CN113599503A - Biological agent for regulating ovarian function and preparation method and application thereof - Google Patents
Biological agent for regulating ovarian function and preparation method and application thereof Download PDFInfo
- Publication number
- CN113599503A CN113599503A CN202110854104.9A CN202110854104A CN113599503A CN 113599503 A CN113599503 A CN 113599503A CN 202110854104 A CN202110854104 A CN 202110854104A CN 113599503 A CN113599503 A CN 113599503A
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- Prior art keywords
- recombinant
- hormone
- follicle
- stimulating hormone
- mullerian
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Abstract
The invention discloses a biological preparation for regulating ovarian function and a preparation method and application thereof, wherein the biological preparation is an oral preparation consisting of one or more components of recombinant anti-mullerian hormone, PEG (pegylated) recombinant anti-mullerian hormone, recombinant follicle-stimulating hormone, PEG (pegylated) recombinant follicle-stimulating hormone, anti-mullerian hormone yolk antibody and follicle-stimulating hormone yolk antibody. In the invention, TAT induction peptide can promote the intestinal absorption of protein, PEG recombinant anti-mullerian hormone and follicle-stimulating hormone can prolong the half-life period, and products containing antibodies of the two proteins can be obtained by immunizing animals with the two recombinant proteins respectively, and can be directly eaten or prepared into granules, tabletting products and the like. The recombinant anti-mullerian hormone and the yolk antibody product thereof, the recombinant follicle stimulating hormone and the yolk antibody product thereof form a system capable of adjusting the ovarian function, can be used for clinically treating ovarian dysfunction and daily health care, and the product has low cost and is convenient for clinical and daily health care use.
Description
Technical Field
The invention relates to the technical field of gene recombination, in particular to a biological agent for regulating ovarian function and a preparation method and application thereof.
Background
The factors influencing the ovarian dysfunction are many, and the factors influencing the ovarian dysfunction are anti-mullerian hormone and follicle stimulating hormone which are the most important from the hormone viewpoint. Anti-mullerian hormone (AMH) belongs to the glycoprotein of members of the transforming growth factor beta superfamily (TGF-beta) and has a molecular weight of 60 kD. AMH promotes the degeneration of the mullerian duct during sexual differentiation to allow the development of the middle renal duct into the male reproductive organs, and the absence of anti-mullerian hormone in female animals allows the mullerian duct to evolve into the uterus and oviduct. In adult women, AMH inhibits the recruitment of primordial follicles and the development of antral follicles, preventing premature follicular depletion, and is the most accurate biomarker for ovarian senescence. Follicle Stimulating Hormone (FSH) is a glycoprotein gonadotropin secreted by the anterior pituitary, and a heterodimer consisting of two subunits, an alpha subunit and a beta subunit, which are non-covalently bonded, has a molecular weight of about 38kD, and plays an important role in the hypothalamic-pituitary-gonadal reproductive axis. FSH causes granulosa cell proliferation, intimal cell differentiation, follicular fluid formation, and enlargement of follicular cavity, thereby promoting growth of ovarian follicles, proliferation of follicular granulosa cells, and synthesis and secretion of estrogen.
TAT transduction protein: the TAT protein transduction domain is a section of basic amino acid polypeptide derived from human immunodeficiency virus TAT protein, can quickly and efficiently transfer biological macromolecules such as polypeptide, protein, nucleic acid and the like which are covalently connected with the TAT protein into cells, and has great application potential in the fields of drug transfer, disease treatment and the like. The TAT protein transduction domain is first attached to the cell membrane by charge interaction and then enters the cell by lipid raft-mediated macropinocytosis.
Eukaryotic expression technology is a method of protein recombination, and refers to a method of expressing an exogenous target gene in a specific eukaryote or cell by constructing an expression vector and introducing the expression vector into an expression cell by a gene cloning technology. Its advantages are short period of time to obtain gene expression product and post-translation modification. The method is simple and suitable for large-scale production of glycoprotein.
