Recombinant fish gth fusion protein, preparation method therefor, and use thereof

Abstract

Disclosed are a recombinant fish GtH fusion protein, a preparation method therefor, and the use thereof. The recombinant fish GtH fusion protein comprises a peptide chain formed by linking a vitamin D-binding protein to at least one of a GtHα subunit, a GtH Iβ subunit, or a GtH IIβ subunit. The recombinant fish GtH fusion protein can achieve spawning induction with one injection, improves the efficiency of fish spawning induction, increases the number of types of fish spawning induction products, reduces irritation to fish, and has application potential in the artificial propagation of fish.

Description

Recombinant fish GtH fusion protein and preparation method and application thereof

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202410703395.5, filed on June 03, 2024, the contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine and animal reproduction technology, and specifically relates to a recombinant fish GtH fusion protein and a preparation method and application thereof. BACKGROUND

[0004] Fishery is an important part of the national economy and plays a significant role in promoting employment and maintaining food security. The substantial contribution of the Chinese-style "mainly based on aquaculture" fishery development model not only makes an important contribution to food security, nutrition security, and ecological security, significantly enhances the food supply function, ecological service function, and cultural service function of fishery, but also overturns the modern fishery development model of "mainly based on capture or capture dominance" since 1840. "Mainly based on aquaculture" has become the main direction of modern fishery development.

[0005] Fish can reproduce on their own in natural environments, but some farmed fish such as silver carp, bighead carp, grass carp, and blue carp can only develop their gonads to the growth maturity (stage IV) in the farming water body and cannot complete the transition to physiological maturity (stage V), i.e., they cannot reproduce by spawning on their own. In addition, some farmed fish, such as carp, crucian carp, and mud carp, can reproduce by spawning on their own in farming water areas, but the spawning time is not concentrated, and the reproduction efficiency is not high. Therefore, artificial spawning is an important means for fish artificial reproduction and improving reproduction efficiency.

[0006] The purpose of artificial spawning is to promote the further development and maturation of the gonads of parent fish through ecological measures and physiological means to achieve the process of ovulation, spawning, and spermiation and fertilization. The basic principle of artificial reproduction is to use ecological principles and methods to meet the conditions required for fish ovulation and spawning, provide spawning pools (such as water temperature, water quality, water flow, spawning sites, and substrates, etc.), and apply physiological principles and measures, such as injecting spawning drugs, to promote the further development and maturation of the gonads of parent fish, thereby achieving the process of ovulation, spawning, and spermiation and fertilization.

[0007] The development of gonad and reproductive activity of fish is mainly regulated and controlled by the brain-pituitary-gonad axis, and the hormone regulation is a double neurohormone system control. When the external conditions (water temperature, water quality, water flow, water level, salinity, substrate attachment and opposite sex) stimulate the sensory organs (eyes, skin, lateral line, etc.), the stimulation is transmitted to the central nervous system, and the central nervous system sends instructions to the endocrine organ hypothalamus. The hypothalamus secretes gonadotropin releasing hormone (GnRH) to stimulate the production and release of gonadotropin hormone (GtH) after combining with the specific receptors of the gonadotropin hormone secreting cells. There are two types of GtHs in fish, GtH I and GtH II, corresponding to the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of mammals. The gonadotropin reaches the gonad and combines with the receptor to produce a physiological effect, stimulates the development and discharge of gametes, and promotes the secretion of sex hormones by the gonad.

[0008] Currently, there are two schemes for fish induced spawning, environmental regulation and drug induced spawning. Drug induced spawning is the most widely used scheme. As early as 1958, China began to use fish pituitary gland (PG) as a fish induced spawning drug, but due to the low content of natural pituitary gland, it cannot be extracted and used on a large scale. After continuous exploration of the mechanism of fish induced spawning, several products have been applied to fish induced spawning, mainly LHRH-A, DOM, HCG and PG, etc. LHRH-A (Luteininzing hormone releasing hormone analogue) is a GnRH analogue, whose function is similar to that of GnRH. The 6th glycine of GnRH is replaced by D-alanine, and the 10th glycine is removed to enhance the affinity of receptor binding and resistance to enzyme digestion, thereby becoming a high-activity analogue. DOM (Domperidone) is a dopamine D-2 receptor antagonist. Dopamine inhibits the release of gonadotropin by blocking GnRH. DOM eliminates the effect of dopamine by competing for dopamine receptors and depleting dopamine. Studies have found that human chorionic gonadotropin (HCG) can also induce fish spawning, which directly acts on the gonads to induce spawning. The hormone that directly acts on the gonads also includes fish pituitary gland, mainly the pituitary gland of adult carp and other fish. The gonadotropin (GtH) contained in fish pituitary gland is a fish-derived hormone, which has good induced spawning effect and is widely applicable to various fish. However, the preparation of pituitary gland is difficult, and the quality of commodity fish is affected after excavation, so pituitary gland cannot be extracted and used for induced spawning on a large scale. In the process of induced spawning, different combinations of induced spawning drugs are used for different species of fish, and the number of injections also varies. Therefore, it is necessary to develop an induced spawning product that meets the needs of most species and is easy to use for fish induced spawning.

