Ovarian-targeting polypeptides and derivatives thereof and applications thereof

Abstract

The present invention discloses the application of ovary targeting polypeptide and its derivatives, which belongs to the biomedicine field.Specifically, it discloses that the polypeptide and its derivatives described in the present invention have the effect of targeting ovary, which further proves its use in the preparation of drugs or reagents for preventing and treating oocyte aging in vivo or in vitro for treating ovarian-related reproductive diseases.The above diseases include menstrual cycle disorder, polycystic ovary syndrome, androgen phenotype, alopecia, metabolic disorder, ovarian insufficiency, premature ovarian failure, ovulation disorder, glucose metabolism disorder, lipid metabolism disorder, and more preferably hyperglycemia and insulin resistance.Otherwise, the polypeptide of the present invention is expected to extend the storage period and storage quality of oocytes or fertilized eggs in the field of assisted reproduction.

Description

[Technical field]

[0001] The present invention is in the field of medical biology and relates to ovarian targeting polypeptides and their derivatives and applications. [Background technology]

[0002] The ovary is a very important reproductive organ for women, consisting of germ cells (oocytes or eggs) and somatic cells (granulosa cells, theca cells and stromal cells), the interaction of which determines the function of the ovary, i.e., secreting hormones necessary for follicular development, menstrual / estrous cycles and maintaining endocrine health, as well as producing mature eggs for fertilization to produce offspring. Abnormalities in ovarian function lead to the development of a series of ovarian reproductive diseases, such as Premature Ovarian Failure (POF), Polycystic Ovary Syndrome (PCOS), ovulation disorders and even ovarian cancer (OC).

[0003] Ovarian reproductive diseases cause biohormonal disorders, and manifest as a series of carbohydrate and lipid metabolic disorders such as obesity, insulin resistance, fatty liver, and osteoporosis, reproductive disorders such as excessive activation of ovarian follicles, decreased ovulation, and infertility, and aging phenomena such as early menopause. High androgens in the female body cause a series of androgen phenotypes such as hirsutism, acne, and alopecia, while high estrogen causes a series of reproductive tumor diseases such as ovarian cancer, breast cancer, and endometrial cancer, which have serious adverse effects on women's physical and mental health and life safety.

[0004] The window period for fertilization after oocyte maturation is very limited. For example, the optimal fertilization and development time for mice is less than 12 hours after ovulation, while for humans, it is within 4-12 hours after ovulation or egg collection. Over time, the released oocytes enter the aging stage and eventually undergo apoptosis and become infertile. At the same time, in assisted reproductive technology, inhibiting postovulatory aging in mammals can improve and enhance fertilization and birth rates.

[0005] At present, the treatment of diseases related to ovarian germ cells and endocrine disorders mainly includes drug therapy, surgical treatment and assisted reproductive technology.The biggest problem with drug therapy is that targeted therapy is impossible, and drugs act on the whole body, which may cause various side effects on other tissues and organs of the body.In addition to the relatively low success rate, surgical treatment and assisted reproductive technology lack a relatively clear non-invasive real-time observation of the development status of ovaries after treatment.Therefore, screening for polypeptides that can specifically target ovaries in vivo, studying their effects in ovarian reproductive diseases, and as drug carriers for targeted treatment of ovarian reproductive diseases, has important social significance and a broad economic market. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the bottleneck problem in the treatment of ovarian reproductive diseases raised in the background art above, the objective of the present invention is to provide the functions and applications of ovarian targeting polypeptides and their derivatives.

[0007] The present invention has discovered the following sequence polypeptides that can specifically target the ovaries and alleviate multiple ovarian-associated reproductive disorders, metabolic disorders and aging symptoms caused by hormonal disorders: [Means for solving the problem]

[0008] One aspect of the invention provides a polypeptide, the polypeptide sequence comprising a sequence of the formula M1-Za-M2 or a sequence of the formula Za, wherein M1, M2 are each independently a single or multiple amino acid polypeptide segment or absent; Za is Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro-Thr; X1 is Asn, Tyr, Asp, or absent; X2 is Asn or Gln or His or Pro or Ser, or is absent; X3 is Gly, Trp, or absent; X4 is Leu or absent, X5 is Pro or Ser, X6 is Ala or Val; X7 is Tyr, Ser or Ala; X8 is Pro or Thr, Furthermore, among them, Za is YLGASVPSPDPLEPT SEQ ID NO:1, YLNNGLGAPAPYPDPLEPT SEQ ID NO:2, YLYQWLGAPVPYPDPLEPT SEQ ID NO:3, YLYQWLGAPVPYPDTLEPT SEQ ID NO:4, YLYQWLGAPAPYPDPLEPT SEQ ID NO:5, YLDHWLGAPAPYPDPLEPT SEQ ID NO:6, YLDPGLGAPAPYPDPLEPT SEQ ID NO:7, YLDHGLGAPAPYPDPLEPT SEQ ID NO:8, YLDQGLGAPAPAPDPLEPT SEQ ID NO:9, YLDSGLGAPVPYPDPLEPT Selected from any one of SEQ ID NO:10.

