CN114656524B

CN114656524B - Target ovarian polypeptide and application thereof

Info

Publication number: CN114656524B
Application number: CN202210329829.0A
Authority: CN
Inventors: 张键; 朱雯; 赵华山; 杨雅莉; 葛磊; 肖天霞
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2023-12-26
Anticipated expiration: 2042-03-31
Also published as: CN114656524A

Abstract

The invention relates to a polypeptide and application thereof, and particularly discloses a polypeptide sequence represented by a formula M1-Za-M2; wherein: m1, M2 are each independently single or multiple amino acid polypeptide segments or are absent; za is His-Val-Gln-X1-Ile-Ser-X2-X3-Gly-X4-Ser-Pro-X5-Ser-X6-Pro-Val-Leu. Derivatives of the above polypeptides are also disclosed. And the use of the polypeptide and the derivative in treating or preventing ovarian diseases, metabolic disorders caused by Gao Xionggao lipid, glycolipid metabolic disorders and hair loss. The polypeptide disclosed by the invention has the advantages of simple structure, high targeting property and excellent treatment effect.

Description

Target ovarian polypeptide and application thereof

Technical Field

The invention discloses a polypeptide capable of targeting an ovary, a derivative thereof and application thereof, belonging to the field of biological medicine. In particular to application of the polypeptide in preparing an ovary targeting drug or a detection reagent; also disclosed are derivatives thereof, which are products of the polypeptides obtained by modification of the terminal or side chain thereof. The polypeptide molecules can specifically target the inside of ovarian tissues, provide a method for treating ovarian diseases, and can also treat diseases caused by metabolic disturbance and aging-related diseases.

Background

Ovarian disease mainly includes premature ovarian failure, ovarian dysfunction, polycystic ovary syndrome. In recent years, the incidence of ovarian diseases is on the rise, which leads to fertility loss and low estrogen status, becomes a factor which can not be ignored for influencing female reproductive health and social stability, and is a disease which seriously affects female physical and mental health. Furthermore, the time that oocytes can be fertilized after maturation is very limited. For example, the time for optimal fertilization and development in mice is not within 12 hours after ovulation, whereas humans are within 4-12 hours after ovulation or egg taking. Over time, the mature oocytes enter the aging stage and eventually degenerate. In assisted reproductive interventions, inhibiting post-ovulatory aging in mammals improves and increases fertility and fertility. The etiology of ovarian disease is complex, the pathogenesis is unknown, and the incidence rate rises year by year. Currently, ovarian disease is treated clinically primarily by hormone replacement therapy. Although the effect is quick, obvious toxic and side effects can occur after long-term use of hormone.

The hormone can regulate metabolism of organism, maintain dynamic balance of physical and chemical factors in organism, regulate growth, development, reproduction and aging functions of organism, and enhance resistance and adaptability of organism to harmful stimulation. Hormonal disorders cause endocrine disorders throughout the body resulting in the development of metabolic diseases such as obesity, diabetes, fatty liver, osteoporosis, etc. In addition, hormonal disorders can lead to premature loss of fertility by the reproductive system and other aging phenotypes such as inflammatory responses, arteriosclerosis, etc. A large number of research literature studies and clinical data indicate that hormonal disorders can lead to the occurrence of diseases such as breast cancer, ovarian cancer, endometrial cancer and the like.

Metabolic disorder is a state of the body, and is a state in which the body is pathological in digestion, absorption and excretion of substances and is not in coordination with supply and demand. Is mainly expressed by glycolipid disorder, and can lead to a series of metabolic diseases such as diabetes, hypertension, hyperlipidemia and the like. Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia due to multiple causes, due to defects in insulin secretion and/or utilization. Long-term carbohydrate, fat, protein metabolism disorders can cause multisystem damage, resulting in chronic progressive lesions, hypofunction and failure of tissues and organs such as eyes, kidneys, nerves, heart, blood vessels, etc. Acute severe metabolic disorders, such as diabetic ketoacidosis, hyperglycemic syndrome, can occur when the condition is severe or stressed. The metformin, sulfonylurea and thiazolidinedione (two or three) can be used singly or in combination, and can be used together with insulin.