[0009] GtH is a fish-derived hormone that has been proven to have good induced spawning effect and is widely applicable to various fish. GtH is a dimeric molecule composed of the same alpha subunit and specific beta subunit. GtH I is a heterodimer composed of GtH alpha and GtH I beta, and GtH II is a heterodimer composed of GtH alpha and GtH II beta. Due to the progress of protein recombination technology, human recombinant gonadotropin products have been applied to assisted reproduction.

[0010] Vitamin D binding protein (DBP) is an albumin-like protein that is the main protein component in fish plasma and plays an important role.

[0011] At present, there is no product of recombinant fish gonadotropin on the market, and the half-life of extracted natural fish gonadotropin is short. Therefore, the development of recombinant fish gonadotropin has important significance for improving the efficiency of fish spawning and promoting fishery production. SUMMARY

[0012] In order to solve the above technical problems, enrich fish spawning products and improve the efficiency of fish spawning, the present application provides the following technical solutions.

[0013] In a first aspect, the present application provides a recombinant fish GtH fusion protein, wherein the recombinant fish GtH fusion protein comprises a peptide chain formed by connecting a vitamin D binding protein (DBP) with at least one of a GtH alpha subunit, a GtH I beta subunit or a GtH II beta subunit.

[0014] Preferably, the vitamin D binding protein (DBP) and the GtH alpha subunit, the GtH I beta subunit or the GtH II beta subunit are connected by a Linker or directly connected.

[0015] Further, the Linker is a flexible polypeptide composed of 2-20 flexible amino acids selected from at least one of Gly, Ser, Ala and Thr.

[0016] Preferably, the Linker is (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer between 2 and 5, and more preferably n is 3.

[0017] Further, the recombinant fish GtH fusion protein comprises the following protein molecules:

[0018] a protein composed of the amino acid sequences shown in SEQ ID No: 1 and SEQ ID No: 4 or SEQ ID No: 6; or

[0019] a protein having 80% or more identity with the amino acid sequence composed of the amino acid sequences shown in SEQ ID No: 1 and SEQ ID No: 4 or SEQ ID No: 6 and having the same function; or

[0020] a protein composed of the amino acid sequences shown in SEQ ID No: 2 and SEQ ID No: 5 or SEQ ID No: 7; or

[0021] a protein having 80% or more identity with the amino acid sequence composed of the amino acid sequences shown in SEQ ID No: 2 and SEQ ID No: 5 or SEQ ID No: 7 and having the same function; or

[0022] a protein consisting of the amino acid sequence shown in any one of SEQ ID No: 8-13; or

[0023] a protein having 80% or more identity with the amino acid sequence shown in any one of SEQ ID No: 8-13 and having the same function; or

[0024] a protein consisting of the amino acid sequence shown in SEQ ID No: 1 combined with the amino acid sequence shown in SEQ ID No: 14 or SEQ ID No: 15; or

[0025] a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 1 combined with the amino acid sequence shown in SEQ ID No: 14 or SEQ ID No: 15 and having the same function; or

[0026] a protein consisting of the amino acid sequence shown in SEQ ID No: 3 combined with the amino acid sequence shown in SEQ ID No: 5 or SEQ ID No: 7; or

[0027] a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 3 combined with the amino acid sequence shown in SEQ ID No: 5 or SEQ ID No: 7 and having the same function; or

[0028] a protein consisting of the amino acid sequence shown in any one of SEQ ID No: 16-21; or

[0029] a protein having 80% or more identity with the amino acid sequence shown in any one of SEQ ID No: 16-21 and having the same function.

[0030] The above-mentioned identity refers to amino acid sequence identity. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage of the NCBI homepage.

[0031] Preferably, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0032] In a second aspect, the present application provides a nucleotide molecule encoding the recombinant fish GtH fusion protein of the first aspect.

[0033] Preferably, the nucleotide molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0034] Further preferably, the nucleotide molecule comprises:

[0035] a combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27;

[0036] a complementary, degenerate or transcribed sequence of the combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27;

[0037] a DNA molecule or mRNA having 75% or more identity to a complementary, degenerate or transcribed sequence of the combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27 and encoding the corresponding protein in the fusion protein;

[0038] a combination of the base sequences represented by SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28;

[0039] a complementary, degenerate or transcribed sequence of the combination of the base sequences represented by SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28;

[0040] a DNA molecule or mRNA having 75% or more identity to a complementary, degenerate or transcribed sequence of the combination of the base sequences represented by SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28 and encoding the corresponding protein in the fusion protein;

[0041] a base sequence represented by any one of SEQ ID NOs: 29-34;

[0042] a complementary, degenerate or transcribed sequence of the base sequence represented by SEQ ID NOs: 29-34;

[0043] a DNA molecule or mRNA having 75% or more identity to a complementary, degenerate or transcribed sequence of the base sequence represented by any one of SEQ ID NOs: 29-34 and encoding the corresponding protein in the fusion protein;