[0009] Another aspect of the present invention provides a derivative of an ovary-targeting polypeptide, the derivative being a product obtained by performing a conventional modification on an amino acid side group, an amino terminus, or a carboxyl terminus of the polypeptide, or by linking a tag for detection or purification of the polypeptide or protein onto the polypeptide, or by isotope-labeling modification, the conventional modifications being fluorescent group modification, phosphorylation modification, disulfide bond cyclization modification, biotin labeling modification, photosensitizer modification, azide modification, PEG modification, methylation modification, fluorescence quenching group modification, protein coupling modification, small molecule compound modification, amination modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, esterification modification, and glycosylation modification; Furthermore, the tag is His 6 , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityeXact.

[0010] Furthermore, the above-mentioned amino-terminal and carboxyl-terminal modifications are selected from acetylation modification at the N-terminus and amination modification at the C-terminus of the polypeptide.

[0011] Furthermore, the side chain modifications are selected from modifications on the R groups of the amino acid side chains in the polypeptide.

[0012] Furthermore, the fluorescent dye used to modify the above fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, and ICG, and the modification can be used for fluorescence detection.

[0013] Furthermore, the phosphorylation modifications are selected from one or more combinations of p-Ser, p-Thr, and p-Tyr.

[0014] Further, the glycosylation modifications are selected from one or more combinations of Ser, Asn, Thr, Tyr.

[0015] Furthermore, the nitration modifications are selected from one or more combinations of Tyr.

[0016] Furthermore, with regard to the biotin label, the biotin is selected from D-biotin, biotin hydrazide, photolabile biotin, and biotin-dUTP.

[0017] Furthermore, the photosensitizer modifications can be used to prepare photosensitive formulations.

[0018] Additionally, the azide modification can be used in secondary conjugation reactions.

[0019] Furthermore, the above PEG modification can be used to prepare drug carriers.

[0020] Further, the isotopes used for the isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I.

[0021] A further aspect of the present invention provides a polynucleotide encoding the above polypeptide or a derivative thereof.

[0022] A further aspect of the present invention provides a vector comprising the above-described polynucleotide.

[0023] A further aspect of the invention provides a host cell transfected with the above vector.

[0024] Yet another aspect of the present invention provides the use of a polypeptide according to the present invention or a derivative thereof in the preparation of an ovarian targeting detection reagent, an ovarian targeting drug or an ovarian targeting tool.

[0025] Furthermore, said tool is selected from a carrier or formulation, for example a carrier that achieves a targeting effect by coupling said polypeptide or its derivative according to the present invention, a formulation such as liposomes or nanoparticles or a reagent kit.

[0026] Another aspect of the present invention provides the use of the above polypeptide or a derivative thereof in the preparation of a drug or detection reagent capable of targeting the ovary.

[0027] Furthermore, the above drugs and detection reagents include the above polypeptides and derivatives based on the polypeptides, detection reagents prepared as antigens, and detection products such as reagent kits and test strips.

[0028] Another aspect of the present invention provides a carrier that targets the ovary, and the above polypeptide and derivatives having the same as the scaffold can be used as a tool for targeted treatment of ovary-related reproductive diseases.

[0029] Further, said carrier is selected from liposomes, nanoparticles, exosomes, biodegradable polymeric substances, antibody-ligands.

[0030] Additionally, the carrier includes a component capable of treating an ovarian-related reproductive disorder.

[0031] The polypeptide and its derivatives can be synthesized autonomously under general chemical laboratory conditions, and recombinant proteins containing the polypeptide obtained by gene expression can also be industrially synthesized by commercial reagent companies. The polypeptide is synthesized by solid-phase synthesis, and different amino acids on the resin can be synthesized in a directional manner to form an amino acid chain by condensation reaction. The polypeptide derivatives can be further labeled with a modifying group after the linkage of amino acids is completed.

[0032] Another aspect of the present invention provides a drug that targets the ovary, which is a conjugate of the above-mentioned polypeptide and its derivative with an active ingredient.The above-mentioned polypeptide and its derivative can treat reproductive, metabolic and aging-related diseases caused by germ cell abnormality and hormonal disorder by targeting the ovary.

[0033] Yet another aspect of the present invention provides a polypeptide probe targeting the ovary, which is obtained by coupling the above polypeptide or a derivative having the same as the backbone with a fluorescent dye by an organic chemical reaction.

[0034] Furthermore, the fluorescent dye is selected from dyes having a fluorescent group in the near infrared region, and preferably, the dye having a fluorescent group in the near infrared region is selected from AIE, Cy5, Cy7, and ICG.

