CN115161328A - Recombinant rat gamma-interferon and preparation method thereof - Google Patents

Recombinant rat gamma-interferon and preparation method thereof Download PDF

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CN115161328A
CN115161328A CN202210402687.6A CN202210402687A CN115161328A CN 115161328 A CN115161328 A CN 115161328A CN 202210402687 A CN202210402687 A CN 202210402687A CN 115161328 A CN115161328 A CN 115161328A
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recombinant
gamma
interferon
rat
ifn
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席勇强
叶健
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Yakoin Wuhan Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention provides a recombinant rat gamma-interferon and a preparation method thereof, wherein the soluble recombinant rat gamma-interferon is obtained by optimizing a gene sequence and adopting escherichia coli to induce, express and purify, the purity of the obtained recombinant rat gamma-interferon is high, and the preparation route is efficient and stable.

Description

Recombinant rat gamma-interferon and preparation method thereof
Technical Field
The invention relates to the technical field of recombinant protein expression, in particular to a recombinant rat gamma-interferon and a preparation method thereof.
Background
Interferon gamma (IFN- γ, also known as type ii interferon) is produced mainly by activated Th cells and NK cells. The IFN-gamma monomer consists of 6 alpha-helices and a C-terminal extended spreading sequence.
IFN-gamma is crucial for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. IFN-gamma can directly inhibit the virus replication ability, promote the activity of NK cells, enhance the cytotoxic effect, and stimulate neutrophils to enhance the phagocytic ability. In addition, IFN-gamma can promote some cells (such as vascular endothelial cells, some epithelial cells and connective tissue cells) which normally do not express MHC II molecules to express MHC II molecules, and play a role in antigen presentation; the intravenous endothelial cells can also have stronger adhesion capability to the neutrophils, and can be differentiated into high endothelial veins to attract circulating lymphocytes.
There are two main expression ideas of recombinant human interferon for injection: coli expression and yeast expression. The Escherichia coli expression system has the two greatest advantages of simple operation and low cost. However, the rat IFN-gamma expressed by the conventional escherichia coli is mainly in an insoluble precipitate expression form, and a small amount of active recombinant protein can be obtained only by complicated inclusion body denaturation and renaturation processes, so that the efficiency is low.
Disclosure of Invention
Therefore, it is necessary to provide a recombinant rat gamma-interferon and a preparation method thereof, which can express recombinant protein in the form of soluble supernatant by using escherichia coli, do not need complicated inclusion body denaturation and renaturation steps, and have higher efficiency and good stability.
The invention adopts the following technical scheme:
the invention provides a gene sequence for coding recombinant rat gamma-interferon, which is shown as SEQ IN NO. 2.
The invention also provides a recombinant rat gamma-interferon coded by the optimized gene sequence.
The present invention can also provide an expression vector comprising the optimized gene sequence described above.
The invention also provides a preparation method of the recombinant rat gamma-interferon, which comprises the following steps: synthesizing a gene sequence for coding recombinant Rat gamma-interferon shown as SEQ IN NO.2, cloning to a prokaryotic expression vector pET-sumo, and constructing a recombinant expression vector pET-sumo-Rat IFN gamma; transforming a recombinant expression vector pET-sumo-Rat IFN gamma into escherichia coli to form recombinant escherichia coli engineering bacteria containing a gene sequence of recombinant Rat IFN-gamma; inducing expression, breaking thallus to obtain bacterial liquid containing soluble recombinant protein; and (5) purifying and carrying out enzyme digestion to remove the label in the recombinant protein.
In some of these embodiments, the step of inducing expression is: inoculating recombinant Escherichia coli engineering bacteria containing recombinant rat IFN-gamma gene sequence into liquid LB culture medium containing kanamycin (preferably 50 mug/mL in mass concentration) for activation, and transferring the activated strain into fresh liquid LB culture medium containing kanamycin (preferably 50 mug/mL in mass concentration) for culture; when OD600 is 0.4-0.6, IPTG is added to make the final concentration 0.3mmol/L, and recombinant protein expressed by recombinant Escherichia coli is induced under the conditions of 16 ℃ and 200 r/min.
In some examples, the centrifuged culture solution is resuspended by using Binding Buffer, the thalli are crushed at 4 ℃ under 1000bar high pressure, the crushing is repeated for 2 times, and the supernatant is collected by centrifuging again to obtain the supernatant containing the soluble recombinant protein.
