A Japanese eel BMP-15 recombinant protein and its expression method and application

Abstract

This invention belongs to, but is not limited to, the field of genetic engineering technology, and discloses a recombinant BMP-15 protein from the Japanese eel, its expression method, and its applications. The recombinant BMP-15 protein comprises: the mature peptide amino acid sequence of Japanese eel BMP-15, an N-terminal 6×histidine tag, and a SUMO lysing polypeptide; the amino acid sequence of the recombinant BMP-15 protein is SEQ ID NO:1, and the DNA sequence of the gene encoding the recombinant BMP-15 protein is SEQ ID NO:3. This invention is the first to construct a prokaryotic expression vector pET-His-SUMO-BMP15 for the recombinant BMP-15 protein from the Japanese eel, which can yield a recombinant BMP-15 protein with an amino acid sequence identical to the mature peptide of Japanese eel BMP-15, and can be applied to the regulation of ovarian development and artificial reproduction research in Japanese eels.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of genetic engineering technology, and particularly relates to a recombinant BMP-15 protein from the Japanese eel, its expression method, and its application. Background Technology

[0002] The Japanese eel (Anguilla japonica) is one of the important migratory fish species in Asia and a significant economic fish in my country. As a migratory fish that spawns at sea, the Japanese eel has a complex life history, which leads to many unknowns, including regarding growth and reproduction. Therefore, artificial breeding technology remains a global challenge. Since the 1930s, significant progress has been made in artificial eel breeding technology, but there is still a considerable gap between the current level and the demand for large-scale industrial-scale production of eel larvae. The main reason is the low maturity rate, spawning rate, and spawning volume of artificially matured parent eels, especially the unsatisfactory quality of the eggs, resulting in very low fertilization, hatching, and survival rates. While the hatched eel larvae can eventually metamorphose into white eel larvae, their growth rate is extremely slow, their survival rate is very low, and their deformity rate is high. As the world's largest producer, processor, and exporter of eels, my country has a huge demand for eel larvae, making the artificial breeding of eels an urgent priority. The key to solving these problems lies in regulating the ovarian development process of Japanese eels in order to improve the quality of eggs from artificially bred eels.

[0003] Bone morphogenetic protein 15 (BMP15) is an ovarian regulatory factor specifically expressed by oocytes. It plays a crucial role in physiological mechanisms such as yolk accumulation and prevention of premature oocyte maturation. It is a key component in coordinating follicle development by sending signals from the oocyte to surrounding follicular cells (granulosa cells and membrane cells). BMP-15 can upregulate the expression levels of genes such as activin A (inhba) and ovarian aromatase (cyp19a1a) through the Smad cell signaling pathway, thus playing a vital regulatory role in ovarian development. Similar to other vertebrates, BMP-15 in the Japanese eel is a classic secretory protein with a typical N-terminal signal peptide sequence. In the cell, once the signal peptide of the nascent polypeptide chain is synthesized, it is recognized by the signal recognition granule (SRP) and translation is suspended. The SRP, along with the polypeptide chain and ribosomes, anchors on the surface of the rough endoplasmic reticulum, allowing translation to continue. As the nascent peptide chain is synthesized, it enters the endoplasmic reticulum while the signal peptide is cleaved. Finally, the peptide chain is folded and processed by the endoplasmic reticulum and Golgi apparatus and secreted into the extracellular space, where it forms a dimer through covalent bonds to perform its function. In addition, the Japanese eel BMP-15 monomer has three disulfide bonds, which are important structures for maintaining its correct molecular conformation and biological activity.

[0004] In the field of genetic engineering, *Escherichia coli* is a commonly used engineered bacterium in prokaryotic expression systems. It is a facultative anaerobic Gram-negative bacterium with a short growth cycle, clear genetic background, and simple and inexpensive culture process, enabling rapid and large-scale production of target proteins. By using strong promoters, exogenous proteins can be expressed at high levels in *E. coli*. However, the rate of exogenous protein expression in *E. coli* is much higher than in eukaryotes, which often leads to misfolding of nascent peptide chains. The exposed hydrophobic portions of these misfolded proteins aggregate to form inactive inclusion bodies.

[0005] Inclusion bodies are a common problem in prokaryotic expression, where misfolding of peptide chains leads to abnormal protein structure and loss of biological activity. To address this issue, inclusion body renaturation can be used to transform misfolded and insoluble inclusion bodies into soluble proteins with the correct molecular structure, restoring their biological activity and yielding bioactive recombinant BMP-15 protein from the Japanese eel. Furthermore, inclusion body renaturation is a more efficient method for obtaining recombinant proteins, achieving 80%–90% purity without the need for purification, saving time and costs in production and possessing significant practical implications.

[0006] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0007] (1) There are currently no reports on the expression of recombinant BMP-15 protein in Japanese eel;

[0008] (2) There are reports of BMP-15 recombinant protein expression in other organisms, but most of the reports use eukaryotic expression, which is inefficient and costly to obtain BMP-15 recombinant protein.