[0035] Yet another aspect of the present invention provides the use of the above polypeptide in the preparation of a medicament or reagent for treating an ovary-related reproductive disorder.

[0036] Further, the ovarian-associated reproductive disorder is selected from ovarian-associated reproductive disorders caused by germ cell abnormalities or hormonal disorders.

[0037] Additionally, the hormonal disorder is one in which androgens are at greater than normal levels.

[0038] Furthermore, the ovary-related reproductive disease caused by the above-mentioned germ cell abnormality is an ovary-related reproductive disease caused by oocyte aging and / or hardening of the oocyte zona pellucida, and preferably, the ovary-related reproductive disease caused by the above-mentioned germ cell abnormality is selected from abnormalities in the number of oocytes, deterioration of oocyte quality, and oocyte aging.

[0039] Furthermore, ovarian-related reproductive diseases caused by the above hormonal disorders include menstrual cycle disorders, polycystic ovarian syndrome, androgen phenotype, alopecia, metabolic disorders, ovarian insufficiency, premature ovarian failure, and ovulatory disorders.

[0040] Furthermore, the metabolic abnormality caused by the above-mentioned hormonal disorder is selected from glucose metabolism abnormality and lipid metabolism abnormality, and preferably hyperglycemia, insulin resistance, obesity, fatty liver, and osteoporosis.

[0041] Further, the androgenic phenotype is selected from acne, hirsutism, and alopecia.

[0042] Furthermore, aging and related conditions caused by the above hormonal disorders include perimenopause, menopausal syndrome, aging, acne, smallpox, hirsutism, and alopecia.

[0043] Yet another aspect of the present invention provides the use of the above-mentioned polypeptide in the preparation of a drug or reagent for preventing and treating oocyte aging in vivo or in vitro.

[0044] Yet another aspect of the present invention provides an assisted reproduction product comprising the above polypeptide or a derivative thereof according to the present invention.

[0045] Furthermore, the above-mentioned assisted reproduction product is a protective liquid for oocytes and / or fertilized eggs during the assisted reproduction process.

[0046] Yet another aspect of the present invention provides a use of the above-mentioned assisted reproduction product in preparing a protective solution for preserving oocytes and / or fertilized eggs in an assisted reproduction process. Effect of the Invention

[0047] The beneficial effects of the present invention are as follows:

[0048] (1) The candidate polypeptides and derivatives based on them provided by the present invention are capable of specifically targeting the ovary in vivo and in vitro.

[0049] (2) The candidate polypeptides and derivatives using the same as the backbone provided by the present invention can enhance the targeting and intervention of drugs or drug carriers to the ovaries and the therapeutic effect as modified polypeptides.

[0050] (3) The candidate polypeptides and derivatives based on them provided by the present invention are easy to synthesize and modify, and are low-cost, so that they have a wide range of applications.

[0051] (4) The candidate polypeptides and derivatives based on them provided by the present invention provide a wide range of strategies for preparing diagnostic compounds or drugs for practical application.

[0052] (5) The candidate polypeptides and derivatives based on them provided by the present invention are suitable for application in targeted therapy of ovary-related reproductive diseases, and are also suitable as research and observation tools in experimental animals to model ovarian diseases. [Brief description of the drawings]

