A Japanese eel Activin A recombinant protein and its expression method and application

Abstract

The present invention belongs to, but is not limited to, the field of genetic engineering technology and discloses a Japanese eel Activin A recombinant protein, its expression method, and application. The Japanese eel Activin A recombinant protein comprises: the amino acid sequence of the Japanese eel Activin A mature peptide, a 6× histidine tag at the N-terminus, and a SUMO solubilizing polypeptide; its amino acid sequence is SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the Japanese eel Activin A recombinant protein is SEQ ID NO: 3. The present invention constructs the prokaryotic expression vector pET-His-SUMO-ActivinA for Japanese eel Activin A for the first time, which can produce an Activin A recombinant protein with an amino acid sequence consistent with the Japanese eel Activin A mature peptide, which can be applied to the research on ovarian development regulation and artificial reproduction of Japanese eels.

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 Activin A recombinant protein and an expression method and application thereof. Background Art

[0002] The Japanese eel (Anguilla japonica) is one of the most important migratory fish species in Asia and an important economic fish species in my country. As a migratory fish that spawns in the sea, the Japanese eel has a complex life history. This complexity leads to many unknowns, including growth and reproduction. Therefore, its artificial propagation technology is a global problem that has not yet been solved. Because the fry required for aquaculture are all caught naturally, this predatory farming method has caused the natural eel fry resources to decline year by year. The shortage of eel fry has restricted the further development of the eel industry. Therefore, research on artificial propagation technology of eels is particularly important for protecting natural resources and my country's eel farming industry. However, there are technical bottlenecks that need to be addressed to successfully achieve artificial propagation of Japanese eels.

[0003] Since the 1930s, significant progress has been made in artificial breeding technology for eels. However, there is still a significant gap between the requirements for large-scale industrial production of eel fry and the current low maturity, spawning rate, and egg production of artificially matured broodstock. In particular, the egg quality is suboptimal, resulting in very low fertilization, hatching, and survival rates. As the world's leading producer, processor, and exporter of eels, my country has a significant demand for eel fry, making artificial breeding of eels a pressing task. The key to addressing these issues lies in regulating the ovarian development of Japanese eels, thereby improving the quality of eggs produced under artificial breeding conditions.

[0004] Activin A, secreted specifically by follicular cells in the ovary, promotes the expression of follicle-stimulating hormone (FSH). It is a crucial ovarian regulatory factor. It plays a crucial role in the transition from primary to secondary follicles and in vitellogenesis. Studies in zebrafish have shown that in the absence of Activin A, yolk granules do not accumulate in oocytes, indicating that Activin A plays a crucial role in ovarian development. Like other vertebrates, Activin A in Japanese eel is a classic secretory protein with a typical N-terminal signal peptide sequence. Once synthesized, the signal peptide of the nascent peptide chain is recognized by the signal recognition particle (SRP), halting translation. The SRP, along with the peptide chain and ribosome, docks on the surface of the rough endoplasmic reticulum, allowing translation to continue while the signal peptide is removed. The nascent peptide chain enters the endoplasmic reticulum (ER) during synthesis, ultimately undergoing folding and processing in the ER and Golgi apparatus before being secreted out of the cell. The two monomers form a homodimer through disulfide bonds, allowing them to function.

[0005] In the field of genetic engineering, Escherichia coli (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, a clear genetic background, simple cultivation operations, and low cost. By using strong promoters, foreign proteins can be expressed at high levels in E. coli, achieving the goal of rapid and large-scale production of foreign proteins. However, because E. coli expresses foreign proteins at a much higher rate than eukaryotic cells, it is prone to misfolding of nascent peptide chains, forming inactive inclusion bodies.

[0006] Inclusion bodies are a common problem in prokaryotic expression. Misfolding of peptide chains can lead to abnormal protein structure and loss of biological activity. To address this problem, inclusion body renaturation can be used to convert misfolded and insoluble inclusion bodies into soluble proteins with the correct molecular structure, restoring their biological activity and obtaining biologically active proteins. Furthermore, inclusion body renaturation is more efficient in obtaining recombinant proteins, and purity can reach 80% to 90% without purification, saving time and costs in production and having important practical significance.

