Anguilla japonica BMP-15 recombinant protein as well as expression method and application thereof

Abstract

The invention belongs to but is not limited to the technical field of gene engineering, and discloses an anguilla japonica BMP-15 recombinant protein and an expression method and application thereof, the anguilla japonica BMP-15 recombinant protein comprises an anguilla japonica BMP-15 mature peptide amino acid sequence, a 6 * histidine tag at the N end and SUMO dissolution promoting polypeptide; the amino acid sequence of the Japanese eel BMP-15 recombinant protein is SEQ ID NO: 1, and the DNA sequence of the coding gene of the Japanese eel BMP-15 recombinant protein is SEQ ID NO: 3. The prokaryotic expression vector pET-His-SUMO-BMP15 of the Japanese eel BMP-15 recombinant protein is constructed for the first time, the Japanese eel BMP-15 recombinant protein of which the amino acid sequence is consistent with the mature peptide of the Japanese eel BMP-15 can be obtained, and the recombinant protein can be applied to ovarian development regulation and artificial propagation research of the Japanese eel.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of genetic engineering technology, and in particular relates to a Japanese eel BMP-15 recombinant protein and an expression method and application thereof. Background Art

[0002] Japanese eel (Anguilla japonica) is one of the important migratory fish in Asia and an important economic fish in my country. As a migratory fish that spawns in the sea, Japanese eel has a complex life history. The complexity of the life history leads to many unknowns, including growth and reproduction. Therefore, its artificial breeding technology is a global problem that has not yet been solved. Since the 1930s, the artificial breeding technology of eels has made great progress, but there is still a big gap from the demand for industrial large-scale breeding and production of eel fry. The main reason is that the maturity rate, spawning rate and egg production of artificially ripened broodstock are low, especially the quality of the eggs produced is not ideal, resulting in low fertilization rate, hatching rate and survival rate. Although the hatched eel fry can eventually grow into white eel fry after metamorphosis, the growth rate is very slow, the survival rate is extremely low, and the deformity rate is high. As the largest country in eel breeding, processing and export, my country has a huge demand for eel fry, so it is urgent to realize artificial breeding of eels. The key to solving these problems lies in regulating the ovarian development process of Japanese eels to improve the quality of eggs in artificially bred eels.

[0003] Bone morphogenetic protein 15 (BMP15) is an ovarian regulatory factor specifically expressed by oocytes. It plays an important role in physiological mechanisms such as yolk accumulation and preventing oocyte precocity. It is a key role in the oocyte sending signals to surrounding follicular cells (granulosa cells and theca cells) to coordinate folliculogenesis. BMP-15 can upregulate the expression levels of genes such as activin A (inhba) and ovarian aromatase (cyp19a1a) through the smad cell signaling pathway, and plays an important regulatory role in the development of the ovary. Like other vertebrates, Japanese eel BMP-15 is a classic secretory protein with a typical N-terminal signal peptide sequence. In cells, once the signal peptide of the nascent peptide chain is synthesized, it will be recognized by the signal recognition particle (SRP) and the translation will be suspended. The SRP, peptide chain and ribosome will be docked on the surface of the rough endoplasmic reticulum together, and translation can continue. The newly synthesized peptide chain enters the endoplasmic reticulum while the signal peptide is removed. Finally, the peptide chain is folded and processed by the endoplasmic reticulum and Golgi apparatus, and is secreted outside the cell, where it forms dimers through covalent bonds to exert its function. In addition, the Japanese eel BMP-15 monomer has three disulfide bonds, which are important structures for maintaining the correct molecular conformation and biological activity.

[0004] In the field of genetic engineering, Escherichia coli is a commonly used engineering bacterium in prokaryotic expression systems. It is a facultative anaerobic Gram-negative bacterium with a short growth cycle, clear genetic background, simple culture operation, low cost, and can quickly and large-scale produce target proteins. By using a strong promoter, foreign proteins can be expressed at high levels in Escherichia coli. However, the rate at which Escherichia coli expresses foreign proteins is much higher than that of eukaryotic organisms, which leads to frequent misfolding of nascent peptide chains, and the hydrophobic parts of the misfolded proteins are exposed and aggregated to form inactive inclusion bodies.