[0053] [Figure 1] Fig. 2 shows the effect of ICG-linked polypeptide on targeting ovaries in vivo, which shows the fluorescent signal distribution and results of mouse ovaries and uterus in three groups: PBS group, ICG group, and ICG-linked peptide group. [Diagram 2] Fig. 2 shows the effect of the polypeptide linked with AIE on targeting the ovary in vivo, which shows the fluorescent signal distribution and results of the mouse ovaries in four groups: PBS group, AIE empty particle group, AIE linked polypeptide group, and AIE linked other polypeptide group. [Diagram 3] This is a diagram showing the localization of polypeptides after AIE linkage in vivo in the ovary. In the diagram, the localization of the fluorescent signal in mouse ovaries of four groups, PBS group, AIE empty particle group, AIE linked polypeptide group, and AIE linked other polypeptide group, is shown. In this figure, the arrow indicates membrane cells, and the star indicates interstitial cells. [Figure 4] The mitigating effect of ovarian targeting polypeptide on follicular development abnormality, estrous cycle disorder, mouse androgen phenotype and blood glucose abnormality caused by high androgen in a mouse model induced by high androgen-dihydrotestosterone (DHT). [Figure 4A] Morphological observation of mouse ovaries in three groups: control group, DHT group, and DHT + ovarian targeting polypeptide group. [Figure 4B] Number of days of estrus for 10 days in three groups of mice. [Figure 4C] Anogenital distance of mice 60 days after modeling (male anogenital distance is relatively large). [Figure 4D] Change in blood glucose after oral administration of glucose to mice 60 days after modeling. [Diagram 5] Fig. 5 shows the mitigating effect of ovarian-targeting polypeptides on weight changes caused by hyperandrogenism in a mouse model induced by hyperandrogenism-dihydrotestosterone (DHT). [Fig. 5A] Change in weight, [Fig. 5B] Change in weight gain, [Fig. 5C] Change in gonadal fat weight, [Fig. 5D] Change in lean body mass excluding fat. [Figure 6] The effect of ovarian targeting polypeptide on high androgen-induced neck and back hair loss in mice in a high androgen-dihydrotestosterone (DHT)-induced mouse model. [Figure 6A] Observation of neck and back hair loss in three groups of mice: control group, DHT group, and DHT + ovarian targeting polypeptide group. [Figure 6B] Immunofluorescence image of androgen receptor AR expression in neck and back skin of three groups of mice. [Figure 7] The ovarian-targeted polypeptides improve oocyte fragmentation and regression, zona pellucida hardening, and in vitro fertilization caused by aging in a postovulatory aging model of oocytes. [Fig. 7A] Regression and fragmentation rates after 12 hours of aging in the control group, aging group, and target polypeptide group. [Fig. 7B] Zona pellucida hardening levels after 12 hours of aging in the control group, aging group, and target polypeptide group. [Fig. 7C] In vitro fertilization rates after 12 hours of in vitro aging in the control group, aging group, and target polypeptide group. [Figure 8] Changes in serum estrogen and progesterone levels in the superovulation model of OCN15 KO mice compared to WT mice. [Figure 8] The left figure shows the changes in estrogen levels during the superovulation process. [Figure 8] The right figure shows the changes in progesterone levels during the superovulation process. [Figure 9] Effects of ovarian-targeting polypeptides on hepatic lipid droplet accumulation caused by hyperandrogenism in a hyperandrogen-dihydrotestosterone (DHT)-induced mouse model. [Figure 10] FIG. 1 is a sequence comparison diagram of metabolites from different species. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] In order to better understand the present invention, the present invention will be further described below with reference to specific examples, but the scope of protection of the present invention is not limited to the following examples.

[0055] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Otherwise, any method or material similar or equivalent to those described herein can be used to carry out the present invention.For the purposes of the present invention, the following terms are defined.

[0056] The terms "subject", "individual" and "patient" can be used interchangeably herein to refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, humans, mice, rats, chimpanzees, rhesus monkeys, cows, sheep, wild boars, moles, and dogs. Also covered are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0057] As used herein, the term "administration" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, oral, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of administration include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0058] The term "treatment" refers to a method for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefit means any treatment-related improvement for one or more diseases, conditions, or symptoms being treated, or any treatment-related effect for one or more diseases, conditions, or symptoms being treated. The term "prevention" refers to a method for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. For a prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0059] The term "therapeutically effective amount" refers to the amount of peptide or composition that is sufficient to achieve beneficial or desired results. Therapeutically effective amount can vary depending on one or more of the following: the subject and disease state to be treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be easily determined by those skilled in the art. The specific amount can vary depending on one or more of the following: the specific agent selected, the type of target cell, the location of the target cell in the subject, the administration regimen to be followed, whether or not it is administered in combination with other compounds, the time of administration, and the solid delivery system that carries it. Example 1: Preparation of Polypeptide Sequences

[0060] Synthesizing a polypeptide sequence using an artificial synthesis method; The polypeptide sequence is as follows:

[0061] YLGASVPSPDPLEPT SEQ ID NO:1, YLNNGLGAPAPYPDPLEPT SEQ ID NO:2, YLYQWLGAPVPYPDPLEPT SEQ ID NO:3, YLYQWLGAPVPYPDTLEPT SEQ ID NO:4, YLYQWLGAPAPYPDPLEPT SEQ ID NO:5, YLDHWLGAPAPYPDPLEPT SEQ ID NO:6, YLDPGLGAPAPYPDPLEPT SEQ ID NO:7, YLDHGLGAPAPYPDPLEPT SEQ ID NO:8, YLDQGLGAPAPAPDPLEPT SEQ ID NO:9, YLDSGLGAPVPYPDPLEPT Sequence number 10.

[0062] Said polypeptide is synthesized by adopting the method of general solid phase synthesis or liquid phase synthesis, among which, by using the solid phase synthesis method of polypeptide, react from the amino acid of C-terminus to N-terminus, and through the steps of resin activation, amino acid linkage, elution protection, detection, etc., complete amino acid linkage one by one, then use excess ether to precipitate and centrifuge, and after HPLC purification of crude peptide, perform mass spectrometry, and then freeze-dry with liquid nitrogen for preparation for use.

[0063] In the present invention, the above peptides are all isolated peptides.