The body develops many aging symptoms with age, and the etiology is complex. Alopecia is a common phenomenon among people in early and middle age, and is also a result of aging. Alopecia not only affects the beauty, but also brings adverse effects to the emotion of people. Arteriosclerosis, bone loss, degenerative diseases, and hypofunction of organs are diseases accompanied with aging. At present, age-related aging symptoms are not overcome well, more and more funds tend to research aging-related diseases, but no good medicines are currently available.

Disclosure of Invention

The invention discovers the polypeptides with the following sequences, which can specifically target the ovary and can treat ovarian diseases, metabolic disorders and diseases caused by aging.

In one aspect, the invention provides a polypeptide comprising a sequence of formula M1-Za-M2 or a sequence of Za, wherein:

m1, M2 are each independently single or multiple amino acid polypeptide segments or are absent;

za is His-Val-Gln-X1-Ile-Ser-X2-X3-Gly-X4-Ser-Pro-X5-Ser-X6-Pro-Val-Leu

X1 is Ala or Ser,

x2 is Ile or Met,

x3 is Gln or Ser or Gly,

x4 is Gln or Thr or Ser,

x5 is Ala or Pro or Leu,

x6 is Glu or Gln.

Further, wherein Za is selected from one of

HVQAIS IQGQSPASEP VL SEQ ID No.1

HVQSIS MSGTSPPSEP VL SEQ ID No.3

HVQSIS MGGSSPPSQP VL SEQ ID No.5

HVQSIS MGGSSPPSEP VL SEQ ID No.7

HVQSIS MSGTSPLSEP VL SEQ ID No.9

Further, wherein M1 is absent and M2 is F, or neither M1 nor M2 is present.

In a further aspect, the present invention provides a derivative of the above polypeptide, wherein the derivative is a product obtained by conventional modification of the amino terminal and the carboxyl terminal of the amino acid side chain group of the polypeptide, or a product obtained by connecting a tag for detecting or purifying a polypeptide or a protein to the polypeptide; the conventional modification is fluorescent group modification, phosphorylation modification, cyclization modification of disulfide bond, biotin labeling modification, photosensitizer, 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;

the tag is His ₆ GST, EGFP, MBP, nus, HA, igG, FLAG, c-Myc or ProfinityeXact.

In a further aspect, the invention provides a polynucleotide encoding a polypeptide as described above or a derivative thereof.

In a further aspect, the invention provides a vector comprising a polynucleotide as described above.

In a further aspect the invention provides a host cell transfected with the vector described above.

In a further aspect, the invention provides the use of a polypeptide of the invention or a derivative thereof in the preparation of an ovarian targeting detection reagent, an ovarian targeting drug or an ovarian targeting tool.

Further, the means is selected from the group consisting of a carrier, a formulation or a kit, e.g. a carrier or a formulation, such as a liposome, a nanoparticle, etc., for achieving a targeting effect by coupling the polypeptide or a derivative thereof according to the invention.

In a further aspect, the invention provides an ovarian targeted assay reagent comprising a polypeptide or derivative of the invention.

In a further aspect, the invention provides the use of a polypeptide or derivative thereof according to the invention in the manufacture of a medicament for the treatment or prophylaxis of an ovarian disease.

In yet another aspect, the invention provides a method of treating or preventing an ovarian disorder, the method comprising administering to a subject a therapeutically effective amount of a polypeptide of the invention or derivative thereof.

In a further aspect, the invention provides a medicament for treating or preventing ovarian disease, which comprises the polypeptide or derivative thereof.

Further, the ovarian disease is selected from premature ovarian failure, polycystic ovary syndrome, functional irregular menstruation, menopausal syndrome, ovarian dysfunction, infertility due to follicular mass, number or difficulty in ovulation.

Further, the premature ovarian failure is caused by genetic factors, chemotherapy or radiotherapy, immunosuppressants, pelvic surgery, viral infections, exposure to heavy metals, organic solvents, pesticides, plasticizers, industrial chemicals.

Further, the treatment or prevention of ovarian disease is to increase ovarian weight, decrease the magnitude of decrease in ovarian weight, decrease ovarian atrophy, increase the number of primordial follicles, primary follicles, secondary follicles, extend the fertilization window period, improve post-ovulation aging, and increase fertility; treating or ameliorating the symptoms of, or inhibiting the progression of: premature ovarian failure, polycystic ovary syndrome, functional irregular menstruation, menopausal syndrome, and ovarian dysfunction.

