CN116003573A - A kind of follicle stimulating hormone beta subunit active peptide and its application - Google Patents

Abstract

The invention discloses a follicle stimulating hormone beta subunit active peptide and application thereof. The active peptide has the following sequence: X _m ‑Y _n ‑Z _c ; X includes at least 3 amino acids in T, R, D and L; Y is the peptide segment LVYKDPARP; Z includes K, I, T, Q and N At least one amino acid of the active peptide, and K at the 3' end of the active peptide; wherein, 0≤m≤4, n=0 or 1, 0≤c≤4, and m and n are not 0 at the same time. The active peptide provided by the invention can be used as a new FSH preparation that is safe, effective, stable in quality, low in price, high in purity and easy to produce. The use of artificially synthesized FSHβ active peptide can avoid the disadvantages of producing FSH through extraction from the urine of postmenopausal women or gene recombination technology.

Description

A follicle stimulating hormone beta subunit active peptide and its application

Technical Field

The invention belongs to the technical field of bioengineering, and in particular relates to a follicle stimulating hormone beta subunit active peptide and application thereof.

Background Art

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein composed of two polypeptides, α and β, synthesized and secreted by the pituitary gland. It plays a key regulatory role in the development of female follicles and spermatogenesis in male animals. In addition to regulating reproductive function, recent studies have confirmed that high levels of FSH are the key cause of metabolic diseases such as obesity, hyperglycemia, hyperlipidemia, and osteoporosis in postmenopausal women.

In clinical practice, FSH is mainly used to treat infertility and as a key drug for superovulation in assisted reproductive technology. At present, the FSH used in clinical practice is mainly extracted from the urine of menopausal women or produced by genetic recombination technology. When extracting FSH from urine, first of all, the source of urine in menopausal women is limited and the FSH content is low, the extraction amount is limited, and the product price is expensive. In addition, urine-derived FSH sometimes contains pathogens or urine protein contaminants, and there is a risk of causing disease after entering the human body. When producing FSH by genetic recombination technology, the complex post-translational glycosylation modification of FSH α and β subunits must be considered. Glycosylation modification has an important influence on the activity and half-life of FSH. Secondly, in clinical use, FSH produced by urine extraction or genetic recombination technology may dissociate from α and β subunits after entering the body, thereby losing biological activity. Third, FSH produced by urine extraction or genetic recombination technology has a high molecular weight and strong immunogenicity, and usually causes adverse immune responses after entering the body. Therefore, a new FSH preparation that is safe, effective, stable in quality, low in price, high in purity and easy to produce has become an urgent need for clinical application.

Summary of the invention

In view of the above-mentioned deficiencies in the prior art, the present invention provides a follicle stimulating hormone β subunit active peptide and its application. The present invention provides a follicle stimulating hormone β subunit active peptide, which can significantly activate FSHR downstream signals, thereby playing the role of FSH analogs. The active peptide provided by the present invention can be used as a new FSH preparation that is safe, effective, stable in quality, low in price, high in purity and easy to produce. The use of artificially synthesized FSH β active peptide can avoid the disadvantages of extracting FSH from the urine of menopausal women or producing FSH by genetic recombination technology.

To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

A follicle stimulating hormone β subunit active peptide, the active peptide having the following sequence: _Xm - _Yn - _Zc ; X includes at least three amino acids of T, R, D and L;

Y is the peptide LVYKDPARP;

Z includes at least one amino acid selected from the group consisting of K, I, T, Q and N, and K located at the 3' end of the active peptide;

Among them, 0≤m≤4, n＝0 or 1, 0≤c≤4, and m and n are not 0 at the same time.

Furthermore, n and c are 0, m is 4, and its active peptide sequence is TRDL (SEQ ID NO.9).

Furthermore, m is 0, n=1, c is 4, and the active peptide sequence is LVYKDPARPZ _c .

Furthermore, the active peptide sequences are shown in SEQ ID NOs. 5-8.

Furthermore, m is 3, n=1, c is 4, and the active peptide sequence is X ₃ -LVYKDPARP-Z ₄ .

Furthermore, the active peptide sequences are shown in SEQ ID NOs. 1-4.

Application of the above active peptides in the preparation of drugs for regulating reproductive function.

Furthermore, the drug may promote follicle development or spermatogenesis.

The application of the active peptide in the preparation of FSH antagonists or FSH activators.

The active peptide is used in the preparation of medicines for treating diseases caused by abnormal FSH secretion.