[0005] Inclusion bodies are a common problem in prokaryotic expression. Due to the misfolding of the peptide chain, the protein structure becomes abnormal and loses its biological activity. To address this problem, the misfolded and insoluble inclusion bodies can be converted into soluble proteins with the correct molecular structure through inclusion body renaturation, thereby restoring their biological activity and obtaining Japanese eel BMP-15 recombinant protein with biological activity. In addition, the inclusion body renaturation method is more efficient in obtaining recombinant proteins, and the purity can reach 80% to 90% without purification, which can save time and cost in production and has important practical significance.

[0006] Through the above analysis, the problems and defects of the prior art are as follows:

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

[0008] (2) There have been reports on the expression of BMP-15 recombinant proteins in other organisms, but most of these reports used eukaryotic expression, which has low efficiency and high cost in obtaining BMP-15 recombinant proteins;

[0009] (3) There are relatively few reports on the prokaryotic expression of BMP-15 recombinant proteins from other organisms. In addition, the inclusion body renaturation effect of BMP-15 recombinant proteins reported in these reports is poor and it is easy to precipitate during dialysis, making it impossible to obtain high-concentration BMP-15 recombinant proteins.

[0010] (4) The recombinant protein expression rate of E. coli is too fast, and there is no endoplasmic reticulum, Golgi apparatus and other organelles for processing and modification, resulting in the failure of the newly formed peptide chains to fold correctly in time, and the hydrophobic parts of the misfolded proteins are exposed and aggregated to form inactive inclusion bodies; Summary of the invention

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

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

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

[0014] Furthermore, a His tag and a connecting peptide were added to the N-terminus of the recombinant protein, and the amino acid sequence was HHHHHHSSGLVPRGSHMAS; the 6×His tag (HHHHHH) was used for nickel column affinity chromatography of the protein, thereby purifying the Japanese eel BMP-15 recombinant protein and further improving the purity of the recombinant protein; 13 amino acids SSGLVPRGSHMAS were added between the His tag and the SUMO soluble polypeptide as a connecting peptide to prevent the His tag from being shielded by the SUMO soluble polypeptide and the Japanese eel BMP-15 mature peptide and to enable it to be fully exposed to the solution, so as to facilitate affinity chromatography purification of the recombinant protein after renaturation.

[0015] Furthermore, a SUMO solubilizing peptide was added to the N-terminus with the sequence of MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG, which was used to inhibit protein aggregation during inclusion body renaturation, further promote the formation of soluble protein during the renaturation process, and improve the efficiency of inclusion body renaturation, which is crucial to improving the efficiency of inclusion body renaturation and the concentration of recombinant protein.

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

[0017] Another object of the present invention is to provide a gene encoding a Japanese eel BMP-15 recombinant protein, comprising: encoding an N-terminal His tag, a connecting peptide between the His tag and a SUMO solubilizing polypeptide, a SUMO solubilizing polypeptide, and a Japanese eel BMP-15 mature peptide; 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, which comprises a gene encoding a Japanese eel BMP-15 recombinant protein and a backbone plasmid, wherein the backbone plasmid is transformed from pET-32a(+).

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

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

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

[0022] (1) constructing the Japanese eel BMP-15 recombinant protein encoding gene, and connecting it to a backbone plasmid to construct an expression vector for the Japanese eel BMP-15 recombinant protein;

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

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

[0025] (4) Obtaining a Japanese eel BMP-15 recombinant protein with higher purity by affinity chromatography based on the His tag in the Japanese eel BMP-15 recombinant protein (the purity of the protein after renaturation can reach 80% to 90%. If the purity requirement is not high, this step can be skipped and the next step can be directly performed);

[0026] (5) The protein was dialyzed into 1× PBS, and bovine serum albumin (BSA) was added to a final concentration of 0.1%. After quick freezing in liquid nitrogen, it was stored at -80°C (after dialyzing into 1× PBS, if a higher concentration of protein is required, 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 Japanese eel BMP-15 recombinant protein in regulating the ovarian development of Japanese eels.