[0044] a combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36;

[0045] a complementary, degenerate or transcribed sequence of the combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36;

[0046] DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences represented by SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36, having 75% or more identity and encoding the corresponding proteins in the fusion protein;

[0047] the combination of the base sequences represented by SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28;

[0048] the complementary, degenerate or transcribed sequences of the combination of the base sequences represented by SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28;

[0049] DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences represented by SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28, having 75% or more identity and encoding the corresponding proteins in the fusion protein;

[0050] the base sequence represented by any one of SEQ ID NO: 37-42;

[0051] the complementary, degenerate or transcribed sequences of the base sequences represented by SEQ ID NO: 37-42;

[0052] DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the base sequences represented by any one of SEQ ID NO: 37-42, having 75% or more identity and encoding the corresponding proteins in the fusion protein.

[0053] Those skilled in the art can easily mutate the nucleotide sequences encoding the above-mentioned fusion proteins of the present application using known methods, such as methods of directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequences encoding the above-mentioned fusion proteins, as long as they encode the above-mentioned fusion proteins and have the same function, are derived from the nucleotide sequences of the present application and are equivalent to the sequences of the present application.

[0054] The identity refers to the sequence similarity with the compared nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or more, having 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence shown in SEQ ID NO: 22-42 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0055] Preferably, the 75% or more identity can be 80%, 85%, 90% or 95% or more identity

[0056] In a third aspect, the present application provides a vector comprising the nucleotide molecule of the second aspect.

[0057] The vector refers to a vector capable of carrying the exogenous DNA, mRNA or target gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In a specific embodiment of the present application, the vector is plasmid pcDNA3.4.

[0058] In a fourth aspect, the present application provides a host cell comprising the nucleotide molecule of the second aspect or the vector of the third aspect.

[0059] Preferably, the host cell (also referred to as recipient cell) is a eukaryotic cell. The host cell can be understood not only to refer to a specific recipient cell, but also to the progeny of such a cell, which can not necessarily be identical to the original parent cell as a result of natural, accidental or deliberate mutation and / or alteration, but still falls within the scope of the host cell.

[0060] Further preferably, the host cell can be a plant cell or an animal cell. Suitable host cells are known in the art. The plant cell can be, but is not limited to, a plant cell of Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc. The animal cell can be a mammalian cell, for example, a human embryonic kidney cell (HEK293 cell), a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a baby hamster kidney cell (BHK cell), a mouse mammary carcinoma cell (C127 cell), a human HeLa cell, a fibroblast cell, a bone marrow cell line, a T cell, an NK cell, a pig kidney cell (PK15 cell), a pig pulmonary alveolar macrophage cell (PAM cell), a pig small intestine epithelial cell (IPEC-1 cell), a pig testis cell (ST cell), an avian cell (for example, a chicken or duck cell), an amphibian cell (for example, a Xenopus laevis cell or an Andrias davidianus cell), a fish cell (for example, a grass carp, a carp, a rainbow trout or a catfish cell), an insect cell (for example, an Sf21 cell or an Sf-9 cell), etc., but is not limited to. In one or more embodiments of the present application, the host cell is a HEK293 cell.

[0061] In a fifth aspect, the present application provides a method for preparing the recombinant fish GtH fusion protein of the first aspect, which comprises introducing the nucleotide molecule of the second aspect or the vector of the third aspect into a host cell, and recovering the fusion protein of the first aspect from the cell or cell culture.

[0062] Preferably, the method for preparing comprises screening a monoclonal cell strain with high and stable expression.

[0063] In a sixth aspect, the present application provides a pharmaceutical composition comprising the recombinant fish GtH fusion protein of the first aspect, or the nucleotide molecule of the second aspect, or the vector of the third aspect.

[0064] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or excipient.

[0065] In a seventh aspect, the present application provides use of any of the recombinant fish GtH fusion protein, the nucleotide molecule, the vector or the host cell described above, which use comprises any of the following:

[0066] (1) use in the preparation of a product containing a recombinant fish GtH;

[0067] (2) use in the preparation of a product for promoting artificial propagation of fish.

[0068] Preferably, the dose of the recombinant fish GtH fusion protein is 5-500 μg / kg / time for male fish and 10-1000 μg / kg / time for female fish.

[0069] Further, the recombinant fish GtH fusion protein is injected only once in the fish induced labor process.

[0070] Preferably, the fish is zebra fish, grass carp, large yellow croaker or yellow catfish.

[0071] Advantages of the present application

[0072] The recombinant fish GtH fusion protein of the present application can realize induced labor by one injection, improves the efficiency of fish induced labor, enriches the types of fish induced labor products, and reduces the stimulation to fish body, and has great application potential in fish artificial propagation. BRIEF DESCRIPTION OF DRAWINGS

[0073] Fig. 1 shows the structural schematic diagram of the recombinant fish GtH fusion protein of the present application, wherein α represents GtH α subunit, β represents GtH I β subunit or GtH II β subunit, and DBP represents vitamin D binding protein.