[0009] (3) There are relatively few reports on prokaryotic expression of BMP-15 recombinant protein in other organisms. Moreover, the inclusion bodies of BMP-15 recombinant protein in the reports have poor renaturation effect and are easily precipitated during dialysis, making it impossible to obtain high concentrations of BMP-15 recombinant protein.

[0010] (4) The recombinant protein of Escherichia coli is expressed too quickly and without the processing and modification of organelles such as endoplasmic reticulum and Golgi apparatus, which often results in the nascent peptide chains not being folded correctly in time. The hydrophobic parts of the misfolded protein are exposed and aggregate together to form inactive inclusion bodies. Summary of the Invention

[0011] To address the problems existing in the prior art, this invention provides a method for inducing the expression of recombinant BMP-15 protein in Japanese eel, particularly relating to a recombinant bone morphogenetic protein 15 (BMP-15) from Japanese eel produced by an Escherichia coli expression system and obtained in large quantities through inclusion body renaturation, and its application in the ovarian development of Japanese eel.

[0012] The present invention is achieved as follows: a recombinant BMP-15 protein from Japanese eel, comprising: the mature peptide amino acid sequence of Japanese eel BMP-15, an N-terminal 6×histidine tag (6×His tag or His tag for short), and a SUMO-promoting polypeptide; the amino acid sequence of the recombinant BMP-15 protein from Japanese eel is shown in SEQ ID NO:1;

[0013] Furthermore, the amino acid sequence of the mature BMP-15 peptide is shown in SEQ ID NO:2.

[0014] Furthermore, a His tag and a linker peptide were added to the N-terminus of the recombinant protein, with the amino acid sequence HHHHHHSSGLVPRGSHMAS. The 6×His tag (HHHHHH) was used for nickel column affinity chromatography to purify the Japanese eel BMP-15 recombinant protein, thereby further improving the purity of the recombinant protein. A 13-amino acid SSGLVPRGSHMAS linker peptide was added between the His tag and the SUMO solubilizing peptide to prevent the His tag from being masked by the SUMO solubilizing peptide and the mature peptide of Japanese eel BMP-15, thus ensuring its full exposure in solution and facilitating affinity chromatography purification of the recombinant protein after refolding.

[0015] Furthermore, a SUMO lysing peptide with the sequence MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG is added to the N-terminus to inhibit protein aggregation during inclusion body refolding, further promote the formation of soluble proteins during refolding, and improve the efficiency of inclusion body refolding. This is crucial for improving the efficiency of inclusion body refolding and the concentration of recombinant proteins.

[0016] The N-terminal His tag and SUMO-promoting peptide do not affect the activity of the Japanese eel BMP-15 recombinant protein, and there is no need to remove the tag.

[0017] Another objective of this invention is to provide a gene encoding a recombinant BMP-15 protein from Japanese eel, comprising: an N-terminal His tag, a linker peptide between the His tag and a SUMO-promoting polypeptide, a SUMO-promoting polypeptide, and a mature peptide of Japanese eel BMP-15; the DNA sequence of the encoding gene is SEQ ID NO:3.

[0018] Another object of the present invention is to provide an expression vector containing a coding gene for the recombinant protein BMP-15 of the Japanese eel and a backbone plasmid, wherein the backbone plasmid is obtained by modifying pET-32a(+).

[0019] Another object of the present invention is to provide a recombinant engineered bacterium containing the expression vector.

[0020] Furthermore, the host bacteria of the recombinant engineered bacteria are selected from Rosetta-gami B(DE3).

[0021] Another object of the present invention is to provide a method for preparing recombinant BMP-15 protein from Japanese eel, comprising the following steps:

[0022] (1) Construct the gene encoding the recombinant protein BMP-15 of the Japanese eel, and connect it to the backbone plasmid to construct the expression vector of the recombinant protein BMP-15 of the Japanese eel;

[0023] (2) The expression vector was transformed into a host bacterium to induce the expression of the Japanese eel BMP-15 recombinant protein;

[0024] (3) By refolding inclusion bodies, misfolded insoluble inclusion bodies are transformed into soluble proteins with the correct structure;

[0025] (4) Based on the His tag affinity chromatography of the Japanese eel BMP-15 recombinant protein, a higher purity Japanese eel BMP-15 recombinant protein was obtained (the protein purity after refolding can reach 80% to 90%. If the purity requirement is not high, this step can be skipped and proceed directly to the next step).

[0026] (5) Dialyze the protein into 1×PBS, add bovine serum albumin (BSA) to a final concentration of 0.1%, freeze in liquid nitrogen, and store at -80°C (if a higher concentration of protein is required after dialysis into 1×PBS, an ultrafiltration tube can be used to enrich and concentrate the protein).

