Small molecule polypeptide and application thereof, and drug for inducing delivery and promoting activity of knife fish

Abstract

This invention relates to the field of fish breeding and discloses a drug for inducing spawning and promoting survival in knife fish. The drug is a polypeptide drug derived from the kisseptin gene discovered in the knife fish brain transcriptome, subsequently synthesized chemically. The technical solution of this invention utilizes transcriptome sequencing technology combined with bioinformatics analysis to discover and synthesize a polypeptide drug containing the amino group of the Kisspeptin2 gene. Intrathoracic injection into the fish effectively promotes ovarian maturation and ovulation in knife fish, while simultaneously increasing the survival rate of induced spawning fish.

Description

Technical Field

[0001] This invention relates to the field of fish breeding technology, specifically, to a drug for inducing spawning and promoting the survival of knife fish. Background Technology

[0002] Fish breeding includes natural and artificial breeding. Natural breeding is the process of mating, spawning, fertilization, and hatching in fish under natural conditions. For fish that cannot spawn naturally, artificial spawning induction is necessary. Commonly used spawning-inducing drugs in fish artificial breeding mainly include human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogues (LRH-A), pituitary gonadotropins (GtH), domperidone (DOM), and different combinations of these drugs. Practice has shown that different fish have different reproductive conditions and gonadal development characteristics. Current drugs are effective for inducing spawning in some fish species but ineffective in others. Furthermore, some aggressive fish species experience stress after artificial spawning induction, leading to mass mortality of fish that have not yet spawned. For valuable fish species where spawning induction causes mass mortality, new drugs with high spawning efficiency, low mortality rates, and normal spawning are needed.

[0003] The anchovy, also known as the knife fish, is considered one of the "Three Delicacies of the Yangtze River" along with pufferfish and shad, prized for its tender flesh and high nutritional value. Anchovies die immediately upon leaving the water, making it difficult to capture live, mature fish for breeding. The best method is to divert water from the Yangtze River into ponds during the breeding season, allowing wild anchovy fry to enter the ponds with the water. These wild anchovies are then captured and raised, subsequently reproducing naturally in the ponds. Despite over a decade of development, artificially raised anchovies exhibit significant asynchronous gonad development, with only a small percentage reaching maturity and spawning naturally during the breeding season, resulting in low yields and severely hindering the development of the anchovy industry.

[0004] Kisspeptin is a neuropeptide that functions via the G protein-coupled receptor GPR54 and is closely related to reproduction. In reproduction, it regulates GnRH secretion and modulates the positive and negative feedback loops of estrogen, participating in reproductive development. Kisspeptin neurons regulate the estradiol positive feedback loop by expressing estrogen receptor α and neurotransmitters such as tyrosine hydroxylase, γ-aminobutyric acid, and glutamate; and influence follicle development by upregulating ovarian anti-Müllerian hormone and downregulating FSH receptor (FSHR) expression. As a key regulatory factor, kisspeptin regulates female follicle development, oocyte maturation, and ovulation in multiple ways. Currently, drugs for inducing spawning and promoting survival after spawning stress in knife fish are very rare, and no research reports have been found. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the purpose of this invention is to provide a drug for inducing spawning and promoting the survival of knife fish.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a drug for inducing spawning and promoting the survival of knife fish, wherein the drug is an amino polypeptide drug derived from the rostral peptide gene found in the brain transcriptome of knife fish through chemical synthesis.

[0007] One technical solution of the present invention is a small molecule polypeptide whose amino acid sequence contains the amino acid sequence shown in SEQ ID No. 1: SEFNINPFGLRF.

[0008] Furthermore, the small molecule polypeptide is composed of the sequence shown in SEQ ID NO.1.

[0009] Furthermore, the small molecule polypeptide has an amino-terminal protecting group and / or a carboxyl-terminal protecting group.

[0010] The amino-terminal protecting group is selected from a selectively substituted C1-C10 acyl group or an optionally substituted C1-C10 amide group.

[0011] The carboxyl-terminal protecting group is selected from optionally substituted C1-C10 acyl groups or optionally substituted C1-C10 amide groups.

[0012] The aforementioned small molecule peptides can effectively promote ovarian maturation and ovulation in knife fish, while also increasing the survival rate of fish that have undergone induced spawning.

[0013] Another technical solution of the present invention is an isolated polynucleotide selected from the group consisting of:

[0014] (a) A small polypeptide or fragment thereof, analogue, or derivative thereof encoding the amino acid sequence shown in SEQ ID NO. 1;

[0015] (b) A polynucleotide complementary to polynucleotide (a);

[0016] Or, (c) a polynucleotide that has at least 70% identity with (a) or (b).