In some of these embodiments, the step of purifying is: under the condition that the recombinant protein is not denatured, an imidazole solution is adopted to elute a nickel column for the first time, and imidazole and foreign protein are removed through dialysis to obtain a recombinant protein solution; and then carrying out enzyme digestion by using sumo protease Ulp1 to remove the label in the recombinant protein, purifying by using a nickel column (the hybrid protein is combined on the nickel column), and obtaining the non-label recombinant rat IFN-gamma by flowing through and washing the column.
In some of these embodiments, the procedure for the first elution is: elution was carried out with 250mM imidazole and 50mM imidazole in this order.
In some of these embodiments, the purification further comprises a step of removing endotoxin from unlabeled recombinant rat IFN- γ.
The recombinant rat gamma-interferon product prepared by the preparation method of the recombinant rat gamma-interferon has the endotoxin content of less than 1 EU/mu g.
The beneficial effects of the invention are:
compared with the prior art, the invention optimizes and provides the coding gene and the expression vector of the recombinant rat gamma-interferon for the first time, realizes the efficient and stable expression of the recombinant protein in the form of soluble supernatant by depending on escherichia coli, and can obtain a high-purity (not less than 95 percent) recombinant rat gamma-interferon product through a specific purification route.
Drawings
FIG. 1 is a graph showing the expression effect of IFN-. Gamma.in rats recombined with the induced genetically engineered bacteria in example 2, in which M represents Marker and "not" represents non-induced control (1) T Represents the whole induction bacteria of No. 1 (1) S Represents the induction supernatant of No. 1 bacterium (1) P Represents the induced precipitation of the bacterium (1), (2) T Representative of the induced whole strain of bacterium No.2 (2) S Representative of the induced supernatant of bacterium (2), (2) P Represents the induction precipitation of the bacterium No. 2.
FIG. 2 is a graph showing the purification effect of recombinant Rat IFN-. Gamma.purified for the first time in example 3, in which M represents Marker, T represents Rat IFN-. Gamma.inducing whole strain, S represents Rat IFN-. Gamma.inducing supernatant, P represents Rat IFN-. Gamma.inducing precipitate, and FT represents Rat IFN-. Gamma.supernatant purified with Ni 2+ The column flows through; 20-1 represents 20mM imidazole wash-mix 1 st tube, 20-3 represents 20mM imidazole wash-mix 3 rd tube, 50-1 represents 50mM imidazole wash-mix 1 st tube, 50-3 represents 50mM imidazole wash-mix 3 rd tube, 50-5 represents 50mM imidazole wash-mix 5 th tube, 250-1 represents 250mM imidazole elution 1 st tube, 250-3 represents 250mM imidazole elution 3 rd tube, 250-5 represents 250mM imidazole elution 5 th tube, 500-1 represents 500mM imidazole elution 1 st tube, and 'Beads' represents Ni after elution 2+ And (4) column packing.
FIG. 3 is a diagram showing the effect of enzyme digestion on purification of recombinant Rat IFN-. Gamma.in example 3, wherein M represents Marker, "Pre-enzyme" represents Rat IFN-. Gamma.after dialysis, "post-enzyme P" represents precipitation of Rat IFN-. Gamma.after enzyme digestion, "FT" represents flow-through of the supernatant of Rat IFN-. Gamma.after enzyme digestion through Ni2+ column, column 1 represents Binding Buffer washing Ni2+ column 1, column 2 represents Binding Buffer washing Ni2+ column 2, 10mM-1 represents 10mM imidazole washing impurity 1, 10mM-2 represents 10mM imidazole washing impurity 2, 15mM imidazole washing impurity, 15mM represents 15mM imidazole washing impurity, 50mM-1 represents 50mM imidazole washing impurity 1, 50mM-2 represents 50mM imidazole washing impurity 2, 250mM represents 250mM imidazole elution, 500mM represents 500mM imidazole elution, and "Beads" represents Ni after elution 2+ And (4) column packing.
FIG. 4 is a graph showing the endotoxin-removing effect of IFN-. Beta.rat in example 3, wherein "2 ug after detoxification of rINFg" represents 2. Mu.g of recombinant rat IFN-. Gamma.protein after endotoxin removal, and M represents Marker.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
Example 1 construction of genetically engineered Strain
The 1-22 amino acid is the signal peptide area and the 23-156 amino acid is the protein functional structure area, and the gene sequence capable of synthesizing the 23-156 amino acid is optimized according to the rat IFN-gamma coding sequence (ACCESSION NUMBER: AF 010466.1) published by NCBI. The optimized gene sequence is synthesized by Nanjing Kingsrei biotech GmbH and cloned to a prokaryotic expression vector pET-sumo to obtain a recombinant expression vector pET-sumo-Rat IFN-gamma.
Wherein, the sequence of the natural expression gene of the rat IFN-gamma is as follows:
aaaggatcca tgagtgctac acgccgcgtc ttggttttgc agctctgcct catggccctc