[0027] Another object of the present invention is to provide an application of the recombinant BMP-15 protein from Japanese eel in the regulation of ovarian development in Japanese eel.

[0028] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0029] First, the difficulty in solving the problems and defects of existing technologies lies in:

[0030] (1) Construct recombinant BMP-15 protein of Japanese eel, and express it in large quantities through Escherichia coli prokaryotic expression system to obtain recombinant BMP-15 protein of Japanese eel with biological activity;

[0031] (2) Escherichia coli lacks the eukaryotic endomembrane system for post-translational modification and processing, and the expressed Japanese eel BMP-15 recombinant protein will lose its activity due to misfolding; moreover, the cytoplasm of Escherichia coli is reducing, which is not conducive to the formation of disulfide bonds in the Japanese eel BMP-15 recombinant protein.

[0032] The significance of addressing the problems and deficiencies of existing technologies lies in obtaining structurally correct and biologically active recombinant BMP-15 protein from the Japanese eel. This protein can be applied to basic research and production processes, providing a theoretical basis and technical support for the development of Japanese eel ovaries and artificial breeding. The recombinant protein of this invention can be induced to express in large quantities by prokaryotic bacteria, and through inclusion body renaturation, a biologically active recombinant protein can be efficiently obtained, saving time and costs. It can be effectively applied to industrial production and has significant commercial value.

[0033] (1) This invention provides a recombinant BMP-15 protein from Japanese eel, comprising: a mature peptide amino acid sequence of Japanese eel BMP-15, an N-terminal 6×histidine tag (6×His tag or simply His tag), a SUMO lysing peptide, and a linker peptide between the 6×histidine tag and the SUMO lysing peptide. The His tag can be used to purify the Japanese eel BMP-15 recombinant protein, improving its purity to meet higher application requirements; the SUMO lysing peptide can inhibit the aggregation of Japanese eel BMP-15 recombinant protein during inclusion body refolding, further promoting the formation of soluble proteins during refolding, improving the efficiency of inclusion body refolding and the concentration of the obtained Japanese eel BMP-15 recombinant protein;

[0034] (2) In this invention, a prokaryotic expression vector pET-His-SUMO-BMP15 for recombinant BMP-15 protein of Japanese eel was constructed, and the vector was transfected into Rosetta-gami B(DE3) Escherichia coli to obtain pET-His-SUMO-BMP15-Rosetta-gami B(DE3) expression strain, which can induce the expression of recombinant BMP-15 protein of Japanese eel in large quantities, and the soluble recombinant protein can be efficiently obtained through inclusion body renaturation, and it has biological activity without the need for tag excision;

[0035] (3) The recombinant BMP-15 protein of Japanese eel provided by the present invention or the recombinant BMP-15 protein of Japanese eel that is consistent with the mature peptide of Japanese eel BMP-15 can be effectively applied to the study of ovarian development and artificial reproduction of Japanese eel.

[0036] Second, considering the technical solution as a whole or from a product perspective, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows:

[0037] (1) This invention provides for the first time a recombinant protein of Japanese eel BMP-15 that is identical to the amino acid sequence of mature peptide BMP-15 of Japanese eel, comprising: mature peptide amino acid sequence of Japanese eel BMP-15, N-terminal 6×histidine tag (6×His tag or His tag for short), SUMO lysing peptide and linking peptide between 6×histidine tag and SUMO lysing peptide.

[0038] (2) This invention first constructs a prokaryotic expression vector pET-His-SUMO-BMP15 for recombinant Japanese eel BMP-15 protein, and transfects this vector into Rosetta-gami B(DE3) Escherichia coli to obtain the pET-His-SUMO-BMP15-Rosetta-gami B(DE3) expression strain, which can induce the expression of Japanese eel BMP15 recombinant protein in large quantities. The recombinant protein with a purity of 80% to 90% can be obtained by inclusion body renaturation (for recombinant Japanese eel BMP-15 protein with higher purity, His tag affinity chromatography can be performed to obtain recombinant protein with a purity higher than 90%).

[0039] (3) The prokaryotic expression vector pET-His-SUMO-BMP15 of the Japanese eel BMP-15 recombinant protein constructed in this invention, with the addition of SUMO lysing peptide, can inhibit the aggregation of Japanese eel BMP-15 recombinant protein during inclusion body refolding, further promote the formation of soluble protein during refolding, improve the efficiency of inclusion body refolding and the concentration of Japanese eel BMP-15 recombinant protein obtained;

[0040] (4) This invention provides for the first time a method for preparing recombinant BMP-15 protein from Japanese eel based on this recombinant protein. The recombinant protein of this invention can be induced to express in large quantities by prokaryotic bacteria, and soluble recombinant protein can be efficiently obtained through inclusion body renaturation, and it has biological activity without the need for tag removal;

[0041] (5) Application of the recombinant protein provided by this invention or the prepared Japanese eel BMP-15 recombinant protein in the study of Japanese eel ovarian development and artificial reproduction.