In a further aspect, the invention provides the use of a polypeptide or derivative thereof according to the invention in the manufacture of a medicament for the treatment of a metabolic disorder caused by Gao Xionggao lipid.

In yet another aspect, the invention provides a method of treating or preventing a metabolic disorder caused by high androgens and hyperlipidemia, the method comprising administering to a subject a therapeutically effective amount of a polypeptide of the invention or a derivative thereof.

In a further aspect, the present invention provides a medicament for treating or preventing a metabolic disorder caused by high-androgenic hyperlipidemia, which comprises the polypeptide or a derivative thereof according to the present invention.

Further, the Gao Xionggao lipid-induced metabolic disorder is a metabolic abnormality, diabetes, insulin resistance, hyperlipidemia, obesity caused by polycystic ovary syndrome with hyperandrogenic features, hyperandrogenism or hyperandrogenism, the method comprising administering to a subject a therapeutically effective amount of a polypeptide of the present invention or a derivative thereof.

In a further aspect, the invention provides the use of a polypeptide or derivative thereof according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a glycolipid metabolic disorder.

In a further aspect, the invention provides a method of treating or preventing a glycolipid metabolic disorder, said method comprising administering to a subject a therapeutically effective amount of a polypeptide or derivative thereof according to the invention

In a further aspect, the present invention provides a medicament for the treatment or prophylaxis of a glycolipid metabolic disorder, which comprises a polypeptide of the present invention or a derivative thereof.

Further, the glycolipid metabolic disorder is caused by a leptin receptor deficiency.

Further, the glycolipid metabolic disorder is selected from the group consisting of fatty liver, diabetes, insulin resistance, hyperlipidemia, obesity.

In a further aspect, the invention provides the use of a polypeptide or derivative thereof according to the invention in the manufacture of a medicament for the treatment or prevention of hair loss.

In yet another aspect, the invention provides a method of treating or preventing hair loss, comprising administering to a subject a therapeutically effective amount of a polypeptide of the invention or a derivative thereof.

In a further aspect, the present invention provides a medicament for treating or preventing hair loss, which comprises the polypeptide or derivative thereof of the present invention.

Further, the hair loss is hair loss due to aging.

In a further aspect the invention provides the use of the polypeptide of the invention as an antigen-producing antibody.

In a further aspect, the invention provides the use of a polypeptide or derivative thereof according to the invention in the preparation of a tool for culturing cells and tissues.

Further, the cells and tissues include ovary, uterus, bone, brain, heart, liver, spleen, lung, kidney, bone, fat at various stages of development.

Further, the means may be, for example, culture fluids, additives, detection reagents, organoids.

The polypeptide medicine has the advantages of accurate targeting and high selectivity, and simultaneously shows the characteristics of a series of small molecular medicines, such as rapid tissue permeability, low immune response activation risk and convenient production. Polypeptide drugs are usually eliminated via the kidneys, avoiding potential hepatotoxicity of small molecule drugs and antibody drugs. Based on the strong binding effect of the specificity of the polypeptide drugs and the target, the polypeptide drugs can use lower dosage, and the occurrence of main side effects is avoided.

Through a plurality of experiments, the targeting ovarian polypeptide provided by the invention can precisely and specifically target the ovary, has a therapeutic effect on ovarian diseases, and can treat impaired glucose tolerance and insulin resistance caused by metabolic disorder caused by Gao Xionggao lipid and alopecia caused by aging.

Drawings

FIG. 1 is a graph showing the results of specific targeting of the ovary-targeting polypeptides of the invention in example 1.

FIG. 2 is a graph showing the results of the heavy changes after treatment of the mice of the different groups in example 2. The left graph shows the weight values of the mice corresponding to different groups, and the right graph shows the weight change values of the mice of different groups.

Figure 3 is a graph showing the results of ovarian weight reduction in the treated mice of the different groups of example 2. The left graph shows the ovarian weight values of mice corresponding to different groups, and the right graph shows the ovarian weight change values of mice of different groups.

FIG. 4 is a chart showing the results of follicle count and histomorphology for the different groups of example 2, with the upper panel showing staining of ovarian tissue and the lower panel showing morphological photographs of ovarian tissue.

Figure 5 shows the results of the counting of follicles at different stages in the ovary of example 2.

FIG. 6 is a graph showing the results of pregnancy occurrence rate in different groups of mice in example 2.

