups, with 2 female animals in each group. The groups were the low-dose group, medium-dose group, and high-dose group of FSH-LH-Fc / Fc heterodimer. The solvent is 1 mg / ml BSA and 0.9% sodium chloride. The FSH-LH-Fc / Fc heterodimer is used as the test product, which is divided into three groups: the low-dose group, medium-dose group, and high-dose group, with doses of 10 μg / kg, 40 μg / kg, and 120 μg / kg respectively. There are two cynomolgus monkeys in each group, and the drug is administered 2 times according to the dose. Timing is carried out according to the drug administration time points: 0 h, 2 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 168 + 2 h, 168 + 6 h, 168 + 12 h, 168 + 24 h, 168 + 48 h, 168 + 72 h, 168 + 96 h, 168 + 120 h, 168 + 120 h, 168 + 168 h. Blood is taken intravenously. After coagulation, the plasma is collected and stored at -20°C, and ELISA detection is carried out at a selected time. ELISA detection method: 1. Coating of ELISA plate: 2 μg / ml anti-FSH antibody + 2 μg / ml anti-LH antibody (1X PB, pH = 7.4) is used for detecting the serum drug concentration of the FSH-LH-Fc / Fc heterodimer group. 2. Blocking: Wash the incubated enzyme-linked immunosorbent assay (ELISA) plate once with the washing solution, spin-dry the microplate using a dehydrator, and add 200 μl / well of blocking solution (2% BSA in PBST) for incubation at 37°C for 2 h. 3. Sample addition: Discard the blocking solution in the blocked ELISA plate, and add 100 μl / well of the prepared samples to the ELISA plate, including the standard curve (concentration gradients are 32 ng / ml, 16 ng / ml, 8 ng / ml, 4 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0 ng / ml, prepared with cynomolgus monkey serum and diluted 10-fold with the blocking solution), and serum samples (diluted 10-fold, 20-fold, and 100-fold with the blocking solution), and incubate at 37°C at 300 rpm for 2 h. 4. Detection reaction: Wash the ELISA plate with the incubated samples three times with the washing solution, add 100 μl / well of the prepared detection reagent to the ELISA plate for incubation, and incubate at 37°C at 300 rpm for 1 h. The detection reagent for the FSH-LH-Fc / Fc heterodimer group is abcam mouse anti-human IgG4 pFc HRP, prepared at 1:30000 in the blocking solution. 5. Color development and termination reaction: Wash the incubated ELISA plate three times with the washing solution, add 100 μl / well of TMB color development solution equilibrated at room temperature to the ELISA plate for light-shielded incubation at 25°C for 10 min. After color development is completed, terminate with 50 μl / well of the termination solution. 6. Reading: Read using an ELISA reader at wavelengths of 450 nm and 630 nm, record and save the data for analysis. 7. Perform non-linear fitting of the standard curve and concentration back-calculation using OriginPro software, and perform curve plotting and data analysis using GraphPad Prism5. II. Results The drug concentrations of the three groups of FSH-LH-Fc / Fc heterodimers are shown in the following table: III. Conclusion The above results indicate that the drug metabolic half-life of the FSH-LH-Fc / Fc heterodimer of this application after a single administration in adult cynomolgus monkeys is about 40 h. The fusion protein of this application has long-acting properties, and the drug release is more gentle and continuous. Example 10 Effect of FSH-LH-Fc / Fc Heterodimer on Promoting Ovarian Weight Gain in Young Female SD Rats I. Grouping and Experimental Design Based on young female SD rats, inject the control or test article subcutaneously, and observe the ovarian weight gain of the animals 24 h after the last administration. A total of 9 groups were set up in the experiment, with 3 animals in each group. That is, the solvent control group, 4 dose groups of Gonal-F, and 4 dose groups of FSH-LH-Fc / Fc heterodimer group. Solvent: 1 mg / ml BSA, 0.9% sodium chloride, 20 IU / ml hCG. Gonal-F group: initial concentration of 44 μg / ml (600 IU / ml), diluted to 6.4 nM, 3.2 nM, 1.6 nM, 0.8 nM respectively using the solvent. Prepare 6.4 nM, 3.2 nM, 1.6 nM, 0.8 nM FSH-LH-Fc / Fc heterodimer solutions for the FSH-LH-Fc / Fc heterodimer group. The grouping situation is shown in the following table. The Gonal-F group was administered once a day for 3 consecutive days. The FSH-LH-Fc / Fc heterodimer group was given the drug once for the first time and the solvent for 3 consecutive days. The solvent group was administered once a day for 3 consecutive days. The volume of each administration was 0.5 ml / animal. 