CN114057897B - Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application - Google Patents
Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application Download PDFInfo
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- CN114057897B CN114057897B CN202111568274.7A CN202111568274A CN114057897B CN 114057897 B CN114057897 B CN 114057897B CN 202111568274 A CN202111568274 A CN 202111568274A CN 114057897 B CN114057897 B CN 114057897B
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a sebastes schlegeli hilgendorf IL-6 recombinant protein, a soluble expression method and application, wherein the sebastes schlegeli hildorf IL-6 recombinant protein comprises an IL-6 mature peptide amino acid sequence, an N-terminal solubilizing short peptide and a C-terminal 6 × histidine tag; preferably, the amino acid sequence of the IL-6 recombinant protein is SEQ ID NO:1, the amino acid sequence of the IL-6 mature peptide is SEQ ID NO:2, the DNA sequences of the coded N-terminal short peptide, the amino acid of the IL-6 mature peptide, the C-terminal connecting peptide and the His tag are SEQ ID NO:3. the invention firstly constructs a prokaryotic expression vector pET-IL6-His of sebastes schlegeli IL-6, can obtain the sebastes schlegeli IL-6 recombinant protein with the amino acid sequence consistent with IL-6 mature peptide, and can be used for spawning induction and seedling raising of sebastes schlegeli.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a sebastes schlegeli IL-6 recombinant protein, a soluble expression method and application.
Background
At present, sebastes schlegelii belongs to the Sebastes schlegelii (Scorpaeniformes), sebastes schlegelii (Sebastes), sebastes (Sebastes), and is one of important cold-warm economic fishes in the north of China. Sebastes schlegeli breeding mode is spawning, the male and female development is asynchronous, the male fish is sexually mature (the female fish is not mature at the moment) in 11 months every year, the male and female fish complete mating in 11-12 months, the sperms can be stored in the female fish body for 4 months, the ovaries of the female fish are mature in 3-4 months in the next year, in-vivo fertilization occurs, the embryos develop in the female fish body for about 1 month, and the juvenile fish is produced in the late 4 months to the early 5 months.
Parturition of oviparous teleost teleosts belongs to typical spontaneous inflammatory reaction, interleukin 6 (interleukin 6, IL-6) belongs to a typical inflammatory factor, and relative expression levels of IL-6 gene (IL 6) and IL-6R gene (IL 6R) of a receptor thereof reach the highest level in parturition and are remarkably reduced after 24 hours of parturition (P < 0.05). IL-6 can directly up-regulate the expression level of prostaglandin synthetase COX2 gene (ptgs 2) through a JAK/STAT3 cell signal path, improve the level of prostaglandin and indirectly regulate the progress of labor. In the breeding of sebastes schlegeli hilgendorf, female fish are in poor state and are easy to dystocia and die. The progress of labor can therefore be facilitated by injection of IL-6 protein. Like other vertebrates, sebastes schlegeli IL-6 is a classical secreted protein with a typical N-terminal signal peptide sequence. In the cell, once synthesized, the signal peptide of the nascent peptide chain is recognized by a Signal Recognition Particle (SRP) and translation is suspended, and the SRP, the peptide chain and the ribosome together anchor to the surface of the rough endoplasmic reticulum, and translation is continued. The nascent peptide chain enters the endoplasmic reticulum as it is synthesized and the signal peptide is cleaved. Finally, the peptide chain is folded and processed by endoplasmic reticulum and Golgi apparatus, and IL-6 without signal peptide is secreted to the outside of cells. In addition, sebastes schlegeli IL-6 has 1 disulfide bond structure.
In the field of genetic engineering, escherichia coli (Escherichia coli) is a commonly used engineering bacterium of a prokaryotic expression system, belongs to facultative anaerobic gram-negative bacteria, and has the advantages of short growth cycle, clear genetic background, simple culture operation, low cost, rapid and large-scale production of target protein and the like. By using a strong promoter, the expression of the foreign protein can be expressed at a high level in the escherichia coli, and the maximum expression amount can reach 40-50% of the total protein of the thalli. However, the rate of expressing the exogenous protein by escherichia coli is far higher than that of eukaryotes, the expression rate of recombinant protein is too high, a nascent peptide chain cannot be correctly folded in time, and hydrophobic parts of misfolded proteins are exposed and aggregated with each other to form an inactive inclusion body.
Inclusion body formation is a common problem in prokaryotic expression. At present, prokaryotic expression of recombinant IL-6 protein of animals such as human, guinea pig and the like has been reported. However, in these reports, the IL-6 recombinant protein is inactive inclusion body, and complicated renaturation operation is required to restore the biological activity. In view of the tedious and time-consuming operation of inclusion body renaturation, the denaturant urea can cause adverse effect on the amino covalent modification of the recombinant protein. If the recombinant protein which can induce escherichia coli to express solubility and activity can greatly simplify the operation flow of protein purification, save production cost and time and have important practical significance.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) The recombinant protein expression rate of the escherichia coli is too high, a new peptide chain cannot be correctly folded in time, and the hydrophobic parts of the protein which is incorrectly folded are exposed and mutually aggregated to form an inactive inclusion body.