FIG. 7 shows the improvement of in vitro fertilization status by improving zona pellucida sclerosis by inhibiting the fragmentation and degeneration of oocytes caused by aging in the aging model of the target polypeptide of example 2. Panel A shows degradation and fragmentation rates after 12 hours of aging in the control, aging, disordered polypeptide aging, and targeted polypeptide groups. Panel B is the zona pellucida hardening level of the control, aging and targeted polypeptide groups aged for 12 hours. Panel C is the in vitro fertilization rates after 12 hours of in vitro aging in the control, aging and targeted polypeptide groups.

FIG. 8 shows the results of intraperitoneal glucose tolerance tests in a mouse pathological model of Gao Xionggao lipid-induced polycystic ovary syndrome (PCOS) for different groups in example 3.

FIG. 9 shows the results of intraperitoneal insulin resistance tests in a mouse pathological model of Gao Xionggao lipid-induced polycystic ovary syndrome (PCOS) for different groups in example 3.

FIG. 10 shows the accumulation of liver lipid droplets in db/db transgenic mice from different groups in example 4.

FIG. 11 shows the results of treatment of aging-induced dehairing in different groups according to example 4.

FIG. 12 is a comparison of polypeptide sequences from different species.

Detailed Description

The following detailed description of the present invention will be made in detail to make the above objects, features and advantages of the present invention more apparent, but should not be construed to limit the scope of the present invention.

Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to those described herein can be used in the practice of the present invention. For the purposes of the present invention, the following terms are defined.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, humans, mice, rats, apes, chickens, cattle, zebra fish, teleost fish, carp, tilapia, large yellow croaker, crucian. Tissues, cells, and progeny of the biological entities obtained in vivo or cultured in vitro are also contemplated.

As used herein, the term "administering" 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, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other modes of delivery include, but are not limited to, use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

The term "treatment" refers to a method for achieving a beneficial or desired result, including but not limited to therapeutic benefit. Therapeutic benefit means any treatment-related improvement in or effect on one or more diseases, conditions or symptoms in treatment. "prevention" refers to a method for achieving a beneficial or desired result, including but not limited to, a prophylactic benefit. For prophylactic benefit, the composition may 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 the disease, even though the disease, condition, or symptom may not have been manifested.

The term "therapeutically effective amount" refers to an amount of a peptide or composition sufficient to achieve a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration, and the like, which can be readily determined by one of ordinary skill in the art. The specific amounts may vary depending on one or more of the following: the particular agent selected, the type of target cell, the location of the target cell in the subject, the dosing regimen to be followed, whether to administer in combination with other compounds, the time of administration, and the entity delivery system in which it is carried.

Polypeptides

In one aspect, the polypeptides of the invention are shown in SEQ ID No.1-SEQ ID No.10, which can be synthesized by methods known in the art.

In the present invention, the peptides are isolated peptides.

The above polypeptide SEQ ID No.1-10 is derived from mouse, human, rat, cow, zebra fish, teleost fish, carp, tilapia, large yellow croaker, and Carassius auratus, and is shown in FIG. 12. SEQ ID Nos. 1 to 10 of the present invention are homologous polypeptide sequences from different species, which have their similar framework sequence structures, respectively compared with SEQ ID No.1, and the difference is not more than 6 amino acid substitutions, so that it can be assumed that these sequences have similar biological functions and are also included in the scope of the present invention.

Composition and medicament

The present application also relates to a medicament or pharmaceutical composition comprising at least one peptide of the invention as described above or one of its pharmaceutically acceptable salts or solvates, and at least one pharmaceutically acceptable carrier.

At least one peptide of the invention is present as an active ingredient in a medicament or pharmaceutical composition of the invention.

The compositions or medicaments of the invention are in a form suitable for mammalian administration.

The compositions or medicaments of the invention may be administered, for example, orally, parenterally, intravenously, rectally, transdermally, topically or by inhalation. In particular, the composition according to the invention is administered by intravenous or subcutaneous route.

According to a specific embodiment, the pharmaceutically acceptable carrier of the composition of the invention is suitably selected from injectable carrier liquids, such as sterile water for injection; and aqueous solutions such as saline.

The medicament or pharmaceutical composition of the invention may comprise at least one peptide of the invention as the sole active ingredient, or may further comprise at least one other active ingredient, provided that said other active ingredient does not interfere with the biological activity of the peptide according to the invention.