Yolk antibody (Immunoglobulin of yolk, IgY): by immunizing egg laying hen (or other egg laying animal), the corresponding antibody can be extracted from egg yolk produced by the egg laying hen, and can be used for preventing and treating corresponding diseases. IgY has properties similar to mammalian IgG. And the IgY has stronger heat resistance, acid resistance, ion resistance and certain enzyme degradation resistance.
At present, recombinant anti-mullerian hormone and follicle-stimulating hormone are developed by related organizations, but the recombinant anti-mullerian hormone and the follicle-stimulating hormone cannot be taken orally. And no preparation technology of anti-mullerian hormone yolk antibody and follicle stimulating hormone yolk antibody exists at present. There is also no therapeutic or health care system consisting of recombinant anti-mullerian hormone and anti-mullerian hormone antibodies, follicle stimulating hormone and follicle stimulating hormone antibodies.
Disclosure of Invention
The invention aims to provide a biological agent for regulating ovarian function, a preparation method and application thereof, which are used for regulating ovarian dysfunction caused by abnormal secretion of anti-mullerian hormone and follicle-stimulating hormone.
The invention realizes the purpose through the following technical scheme:
a biological agent for regulating ovarian function, wherein the biological agent is an oral preparation consisting of one or more components of recombinant anti-mullerian hormone, pegylated recombinant anti-mullerian hormone, recombinant follicle-stimulating hormone, pegylated recombinant follicle-stimulating hormone, anti-mullerian hormone yolk antibody and follicle-stimulating hormone yolk antibody; wherein the recombinant anti-mullerian hormone consists of an amino acid sequence of anti-mullerian hormone N-terminally linked to a TAT transduction sequence; the recombinant follicle-stimulating hormone consists of an N end of an amino acid sequence of the follicle-stimulating hormone connected with a TAT transduction sequence; the anti-mullerian hormone yolk antibody is obtained by immunizing an oviparous animal through recombinant anti-mullerian hormone and then extracting yolk produced by the oviparous animal; the follicle stimulating hormone egg yolk antibody is obtained by immunizing an oviparous animal by recombinant follicle stimulating hormone and then extracting egg yolk produced by the oviparous animal.
The further improvement is that the gene sequence of the amino acid sequence for coding the recombinant anti-mullerian hormone is shown as SEQ ID NO. 1, and the gene sequence of the amino acid sequence for coding the recombinant follicle-stimulating hormone is shown as SEQ ID NO. 2.
The further improvement is that the pegylated recombinant anti-mullerian hormone and the pegylated recombinant follicle-stimulating hormone are prepared by modifying the recombinant anti-mullerian hormone and the recombinant follicle-stimulating hormone by methoxy polyethylene glycol succinyl-imide propionate mPEG-SPA with the molecular weight of 5 kD.
In a further improvement, the oviparous animal is a chicken.
The invention also provides a preparation method of the biological preparation, and the preparation method of the recombinant anti-mullerian hormone or the recombinant follicle-stimulating hormone comprises the following steps:
taking a human anti-mullerian hormone amino acid sequence or a follicle-estrogen amino acid sequence, connecting a TAT transduction sequence at the N end, performing reverse translation to obtain a target gene sequence, and connecting the target gene sequence to a pYES2/CT plasmid;
secondly, introducing the plasmid into Invscv1 saccharomyces cerevisiae by a PEG/LiCl method to obtain an AMH engineering bacterium or an FSH engineering bacterium;
and thirdly, carrying out fermentation culture and purification on the AMH engineering bacteria or the FSH engineering bacteria to obtain the recombinant target protein AMH or FSH.
In a further improvement, the preparation method of the pegylated recombinant anti-mullerian hormone and the pegylated recombinant follicle stimulating hormone comprises the following steps:
carrying out vacuum freeze drying on the recombinant anti-mullerian hormone or the recombinant follicle-stimulating hormone to obtain powder;
PEG-SPA is utilized to carry out PEG treatment in a reaction buffer system to obtain the target protein modified by PEG.
In a further improvement, the preparation method of the anti-mullerian hormone yolk antibody or the follicle stimulating hormone yolk antibody comprises the following steps:
step one, immunizing an oviparous animal by utilizing recombinant anti-mullerian hormone or recombinant follicle-stimulating hormone, and collecting eggs when the serum antibody titer of the oviparous animal is more than 32;
step two, processing egg yolk liquid of the eggs into a powdery preparation and storing the powdery preparation.