[0064] Among them, SEQ ID NO:1 is a mouse metabolite, SEQ ID NO:2 is a rat metabolite, SEQ ID NO:3 is a human metabolite, SEQ ID NO:4 is a chimpanzee metabolite, SEQ ID NO:5 is a rhesus monkey metabolite, SEQ ID NO:6 is a cow metabolite, SEQ ID NO:7 is a sheep metabolite, SEQ ID NO:8 is a wild boar metabolite, SEQ ID NO:9 is a mole metabolite, and SEQ ID NO:10 is a dog metabolite (as shown in Figure 10). Example 2: Preparation of Polypeptide Probes

[0065] This example employs the polypeptide prepared in Example 1 to provide a polypeptide probe that is bound to ICG / AIE through an organic chemical reaction.

[0066] The specific method is custom synthesis or self-synthesis. In this embodiment, the polypeptide is linked to ICG or AIE by click chemistry to complete the preparation of the probe, of which the linker is the universal DBCO. For ICG / AIE with an activated functional group, the activated functional group is an amino group NH 2 , carboxyl group COOH, activated lipid NHS, maleimide MAL, mercapto group SH, azide N 3 , containing the alkyne ALK, which is then coupled with the polypeptide prepared in Example 1 to obtain the polypeptide ICG / AIE. Example 3: Detection of the ability of polypeptide ICG probes to target mouse ovaries

[0067] The polypeptide ICG probe (containing sequence number 1) solution prepared in Example 2, the ICG solution (2 mM) and PBS were injected into the tail vein of adult mice, 100 μL was injected into each mouse, and 24 hours later, the results were detected by a small animal imaging device.

[0068] The experimental results are shown in Figure 1. Comparing the results of the three groups, it can be seen that the polypeptide ICG probe group (OCN15-ICG, an ICG probe containing sequence number 1) had a specific signal in the ovarian area, whereas the other two groups (ICG solution group and PBS group) did not show any fluorescent signal in the ovarian area.

[0069] As can be seen from the above experimental results, the polypeptide of the present invention has a specific response to the ovary and can achieve an action of targeting the ovary in vivo. Example 4: Detection of the ability of polypeptide AIE probes to target mouse ovaries

[0070] The polypeptide AIE probe (including sequence number 1), disordered polypeptide AIE probe, AIE empty particles and PBS prepared in Example 2 are injected into the tail vein of adult mice, 100 μL is injected into each mouse, and 24 hours later, the antibodies are detected by a small animal imaging device.

[0071] The experimental results are shown in Figure 2. Comparing the results of the four groups, it can be seen that the polypeptide AIE probe group (OCN15, AIE probe containing sequence number 1) had a specific signal in the ovarian region, while the disordered polypeptide AIE probe group (OCN22) and the PBS group did not show any fluorescent signal in the ovarian region, and the AIE empty particle group (NP) had a weak signal.

[0072] As can be seen from the above experimental results, the polypeptide of the present invention has a specific response to the ovary and can achieve an action of targeting the ovary in vivo.

[0073] As can be seen by comparing Examples 3 and 4, the polypeptides of the present invention have a specific response to the ovary, and modifications thereto do not affect their targeting activity, suggesting that the polypeptides of the present invention can be used to prepare drugs, detection reagents, formulations, etc. that target the ovary. Example 5: Targeted localization of polypeptides to mouse ovaries

[0074] To further determine the ovarian targeting localization of the polypeptide, the ovaries of the four mouse groups in Example 4 were fixed with 4% PFA, then frozen sectioned (10 μm), stained with nuclei (DAPI), and observed for fluorescent localization by laser confocal microscopy.

[0075] The experimental results are shown in FIG. 3, in which the arrows indicate membranous cells and the asterisks indicate interstitial cells.

[0076] As can be seen by comparing the results of the four groups, the polypeptide AIE probe group (OCN15-AIE) mainly targets mouse ovarian stromal cells and thecal cells.

[0077] In summary, the mediator polypeptides provided by the present invention can be used for targeting the ovary, particularly to target ovarian stromal cells and thecal cells in the ovary. Modifications on the polypeptide do not affect the action of the polypeptide.

[0078] The results of Example 5 demonstrate that OCN15 targets ovarian stromal cells and thecal cells, and in combination with the results of the following Examples, it is expected that the results will be applicable to ovarian diseases associated with these two cellular abnormalities, such as thecal cell hyperplasia and stromal cell proliferation caused by high androgen, ovulation disorders due to thecal cell hyperplasia and stromal proliferation, ovarian dysfunction due to ovulation disorders, menstrual cycle disorders, and polycystic ovary syndrome.