[0074] Fig. 2 shows the purity result of the recombinant fish GtH fusion protein detected by SDS-PAGE electrophoresis, wherein 1-10 represents recombinant GtH I fusion protein, 11-20 represents recombinant GtH II fusion protein, and M represents protein marker.

[0075] Fig. 3 shows the result of wild-type recombinant fish GtH protein detected by SDS-PAGE electrophoresis, wherein M represents protein marker, 1 is GtH α and GtH II β recombinant, and 2 is GtH α and GtH I β recombinant. DETAILED DESCRIPTION

[0076] The technical solutions of the present application will be further described below in combination with examples and drawings, and the advantages and characteristics of the present application will be more clear with the description. However, it should be understood that the examples are only exemplary and do not limit the scope of the present application.

[0077] It should be noted that, unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application relates.

[0078] Example 1 Construction of expression vector

[0079] The mature peptide sequence of the α subunit of GtH of Cyprinus carpio was derived from P18857 (Uniprot database), the mature peptide sequence of the β subunit of GtH I of Cyprinus carpio was derived from O13050 (Uniprot database), the mature peptide sequence of the β subunit of GtH II of Cyprinus carpio was derived from P01235 (Uniprot database), and the mature peptide sequence of the vitamin D binding protein (DBP) of Cyprinus carpio, which is a member of the albumin family, was derived from A0A8C1HIT6 (Uniprot database). The α, β, and β subunits of GtH of Cyprinus carpio were combined with the DBP protein through a linker (GGGGS) 3, respectively, to form a series of new recombinant GtH fusion proteins of Cyprinus carpio. The structures of the recombinant GtH I and GtH II fusion proteins are shown in FIG. 1.

[0080] The amino acid sequences of the mature peptides or fusion proteins were codon-optimized, and then the corresponding nucleotide coding sequences were synthesized and inserted into the expression vector pcDNA3.4. The names of the fusion proteins, the names of the expression vectors, and the corresponding amino acid sequence numbers and nucleotide sequence numbers are shown in Tables 1-6.

[0081] Table 1: Names of GtH I DBP fusion proteins, names of expression vectors, and amino acid sequence numbers

[0082] Table 2: Names of GtH I DBP fusion proteins of Table 1, names of expression vectors, and nucleotide sequence numbers

[0083] Table 3: Names of GtH II DBP fusion proteins, names of expression vectors, and amino acid sequence numbers

[0084] Table 4: Names of GtH II DBP fusion proteins of Table 3, names of expression vectors, and nucleotide sequence numbers

[0085] Table 5: Names of wild-type GtH I and II recombinant proteins, names of expression vectors, and amino acid sequence numbers

[0086] Table 6: Names of wild-type GtH I and II recombinant proteins of Table 5, names of expression vectors, and nucleotide sequence numbers

[0087] Example 2: Expression and purification of recombinant GtH proteins

[0088] First, the fusion proteins were expressed using HEK293 cells. For the fusion proteins with only one expression vector, expression vector 1 was transfected into HEK293 cells using PEI. For the heterodimeric fusion proteins composed of two expression vectors, expression vectors 1 and 2 were mixed at equal proportions and then transfected into HEK293 cells using PEI. Then, the cell supernatant was collected on the sixth day after cell transfection, and the recombinant GtH proteins were purified from the cell supernatant by two-step chromatography (anion chromatography and cation chromatography). Finally, the purity of the recombinant GtH proteins was detected by SDS-PAGE electrophoresis, and the results showed that the purity was high (Figures 2 and 3).

[0089] Example 3 Determination of the biological activity of recombinant GtH I and GtH II fusion proteins

[0090] The biological activity of 20 kinds of recombinant GtH I and GtH II fusion proteins and recombinant GtH I and GtH II was determined using sexually mature zebrafish. 230 sexually mature zebrafish males and females were selected, respectively, and randomly divided into 23 groups, 10 groups for recombinant GtH I fusion protein groups, 10 groups for recombinant GtH II fusion protein groups, 1 group for a recombinant GtH I group, 1 group for a recombinant GtH II group, and 1 group for a blank control group, 10 zebrafish per group. Before injection of the drugs, the abdomen of the male and female zebrafish was gently pressed to determine whether sperm and eggs had been released.

[0091] On the day of drug injection, 0.5 μg / g of the corresponding recombinant GtH I fusion protein was injected into the 10 groups of male zebrafish in the recombinant GtH I fusion protein group, 0.5 μg / g of the corresponding recombinant GtH II fusion protein was injected into the 10 groups of male zebrafish in the recombinant GtH II fusion protein group, 0.5 μg / g of recombinant GtH I was injected into the male zebrafish in the recombinant GtH I group, 0.5 μg / g of recombinant GtH II was injected into the male zebrafish in the recombinant GtH II group, and no treatment was performed on the male zebrafish in the blank control group; 1 μg / g of the corresponding recombinant GtH I fusion protein was injected into the 10 groups of female zebrafish in the recombinant GtH I fusion protein group, 1 μg / g of the corresponding recombinant GtH II fusion protein was injected into the 10 groups of female zebrafish in the recombinant GtH II fusion protein group, 1 μg / g of recombinant GtH I was injected into the female zebrafish in the recombinant GtH I group, 1 μg / g of recombinant GtH II was injected into the female zebrafish in the recombinant GtH II group, and no treatment was performed on the female zebrafish in the blank control group. After drug injection, the number of sperm-releasing male zebrafish was recorded by gently pressing the abdomen of the male zebrafish 24 h later, and the results are shown in Table 7. The female zebrafish were stimulated using male zebrafish, and the number of ovipositing female zebrafish was recorded, and the results are shown in Table 8.