(2) At present, prokaryotic expression of recombinant IL-6 protein of animals such as human beings, guinea pigs and the like is reported, but in the report, the recombinant IL-6 protein is an inactive inclusion body, and the bioactivity can be recovered by fussy renaturation operation.
(3) In view of the tedious and time-consuming operation of inclusion body renaturation, the denaturant urea can cause adverse effect on the amino covalent modification of the recombinant protein.
The difficulty in solving the above problems and defects is: coli lacks the endomembrane system of eukaryotes and post-translational modifications, and the expressed IL-6 recombinant protein is often misfolded without activity. Moreover, the cytoplasm of Escherichia coli is reductive, which is not favorable for formation of IL-6 disulfide bond.
The significance for solving the problems and the defects is as follows: disulfide bonds are critical to the structure of IL-6, and if disulfide bonds can be formed correctly and soluble, active recombinant IL-6 can be induced, the protein purification steps can be greatly shortened, and the time and reagent cost can be greatly saved in production.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a sebastes schlegeli hilgendorf IL-6 recombinant protein, a soluble expression method and application thereof, and particularly relates to a sebastes schlegeli hilgendorf IL-6 (IL-6) recombinant protein which is produced by an escherichia coli expression system and has solubility and bioactivity, and application thereof in sebastes schlegeli hilgendorf breeding.
The invention is realized in such a way, the sebastes schlegeli IL-6 recombinant protein comprises an IL-6 mature peptide amino acid sequence, an N-terminal solubilizing short peptide and a C-terminal 6 × histidine tag (6 × His tag or His tag for short); preferably, the amino acid sequence of the IL-6 recombinant protein is SEQ ID NO:1.
further, the amino acid sequence of the mature IL-6 peptide is SEQ ID NO:2.
further, the shorter, negatively charged short peptide; preferably, the N-terminal short peptide sequence is MGDEDNSG; more preferably, the C-terminal connecting peptide and His-tag sequence is gtsgngsghhhhhhhhhhhhhh.
Further, the DNA sequences of the coded N-terminal short peptide, IL-6 mature peptide amino acid, C-terminal connecting peptide and His label are SEQ ID NO:3.
the invention also aims to provide an expression vector for expressing the IL-6 recombinant protein of sebastes schlegeli, which comprises a coding gene of the IL-6 recombinant protein and a skeleton plasmid, wherein the skeleton plasmid is obtained by modifying pET-32a (+).
The invention also aims to provide the recombinant engineering bacterium for expressing the IL-6 recombinant protein of sebastes schlegeli hilgendorf, and the recombinant engineering bacterium comprises the expression vector.
Further, the host bacteria of the recombinant engineering bacteria are selected from Rosetta-gami B (DE 3). Rosetta-gami B (DE 3) is the name of the complete expression strain and refers to the bacterium Escherichia coli Rosetta-gami B, which has the lysogenic properties of the DE3 segment of the lambda phage genome. DE3 in parentheses is semantically a supplementary description rather than a paraphrase and cannot be said to be Rosetta-gami B, DE3.
Another object of the present invention is to provide a method for solublizing expression of sebastes schlegeli IL-6 recombinant protein using said sebastes schlegeli IL-6 recombinant protein, said method for solublizing expression of sebastes schlegeli IL-6 recombinant protein comprising the following steps:
(1) After constructing the IL-6 recombinant protein coding gene, connecting the coding gene to a skeleton plasmid to construct an expression vector of the IL-6 recombinant protein;
(2) Transforming the expression vector into host bacteria, and inducing and expressing the IL-6 recombinant protein;
(3) Based on the His label affinity chromatography separation of the IL-6 recombinant protein, obtaining the IL-6 recombinant protein;
(4) Dialyzing the purified protein solution to 1 XPBS, adding bovine serum albumin, and preserving at-80 ℃; or desalting, lyophilizing, and storing at-20 deg.C.
Further, the final concentration of bovine serum albumin BSA in step (3) is 0.1%.
The invention also aims to provide application of the sebastes schlegeli IL-6 recombinant protein in sebastes schlegeli hilgendorf breeding.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a sebastes schlegeli IL-6 recombinant protein, which comprises the following components: the mature peptide amino acid sequence of IL-6, short peptide for promoting solubility connected to N end, and 6 × histidine tag peptide segment connected to C end. The invention also provides a gene for encoding the recombinant protein, a vector for expressing the recombinant protein, recombinant engineering bacteria, a method for preparing sebastes schlegeli hilgendorf IL-6 protein based on the recombinant protein, and application of the recombinant protein or the prepared sebastes schlegeli hilgendorf IL-6 protein in sebastes schlegeli hilgendorf breeding. The recombinant protein can be used for obtaining the soluble recombinant protein through large-scale induction expression of prokaryotic bacteria, is favorable for subsequent industrialized extraction and purification, has biological activity without removing a label after purification, and can be effectively used for breeding sebastes schlegeli.