Furthermore, the active peptide can alleviate or treat diseases such as obesity, hyperlipidemia, hyperglycemia, osteoporosis, etc. caused by high concentrations of FSH.

Beneficial effects of the present invention:

The physiological function of FSH is mediated by its receptor (FSH receptor, FSR). Among them, the FSHβ subunit plays a core role in mediating the binding of FSH to FSHR and activating downstream signals. The present invention screened the key amino acid site sequence for the binding of FSHβ to FSHR-FSHβ active peptide. The artificially synthesized FSHβ active peptide can significantly activate the downstream signal of FSHR, thereby playing the role of FSH analogs. FSHβ active peptide can be used as a new FSH preparation that is safe, effective, stable in quality, low in price, high in purity and easy to produce. The use of artificially synthesized FSHβ active peptide can avoid the disadvantages of extracting FSH from the urine of menopausal women or producing FSH by genetic recombination technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the effect of FSHβ active polypeptide on the proliferation of mouse granulosa cells;

FIG2 shows the effect of FSHβ active polypeptide on estradiol synthesis in mouse granulosa cells;

FIG3 shows the effect of FSHβ active polypeptide on the initiation of puberty and spermatogenesis in male young mice;

FIG4 shows the effect of FSHβ active polypeptide on the initiation of puberty and ovarian follicle development in female young mice;

FIG. 5 shows the effect of FSHβ active polypeptide on reproductive function of adult female mice.

DETAILED DESCRIPTION

The specific implementation modes of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

Example 1 Synthesis of FSHβ Active Peptide Sequence

The FSHβ active peptide sequence was synthesized using CTC Resin resin as raw material by Fmoc-based solid phase peptide synthesis. The peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The specific sequence is shown in Table 1.

Table 1 FSHβ active peptide sequence

Example 2 Functional verification of FSHβ active peptide

1. In vitro cell culture confirmed that FSHβ active peptide can bind to FSHR and activate its downstream signaling

25-day-old female C57BL/6 mice were selected, killed by cervical dislocation, and the mouse ovaries were quickly isolated and placed in a 4°C pre-cooled PBS solution (containing a 2% penicillin-streptomycin mixture). Under a microscope, the fat around the ovaries was removed with a needle, and the ovaries were rinsed with PBS solution 2-3 times and then transferred to DMEM/F12 medium (supplemented with a 3% FBS and 1% penicillin-streptomycin mixture). The ovaries were punctured with a needle to fully release the granulosa cells in the follicles. The DMEM/F12 medium containing granulosa cells was filtered with a 40μm cell sieve and collected into a 15mL centrifuge tube. Centrifuge at 1000r/min for 5min, discard the supernatant, and repeat 2-3 times to thoroughly wash the impurities. The washed cells were resuspended in a DMEM/F12 medium containing 10% FBS. The isolated ovarian granulosa cells were inoculated into a 96-well cell culture plate at a concentration of 5×10 ³ /well and cultured overnight at 37°C in an incubator containing 5% CO2. After the cells adhered, the culture medium was removed, and then DMEM/F12 culture medium containing 100 ng/mL different FSHβ active peptides (Table 1) was added, and DMEM/F12 culture medium (Vehicle) without peptide was used as a control. 10 μL CCK-8 analysis solution (purchased from Boster, catalog number AR1160) was added to each well, and the absorbance of each well was measured with an ELISA reader after treatment for 0, 6, 12, 18, 24, 30 and 36 hours, and the measurement wavelength was 450 nm.

The results showed that the addition of 9 different FSHβ active peptides (Table 1) can significantly promote the proliferation of mouse ovarian granulosa cells (Figure 1A); further study of the mechanism found that FSHβ active peptides promoted the proliferation of ovarian granulosa cells by upregulating the expression of Ccnd2, a key regulatory gene for ovarian granulosa cell proliferation (Figure 1B). Ccnd2 is a cell cycle regulatory protein and an important downstream target gene of FSH-FSHR signaling, indicating that the 9 different FSHβ active peptides disclosed in the present invention can bind to FSHR and activate the FSHR signaling pathway, and can be used as FSH activators or analogs for commercial applications. Note: * indicates significant difference (P<0.05); Ccnd2, cyclin D2; ^ac different letters indicate significant differences between groups (P<0.05).

