CN117143186B

CN117143186B - Polypeptide for promoting ovulation of female pelteobagrus fulvidraco and application

Info

Publication number: CN117143186B
Application number: CN202310978947.9A
Authority: CN
Inventors: 梅洁; 王旭; 熊阳; 皇培培; 孙瑞东; 蒋有渤
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2023-08-04
Filing date: 2023-08-04
Publication date: 2024-05-28
Anticipated expiration: 2043-08-04
Also published as: CN117143186A

Abstract

The application relates to the technical field of pelteobagrus fulvidraco, in particular to a polypeptide for promoting ovulation of female pelteobagrus fulvidraco and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1 or 2. The polypeptide can effectively promote synchronous ovulation of pelteobagrus fulvidraco. The polypeptide is used as an injection medicine, so that the spawning quantity of pelteobagrus fulvidraco can be obviously improved, the postpartum mortality rate can be reduced, and an obvious economic effect can be achieved.

Description

Polypeptide for promoting ovulation of female pelteobagrus fulvidraco and application

Technical Field

The application relates to the technical field of pelteobagrus fulvidraco, in particular to a polypeptide for promoting ovulation of female pelteobagrus fulvidraco and application thereof.

Background

Pelteobagrus fulvidraco is one of important characteristic freshwater aquaculture fishes in China, and the annual output of pelteobagrus fulvidraco aquaculture in China is counted to reach 60 ten thousand tons according to the annual identification of the fishery in 2022. The problems that the intestinal membrane fat of the artificially cultured pelteobagrus fulvidraco is high, the gonad index is low, the spawning quantity of part of pelteobagrus fulvidraco is small, even the pelteobagrus fulvidraco does not spawn at all after artificial spawning is carried out by using common spawning promoting medicaments such as LHRH, DOM, HCG and the like, the postpartum mortality of female parent is high and the like still exist. Therefore, development of novel oxytocic drugs for artificial reproduction of fish is needed.

Disclosure of Invention

The application provides a polypeptide with high solubility and high activity, which can effectively promote synchronous ovulation of pelteobagrus fulvidraco. In order to achieve the above purpose, the present application at least discloses the following technical solutions:

in a first aspect, the examples disclose a polypeptide having an amino acid sequence as set forth in SEQ ID NO.1 or 2.

In a second aspect, embodiments disclose a pharmaceutical composition comprising a polypeptide according to the first aspect and a pharmaceutically acceptable diluent, excipient, carrier or luteinizing hormone releasing A2.

In a third aspect, embodiments disclose a method for preparing a pharmaceutical composition of the second aspect, comprising mixing a polypeptide of the second aspect with at least one pharmaceutically acceptable diluent or carrier.

In a fourth aspect, the embodiment discloses the use of the polypeptide of the first aspect for preparing a medicament for promoting ovulation and/or oxytocic function of female pelteobagrus fulvidraco.

Drawings

FIG. 1 is a YC-9aa high performance liquid chromatogram provided in the example.

FIG. 2 is a diagram showing the comparison of YC-9aa and YC-9aaL according to the present embodiment.

Fig. 3 shows the induced spawning rate of pelteobagrus fulvidraco after injection of YC-9aa and YC-9aaL in the examples.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that, the terms "first," "second," and the like in the description and the claims of the present application and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the application described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

For a better understanding of the present application, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Currently, there are some natural unknown proteins that play an important role in the fish reproduction process. Therefore, the inventor designs a polypeptide with high solubility and strong activity, and the polypeptide can effectively promote synchronous ovulation of pelteobagrus fulvidraco.

In some embodiments, the polypeptide as set forth in SEQ ID NO.1 is synthesized by chemical combination.

In some embodiments, the polypeptide as set forth in SEQ ID NO.2 is synthesized by chemical combination.

In some embodiments, a short peptide comprising 9 amino acids (aa) is isolated from the pituitary of pelteobagrus fulvidraco in the breeding season and sequenced by mass spectrometry Cys¹-Tyr²-Ile³-Ser⁴-Asn⁵-Cys⁶-Pro⁷-Ile⁸-Gly⁹,SEQ ID NO.1.