The invention constructs a prokaryotic expression vector pET-IL6-His of sebastes schlegeli IL-6 for the first time, and the vector is transfected into Rosetta-gami B (DE 3) escherichia coli to obtain pET-IL6-His/Rosetta-gami B (DE 3) expression strain, and soluble recombinant protein can be obtained through large-scale inducible expression through 37 ℃ amplification, 0 ℃ cold shock and 16 ℃ inducible expression; after affinity purification by histidine tag, recombinant protein with purity up to 90% can be obtained, and the purified protein has bioactivity without cutting off the tag. By using the method, the sebastes schlegeli IL-6 recombinant protein with the amino acid sequence consistent with IL-6 mature peptide can be obtained; in production, the operation of purifying soluble protein is simple and convenient, time and reagent are saved, compared with the conventional denaturation and dilution renaturation of inclusion bodies, the time for purifying IL-6 soluble protein can be saved by 1 to 2 days, a large amount of urea is not required to be consumed for renaturation, and the yield is about 20mg/L of culture medium.
In the breeding of the sebastes schlegeli hilgendorf offspring, because the sebastes schlegeli hilgendorf are fertilized in vivo and spawned fish in ova fetuses, the breeding synchronization is difficult to achieve in production, and the labor and material cost is increased due to the asynchronous spawning of female fish. By injecting IL-6 recombinant protein, the effect of induced spawning is achieved, the synchronization of farrowing is realized, and the seedling raising cost is greatly saved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic diagram of the structure of sebastes schlegeli IL-6 protein and the cloning of its gene Open Reading Frame (ORF) provided in the embodiment of the present invention.
Fig. 1A is a schematic diagram of sebastes schlegeli IL-6 composed of a signal peptide of 26 amino acids and a mature peptide of 211 amino acids provided by the embodiment of the present invention; wherein, aa: an amino acid.
FIG. 1B is an ORF sequence of IL-6 and its translated amino acid sequence provided by the present invention, and the light gray region is a schematic diagram of the amino acid sequence of the mature peptide.
FIG. 2 is a map of the general vector pET-HTH provided in the examples of the present invention, which was modified from pET-32a (+).
FIG. 2A is a schematic diagram of the composition of the pET-HTH vector provided in the examples of the present invention.
FIG. 2B is a schematic diagram showing the sequence around the pET-HTH polyclonal cleavage site provided in the examples of the present invention;
in the figure: MCS (modulation and coding scheme): a polyclonal cleavage site; lacO: a lactose operon manipulation sequence; RBS: a ribosome binding site; and (4) LacI: lactose operon regulatory gene I; rop: a primer repressor gene; pBR322 Origin: pBR322 origin of replication; ampR: ampicillin resistance gene.
Fig. 3 is a schematic diagram of an expression vector map of sebastes schlegeli IL-6 recombinant protein, a recombinant protein composition and a recombinant protein ORF sequence provided by the embodiment of the invention.
Fig. 3A is a map of an IL-6 recombinant protein expression vector of sebastes schlegeli according to the embodiment of the present invention.
FIG. 3B is a schematic diagram of the structure of the IL-6 recombinant protein provided in the embodiment of the present invention.
FIG. 3C is a schematic diagram of the ORF of the recombinant protein and its translated amino acid sequence (with part of the codons optimized) provided by the embodiment of the invention; the light gray area is IL-6 mature peptide, the dark gray area is His label;
in the figure: lacO: a lactose operon manipulation sequence; RBS: a ribosome binding site; lacI: lactose operon regulatory gene I; rop: a primer repressor gene; pBR322 Origin: pBR322 origin of replication; ampR: ampicillin resistance gene.
FIG. 4 is an SDS-PAGE gel electrophoresis image of IL-6 recombinant protein expression and purification provided by the embodiment of the invention;
in the figure: m: protein molecular weight standards; 1: IPTG induces pre-bacterial total protein; 2: bacterial total protein after induction for 14h at 0.3mM IPTG16 ℃;3: precipitating the bacterial lysate after induction; 4: supernatant of the induced bacterial lysate; 5: NTA-Ni affinity purification flow-through; 6: washing the impurity liquid; 7: eluent (containing target protein, purity > 90%); the black arrow indicates the IL-6 recombinant protein band (molecular weight 26 kDa).
