Anguilla japonica Activin B recombinant protein as well as expression method and application thereof
By adding a His tag and SUMO lysing peptide to the recombinant Activin B protein from Japanese eels, a prokaryotic expression vector was constructed and expressed in E. coli. Combined with inclusion body renaturation technology, the problems of low expression efficiency and insufficient activity of the recombinant Activin B protein from Japanese eels were solved, and high-purity, high-concentration active protein was obtained for application in ovarian development and artificial reproduction research.
Patent Information
- Application Number
- CN202511047681.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-10-31
AI Technical Summary
In existing technologies, the recombinant Activin B protein from Japanese eels has low expression efficiency and high cost. Furthermore, the protein expressed through the E. coli prokaryotic expression system is misfolded and loses its activity, making it difficult to obtain high concentrations of active protein.
By adding a 6×His tag and a SUMO lysing peptide to the recombinant Activin B protein from Japanese eel, a pET-His-SUMO-ActivinB prokaryotic expression vector was constructed. The protein was then expressed using Rosetta-gami B(DE3) E. coli, and combined with inclusion body renaturation technology, a biologically active recombinant protein was obtained.
This study achieved efficient and low-cost acquisition of structurally correct and biologically active recombinant Activin B protein from Japanese eels, suitable for research on ovarian development and artificial breeding of Japanese eels, and improved the purity and concentration of the recombinant protein.
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Abstract
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 Activin B protein from Japanese eels, its expression method, and its application. Background Technology
[0002] The Japanese eel (Anguilla japonica) is an important economic fish species in my country, holding a significant position in the aquaculture industry of East Asia. As a migratory fish that grows in freshwater and reproduces in seawater, the Japanese eel has a highly complex life cycle, making it difficult to fully understand the natural conditions required for its gonadal development, fertilized egg hatching, and juvenile growth. Therefore, artificial breeding technology for the Japanese eel remains a global challenge that has yet to be solved. my country is the world's largest producer and exporter of artificially farmed eels and processed eel products. Currently, the seedlings needed for eel farming all come from wild eel fry caught in estuaries. However, due to overfishing of wild eel fry in recent years, wild eel fry resources have been declining year by year, and the shortage of seedlings has limited the further development of the eel farming and processing industries. Therefore, achieving fully artificial breeding of eels is particularly important for the protection of eel natural resources and the development of my country's eel industry. However, several technical challenges still restrict research into artificial eel breeding technology.
[0003] Researchers worldwide began exploring artificial eel breeding in the 1930s, and nearly a century of continuous effort has yielded significant progress. However, industrialization remains a long way off, primarily due to the unsatisfactory quality of eggs produced by broodstock eels under artificial maturation conditions, resulting in low fertilization, hatching, and survival rates. As the world's largest producer, processor, and exporter of eels, my country has an enormous demand for eel larvae, thus urgently needing to overcome the challenges of artificial eel breeding technology. One key to solving this problem lies in regulating the development of eel ovaries to improve the quality of eel eggs produced under artificial breeding conditions.
[0004] Activin B, a crucial ovarian regulatory factor, promotes the expression of follicle-stimulating hormone (FSH) and is specifically secreted by follicular cells in the ovary. As a local factor, Activin B regulates gamete production and steroid production in the gonads through autocrine and / or paracrine mechanisms. In mice, Activin promotes primordial follicle formation by stimulating germline cyst rupture or follicle assembly. Studies in zebrafish have shown a dramatic increase in Activin B expression in the ovary prior to spawning, indicating its important role in oocyte maturation and ovulation. Similar to other vertebrates, Activin B in the Japanese eel is a classic secreted protein with a typical N-terminal signal peptide sequence. Once synthesized, the signal peptide of the nascent polypeptide chain is recognized by the signal recognition particle (SRP), pausing translation. The SRP, along with the polypeptide chain and ribosomes, anchors on the surface of the rough endoplasmic reticulum, continuing translation while the signal peptide is cleaved. The newly synthesized peptide chains continue to be synthesized in the endoplasmic reticulum. After folding and processing in the endoplasmic reticulum and Golgi apparatus, they are eventually secreted into the extracellular space. The two monomers form homodimers through disulfide bonds to perform their functions.