In some embodiments, the polypeptide shown in SEQ ID NO.1 is modified to give a polypeptide body designated YC-9aaL, the amino acid sequence of which is Cys¹-Tyr²-Ile³-Gln⁴-Asn⁵-Cys⁶-Pro⁷-Ile⁸-Gly⁹-Glu¹⁰-Ala¹¹-Ala¹²-Ala¹³-lys¹⁴,SEQ ID NO.2.

In some embodiments, the hydrophilicity of the polypeptide is 0.71, the solubility after ultrasonic crushing is 0.03g, and the spawning induction rate of pelteobagrus fulvidraco after 100 μg/kg high concentration dose is only 10%.

In some embodiments, the hydrophilicity of the polypeptide is 0.12, the polypeptide is extremely soluble in water, the spawning induction rate of pelteobagrus fulvidraco after only injection of 10 mug/kg dose is improved to 80%, and the pelteobagrus fulvidraco spawn can be promoted to mature and synchronously ovulate after being matched with LHRH-A2 injection, so that the polypeptide is suitable for large-scale artificial propagation of pelteobagrus fulvidraco.

In some embodiments, the polypeptide is for use as a medicament. The polypeptide provided by the embodiment has the function of obviously promoting the ovulation of female pelteobagrus fulvidraco, reduces the dosage of the polypeptide, and has lower price than the conventional oxytocic.

In some embodiments, the polypeptide is used to promote ovulation and/or oxytocic function in female pelteobagrus fulvidraco.

In a second aspect, a pharmaceutical composition comprising said polypeptide and a pharmaceutically acceptable diluent, excipient, carrier or another active ingredient which, together with said polypeptide, promotes ovulation and/or oxytocic activity in female pelteobagrus fulvidraco. In some embodiments, the polypeptide is matched with LHRH-A2 to obviously improve the spawning quantity of pelteobagrus fulvidraco and reduce the postpartum mortality by using the artificial breeding process, so that obvious economic effects are achieved.

In some embodiments, the pharmaceutical composition may be formulated for oral, intramuscular, subcutaneous, or intravenous delivery.

In some embodiments, the pharmaceutical composition is formulated as a liquid dosage form, a semi-solid dosage form, or a solid dosage form.

In some embodiments, the pharmaceutical composition is selected from the group consisting of, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Solid dosage forms are preferred. The polypeptides, pharmaceutical compositions or constructs of the application may incorporate excipients and be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. For the treatment of eosinophilic esophagitis, delivery in the form of lozenges is particularly preferred. For the treatment of atopic dermatitis, delivery in the form of a cream is particularly preferred.

Typically, the pharmaceutical composition comprises a polypeptide of the application and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers which enhance shelf life or effectiveness of the polypeptides of the present application. The pharmaceutical composition may comprise an anti-sticking agent, a binder, a coating, a disintegrant, a flavoring agent, a coloring agent, a lubricant, an adsorbent, a preservative, a sweetener, a lyophilization excipient (including lyoprotectants), or a compression aid.

The pharmaceutical compositions provided by the examples may also provide an enteric coating for the polypeptide. An enteric coating is a polymeric barrier applied to an oral drug that helps to protect the polypeptide from the low pH of the stomach. Materials for enteric coatings include fatty acids, waxes, shellac, plastics and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl phthalate cellulose, hydroxypropyl methyl succinate cellulose acetate (hydroxypropyl methyl cellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, sodium alginate, and stearic acid. Suitable enteric coatings include pH dependent release polymers. These are polymers that are insoluble at the high acidic pH present in the stomach but dissolve rapidly at the lower acidic pH. Thus, suitably, the enteric coating is insoluble in the acidic gastric juice of the stomach (pH-3), but soluble in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH-dependent release polymer is selected such that the polypeptide of the application is released at about the time the dose reaches the small intestine.