Fig. 5 is a schematic diagram of changes in the relative expression levels of the downstream gene ptgs2 after the primary cells of sebastes schlegeli hilgendorf and the stimulation of different concentrations of IL-6 recombinant protein, which are provided by the embodiment of the invention.
Fig. 5A is a morphological schematic diagram of spleen primary cells of sebastes schlegeli provided by the embodiment of the present invention.
FIG. 5B is a schematic diagram showing the relative expression level change of the downstream gene ptgs2 after stimulation by IL-6 recombinant protein of 0-500 ng/mL in the embodiment of the present invention.
Fig. 6 is a flowchart of a solubilizing expression method of sebastes schlegeli IL-6 recombinant protein according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a sebastes schlegeli hilgendorf IL-6 recombinant protein, a soluble expression method and application thereof, and the invention is described in detail with reference to the accompanying drawings.
As shown in fig. 6, the soluble expression method of sebastes schlegeli IL-6 recombinant protein provided by the embodiment of the present invention comprises the following steps:
s101, after constructing the IL-6 recombinant protein coding gene, connecting the coding gene to a skeleton plasmid to construct an expression vector of the IL-6 recombinant protein;
s102, transforming the expression vector into host bacteria, and carrying out induced expression on the IL-6 recombinant protein at 16 ℃;
s103, separating based on His label affinity chromatography of the IL-6 recombinant protein to obtain the IL-6 recombinant protein;
s104, dialyzing the purified protein solution to 1 XPBS, adding bovine serum albumin with the final concentration of 0.1 percent, and preserving at-80 ℃; or desalting, lyophilizing, and storing at-20 deg.C.
The invention provides a method for performing soluble induction expression on an IL-6 recombinant protein of sebastes schlegeli hilgendorf, which comprises the following steps: IL-6 recombinant protein amino acid sequence, and IL-6 prokaryotic expression vector construction method and protein purification mode. Preferably, the recombinant amino acid sequence of IL-6 is SEQ ID NO:1, mgdedngsveddvidptpvsgeggeblevwpsdlssqywdivietkrrhqqqefenfqneceneveyifyinspalmipcpsnfskeacllhrllaqglylvlylkvhvekeypssqarlylinlikmrnqpvtatqtqetlqlyldndfdmahsffldragiskruskratglcnldsrstnsfrxhtmihslldihshyhlhhh 1
The amino acid sequence of the mature IL-6 peptide is SEQ ID NO:2: <xnotran> vedlptdspvsgepsgeeevwpsdllsssqywdivieatkrhqqefenefqneveyiflehyrisslpagcppsnfskeaclhrlaqgllvytvllkhvekeypsssihsqaryysnilinlikekmrnpeqvtaptssqetqllrdldnsdtfqrrmtahsimrklhfflidgkraiskrektrgsmanrstptisfyiqklkdgsinql. </xnotran>
In one embodiment of the invention, the negative charge short peptide solubility promoting sequence MGDEDNSG is added at the N end of the recombinant protein, so that protein coagulation can be inhibited, soluble expression of the protein can be promoted, and the expression level can be improved.
In one embodiment according to the invention, a linking peptide is added to the C-terminus of the recombinant protein along with a His-tag, and the amino acid sequence is gtsgngsghhhhhhhhhhhhhhhh. The 6 × His tag (HHHHHHHH) can be used for protein nickel column affinity chromatography purification; the 8 amino acids GTSGNGSG is added between the IL-6 and the His tag to serve as a connecting peptide, so that the His tag is prevented from being shielded by the IL-6 and can be sufficiently exposed to a solution, and affinity purification of the recombinant protein is facilitated under non-denaturing conditions.
The N-terminal short peptide label and the C-terminal short peptide label are small, protein activity is not affected, and the labels do not need to be cut off.
The invention also provides the coding gene of the IL-6 recombinant protein, which comprises the following components: the peptide comprises N-terminal short peptide, IL-6 mature peptide, C-terminal connecting peptide and His label; the nucleotide sequence of the amino acid of the coding IL-6 mature peptide is SEQ ID NO:3.
The invention further provides an expression vector for expressing the IL-6 recombinant protein, which comprises the coding gene of the IL-6 recombinant protein and a skeleton plasmid, wherein the skeleton plasmid is obtained by modifying pET-32a (+).
The invention further provides a recombinant engineering bacterium for expressing the IL-6 recombinant protein, which comprises the expression vector; preferably, the host bacterium of the recombinant engineered bacterium is selected from Rosetta-gami B (DE 3). The Rosetta-gami B (DE 3) gor gene and TrxB gene are double mutated to make the cytoplasm of the cells oxidized, which is favorable for the formation of disulfide bonds of the recombinant IL-6. Rosetta-gami B (DE 3) Escherichia coli also contains pRARE plasmid with chloramphenicol resistance, provides 6 kinds of rare codons of Escherichia coli, and can improve the expression level of exogenous genes (or artificially synthesize IL-6 gene to improve the expression level after optimizing IL-6 codon).