[0042] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0043] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: The method of the present invention can be used to obtain recombinant protein of Japanese eel BMP-15 with amino acid sequence consistent with mature peptide of Japanese eel BMP-15. The recombinant protein with biological activity can be obtained in large quantities and efficiently through the method, which can be effectively applied to industrial production and has important commercial value.

[0044] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad: There are no reports on the recombinant protein of Japanese eel BMP-15 in relevant studies at home and abroad. The present invention constructs for the first time the prokaryotic expression vector pET-His-SUMO-BMP15 of Japanese eel BMP-15, and transfects the vector into Rosetta-gami B(DE3) Escherichia coli. The resulting pET-His-SUMO-BMP15-Rosetta-gami B(DE3) expression strain can induce the expression of Japanese eel BMP-15 recombinant protein in large quantities, and obtain Japanese eel BMP-15 recombinant protein efficiently and in large quantities through inclusion body renaturation.

[0045] (3) The technical solution of this invention solves a long-standing but unsolved technical problem: artificial breeding of Japanese eels remains a global challenge, and the low quality of eggs obtained through artificial spawning is one of the main difficulties. Therefore, research on improving the quality of Japanese eel eggs is urgently needed. BMP-15 plays an important role in fish ovarian development and is a key factor in inducing the transition from secondary follicles to yolk sacs. Therefore, exploring the role of BMP-15 in Japanese eel ovarian development can provide a theoretical basis and technical support for achieving artificial eel breeding. The recombinant BMP-15 protein from Japanese eels obtained by the method described in this invention can be effectively applied to the research on the regulation of Japanese eel ovarian development and artificial breeding, which has significant scientific research value. Attached Figure Description

[0046] Figure 1 This is a flowchart of the preparation method of recombinant BMP-15 protein from Japanese eel provided in an embodiment of the present invention;

[0047] Figure 2 This is a schematic diagram of the structure of the Japanese eel BMP-15 protein and its open reading frame (ORF) cloning provided in an embodiment of the present invention;

[0048] Figure 2 A is a schematic diagram of the composition of Japanese eel BMP-15 containing a signal peptide of 28 amino acids, a precursor peptide of 278 amino acids, and a mature peptide of 126 amino acids, provided in an embodiment of the present invention; wherein, aa: amino acid;

[0049] Figure 2 B is the ORF sequence of Japanese eel BMP-15 and its translated amino acid sequence provided in the embodiments of the present invention. The dark gray area is a schematic diagram of the mature peptide amino acid sequence.

[0050] Figure 3 This is a schematic diagram of the Japanese eel BMP-15 recombinant protein expression vector, the recombinant protein composition, and the recombinant protein ORF sequence provided in this embodiment of the invention.

[0051] Figure 3 A is a map of the recombinant protein expression vector of Japanese eel BMP-15 provided in this embodiment of the invention. Wherein, LacO: lactose operon operator sequence; RBS: ribosome binding site; LacI: lactose operon regulatory gene I; rop: primer repressor gene; pBR322 Origin: pBR322 origin of replication; AmpR: ampicillin resistance gene;

[0052] Figure 3 B is a schematic diagram of the recombinant BMP-15 protein structure of Japanese eel provided in an embodiment of the present invention;

[0053] Figure 3 C is a schematic diagram of the recombinant protein ORF of Japanese eel BMP-15 and its translated amino acid sequence provided in the embodiments of the present invention; the gray area is the His tag, the dark gray area is the SUMO-promoting polypeptide, and the light gray area is the mature peptide of Japanese eel BMP-15;

[0054] Figure 4 This is an SDS-PAGE gel electrophoresis image of the expression, inclusion body renaturation, and concentration of recombinant BMP-15 protein from Japanese eel provided in this embodiment of the invention; In the figure: M: protein molecular weight standard; 1: total bacterial protein before IPTG induction; 2: total bacterial protein after IPTG induction; 3: supernatant of bacterial lysate after induction; 4: precipitate of bacterial lysate after induction; 5: total protein after inclusion body renaturation (including target protein); the black arrow indicates the BMP-15 recombinant protein band (molecular weight ~29kDa);

[0055] Figure 5 This is a qPCR result of cox gene expression in in vitro cultured follicular cells of Japanese eel regulated by the recombinant protein BMP-15 of Japanese eel, provided in an embodiment of the present invention. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0057] This invention provides a recombinant BMP-15 protein from Japanese eel, comprising: the mature peptide amino acid sequence of Japanese eel BMP-15, an N-terminal 6×histidine tag (6×His tag or His tag for short), and a SUMO lysing polypeptide; the amino acid sequence of the recombinant BMP-15 protein is shown in SEQ ID NO:1; the mature peptide amino acid sequence of Japanese eel BMP-15 is shown in SEQ ID NO:2.