[0028] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0029] First, the difficulty of solving the problems and defects of the existing technology is:

[0030] (1) constructing a Japanese eel BMP-15 recombinant protein, expressing it in large quantities through an Escherichia coli prokaryotic expression system, and obtaining a Japanese eel BMP-15 recombinant protein with biological activity;

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

[0032] The significance of solving the problems and defects of the prior art is to obtain a Japanese eel BMP-15 recombinant protein with correct structure and biological activity, which can be applied to basic research and production processes, and provide a theoretical basis and technical support for the ovarian development and artificial reproduction of Japanese eels. The recombinant protein of the present invention can be induced to express in large quantities by prokaryotic bacteria, and a recombinant protein with biological activity can be efficiently obtained by inclusion body renaturation, which can save time and cost, can be effectively applied to industrial production, and has important commercial value.

[0033] (1) The present invention provides a Japanese eel BMP-15 recombinant protein, comprising: a Japanese eel BMP-15 mature peptide amino acid sequence, an N-terminal 6×histidine tag (6×His tag or simply His tag), a SUMO solubilizing polypeptide, and a connecting peptide between the 6×histidine tag and the SUMO solubilizing polypeptide. Among them, the His tag can be used to purify the Japanese eel BMP-15 recombinant protein, which can improve the purity of the recombinant protein and meet higher application requirements; the SUMO solubilizing polypeptide can be used to inhibit the aggregation of the Japanese eel BMP-15 recombinant protein during the inclusion body renaturation process, further promote the formation of soluble protein during the renaturation process, and improve the efficiency of the inclusion body renaturation and the concentration of the obtained Japanese eel BMP-15 recombinant protein;

[0034] (2) The present invention constructs a prokaryotic expression vector pET-His-SUMO-BMP15 of a Japanese eel BMP-15 recombinant protein, and transfects the vector into Rosetta-gami B (DE3) Escherichia coli to obtain a pET-His-SUMO-BMP15-Rosetta-gami B (DE3) expression strain, which can induce the expression of a large amount of Japanese eel BMP-15 recombinant protein, and efficiently obtain soluble recombinant protein by inclusion body renaturation, and has biological activity without the need to remove the tag;

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

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

[0037] (1) The present invention provides for the first time a Japanese eel BMP-15 recombinant protein having the same amino acid sequence as the Japanese eel BMP-15 mature peptide, comprising: the Japanese eel BMP-15 mature peptide amino acid sequence, an N-terminal 6×histidine tag (6×His tag or simply His tag), a SUMO soluble polypeptide, and a connecting peptide between the 6×histidine tag and the SUMO soluble polypeptide;

[0038] (2) The present invention is the first to construct a prokaryotic expression vector pET-His-SUMO-BMP15 of a Japanese eel BMP-15 recombinant protein, and transfect 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 a large amount of Japanese eel BMP15 recombinant protein, and a recombinant protein with a purity of 80% to 90% can be obtained through inclusion body renaturation (if a Japanese eel BMP-15 recombinant protein with a higher purity is required, His tag affinity chromatography purification can be performed to obtain a recombinant protein with a purity higher than 90%).

[0039] (3) Adding SUMO solubilizing polypeptide to the prokaryotic expression vector pET-His-SUMO-BMP15 of the Japanese eel BMP-15 recombinant protein constructed by the present invention can inhibit the aggregation of the Japanese eel BMP-15 recombinant protein during the inclusion body renaturation process, further promote the formation of soluble protein during the renaturation process, and improve the efficiency of inclusion body renaturation and the concentration of the obtained Japanese eel BMP-15 recombinant protein;