[0007] In view of the above analysis, the technical problems that need to be solved urgently in the existing technology are:

[0008] (1) There are currently no reports on the expression of recombinant Activin A protein in Japanese eel;

[0009] (2) There have been reports on the expression of recombinant Activin A proteins from other organisms, but most of these reports used eukaryotic expression, which is inefficient and costly to obtain.

[0010] (3) Currently, there are relatively few reports on other organisms expressing Activin A recombinant protein through prokaryotic expression systems. In addition, the inclusion body renaturation effect of Activin A recombinant protein in these reports is poor, and it is easy to precipitate during dialysis, making it impossible to obtain high concentrations of Activin A recombinant protein.

[0011] (4) The recombinant protein expression rate of E. coli is too fast, and there is no organelle such as the endoplasmic reticulum and Golgi apparatus for processing and modification, which easily leads to the inability of the newly formed peptide chain to fold correctly, thereby forming inactive inclusion bodies. Summary of the Invention

[0012] In response to the problems existing in the prior art, the present invention provides a method for inducing the expression of a Japanese eel Activin A recombinant protein, and in particular relates to a Japanese eel Activin A recombinant protein produced by an Escherichia coli expression system and obtainable in large quantities by inclusion body renaturation, and its application in the study of Japanese eel ovarian development.

[0013] The present invention is achieved as follows: a Japanese eel Activin A recombinant protein comprises: an amino acid sequence of a Japanese eel Activin A mature peptide, an N-terminal 6×histidine tag (6×His tag or simply His tag) and a SUMO solubility-promoting polypeptide; the amino acid sequence of the Japanese eel Activin A recombinant protein is shown in SEQ ID NO: 1; the amino acid sequence of the Japanese eel Activin A 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, with an amino acid sequence of HHHHHHSSGLVPRGSHMAS; the 6×His tag (HHHHHH) was used to perform nickel column affinity chromatography on the protein to purify the Japanese eel Activin A recombinant protein, further improving the purity of the recombinant protein; 13 amino acids SSGLVPRGSHMAS were added as a connecting peptide between the His tag and the SUMO soluble peptide to prevent the His tag from being shielded by the SUMO soluble peptide and the Japanese eel Activin A mature peptide, allowing 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 solubility-promoting polypeptide will not affect the activity of the Japanese eel Activin A 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 recombinant protein of Japanese eel Activin A, comprising: a nucleotide sequence encoding an N-terminal His tag, a connecting peptide between the His tag and the SUMO solubilizing polypeptide, a SUMO solubilizing polypeptide, and a mature peptide of Japanese eel Activin A; the nucleotide 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 the coding gene of the Japanese eel Activin A 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 Activin A recombinant protein, comprising the following steps:

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

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

[0024] (3) By renaturing inclusion bodies, misfolded insoluble inclusion bodies are converted into soluble proteins with correct structures;

[0025] (4) Affinity chromatography based on the His tag in the Japanese eel Activin A recombinant protein to obtain a recombinant protein of higher purity (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, the protein can be enriched and concentrated using an ultrafiltration tube).

[0027] Another object of the present invention is to provide an application of the Japanese eel Activin A 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 technical problems and defects in the relevant existing technologies is:

[0030] (1) Constructing a Japanese eel Activin A recombinant protein expression vector and expressing it in an Escherichia coli prokaryotic expression system to obtain a Japanese eel Activin A recombinant protein with biological activity;

[0031] (2) E. coli lacks the eukaryotic endomembrane system for post-translational modification and processing, and the expressed Activin A recombinant protein loses its activity due to misfolding;

[0032] (3) The cytoplasm of E. coli is reducing, which is not conducive to the formation of disulfide bonds in the recombinant Activin A protein.

[0033] The significance of overcoming the problems and defects of the related prior art lies in obtaining a structurally correct, biologically active recombinant protein of Japanese eel Activin A, which can be applied to basic molecular biology research and the artificial propagation and production of Japanese eels, providing a theoretical basis and technical support for ovarian development and artificial propagation of Japanese eels. The Japanese eel Activin A recombinant protein of the present invention can be induced to express in large quantities in Escherichia coli and, through inclusion body renaturation, rapidly and efficiently obtain the biologically active recombinant protein, saving time and costs, and can be effectively applied to industrial production, thus possessing significant commercial value.