[0058] SEQ ID NO:1:

[0059] MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGRPRR SKEPGSIGADIPNYVRQNSVAKNQCKLHSYRVTFQDLGWDHWIIAPHKYNPRYCRGDCPRILHYGYNSPNHAIVQNFINEMGVGEVPPPACVPYKYKPISVLMLEKNGSIVYKEYEDMIAESCTCR

[0060] SEQ ID NO:2:

[0061] RPRRSKEPGSIGADIPNYVRQNSVAKNQCKLHSYRVTFQDLGWDHWIIAPHKYNPRYCRGDCPRILHYGYNSPNHAIVQNFINEMGVGEVPPPACVPYKYKPISVLMLEKNGSIVYKEYEDMIAESCTCR

[0062] A His tag and a linker peptide were added to the N-terminus of the recombinant protein, with the amino acid sequence HHHHHHSSGLVPRGSHMAS (SEQ ID NO:4). The 6×His tag (HHHHHH) was used for nickel column affinity chromatography to purify the Japanese eel BMP-15 recombinant protein and further improve the purity of the recombinant protein. A 13-amino acid SSGLVPRGSHMAS (SEQ ID NO:5) was added between the His tag and the SUMO solubilizing peptide as a linker peptide to prevent the His tag from being masked by the SUMO solubilizing peptide and the mature peptide of Japanese eel BMP-15, thus ensuring that the His tag is fully exposed in solution to facilitate affinity chromatography purification of the recombinant protein after refolding.

[0063] The N-terminus of the SUMO-promoting polypeptide, with the sequence MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:6), is used to inhibit protein aggregation during inclusion body refolding, further promote the formation of soluble proteins during refolding, and improve the efficiency of inclusion body refolding.

[0064] The N-terminal His tag and SUMO-promoting peptide do not affect the activity of the Japanese eel BMP-15 recombinant protein, and there is no need to remove the tag.

[0065] This invention provides a gene encoding a recombinant BMP-15 protein from Japanese eel, comprising: DNA encoding an N-terminal His tag, a linker peptide between the His tag and a SUMO-promoting polypeptide, the SUMO-promoting polypeptide, and a mature peptide of Japanese eel BMP-15; the DNA sequence of the encoding gene is SEQ ID NO:3.

[0066] SEQ ID NO:3:

[0067] ATGGGCAGCAGCCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAA AAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGAACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGTCGCCCGCGCAGGTCG AAAGAACCGGGTAGCATTGGTGCAGACATCCCCAACTACGTACGCCAAAACAGTGTGGCTAAGAACCAGTGCAAGCTTCACTCCTACAGAGTGACCTTCCAGGACTTGGGTTGGGATCACTGGATCATCGCCCCACATAAGTACAACCCTCGGTACTGCAGAGGAGACTGCCCACGTATCCTCCACTAT GGGTACAATTCTCCAAACCATGCCATCGTGCAGAACTTCATCAATGAGATGGGTGTGGGGGAGGTGCCCCCGCCCGCTTGTGTACCTTACAAGTACAAGCCAATCAGCGTCCTCATGTTGGAGAAGAATGGCAGCATTGTATACAAAGAGTATGAGGACATGATCGCAGAGTCGTGCACCTGCCGCTAA

[0068] This invention provides an expression vector containing the coding gene for the recombinant protein BMP-15 of the Japanese eel and a backbone plasmid, wherein the backbone plasmid is obtained by modifying pET-32a(+).

[0069] This invention provides a recombinant engineered bacterium containing the expression vector.

[0070] Furthermore, the host bacteria of the recombinant engineered bacteria are selected from Rosetta-gami B(DE3).

[0071] like Figure 1 As shown in the embodiment of the present invention, a method for preparing recombinant BMP-15 protein from Japanese eel includes the following steps:

[0072] (1) Construct the gene encoding the recombinant protein BMP-15 of the Japanese eel, and connect it to the backbone plasmid to construct the expression vector of the recombinant protein BMP-15 of the Japanese eel;

[0073] (2) The expression vector was transformed into a host bacterium to induce the expression of the Japanese eel BMP-15 recombinant protein;

[0074] (3) By refolding inclusion bodies, misfolded insoluble inclusion bodies are transformed into structurally correct soluble proteins;

[0075] (4) Based on the His tag in the Japanese eel BMP-15 recombinant protein, a higher purity Japanese eel BMP-15 recombinant protein was obtained by affinity chromatography (the purity of the protein after refolding can reach 80% to 90%. If the purity requirement is not high, this step can be skipped and proceed directly to the next step).

[0076] (5) Dialyze the protein into 1×PBS, add bovine serum albumin (BSA) to a final concentration of 0.1%, freeze in liquid nitrogen, and store at -80°C (if a higher concentration of protein is required after dialysis into 1×PBS, an ultrafiltration tube can be used to enrich and concentrate the protein).