[0079] Example 6: Effects of ovarian-targeted polypeptides on hyperandrogen-induced reproductive and metabolic symptoms in mice

[0080] Wild-type female mice were randomly divided into a control group (CTRL), a DHT group, and a DHT+OCN15 (OCT15, i.e., the polypeptide shown in SEQ ID NO: 1) group on postnatal day 25 (D25), with at least 8 mice in each group. In the control group, an empty pump was subcutaneously implanted and 200 μL of saline was intragastrically injected daily; in the DHT group, a sustained-release pump containing 5 mg of dihydrotestosterone (DHT) powder was subcutaneously implanted and 200 μL of saline was intragastrically injected daily; in the DHT+OCN15 group, a sustained-release pump containing 5 mg of DHT powder was subcutaneously implanted and 200 μL of saline containing the polypeptide was intragastrically injected daily (500 μg / Kg); the modeling time was 60 days.

[0081] The experimental results are shown in Figure 4. Comparing the three groups of mice, it can be seen that high androgens induce polycystic changes in mouse ovaries and a decrease in estrus. The number of days of estrus in normal mice is more than 4 days, while the number of days of estrus in the high androgen group is about 2 days. However, the concomitant use of OCN15 can alleviate these phenomena to a certain extent, with the number of days of estrus approaching 3 days and the polycystic changes also decreasing (Figure 4A, B). High androgens cause the phenomenon of androgenization in female mice, i.e., the anogenital distance increases, while OCN15 can significantly improve this symptom (Figure 4C), restoring the anogenital distance to a level close to that of normal mice, among which significant differences are shown between the control group and the DHT group, and between the DHT group and the DHT+OCN15 group.

[0082] The results of the blood glucose experiment are shown in Figure 4D. The peak value of blood glucose should be 15 minutes after oral glucose administration. At 30 minutes, the control group had already dropped, and the DHT group was still very high, with a difference of 3 stars from the control group. However, after OCN15 treatment, it dropped significantly compared with the DHT group alone, with no difference from the control group. OCN15 tends to normalize the blood glucose abnormality induced by DHT at 30 minutes.

[0083] The results of body weight change are shown in Figure 5. Figure 5A and B illustrate that OCN15 can alleviate the increase in body weight and body weight gain of mice caused by DHT to some extent, and mainly reduce the weight of gonadal fat of mice (Figure 5C). The lean body mass of mice after combined treatment with OCN15 and DHT increased, and lean body mass is mainly bone weight, illustrating that OCN15 can alleviate osteoporosis caused by hormonal disorders.

[0084] The results of this example demonstrate that OCN15 can be applied to ovarian-related reproductive diseases caused by hyperandrogens, such as menstrual cycle disorders, polycystic ovary syndrome, and androgenic phenotype, as well as metabolic disorders caused by hormonal disorders, such as glucose metabolic disorders, obesity, and osteoporosis.

[0085] In addition, we observed the hair status of mice on the 60th day of modeling and the expression status of androgen receptor in the epidermis layer of mice. The experimental results are shown in Figure 6. As can be seen by comparing the three groups of mice, high androgen can cause hair loss in the neck and back skin of mice, and OCN15 can significantly improve this phenomenon (Figure 6A). This is probably because high androgen causes a significant increase in the expression of androgen receptor AR in the epidermis layer in the neck and back skin of female mice, which leads to hair loss, while OCN15 can significantly reduce the significant increase in the expression of androgen receptor AR in the epidermis layer in the neck and back skin of female mice induced by DHT (Figure 6B), which is also consistent with the hair loss situation on the neck and back of mice.

[0086] The results of this example demonstrate that OCN15 can be applied to related conditions caused by hyperandrogenism, such as female alopecia. Example 7: Effect of ovarian-targeted polypeptides on postovulatory aging of oocytes

[0087] Wild-type female mice were intraperitoneally injected with 5IU PMSG (Pregnant Mare Serum Gonadotropin), and 48 hours later, 5IU hCG (Human Chorionic Gonadotropin) was intraperitoneally injected to induce superovulation. After 14 hours, oocytes were collected, cumulus cells were removed using hyaluronidase, and the mice were randomly divided into the Aging group and the Aging+OCN15 group, and cultured and aged in M16 medium. The aging time for the control group was 0 hours, the aging time for the Aging group was 12 hours, and the Aging+OCN15 group was aged for 12 hours in M16 (1μg / mL) containing polypeptide.

[0088] The experimental results are shown in Figure 7. By comparing the three groups, OCN15 can improve the fragmentation and regression caused by in vitro aging of oocytes (Figure 7A). OCN15 can improve the hardening of the zona pellucida caused by in vitro aging of oocytes (Figure 7B). OCN15 can improve the decline in fertilization rate caused by in vitro aging of oocytes, which is close to the fertilization rate at 0 hours of aging and has no significant difference.

[0089] The results of this embodiment prove that OCN15 can be applied to protect the quality of oocytes, delay their aging process, and improve the decline in fertilization rate caused by aging. It can be applied to the field of assisted reproduction to improve the storage period and storage quality of oocytes and fertilized eggs. At the same time, it has the effect of improving the in vitro aging of oocytes, so it is expected to be used for diseases induced by abnormalities caused by oocyte aging, such as abnormality in oocyte number, decline in oocyte quality, and oocyte aging.