[0005] 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. By using a strong promoter, foreign proteins can be expressed at high levels in *E. coli*, achieving rapid and large-scale production. However, because the rate of foreign protein expression in *E. coli* is much higher than in eukaryotic cells, it easily leads to misfolding of nascent peptide chains, forming inactive inclusion bodies.
[0006] Misfolding of nascent peptide chains into insoluble inclusion bodies is a common problem in prokaryotic expression. This misfolding leads to abnormal protein structure, loss of biological activity, and precipitation. To address this issue, inclusion body renaturation can be used to transform structurally incorrect and insoluble inclusion bodies into soluble proteins with the correct molecular structure, restoring their biological activity and yielding functional proteins. Furthermore, inclusion body renaturation is more efficient in obtaining recombinant proteins, achieving 80%–90% purity without the need for purification, saving time and costs in production and possessing significant practical implications.
[0007] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:
[0008] (1) There are currently no reports on the expression of recombinant Activin B protein in Japanese eel;
[0009] (2) There are reports on the expression of recombinant Activin B protein in other organisms, but most of the reports use eukaryotic expression, which is inefficient and costly to obtain recombinant Activin B protein.
[0010] (3) There are relatively few reports on other organisms expressing recombinant Activin B protein through prokaryotic expression systems. Moreover, the recombinant Activin B protein inclusion bodies in the reports have poor renaturation effects and are easily precipitated during dialysis, making it impossible to obtain high concentrations of recombinant Activin B protein.
[0011] (4) The recombinant protein of Escherichia coli is expressed too quickly and there are no organelles such as endoplasmic reticulum and Golgi apparatus to process and modify the nascent peptide chain, which leads to the nascent peptide chain being unable to fold correctly and thus forming inactive inclusion bodies.
[0012] The difficulty in solving the above problems and defects is as follows:
[0013] (1) A recombinant expression vector for Japanese eel Activin B was constructed and expressed using an Escherichia coli prokaryotic expression system to obtain biologically active Japanese eel Activin B recombinant protein;
[0014] (2) Escherichia coli lacks the eukaryotic endomembrane system for post-translational modification and processing, and the expressed Activin B recombinant protein loses its activity due to misfolding;
[0015] (3) The cytoplasm of Escherichia coli is reducing, which is not conducive to the formation of disulfide bonds in the recombinant Activin B protein.
[0016] The significance of solving the above problems and defects is that it allows for the acquisition of a structurally correct and biologically active recombinant Activin B protein from Japanese eels, which can be applied to basic molecular biology research and the artificial breeding and production process of Japanese eels, providing a theoretical basis and technical support for ovarian development and artificial breeding of Japanese eels. The recombinant Activin B protein from Japanese eels of this invention can be induced to express in large quantities by E. coli, and through inclusion body renaturation, a biologically active recombinant protein can be obtained rapidly and efficiently, saving time and costs, and can be effectively applied to industrial production, thus possessing significant commercial value. Summary of the Invention
[0017] To address the problems existing in the prior art, this invention provides a method for inducing the expression of recombinant Activin B protein from Japanese eels, particularly relating to a recombinant Activin B protein from Japanese eels produced by an Escherichia coli prokaryotic expression system, which can be obtained in large quantities through inclusion body renaturation and its application in the study of ovarian development in Japanese eels.
[0018] This invention is achieved as follows: a recombinant Activin B protein from Japanese eel, comprising: the mature peptide amino acid sequence of Activin B from Japanese eel, 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 Activin B protein from Japanese eel is shown in SEQ ID NO:1.
[0019] Furthermore, the gene encoding the recombinant Activin B protein of Japanese eel includes: nucleotide sequences encoding an N-terminal His tag, a linker peptide between the His tag and the SUMO lysing peptide, the SUMO lysing peptide, and the mature peptide of Japanese eel Activin B; the nucleotide sequence of the encoding gene is SEQ ID NO:3.