The invention also provides application of the biological agent in preparing a medicine for regulating ovarian dysfunction caused by abnormal secretion of anti-mullerian hormone and follicle-stimulating hormone.
The invention has the beneficial effects that:
(1) the recombinant protein adopted by the biological preparation is a fusion protein of TAT-induced peptide and anti-mullerian hormone or follicle-stimulating hormone, the TAT-induced peptide can promote intestinal absorption of the protein, the PEG-modified recombinant anti-mullerian hormone and follicle-stimulating hormone can prolong half-life period, and the problem of oral administration of the anti-mullerian hormone and follicle-stimulating hormone can be solved.
(2) The biological preparation provides a yolk antibody product for resisting mullerian hormone and follicle-stimulating hormone, fresh eggs can be directly eaten, yolk electuary can be orally taken, the problem of high cost of antibody application is solved, and the yolk antibody product can be widely applied to clinical and daily health care.
(3) The biological preparation can solve the problem of AMH and FSH abnormality targeted therapy and health promotion.
Drawings
Figure 1 is a validation scheme of recombinant anti-mullerian hormone wb; in the figure, lane M: protein Marker, lane 1: empty bacteria control, lane 2: recombinant anti-mullerian hormone, lane 3: pegylated recombinant anti-mullerian hormone.
FIG. 2 is a graph showing the verification of recombinant follicle stimulating hormone wb; in the figure, lane M: protein Marker, lane 1: empty bacteria control, lane 2: recombinant follicle stimulating hormone, lane 3: pegylated recombinant follicle stimulating hormone.
Figure 3 is an agar double diffusion plot of recombinant anti-mullerian hormone and recombinant follicle stimulating hormone immunized laying hens;
in the figure, A is the result of the serum titer test of the recombinant anti-mullerian hormone immunized laying hens; and B is a result of the serum titer test of the recombinant follicle-stimulating hormone immunized laying hens. From 1 to 6 wells were diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold and 64-fold with serum, respectively. The central hole is antigen, the antigen of A is recombinant anti-mullerian hormone, and the antigen of B is recombinant follicle-stimulating hormone.
Fig. 4 is a partial preparation display of yolk antibodies: a is fresh egg, B is yolk powder, C is yolk powder tablet, and D is IgY antibody preparation (lyophilized or liquid).
Detailed Description
The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.
Example 1: preparation of recombinant anti-mullerian hormone
(1) Acquisition of gene sequence of recombinant anti-mullerian hormone:
referring to GENBANK accession No. NM-000479.5, the amino acid sequence of human anti-Mullerian hormone is taken, and the N end is connected with a TAT transduction sequence, and the target gene is obtained by reverse translation. The target gene sequence is sent to Huahua large gene to synthesize pYES2/CT plasmid containing the target gene, the sequencing is qualified, and the target gene sequence shown as SEQ ID NO. 1 is obtained.
(2) Construction of engineering bacteria:
introducing plasmids into Invscv1 saccharomyces cerevisiae by a PEG/LiCl method, coating the Invscv1 saccharomyces cerevisiae on an ampicillin SD-Ura plate, culturing for 72h at constant temperature of 30 ℃, selecting colonies growing on the SD-Ura plate, preserving the seeds, sending the colonies to Huada gene sequencing to identify target genes, and marking the obtained sequencing result as the same as the target genes, which indicates that the expression vector is successfully constructed as AMH engineering bacteria.
(3) Expression by fermentation
Culturing AMH engineering bacteria in a SD-Ura glucose fermentation culture medium at 30 ℃ and 220r/min in a shaking table until OD600 is about 5.0, adding 5 times of SD-Ura galactose induction culture medium, and continuously performing induction culture for 96h to obtain the strain. Centrifuging at 7000rpm and 4 deg.C for 15min, and collecting the supernatant as crude product of the target protein AMH.