2. In vitro cell culture confirmed that FSHβ peptide can activate FSHR and promote ovarian granulosa cells to synthesize estradiol

Granulosa cells of ovaries of 25-day-old female C57BL/6 mice were isolated in the same manner as above. The isolated granulosa cells were inoculated into a 48-well cell culture plate at a concentration of 3×10 ⁴ /well and cultured overnight at 37°C in an incubator containing 5% CO _2. After the cells adhered to the wall, the culture medium was removed and treated with a culture medium containing 100 ng/mL of different FSHβ active peptides (Table 1) for 24 hours, and a DMEM/F12 culture medium without active peptides was used as a control (Vehicle). After the treatment was completed, the culture supernatant was collected and the estradiol (17β-estradiol) content in the cell culture supernatant was determined using a mouse estradiol ELISA kit (KGE014) produced by R&D Company of the United States. The results showed that the addition of 9 different FSHβ active peptides (Table 1) can significantly promote the synthesis of 17β-estradiol in granulosa cells (Figure 2A); further study of the mechanism found that FSHβ active peptides promote estradiol synthesis in granulosa cells by upregulating the expression of aromatase (Cyp19a1), a key gene for estradiol synthesis in ovarian granulosa cells (Figure 2B). The experimental results once again confirmed that the 9 different FSHβ active peptides (Table 1) disclosed in this patent can bind to FSHR and significantly activate FSHR downstream signals, and can be used as FSH activators or analogs for commercial applications. Note: Different letters ^ab indicate significant differences between groups (P<0.05).

The results of in vitro cell culture experiments showed that the activities of the four different FSHβ13AA peptides (i.e., FSHβ13AA1, FSHβ13AA2, FSHβ13AA3, and FSHβ13AA4) were comparable, and the activities of the four different FSHβ16AA peptides (i.e., FSHβ16AA1, FSHβ16AA2, FSHβ16AA3, and FSHβ16AA4) were also comparable; at the same time, the activities of the four different FSHβ13AA and FSHβ16AA were higher than those of FSHβ4AA. Based on this, in subsequent experiments, only three peptides, FSHβ13AA1, FSHβ16AA1, and FSHβ4AA, were selected for in vivo activity evaluation.

3. FSHβ active peptide injection significantly promotes the onset of puberty and testicular spermatogenesis in male mice

25-day-old C57BL/6J male pups were intraperitoneally injected with a single dose of 200 μg/g body weight of different FSHβ active peptides, and mice injected with normal saline were used as controls (Vehicle). Starting from the second day of injection, the separation of the mouse penis foreskin was observed every day, and the age of the mouse penis foreskin separation was recorded. The separation of the penis foreskin is a sign of the onset of puberty in mice. The results showed that a single dose of 200 μg/g body weight of different FSHβ active peptides (FSHβ13AA1, FSHβ16AA1 and FSHβ4AA) can significantly promote the onset of puberty in male mice (Figure 3A).

Starting from the 25th day of age, C57BL/6J male mice were intraperitoneally injected with 200 μg/g body weight of different FSHβ active peptides (Table 1) every day for 4 consecutive days. The mice were killed at the 30th day of age, and the blood was collected to separate the serum. The serum testosterone concentration was determined using the testosterone ELISA kit (KGE010) of R&D Company in the United States; the testes were isolated and embedded in paraffin, sectioned, and H&E stained to detect the spermatogenesis of the mouse testis. The results showed that the injection of three different FSHβ active peptides, FSHβ13A/1, FSHβ16AA1 and FSHβ4AA, could significantly promote the testicular testosterone synthesis of mice (Figure 3B); it was particularly obvious that the testes of mice without FSHβ active peptide injection had no sperm production at 30 days of age (Figure 3C), while the testicular sperm production of mice injected with FSHβ active peptide was clearly visible at 30 days of age (Figure 3C, as shown in the box), indicating that FSHβ active peptide can significantly promote the testicular sperm production of mice. Note: BSP stands for separation of the penis prepuce, which is a sign of the onset of puberty in male mice; *P<0.05;**P<0.01; different letters ^{in ab} indicate significant differences among the groups (P<0.05); in Figure C: short arrows indicate spermatogonia, long arrows indicate secondary spermatocytes, and squares indicate mature sperm.

4. FSHβ active peptide injection significantly promotes the onset of puberty and ovarian follicle development in female mice

A single dose of 200 μg/g body weight of different FSHβ active peptides was intraperitoneally injected into 25-day-old C57BL/6J female mice, and mice injected with normal saline were used as controls (Vehicle). From the second day of injection, the age of the first estrous (First estrous) of the mice was tracked and detected every day by vaginal smear. The first estrous is a sign of the end of puberty in mice. The results showed that a single dose of three different FSHβ active peptides (FSHβ13AA1, FSHβ16AA1 and FSHβ4AA) can significantly promote the onset of puberty in female mice (Figure 4A).