[0020] Furthermore, the amino acid sequence of the mature Activin B peptide from Japanese eel is shown in SEQ ID NO:2.
[0021] 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 recombinant Japanese eel Activin B protein, further improving the purity of the recombinant protein. A 13-amino acid linker peptide SSGLVPRGSHMAS 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 Japanese eel Activin B peptide, thus ensuring its full exposure in solution and facilitating affinity chromatography purification of the recombinant protein after refolding.
[0022] 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.
[0023] The N-terminal His tag and SUMO-promoting peptide do not affect the activity of the Japanese eel Activin B recombinant protein, and there is no need to remove the tag.
[0024] Another object of the present invention is to provide an expression vector for expressing the recombinant Activin B protein of Japanese eel, the expression vector comprising the coding gene of the recombinant Activin B protein of Japanese eel and a backbone plasmid, the backbone plasmid being obtained by modifying pET-32a(+).
[0025] Another object of the present invention is to provide a recombinant engineered bacterium expressing the recombinant protein Activin B from the Japanese eel, the recombinant engineered bacterium comprising the expression vector.
[0026] Furthermore, the host bacteria of the recombinant engineered bacteria are selected from Rosetta-gami B(DE3).
[0027] Another object of the present invention is to provide a method for preparing recombinant Activin B protein from Japanese eel, comprising the following steps:
[0028] (1) Construct the recombinant protein encoding gene of Japanese eel Activin B, and connect it to a backbone plasmid to construct an expression vector for the recombinant protein of Japanese eel Activin B;
[0029] (2) The expression vector was transformed into a host bacterium to induce the expression of recombinant Activin B protein from Japanese eel;
[0030] (3) By refolding inclusion bodies, misfolded insoluble inclusion bodies are transformed into soluble proteins with the correct structure;
[0031] (4) Based on the His tag affinity chromatography of the Japanese eel Activin B recombinant protein, a higher purity recombinant protein was obtained (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).
[0032] (5) Dialyze the protein into 1×PBS, add bovine serum albumin (BSA) to a final concentration of 0.1% to facilitate the preservation of the recombinant protein, freeze it in liquid nitrogen, and store it 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).
[0033] Another object of the present invention is to provide an application of the recombinant Activin B protein from Japanese eel in the regulation of ovarian development in Japanese eel.
[0034] 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:
[0035] First, regarding the technical problems existing in the aforementioned prior art, the creative technical effects resulting from solving these problems are described in detail below:
[0036] (1) This invention provides a recombinant Activin B protein from Japanese eel, comprising: a mature peptide amino acid sequence of Activin B from Japanese eel, 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 is used for the purification of the recombinant Activin B protein from Japanese eel, which can improve the purity of the recombinant protein to meet higher application requirements; the SUMO lysing peptide can be used to inhibit the aggregation of the recombinant Activin B protein from Japanese eel 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 recombinant protein;
[0037] (2) In this invention, a prokaryotic expression vector pET-His-SUMO-ActivinB for recombinant Activin B protein of Japanese eel was constructed, and the vector was transfected into Rosetta-gami B(DE3) Escherichia coli to obtain the pET-His-SUMO-ActivinB-Rosetta-gami B(DE3) expression strain, which can induce the expression of recombinant Activin B 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;
[0038] (3) The recombinant Activin B protein of Japanese eel provided by the present invention can be effectively applied to the study of ovarian development and artificial reproduction of Japanese eel.
[0039] 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 this invention are specifically described as follows:
[0040] (1) This invention provides a recombinant Japanese eel Activin B protein that is identical to the amino acid sequence of the mature peptide of Japanese eel Activin B, comprising: the amino acid sequence of the mature peptide of Japanese eel Activin B, an N-terminal 6×histidine tag (6×His tag or His tag for short), a SUMO lysing peptide, and a linking peptide between the 6×histidine tag and the SUMO lysing peptide.