[0090] In summary, the mediator polypeptides provided by the present invention can be used to treat conditions such as ovarian-related reproductive disorders, metabolic disorders and hair loss caused by hormonal disorders that prolong the fertilization window time of oocytes. Example 8: Effects of ovarian targeted polypeptide knockout on serum hormone levels during superovulation in mice

[0091] Wild-type female mice and OCN15 KO mice were intraperitoneally injected with 5IU PMSG and 48 hours later with 5IU hCG to induce superovulation. Mice were killed before injection, 24h after PMSG, 48h after PMSG, and 12h after hCG, and serum was collected to detect sex hormone levels.

[0092] The experimental results are shown in Figure 8. By comparing with WT mice, it is found that the estrogen level does not change during the superovulation process (left panel of Figure 8), but the progesterone level of OCN15 KO mice is significantly improved compared to WT mice at PMSG48h (right panel of Figure 8). PMSG48h is the stage at which most follicles are stimulated to develop into dominant follicles ready for ovulation, and progesterone at this stage is mainly secreted by granulosa cells of large follicles, explaining that at this stage, more follicles respond to PMSG stimulation and develop to the stage of waiting for ovulation in OCN15 KO mice compared to WT. As a result, OCN15 KO may cause excessive activation of mouse follicles and cause premature ovarian failure, which proves that the ovarian targeting polypeptide of the present invention can be used to treat premature ovarian failure. Example 9: Effect of ovarian-targeting polypeptide on high-androgen-induced lipid droplet accumulation in mouse liver

[0093] The method of high androgen DHT-induced mouse PCOS model is shown in Example 6. After modeling is completed, the mouse liver is taken, fixed overnight in 4% paraformaldehyde (PFA), dehydrated in gradient alcohol, embedded in paraffin, sectioned (5 um), and stained with HE for morphological observation.

[0094] The experimental results are shown in Figure 9 , which shows that, compared with the liver morphology of WT mice, the hepatocytes of DHT model mice had a larger volume and more lipid droplets accumulated in the cytoplasm, whereas this phenomenon was alleviated in the DHT+OCN15 treatment group.

[0095] This demonstrates that the ovary-targeting polypeptide of the present invention can alleviate high androgen-induced lipid droplet accumulation in mouse liver and can be used to treat fatty liver.

[0096] The above description is merely a form for implementing the present invention, and is not all of the embodiments. Any equivalent transformations that a person skilled in the art adopts to the technical solutions of the present invention upon reading this specification are all encompassed in the scope of the claims of the present invention.

Claims

1. 1. A polypeptide for targeting the ovary, the polypeptide sequence comprising a sequence represented by the formula M1-Za-M2 or a sequence represented by Za, wherein: M1, M2 are each independently a single or multiple amino acid polypeptide segment or absent; Za is Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro-Thr, X1 is Asn, Tyr, Asp, or absent; X2 is Asn or Gln or His or Pro or Ser, or is absent; X3 is Gly, Trp, or absent; X4 is Leu or absent; X5 is Pro or Ser; X6 is Ala or Val; X7 is Tyr, Ser or Ala; X8 is Pro or Thr, Preferably, Za is YLGASVPSP DPLEPT SEQ ID NO: 1, YLNNGLGAPAPYPDPLEPT SEQ ID NO:2, YLYQWLGAVPYPDPLEPT SEQ ID NO:3, YLYQWLGAVPYPDTLEPT SEQ ID NO:4, YLYQWLGAPAPYPDPLEPT SEQ ID NO:5, YLDHWLGAPAPYPDPLEPT SEQ ID NO:6, YLDPGLGAPAPYPDPLEPT SEQ ID NO:7, YLDHGLGAPAPYPDPLEPT SEQ ID NO:8, YLDQGLGAPAPAPDPLEPT SEQ ID NO:9, YLDSGLGAPVPYPDPLEPT Selected from any one of SEQ ID NO: 10; Polypeptides.