GE XK50 self-contained column was packed with GE Chelating Sepharose Fast Flow Ni2+ chelate packing, eluted at pH8.0, and dialyzed for 12 hours in 20 column volumes of PBS buffer. And (5) carrying out sterile filtration treatment on the protein with the diameter of 0.22 mu m, wherein a treated sample is the purified target protein and is marked as AMH.
Example 2: PEGylation modification of recombinant anti-mullerian hormone
And (4) carrying out vacuum freeze drying on the purified target protein AMH to obtain powder.
Preparing a reaction buffer system, wherein the buffer system can be selected from acetate buffer solution with pH4.0 and 5.0, and phosphate buffer solution with pH6.0, 7.0 and 8.0.
And (3) taking the freeze-dried AMH and mPEG-SPA according to the mol ratio of 1: 2-1: 12, and stirring the mixture for 2-12 hours at room temperature in a reaction buffer system in a dark place by using a magnetic stirrer. Dialyzed against 20 volumes of PBS buffer for 12 h. And (3) carrying out sterile filtration treatment on the protein with the diameter of 0.22 mu m, wherein the treated sample is the target protein modified by PEG and is marked as mPEG-AMH.
WB was verified by adjusting the concentrations of AMH and mPEG-AMH to 1mg/mL, using mouse-anti-AMH (cat # ab239491) from abcam and goat-anti-mouse IgG (HRP) as a secondary antibody (cat # ab 6789). As shown in FIG. 1, a significant positive band at 60kD for AMH in lane 2 and a significant positive band at 65kD for 3mPEG-AMH in lane 3 indicate that the recombinant target protein is anti-Mullerian hormone and that the PEGylation modification is successful.
Example 3: preparation of recombinant follicle stimulating hormone
(1) Acquisition of gene sequence of recombinant follicle-stimulating hormone:
the amino acid sequences of GENBANK accession AAA52476.1 and GENBANK accession ACM91588.1 are referred, the flexible linker is used for connection between the amino acid sequences, the TAT transduction sequence is connected at the N end, and the target gene is obtained by reverse translation. The target gene sequence is sent to Huahua large gene to synthesize pYES2/CT plasmid containing the target gene, the sequencing is qualified, and the target gene sequence shown as SEQ ID NO. 2 is obtained.
(2) Construction of engineering bacteria:
introducing plasmids into Invscv1 saccharomyces cerevisiae by a PEG/LiCl method, coating the Invscv1 saccharomyces cerevisiae on an ampicillin SD-Ura plate, culturing for 72h at constant temperature of 30 ℃, selecting bacterial colonies growing on the SD-Ura plate, preserving the seeds, sending a Huada gene to sequence and identify a target gene, wherein the sequencing result is consistent with the target gene, which indicates that an expression vector is successfully constructed and is marked as FSH engineering bacteria.
(3) Expression by fermentation
Culturing FSH engineering bacteria in a SD-Ura glucose culture medium at 30 ℃ and 220r/min in a shaking table until the OD600 is about 5.0, adding 5 times of the SD-Ura galactose induction culture medium, and continuously performing induction culture for 96h to obtain the FSH engineering bacteria. Centrifuging at 7000rpm and 4 deg.C for 15min, and collecting the supernatant as the crude product of the target protein FSH.
GE XK50 self-contained column was packed with GE Chelating Sepharose Fast Flow Ni2+ chelate packing, eluted at pH8.0, and dialyzed for 12 hours in 20 column volumes of PBS buffer. Sterile filtration treatment is carried out at 0.22 mu m, and the treated sample is the purified target protein which is marked as FSH.
Example 4: PEGylation modification of recombinant follicle stimulating hormone
And (4) carrying out vacuum freeze drying on the purified FSH to obtain powder.
Preparing a reaction buffer system, wherein the buffer system can be selected from acetate buffer solution with pH4.0 and 5.0, and phosphate buffer solution with pH6.0, 7.0 and 8.0.
And (3) taking the freeze-dried AMH and mPEG-SPA according to the mol ratio of 1: 2-1: 12, and stirring the mixture for 2-12 hours at room temperature in a reaction buffer system in a dark place by using a magnetic stirrer. Dialyzed against 20 volumes of PBS buffer for 12 h. And (3) carrying out sterile filtration treatment on the mixture with the diameter of 0.22 mu m, wherein the treated sample is the target protein modified by PEG and is marked as mPEG-FSH.