[0041] (2) This invention first constructs a prokaryotic expression vector pET-His-SUMO-ActivinB for recombinant Activin B protein of Japanese eel, and transfects this vector into Rosetta-gami B(DE3) Escherichia coli. The resulting pET-His-SUMO-ActivinB-Rosetta-gami B(DE3) expression strain can be induced to express recombinant Activin B protein of Japanese eel in large quantities. The recombinant protein with a purity of 80% to 90% can be obtained by inclusion body renaturation (if a higher purity recombinant protein is required, His tag affinity chromatography can be performed to purify the recombinant protein with a purity higher than 90%).
[0042] (3) In the prokaryotic expression vector pET-His-SUMO-ActivinB of the Japanese eel Activin B recombinant protein constructed in this invention, the addition of SUMO soluble peptide can inhibit the aggregation of 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 the obtained Japanese eel Activin B recombinant protein.
[0043] (4) This invention provides for the first time a method for preparing recombinant Activin B protein from Japanese eel based on this recombinant protein. The recombinant protein of this invention can be induced to be expressed in large quantities through an Escherichia coli prokaryotic expression system, and soluble recombinant protein can be efficiently obtained through inclusion body renaturation, while retaining biological activity when the tag is retained;
[0044] (5) Application of the recombinant protein provided by this invention or the prepared Japanese eel Activin B recombinant protein in the study of Japanese eel ovarian development and artificial reproduction.
[0045] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:
[0046] (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 Japanese eel Activin B protein with an amino acid sequence consistent with the mature peptide of Japanese eel Activin B. The biologically active target protein can be obtained in large quantities and efficiently through the method, which can be effectively applied to industrial production and has important commercial value.
[0047] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad: There are no reports on recombinant Activin B protein of Japanese eel in relevant studies at home and abroad. The present invention constructs a prokaryotic expression vector pET-His-SUMO-ActivinB of recombinant Activin B protein of Japanese eel for the first time, and transfects the vector into Rosetta-gami B(DE3) Escherichia coli. The resulting pET-His-SUMO-ActivinB-Rosetta-gami B(DE3) expression strain can induce the expression of recombinant Activin B protein of Japanese eel in large quantities, and obtain recombinant Activin B protein of Japanese eel efficiently and in large quantities through inclusion body renaturation.
[0048] (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 produced by artificially induced spawning broodstock is one of the main difficulties currently faced. Therefore, research on the regulatory mechanism of ovarian development in Japanese eels is urgently needed. Recent studies have shown that ovarian development is not only passively regulated by gonadotropins (GtH), but also involves various growth factors produced by oocytes and surrounding follicular cells, which play important roles. Activin B is a key influencing factor in regulating ovarian development and has a certain regulatory function throughout the entire oocyte development process. It plays an indispensable role in the final maturation and ovulation of eggs. Therefore, exploring the role of Activin B in the ovarian development of Japanese eels can provide a theoretical basis and technical support for the artificial breeding of eels. The recombinant Activin B protein of Japanese eels obtained by the method described in this 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. Attached Figure Description
[0049] Figure 1 This is a schematic diagram of the structure of the Japanese eel Activin B protein and its open reading frame (ORF) cloning provided in an embodiment of the present invention;
[0050] Figure 1 A is a schematic diagram of the composition of Japanese eel Activin B, which includes a signal peptide of 20 amino acids, a precursor peptide of 258 amino acids, and a mature peptide of 115 amino acids, provided in an embodiment of the present invention; wherein, aa: amino acid;
[0051] Figure 1 B is a schematic diagram of the ORF sequence and the translated amino acid sequence of Activin B of Japanese eel provided in the embodiments of the present invention, with the dark gray area representing the mature peptide amino acid sequence;
[0052] Figure 2 This is a schematic diagram of the Japanese eel Activin B recombinant protein expression vector, recombinant protein composition, and recombinant protein ORF sequence provided in this embodiment of the invention;
[0053] Figure 2 A is a map of the Japanese eel Activin B recombinant protein expression vector 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;
[0054] Figure 2 B is a schematic diagram of the recombinant Activin B protein from Japanese eel provided in an embodiment of the present invention;
[0055] Figure 2 C is a schematic diagram of the recombinant Activin B protein ORF of Japanese eel 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 Activin B peptide of Japanese eel;
[0056] Figure 3 This is an SDS-PAGE gel electrophoresis image of the expression, inclusion body renaturation, and concentration of recombinant Activin B protein from Japanese eel provided in this embodiment of the invention; wherein: 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, 6, 7: total protein after inclusion body renaturation (including target protein); the black arrow indicates the band of recombinant Activin B protein from Japanese eel (theoretical molecular weight is 26.4 kDa);
[0057] Figure 4 This is a qPCR result of cox gene expression in Japanese eel ovary cells regulated by recombinant Activin B protein provided in an embodiment of the present invention.