[0077] Example 1: Construction of the Japanese eel BMP-15 recombinant protein expression vector pET-His-SUMO-BMP15

[0078] Using Japanese eel ovarian cDNA as a template, PCR amplification was performed to obtain the DNA sequence of the open reading frame (ORF) of the Japanese eel BMP-15 gene (bmp15). Figure 2The DNA was ligated into the pCE2 TA / Blunt Zero vector (Norvoza, Nanjing) via TOPO ligation and transfected into *E. coli* (Norvoza, Nanjing). Single colonies were screened on ampicillin solid medium, and the sequences were confirmed to be correct using Sanger sequencing and NCBIBLAST. PCR amplification was performed using the above-mentioned *E. japonica* bmp15 monoclonal bacterial culture as a template to obtain the double-stranded DNA fragment corresponding to the mature peptide of *E. japonica* BMP-15 with homologous arms. The modified backbone plasmid pET-HS (pET-32a(+) plasmid containing a His tag and SUMO lysing peptide) was linearized by double restriction endonuclease digestion. The PCR products and double-enzyme digestion products were purified using the FastPure Gel DNA Extraction Mini Kit (Novizan, Nanjing). The insert fragment and linear vector were ligated using a homologous recombination kit (Novizan, Nanjing), and transfected into DH5α competent *E. coli*. Single colonies were screened on ampicillin solid medium, and after confirmation by Sanger sequencing, the plasmid was extracted using a plasmid mini-extraction kit (Tiangen, Beijing), which yielded the *Eel japonicus* BMP-15 prokaryotic expression vector pET-His-SUMO-BMP15. Figure 3 ).

[0079] Example 2: Preparation and Induction of Japanese Eel BMP-15 Recombinant Protein Escherichia coli Expression Strain. The expression vector pET-His-SUMO-BMP15 was transfected into Rosetta B(DE3) competent Escherichia coli (Angyu, Shanghai). Single colonies were screened by overnight culture in ampicillin-chloramphenicol double antibiotic LB solid medium at 37°C, which is the Japanese eel BMP-15 recombinant protein prokaryotic expression engineered strain pET-His-SUMO-BMP15-Rosetta-gami B(DE3). Single colonies were picked and cultured overnight at 37°C and 220 rpm in 10 mL of ampicillin-chloramphenicol LB broth. Then, they were inoculated at a 1:100 ratio into 200 mL of LB broth containing ampicillin-chloramphenicol and 0.2% glucose, and cultured at 37°C and 220 rpm for 6-7 h. IPTG was then added to a final concentration of 0.6 mM (10 mL of the bacterial culture was used as a control before induction). After culturing at 37°C and 220 rpm for 4 h, the cells were collected by centrifugation at 3500 g for 10 min. The supernatant was discarded, and the cells were resuspended in 20 mL of lysis buffer (500 mM NaCl, 20 mM PB, 10 mM imidazole, pH 7.4). The cells were sonicated at 60 W for 30 min, with a 5-second break after 5 seconds of sonication. The cells were then centrifuged at 12000 g for 10 min, and the supernatant and precipitate were collected separately. The precipitate was resuspended in 20 mL of lysis buffer, and 80 μL and 20 μL of the sample were taken respectively. Mix 5× protein loading buffer, incubate at 95℃ for 10 min in a PCR instrument, then perform SDS-PAGE electrophoresis and Coomassie brilliant blue staining to observe protein bands. Figure 4 (Samples 1-4). The results showed that the recombinant BMP-15 protein of Japanese eel could be expressed in large quantities and existed in the precipitate as misfolded inclusion bodies, and was an insoluble protein.

[0080] Example 3: Washing, refolding, protein purification, and enrichment / concentration of recombinant BMP-15 protein inclusion bodies from Japanese eel

[0081] The precipitate obtained in Example 2 (the recombinant BMP-15 protein of the Japanese eel exists in the precipitate as insoluble inclusion bodies due to structural errors caused by misfolding) was washed with 20 mL of inclusion body washing buffer ① (NaCl 500 mM, Tris-HCl 20 mM, Triton X-100 2% (V / V), pH=7.4). After shaking and resuspending, the precipitate was sonicated at 60 W for 10 min, with a 5 s break for 5 s, and centrifuged at 12000 g for 10 min. The supernatant was discarded and the precipitate was retained. The precipitate was then washed with 20 mL of inclusion body washing buffer ② (NaCl 500 mM, Tris-HCl 20 mM, urea 2 M, pH=7.4). After shaking and resuspending, the precipitate was sonicated at 60 W for 10 min, with a 5 s break for 5 s, and centrifuged at 12000 g for 10 min. The supernatant was discarded and the precipitate was retained. After two washings, some hydrophobic lipoproteins, bacterial fragments, genomic DNA, etc., can be removed. At this time, the purity of the inclusion bodies can reach 80% to 90%.