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

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

[0042] Third, as auxiliary evidence of the inventiveness of the claims of the present 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 a Japanese eel BMP-15 recombinant protein whose amino acid sequence is consistent with the Japanese eel BMP-15 mature peptide. The method can be used to obtain a large amount of recombinant protein with biological activity in an efficient manner, 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 is no relevant report on the recombinant protein of Japanese eel BMP-15 in related research at home and abroad. The present invention is the first to construct the prokaryotic expression vector pET-His-SUMO-BMP15 of the recombinant protein of Japanese eel BMP-15, and transfect the vector into Rosetta-gami B (DE3) Escherichia coli. The obtained 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 the present invention solves a technical problem that people have been eager to solve but have never been successful: the artificial breeding technology of Japanese eels is a global problem that has not yet been solved. The low quality of eggs obtained by artificial induction of ovulation is one of the main difficulties currently faced. Therefore, it is urgent to carry out research on improving the quality of Japanese eel eggs. BMP-15 has an important function in the development of fish ovaries and is a key factor in inducing the transition of secondary follicles to vitellogenic follicles. Therefore, exploring the role of BMP-15 in the development of Japanese eels' ovaries can provide a theoretical basis and technical support for the realization of artificial breeding of eels. The Japanese eel BMP-15 recombinant protein obtained by the method described in the present invention can be effectively applied to the research on the regulation of ovarian development and artificial breeding of Japanese eels, which has important scientific research significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a flow chart of a method for preparing Japanese eel BMP-15 recombinant protein provided by an embodiment of the present invention;

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

[0048] Figure 2 A is a schematic diagram of the composition of Japanese eel BMP-15 provided in an embodiment of the present invention, which includes a signal peptide of 28 amino acids, a precursor peptide of 278 amino acids, and a mature peptide of 126 amino acids; 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 examples of the present invention, and the dark grey area is a schematic diagram of the amino acid sequence of the mature peptide;

[0050] Figure 3 It is a diagram of the Japanese eel BMP-15 recombinant protein expression vector, the recombinant protein composition and the recombinant protein ORF sequence provided by the embodiment of the present invention;

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

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

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

[0054] Figure 4 This is an SDS-PAGE gel electrophoresis diagram of the expression, inclusion body renaturation and concentration enrichment of the Japanese eel BMP-15 recombinant protein provided by the embodiment of the present 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 graph showing the qPCR results of regulating the expression of cox gene in in vitro cultured Japanese eel follicular layer cells by the Japanese eel BMP-15 recombinant protein provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0057] An embodiment of the present invention provides a Japanese eel BMP-15 recombinant protein, comprising: a Japanese eel BMP-15 mature peptide amino acid sequence, an N-terminal 6×histidine tag (6×His tag or His tag for short) and a SUMO solubilizing polypeptide; the amino acid sequence of the Japanese eel BMP-15 recombinant protein is shown in SEQ ID NO: 1; the Japanese eel BMP-15 mature peptide amino acid sequence 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 connecting peptide were added to the N-terminus of the recombinant protein, and the amino acid sequence was HHHHHHSSGLVPRGSHMAS (SEQ ID NO: 4); the 6×His tag (HHHHHH) was used for protein nickel column affinity chromatography to purify the Japanese eel BMP-15 recombinant protein, thereby further improving the purity of the recombinant protein; 13 amino acids SSGLVPRGSHMAS (SEQ ID NO: 5) were added between the His tag and the SUMO solubilizing polypeptide as a connecting peptide to prevent the His tag from being shielded by the SUMO solubilizing polypeptide and the Japanese eel BMP-15 mature peptide and to enable it to be fully exposed to the solution, so as to facilitate affinity chromatography purification of the recombinant protein after renaturation.

[0063] A SUMO solubilizing peptide was added to the N-terminus, and the sequence was MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO: 6), which was used to inhibit protein aggregation during inclusion body renaturation, further promote the formation of soluble protein during renaturation, and improve the efficiency of inclusion body renaturation.

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

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

[0066] SEQ ID NO:3:

[0067] ATGGGCAGCAGCCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAA AAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGAACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGTCGCCCGCGCAGGTCG AAAGAACCGGGTAGCATTGGTGCAGACATCCCCAACTACGTACGCCAAAACAGTGTGGCTAAGAACCAGTGCAAGCTTCACTCCTACAGAGTGACCTTCCAGGACTTGGGTTGGGATCACTGGATCATCGCCCCACATAAGTACAACCCTCGGTACTGCAGAGGAGACTGCCCACGTATCCTCCACTAT GGGTACAATTCTCCAAACCATGCCATCGTGCAGAACTTCATCAATGAGATGGGTGTGGGGGAGGTGCCCCCGCCCGCTTGTGTACCTTACAAGTACAAGCCAATCAGCGTCCTCATGTTGGAGAAGAATGGCAGCATTGTATACAAAGAGTATGAGGACATGATCGCAGAGTCGTGCACCTGCCGCTAA

[0068] The embodiment of the present invention provides an expression vector, which comprises a coding gene of a Japanese eel BMP-15 recombinant protein and a backbone plasmid, wherein the backbone plasmid is transformed from pET-32a(+).