[0058] Figure 5 This is a graph showing the results of the Japanese eel Activin B recombinant protein improving the estradiol (E2) level in the serum of Japanese eels, as provided in the embodiments of the present invention. Detailed Implementation
[0059] 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.
[0060] This invention provides a recombinant Activin B protein from Japanese eel, comprising: a mature peptide amino acid sequence of Activin B from Japanese eel, 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 Activin B protein from Japanese eel is shown in SEQ ID NO:1;
[0061] The gene encoding the recombinant Activin B protein of Japanese eel includes: nucleotide sequences encoding an N-terminal His tag, a linker peptide between the His tag and the SUMO lysing peptide, the SUMO lysing peptide, and the mature peptide of Japanese eel Activin B; the nucleotide sequence of the encoding gene is SEQ ID NO:3.
[0062] Furthermore, the amino acid sequence of the mature Activin B peptide from Japanese eel is shown in SEQ ID NO:2.
[0063] The amino acid sequence of the recombinant Activin B protein from Japanese eel is SEQ ID NO:1
[0064] MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQ IGGGLECDGTGGLCCRQQFYIDFRLIGWNDWIIAPSGYFGNYCEGSCPAYMAGVPSSASSFHTAVVNQYRMRGMSPGSMNSCCIPTRLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA
[0065] The amino acid sequence of the mature peptide Activin B from Japanese eel is SEQ ID NO:2
[0066] GLECDGTGGLCCRQQFYIDFRLIGWNDWIIAPSGYFGNYCEGSCPAYMAGVPSSASSFHTAVVNQYRMRGMSPGSMNSCCIPTRLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA
[0067] The nucleotide sequence of the gene encoding the recombinant Activin B protein from the Japanese eel is SEQ ID NO:3.
[0068] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTC TTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGA TTGGTGGTGGCCTGGAATGCGACGGCACCGGCGGCCTCTGCTGCCGCCAGCAGTTCTACATCGACTTCCGGCTCATCGGCTGGAACGACTGGATCATCGCCCCCTCCGGCTACTTCGGGAACTACTGCGAGGGCAGCTGCCCCGCCTACATGGCGGGCGTGCCCAGCTCGGCCTCCTC CTTCCACACGGCGGTGGTGAACCAGTACCGCATGCGCGGCATGAGCCCCGGGTCCATGAACTCCTGCTGCATCCCCACGGCCTCAGCACCATGTCCATGCTCTACTTCGACGACGAGTACAACATCGTCAAGCGGGACGTGCCCAACATGATCGTGGAGGAGTGCGGCTGCGCCTAA
[0069] Furthermore, 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 affinity chromatography of the recombinant protein via a nickel column to purify the Japanese eel Activin B recombinant protein, further improving the purity of the recombinant protein. A 13-amino acid linker peptide SSGLVPRGSHMAS (SEQ ID NO:5) 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 Japanese eel Activin B peptide, thus ensuring its full exposure in solution and facilitating affinity chromatography purification of the recombinant protein after refolding.