24 hours after the last administration, the animals were sacrificed, weighed, dissected, the ovaries were removed, the attached tissues were dissected, the fallopian tubes were removed, the surrounding liquid was blotted with filter paper, weighed and converted to the ovarian weight per 10 g body weight (mg). After collecting the ovarian tissues and fixing them sufficiently with 4% PFA, they were dehydrated with 15%, 20%, 30% sucrose solutions, embedded in OCT as frozen tissue samples, and subjected to frozen section HE staining, and observed and photographed under a microscope. II. Results The ovarian weight per 10 g body weight of the solvent control group, 4 dose groups of Gonal-F, and 4 dose groups of FSH-LH-Fc / Fc heterodimer group is shown in the following table and Figure 12. Among them, the HE sections of the ovarian tissues of the solvent control group, 6.4 nM of the Gonal-F group and 6.4 nM of the FSH-LH-Fc / Fc heterodimer group are shown in Figure 11. Compared with the solvent control group, the number of ovarian follicles in the Gonal-F group and the FSH-LH-Fc / Fc heterodimer group increased, the granulosa cells increased, the proportion of multilayered follicles increased, and the ovarian tissue space was loose, which was in line with the ovarian signs after superovulation stimulation. III. Conclusion The Chinese Pharmacopoeia uses the method of increasing ovarian weight in rats to determine the biological activity of FSH. The above results show that the FSH-LH-Fc / Fc heterodimer of this application has an obvious effect of promoting ovarian weight gain after only one administration in young female SD rats, and shows a certain dose-dependence. And the HE staining results show that the ovary has signs of superovulation, and the activity of the FSH part is not affected by the addition of LH and Fc fragments, and it has a long-acting effect compared with Gonal-F. Example 11 Effect of FSH-LH-Fc / Fc heterodimer on promoting seminal vesicle weight gain in young male SD rats I. Grouping and experimental design Based on young male SD rats, the reference substance or the test article was administered by subcutaneous injection, and the seminal vesicle weight gain of the animals was observed 24 hours after the last administration. A total of 9 groups were set up in the experiment, with 3 animals in each group. That is, the solvent control group, 4 dose groups of Luveris, and 4 dose groups of FSH-LH-Fc / Fc heterodimer. Solvent: 1 mg / ml BSA, 0.9% sodium chloride. The Luveris group was prepared according to a concentration of 2 IU / ml = 6.4 nM, and FSH-LH-Fc / Fc heterodimer solutions of 51.2 nM, 25.6 nM, 12.8 nM, and 6.4 nM were prepared. The grouping is shown in the following table. In the FSH-LH-Fc / Fc heterodimer group, the drug was administered once for the first time, and the solvent was administered continuously for 3 days. In the solvent group, it was administered once a day for 3 days, and the volume of each administration was 0.5 ml / animal. In the Luveris group, it was administered once a day for 3 days, and the volume of each administration was 0.5 ml / animal. 