[0092] Table 7 Effect of recombinant GtH I and GtH II fusion proteins on sperm release in male zebrafish

[0093] As shown in Table 7, for male zebrafish, both recombinant GtH I and GtH II fusion proteins can promote spermiation of male zebrafish, wherein the effective rate of spermiation induced by GtH I β-DBP & GtH α, DBP-GtH I β & GtH α, DBP-GtH α & GtH I β is 100%, the effective rate of spermiation induced by GtH α-DBP-GtH I β, GtH α-GtH II β-DBP, DBP-GtH α-GtH II β, GtH II β-DBP-GtH α, GtH α-DBP-GtH II β is below 50%, the effective rate of spermiation induced by other recombinant GtH I and GtH II fusion proteins is above 50% (including 50%), and both recombinant GtH I and recombinant GtH II can effectively induce spermiation of male zebrafish.

[0094] Table 8 Effect of recombinant GtH I and GtH II fusion proteins on ovulation of female zebrafish

[0095] As shown in Table 8, for female zebrafish, the effective rate of ovulation induced by recombinant GtH I fusion protein and recombinant GtH I is lower than that of recombinant GtH II fusion protein, the effective rate of ovulation induced by GtH II β-DBP & GtH α and DBP-GtH II β & GtH α is 100%, the effective rate of ovulation induced by GtH II β-DBP-GtH α and GtH α-DBP-GtH II β is 50%, and the effective rate of ovulation induced by other recombinant GtH II fusion protein and recombinant GtH II is above 50%, which indicates that ovulation of female zebrafish depends more on recombinant GtH II fusion protein.

[0096] Example 4 Effect of recombinant GtH I fusion protein on parturition of grass carp

[0097] The brood fish was intensively cultured before spawning, and the regular flushing, timely water injection, scientific oxygenation and bait feeding were carried out. The 5-6 year old brood fish with the weight of 5-10 kg, complete fins and scales, bright color, no disease and injury, active swimming, strong constitution and normal growth were selected. The male grass carp was selected by the fin bar with thick and long shape, natural opening with sharp knife shape, very dense fine granular protrusions called "pearl stars" arranged on the inner side of pectoral fin and gill cover, rough feeling when touched, soft at one-finger distance from the genital pore, slightly purple, thick and white semen flowed out after the abdomen was pressed, and scattered in water. The female grass carp was selected by the fin bar with thin and short shape, natural opening with fan shape, generally no pearl stars or only a small amount of protrusions at the end of pectoral fin, no rough feeling when touched, elastic abdomen, obvious ovary outline, loose genital pore, and soft feeling when the abdomen was tapped. 240 female fish and 240 male fish were randomly selected, and the female and male brood fish were mixed at a ratio of 1:1 and randomly divided into 12 groups. The control group was injected with LHRH-A2 7 μg+DOM 5 mg per kg of body weight, and the female fish in experimental groups 1-11 were injected with recombinant GtH I fusion protein at a dose of 10 μg / kg of body weight, and the dose of male brood fish was halved. The treated brood fish was put into the spawning pool for spawning, and the fish eggs were collected and put into the ring channel for flow water hatching. The spawning rate, fertilization rate and hatching rate were recorded and counted during the experiment. The results are shown in Table 9.

[0098] Table 9 Spawning effect of recombinant GtH I fusion protein on grass carp

[0099] As can be seen from Table 9, compared with the control group, the number of fry in the other experimental groups increased to different degrees, except for experimental groups 9 and 10. The number of fry in experimental groups 1, 3, 4, 6, 7 and 11 increased by 29.6%, 43.3%, 41.8%, 29.6%, 30.1% and 33.1% respectively compared with the control group. It is shown that the recombinant GtH I fusion protein has good spawning effect on grass carp, and even higher than the wild type GtH I.