WB assay was performed by adjusting the concentrations of FSH and mPEG-FSH to 1mg/mL, using rabbit anti-FSH-. beta.from abcam as the primary antibody (cat No. ab150425) and goat anti-rabbit IgG (HRP) from abcam as the secondary antibody (cat No. ab 6721). As shown in FIG. 2, a significant positive band at 29kD in FSH in lane 2 and a significant positive band at 34kD in 3mPEG-FSH in lane 3 indicate that the recombinant target protein is follicle stimulating hormone and that the PEGylation modification is successful.
Example 5: preparation of anti-mullerian hormone yolk antibody
Animal immunization
4-month-old healthy laying hens are selected for immunization. Diluting anti-mullerian hormone to 2mg/mL by PBS, emulsifying by Freund's complete adjuvant 1:1, and performing primary immunization by intramuscular multi-point injection, wherein the total injection amount of each chicken is 2 mL; the booster immunization was performed in the same manner after 14 days. Collecting blood from vein of wing on day 21, measuring serum antibody titer by agar double diffusion method, collecting ovum gallus Domesticus when titer is not less than 32 (shown in figure 3), and supplementing immunity according to the above method until titer meets the requirement.
Yolk antibody preparation
Firstly, preparing yolk powder: selecting intact egg, cleaning with warm water, sterilizing with sodium hypochlorite, air drying with compressed air, separating yolk from egg white with full-automatic method, and automatically detecting loose yolk. Egg yolk liquid is collected and conveyed to a plate heat exchanger by a rotor pump to be instantly cooled to 4 ℃. The temperature was kept at 4 ℃ using a temporary storage tank with stirring and ice water jacket. The soft sterilization mode is adopted, the temperature is 63 ℃ for 3.5 minutes, the virus and the harmful bacteria are killed, and the activity of the IgY is kept. Spray drying at low temperature under pressure for 1 s, evaporating at 60 deg.C for 5% water, and sieving with 30 mesh sieve to obtain yolk powder.
Preparing an IgY antibody: selecting intact egg, cleaning with warm water, sterilizing with sodium hypochlorite, air drying with compressed air, separating yolk from egg white with full-automatic method, and automatically detecting loose yolk. Collecting egg yolk liquid. Diluting yolk with 1:9 times of distilled water, adjusting pH to 5.4, stirring for 15min, standing at 4 deg.C overnight for natural precipitation, centrifuging at 10000rpm at 4 deg.C for 30min to remove lipid, and collecting supernatant. Adding PBS with the same volume as the PBS for dilution, adding saturated ammonium sulfate according to the volume of 1:1, standing for 2h at 4 ℃, centrifuging for 15min at 10000rpm, and removing the supernatant. Dissolving the precipitate with PBS, centrifuging to remove insoluble substances, collecting supernatant, adding 100m saturated ammonium sulfate to 33% saturation, standing at 4 deg.C for 2 hr, centrifuging at 10000rpm for 15min, and discarding supernatant. Repeating the above steps for 3 times to obtain relatively pure IgY, dialyzing with PBS (20 times volume) overnight, performing sterile filtration with 0.22 μm to obtain liquid IgY preparation of AMH, and lyophilizing after compatibility.
Preparation of finished product
The obtained egg can be used as fresh egg product after sterilization. The egg powder can be made into granule or tablet with sugar and milk powder, and the liquid IgY antibody can be made into injection preparation, as shown in figure 4.
Example 6: preparation of follicle stimulating hormone egg yolk antibody
(1) Animal immunization
4-month-old healthy laying hens are selected for immunization. Diluting follicle stimulating hormone to 2mg/mL by using PBS, emulsifying by Freund's complete adjuvant 1:1, and performing primary immunization by intramuscular multi-point injection, wherein the total injection amount of each chicken is 2 mL; the booster immunization was performed in the same manner after 14 days. Collecting blood from vein of wing on day 21, measuring serum antibody titer by agar double diffusion method, collecting ovum gallus Domesticus when titer is not less than 32 (shown in figure 3), and supplementing immunity according to the above method until titer meets the requirement.