[0017] The carboxyl-terminal protecting group is selected from an optionally substituted C2-10 acyl group or an optionally substituted C2-10 amide group.

[0018] The aforementioned small molecule polypeptides can be used to prepare fish medicines. Preferably, the fish medicine is used for cuttlefish.

[0019] The fish medicine is applicable for conditions such as inducing spawning, promoting survival after spawning stress, or conditions related to abnormal estrogen secretion.

[0020] A fish medicine whose active ingredient includes the aforementioned small molecule polypeptides. This fish medicine is used to induce spawning and promote the survival of knife fish.

[0021] The preparation method of the fish medicine includes the following steps: using transcriptome sequencing technology, analyzing genes (DEGs) with significant differential expression between phase IV and phase II of ovarian development in knife fish during the same breeding period, screening DEGs that regulate ovarian development, and identifying the CDS sequence of the Kisspeptin2 gene. The 12-amino polypeptide of the Kisspeptin2 gene is chemically synthesized with a purity >90%, dissolved in 0.75% NaCl physiological saline to prepare an injection solution, and induced spawning by intrathoracic injection.

[0022] The advantages of this invention are:

[0023] Using transcriptome sequencing technology combined with bioinformatics analysis, a 12-amino peptide drug of the Kisspeptin2 gene was discovered and synthesized. When injected into the pleural cavity of fish, it can effectively promote ovarian maturation and ovulation in knife fish, while improving the survival rate of fish that have been induced to spawn. Attached Figure Description

[0024] Figure 1 Anatomical morphological characteristics of the ovaries in the 0.1 μg / g injection group (A), the 0.5 μg / g injection group (B), and the control group (C);

[0025] Figure 2 The study showed the hormone levels in the serum of knife fish injected with different concentrations of chemically synthesized rostral peptide. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0027] Experimental methods in the following examples that do not specify specific conditions should be performed according to conventional methods and conditions, or according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0028] The present invention will be further described below with reference to the embodiments:

[0029] Example 1

[0030] 1. Experimental Design:

[0031] This invention analyzes significantly differentially expressed genes in the ovaries of knife fish at two different developmental stages during the breeding season, screens for the CDS (Cellular Data Set) of the rostral peptide gene, predicts the amino acid sequence and functional motif encoded by the CDS, chemically synthesizes the peptide chain of the functional motif, dissolves it in DMSO, dilutes it with 0.75% physiological saline, injects it into the pleural cavity, and draws blood 14 hours later. The ovary is then dissected and removed. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), and estradiol (E2) are detected using an ELISA kit.

[0032] 1.1 Experimental Materials:

[0033] During the breeding season of anchovies in mid-April, 30 adult anchovies weighing 66g-131.6g and measuring 26-31.5cm in length were purchased from a fish farm in Jiangsu Province. They were randomly divided into 3 groups of 10 fish each. After being injected with medication, they were temporarily kept in an indoor cement tank (5m×10m×1.2m).

[0034] 1.2 Drug Synthesis and Formulation

[0035] The 12-amino polypeptide of the kiss peptide, synthesized by Beijing Qingke Biotechnology Co., Ltd. with a purity >90, was first dissolved in DMSO and then prepared into an injection solution with 0.75% NaCl physiological saline.

[0036] The sequence is: SEFNINPFGLRF-CONH2.

[0037] 1.3 Injection Method and Dosage

[0038] The drugs were administered via intrapleural injection at doses of 0.1 μg / g and 0.5 μg / g, respectively, while the control group received 0.75% NaCl saline.

[0039] 2 Experimental Results

[0040] 2.1 Observe ovarian development

[0041] Fourteen hours after injection, the experimental fish were anesthetized with MS-222, and 0.5 ml of blood was drawn from the tail vein, added to heparin sodium, and stored for later use. First, a transverse incision was made at the anus, then the abdomen was cut along the midline from the anus towards the head, and the ovaries were removed for observation.

[0042] See the observation results. Figure 1 . Figure 1 Anatomical morphological characteristics of the ovaries in the 0.1 μg / g injection group (A), the 0.5 μg / g injection group (B), and the control group (C).

[0043] like Figure 1As shown, 14 hours after drug injection in different injection groups, 8 tails survived in the 0.1 μg / g injection group. Among them, 5 tails had plump, bluish-gray ovaries with visible oocytes, indicating late stage IV (see details). Figure 1 A).