The pharmaceutical composition or medicament according to the invention may further comprise at least one antioxidant, dispersant, emulsifier, defoamer, flavoring agent, preservative, solubilizer and/or colorant, provided that this/these other substance(s) do not interfere with the biological properties of the peptide according to the invention.

The sterile compositions of the present invention for parenteral administration can be in particular aqueous or non-aqueous solutions, suspensions or emulsions. Solvents or vehicles that may be used include water, propylene glycol, polyethylene glycol, vegetable oils, particularly olive oil, injectable organic esters such as ethyl oleate, or other suitable organic solvents. These compositions may also contain adjuvants, in particular wetting agents, tonicity agents, emulsifying agents, dispersing agents and stabilizing agents. Sterilization can be performed in several ways, for example by sterile filtration, by incorporating sterilizing agents into the composition, by irradiation or by heating. They may also be prepared in the form of sterile solid compositions which may be dissolved in sterile water or any other injectable sterile medium at the time of use.

The composition for topical administration may be, for example, nasal drops or aerosols.

For subcutaneous, intramuscular or intravenous administration, the peptides of the invention used are converted into solutions, suspensions or emulsions with substances conventionally used for this purpose, such as solubilizers, emulsifiers or other excipients. Suitable solvents, for example: water, physiological saline or alcohol, such as ethanol, propanol, glycerol, and sugar solutions (e.g., dextrose or mannitol solutions), or mixtures of the above various solvents.

In a particular embodiment, the composition of the invention, the medicament of the invention, or one of the peptides of the invention or pharmaceutically acceptable salts or solvates thereof is administered to a subject by parenteral route, and in particular transdermal, intravenous, subcutaneous or intramuscular, in particular intravenous or subcutaneous.

Application of

Example 1 Targeted Studies

The modified product is selected from a product obtained by modification of a fluorescent group, a product obtained by phosphorylation, a product obtained by cyclization modification based on disulfide bonds, a product obtained by biotin labeling modification, a product obtained by modification of a photosensitizer, a product obtained by azide modification, a product obtained by PEG modification, a product obtained by methylation modification, a product obtained by modification of a fluorescence quenching group, a product obtained by modification of a protein coupling modification, or a product obtained by modification of a small molecular compound;

the modified polypeptide Peptide (shown as SEQ ID No. 1) is connected with a fluorescent group, so that the polypeptide is fluorescent.

Mice were divided into four groups, peptide, PBS, flu, SCR. The polypeptide Peptide (Peptide group), PBS (PBS group), single fluorescent material (Flu group) and disordered polypeptide (SCR group) connected with the fluorescent material are respectively injected by tail vein injection, the injection amount is 0.325mM, each mouse is injected with 100 mu L, and after the mice are metabolized for a period of time in vivo, the fluorescent brightness of each organ of the mice is observed by a small animal imager.

The results are shown in fig. 1, and the experimental results show that the ovaries of mice injected with the Peptide with the fluorescent group are strong in fluorescence compared with other groups, the other tissues do not observe strong fluorescence reaction, and the other control groups, namely PBS group, flu group and SCR group, do not observe strong fluorescence reaction at the positions of the ovaries, so that the Peptide can specifically target the ovaries.

According to the experimental results, the polypeptide provided by the invention has a specific ovarian targeting effect, and due to the fact that the polypeptide is short, the polypeptide can be expected to be further developed into an ovarian detection reagent through a coupling tag or a fluorescent preparation, and the targeting and bioavailability of the therapeutic drug for ovarian diseases can be further improved through coupling other active ingredients or serving as a targeting ingredient in a pharmaceutical preparation.

EXAMPLE 2 treatment experiments on premature ovarian failure

Establishing an premature ovarian failure model: 8 weeks of mice were selected and the mice were divided into 4 experimental groups CTX (cyclophosphamide), experimental group peptide+ctx, control group CTRL and control group Peptide.

The CTX group of mice was injected with 75mg/kg Cyclophosphamide (CTX) on the first day, followed by 30mg/mL cyclophosphamide every other day for 3 weeks.

The pepide + CTX group mice were injected with cyclophosphamide at a dose of 750nM/g, daily, and three weeks continuously, except for the dose and interval of the CTX group mice, starting with the pepide one day before the first injection of cyclophosphamide.