(2) Yolk antibody preparation
Firstly, preparing yolk powder: selecting intact egg, cleaning with warm water, sterilizing with sodium hypochlorite, air drying with compressed air, separating yolk from egg white with full-automatic method, and automatically detecting loose yolk. Egg yolk liquid is collected and conveyed to a plate heat exchanger by a rotor pump to be instantly cooled to 4 ℃. The temperature was kept at 4 ℃ using a temporary storage tank with stirring and ice water jacket. The soft sterilization mode is adopted, the temperature is 63 ℃ for 3.5 minutes, the virus and the harmful bacteria are killed, and the activity of the IgY is kept. Spray drying at low temperature under pressure for 1 s, evaporating at 60 deg.C for 5% water, and sieving with 30 mesh sieve to obtain yolk powder.
Preparing an IgY antibody: selecting intact egg, cleaning with warm water, sterilizing with sodium hypochlorite, air drying with compressed air, separating yolk from egg white with full-automatic method, and automatically detecting loose yolk. Collecting egg yolk liquid. Diluting yolk with 1:9 times of distilled water, adjusting pH to 5.4, stirring for 15min, standing at 4 deg.C overnight for natural precipitation, centrifuging at 10000rpm at 4 deg.C for 30min to remove lipid, and collecting supernatant. Adding PBS with the same volume as the PBS for dilution, adding saturated ammonium sulfate according to the volume of 1:1, standing for 2h at 4 ℃, centrifuging for 15min at 10000rpm, and removing the supernatant. Dissolving the precipitate with PBS, centrifuging to remove insoluble substances, collecting supernatant, adding 100m saturated ammonium sulfate to 33% saturation, standing at 4 deg.C for 2 hr, centrifuging at 10000rpm for 15min, and discarding supernatant. Repeating the above steps for 3 times to obtain relatively pure IgY, dialyzing with PBS (20 times volume) overnight, sterile filtering with 0.22 μm to obtain liquid IgY preparation of FSH, mixing, and freeze drying to obtain lyophilized preparation.
(3) Preparation of finished product
The obtained egg can be used as fresh egg product after sterilization. The egg powder can be made into granule or tablet with sugar and milk powder, and the liquid IgY antibody can be made into injection.
Example 7: recombinant protein for regulating ovarian function and application of system of yolk antibody product thereof
The recombinant protein for regulating the ovarian function and the system of the yolk antibody product thereof can be selected according to the actual clinical requirements and can be prepared by 1 or more of 6 substances of anti-mullerian hormone, pegylated anti-mullerian hormone, anti-mullerian hormone yolk antibody, follicle stimulating hormone, pegylated follicle stimulating hormone and follicle stimulating hormone yolk antibody. This example was studied as a single species.
60 female volunteers with non-organic ovulation failure were selected from sterile departments of the maternal and child care institute of Anhui province, wherein 30 (15 higher and 15 lower) serum AMH and 30 (15 higher and 15 lower) serum FSH were abnormal. As shown in table 1 below.
Table 1: 60 patients were selected from the experiment and tested for initial serum AMH and FSH (normal range of serum AMH: 2.0-6.8ng/mL, normal range of serum FSH: 5-40mIU/mL)
10 patients with low AMH were orally administered with AMH at a dose of 100 mg/time/day for 90 days, and recorded as group A. The 5 saline oral controls were given and are designated group B. 10 patients with higher AMH were administered with AMH yolk antibody, the dose was 200 mg/time/day, and the administration time was 90 days continuously, and the administration time was recorded as group C. The 5 saline oral controls were given and scored as group D. Blood was collected each month for serum AMH, and the dose of AMH or AMH yolk antibody was adjusted according to the physician's advice.
10 patients with low FSH were orally administered with FSH at a dose of 100 mg/time/day for 90 consecutive days, and the dose was recorded as group E. The 5 saline oral controls were given and scored as group F. The FSH yolk antibody is administered to 10 patients with higher FSH, the dose is 200 mg/time/day, and the administration is continued for 90 days, which is marked as group G. The 5 saline oral controls were given and scored as group H. Serum FSH was measured every month and the amount of FSH or FSH yolk antibody was adjusted with the physician's advice.
