Anguilla japonica Activin A recombinant protein as well as expression method and application thereof

Abstract

The invention belongs to but not limited to the technical field of genetic engineering, and discloses an Anguilla japonica Activin A recombinant protein, and an expression method and application thereof. The anguilla japonica Activin A recombinant protein comprises an anguilla japonica Activin A mature peptide amino acid sequence, a 6 * histidine tag at the N end and SUMO dissolution promoting polypeptide; the amino acid sequence of the anguilla japonica Activin A recombinant protein is SEQ ID NO: 1, and the nucleotide sequence of the coding gene of the anguilla japonica Activin A recombinant protein is SEQ ID NO: 3. The prokaryotic expression vector pET-His-SUMO-Activin A of the Japanese eel Activin A is constructed for the first time, the Activin A recombinant protein of which the amino acid sequence is consistent with the mature peptide of the Japanese eel Activin A can be obtained, and the Activin A 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 Activin A 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 all the seedlings required for breeding rely on natural fishing, this predatory breeding method has caused the natural eel seedling resources to decline year by year. The shortage of eel seedlings has restricted the further development of the eel industry. Therefore, it is particularly important to carry out research on artificial breeding technology of eels to protect natural resources and my country's eel farming industry. However, there are technical bottlenecks that need to be solved in the successful artificial breeding of Japanese eels.

[0003] Since the 1930s, artificial breeding technology for 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. 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, so as to improve the quality of eel eggs under artificial breeding.

[0004] Activin A can promote the expression of follicle-stimulating hormone. It is specifically secreted by follicular layer cells in the ovary and is a very important ovarian regulatory factor. Activin A plays a vital role in the transition from primary follicles to secondary follicles and in the process of vitellogenesis. In the study of zebrafish, it was found that in the absence of Activin A, there was no accumulation of yolk granules in zebrafish oocytes, indicating that Activin A plays an important regulatory role in the development of the ovary. Like other vertebrates, Japanese eel Activin A is a classic secretory protein with a typical N-terminal signal peptide sequence. 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 parked on the surface of the rough endoplasmic reticulum together and continue to translate, while the signal peptide is removed. The nascent peptide chain enters the endoplasmic reticulum while being synthesized, and finally folded and processed by the endoplasmic reticulum and Golgi apparatus, and secreted outside the cell. The two monomers form homodimers through disulfide bonds to function.

[0005] 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 and low cost. By using a strong promoter, foreign proteins can be expressed at high levels in Escherichia coli, thereby achieving the purpose of rapid and large-scale production of foreign proteins. However, since the rate at which Escherichia coli expresses foreign proteins is much higher than that of eukaryotic cells, it is easy to cause the nascent peptide chain to misfold and form inactive inclusion bodies.

[0006] Inclusion bodies are a common problem in prokaryotic expression. The misfolding of the peptide chain causes the protein structure to become abnormal and lose 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 biologically active proteins. 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.

[0007] In view of the above analysis, the technical problems that need to be solved urgently in the prior art 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 recombinant Activin A proteins;

[0010] (3) 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-concentration Activin A recombinant protein.

[0011] (4) The recombinant protein expression rate of Escherichia coli is too fast, and there is no endoplasmic reticulum, Golgi apparatus and other organelles for processing and modification, which can easily lead to the inability of the newly formed peptide chain to fold correctly, thereby forming inactive inclusion bodies. Summary of the invention

[0012] In view of 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 more particularly relates to a Japanese eel Activin A recombinant protein produced by an Escherichia coli expression system and obtainable in large quantities by means of inclusion body renaturation, and its application in the study of Japanese eel ovarian development.

[0013] The present invention is achieved in this way: a Japanese eel Activin A recombinant protein comprises: a Japanese eel Activin A 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 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, and the amino acid sequence was 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, thereby 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 Activin A 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 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 soluble polypeptide, a SUMO soluble 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 a gene encoding 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 Japanese eel Activin A 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 recombinant protein with higher purity based on the His tag affinity chromatography in the Japanese eel Activin A 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 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, expressing it through an Escherichia coli prokaryotic expression system, and obtaining a Japanese eel Activin A recombinant protein with biological activity;

[0031] (2) E. coli lacks the eukaryotic inner membrane 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 solving the problems and defects in the related prior art is to obtain a Japanese eel Activin A recombinant protein with correct structure and biological activity, which can be applied to basic research in molecular biology and the artificial breeding and production process of Japanese eels, and provide a theoretical basis and technical support for the ovarian development and artificial breeding of Japanese eels. The Japanese eel Activin A recombinant protein of the present invention can be induced to express in large quantities by Escherichia coli, and a recombinant protein with biological activity can be obtained quickly and efficiently by inclusion body renaturation, which can save time and cost, can be effectively applied to industrial production, and has important 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, a 6×histidine tag at the N-terminus (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 is used to purify the Japanese eel Activin A recombinant protein, which can improve 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 of Japanese eel Activin A recombinant protein, and transfects the vector into Rosetta-gami B (DE3) Escherichia coli to obtain a 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 obtain soluble recombinant protein by inclusion body renaturation, and has biological activity without the need to remove the tag;

[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 breeding 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 soluble polypeptide, and a connecting peptide between the 6×histidine tag and the SUMO soluble polypeptide;

[0039] (2) The present invention is the first to construct a prokaryotic expression vector pET-His-SUMO-ActivinA of 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, and a recombinant protein with a purity of 80% to 90% can be obtained through inclusion body renaturation (if a 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%).