The invention provides an optimized soluble protein induction method, which comprises the steps of adding 0.2% of glucose into a culture medium to antagonize a lactose metabolic pathway of escherichia coli, inhibiting background expression of the escherichia coli at the stage of 37 ℃ spread culture, and preventing the expression from generating insoluble inclusion bodies; the bacterial liquid is subjected to cold shock at 0 ℃ to promote the intracellular generation of components such as mannose and the like, so that the folding of the protein is facilitated, the induced expression at 16 ℃ is performed, the protein folding rate can be delayed at a lower temperature, and the generation of soluble protein by correct folding is facilitated.
The open reading frame sequence of sebastes schlegeli hilgendorf IL-6cDNA provided by the invention is SEQ ID NO:4: <xnotran> atgccttctcatctcaactcgtcctggctctctgcggtgatgctggcagctctgttgctgtgtgctcccggtgctccggttgaagacttgcccaccgacagcccggtgtcaggtgagccctcaggtgaggaggaggtgtggccctctgacctgctgagctcctctcagtactgggacatagtcatcgaagcaaccaaacgccaccagcaggagtttgaaaatgaattccaaaatgaggtggaatatatttttctggagcactacaggatctcctcacttccagcaggctgccctccctccaacttcagcaaggaggcttgtctccacaggttggcacaaggcctgctggtttacacggttcttctcaagcatgtggagaaggagtaccccagcagctccatccactcacaggccaggtactacagcaacatcctgatcaacctcatcaaagaaaagatgaggaaccctgaacaggtcacggcaccgaccagcagccaggagacgcagctgctgagggacctcgacaactccgacactttccagagaaggatgaccgcacacagcatcatgcgcaagctccacttcttcctcatcgacggcaaaagagcgatctctaaaagggagaagaccagaggaagtatggccaacaggagcacgccaaccatcagtttctatatccaaaagttaaaagatgggagcatcaaccagttatga; </xnotran> Sebastes schlegeli IL-6 signal peptide + mature peptide sequence is SEQ ID NO:5: <xnotran> mpshlnsswlsavmlaalllcapgapvedlptdspvsgepsgeeevwpsdllsssqywdivieatkrhqqefenefqneveyiflehyrisslpagcppsnfskeaclhrlaqgllvytvllkhvekeypsssihsqaryysnilinlikekmrnpeqvtaptssqetqllrdldnsdtfqrrmtahsimrklhfflidgkraiskrektrgsmanrstptisfyiqklkdgsinql. </xnotran>
The technical solution of the present invention is further described below with reference to specific examples.
Example 1: construction of Sebastes schlegeli IL-6 expression vector pET-IL6-His
With sebastes schlegeli hilgendorus cDNA as a template, PCR was performed using a primer O-IL6-F/R (Producer, shanghai) and a reagent 2 × Phanta Max Master Mix (Nodezaar, nanjing) to amplify a DNA sequence of the Open Reading Frame (ORF) of sebastes schlegeli hilgendorus IL-6 gene (IL 6) (see FIG. 1). This was ligated into pCE2 TA/Blunt Zero vector (Novozan, nanjing) by TOPO ligation to transfect DH 5. Alpha. E.coli (Novozan, nanjing), ampicillin solid medium was screened for single colonies, sanger sequencing, NCBI BLAST to identify the sequence as correct. The sebastes schlegeli hilgendorf IL6 monoclonal bacterial liquid is used as a template, primers PE-IL6-F/R and 2 x Phanta Max Master Mix are used for PCR amplification, and a double-stranded DNA fragment corresponding to the IL-6 mature peptide with the homology arm is obtained (except for adopting a cloning technical means, a corresponding sequence can also be artificially synthesized after codon optimization according to an amino acid sequence). The backbone plasmid pET-HTH (see FIG. 2) was linearized by a double-restriction with the restriction endonucleases Nde I and Kpn I. Using FastPure Gel DNA Extraction Mini Kit (Novozan, nanjing) to recover PCR products and double enzyme digestion product Gel, using a homologous recombination Kit (Novozan, nanjing) to connect an insert fragment and a linear vector, transfecting DH5 alpha competent escherichia coli, screening single colonies on an ampicillin solid culture medium, using a plasmid miniprep Kit (Tiangen, beijing) to extract plasmids (D in figure 5) after confirming no errors by Sanger sequencing, namely obtaining the Schleichert IL-6 prokaryotic expression vector pET-IL6-His (see figure 3).