2. A derivative of an ovary-targeting polypeptide, the derivative being a product obtained by performing a conventional modification on an amino acid side group, an amino terminus, or a carboxyl terminus of the polypeptide according to claim 1, or a product obtained by linking a tag for detection or purification of a polypeptide or protein onto the polypeptide according to claim 1, or a product obtained by isotope-labeling modification, the conventional modifications being a fluorescent group modification, a phosphorylation modification, a cyclization modification of a disulfide bond, a modification by biotin labeling, a photosensitizer, an azide modification, a PEG modification, a methylation modification, a fluorescence quenching group modification, a protein coupling modification, a small molecule compound modification, an amination modification, an amidation modification, a hydroxylation modification, a carboxylation modification, a carbonylation modification, an alkylation modification, an acetylation modification, an esterification modification, or a glycosylation modification; Preferably, the tag is His ６ , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityXact; Preferably, the amino-terminal, carboxyl-terminal modification is selected from an acetylation modification at the N-terminus of the polypeptide and an amination modification at the C-terminus; Preferably, the side chain modifications are selected from modifications on the R groups of the amino acid side chains in the polypeptide, Preferably, the fluorescent dye used for the modification of the fluorescent group is selected from AMCA, FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, AIE, ICG, and said modification can be used for fluorescence detection, Preferably, the phosphorylation modifications are selected from one or more combinations of p-Ser, p-Thr, p-Tyr; Preferably, the glycosylation modifications are selected from one or more combinations of Ser, Asn, Thr, Tyr; Preferably, the nitration modification is selected from one or more combinations of Tyr, Preferably, for the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photolabile biotin, and biotin-dUTP; Preferably, the isotopes used for the isotope labeling are selected from one or more combinations of 13C, 14C, 14N, 15N, 2H, 3H, 18O, 32P, 32S, 34S, 35S, 36S, 35Cl, 37Cl, 125I, and 131I; derivative.

3. A polynucleotide encoding the polypeptide of claim 1 or the derivative of claim 2.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell transfected with the vector of claim 4.

6. 13. Use of a polypeptide according to claim 1 or a derivative according to claim 2 in the preparation of an ovarian targeting detection reagent, an ovarian targeting drug or an ovarian targeting tool, comprising: Preferably, the tool is selected from a carrier or a formulation, more preferably a carrier, formulation or reagent kit that achieves a targeting effect by coupling the polypeptide or its derivative according to the present invention, and even more preferably, the formulation is a liposome or nanoparticle. use.

7. Use of a polypeptide according to claim 1 or a derivative according to claim 2 in the preparation of a drug or detection reagent capable of targeting the ovary, comprising Preferably, the drug and detection reagent include the polypeptide according to claim 1 and / or the derivative according to claim 2, a detection product prepared as an antigen, more preferably, the detection product is a detection reagent, a reagent kit, a test strip; use.

8. A carrier targeted to the ovary, comprising a polypeptide according to claim 1 and / or a derivative according to claim 2 as a targeting agent, Preferably, the carrier is selected from a liposome, a nanoparticle, an exosome, a biodegradable polymeric substance, an antibody-ligand, Preferably, the carrier comprises a component capable of treating an ovarian-related reproductive disorder. Carrier.

9. A drug targeting the ovary, said drug being a conjugate of the polypeptide according to claim 1 and / or the derivative according to claim 2 with an active ingredient. Drugs.

10. A polypeptide probe targeted to the ovary, said polypeptide probe being obtained by coupling the polypeptide according to claim 1 and / or the derivative according to claim 2 with a fluorescent dye by organic chemical reaction, Preferably, the fluorescent dye is a dye having a fluorescent group in the near infrared region, and more preferably, the dye having a fluorescent group in the near infrared region is selected from AIE, Cy5, Cy7, and ICG. Polypeptide probes.

11. Use of the polypeptide according to claim 1 and / or the derivative according to claim 2 in the preparation of a drug or reagent for treating or alleviating symptoms of an ovarian associated reproductive disease, said ovarian associated reproductive disease being selected from ovarian associated reproductive diseases caused by germ cell abnormalities, ovarian associated reproductive diseases caused by hormonal disorders, aging and related conditions caused by hormonal disorders, Alternatively, the present invention relates to a method for the preparation of a drug or reagent for preventing oocyte aging in vivo or in vitro, comprising the steps of: Preferably, the hormonal disorder is above normal levels of androgens; Preferably, the ovary-associated reproductive disease caused by the germ cell abnormality is an ovary-associated reproductive disease caused by oocyte aging and / or oocyte zona pellucida hardening, more preferably, the ovary-associated reproductive disease caused by the germ cell abnormality is selected from abnormality in oocyte number, deterioration of oocyte quality, and oocyte aging; Preferably, the ovarian-related reproductive disease caused by said hormonal disorder is selected from menstrual cycle disorder, polycystic ovarian syndrome, androgen phenotype, alopecia, metabolic disorder, ovarian insufficiency, premature ovarian failure, and ovulation disorder, more preferably, the androgen phenotype is selected from acne, hirsutism, and alopecia; Preferably, the metabolic disorder caused by the hormone disorder is selected from glucose metabolism disorder and lipid metabolism disorder, more preferably hyperglycemia, insulin resistance, obesity, fatty liver, and osteoporosis; Preferably, the aging and related symptoms caused by hormonal disorders include perimenopause and menopausal syndromes; use.

12. An assisted reproductive product comprising a polypeptide according to claim 1 and / or a derivative according to claim 2, Preferably, the reproductive aid is a protective liquid for oocytes and / or fertilized eggs during the assisted reproduction process. Reproductive aids.

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