[0034] (1) The present invention provides a Japanese eel Activin A recombinant protein, comprising: an amino acid sequence of a Japanese eel Activin A mature peptide, 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. The His tag is used to purify the Japanese eel Activin A recombinant protein, thereby improving the purity of the recombinant protein to meet higher application requirements; the SUMO solubilizing polypeptide can be used to inhibit the aggregation of the Japanese eel Activin A 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 recombinant protein;

[0035] (2) The present invention constructs a prokaryotic expression vector pET-His-SUMO-ActivinA for the Japanese eel Activin A recombinant protein, and transfects the vector into Rosetta-gami B (DE3) Escherichia coli to obtain the pET-His-SUMO-ActivinA-Rosetta-gami B (DE3) expression strain, which can induce the expression of Japanese eel Activin A recombinant protein in large quantities, and efficiently obtains soluble recombinant protein through inclusion body renaturation, and has biological activity without the need for tag removal;

[0036] (3) The Japanese eel Activin A recombinant protein provided by the present invention can be effectively applied to the research on ovarian development and artificial reproduction of Japanese eels.

[0037] 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:

[0038] (1) The present invention provides for the first time a Japanese eel Activin A recombinant protein having the same amino acid sequence as the Japanese eel Activin A mature peptide, comprising: the Japanese eel Activin A mature peptide amino acid sequence, an N-terminal 6×histidine tag (6×His tag or simply His tag), a SUMO solubilizing peptide, and a connecting peptide between the 6×histidine tag and the SUMO solubilizing peptide;

[0039] (2) The present invention is the first to construct a prokaryotic expression vector pET-His-SUMO-ActivinA for the Japanese eel Activin A recombinant protein, and transfect the vector into Rosetta-gami B (DE3) Escherichia coli. The resulting pET-His-SUMO-ActivinA-Rosetta-gami B (DE3) expression strain can induce the expression of Japanese eel Activin A recombinant protein in large quantities. The recombinant protein with a purity of 80% to 90% can be obtained through inclusion body renaturation (if a higher purity recombinant protein is required, His tag affinity chromatography can be performed to obtain a recombinant protein with a purity higher than 90%).

[0040] (3) The addition of SUMO solubilizing peptide to the prokaryotic expression vector pET-His-SUMO-ActivinA of the Japanese eel Activin A recombinant protein constructed by the present invention can inhibit the aggregation of the 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 Activin A recombinant protein;

[0041] (4) The present invention provides for the first time a method for preparing a recombinant protein of Japanese eel Activin A based on the recombinant protein. The recombinant protein of the present invention can be induced to express in large quantities through an Escherichia coli prokaryotic expression system, and a soluble recombinant protein can be efficiently obtained through inclusion body renaturation, and the protein exhibits biological activity without the need for tag removal.

[0042] (5) Application of the recombinant protein provided by the present invention or the prepared Japanese eel Activin A recombinant protein in the study of Japanese eel ovarian development and artificial reproduction.

[0043] Third, as auxiliary evidence for the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0044] (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 Activin A recombinant protein having an amino acid sequence consistent with the Japanese eel Activin A mature peptide, and the method can be used to obtain a target protein with biological activity in large quantities and efficiently, which can be effectively applied to industrial production and has important commercial value.

[0045] (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 Activin A in related research at home and abroad. The present invention is the first to construct the prokaryotic expression vector pET-His-SUMO-ActivinA of the recombinant protein of Japanese eel Activin A, and transfect the vector into Rosetta-gami B (DE3) Escherichia coli. The obtained pET-His-SUMO-ActivinA-Rosetta-gami B (DE3) expression strain can induce the expression of Japanese eel Activin A recombinant protein in large quantities, and obtain Japanese eel Activin A recombinant protein efficiently and in large quantities through inclusion body renaturation.