CTRL group (PBS group) mice were injected at the dose and interval of CTX group mice, differing only in the substitution of cyclophosphamide to PBS.

Peptides mice the same procedure was used for Peptides+CTX mice except that cyclophosphamide was replaced with PBS.

Female mice were subcutaneously injected with 5IU PMSG and 5IU hCG was intraperitoneally injected 48 hours later for superovulation. The random groups are divided into a control group, an Aging group and an aging+peptide group.

Oocytes were collected after 14 hours, cumulus cells were removed using hyaluronidase, randomly divided into an Aging group and an aging+peptide group, and aged in M16 medium. The control group was freshly obtained mature ova for 0 hours.

Aging time of the Aging group was 12 hours, and Aging of the aging+Peptide group was performed in M16 containing Peptide (1 ug/ML) for 12 hours.

The mice were examined for various indicators during the course of the experiment.

1) Study of body weight and ovarian weight

The weight of each group of mice was weighed before and three weeks after administration and the weight of ovaries after dissection. The results are shown in FIG. 2 and FIG. 3.

Compared with the control group, the weight and the weight of the ovaries of the mice in the experimental group have a decreasing trend, which indicates that the ovaries of the cyclophosphamide mice are atrophic and conform to the characteristics of premature ovarian failure.

After cyclophosphamide mice were given Peptide treatment, no significant weight loss of ovaries occurred in the mice compared to the control group. While there was no difference between the Peptides injected alone and the control group, the above results indicate that Peptides can improve ovarian atrophy and that Peptides alone have no toxicity.

2) Follicular counting and histomorphometric analysis

The ovaries of mice were fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded and cut into 5 μm thick sections and the ovaries were stained with hematoxylin and eosin and observed. As shown in fig. 4 and 5, compared with the experimental group, the ovarian premature senility model group mice have obviously reduced ovarian primordial follicles and obviously reduced slice areas. The reduction trend of the number of the primordial follicles of the premature ovarian failure making module after the treatment by the Peptide is obviously relieved, and the reduction trend of the area of the ovarian slice is improved. Whereas the Peptide alone group was not different from the control group.

3) Evaluation of the influence of CTX treatment and Peptides combination treatment on fertility

At 8 weeks after the last treatment, female mice were mated with normal and fertility-proven male mice, and then mated again every 7 weeks for 3 consecutive rounds of mating. The number of pups per pregnancy was counted for each group. The measurement results show that CTX reduced fertility in mice, but saved fertility after treatment with Peptide. Although the CTX-treated mice were not sterilized by the dose administered, the incidence of pregnancy was lower than that of the control group, but the incidence of pregnancy was recovered by co-treatment with pepide, and the pepide alone was not different from the PBS group of the control group, and the results are shown in fig. 6.

4) The experimental results are shown in FIG. 7, and by three groups of comparison, peptide can inhibit the fragmentation and degradation caused by in vitro aging of oocytes after ovulation (A). The Peptide can improve zona pellucida hardening (B) caused by in vitro aging of the oocyte, thereby improving the fertilization rate reduction caused by in vitro aging of the oocyte.

Example 3 therapeutic experiments on amelioration of elevated blood glucose due to metabolic disorders

A mouse pathological model of Gao Xionggao lipid-induced polycystic ovary syndrome (PCOS) was established, and the specific experimental procedure was as follows: c57 female mice (21-day-old) experimental groups were subcutaneously injected daily with DHEA (6 mg/100g body weight, 100 μl of mice, sesame oil plus 95% ethanol) for 20 consecutive days, and fed with high fat (dhea+hfd), a portion of which was simultaneously injected with Peptide (dhea+hfd+peptide). The control group was injected with 0.09mL sesame oil and 0.01mL95% ethanol daily, continuously for 20d, fed with normal feed, and injected with physiological saline (CTRL). The intraperitoneal injection glucose tolerance and insulin tolerance were tested after the end of the experiment. Polycystic ovary syndrome is both a reproductive and endocrine disease, mainly because ovarian dysfunction leads to estrogen reduction and marked androgen elevation, leading to endocrine dysfunction. Endocrine disorders are mainly manifested by acne, obesity, insulin resistance, diabetes, etc. From the results shown in fig. 8 and 9, the metabolic disorder caused by Gao Xionggao lipid was significantly improved after treatment with Peptide polypeptide, which is mainly represented by improvement of glucose tolerance and insulin tolerance.