tctggctgtt actgccaagg cacactcatt gaaagcctag aaagtctgaa gaactatttt
aactcaagta gcatggatgc tatggaagga aagagcctcc tcttggatat ctggaggaac
tggcaaaagg acggtaacac gaaaatactt gagagccaga ttatctcttt ctacctcaga
ctctttgaag tcttgaaaga caaccaggcc atcagcaaca acataagtgt catcgaatcg
cacctgatca ctaacttctt cagcaacagt aaagcaaaaa aggatgcatt catgagcatc
gccaagttcg aggtgaacaa cccacagatc cagcacaaag ctgtcaatga actcatcaga
gtgattcacc agctgtcacc agaatctagc ctaaggaagc ggaaaaggag tcggtgctga
ttctggggta gagagtgtgc caataagaag aattctggat ccttt(SEQ ID NO.1)
the gene sequence of the artificial synthetic rat expressing 23-156 amino acids is as follows:
caaggaacac taatagagag tttagaatca ttgaagaact atttcaacag cagcagcatg
gacgctatgg aaggtaaaag cttgctgctg gatatttggc gtaattggca aaaagacggc
aacacgaaaa tcttggagtc tcagattatc tctttttacc tgcgcctgtt cgaggtgctg
aaggacaacc aggcaatttc aaataacatc tccgtgatcg agtcccatct tatcaccaat
tttttcagca acagcaaagc caaaaaggac gcgtttatga gcattgcgaa gttcgaggtt
aacaacccgc aaatccaaca caaagcggtt aatgaactga ttcgtgtcat ccaccagctg
agcccggaat cgagcttacg taagcgcaag agaagccgtt gc(SEQ ID NO.2)
and transforming the recombinant expression vector pET-sumo-Rat IFN-gamma into competent escherichia coli BL21 (DE 3) to obtain recombinant engineering bacteria E.coli BL21 (DE 3)/pET-sumo-Rat IFN gamma containing Rat IFN-gamma genes.
Example 2 Induction of genetically engineered Strain to express recombinant proteins
Coli BL21 (DE 3)/pET-sumo-Rat IFN γ obtained in example 1 was inoculated into a liquid LB medium containing 50. Mu.g/mL kanamycin and cultured and activated at 37 ℃ overnight at 200 r/min. The activated strain was transferred to a fresh liquid LB medium containing 50. Mu.g/mL kanamycin for culture. When OD is reached 600 When the concentration is 0.4-0.6, IPTG is added to make the final concentration be 0.3mmol/L, and the mixture is induced at 16 ℃ and 200r/min for 18h to obtain the bacterial liquid of the fermented rat IFN-gamma fusion protein. Meanwhile, the test is also provided with a contrast, and the result of inducing and expressing the recombinant protein is shown inFig. 1. As can be seen from fig. 1, recombinant engineered bacterium e.coli BL21 (DE 3)/pET-sumo-Rat IFN γ containing optimized IFN- γ gene can express recombinant protein (including Rat IFN- γ fusion protein) in large amount in form of soluble supernatant in e.coli.
Example 3 purification of rat IFN-. Gamma.fusion proteins
The bacterial liquid of the fermented rat IFN-gamma fusion protein prepared in example 2 was centrifuged, the collected thallus was resuspended in Binding Buffer, the thallus was crushed at 4 ℃ and 1000bar under high pressure, and the crushing was repeated 2 times. Centrifuging at 4 deg.C and 8000/min for 30min to collect supernatant, and eluting Ni with 250mM imidazole and 50mM imidazole 2+ Purifying and collecting the eluent containing the target protein by a column to obtain the primarily purified rat IFN-gamma protein.
Removing imidazole from primarily purified rat IFN-gamma protein through dialysis, adding 2U/mg sumo protease Ulp1 at 4 ℃ for enzyme digestion for 2h to remove labels, and then performing Ni-based labeling 2+ Purifying the column, and collecting the components flowing through and washing the column to obtain the recombinant rat IFN-gamma protein with high purity and no label.
Endotoxin of recombinant rat IFN-gamma protein is removed through an endo-Removal Kit, and the Endotoxin content is detected to be less than 1 EU/mu g through an LAL method.
The purification results are shown in FIGS. 2 to 4. FIG. 2 shows that a large amount of recombinant rat INF-gamma can be obtained by purification, and a large amount of recombinant rat IFN-gamma with purity greater than or equal to 95% can be obtained by pictures 3 and 4.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Biotechnology Limited for subfamily (Wuhan)
<120> recombinant rat gamma-interferon and preparation method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 525
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaggatcca tgagtgctac acgccgcgtc ttggttttgc agctctgcct catggccctc 60
tctggctgtt actgccaagg cacactcatt gaaagcctag aaagtctgaa gaactatttt 120
aactcaagta gcatggatgc tatggaagga aagagcctcc tcttggatat ctggaggaac 180
tggcaaaagg acggtaacac gaaaatactt gagagccaga ttatctcttt ctacctcaga 240
ctctttgaag tcttgaaaga caaccaggcc atcagcaaca acataagtgt catcgaatcg 300
cacctgatca ctaacttctt cagcaacagt aaagcaaaaa aggatgcatt catgagcatc 360
gccaagttcg aggtgaacaa cccacagatc cagcacaaag ctgtcaatga actcatcaga 420
gtgattcacc agctgtcacc agaatctagc ctaaggaagc ggaaaaggag tcggtgctga 480
ttctggggta gagagtgtgc caataagaag aattctggat ccttt 525
<210> 2
<211> 402
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caaggaacac taatagagag tttagaatca ttgaagaact atttcaacag cagcagcatg 60
gacgctatgg aaggtaaaag cttgctgctg gatatttggc gtaattggca aaaagacggc 120
aacacgaaaa tcttggagtc tcagattatc tctttttacc tgcgcctgtt cgaggtgctg 180
aaggacaacc aggcaatttc aaataacatc tccgtgatcg agtcccatct tatcaccaat 240
tttttcagca acagcaaagc caaaaaggac gcgtttatga gcattgcgaa gttcgaggtt 300
aacaacccgc aaatccaaca caaagcggtt aatgaactga ttcgtgtcat ccaccagctg 360
agcccggaat cgagcttacg taagcgcaag agaagccgtt gc 402