[0069] An embodiment of the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the expression vector.

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

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

[0072] (1) constructing the Japanese eel BMP-15 recombinant protein encoding gene, and connecting it to a backbone plasmid to construct an expression vector for the Japanese eel BMP-15 recombinant protein;

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

[0074] (3) Converting misfolded insoluble inclusion bodies into structurally correct soluble proteins through inclusion body renaturation;

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

[0076] (5) The protein was dialyzed into 1× PBS, and bovine serum albumin (BSA) was added to a final concentration of 0.1%. After quick freezing in liquid nitrogen, it was stored at -80°C (after dialyzing into 1× PBS, if a higher concentration of protein is required, an ultrafiltration tube can be used to enrich and concentrate the protein).

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

[0078] Using Japanese eel ovary 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 2). It was connected to the pCE2 TA / Blunt Zero vector (Novagen, Nanjing) by TOPO ligation and transfected into Escherichia coli (Novagen, Nanjing). Single colonies were screened on ampicillin solid medium, and the sequence was verified by Sanger sequencing and NCBIBLAST. PCR amplification was performed using the above Japanese eel bmp15 monoclonal bacterial solution as a template to obtain a double-stranded DNA fragment corresponding to the mature peptide of Japanese eel BMP-15 with homology arms. The modified backbone plasmid pET-HS (His tag and SUMO soluble peptide were embedded in the pET-32a(+) plasmid) was double-digested and linearized using restriction endonucleases. The PCR products and double-enzyme digestion products were purified using FastPure Gel DNA Extraction Mini Kit (Novuzan, Nanjing), and the insert fragments and linear vectors were connected using a homologous recombination kit (Novuzan, Nanjing). The fragments were transfected into DH5α competent Escherichia coli, and single colonies were screened by ampicillin solid medium. After confirmation by Sanger sequencing, the plasmid was extracted using a plasmid mini-extraction kit (Tiangen, Beijing), which was the Japanese eel BMP-15 prokaryotic expression vector pET-His-SUMO-BMP15 ( Figure 3 ).

[0079] Example 2: Preparation of Escherichia coli expression strain for Japanese eel BMP-15 recombinant protein and induced expression The expression vector pET-His-SUMO-BMP15 was transfected into Rosetta B (DE3) competent Escherichia coli (Angyu, Shanghai), and cultured overnight at 37°C in ampicillin-chloramphenicol double-resistance LB solid medium to screen single colonies, which was the Japanese eel BMP-15 recombinant protein prokaryotic expression engineering bacteria pET-His-SUMO-BMP15-Rosetta-gami B (DE3). A single colony was picked and cultured in 10 mL of ampicillin-chloramphenicol dual-resistance LB liquid medium at 37°C, 220 rpm / min overnight, and then inoculated into 200 mL of LB liquid medium containing ampicillin-chloramphenicol dual-resistance and 0.2% glucose at a ratio of 1:100, and cultured at 37°C, 220 rpm / min for 6-7 h, and then IPTG was added to make its final concentration 0.6 mM (10 mL of bacterial solution was taken out as a control before induction), and after culturing at 37°C, 220 rpm / min for 4 h, the bacteria were collected by centrifugation at 3500g for 10 min, the supernatant was discarded, and the bacteria were resuspended with 20 mL of bacterial lysis buffer (500 mM NaCl, 20 mM PB, 10 mM imidazole, pH 7.4), 60 W ultrasonic disruption for 30 min, disruption for 5 s and stop for 5 s, 12000 g centrifugation for 10 min, the supernatant and precipitate were collected respectively, and the precipitate was resuspended with 20 mL of bacterial lysis buffer, and 80 μL of sample and 20 μL were taken respectively. 5× protein loading buffer was mixed, incubated at 95°C for 10 min, and protein bands were observed by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining ( Figure 4 The results showed that the Japanese eel BMP-15 recombinant protein could be expressed in large quantities and existed in the precipitate in the form of misfolded inclusion bodies, which was an insoluble protein.