[0040] (3) Adding SUMO solubilizing polypeptide 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 Japanese eel Activin A recombinant protein based on the recombinant protein. The recombinant protein of the present invention can be induced to express in large quantities through the Escherichia coli prokaryotic expression system, and the soluble recombinant protein can be efficiently obtained through inclusion body renaturation, and has biological activity without the need to remove the tag;

[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 ovarian development and artificial reproduction of Japanese eels.

[0043] Third, as auxiliary evidence of 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 whose amino acid sequence is consistent with the Japanese eel Activin A mature peptide, and the method can be used to obtain a large amount of target protein with biological activity in a high-efficiency manner, 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 is no relevant report 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 the 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 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 produced by artificially induced spawning 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 occurrence 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 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 The structure of the Japanese eel Activin A protein and the schematic diagram of the gene open reading frame (ORF) cloning provided in the embodiments of the present invention are shown;

[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 the examples of the present invention, and the dark gray area is the mature peptide amino acid sequence;

[0051] Figure 3 It 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 the embodiments 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 The figure is an SDS-PAGE gel electrophoresis diagram of the expression, inclusion body renaturation and concentration enrichment of the Japanese eel Activin A recombinant protein provided by the 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 target protein) after inclusion body renaturation and concentration; the black arrow indicates the Japanese eel Activin A recombinant protein band (molecular weight ~27kDa);

[0056] Figure 5The results of the experiment in which the Japanese eel Activin A recombinant protein provided by the embodiment of the present invention promotes FITC fluorescently labeled vitellogenin to enter the oocyte are observed under a fluorescence microscope; wherein: the first row is a complete Japanese eel oocyte in the vitellogenetic stage, and the second row is a ruptured Japanese eel oocyte in the vitellogenetic stage (punctured with a pin to facilitate observation of the FITC fluorescence quantity); Control: oocytes in the control group, no FITC fluorescently labeled vitellogenin was added to the culture medium; T: three treatment groups, FITC fluorescently labeled vitellogenin was added to the culture medium; PBS: oocytes in the PBS group, 1×PBS was added to the culture medium as a negative treatment; hCG: oocytes in the hCG (human chorionic gonadotropin) group, hCG was added to the culture medium as a positive treatment; Activin A: oocytes in the Activin A (Japanese eel Activin A recombinant protein provided by the present invention) group, and the Japanese eel Activin A recombinant protein provided by the present invention was added to the culture medium;

[0057] Figure 6 It 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 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.

[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 His tag for short) 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, and the amino acid sequence was HHHHHHSSGLVPRGSHMAS (SEQID NO: 4); the 6×His tag (HHHHHH) was used for affinity chromatography of the recombinant protein through a nickel column to purify the Japanese eel Activin A 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 soluble polypeptide as a connecting peptide to prevent the His tag from being shielded by the SUMO soluble polypeptide and the Japanese eel Activin A mature peptide so that the His tag can be fully exposed to the solution, so as to facilitate affinity chromatography purification of the recombinant protein after renaturation.

[0065] 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.

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

[0067] The embodiment of the present invention provides a gene encoding a Japanese eel Activin A recombinant protein, 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 Japanese eel Activin A mature peptide; the nucleotide sequence of the encoding gene is SEQ ID NO: 3.

[0068] SEQ ID NO:3:

[0069] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCT TCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATT GGTGGTGGCCTGGAATGCGACGGCAAGATCCGCATCTGCTGCAAGAAGCAGTTCCACGTCAACTTCAAGGACATCGGCTGGAGCGACTGGATCATAGCGCCGCCAGGGTACCACGCCAACTACTGCGAGGGCGACTGCCCGAGCCACGTGGCCAGCATCACGGGTCTCCTCCCTTTCCT TCCACTCCACCGTCATCAACCATTACCGCATGCGGGGCTACACCCCCTTCACAAACATCAAGTCCTGCTGCGTGCCCACGCGGCTGCGGGCCATGTCCATGCTCTACTACAACGAGGAGCACAAGATCGTCAAAAAGGACATCCAGAACATGGTGGTGGAGGAATGCGGCTGCTCGTAA

[0070] The embodiment of the present invention provides an expression vector, which comprises a coding gene of 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, wherein the recombinant engineered bacterium 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 Japanese eel Activin A recombinant protein;

[0076] (3) The misfolded insoluble inclusion bodies are converted 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 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);

[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, an ultrafiltration tube can be used to enrich and concentrate the protein).