[0082] Add 10 mL of inclusion body dissolution solution (NH4Cl 100 mM, Tris-HCl 50 mM, urea 8 M, glycerol 10% (V / V), pH = 8.0) to the washed inclusion bodies, and add DTT solution to make the final concentration 10 nM. After shaking and resuspending, sonicate at 60 W for 10 min, break up for 5 s, stop for 5 s, centrifuge at 12000 g for 15 min, and retain the supernatant, which is the denatured and dissolved Japanese eel BMP-15 recombinant protein. Pour 20-50 mL of diluted refolding buffer (NaCl 50 mM, Tris-HCl 50 mM, L-arginine 0.4 M, urea 1 M, reduced glutathione 5 mM, oxidized glutathione 1 mM, 10% glycerol (V / V), pH 8.0) into a glass bottle. Add a magnetic stir bar and add dissolved inclusion bodies dropwise until the final concentration is approximately 0.05 mg / mL (during this process, continuous stirring with a magnetic stir bar is required to quickly disperse the protein and prevent aggregation). Incubate the glass bottle at 4°C for 4 hours to allow the denatured protein to fully fold. Continue adding dissolved inclusion bodies until the final concentration is approximately 0.1 mg / mL… repeat this process, adding a total of 4 times, increasing the concentration by 0.05 mg / mL each time. After the final refolding, incubate at 4°C overnight to allow the protein to fully fold, thus obtaining the structurally correct soluble recombinant Japanese eel BMP-15 protein.

[0083] To obtain higher purity Japanese eel BMP-15 recombinant protein, insoluble microparticles can be removed by filtration using a 0.45μm or 0.22μm filter membrane, followed by His-tag affinity purification using NTA-Ni packing material (Beyotime, Shanghai). This will yield Japanese eel BMP-15 recombinant protein with a purity >90%. The obtained recombinant protein solution is then poured into a protein dialysis bag (molecular weight cutoff: 3500D) and dialyzed into 1×PBS solution. A small amount of protein is taken and its concentration is determined using the BCA method. The remaining protein solution is then treated with 0.1% bovine serum albumin (BSA) to stabilize the recombinant protein and prevent aggregation. After aliquoting, the solution is flash-frozen in liquid nitrogen and stored at -80℃. For higher concentrations of Japanese eel BMP-15 recombinant protein, the protein can be enriched and concentrated using ultrafiltration after dialysis. Figure 4 Medium sample 5).

[0084] Example 4: Regulation of cox gene expression in follicular cells cultured in vitro by recombinant BMP-15 protein from Japanese eel

[0085] Follicular layer cells of Japanese eels before vitelline development were isolated and cultured for 48 hours. The treatment group (BMP-15) was stimulated with recombinant BMP-15 protein obtained using the method described in this invention, while the control group (Control) received an equal volume of 1×PBS buffer. RNA was extracted and reverse transcribed after 6 hours. The relative expression levels of the Cox gene in the two groups were detected using qPCR. It was found that the expression level of the Cox gene in the treatment group was significantly higher than that in the control group (…). Figure 5 This indicates that the recombinant BMP-15 protein from Japanese eel obtained by the method described in this invention has biological activity and can regulate the expression of downstream genes in the follicular layer cells of Japanese eel.

[0086] Example 5: Study on ovarian development in fish using recombinant BMP-15 protein from Japanese eel

[0087] In this embodiment, the recombinant BMP-15 protein from the Japanese eel can be applied to research related to fish ovarian development. Researchers can add this recombinant protein to Japanese eel secondary ovarian cell cultures to evaluate the effects of BMP-15 on follicle growth, oocyte maturation, and related hormone secretion in Japanese eels. This research contributes to understanding the reproductive biology of Japanese eels and other related fish, providing important biological information for fish ovarian development and reproduction.

[0088] The specific operating steps include:

[0089] 1. Isolation and culture of secondary ovarian cells from Japanese eel.

[0090] 2. Add different concentrations of Japanese eel BMP-15 recombinant protein to the cell culture medium.

[0091] 3. Observe and record the growth, differentiation and hormone secretion of cells in the BMP-15 treatment group and the control group.

[0092] 4. Perform statistical analysis on the collected data to assess the impact of BMP-15 on ovarian development.

[0093] Example 6: Development of a fish reproductive health detection kit based on recombinant BMP-15 protein from Japanese eel

[0094] In this embodiment, recombinant BMP-15 protein from the Japanese eel was used to develop a fish reproductive health testing kit. This kit can detect the level of BMP-15 in fish, helping fish farmers assess the reproductive health of their fish and adjust their aquaculture management strategies accordingly.

[0095] The specific operating steps include:

[0096] 1. Prepare a specific antibody for detecting BMP-15.

[0097] 2. Develop detection methods based on ELISA or other appropriate technologies for use in the construction of reagent kits.