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

[0081] The precipitate obtained in Example 2 was washed with 20 mL of inclusion body washing solution ① (NaCl 500 mM, Tris-HCl 20 mM, Triton X-100 2% (V / V), pH=7.4) (the Japanese eel BMP-15 recombinant protein was present in the precipitate in the form of insoluble inclusion bodies due to structural errors caused by misfolding), and then the mixture was resuspended by shaking and ultrasonically disrupted at 60 W for 10 min, and then the mixture was broken for 5 s and then stopped for 5 s. After centrifugation at 12000 g for 10 min, the supernatant was discarded and the precipitate was retained; the precipitate was washed with 20 mL of inclusion body washing solution ② (NaCl 500 mM, Tris-HCl 20 mM, urea 2 M, pH=7.4), and then the mixture was resuspended by shaking and ultrasonically disrupted at 60 W for 10 min, and then the mixture was broken for 5 s and then stopped for 5 s. After centrifugation at 12000 g for 10 min, the supernatant was discarded and the precipitate was retained. After two washes, some hydrophobic lipoproteins, bacterial fragments, genomic DNA, etc. can be removed, and the purity of the inclusion bodies can reach 80% to 90%.

[0082] To the washed inclusion bodies, 10 mL of inclusion body dissolution solution (100 mM NH4Cl, 50 mM Tris-HCl, 8 M urea, 10% glycerol (V / V), pH = 8.0) was added, and DTT solution was added to make the final concentration 10 nM. After oscillation and resuspending, ultrasonic disruption was performed at 60 W for 10 min, and the disruption was stopped for 5 s for 5 s. After centrifugation at 12000 g for 15 min, the supernatant was retained, which was the denatured and dissolved Japanese eel BMP-15 recombinant protein. Take 20-50mL of dilution refolding solution (NaCl 50mM, Tris-HCl 50mM, L-arginine 0.4M, urea 1M, reduced glutathione 5mM, oxidized glutathione 1mM, 10% glycerol (V / V), pH 8.0) and pour it into a glass bottle, add a magnetic rotor, and add the dissolved inclusion bodies dropwise to a final concentration of about 0.05mg / mL (a magnetic stirrer should be used to stir continuously during this process to quickly disperse the protein and prevent coagulation). The glass bottle is placed in a 4°C refrigerator for 4h to allow the denatured protein to be completely folded, and the dissolved inclusion bodies are continued to be added to a final concentration of about 0.1mg / mL..., and so on, a total of 4 times, each time the concentration is increased by 0.05mg / mL, and the last refolding is placed at 4°C overnight to allow the protein to be fully folded, and the structurally correct soluble Japanese eel BMP-15 recombinant protein can be obtained.

[0083] If you need to obtain a higher purity of Japanese eel BMP-15 recombinant protein, you can use a 0.45μm or 0.22μm filter membrane to remove insoluble microparticles, and then use NTA-Ni filler (Biyuntian, Shanghai) for His tag affinity purification to obtain Japanese eel BMP-15 recombinant protein with a purity of >90%. Pour the obtained recombinant protein solution into a protein dialysis bag (molecular weight cutoff: 3500D), dialyze into 1×PBS solution, take a small amount of protein and determine the protein concentration by BCA method, add bovine serum albumin (BSA) with a final concentration of 0.1% to the remaining protein solution to stabilize the recombinant protein and prevent precipitation. After aliquoting, use liquid nitrogen to freeze and store at -80°C. If you need to obtain a high concentration of Japanese eel BMP-15 recombinant protein, you can use an ultrafiltration tube to enrich and concentrate the recombinant protein after dialysis ( Figure 4 Sample 5).