24 hours after the last administration, the animals were sacrificed, weighed, dissected, the entire prostate was removed, the seminal vesicles were dissected from the junction of the anterior lobe and the seminal vesicles, the attached tissues were removed, the surrounding liquid was blotted with filter paper, weighed, and converted into the seminal vesicle weight per 10 g body weight. After the seminal vesicle tissue was collected and fixed sufficiently with 4% PFA, it was dehydrated with 15%, 20%, and 30% sucrose solutions, embedded in OCT to form frozen tissue samples, and subjected to frozen section HE staining, and observed and photographed under a microscope. II. Results The seminal vesicle weight per 10 g body weight of the animals in each group is shown in the following table and Figure 14. Among them, the HE sections of the seminal vesicle tissues in the solvent control group and the 51.2 nM FSH-LH-Fc / Fc heterodimer group are shown in Figure 13. Compared with the solvent control group, the seminal vesicle gland ducts in the FSH-LH-Fc / Fc heterodimer group were hyperplastic and tortuous, and even overly crowded so that the gland duct structure was unclear, and the single-layer epithelium became stratified epithelium, which was in line with the sign of seminal vesicle weight gain. III. Conclusion In the art, the seminal vesicle weight gain method is commonly used to determine the biological activity of LH. The above results show that the FSH-LH-FC / FC heterodimer of the present application has an obvious effect of promoting seminal vesicle weight gain after only one administration in young male SD rats, and shows a certain dose-dependence. The activity of the LH part is not affected by the addition of FSH and Fc fragments, and it has the effect of promoting the hyperplasia of seminal vesicle gland ducts. Example 12 FSH-LH-Fc / Fc heterodimer promotes the development of multiple follicles in the rat ovary and the proliferation of FSH and LH target cells I. Grouping and experimental design Based on young female SD rats, the test article was administered by subcutaneous injection, and the ovarian weight gain of the animals was observed 24 hours after the last administration. A total of 4 groups were set up in the experiment, namely 4 dose groups of the FSH-LH-Fc / Fc heterodimer, with 3 animals in each group. Solvent: 1 mg / ml BSA, 0.9% sodium chloride, 20 IU / ml hCG. FSH-LH-Fc / Fc heterodimer solutions with concentrations of 6.4 nM, 3.2 nM, 1.6 nM, and 0.8 nM were prepared. The drug was administered once for the first time, and the solvent was administered continuously for 3 days. 24 hours after the last drug administration, the animals were sacrificed, weighed, dissected, the ovaries were removed, the attached tissues were stripped, the fallopian tubes were removed, the surrounding liquid was blotted with filter paper, and then weighed and converted to ovarian weight per 10 g body weight (mg). After the ovarian tissues were collected and fixed sufficiently with 4% PFA, paraffin tissue samples were prepared after gradient dehydration, and paraffin sections were stained with HE and IHC, and observed and photographed under a microscope. II. Results As shown in Figures 15 - 16, after the rats were treated with the FSH-LH-Fc / Fc recombinant protein, HE staining showed multiple follicle developments in the ovaries. The cross-sectional diameter of the ovaries increased with the increase in the drug treatment dose, and the number of antral follicles with a diameter greater than 5 μm in the ovaries increased with the increase in the drug treatment dose. Immunohistochemical staining showed that the FSH-LH-Fc / Fc recombinant protein could promote the proliferation of target cells. The FSHR(+)-granulosa cell layer downstream of FSH and the 3β-HSD(+)-theca cell layer downstream of LH thickened with the dose, proving that the FSH-LH-Fc / Fc recombinant protein could simultaneously exert the functions of FSH and LH in rats. Example 13: FSH-LH-Fc / Fc heterodimer activates the downstream cAMP pathway expression respectively Both FSH and LH are G protein-coupled receptors, which can promote the synthesis of downstream cAMP and the generation of downstream signaling pathways. The CHO-FSHR cell line and the CHO-LHCGR cell line were cultured. After the cells were treated with concentration gradients of 1 nM, 10 nM, 100 nM, and 1000 nM for 2 hours, the cell lysates were collected for cAMP ELISA detection, and the results are shown in Figure 17. Conclusion: In the CHO-FSHR cell line and the CHO-LHCGR cell line, the FSH-LH-Fc / Fc heterodimer can promote the production of cAMP, and it increases with the