TABLE 1 sequence chart of primers used for vector construction
Wherein the nucleotide sequence of the primer O-il6 is SEQ ID NO:6: ttcttccagcagcagcagcagcagctc
ttcttccagcagcagcagcagcagctc, primer PE-il6 has the nucleotide sequence of SEQ ID NO:7: agaaggagatatagatatgggtgatgaagagatactctgggttggaagactttgcacccacc
gttaccagaggtacctaactggttgatgctcccatc。
Note: the primer lower case positive body shows a homologous arm and is used for constructing a prokaryotic expression vector by homologous recombination connection; the lower case italics shows the corresponding codon of the short peptide for promotion of solubility, which is supplemented at the 5' end of the gene sequence of the mature peptide of IL-6.
Example 2: preparation and small-amount induction expression of sebastes schlegeli IL-6 escherichia coli expression strain
The expression vector pET-IL6-His is transfected into Rosetta B (DE 3) competent Escherichia coli (On Yue, shanghai), and is cultured by using an ampicillin-chloramphenicol double-antibody LB solid culture medium at 37 ℃ overnight to screen a single colony, namely, sebastes schlegeli IL-6 prokaryotic expression engineering bacteria pET-IL6-His-Rosetta-gami B (DE 3). Single colonies were picked in 10mL of ampicillin-chloramphenicol double-resistant LB liquid medium at 37 ℃ overnight at 220 rpm, and then cultured according to the following protocol 1:100 percent of the total amount of the cells were inoculated into 100mL of LB liquid medium containing double antibody and 0.2 percent of glucose, cultured at 37 ℃ for 3-4 h at 220 rpm until OD600 is 0.4-0.6, then the cell liquid was placed in an ice water mixture for quenching, IPTG was added to make the final concentration 0.3mM (50 mL of the cell liquid was taken out as a control before induction), after 14h culture at 16 ℃ at 90 rpm, 3500g of the cell liquid was centrifuged for 10min, the cell liquid was collected, the supernatant was discarded, the cell liquid was resuspended in 5mL of a lysis buffer (500mM NaCl, 2mM PB,10mM imidazole, pH 7.4), 60W was sonicated for 30min, 5s of a superscalar was stopped, 120g of the cell liquid was centrifuged for 10min, the supernatant and the precipitate were collected separately, 5mL of the lysis buffer was resuspended in 5mL of a lysis buffer, 80. Mu.L of the sample and 20. Mu.L of 5 Xprotein loading buffer were collected separately, and the precipitate was mixed in metal bath for 95 ℃ 10min, SDS-PAGE and Coomassie blue staining was performed to observe protein bands (sample 1-4 in FIG. 4). The results show that: the IL-6 recombinant protein can be correctly expressed, and is mainly present in supernatant and is soluble protein.
Example 3: sebastes schlegeli IL-6 recombinant protein soluble expression and protein purification
According to the following steps of 1:100 percent of the total protein is inoculated into 500mL LB liquid culture medium containing double antibody and 0.2 percent of glucose, the culture is carried out for 3-4 h at 37 ℃ and 220 r/min until the OD600 is 0.4-0.6, IPTG is added to lead the final concentration to be 0.3mM,16 ℃ and 12h at 90 r/min, then 3500g is centrifuged for 10min to collect thalli, the thalli are discarded, the thalli are resuspended by 50mL of non-denaturing schizomycete buffer solution, 60W is ultrasonically crushed for 30min, 5s of ultrasound is stopped for 5s,12000g is centrifuged for 10min to discard precipitates, the supernatant is retained, a 0.45 mu m or 0.22 mu m filter membrane is used for filtering to remove insoluble micro-particles, and the His label affinity purification is carried out by NTA-Ni filler (Biyun, shanghai), thus obtaining the IL-6 recombinant protein with the purity of more than 90 percent (shown as samples 4-7 in figure 4).
Filling the purified IL-6 recombinant protein solution into a protein dialysis bag (with an aperture MWCO 500), dialyzing to 1 XPBS solution, taking a small amount of protein, determining the protein concentration by using a BCA method, adding Bovine Serum Albumin (BSA) with the final concentration of 0.1% into the residual protein solution to stabilize the recombinant protein and prevent coagulation, and storing at-80 ℃ after subpackaging. If the storage conditions are limited, the protein can also be desalted by using a desalting column and then freeze-dried, and the protein can be stored at the temperature of minus 20 ℃.
Example 4: sebastes schlegeli IL-6 recombinant protein activated inflammation-related cell pathway NF-kappa B
After primary cells of spleen of sebastes schlegeli hilgendorf were isolated, the cell concentration was adjusted to 5X 10 using L-15 medium containing 10% Fetal Bovine Serum (FBS) 5 Cells/ml, then 1ml per well in 12-well plates, and incubated in an incubator at 25 ℃ (see A in FIG. 5). Culturing for 24-48 h, changing to 1mL FBS-free L15 culture medium for starvation for 8, adding 10% FBS culture medium, adding IL-6 solution into each well of the treated group respectively to a final concentration of 0/5/50/500ng/mL, culturing for 3h in an incubator, extracting cell RNA by TRIzol method, reverse transcription, and fluorescenceThe relative expression level of the mRNA of the downstream gene ptgs2 regulated by the IL-6 is quantitatively measured, and the result shows that the expression level of the ptgs2 is remarkably increased after IL-6 recombinant protein with the concentration of 50-500 ng/mL stimulates cells (see B in figure 5).