[0046] (3) The technical solution of the present invention solves a technical problem that people have always been eager to solve but have never been able to achieve success: the artificial breeding technology of Japanese eels is a global problem that has not yet been solved. The low quality of eggs produced by artificially induced broodstock is one of the main difficulties currently faced. Therefore, it is urgent to carry out research on improving the quality of Japanese eel eggs. The quality of eggs depends on the accumulation of yolk substances during folliculogenesis, and Activin A is a key influencing factor in regulating yolk development and plays a decisive role in the accumulation of yolk substances. This shows that Activin A has an important function in the oogenesis and ovarian development of fish. Therefore, exploring the role of Activin A in the ovarian development of Japanese eels can provide a theoretical basis and technical support for the realization of artificial breeding of eels. The Japanese eel Activin A recombinant protein obtained by the method of 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

[0047] Figure 1 This is a flow chart of a method for preparing Japanese eel Activin A recombinant protein provided by an embodiment of the present invention;

[0048] Figure 2 Schematic diagram of the structure of the Japanese eel Activin A protein and its gene open reading frame (ORF) cloning provided in an embodiment of the present invention;

[0049] Figure 2A is a schematic diagram of the composition of Japanese eel Activin A provided in an embodiment of the present invention, which includes a signal peptide of 19 amino acids, a precursor peptide of 273 amino acids, and a mature peptide of 116 amino acids; wherein, aa: amino acid;

[0050] Figure 2 B is a schematic diagram of the ORF sequence of Japanese eel Activin A and its translated amino acid sequence provided in an embodiment of the present invention, wherein the dark gray area is the mature peptide amino acid sequence;

[0051] Figure 3 This is a diagram of the Japanese eel Activin A recombinant protein expression vector, the composition of the recombinant protein, and the ORF sequence of the recombinant protein provided in an embodiment of the present invention;

[0052] Figure 3 A is a map of the Japanese eel Activin A recombinant protein expression vector provided in an embodiment of the present invention; wherein, 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;

[0053] Figure 3 B is a schematic diagram of the structure of the Japanese eel Activin A recombinant protein provided in an embodiment of the present invention;

[0054] Figure 3 C is a schematic diagram of the Japanese eel Activin A 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 Activin A mature peptide;

[0055] Figure 4 This is an SDS-PAGE gel electrophoresis diagram of the expression, inclusion body renaturation, and enrichment of the Japanese eel Activin A recombinant protein provided in an embodiment of the present 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: total protein (including the target protein) after inclusion body renaturation and enrichment; the black arrow indicates the Japanese eel Activin A recombinant protein band (molecular weight ~27 kDa);

[0056] Figure 5The present invention provides an embodiment of the Japanese eel Activin A recombinant protein to promote FITC fluorescently labeled vitellogenin into the oocyte experiment results obtained by observing under a fluorescence microscope; wherein: the first row is an intact Japanese eel oocyte in the vitellogenic stage, and the second row is a ruptured Japanese eel oocyte in the vitellogenic stage (punctured with a pin to facilitate observation of the FITC fluorescence amount); Control: control group oocytes, FITC fluorescently labeled vitellogenin was not added to the culture medium; T: three treatment groups, FITC fluorescently labeled vitellogenin was added to the culture medium; PBS: PBS group oocytes, 1×PBS was added to the culture medium as a negative treatment; hCG: hCG (human chorionic gonadotropin) group oocytes, hCG was added to the culture medium as a positive treatment; Activin A: Activin A (Japanese eel Activin A recombinant protein provided by the present invention) group oocytes, Japanese eel Activin A recombinant protein provided by the present invention was added to the culture medium;

[0057] Figure 6 This is a FITC / β-actin grayscale statistical graph corresponding to the Western blot provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions 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 intended to limit the present invention.

[0059] An embodiment of the present invention provides a Japanese eel Activin A recombinant protein, comprising: a Japanese eel Activin A mature peptide amino acid sequence, an N-terminal 6×histidine tag (6×His tag or simply His tag) and a SUMO solubilizing polypeptide; the amino acid sequence of the Japanese eel Activin A recombinant protein is shown in SEQ ID NO: 1; the amino acid sequence of the Japanese eel Activin A mature peptide is shown in SEQ ID NO: 2.