And B ultrasonic observation is carried out on the experimental subject whether the experimental subject has ovulation or not within 90-120 days, and the record is shown in the table 2. The result shows that the recombinant anti-mullerian hormone, the recombinant anti-mullerian hormone yolk antibody, the recombinant follicle stimulating hormone and the recombinant follicle stimulating hormone yolk antibody can regulate the ovarian function and promote ovulation, and the effective rate reaches 92.5%. Reverse use of the present system may also be developed as a contraceptive.
Table 2: enrollment experiment 60 patients were treated within one month for ovulation recording (+ for ovulation, -for non-ovulation)
| Serial number | A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 |
| Whether or not to ovulate | + | + | + | + | + | + | + | + |
| Serial number | A9 | A10 | B1 | B2 | B3 | B4 | B5 | |
| Whether or not to ovulate | + | + | - | - | - | - | - | |
| Serial number | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
| Whether or not to ovulate | + | + | - | + | - | + | + | + |
| Serial number | C9 | C10 | D1 | D2 | D3 | D4 | D5 | |
| Whether or not to ovulate | + | + | - | - | - | - | - | |
| Serial number | E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 |
| Whether or not to ovulate | + | + | + | + | + | + | + | + |
| Serial number | E9 | E10 | F1 | F2 | F3 | F4 | F5 | |
| Whether or not to ovulate | + | + | - | - | - | - | - | |
| Serial number | G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 |
| Whether or not to ovulate | - | + | + | + | + | + | + | + |
| Serial number | G9 | G10 | H1 | H2 | H3 | H4 | H5 | |
| Whether or not to ovulate | + | + | - | - | - | - | - |
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Onhui Zhongzhi Biotech Co Ltd
<120> biological preparation for regulating ovarian function and preparation method and application thereof
<141> 2021-07-28
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<170> SIPOSequenceListing 1.0
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cttcgggtgg ttggcgcgct ctccgcttac gagcaagcct tcctaggagc agtccagaga 300
gcgaggtggg ggccgcgtga tctggctacg tttggtgtat gcaacactgg cgaccgccaa 360
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ctagtggttc tgcatttaga agaggtcacc tgggaaccaa caccgtcgtt gcgtttccaa 480
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cttcaggccc tcctatttgg tgatgaccac cggtgcttca ctagaatgac ccctgcactg 780
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ccctttccac cgcctcgtcc cagtgccgaa ctggaggaaa gcccaccgtc tgcagaccct 900
ttcttagaga cgttgactcg ccttgttcga gcgctccggg tccccccagc tagagcctcc 960
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gcgtatgctg gaaaacttct catttcgcta agtgaggaac gaatcagcgc ccaccatgtg 1680
cccaacatgg ttgcaacaga gtgtgggtgc cgg 1713
<210> 2
<211> 792
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tatggtcgta aaaagcgccg acaacggaga aggatggatt actatcgtaa atacgctgcc 60
atttttttag ttactttgtc tgtcttcctt catgtactcc actccgcacc tgacgtgcag 120
gattgtcccg aatgcaccct acaagagaat ccatttttct cacagccggg cgcgcctatc 180
ctgcaatgta tgggatgctg tttttcgcgc gcttatccca caccattacg aagtaagaaa 240
acgatgttgg ttcagaagaa cgtcactagc gaatctacct gctgtgtagc caaatcctac 300
aatcgggtga cagttatggg gggtttcaag gtcgagaacc atacggcatg ccactgttca 360
acttgctatt accataaatc gggcggaggg ggtggcggag ggggtatgaa gacccttcaa 420
tttttctttc tcttctgttg ctggaaagcg atatgttgca atagttgtga actaacaaac 480
attacgatcg ctatagagaa ggaagagtgc agattttgta ttagcatcaa tactacctgg 540
tgcgccggct attgttacac aagggacctg gtatataaag atccggcacg tcctaagata 600
cagaaaacgt gcactttcaa ggaattagtg tacgagaccg ttcgcgtccc cggatgtgcg 660
caccatgctg actctttgta tacataccca gtagccacgc aatgccactg tgggaaatgc 720
gattccgact caactgattg taccgtgcga ggtcttggcc cgtcgtattg cagttttgga 780
gaaatgaagg ag 792
Claims (8)