dose, proving that the FSH-LH-Fc / Fc heterodimer has both FSH and LH activities in vitro and has bifunctionality. Example 14: FSH-LH-Fc / Fc heterodimer can promote multiple follicle developments in ICR mice I. Grouping and experimental design ICR female mice were randomly assigned to 2 groups. FSH-LH-Fc / Fc heterodimer was used as the test article, and PMSG was used as the control article, with 10 IU per mouse. Ovulation induction was performed by injection at 19:00 on D1. A trigger dose of 10 IU was given at 19:00 on D3. The mice were dissected at 9:30 on D4, and the multiple COCs clusters in the ampulla of the fallopian tube were taken for counting and statistical analysis. II. Results As shown in the following table and Figure 18, the number of eggs obtained with FSH-LH-Fc / Fc heterodimer was evaluated. Superovulation can be carried out in the ICR mouse animal model, and multiple follicle development can be obtained. FSH-LH-Fc / Fc heterodimer can effectively exert the function of FSH in mice and has the potential to be used as a superovulation drug. Example 15 FSH-LH-Fc / Fc heterodimer can promote ovulation in ICR mice to obtain healthy embryos I. Grouping and experimental design ICR female mice were randomly assigned to 2 groups. FSH-LH-Fc / Fc heterodimer was used as the test article, with 10 IU per mouse. Ovulation induction was performed by injection at 19:00 on D1. A trigger dose of 10 IU HCG was given at 19:00 on D3, and the mice were caged with male mice. The mice were dissected at 12:30 on D5, and the embryos in the ampulla were taken for culture. The culture medium was KSOM + 1% FBS. II. Results FSH-LH-Fc / Fc heterodimer has a good ovulation-promoting function and good potential for embryo development. Figure 19 shows the blastocyst diagram obtained by in vitro culture. Example 16 FSH-LH-Fc / Fc heterodimer can cause an increase in serum-related hormone levels, ovarian weight gain, and follicle development in cynomolgus monkeys I. Grouping and experimental design Six female cynomolgus monkeys were used as experimental animals. The experiment was divided into 3 groups, namely the low-dose group (10 μg / kg), the medium-dose group (40 μg / kg), and the high-dose group (120 μg / kg), with 2 animals in each group. FSH-LH-Fc / Fc heterodimer was administered once a week for 2 consecutive weeks (a total of 3 administrations). During the experiment, the animals in each group were clinically observed, and their body weight, food intake, body temperature, and electrocardiogram were monitored. Blood samples were collected and serum was separated at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 168 h after the first administration to detect the serum drug concentration. Blood samples were collected and serum was separated at 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after the first administration to detect the concentrations of estradiol, testosterone, progesterone, and luteinizing hormone in the serum. After the animals were euthanized, ovarian tissues were collected and the ovarian weight was measured. The grouping and dosing information are shown in the following table: Note: a: Number of animals. II. Results During the experiment, no animal deaths / near-deaths related to the test article were observed. No changes related to the test article in clinical symptoms, body weight, food intake, body temperature, and electrocardiogram were observed. Subcutaneous administration could cause a rapid increase in the levels of estradiol, luteinizing hormone, and progesterone in the serum of cynomolgus monkeys, which gradually decreased with drug metabolism within 168 h after administration, and there was a certain dose-effect relationship. Subcutaneous administration of FSH-LH-Fc / Fc heterodimer had no obvious effect on the change in serum testosterone level. The concentrations of estradiol, testosterone, progesterone, and luteinizing hormone in the serum of the animals in each group during the experiment are shown in the following table. Serum estradiol concentration (pg / mL) of each group of animals Serum testosterone concentration (pg / mL) of each