The above description is only for the purpose of illustrating the embodiments of the present invention, and the scope of the present invention should not be limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed in the present invention should be covered by the scope of the present invention.
Sequence listing
<110> China oceanic university
<120> Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application
<141> 2021-12-21
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Leu Arg Asp Leu Asp Asn Ser Asp Thr Phe Gln Arg Arg Met Thr Ala
145 150 155 160
His Ser Ile Met Arg Lys Leu His Phe Phe Leu Ile Asp Gly Lys Arg
165 170 175
Ala Ile Ser Lys Arg Glu Lys Thr Arg Gly Ser Met Ala Asn Arg Ser
180 185 190
Thr Pro Thr Ile Ser Phe Tyr Ile Gln Lys Leu Lys Asp Gly Ser Ile
195 200 205
Asn Gln Leu
210
<210> 3
<211> 684
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgggtgttg aagatctgcc aactgattct ccggtgagcg gcgaaccgag cggtgaagag 60
gaggtgtggc cctctgacct gctgagctcc tctcagtact gggacatagt catcgaagca 120
accaaacgcc accagcagga gtttgaaaat gaattccaaa atgaggtgga atatattttt 180
ctggagcact acaggatctc ctcacttcca gcaggctgcc ctccctccaa cttcagcaag 240
gaggcttgtc tccacaggtt ggcacaaggc ctgctggttt acacggttct tctcaagcat 300
gtggagaagg agtaccccag cagctccatc cactcacagg ccaggtacta cagcaacatc 360
ctgatcaacc tcatcaaaga aaagatgagg aaccctgaac aggtcacggc accgaccagc 420
agccaggaga cgcagctgct gagggacctc gacaactccg acactttcca gagaaggatg 480
accgcacaca gcatcatgcg caagctccac ttcttcctga tcgacggcaa aagagcgatt 540
agcaaacgcg aaaaaacccg cggcagcatg gcgaaccgta gcaccccgac cattagcttt 600
tatattcaga aactgaaaga tggcagcatc aaccagctgg gtacctctgg taacggttct 660
ggccatcacc atcaccacca ctaa 684
<210> 4
<211> 714
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgccttctc atctcaactc gtcctggctc tctgcggtga tgctggcagc tctgttgctg 60
tgtgctcccg gtgctccggt tgaagacttg cccaccgaca gcccggtgtc aggtgagccc 120
tcaggtgagg aggaggtgtg gccctctgac ctgctgagct cctctcagta ctgggacata 180
gtcatcgaag caaccaaacg ccaccagcag gagtttgaaa atgaattcca aaatgaggtg 240
gaatatattt ttctggagca ctacaggatc tcctcacttc cagcaggctg ccctccctcc 300
aacttcagca aggaggcttg tctccacagg ttggcacaag gcctgctggt ttacacggtt 360
cttctcaagc atgtggagaa ggagtacccc agcagctcca tccactcaca ggccaggtac 420
tacagcaaca tcctgatcaa cctcatcaaa gaaaagatga ggaaccctga acaggtcacg 480
gcaccgacca gcagccagga gacgcagctg ctgagggacc tcgacaactc cgacactttc 540
cagagaagga tgaccgcaca cagcatcatg cgcaagctcc acttcttcct catcgacggc 600
aaaagagcga tctctaaaag ggagaagacc agaggaagta tggccaacag gagcacgcca 660
accatcagtt tctatatcca aaagttaaaa gatgggagca tcaaccagtt atga 714
<210> 5
<211> 237
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Pro Ser His Leu Asn Ser Ser Trp Leu Ser Ala Val Met Leu Ala
1 5 10 15
Ala Leu Leu Leu Cys Ala Pro Gly Ala Pro Val Glu Asp Leu Pro Thr
20 25 30
Asp Ser Pro Val Ser Gly Glu Pro Ser Gly Glu Glu Glu Val Trp Pro
35 40 45
Ser Asp Leu Leu Ser Ser Ser Gln Tyr Trp Asp Ile Val Ile Glu Ala
50 55 60
Thr Lys Arg His Gln Gln Glu Phe Glu Asn Glu Phe Gln Asn Glu Val
65 70 75 80
Glu Tyr Ile Phe Leu Glu His Tyr Arg Ile Ser Ser Leu Pro Ala Gly
85 90 95
Cys Pro Pro Ser Asn Phe Ser Lys Glu Ala Cys Leu His Arg Leu Ala
100 105 110
Gln Gly Leu Leu Val Tyr Thr Val Leu Leu Lys His Val Glu Lys Glu
115 120 125
Tyr Pro Ser Ser Ser Ile His Ser Gln Ala Arg Tyr Tyr Ser Asn Ile
130 135 140
Leu Ile Asn Leu Ile Lys Glu Lys Met Arg Asn Pro Glu Gln Val Thr
145 150 155 160
Ala Pro Thr Ser Ser Gln Glu Thr Gln Leu Leu Arg Asp Leu Asp Asn
165 170 175
Ser Asp Thr Phe Gln Arg Arg Met Thr Ala His Ser Ile Met Arg Lys
180 185 190
Leu His Phe Phe Leu Ile Asp Gly Lys Arg Ala Ile Ser Lys Arg Glu
195 200 205