The polypeptides or pharmaceutical compositions of the application may be delivered locally. Such pharmaceutical compositions may suitably be in the form of creams, ointments, lotions, gels, foams, transdermal patches, powders, pastes or tinctures, and may suitably include vitamin D3 analogues (e.g. calcipotriol and maxacalcitol), steroids (e.g. fluticasone propionate, betamethasone valerate and clobetasol propionate), retinoic acid (e.g. tazarotene), coal tar and dithranol. Topical agents are often used in combination with each other (e.g., vitamin D3 and a steroid) or with other agents such as salicylic acid.

The polypeptides or pharmaceutical compositions of the application may be formulated for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oils or other similar oils, fatty acid glycerides, esters of higher fatty acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. Acceptable carriers, excipients, and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlorom-cresol, methyl or propyl parahydroxybenzoate, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; a low molecular weight (less than about 10 residues) polypeptide; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methyl glucosamine, galactosamine, and neuraminic acid; and/or nonionic surfactants such as polysorbates, POE ethers, poloxamers, triton-X or polyethylene glycols.

For all modes of delivery, the polypeptides, pharmaceutical compositions or constructs of the application may be formulated in a buffer so as to stabilize the pH of the composition at a concentration of between 5 and 50g/l, or more suitably between 15 and 40g/l, or more suitably between 25 and 30 g/l. Examples of suitable buffer components include physiological salts, such as sodium citrate and/or citric acid. Suitably, the buffer contains 100-200mM, more suitably 125-175mM physiological salt, such as sodium chloride. Suitably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of pelteobagrus fulvidraco.

Exemplary polypeptide concentrations in the pharmaceutical composition may range from about 1mg/mL to about 200mg/mL, or from about 50mg/mL to about 200mg/mL, or from about 150mg/mL to about 200mg/mL.

The aqueous formulation of the polypeptide or pharmaceutical composition of the application may be prepared in a pH buffered solution, for example at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate, histidine, citrate, succinate, acetate buffers and other organic acid buffers. The buffer concentration may be about 1mM to about 100mM, or about 5mM to about 50mM, depending on, for example, the desired tonicity of the buffer and formulation.

The tonicity of the pharmaceutical composition may be altered by the inclusion of tonicity adjusting agents. Such tonicity adjusting agents may be charged or uncharged chemicals. Typical uncharged tonicity adjusting agents include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1, 2-propanediol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1, 2-propanediol). Typical charged tonicity modifiers include salts such as sodium, potassium or calcium ions in combination with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulfate); or an amino acid such as arginine or histidine. Suitably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" means a solution having the same tonicity as some other solution to which it is compared, such as a physiological saline solution or serum. The tonicity agent may be used in an amount of about 5mM to about 350mM, for example in an amount of 1mM to 500 nM. Suitably, at least one isotonic agent is included in the composition.

Surfactants may also be added to the pharmaceutical composition to reduce aggregation of the formulated polypeptide and/or to minimize particle formation and/or reduce adsorption in the formulation. Exemplary surfactants include polyoxyethylene sorbitol fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer), pluronic (Pluronic), and Sodium Dodecyl Sulfate (SDS). Examples of suitable polyoxyethylene sorbitol-fatty acid esters are polysorbate 20 and polysorbate 80. Exemplary concentrations of surfactant range from about 0.001% to about 10% w/v.

Lyoprotectants may also be added to the pharmaceutical composition in order to protect the polypeptide of the application from destabilizing conditions during the freezing process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose, and trehalose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants may be included in amounts of about 10mM to 500 mM.

The dosage ranges for administration of the polypeptides of the application, the pharmaceutical compositions of the application are those that produce the desired therapeutic effect. The required dosage range is judged depending on the exact nature of the polypeptide of the application, the route of administration, the nature of the formulation, the age of pelteobagrus fulvidraco, the nature, extent or severity of the pelteobagrus fulvidraco condition. These changes in dosage levels can be accommodated for optimization using standard empirical practices.

Suitably, the daily dose of the polypeptide or pharmaceutical composition of the application is in the range of 50ng-50 μg/kg, such as 1-40 μg/kg.