[0060] SEQ ID NO: 1:

[0061] MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQ IGGGLECDGKIRICCKKQFHVNFKDIGWSDWIIAPPGYHANYCEGDCPSHVASITGSSLSFHSTVINHYRMRGYTPFTNIKSCCVPTRLRAMSMLYYNEEHKIVKKDIQNMVVEECGCS

[0062] SEQ ID NO: 2:

[0063] GLECDGKIRICCKKQFHVNFKDIGWSDWIIAPPGYHANYCEGDCPSHVASITGSSLSFHSTVINHYRMRGYTPFTNIKSCCVPTRLRAMSMLYYNEEHKIVKKDIQNMVVEECGCS

[0064] A His tag and a connecting peptide were added to the N-terminus of the recombinant protein, with an amino acid sequence of HHHHHHSSGLVPRGSHMAS (SEQ ID NO: 4); the 6×His tag (HHHHHH) was used to purify the Japanese eel Activin A recombinant protein by affinity chromatography on a nickel column, 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-soluble peptide to prevent the His tag from being shielded by the SUMO-soluble peptide and the Japanese eel Activin A mature peptide, allowing it to be fully exposed to the solution, thereby facilitating affinity chromatography purification of the recombinant protein after renaturation.

[0065] A SUMO solubilizing peptide was added to the N-terminus, with the sequence of 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 the renaturation process, and improve the efficiency of inclusion body renaturation.

[0066] The N-terminal His tag and SUMO solubility-promoting polypeptide will not affect the activity of the Japanese eel Activin A recombinant protein, and there is no need to remove the tag.

[0067] An embodiment of the present invention provides a gene encoding a recombinant protein of Japanese eel Activin A, comprising: a nucleotide sequence encoding an N-terminal His tag, a connecting peptide between the His tag and a SUMO solubilizing polypeptide, a SUMO solubilizing polypeptide, and a mature peptide of Japanese eel Activin A; the nucleotide sequence of the encoding gene is SEQ ID NO: 3.

[0068] SEQ ID NO: 3:

[0069] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCT TCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATT GGTGGTGGCCTGGAATGCGACGGCAAGATCCGCATCTGCTGCAAGAAGCAGTTCCACGTCAACTTCAAGGACATCGGCTGGAGCGACTGGATCATAGCGCCGCCAGGGTACCACGCCAACTACTGCGAGGGCGACTGCCCGAGCCACGTGGCCAGCATCACGGGTCTCCTCCCTTTCCT TCCACTCCACCGTCATCAACCATTACCGCATGCGGGGCTACACCCCCTTCACAACATCAAGTCCTGCTGCGTGCCCACGCGGCTGCGGGCCATGTCCATGCTCTACTACAACGAGGAGCACAAGATCGTCAAAAAGGACATCCAGAACATGGTGGTGGAGGAATGCGGCTGCTCGTAA

[0070] An embodiment of the present invention provides an expression vector, which comprises a gene encoding the Japanese eel Activin A recombinant protein and a backbone plasmid, wherein the backbone plasmid is transformed from pET-32a(+).

[0071] An embodiment of the present invention provides a recombinant engineered bacterium, which comprises the expression vector.

[0072] Furthermore, the host bacteria of the recombinant engineered bacteria is selected from Rosetta-gami B (DE3).

[0073] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing Japanese eel Activin A recombinant protein, comprising the following steps:

[0074] (1) constructing the Japanese eel Activin A recombinant protein encoding gene, and connecting it to a backbone plasmid to construct an expression vector for the Japanese eel Activin A recombinant protein;

[0075] (2) transforming the expression vector into a host bacterium to induce expression of the Japanese eel Activin A recombinant protein;

[0076] (3) Converting misfolded insoluble inclusion bodies into structurally correct, biologically active soluble proteins through inclusion body renaturation;

[0077] (4) Based on the His tag in the Japanese eel Activin A recombinant protein, a higher purity recombinant protein 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);

[0078] (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, the protein can be enriched and concentrated using an ultrafiltration tube).