[0098] 3. Verify the sensitivity, specificity, and stability of the kit.

[0099] 4. Apply the kit to actual fish reproductive health testing to provide fish farmers or researchers with rapid and accurate test results.

[0100] Example 7: Expression and purification of recombinant BMP-15 protein from Japanese eel

[0101] Total RNA was extracted from the ovarian tissue of Japanese eel, and the cDNA sequence of the BMP-15 gene was obtained by reverse transcription. The BMP-15 gene was amplified by PCR using specific primers, and restriction endonuclease sites were added to both ends of the amplification product. The amplified BMP-15 gene was inserted into a backbone plasmid modified from pET-32a(+) to construct an expression vector containing the T7 promoter. The ligation product was transformed into DH5α competent cells, positive clones were selected, and the correctness of the construction was verified by restriction enzyme digestion and sequencing.

[0102] The validated expression vector was transformed into Rosetta-gami B host bacteria using a heat shock transformation method. After transformation, the cells were plated on LB agar plates containing ampicillin and chloramphenicol and incubated overnight at 37°C. Resistant positive clones were screened. Single colonies were selected, and successfully transformed recombinant strains were identified by PCR.

[0103] The selected positive recombinant bacteria were inoculated into LB liquid medium containing antibiotics and cultured at 37°C with shaking until the OD600 value reached 0.6. IPTG was added to a final concentration of 0.5 mM for induction, and the culture was continued at 16°C for 12 hours. After induction, the bacterial cells were collected, and the expression of the recombinant protein was analyzed by SDS-PAGE.

[0104] The induced bacterial cells were sonicated to obtain soluble protein solution and inclusion body protein. The His-tagged soluble recombinant protein was purified using a Ni-NTA affinity column, and the inclusion body protein was processed using a denaturation-renaturation method to obtain the soluble protein. Impurities were removed by dialysis and the buffer was replaced to finally obtain the purified BMP-15 recombinant protein.

[0105] The purified protein sample was analyzed using SDS-PAGE to confirm that its molecular weight was consistent with the theoretical value. Further validation using Western blot with a BMP-15 specific antibody confirmed the correctness and purity of the target protein. In addition, the biological activity of the recombinant protein was tested using ELISA.

[0106] The purified recombinant BMP-15 protein was applied to in vitro ovarian cell culture experiments to examine its effect on oocyte development. The results showed that BMP-15 significantly promoted oocyte maturation and follicle growth. This recombinant protein has potential application value in aquaculture and provides an important tool for studying the reproductive physiology of the Japanese eel.

[0107] The strain is publicly available and commercially available; it can be purchased from reagent companies.

[0108] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A recombinant BMP-15 protein from the Japanese eel, characterized in that, It contains: the mature peptide amino acid sequence of Japanese eel BMP-15, an N-terminal 6×histidine tag, and a SUMO-promoting polypeptide; the amino acid sequence of the recombinant Japanese eel BMP-15 protein is shown in SEQ ID NO:

1.

2. A gene encoding the recombinant BMP-15 protein of the Japanese eel as described in claim 1, characterized in that, It contains: an N-terminal His tag, a linker peptide between the His tag and the SUMO-promoting peptide, the SUMO-promoting peptide, and a mature BMP-15 peptide from the Japanese eel; the DNA sequence of the encoding gene is SEQ ID NO:

3.

3. An expression carrier, characterized in that, The expression vector contains the encoding gene of the Japanese eel BMP-15 recombinant protein as described in claim 2 and a backbone plasmid, wherein the backbone plasmid is obtained by modifying pET-32a(+).

4. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria comprises the expression vector described in claim 3.

5. The recombinant engineered bacteria as described in claim 4, characterized in that, The host bacteria of the recombinant engineered bacteria were selected from Rosetta-gami B.

6. A method for preparing the recombinant BMP-15 protein from the Japanese eel as described in claim 1, characterized in that, Includes the following steps: (1) Construct the gene encoding the recombinant protein BMP-15 of the Japanese eel, and connect it to the backbone plasmid to construct the expression vector of the recombinant protein BMP-15 of the Japanese eel; (2) The expression vector was transformed into a host bacterium to induce the expression of the Japanese eel BMP-15 recombinant protein; (3) By refolding inclusion bodies, misfolded insoluble inclusion bodies are transformed into soluble proteins with the correct structure; (4) Based on His tag affinity chromatography of recombinant protein BMP-15 from Japanese eel, a higher purity GDF-9 recombinant protein was obtained; (5) Dialyze the protein into 1×PBS, add bovine serum albumin (BSA) to a final concentration of 0.1%, freeze in liquid nitrogen, and store at -80°C.

7. The method for preparing recombinant BMP-15 protein from Japanese eel as described in claim 6, characterized in that, After dialysis to 1×PBS, the protein can be enriched and concentrated using an ultrafiltration tube to obtain a higher concentration of protein.

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