The pharmaceutical compositions of the application may also comprise one or more active agents (e.g., active agents suitable for treating the diseases mentioned herein). Within the scope of the present application is the use of the pharmaceutical composition of the application in addition to or in combination with other established therapies commonly used in the treatment of autoimmune diseases in the promotion of ovulation and/or oxytocic activity in female pelteobagrus fulvidraco. For example, the polypeptide provided in the first aspect is combined with lutein-releasing A2 or chorionic gonadotrophin to promote ovulation and/or oxytocic function in female pelteobagrus fulvidraco.

The application will now be illustrated by the following more specific examples, which are not to be construed as limiting the embodiments of the application.

Example 1: development, preparation and modification of polypeptide YC-9aa

A. And (3) determining pituitary transcriptomes corresponding to different stages of pelteobagrus fulvidraco egg nest development, screening ovulation related differential expression genes, and translating candidate genes into amino acid sequences. Extracting polypeptide from pituitary gland of female pelteobagrus fulvidraco in breeding season, separating the polypeptide by chromatography, performing mass spectrometry to obtain polypeptide sequence, and comparing the polypeptide sequence with amino acid sequence of candidate gene to find short peptide YC-9aa, wherein the amino acid sequence is Cys¹-Tyr²-Ile³-Ser⁴-Asn⁵-Cys⁶-Pro⁷-Ile⁸-Gly⁹, high performance liquid chromatogram shown in figure 1.

B. The YC-9aa has a hydropathic index of 0.71, and the artificially synthesized polypeptide is poorly soluble in water, and the solubility measured after ultrasonication is 0.03, and is still poorly soluble in water.

C. Modification design is carried out on specific sites of the core 9 peptide: the fourth serine (Ser) is replaced by glutamine (Gln), glu-Ala-Ala-Ala-lys is added after the ninth amino acid, and the YC-9aaL polypeptide designed by artificial synthesis and modification has a structural formula Cys¹-Tyr²-Ile³-Gln⁴-Asn⁵-Cys⁶-Pro⁷-Ile⁸-Gly⁹-Glu¹⁰-Ala¹¹-Ala¹²-Ala¹³-lys¹⁴, as shown in figure 2.

D. The modified polypeptide YC-9aaLL has a hydrophilicity of 0.12, is easily dissolved in water, and the solubility is also remarkably improved (Table 1).

TABLE 1 comparison of physicochemical Properties of polypeptide YC-9aa before and after modification

	YC-9aa	YC-9aaL
			Molecular mass (g/mol)	996.1	1477.68
Isoelectric point (pH)	5.25	6.13
			Hydrophilicity index	0.71	0.12
Solubility (g)	0.03	＞0.1

Example 2: comparison of the oxytocic efficiency of YC-9aa and YC-9aaL on pelteobagrus fulvidraco

In the breeding season of pelteobagrus fulvidraco, selecting 60 female pelteobagrus fulvidraco with water temperature of 25 ℃ and weight of about 80 g, randomly dividing the pelteobagrus fulvidraco into two groups, wherein each group of 30 female pelteobagrus fulvidraco. The two-needle injection method is adopted to hasten parturition, and the two needles are separated by 12h.

A first group: the first needle and the second needle were injected with YC-9aa at 1. Mu.g/kg, 10. Mu.g/kg and 40. Mu.g/kg, respectively.

Second group: the first needle and the second needle were injected with 1. Mu.g/kg, 10. Mu.g/kg and 40. Mu.g/kg of YC-9aaL, respectively.

And counting the spawning induction efficiency of the YC-9aa and the YC-9aaL on the pelteobagrus fulvidraco within 24 hours from the second needle.