[0100] Example 5 Spawning effect of recombinant GtH II fusion protein on grass carp

[0101] The parent fish is intensively cultured before spawning, and the regular flushing, timely water injection, scientific oxygenation and bait feeding are carried out. The 5-6 year old parent fish with the weight of 5-10 kg, complete fins and scales, bright color, no disease and injury, active swimming, strong constitution and normal growth are selected. The male parent fish is selected with the thick and long fin bar, the natural opening of the sharp knife shape, the very dense fine granular protrusions called "pearl stars" arranged on the inner side of the pectoral fin and the gill cover, the rough feeling when touched, the soft one-finger distance from the genital pore, the slight purple color, the thick and white sperm flowing out after the light pressure on the abdomen, and the dispersion in water. The female parent fish is selected with the thin and short fin bar, the natural opening of the fan shape, no pearl stars or only a small amount of protrusions at the end of the pectoral fin, no rough feeling when touched, the elastic abdomen, the obvious ovary outline, the loose genital pore, and the soft feeling when the abdomen is tapped. 240 female fish and 240 male fish are randomly selected, and the female and male parent fish are matched at the ratio of 1:1 and randomly divided into 12 groups. The control group uses the LRH-A3+DOM mixed spawning agent, and the LHRH-A3 7 μg+DOM 5 mg is injected into the chest cavity according to the weight of 1 kg. The female fish in the experimental groups 1-11 is injected with the recombinant GtH II fusion protein at the dose of 10 μg / kg of the weight, and the dose of the male parent fish is halved. The parent fish after the treatment is put into the spawning pool for spawning, and the fish eggs are collected and put into the ring channel for the flow water hatching. The spawning rate, fertilization rate and hatching rate and other data are recorded and counted during the experiment. The results are shown in Table 10.

[0102] Table 10 Spawning effect of the recombinant GtH II fusion protein on grass carp

[0103] As shown in Table 10, compared with the control group, the number of fry in the experimental groups is increased in different degrees. The number of fry in the experimental group 11 is increased by 45.1% than the control group, the number of fry in the experimental group 3 is higher than that in the experimental group 11, and is increased by 50.0% than the control group, and the number of fry in the experimental group 4 is not obviously different from that in the experimental group 11, and is increased by 44.0% than the control group. It is shown that the recombinant GtH II fusion protein has good spawning effect on grass carp, and is even higher than the wild type GtH II.

[0104] Example 6 Spawning effect of the recombinant GtH I fusion protein on the sea fish large yellow croaker

[0105] Select 3 years old, bright color, good vitality, healthy and no injury of the parent fish. Select the abdomen swelling soft and obvious female fish (abdominal circumference 14 cm or more), and the light pressure abdomen can flow out of the white, into the water immediately scattered out of the sperm of male fish for induced spawning. Randomly select 240 female fish and 120 male fish, female and male parent fish according to the ratio of 2:1, randomly divided into 12 groups. The spawning pool requires weak light, small noise, and the water temperature is maintained at 20-24℃. The control group of female fish is injected with LHRH-A3 4 μg per kg of body weight, and the experimental 1-11 groups of female fish are injected with recombinant GtH I fusion protein at a dose of 10 μg / kg of body weight, and the dose of male parent fish in each group is halved. The treated parent fish are put into the spawning pool for spawning, and the flow water method is used to collect the fish fertilized eggs after spawning and sperm, and the hatching pool is moved for hatching. Record and count the data of yield, fertilization rate and hatching rate during the experiment. The results are shown in Table 11.

[0106] Table 11 Effect of recombinant GtH I fusion protein on induced spawning of large yellow croaker

[0107] As can be seen from Table 10, compared with the control group, the number of fry in the other experimental groups increased to varying degrees, except for the experimental group 10. The number of fry in the experimental group 11 increased by 23.7% compared with the control group, the number of fry in the experimental group 3 was higher than that in the experimental group 11, and increased by 27.7% compared with the control group. The number of fry in the experimental group 4 was not significantly different from that in the experimental group 11, and increased by 23.9% compared with the control group. It is shown that the effect of recombinant GtH I fusion protein on induced spawning of large yellow croaker is good, and even higher than that of wild type GtH I.

[0108] Example 7 Effect of recombinant GtH II fusion protein on induced spawning of large yellow croaker

[0109] Select 3 years old, bright color, good vitality, healthy and no injury of the parent fish. Select the abdomen swelling soft and obvious female fish (abdominal circumference 14 cm or more), and the light pressure abdomen can flow out of the white, into the water immediately scattered out of the sperm of male fish for induced spawning. Randomly select 240 female fish and 120 male fish, female and male parent fish according to the ratio of 2:1, randomly divided into 12 groups. The spawning pool requires weak light, small noise, and the water temperature is maintained at 20-24℃. The control group of female fish is injected with LHRH-A3 4 μg per kg of body weight, and the experimental 1-11 groups of female fish are injected with recombinant GtH I fusion protein at a dose of 10 μg / kg of body weight, and the dose of male parent fish in each group is halved. The treated parent fish are put into the spawning pool for spawning, and the flow water method is used to collect the fish fertilized eggs after spawning and sperm, and the hatching pool is moved for hatching. Record and count the data of yield, fertilization rate and hatching rate during the experiment. The results are shown in Table 11.