[0079] Example 1: Construction of Japanese eel Activin A recombinant protein expression vector pET-His-SUMO-ActivinA

[0080] 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 Activin A gene (inhbaa) Figure 2The fragment was ligated into the pCE2 TA / BluntZero vector (Novagen, Nanjing) via TOPO ligation and transfected into Escherichia coli (Novagen, Nanjing). Single colonies were screened on ampicillin LB solid medium, and sequence accuracy was verified by Sanger sequencing and DNA MAN. PCR amplification using the aforementioned Japanese eel inhbaa monoclonal culture as a template yielded a double-stranded DNA fragment corresponding to the mature peptide of Japanese eel Activin A with homology arms. The modified backbone plasmid pET-HS (pET-32a(+) plasmid containing a His tag and SUMO solubility-promoting peptide) was linearized using restriction endonucleases. The PCR products and double enzyme digestion products were purified using FastPure Gel DNA Extraction Mini Kit (Novagen, Nanjing). The insert fragment and linear vector were connected using a homologous recombination kit (Novagen, Nanjing) and transfected into DH5α competent Escherichia coli. Single colonies were selected in ampicillin LB solid medium. After confirmation by Sanger sequencing and DNA MAN comparison, the plasmid was extracted using a plasmid mini-extraction kit (Tiangen, Beijing), which is the prokaryotic expression vector pET-His-SUMO-ActivinA ( Figure 3 ).

[0081] Example 2: Preparation and induction of expression of Escherichia coli expression strain of Japanese eel Activin A recombinant protein

[0082] The expression vector pET-His-SUMO-ActivinA was transfected into Rosetta-gami B (DE3) competent Escherichia coli (Angyu, Shanghai), and cultured overnight in ampicillin-chloramphenicol double-resistance LB solid medium at 37°C to select single colonies, which were the prokaryotic expression engineering bacteria pET-His-SUMO-ActivinA-Rosetta-gami B (DE3) of Japanese eel Activin A recombinant protein. A single colony was picked and cultured in 10 mL of ampicillin-chloramphenicol double-antibody LB liquid medium at 37 ° C, 220 rpm / min overnight, and then inoculated into 200 mL of LB liquid medium containing ampicillin-chloramphenicol double-antibody 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 a final concentration of 0.6 mM (10 mL of bacterial solution was taken out as a control before induction), and cultured at 37 ° C, 220 rpm / min for 4 h. After centrifugation at 3500g for 10 min, the bacteria were collected and the supernatant was discarded. The bacteria were resuspended with 20 mL of lysis buffer (500 mM NaCl, 20 mM sodium phosphate, 10 mM imidazole, pH 7.4), 60W ultrasonic disruption for 30 min, disruption for 5 s and stop for 5 s, and centrifuged at 12000g for 10 min to collect the supernatant and precipitate, respectively. The precipitate was resuspended with 20 mL of lysis buffer, and 80 μL sample and 20 μL were taken respectively. Mix with 5× protein loading buffer, incubate at 95℃ for 10min, perform SDS-PAGE and Coomassie brilliant blue staining to observe protein bands ( Figure 4 The results showed that the Japanese eel Activin A recombinant protein could be expressed in large quantities and existed in the precipitate as misfolded inclusion bodies, which were insoluble proteins.

[0083] Example 3: Washing, renaturation, protein purification and enrichment of Japanese eel Activin A recombinant protein inclusion bodies

[0084] The precipitate obtained in Example 2 (the Japanese eel Activin A recombinant protein was present in the precipitate as insoluble inclusion bodies due to structural errors caused by misfolding) was washed with 20 mL of inclusion body washing solution ① (500 mM NaCl, 20 mM Tris-HCl, 2% (V / V), pH = 7.4). After resuspending by vortexing, the protein was ultrasonically disrupted at 60 W for 10 min, followed by disruption for 5 s and rest for 5 s. The protein was centrifuged at 12,000 g for 10 min, and the supernatant was discarded. The precipitate was washed with 20 mL of inclusion body washing solution ② (500 mM NaCl, 20 mM Tris-HCl, 2 M urea, pH = 7.4). After resuspending by vortexing, the protein was ultrasonically disrupted at 60 W for 10 min, followed by disruption for 5 s and rest for 5 s. The protein was centrifuged at 12,000 g for 10 min, and the supernatant was discarded. These two washes can remove some hydrophobic lipoproteins, bacterial debris, genomic DNA, etc., and the purity of the inclusion bodies can reach 80% to 90%.