[0070] Furthermore, a SUMO-promoting polypeptide with the sequence MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:6) 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.
[0071] Furthermore, the N-terminal His tag and SUMO-promoting peptide do not affect the activity of the Japanese eel Activin B recombinant protein, and there is no need to remove the tag.
[0072] This invention provides an expression vector for expressing recombinant Activin B protein from Japanese eel. The expression vector includes the coding gene for recombinant Activin B protein from Japanese eel and a backbone plasmid, wherein the backbone plasmid is obtained by modifying pET-32a(+).
[0073] This invention provides a recombinant engineered bacterium expressing the recombinant protein Activin B from the Japanese eel, wherein the recombinant engineered bacterium contains the expression vector.
[0074] Furthermore, the host bacteria of the recombinant engineered bacteria are selected from Rosetta-gami B(DE3).
[0075] This invention provides a method for preparing recombinant Activin B protein from Japanese eel, comprising the following steps:
[0076] (1) Construct the recombinant protein encoding gene of Japanese eel Activin B, and connect it to a backbone plasmid to construct an expression vector for the recombinant protein of Japanese eel Activin B;
[0077] (2) The expression vector was transformed into a host bacterium to induce the expression of recombinant Activin B protein from Japanese eel;
[0078] (3) Misfolded insoluble inclusion bodies are transformed into structurally correct and biologically active soluble proteins through inclusion body renaturation.
[0079] (4) Based on the His tag in the Japanese eel Activin B recombinant protein, a higher purity recombinant protein is 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).
[0080] (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).
[0081] Example 1: Construction of the recombinant expression vector pET-His-SUMO-ActivinB for Japanese eel Activin B
[0082] 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 Activin B gene (inhbb). Figure 1 The 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 LB solid medium, and the sequence was verified by Sanger sequencing and DNAMAN assay. Using the above-mentioned *Inhbb* Japanese eel monoclonal culture as a template, PCR amplification was performed to obtain the double-stranded DNA fragment corresponding to the mature peptide of *Activin B* from *Inhbb* Japanese eel 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. 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 LB agar, and confirmed by Sanger sequencing and DNA MAN comparison. Plasmids were then extracted using a plasmid mini-extraction kit (Tiangen, Beijing), yielding the prokaryotic expression vector pET-His-SUMO-ActivinB, a recombinant protein of the Japanese eel. Figure 2 ).
[0083] Example 2: Preparation and Induction of Expression of Recombinant Activin B Protein in Escherichia coli from Japanese Eel
[0084] The expression vector pET-His-SUMO-ActivinB was transfected into Rosetta-gami B(DE3) competent Escherichia coli (Angyu, Shanghai). Single colonies were screened by incubating overnight on ampicillin-chloramphenicol double antibiotic LB solid medium at 37°C. These colonies were the prokaryotic expression engineered strain pET-His-SUMO-ActivinB-Rosetta-gami B(DE3) of recombinant Activin B protein from Japanese eel. Single colonies were picked and incubated 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 incubated 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 incubation 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 cell pellet was treated with 20 mL of lysis buffer (500 mM NaCl, 20 mM sodium phosphate, 10 mM imidazole, pH 10). 7.4) Resuspend, sonicate at 60W (5s for 5s, 5s for 30min total), centrifuge at 12,000×g for 10min, collect the supernatant and precipitate separately, resuspend the precipitate in 20mL of lysis buffer, take 80μL of sample and 20μL of 5× protein loading buffer, mix well, incubate at 95℃ for 10min in a PCR instrument, and perform SDS-PAGE and Coomassie Brilliant Blue staining to observe protein bands. Figure 3 (Samples 1-4). The results showed that the recombinant Activin B protein of Japanese eel was expressed in large quantities and existed in the precipitate as misfolded inclusion bodies, and was an insoluble protein.
[0085] Example 3: Washing, refolding, protein purification, and enrichment / concentration of recombinant Activin B protein inclusion bodies from Japanese eel.