Lys Thr Arg Gly Ser Met Ala Asn Arg Ser Thr Pro Thr Ile Ser Phe
210 215 220
Tyr Ile Gln Lys Leu Lys Asp Gly Ser Ile Asn Gln Leu
225 230 235
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttcttccagc agcagcagct cttcttccag cagcagcagc tc 42
<210> 7
<211> 94
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
agaaggagat atacatatgg gtgatgaaga taactctggt gttgaagact tgcccaccgt 60
taccagaggt acctaactgg ttgatgctcc catc 94
Claims (6)
1. Sebastes schlegeli IL-6 recombinant protein is characterized in that the Sebastes schlegeli IL-6 recombinant protein comprises an IL-6 mature peptide amino acid sequence, an N-terminal lytic short peptide and a C-terminal 6 × histidine tag;
the amino acid sequence of the IL-6 mature peptide is SEQ ID NO:2.
2. the expression vector of sebastes schlegeli hilgendorf IL-6 recombinant protein as claimed in claim 1, wherein said expression vector comprises coding gene of said IL-6 recombinant protein and skeleton plasmid, and said skeleton plasmid is obtained by modifying pET-32a (+).
3. A recombinant engineering bacterium for expressing the Sebastes schlegeli IL-6 recombinant protein as claimed in claim 1, wherein the recombinant engineering bacterium comprises the expression vector.
4. The recombinant engineering bacterium of sebastes schlegeli IL-6 recombinant protein of claim 3, wherein the host bacterium of said recombinant engineering bacterium is selected from Rosetta-gami B (DE 3).
5. The method for solubly expressing sebastes schlegeli hilgendorf IL-6 recombinant protein as claimed in claim 1, wherein said method for solubly expressing sebastes schlegeli hilgendorf IL-6 recombinant protein comprises the following steps:
(1) After constructing the IL-6 recombinant protein coding gene, connecting the coding gene to a skeleton plasmid to construct an expression vector of the IL-6 recombinant protein;
(2) Transforming the expression vector into host bacteria, and inducing and expressing the IL-6 recombinant protein;
(3) Based on the His label affinity chromatography separation of the IL-6 recombinant protein, obtaining the IL-6 recombinant protein;
(4) Dialyzing the purified protein solution to 1 XPBS, adding bovine serum albumin, and preserving at-80 ℃; or desalting, lyophilizing, and storing at-20 deg.C.
6. The method for solubly expressing sebastes schlegeli hilgendorf IL-6 recombinant protein of claim 5, wherein the final concentration of bovine serum albumin BSA in step (4) is 0.1%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000166539A (en) * | 1998-12-03 | 2000-06-20 | Tosoh Corp | Il-6 receptor.il-6 fused protein |
| CN101805404A (en) * | 2009-12-31 | 2010-08-18 | 大连水产学院 | Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof |
| CN102735841A (en) * | 2011-04-13 | 2012-10-17 | 苏州卫生职业技术学院 | Method for detecting content of soluble CD28 in blood of patients suffering Graves disease |
| CN112979781A (en) * | 2021-03-18 | 2021-06-18 | 中国海洋大学 | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof |
-
2021
- 2021-12-21 CN CN202111568274.7A patent/CN114057897B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000166539A (en) * | 1998-12-03 | 2000-06-20 | Tosoh Corp | Il-6 receptor.il-6 fused protein |
| CN101805404A (en) * | 2009-12-31 | 2010-08-18 | 大连水产学院 | Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof |
| CN102735841A (en) * | 2011-04-13 | 2012-10-17 | 苏州卫生职业技术学院 | Method for detecting content of soluble CD28 in blood of patients suffering Graves disease |
| CN112979781A (en) * | 2021-03-18 | 2021-06-18 | 中国海洋大学 | Sebastes schlegeli IL-1 beta recombinant protein and preparation method and application thereof |
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