[0085] To the washed inclusion bodies, 10 mL of inclusion body solubilization solution (100 mM NH4Cl, 50 mM Tris-HCl, 8 M urea, 10% (V / V), pH = 8.0) was added, and DTT solution was added to a final concentration of 10 nM. After resuspending by oscillation, the mixture was ultrasonically disrupted at 60 W for 10 min, disrupted for 5 s and rested for 5 s. After centrifugation at 12000 g for 15 min, the supernatant was retained to obtain the denatured and solubilized Japanese eel Activin A recombinant protein. Pour 20-50 mL of the diluted refolding solution (50 mM NaCl, 50 mM Tris-HCl, 0.4 M L-arginine, 1 M urea, 5 mM reduced glutathione, 1 mM oxidized glutathione, 10% glycerol (V / V), pH 8.0) into a glass bottle. Place a magnetic rotor and add the dissolved inclusion bodies dropwise to a final concentration of approximately 0.05 mg / mL. (Use a magnetic stirrer to continuously stir during this process to quickly disperse the protein and prevent aggregation.) Place the glass bottle in a 4°C refrigerator for 4 hours to allow the denatured protein to completely fold. Continue adding the dissolved inclusion bodies to a final concentration of approximately 0.1 mg / mL. Repeat this process for a total of four additions, increasing the concentration by 0.05 mg / mL each time. After the final refolding step, place the solution at 4°C overnight to allow the protein to fully fold. This will yield a structurally correct soluble recombinant protein of Japanese eel Activin A.

[0086] If you need to obtain a higher purity of Japanese eel Activin A 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) to perform His tag affinity purification to obtain Japanese eel Activin A recombinant protein with a purity of >90%. The obtained recombinant protein solution is poured into a protein dialysis bag (molecular weight cutoff: 3500D), dialyzed into 1×PBS solution, and a small amount of protein is taken to determine the protein concentration by BCA method. The remaining protein solution is added with bovine serum albumin (BSA) at a final concentration of 0.1% to stabilize the recombinant protein and prevent precipitation. After aliquoting, it is quickly frozen with liquid nitrogen and stored at -80°C. If you need to obtain a high concentration of Japanese eel Activin A recombinant protein, you can use an ultrafiltration tube to enrich and concentrate the recombinant protein after dialysis ( Figure 4 Sample 5).

[0087] Example 4: Japanese eel Activin A recombinant protein promotes Japanese eel yolk accumulation

[0088] After isolating Japanese eel vitellogenic oocytes, FITC fluorescently labeled vitellogenin was not added to the culture medium of the control group (Control group), FITC fluorescently labeled vitellogenin was added to the treatment group (T), and 1×PBS (PBS group) was added as a negative treatment, human chorionic gonadotropin hCG (hCG group) was added as a positive treatment, and the Japanese eel Activin A recombinant protein provided by the present invention (Activin A group) was added. After culturing for 24 hours, the fluorescence intensity in the oocytes of each group was observed under a fluorescence microscope. The results are as follows: Figure 5 As shown, there was no FITC fluorescence in the Control group, and FITC fluorescence was observed in all three treatment groups. However, the FITC fluorescence in the PBS group was weaker than that in the hCG group and the Activin A group, indicating that the Japanese eel Activin A recombinant protein provided by the present invention can promote the accumulation of Japanese eel yolk.

[0089] Protein was extracted from oocytes of the Control group, PBS group, and Activin A group. Western Blot experiments were performed with FITC as the target protein and β-actin as the internal reference. Statistical analysis of the FITC / β-actin grayscale was performed. The results showed that the FITC / β-actin grayscale in the Activin A group was significantly higher than that in the Control group and PBS group ( Figure 6 ), which also shows that the Japanese eel Activin A recombinant protein provided by the present invention can promote the accumulation of Japanese eel yolk.

[0090] Example 5: Development of a fish reproductive health detection kit based on Japanese eel Activin A recombinant protein

[0091] In this example, recombinant Activin A protein from Japanese eel was used to develop a fish reproductive health test kit. This kit can detect Activin A levels in fish, helping farmers assess fish reproductive health and adjust their management strategies in a timely manner.

[0092] The specific steps include:

[0093] 1. Prepare specific antibodies for detecting Activin A.

[0094] 2. Develop detection methods based on ELISA or other appropriate technologies for use in the construction of test kits.

[0095] 3. Verify the sensitivity, specificity and stability of the kit.