Starting from 25 days of age, C57BL/6J female mice were intraperitoneally injected with 200 μg/g body weight of different FSHβ active peptides every day for 4 consecutive days. Mice were killed at 30 days of age, blood was collected to separate serum, and serum estradiol (17-βestradiol) concentration was measured using the estradiol ELISA kit (KGE014) produced by R&D Company of the United States; ovaries were separated, paraffin-embedded and sectioned, and the development of mouse ovarian follicles was detected by H&E staining. The results showed that injection of three different FSHβ active peptides, FSHβ13AA1, FSHβ16AA1 and FSHβ4AA, could significantly promote the synthesis of estradiol in mouse ovaries (Figure 4B) and follicle development (Figure 4C and D). Note: First estrous is the first estrous, which is the sign of the end of puberty in female mice; Antral follicle is the antral follicle; * indicates significant difference (P<0.05); different letters ^{in ab} indicate significant difference between groups (P<0.05); arrows indicate antral follicles.

5. FSHβ active peptide antagonizes endogenous FSH activity in adult female mice

The estrus cycle of female adult mice was detected by vaginal smear. The mice were intraperitoneally injected with a single dose of 200 μg/g body weight of different FSHβ active peptides during the estrus period, and the mice were killed in the proestrus period immediately thereafter. After the mice were killed, blood was collected, and the ovaries were separated and weighed. After the blood was centrifuged to separate the serum, the ELISA kit (KGE014) of R&D Company in the United States was used to determine the serum estradiol concentration of the mice.

The results showed that compared with the blank injection control (Vehicle), the injection of three different FSHβ active peptides, FSHβ13AA1, FSHβ16AA1 and FSHβ4AA, could significantly reduce the ovarian weight of mice (Figure 5A) and serum estradiol concentration (Figure 5B). It can be seen that the biological activity of FSHβ active peptide is lower than that of endogenous FSH. When the endogenous FSH content in humans or animals is high, the injection of FSHβ active peptide can competitively bind to FSHR, thereby hindering the binding of endogenous FSH to FSHR, thereby reducing the physiological activity and effect of endogenous FSH. Therefore, in the presence of endogenous FSH, FSHβ active peptide can be used as an FSH antagonist for commercial applications. Note: Different letters ^ab indicate significant differences between groups (P<0.05).

Sequence Listing

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Claims (10)

1. A follicle stimulating hormone beta subunit active peptide, characterized in that the active peptide has the sequence: x is X _m -Y _n -Z _c The method comprises the steps of carrying out a first treatment on the surface of the The X comprises at least 3 amino acids of T, R, D and L;

the Y is a peptide LVYKDPARP;

the Z comprises at least 1 amino acid in K, I, T, Q and N and K positioned at the 3' -end of the active peptide;

wherein m is more than or equal to 0 and less than or equal to 4, n is more than or equal to 0 or 1, c is more than or equal to 0 and less than or equal to 4, and m and n are not simultaneously 0.

2. The follicle stimulating hormone β subunit active peptide of claim 1, wherein n and c are 0, m is 4, and the active peptide sequence is TRDL.

3. The follicle stimulating hormone beta subunit active peptide of claim 1, wherein m is 0, n=1, c is 4, and the active peptide sequence is lvykdprpz _c 。

4. The follicle stimulating hormone beta subunit active peptide of claim 3, wherein the active peptide sequence is shown in SEQ ID nos. 5-8.

5. The follicle stimulating hormone beta subunit active peptide of claim 1, wherein m is 3, n=1, c is 4, and the active peptide sequence is X ₃ -LVYKDPARP-Z ₄ 。

6. The follicle stimulating hormone beta subunit active peptide of claim 5, wherein the active peptide sequence is shown in SEQ ID nos. 1-4.

7. Use of an active peptide according to any one of claims 1 to 6 for the preparation of a medicament for modulating reproductive function.

8. The use of claim 7, wherein the medicament promotes follicular development or spermatogenesis.

9. Use of an active peptide according to any one of claims 1 to 6 for the preparation of an FSH antagonist or FSH activator.

10. Use of an active peptide according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of a disease caused by abnormal secretion of FSH.

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