1. A biological agent for regulating ovarian function, which is an oral preparation consisting of one or more components selected from recombinant anti-mullerian hormone, pegylated recombinant anti-mullerian hormone, recombinant follicle-stimulating hormone, pegylated recombinant follicle-stimulating hormone, anti-mullerian hormone yolk antibody and follicle-stimulating hormone yolk antibody; wherein the recombinant anti-mullerian hormone consists of an amino acid sequence of anti-mullerian hormone N-terminally linked to a TAT transduction sequence; the recombinant follicle-stimulating hormone consists of an N end of an amino acid sequence of the follicle-stimulating hormone connected with a TAT transduction sequence; the anti-mullerian hormone yolk antibody is obtained by immunizing an oviparous animal through recombinant anti-mullerian hormone and then extracting yolk produced by the oviparous animal; the follicle stimulating hormone egg yolk antibody is obtained by immunizing an oviparous animal by recombinant follicle stimulating hormone and then extracting egg yolk produced by the oviparous animal.
2. The biological agent for regulating ovarian function according to claim 1, wherein the gene sequence encoding the amino acid sequence of the recombinant anti-mullerian hormone is shown as SEQ ID NO. 1, and the gene sequence encoding the amino acid sequence of the recombinant follicle-stimulating hormone is shown as SEQ ID NO. 2.
3. The biological preparation for regulating ovarian function according to claim 1, wherein the pegylated recombinant anti-mullerian hormone and the pegylated recombinant follicle-stimulating hormone are prepared by modifying the recombinant anti-mullerian hormone and the recombinant follicle-stimulating hormone with methoxypolyethyleneglycol succinyl-imine propionate mPEG-SPA having a molecular weight of 5 kD.
4. The biological agent for regulating ovarian function of claim 1, wherein said oviparous animal is a chicken.
5. A method for preparing a biological agent as claimed in any one of claims 1 to 4, wherein the method for preparing the recombinant anti-Mullerian hormone or recombinant follicle-stimulating hormone comprises the steps of:
taking a human anti-mullerian hormone amino acid sequence or a follicle-estrogen amino acid sequence, connecting a TAT transduction sequence at the N end, performing reverse translation to obtain a target gene sequence, and connecting the target gene sequence to a pYES2/CT plasmid;
secondly, introducing the plasmid into Invscv1 saccharomyces cerevisiae by a PEG/LiCl method to obtain an AMH engineering bacterium or an FSH engineering bacterium;
and thirdly, carrying out fermentation culture and purification on the AMH engineering bacteria or the FSH engineering bacteria to obtain the recombinant target protein AMH or FSH.
6. The method for preparing a biological agent according to claim 5, wherein the steps of preparing the pegylated recombinant anti-mullerian hormone and the pegylated recombinant follicle-stimulating hormone comprise:
(1) carrying out vacuum freeze drying on the recombinant anti-mullerian hormone or the recombinant follicle-stimulating hormone to obtain powder;
(2) PEG-SPA is utilized to carry out PEG treatment in a reaction buffer system to obtain the target protein modified by PEG.
7. The method for preparing a biological agent according to claim 5, wherein the method for preparing the anti-mullerian hormone yolk antibody or the follicle stimulating hormone yolk antibody comprises the steps of:
step one, immunizing an oviparous animal by utilizing recombinant anti-mullerian hormone or recombinant follicle-stimulating hormone, and collecting eggs when the serum antibody titer of the oviparous animal is more than 32;
step two, processing egg yolk liquid of the eggs into a powdery preparation and storing the powdery preparation.
8. Use of a biological agent as claimed in any of claims 1 to 4 as a medicament for the modulation of ovarian dysfunction caused by abnormal secretion of anti-mullerian hormone and follicle stimulating hormone.
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