[0096] 4. Apply the kit to actual fish reproductive health testing, providing farmers or researchers with fast and accurate test results.

[0097] Example 6: Construction of recombinant engineered bacteria expressing recombinant proteins

[0098] Recombinant engineered bacteria are used to produce specific recombinant proteins for medical or industrial applications.

[0099] The target gene (eg, a gene encoding a specific enzyme or functional protein) is inserted into the expression vector of the present invention, digested with restriction endonucleases, and ligated with T4 DNA ligase to form a complete expression vector.

[0100] The expression vector contains a strong promoter (such as T7 promoter) and a fusion tag (such as His tag) for efficient expression and protein purification.

[0101] Rosetta-gami B was used as the host bacteria, the expression vector was electroporated into the host bacteria, and positive clones were screened in LB medium containing antibiotics.

[0102] Positive clones were selected and cultured until OD600 reached 0.6, and 0.5 mM IPTG was added to induce expression.

[0103] After 4 hours of induction, the expression of the target protein was confirmed by SDS-PAGE and Western blot.

[0104] The recombinant protein was purified using a Ni-NTA affinity column, and the purity and activity were verified.

[0105] The target protein is efficiently expressed in a soluble form, with a purity of over 95% after purification, and the enzyme activity is basically consistent with that of the natural protein.

[0106] Example 7: Application of recombinant engineered bacteria to produce biologically active small molecules

[0107] Use recombinant engineered bacteria to synthesize biologically active small molecules for use in pharmaceutical research and development (such as anti-tumor drugs).

[0108] The expression vector according to claim 3 is constructed, and multiple key enzyme genes (such as acyltransferase, polyketide synthase, etc.) in the target metabolic pathway are loaded to achieve synthetic metabolism through multi-gene tandem connection.

[0109] The expression vector was electroporated into the Rosetta-gami B host bacteria, and the successfully transformed recombinant strain was obtained by antibiotic screening and PCR verification.

[0110] The positive recombinant bacteria were inoculated into the optimized culture medium, cultured at 37°C until the logarithmic growth phase, and an inducer (such as IPTG) was added to induce the expression of enzymes related to the metabolic pathway.

[0111] After culturing for 24 hours, the culture medium was collected and extracted.

[0112] The target bioactive small molecule products in the culture medium were detected by HPLC and mass spectrometry, and their chemical structure and purity were confirmed.

[0113] The recombinant engineered bacteria successfully synthesized the target bioactive small molecules (such as antibiotics or anti-tumor drug precursors) with a yield of 1g / L, which is significantly higher than the traditional fermentation method.

[0114] The above examples demonstrate the wide application of the expression vector described in claim 3 and the Rosetta gami B host bacteria, showing excellent performance from efficient recombinant protein expression to complex metabolite synthesis, providing a reliable tool for the medical and industrial fields.

[0115] The strain is public and commercialized and can be purchased from reagent companies.

[0116] The above description is only 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 any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A Japanese eel Activin A recombinant protein, characterized in that: The invention comprises: an amino acid sequence of a mature peptide of Japanese eel Activin A, a 6×histidine tag at the N-terminus, and a SUMO solubility-promoting polypeptide; the amino acid sequence of the Japanese eel Activin A recombinant protein is shown in SEQ ID NO:

1.

2. A gene encoding the Japanese eel Activin A recombinant protein according to claim 1, characterized in that: The invention comprises: a nucleotide sequence 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 Activin A 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 ActivinA 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, comprising 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 Activin A recombinant protein according to claim 1, characterized in that: The following steps are involved: (1) constructing the Japanese eel Activin A recombinant protein encoding gene, and connecting it to a backbone plasmid to construct an expression vector for the Japanese eel Activin A recombinant protein; (2) transforming the expression vector into a host bacterium to induce expression of the Japanese eel Activin A recombinant protein; (3) By renaturing inclusion bodies, misfolded insoluble inclusion bodies are converted into soluble proteins with correct structures; (4) Affinity chromatography of the His tag in the Japanese eel Activin A recombinant protein was used to obtain a higher purity 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 Activin A recombinant protein according to claim 6, wherein: After dialysis into 1× PBS, the protein can be enriched and concentrated using an ultrafiltration tube to obtain a higher concentration of protein.

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