group of animals Serum progesterone concentration (pg / mL) of each group of animals Serum luteinizing hormone concentration (mIU / mL) of each group of animals After administration in the high-dose group (120 μg / kg), the ovarian volume and body weight of the animals increased significantly, as shown in the following table. Ovarian weight (g) of each group of animals II. Discussion During the experiment, no test article-related adverse reactions were observed in the animals of the low, medium, and high-dose groups (10, 40, and 120 μg / kg) of FSH-LH-Fc / Fc heterodimer; FSH-LH-Fc / Fc heterodimer could increase the levels of estradiol, luteinizing hormone, and progesterone in the serum of cynomolgus monkeys, increase ovarian weight, and promote follicle development. It is suggested that FSH-LH-Fc / Fc heterodimer has an obvious ovulation-promoting effect, and its long-acting characteristic reduces the administration frequency and improves patient compliance, having great development value and clinical application prospects. Example 17 FSH-LH-Fc / Fc heterodimer can promote the development of multiple follicles in cynomolgus monkeys and obtain mature MII-stage eggs I. Grouping and experimental design Two adult sexually mature cynomolgus monkeys were administered FSH-LH-Fc / Fc heterodimer three times at doses of 107 μg, 214 μg, and 214 μg, respectively, by intramuscular injection every 72 h, and 120 μg HCG was given for triggering 84 h after the third injection. Ovarian puncture was performed 26 h after triggering for evaluation, and serum hormone detection and abdominal B-ultrasound detection were performed for evaluation during the period. III. Results Multiple follicle development could be achieved in both of the two cynomolgus monkeys. Estrogen could respond and double during superovulation, progesterone also increased accordingly, ovarian volume increased, dominant follicles and the volume of dominant follicles increased, and finally a certain number of eggs and a certain proportion of mature MII-stage eggs could be obtained, proving that FSH-LH-Fc / Fc can promote the development of multiple follicles in cynomolgus monkeys and obtain mature MII-stage eggs, having the potential as a superovulation drug. Figure 20 shows the changes in hormone levels during superovulation in cynomolgus monkeys, and Figure 21 shows the ovarian puncture results in cynomolgus monkeys. References: [1] Fevold HL. Synergism of the follicle stimulating and luteinizing hormones in producing estrogen secretion. Endocrinology. 1941; 28: 33-6. [2] Greep RO, Van Dyke HB, Chow BF. Gonadotropins of the swine pituitary: Various biological effects of purified thylakrntrin (FSH) and pure metakentrin (ICSH). Endocrinology. 1942; 30: 635-49. [3] Casarini L, Santi D, Brigante G, Simoni M. Two Hormones for One Receptor: Evolution, Biochemistry, Actions, and Pathophysiology of LH and hCG. Endocr Rev. 2018 Oct 1; 39(5): 549 - 592. [4] Tian Lifeng, Wu Qiongfang, Tan Jun, et al. Clinical outcome analysis of adding r - LH in patients with low serum LH in the long protocol during the early follicular phase [J]. Journal of Reproductive Medicine, 2019, 27(4): 310 - 315. [5] Ernesto B, Elena L, Juana C, Carlos S, Jose R, Antonio P. Impact of luteinizing hormone administration on gonadotropin - releasing hormone antagonist cycles: an age - adjusted analysis. Fertility and Sterility. 2011; 95: 1031 - 1036. [6] Klein NA, Battaglia DE, Fujimoto VY, Davis GS, Bremner WJ, Soules MR. Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle - stimulating hormone rise in normal older women. J Clin Endocrinol Metab. 1996; 82: 1038 - 45. [7] Zumoff B, Strain GW, Miller LK, Rosner W. Twenty - four - hour mean plasma testosterone concentration declines with age in normal premenopausal women. J Clin Endocrinol Metab. 1995; 80: 1429 - 30. [8]Davison SL,Bell R,Donath S,Montalto JG,Davis SR.Androgen levels in adult females:changes with age,menopause,and oophorectomy.J Clin Endocrinol Metab.2005;90:3847-53. [9]Welt CK,Jimenez Y,Sluss PM,Smith PC,Hall JE.Control of estradiol secretion in reproductive ageing.Hum Reprod.2006;21:2189-93.