Example 4

And selecting db/db transgenic mice of 3 months of age for experiments, wherein the db/db transgenic mice are leptin receptor gene defective mice. The phenotype of the strain is obesity, diabetes mellitus, fatty liver and the like, and the strain is a classical model for researching glycolipid metabolic disturbance. Db/db mice were divided into two groups, one group was injected daily with Peptide at a dose of 750nM/g, the other group was injected with equal amounts of PBS, daily, and one month of continuous injection. As a result, as shown in fig. 10, accumulation of lipid droplets in the liver of Peptide group was significantly reduced as compared with the control group. Leptin is involved in the regulation of sugar, fat and energy metabolism after entering the blood circulation, and promotes the body to reduce ingestion and inhibit synthesis of fat cells, thereby reducing body weight and improving diabetes, fatty liver and the like caused by metabolic disorder. It acts upon binding to leptin receptors and leptin does not act upon receptor knockout. From the results, the polypeptide Peptide can obviously improve fatty liver symptoms of db/db transgenic mice, which indicates that the polypeptide can treat diseases caused by glycolipid metabolic disorder.

EXAMPLE 5 treatment experiment on dehairing

C57 female mice over eight months of age are selected, the mice have serious dehairing phenomenon, dehairing mice caused by the aging are randomly divided into two groups, one group is injected with Peptide polypeptide, the injection dose is 750nM/g, the injection is daily, the other group is injected with PBS with equal quantity, the injection is daily, the observation is carried out after one month of continuous treatment, the experimental result is shown in figure 11, the hair of the Peptide group mice starts to grow, and the dehairing of the PBS group is more serious. This result indicates that Peptide can treat the depilation phenomenon caused by aging.

SEQUENCE LISTING

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<120> a targeted ovarian polypeptide and uses thereof

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<170> PatentIn version 3.3

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Claims

1. Application of polypeptide with an amino acid sequence shown as SEQ ID No.1 or derivatives thereof in preparing detection reagents targeting ovaries, medicaments targeting ovaries or tools targeting ovaries;

the derivative is a product obtained by conventional modification of amino terminal and carboxyl terminal of amino acid side chain groups of the polypeptide; or a product obtained by attaching a tag for detection or purification of a polypeptide or protein to the polypeptide;

the conventional modification is fluorescent group modification, biotin labeling modification and fluorescence quenching group modification;

the tag is His ₆ GST, EGFP, MBP, nus, HA, igG, FLAG, c-Myc or proficiency eXact.

2. The use according to claim 1, wherein the means is selected from the group consisting of a carrier, a formulation or a kit.

3. The application of the polypeptide with the amino acid sequence shown as SEQ ID No.1 in preparing medicines for treating or preventing premature ovarian failure, metabolic disorder caused by Gao Xionggao lipid, glycolipid metabolic disorder and hair loss caused by aging.

4. The use according to claim 3, wherein the treatment or prevention of premature ovarian failure is an increase in ovarian weight, a decrease in the magnitude of decrease in ovarian weight, a decrease in ovarian atrophy, an increase in the number of primordial follicles, an improvement in post-ovulatory aging and an increase in fertility and fertility rate;

the Gao Xionggao lipid-induced metabolic disorder is Gao Xionggao lipid-induced polycystic ovary syndrome;

the glycolipid metabolic disorder is caused by a leptin receptor deficiency.

5. The use according to claim 3, wherein the glycolipid metabolism disorder is selected from the group consisting of fatty liver caused by glycolipid metabolism disorder.

6. A medicament comprising a therapeutically effective amount of the polypeptide of claim 1;

the medicine has the functions of targeting the ovary, treating or preventing premature ovarian failure, treating or preventing metabolic disorder caused by high male lipid, treating or preventing glycolipid metabolic disorder, and treating or preventing hair loss caused by aging.

7. Use of the polypeptide of claim 1 and/or the derivative of claim 1 as an antigen for the production of antibodies or in the preparation of a detection kit for non-disease diagnostic or therapeutic purposes.

8. Use of the polypeptide of claim 1 and/or the derivative of claim 1 for the preparation of a means for culturing cells and tissues;

the cells and tissues include ovaries at various stages of development.