[0044] like Figure 1 As shown in Figure B, the ovaries of the three samples were flesh-colored, with visible oocytes, indicating late stage III. Nine fish survived in the 0.5 ug / g injection group, among which seven samples had very full, jade-green ovaries filled with clearly identifiable oocytes, including free oocytes, indicating early stage V.

[0045] like Figure 1 As shown in C, the ovaries of 3 samples were in late stage IV. In the control group, 6 tails survived; of these, 1 sample had an ovary in early stage V, and 5 samples had ovaries in late stage III. Figure 1 C). It can be seen that the rate of artificial labor induction under the condition of 0.5ug / g drug injection is 77.8%.

[0046] 2.2 Measurement of serum hormone concentrations

[0047] Blood was drawn from the tail vein, centrifuged at 2000 rpm for 3 min, and the supernatant was collected. The serum levels of LH, FSH, GnRH, and E2 hormones in the experimental fish were measured using a Readmax 1900 microplate reader (MLBIO) with the following kits: Fish Luteinizing Hormone (LH) ELISA Kit (ml003456-48T), Fish Follicle-Stimulating Hormone (FSH) ELISA Kit (MLBIO, ml003455-48T), Fish Gonadotropin-Releasing Hormone (GnRH) ELISA Kit (ml003465-48T), and Fish Estradiol (E2) ELISA Kit (MLBIO, ml003452-48T).

[0048] See relevant results Figure 2 And Table 1. Table 1 shows the hormone levels in the serum of knife fish and the statistical differences between groups.

[0049] Table 1.

[0050]

[0051] In Table 1, * indicates a significant difference (P < 0.05); ** indicates a highly significant difference (P < 0.01).

[0052] Figure 2 The hormone levels in the serum of knife fish injected with different concentrations of chemically synthesized rostrum peptides are shown. Subplots A, B, C, and D represent the hormone levels of LH, FSH, E2, and GnRHd, respectively. The injection dosages for each subplot can be found in Table 1. Figure 2In the indices, * indicates a significant difference (P < 0.05); ** indicates an extremely significant difference (P < 0.01); ns indicates no significant difference.

[0053] Combining Table 1 and Figure 2 It was found that, compared with the control group, the LH levels in the 0.1 μg / g injection group and the 0.5 μg / g injection group were significantly increased (P < 0.05); although the levels of FSH, GnRH, and E2 were also increased, only the 0.5 μg / g injection group showed significant (P < 0.05) or highly significant (P < 0.01) increases. The increase in serum LH, FSH, GnRH, and E2 hormone levels was drug dose-dependent, and the effective dose of chemically synthesized rostral peptide for artificial spawning in cod was 0.5 μg / g.

[0054] As shown in Table 1, the initial experimental fish in the 0.1 ug / g injection group, the 0.5 ug / g injection group, and the control group were all 10 fish. After 14 hours of temporary rearing, the surviving experimental fish were 8, 9, and 6 fish, respectively, with survival rates of 80%, 90%, and 60%. This demonstrates that the drug significantly improves the survival rate of experimental fish while artificially inducing spawning.

[0055] In summary, it can be seen that the roefish kiss peptide, as a spawning-inducing drug, affects the ovarian development and maturation of roefish and their survival after spawning. The small molecule polypeptide prepared in this case also has the same related activity, and it can be applied to the reproduction and spawning of roefish, especially to effectively induce spawning in roefish and improve the survival rate of roefish during the spawning process.

[0056] It should be noted that the scope of protection of the prior art in this invention is not limited to the embodiments given in this application. All prior art that does not contradict the solution of this invention, including but not limited to prior patent documents, prior publications, prior public uses, etc., can be included in the scope of protection of this invention.

[0057] Furthermore, the combination of the technical features in this case is not limited to the combination methods described in the claims of this case or the combination methods described in the specific embodiments. All technical features described in this case can be freely combined or combined in any way, unless they contradict each other.

[0058] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A small molecule polypeptide, characterized in that, The amino acid sequence of the small molecule polypeptide is as shown in SEQ ID No. 1, or the amino acid sequence of the small molecule polypeptide is SEFNINPFGLRF-CONH2.

2. An isolated polynucleotide, characterized in that, The polynucleotide is selected from one of the following groups: (a) A polynucleotide encoding a small polypeptide with the amino acid sequence shown in SEQ ID NO. 1; (b) A polynucleotide complementary to polynucleotide (a).

3. The application of the small molecule polypeptide as described in claim 1 in the preparation of fish medicine, characterized in that, The fish medicine is applicable to the following conditions: inducing spawning in knife fish and promoting survival after spawning stress.

4. A fish medicine, characterized in that, The active ingredient of the fish medicine includes the small molecule polypeptide as described in claim 1.

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