[0010] Bosch E,Pau E,Albert C,Zuzuarregui JL,Remohi J,Pellicer A.Impact of different amounts of LH in controlled ovarian hyperstimulation oocyte donation cycles.Fertil Sertil.2006;86(Suppl):S425.

[0011] Ruvolo G,Bosco L,Pane A,Morici G,Cittadini E,Roccheri MC.Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures.Fertil Sertil.2007;87:542-6.

Claims

1. A fusion protein composed of a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide; wherein, The first polypeptide from the N-terminus to the C-terminus contains: the β subunit of FSH, the β subunit of LH, a hinge, a CH2 domain, a CH3 domain, wherein the β subunit of FSH is directly or indirectly connected to the β subunit of LH through a linker peptide; the β subunit of LH is directly or indirectly connected to the hinge through a linker peptide; Or the first polypeptide from the N-terminus to the C-terminus contains: the β subunit of LH, the β subunit of FSH, a hinge, a CH2 domain, a CH3 domain, wherein the β subunit of LH is directly or indirectly connected to the β subunit of FSH through a linker peptide, and the β subunit of FSH is directly or indirectly connected to the hinge through a linker peptide; The second polypeptide from the N-terminus to the C-terminus contains: a hinge, a CH2 domain, a CH3 domain; The third polypeptide is composed of the α subunit of FSH; The fourth polypeptide is composed of the α subunit of LH; There is a non-covalent interaction between the first polypeptide and the third polypeptide; there is a non-covalent interaction between the first polypeptide and the fourth polypeptide; the first polypeptide and the second polypeptide are connected by a disulfide bond.

2. The fusion protein according to claim 1, wherein there is a non-covalent interaction between the β subunit of FSH in the first polypeptide and the third polypeptide; there is a non-covalent interaction between the β subunit of LH in the first polypeptide and the fourth polypeptide; the first polypeptide and the second polypeptide are connected by a disulfide bond in the hinge region.

3. The fusion protein according to claim 1, wherein the linking peptide is a flexible linking peptide (GGGGS) n , where n is an integer from 1 to 4, or a rigid linking peptide (EAAAK) n , where n is an integer from 2 to 5.

4. The fusion protein according to claim 3, wherein the amino acid sequence of the linker peptide is as shown in SEQ ID NO:

2.

5. The fusion protein according to claim 1, wherein the hinge, CH2 domain, and CH3 domain are the hinge, CH2 domain, and CH3 domain of natural IgG1, IgG2, or IgG4, or mutants thereof.

6. The fusion protein according to claim 1 or 5, wherein the hinge in the first polypeptide or the second polypeptide contains an S228P mutation based on the hinge of natural IgG4; And / or, the CH3 domain in the first polypeptide or the second polypeptide contains S375C and T389W mutations, and the corresponding CH3 domain in the second polypeptide or the first polypeptide contains Y370C, T389S, L391A, and Y438V mutations.

7. The fusion protein according to claim 6, wherein the amino acid sequence of the hinge, CH2 domain, and CH3 domain of the first polypeptide is as shown in SEQ ID NO: 5, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 6; or the amino acid sequence of the hinge, CH2 domain, and CH3 domain of the first polypeptide is as shown in SEQ ID NO: 6, and the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 5; 0 or 1 lysine is deleted at the C-terminus of the first polypeptide and the second polypeptide.

8. The fusion protein according to claim 1, wherein the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 11; 0 or 1 lysine is deleted at the C-terminus of the first polypeptide and the second polypeptide.

9. A method for preparing the fusion protein according to claim 1, comprising the following steps: (I). Vector construction Insert the coding gene of the first polypeptide into the AvrII and BstZ17I sites of pZHK1.3 to obtain the pZHK1.3-FSHβ-LHβ-Fc expression vector; Insert the coding gene of the third polypeptide / fourth polypeptide into the EcoRV and PacI sites of pZHK5.18, and insert the coding gene of the second polypeptide into the AvrII and BstZ17I sites to obtain the pZHK5.18-FSHα / Fc expression vector; (II) Expression in CHO-S cells Electroporate the expression vector into CHO-S cells, and screen for a high-expression stable cell line by the pressure method of MTX + puromycin + hygromycin, The amplified high-expressing and stable-expressing mammalian cell lines obtained by screening were inoculated into Dynamis medium at an inoculation density of 0.5×10 6 cells / ml, and batch-fed perfusion culture was carried out at 37°C, 8% CO2, and 130 rpm. The feeding medium was Efficient Feed C + supplement. After 12-17 days of expression, the expression supernatant was collected, filtered and clarified through a 0.22-μm filter, and then purified to obtain the fusion protein.

10. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 9.

11. Use of the fusion protein according to any one of claims 1 to 9 in stimulating follicular development, assisted reproduction, diseases related to FSH and LH deficiency, and menopausal syndrome.

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