The test results are shown in FIG. 3. The results show that the low concentration (1 mug/kg and 10 mug/kg) of YC-9aa can not induce spawning of pelteobagrus fulvidraco, and the production rate of pelteobagrus fulvidraco of 40 mug/kg is only 13.3%. The oxytocic efficiency of injecting 1 mug/kg and 10 mug/kg of pelteobagrus fulvidraco in YC-9aaL group is respectively 10% and 66.7%, and the oxytocic efficiency of injecting 100 mug/kg is as high as 80.0%, so that the oxytocic effect is obvious. In conclusion, YC-9aa has ovulation promoting effect, and the modified polypeptide YC-9aaL has better spawning induction effect on pelteobagrus fulvidraco.

Example 3: different compound oxytocic drugs are used for artificial oxytocic of pelteobagrus fulvidraco

The commercial oxytocin chorionic gonadotrophin (HCG) is used as a control, and the difference of the effects of YC-9aaL designed by modification and HCG on the maturation and ovulation of pelteobagrus fulvidraco is compared. In the breeding season of pelteobagrus fulvidraco, 60 female pelteobagrus fulvidraco with well developed gonads is selected, and the weight is about 80 g. The method is divided into a control group and an experimental group at random, 30 tails of each group are placed in a barrel with the diameter of 1m, the water depth is 0.7 m, the water temperature is 26 ℃, the two-needle injection method is adopted to hasten parturition, and the two needles are separated by 12h.

Control group: the first needle injected with 10. Mu.g/kg LHRH-A2 and the second needle injected with 10. Mu.g/kg LHRH-A2 and 2000IU/kg HCG. The control group is an oxytocic drug combination for large-scale breeding of pelteobagrus fulvidraco.

Experimental group: the first needle injected with 10. Mu.g/kg LHRH-A2 and the second needle injected with 10. Mu.g/kg LHRH-A2 and 10. Mu.g/kg YC-9aaL.

The results are shown in Table 2. The experiment group injected with YC-9aaL had 23 pelteobagrus fulvidraco spawning, the spawning rate was 76.7%, the average spawning amount was 7.1g, while the control group injected with HCG had 20 pelteobagrus fulvidraco spawning, the spawning rate was 66.7%, and the average spawning amount was 5.9g. One week after the two groups, the post-partum survival rate of the pelteobagrus fulvidraco in the experimental group is 83.3%, and the control group is only 63.3%. In conclusion, YC-9aaL and LHRH-A2 combined use has the advantages of increasing ovulation quantity and improving postpartum survival rate of female parent.

TABLE 2 comparison of reproductive performance of female Pelteobagrus fulvidraco injected with YC-9aaL or HCG

	Catalytic yield (%)	Egg laying amount (g)	Fertilization rate (%)	Post partum survival (%)
					Control Group (HCG)	66.7±5.8	5.9±1.0	73.6±5.6	63.3±5.8
Experiment set (YC-9 aaL)	76.7±5.8	7.1±0.9*	77.0±9.3	83.3±5.8*

In table 2, "x" represents a significant difference from the control group

In addition, as shown in Table 3, the control group had a cost of 14.88 yuan per kg of LHRH-A2 and HCG injected, whereas the experimental group had a cost of 2.24 yuan per kg of LHRH-A2 and YC-9aaL, which was 6.6 times cheaper than the control group.

Table 3 price comparison of injected drug per kg female pelteobagrus fulvidraco in experimental and control group

	LHRH-A2 (Yuan/kg)	HCG (Yuan/kg)	YC-9aaL (Yuan/kg)	Total price (Yuan/kg)
					Control Group (HCG)	1.68	13.20	—	14.88
Experiment set (YC-9 aaL)	1.68	—	0.56	2.24

In Table 3, "-" represents that the group was not injected with the drug, and no price is shown

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. The application of the polypeptide in preparing a medicament for promoting ovulation, induced spawning and improving postpartum survival rate of female pelteobagrus fulvidraco, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.2, and the medicament comprises the polypeptide shown as SEQ ID NO.2 and LHRH-A2.

2. The use of claim 1, wherein the medicament is formulated for oral, intramuscular, subcutaneous or intravenous delivery.

3. The use according to claim 1, wherein the medicament is formulated as a liquid, semi-solid or solid dosage form.