[0110] Table 12 Effect of recombinant GtH II fusion protein on induced spawning of large yellow croaker

[0111] As shown in Table 12, compared with the control group, the number of fry in the other experimental groups increased to different degrees, except for a slight decrease in the experimental group 10. The number of fry in the experimental group 11 increased by 22.3% compared with the control group, and the number of fry in the experimental group 3 was higher than that in the experimental group 11, which increased by 25.2% compared with the control group. The number of fry in the experimental groups 1 and 4 was not significantly different from that in the experimental group 11, which increased by 16.1% and 18.3% compared with the control group, respectively. It is shown that the recombinant GtH II fusion protein has good effects on inducing spawning of Pseudosciaena crocea, and even higher than the wild type GtH II.

[0112] Example 8 Effects of recombinant GtH I fusion protein on inducing spawning of Pseudobagrus fulvidraco

[0113] Three winter Pseudobagrus fulvidraco broodstocks were selected, and the average weight of female fish was more than 140 g per tail, and the average weight of male fish was more than 280 g per tail. The fish were full-bodied, normal in color, smooth in surface, robust in body, and had no disease, injury, or deformity and had reached sexual maturity. 240 female fish and 240 male fish were randomly selected, and the female and male broodstocks were matched at a ratio of 1:1 and randomly divided into 12 groups. The water temperature was maintained at 22-28°C. The control group of female fish used 10 μg of LHRH-A2, 5 mg of DOM, and 4000 IU of HCG mixture per kilogram of body weight, which was injected intramuscularly in two times. The first injection was 1 / 5 of the total dose, and the remaining amount was injected after 20 hours. The male fish was injected half of the dose and was injected synchronously with the second injection of the female fish. The female fish in the experimental groups 1-10 was injected with the recombinant GtH I fusion protein at a dose of 10 μg / kg of body weight, and the male broodstock was injected half of the dose. The female fish in the experimental group 11 was injected with the recombinant GtH I fusion protein at a dose of 10 μg / kg of body weight in two times. The first injection was 1 / 5 of the total dose, and the remaining amount was injected after 20 hours. The male broodstock was injected half of the dose and was injected synchronously with the second injection of the female fish. The broodstocks after inducing spawning were placed in the spawning pool, and the water was slightly flowing or aerated to maintain sufficient dissolved oxygen and fresh water quality. After spawning, the eggs were hatched, and the water was slightly flowing, changed, and aerated to maintain fresh water quality and sufficient dissolved oxygen. The data of the production rate, fertilization rate, and hatching rate were recorded and counted during the experiment, and the results are shown in Table 13.

[0114] Table 13 Effects of recombinant GtH I fusion protein on inducing spawning of Pseudobagrus fulvidraco

[0115] As shown in Table 13, compared with the control group, the number of fry in the other experimental groups increased to different degrees, except for a slight decrease in the experimental group 10. It is shown that the recombinant GtH I fusion protein has good effects on inducing spawning of Pseudobagrus fulvidraco, which is higher than the wild type GtH I.

[0116] Example 9 Effects of recombinant GtH II fusion protein on inducing spawning of Pseudobagrus fulvidraco

[0117] Select 3 winter age of yellow fish parent fish, female fish average weight 140 g / tail above, male fish average weight 280 g / tail above, full body, normal body color, smooth surface, robust, no disease, no injury, no deformity and have reached sexual maturity of the species. Randomly selected 240 female and 240 male, female and male parents were matched in a ratio of 1:1, and randomly divided into 12 groups. The water temperature was maintained at 22-28℃. The control group of female fish used 10 μg LHRH-A2, 5 mg DOM and 4000 IU HCG mixture per kilogram of body weight, and was injected intramuscularly, twice, with the first injection being 1 / 5 of the total dose, and the remaining amount being injected 20 hours apart; the male fish dose was halved and injected synchronously with the second injection of the female fish. The female fish in experiments 1-10 were injected with recombinant GtH II fusion protein at a dose of 10 μg / kg body weight, and only once, and the male parent dose was halved. The female fish in experiment 11 were injected with recombinant GtH II fusion protein at a dose of 10 μg / kg body weight, twice, with the first injection being 1 / 5 of the total dose, and the remaining amount being injected 20 hours apart, and the male parent dose was halved and injected synchronously with the second injection of the female fish. The induced parents were placed in the spawning pool, with micro-flow water or aeration, to maintain sufficient dissolved oxygen and fresh water quality. After spawning, incubation was carried out, with micro-flow water, water change and aeration, to maintain fresh water quality and sufficient dissolved oxygen. The yield, fertilization rate and hatching rate were recorded and counted during the experiment, and the results are shown in Table 14.

[0118] Table 14 Effect of recombinant GtH II fusion protein on the induced spawning of yellow fish

[0119] As can be seen from Table 14, compared with the control group, the number of fry in experiments 10 and 11 was slightly lower than that of the control group, and the number of fry in the other experimental groups was increased to varying degrees. It shows that the recombinant GtH II fusion protein has good effect on the induced spawning of yellow fish, and is higher than that of the wild type GtH II.

[0120] The results of Examples 3-9 show that the recombinant fish GtH fusion protein of the present application can effectively improve the effect of artificial induced spawning in fishery, and the effect of some recombinant fish GtH fusion protein is higher than that of the wild type. The present application enriches the products of fish induced spawning agent and improves the application range of fish artificial induced spawning technology.