[0086] The precipitate obtained in Example 2 (the recombinant Activin B protein from the Japanese eel exists as insoluble inclusion bodies in the precipitate 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 resuspension with shaking, the precipitate was sonicated at 60 W (5 s for 5 s, 5 s for 10 min), centrifuged at 12,000 × g for 10 min, and the supernatant was discarded, retaining the precipitate. The precipitate was then washed with 20 mL of inclusion body washing buffer 2 (NaCl 500 mM, Tris-HCl 20 mM, Triton X-100 2% (v / v), pH 7.4). The precipitate was washed with 500 mM, Tris-HCl 20 mM, and urea 2 M (pH 7.4), then resuspended by shaking and sonicated at 60 W (5 s for 5 s, 5 s for 10 min). After centrifugation at 12,000 × 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 point, the purity of the inclusion bodies can reach 80% to 90%.
[0087] Add 10 mL of inclusion body dissolving 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 bring the final concentration to 10 nM. After shaking and resuspending, sonicate at 60 W (disrupt for 5 seconds, stop for 5 seconds, for a total of 10 min). Centrifuge at 12,000 × g for 15 min and retain the supernatant, which is the denatured and dissolved Japanese eel Activin B recombinant protein. Take 20-50 mL of dilution and refolding solution (NaCl 50 mM, Tris-HCl 50 mM, L-arginine 0.4 M, urea 1 M, reduced glutathione 5 mM, oxidized glutathione 1 mM, glycerol 10% (v / v), pH 8.0) 8.0) Pour into a glass bottle, add a magnetic stir bar, and add the dissolved inclusion bodies dropwise until the final concentration is approximately 0.05 mg / mL (during this process, the magnetic stir bar must be used to continuously stir 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 the dissolved inclusion bodies until the final concentration is approximately 0.1 mg / mL... Repeat this process for a total of 4 additions, increasing the concentration by 0.05 mg / mL each time. For the last addition, incubate at 4°C overnight to allow the protein to fully fold, thus obtaining the structurally correct soluble Japanese eel Activin B recombinant protein.
[0088] To obtain higher purity recombinant Activin B protein from Japanese eels, insoluble microparticles can be removed by filtration using a 0.45 μm or 0.22 μm filter membrane. Then, His-tag affinity purification can be performed using NTA-Ni packing material (Beyotime, Shanghai) to obtain recombinant Activin B protein with a purity >90%. The obtained recombinant protein solution is poured into a protein dialysis bag (molecular weight cutoff: 3.5 kDa) 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 added to a final concentration of 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°C. To obtain a high concentration of recombinant Activin B protein from Japanese eels, the recombinant protein can be enriched and concentrated using an ultrafiltration tube after dialysis. Figure 3 (Samples 5, 6, and 7).
[0089] Example 4: Regulating the expression of the cox gene in follicular layer cells cultured in vitro using recombinant Activin B protein from Japanese eel.
[0090] Ovarian cells from Japanese eels during vitelline development were isolated. The treatment group (Activin B group) was stimulated with recombinant Activin B protein obtained using the method described in this invention, while the control group (Control group) 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 4 This indicates that the recombinant Activin B protein from Japanese eel obtained by the method described in this invention has biological activity and can regulate the expression of downstream genes in Japanese eel ovarian cells.
[0091] Example 5: Recombinant Activin B protein from Japanese eel increases E2 levels in Japanese eel serum.
[0092] The recombinant Activin B protein obtained using the method described in this invention was injected into female Japanese eels weighing approximately 1 kg. In addition to artificially inducing maturation of female Japanese eels using human chorionic gonadotropin analogs and luteinizing hormone-releasing hormone analogs, the treatment group received weekly injections of the recombinant Activin B protein at a dose of 1 mg / kg, while the control group received an equal volume of physiological saline. After 10 weeks, the serum E2 level of the female Japanese eels was measured. The results showed that compared with the control group, the serum E2 level of the female Japanese eels in the experimental group was significantly increased, indicating that the recombinant Activin B protein obtained by the method described in this invention can effectively promote ovarian development in Japanese eels. Figure 5 ).