[0084] Example 4: Japanese eel BMP-15 recombinant protein regulates the expression of cox gene in in vitro cultured follicular layer cells

[0085] Japanese eel follicular layer cells before the yolk formation period were isolated and cultured for 48 hours. The treatment group (BMP-15) used the Japanese eel BMP-15 recombinant protein obtained by the method described in the invention to stimulate the follicular layer cells, and the control group (Control) added an equal amount of 1×PBS buffer. RNA was extracted and reverse transcribed after 6 hours. The relative expression of the cox gene in the two groups was detected by qPCR, and it was found that the expression of the cox gene in the treatment group was significantly higher than that in the control group ( Figure 5 ), indicating that the Japanese eel BMP-15 recombinant protein obtained by the method of the present 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 fish ovarian development using Japanese eel BMP-15 recombinant protein

[0087] In this example, the Japanese eel BMP-15 recombinant protein can be applied to fish ovarian development-related research. Researchers can add the recombinant protein to Japanese eel secondary ovarian cell culture to evaluate the effects of BMP-15 on Japanese eel follicle growth, oocyte maturation, and related hormone secretion. This study helps to understand the reproductive biology of Japanese eels and other related fish, and provides important biological information for fish ovarian development and reproduction.

[0088] The specific steps include:

[0089] 1. Isolation and culture of secondary ovarian cells of 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 evaluate the effect of BMP-15 on ovarian development.

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

[0094] In this example, Japanese eel BMP-15 recombinant protein was used to develop a fish reproductive health detection kit. This kit can detect the level of BMP-15 in fish, help farmers assess the reproductive health of fish, and adjust the breeding management strategy in time.

[0095] The specific steps include:

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

[0097] 2. Develop detection methods based on ELISA or other appropriate technologies for use in constructing test 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 breeders or researchers with fast and accurate test results.

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

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

[0102] The verified expression vector was transformed into Rosetta-gami B host bacteria and transformed by heat shock transformation. After transformation, it was spread on LB plates containing ampicillin and chloramphenicol, cultured at 37°C overnight, and resistant positive clones were screened. Single colonies were selected and the successfully transformed recombinant strains were identified by PCR.

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

[0104] The induced expression bacteria were broken by ultrasound to obtain soluble protein solution and inclusion body protein. The soluble recombinant protein containing the His tag was purified using a Ni-NTA affinity column, and the inclusion body protein was treated by a denaturation-renaturation method to obtain 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 samples were analyzed by SDS-PAGE to confirm that the molecular weight was consistent with the theoretical value. Western blot was further used to verify the accuracy and purity of the target protein using a BMP-15-specific antibody. In addition, the biological activity of the recombinant protein was tested by ELISA.

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

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

[0108] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

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

1.

2. A gene encoding the Japanese eel BMP-15 recombinant protein as claimed in claim 1, characterized in that: The invention comprises: a His tag encoding an N-terminal, a connecting peptide between the His tag and a SUMO solubilizing polypeptide, a SUMO solubilizing polypeptide, and a Japanese eel BMP-15 mature peptide; the DNA sequence of the encoding gene is SEQ ID NO:

3.

3. An expression vector, characterized in that: The expression vector comprises the coding gene of the Japanese eel BMP-15 recombinant protein according to claim 2 and a backbone plasmid, and the backbone plasmid is obtained by transforming pET-32a(+).

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

5. The recombinant engineered bacterium according to claim 4, characterized in that The host bacteria of the recombinant engineering bacteria is selected from Rosetta-gami B.

6. A method for preparing the Japanese eel BMP-15 recombinant protein according to claim 1, characterized in that: The following steps are involved: (1) constructing the Japanese eel BMP-15 recombinant protein encoding gene, and connecting it to a backbone plasmid to construct an expression vector for the Japanese eel BMP-15 recombinant protein; (2) transforming the expression vector into a host bacterium to induce expression of Japanese eel BMP-15 recombinant protein; (3) Through inclusion body refolding, the misfolded insoluble inclusion bodies are converted into soluble proteins with correct structure; (4) Affinity chromatography of the His tag in the Japanese eel BMP-15 recombinant protein was used to obtain a higher purity GDF-9 recombinant protein; (5) The protein was dialyzed into 1× PBS, and bovine serum albumin (BSA) was added to a final concentration of 0.1%. After quick freezing in liquid nitrogen, it was stored at -80°C.

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

8. Use of the Japanese eel BMP-15 recombinant protein as claimed in claim 1 in regulating the ovarian development of Japanese eel.

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