[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 2). It was connected to the pCE2 TA / BluntZero vector (Novagen, Nanjing) by TOPO ligation and transfected into Escherichia coli (Novagen, Nanjing). Single colonies were screened in ampicillin LB solid medium. Sanger sequencing and DNA MAN were used to identify whether the sequence was correct. PCR amplification was performed using the above Japanese eel inhbaa monoclonal bacterial solution as a template to obtain a double-stranded DNA fragment corresponding to the mature peptide of Japanese eel Activin A 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 product and double-enzyme digestion product were purified using FastPure Gel DNA Extraction Mini Kit (Novuzan, Nanjing), and the insert fragment and linear vector were connected using a homologous recombination kit (Novuzan, Nanjing) and transfected into DH5α competent Escherichia coli. Single colonies were screened with ampicillin LB solid medium. After Sanger sequencing and DNA MAN comparison, the plasmid was extracted using a plasmid mini-extraction kit (Tiangen, Beijing), which is the Japanese eel Activin A recombinant protein prokaryotic expression vector pET-His-SUMO-ActivinA ( Figure 3 ).

[0081] Example 2: Preparation and induction of expression of Escherichia coli strain expressing 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 at 37°C in ampicillin-chloramphenicol double-resistance LB solid medium to select single colonies, which was the Japanese eel Activin A recombinant protein prokaryotic expression engineering bacteria pET-His-SUMO-ActivinA-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 3500 g for 10 min, and the supernatant was discarded. The bacteria were resuspended with 20 mL of bacterial lysis buffer (500 mM NaCl, 20 mM sodium phosphate, 10 mM imidazole, pH 7.4), and ultrasonically disrupted at 60 W for 30 min, disrupted for 5 s and stopped for 5 s, and centrifuged at 12000 g for 10 min to collect the supernatant and precipitate, 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 and Coomassie Brilliant Blue staining ( Figure 4 The results showed that the Japanese eel Activin A 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.

[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 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 Activin A 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 suspended 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 suspended 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%.

[0085] To the washed inclusion bodies, 10 mL of inclusion body dissolution 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 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 ActivinA 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, glycerol 10% (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). Place the glass bottle in a 4°C refrigerator for 4h to allow the denatured protein to completely fold, and continue to add the dissolved inclusion bodies to a final concentration of about 0.1mg / mL..., repeat this process, add a total of 4 times, increase the concentration by 0.05mg / mL each time, and place the last refolding at 4°C overnight to allow the protein to fully fold, and you can get a soluble Japanese eel Activin A recombinant protein with the correct structure.

[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) for His tag affinity purification to obtain Japanese eel Activin A 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 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 the Japanese eel vitellogenic oocytes were isolated, 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 respectively. 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, but 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 ActivinA 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 embodiment, the Japanese eel Activin A recombinant protein is used to develop a fish reproductive health detection kit. This kit can detect the level of Activin A in fish, help farmers assess the reproductive health of fish, and adjust the breeding management strategy in time.

[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 constructing test kits.

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

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

[0097] Example 6: Construction of recombinant engineering bacteria expressing recombinant protein

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

[0099] The target gene (such as a gene encoding a specific enzyme or functional protein) is inserted into the expression vector of the present invention, and the restriction endonuclease is used to cut it and the T4 DNA ligase is used to connect it 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 more than 95% after purification, and the enzyme activity is basically consistent with the natural protein.

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

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

[0108] The expression vector of claim 3 is constructed, and multiple key enzyme genes (such as acyltransferase, polyketide synthase, etc.) in the target metabolic pathway are loaded to achieve anabolism 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 24 hours of cultivation, the culture fluid 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 and Rosetta-gami B host bacteria described in claim 3, which exhibit excellent performance from efficient recombinant protein expression to complex metabolite synthesis, and provide a reliable tool for the medical and industrial fields.

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

[0116] 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 modifications, equivalent substitutions and improvements 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 Activin A recombinant protein, characterized in that: It comprises: an amino acid sequence of Japanese eel Activin A mature peptide, a 6×histidine tag at the N-terminus and a SUMO solubilizing 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 as claimed in 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; and 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 as claimed in 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 Japanese eel Activin A recombinant protein; (3) Through inclusion body refolding, the misfolded insoluble inclusion bodies are converted into soluble proteins with correct structure; (4) Affinity chromatography based on the His tag in the Japanese eel Activin A recombinant protein was used to obtain a recombinant protein with higher purity; (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, characterized in that: After dialysis into 1× PBS, the protein can be enriched and concentrated using an ultrafiltration tube to obtain a higher protein concentration.

8. Use of the Japanese eel Activin A recombinant protein as claimed in claim 1 in regulating the ovarian development of Japanese eel.

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