[0093] Example 6: Study on ovarian development in fish using recombinant Activin B protein from Japanese eel
[0094] In this embodiment, the recombinant Activin B protein from the Japanese eel can be applied to research related to fish ovarian development. Researchers can add this recombinant protein to ovarian cell cultures during the vitellogenesis phase of the Japanese eel to assess the effects of Activin B on follicle development, yolk accumulation, and oocyte maturation in the Japanese eel. This research contributes to understanding the reproductive biology of the Japanese eel and other related fish species, providing important biological information for fish ovarian development and reproduction.
[0095] The specific operating steps include:
[0096] 1. Isolation and culture of vitellogenic follicles from Japanese eels.
[0097] 2. Add different concentrations of Japanese eel Activin B recombinant protein to the cell culture medium.
[0098] 3. Observe and record the development of follicles, yolk accumulation, and hormone secretion in the Activin B treatment group and the control group.
[0099] 4. Perform statistical analysis on the collected data to evaluate the impact of Activin B on ovarian development.
[0100] The three examples above demonstrate the potential value of the recombinant Activin B protein from the Japanese eel in basic biological research and practical applications. These studies can lead to a better understanding of the function of Activin B in fish reproductive processes and provide strong technical support for the aquaculture industry.
[0101] 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 Activin B protein from Japanese eel, characterized in that, It comprises: the mature amino acid sequence of Japanese eel Activin B peptide, an N-terminal 6×histidine tag, and a SUMO-promoting polypeptide; the amino acid sequence of the recombinant Japanese eel Activin B protein is shown in SEQ ID NO:
1.
2. The recombinant Activin B protein from Japanese eel as described in claim 1, characterized in that, The amino acid sequence of the mature Activin B peptide from Japanese eel is shown in SEQ ID NO:
2.
3. The recombinant Activin B protein from Japanese eel as described in claim 1, characterized in that, A His tag and a linker peptide were added to the N-terminus of the recombinant protein, with the amino acid sequence HHHHHHSSGLVPRGSHMAS; a 13-amino acid SSGLVPRGSHMAS linker peptide was added between the His tag and the SUMO lysing peptide.
4. The recombinant Activin B protein from Japanese eel as described in claim 1, characterized in that, The N-terminus is augmented with SUMO-promoting peptide, the amino acid sequence of which is MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.
5. An expression vector for expressing the recombinant Activin B protein of the Japanese eel as described in any one of claims 1 to 4, characterized in that, The expression vector includes the encoding gene of the Japanese eel Activin B recombinant protein and a backbone plasmid, the backbone plasmid being modified from pET-32a(+).
6. A recombinant engineered bacterium expressing the recombinant Activin B protein of Japanese eel as described in any one of claims 1 to 4, wherein the recombinant engineered bacterium comprises the expression vector.
7. The recombinant engineered bacteria expressing the recombinant protein Activin B from Japanese eel as described in claim 6, characterized in that, The host bacteria of the recombinant engineered bacteria were selected from Rosetta-gami B.
8. A method for preparing recombinant Activin B protein from Japanese eel as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Construct the recombinant protein encoding gene of Japanese eel Activin B, and connect it to a backbone plasmid to construct an expression vector for the recombinant protein of Japanese eel Activin B; (2) The expression vector was transformed into a host bacterium to induce the expression of recombinant Activin B protein from Japanese eel; (3) By refolding inclusion bodies, misfolded insoluble inclusion bodies are transformed into soluble proteins with the correct structure; (4) Affinity chromatography based on the His tag in the recombinant protein of Japanese eel Activin B was used to obtain recombinant protein with higher purity; (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.
9. The method for preparing recombinant Activin B protein from Japanese eel as described in claim 8, 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.
10. The application of the recombinant Activin B protein from Japanese eel as described in any one of claims 1 to 4 in the regulation of ovarian development in Japanese eel.