[0121] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, which all belong to the protection scope of the present application.

Claims

1. A recombinant fish GtH fusion protein, characterized in that: The peptide chain in the recombinant fish GtH fusion protein comprises a vitamin D binding protein and a subunit, and the subunit is at least one of GtH α subunit, GtH I β subunit or GtH II β subunit.

2. The recombinant fish GtH fusion protein of claim 1, wherein: The recombinant fish GtH fusion protein comprises the following protein molecules: a protein comprising the amino acid sequence shown in SEQ ID No: 1 and SEQ ID No: 4 or SEQ ID No: 6; or a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 1 and SEQ ID No: 4 or SEQ ID No: 6 and having the same function; or a protein comprising the amino acid sequence shown in SEQ ID No: 2 and SEQ ID No: 5 or SEQ ID No: 7; or a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 2 and SEQ ID No: 5 or SEQ ID No: 7 and having the same function; or a protein comprising the amino acid sequence shown in any one of SEQ ID No: 8-13; or a protein having 80% or more identity with the amino acid sequence shown in any one of SEQ ID No: 8-13 and having the same function; or a protein comprising the amino acid sequence shown in SEQ ID No: 1 and SEQ ID No: 14 or SEQ ID No: 15; or a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 1 and SEQ ID No: 14 or SEQ ID No: 15 and having the same function; or a protein comprising the amino acid sequence shown in SEQ ID No: 3 and SEQ ID No: 5 or SEQ ID No: 7; or a protein having 80% or more identity with the amino acid sequence shown in SEQ ID No: 3 and SEQ ID No: 5 or SEQ ID No: 7 and having the same function; or a protein comprising the amino acid sequence shown in any one of SEQ ID No: 16-21; or a protein having 80% or more identity with the amino acid sequence shown in any one of SEQ ID No: 16-21 and having the same function.

3. A nucleotide molecule, characterized in that: The nucleotide molecule encodes the recombinant fish GtH fusion protein according to any one of claims 1-2.

4. The nucleotide molecule of claim 3, wherein: The nucleotide molecule comprises: a combination of the base sequence shown in SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27; a complementary, degenerate or transcribed sequence of the combination of the base sequence shown in SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27; and a combination of the base sequence shown in SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO:

28. a complementary, degenerate or transcribed sequence of the combination of the base sequence shown in SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO:

28. DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 22 and SEQ ID NO: 25 or SEQ ID NO: 27, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; the combination of the base sequences shown in SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28; the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28; DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 23 and SEQ ID NO: 26 or SEQ ID NO: 28, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; the base sequence shown in any one of SEQ ID NO: 29-34; the complementary, degenerate or transcribed sequences of the base sequences shown in SEQ ID NO: 29-34; DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the base sequences shown in any one of SEQ ID NO: 29-34, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; the combination of the base sequences shown in SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36; the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36; DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 22 and SEQ ID NO: 35 or SEQ ID NO: 36, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; the combination of the base sequences shown in SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28; the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28; DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the combination of the base sequences shown in SEQ ID NO: 24 and SEQ ID NO: 26 or SEQ ID NO: 28, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; the base sequence shown in any one of SEQ ID NO: 37-42; the complementary, degenerate or transcribed sequences of the base sequences shown in SEQ ID NO: 37-42; DNA molecules or mRNAs defined by the complementary, degenerate or transcribed sequences of the base sequences shown in any one of SEQ ID NO: 37-42, which have 75% or more identity to the fusion protein and encode the corresponding protein in the fusion protein; DNA molecules or mRNAs having 75% or more identity with the complementary, degenerate or transcribed sequence of any one of the base sequences shown in SEQ ID NO: 37-42 and encoding the corresponding protein in the fusion protein.

5. A vector, characterized by: The vector comprises the nucleotide molecule according to any one of claims 3-4.

6. A host cell, characterized in that: The host cell comprises the nucleotide molecule according to any one of claims 3-4 or the vector according to claim 5.

7. A method for preparing the recombinant fish GtH fusion protein according to any one of claims 1-2, characterized by: The preparation method comprises introducing the nucleotide molecule according to any one of claims 3-4 or the vector according to claim 5 into a host cell, and recovering the fusion protein according to any one of claims 1-2 from the cell or cell culture.

8. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises the recombinant fish GtH fusion protein according to any one of claims 1-2, the nucleotide molecule according to any one of claims 3-4, or the vector according to claim 5.

9. Use of the recombinant fish GtH fusion protein according to any one of claims 1-2, the nucleotide molecule according to any one of claims 3-4, the vector according to claim 5 or the host cell according to claim 6, which use comprises any one of the following: (1) use in the preparation of a product containing a recombinant fish GtH; (2) use in the preparation of a product for promoting artificial reproduction of fish.

10. Use according to claim 9, characterized in that: The dose of the recombinant fish GtH fusion protein is 5-500 μg / kg / time for male fish and 10-1000 μg / kg / time for female fish.

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