Claims (9)

1. A gene sequence of coding recombinant rat gamma-interferon is shown IN SEQ IN NO. 2.
2. A recombinant rat interferon-gamma encoded by the gene sequence encoding a recombinant rat interferon-gamma of claim 1.
3. An expression vector comprising the gene sequence of claim 1.
4. A preparation method of recombinant rat gamma-interferon is characterized by comprising the following steps:
synthesizing a gene sequence of the coding recombinant Rat gamma-interferon shown IN SEQ IN NO.2, cloning to a prokaryotic expression vector pET-sumo, and constructing a recombinant expression vector pET-sumo-Rat IFN gamma;
transforming a recombinant expression vector pET-sumo-Rat IFN gamma into escherichia coli to form recombinant escherichia coli engineering bacteria containing a gene sequence of recombinant Rat IFN-gamma;
inducing expression, breaking thallus to obtain bacterial liquid containing soluble recombinant protein;
purifying and enzyme cutting to remove the label in the recombinant protein, thus obtaining the recombinant rat gamma-interferon.
5. The method of claim 4, wherein the step of inducing expression comprises:
inoculating recombinant Escherichia coli engineering bacteria containing recombinant rat IFN-gamma gene sequences into a liquid LB culture medium containing kanamycin for activation, and transferring the activated strains into a fresh liquid LB culture medium containing kanamycin for culture;
when OD600 is 0.4-0.6, IPTG is added to make the final concentration 0.3mmol/L, and recombinant colibacillus is induced to express recombinant protein under the conditions of 16 ℃ and 200 r/min.
6. The method for preparing recombinant rat gamma-interferon according to claim 5, wherein the purification step is:
under the condition that the recombinant protein is not denatured, an imidazole solution is adopted to elute a nickel column for the first time, and imidazole is removed through dialysis to obtain a recombinant protein solution;
and then carrying out enzyme digestion on the recombinant protein by sumo protease Ulp1 to remove the label, purifying by using a nickel column, and obtaining the flow-through component which is the non-label recombinant rat IFN-gamma.
7. The method of claim 6, wherein the first elution is performed by sequentially eluting with 250mM imidazole and 50mM imidazole.
8. The method of claim 7, further comprising the step of removing endotoxin from the unlabeled recombinant rat IFN- γ.
9. The recombinant rat interferon-gamma product prepared by the method of claim 8, having an endotoxin content of <1EU/μ g.
CN202210402687.6A 2022-04-18 2022-04-18 Recombinant rat gamma-interferon and preparation method thereof Pending CN115161328A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103014015A (en) * 2010-03-26 2013-04-03 华南农业大学 Gene segment for coding porcine interferon-gamma and application of gene segment
CN107056941A (en) * 2017-05-09 2017-08-18 杨凌博德越生物科技有限公司 A kind of specific recognition goat IFN γ method for preparing monoclonal antibody

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103014015A (en) * 2010-03-26 2013-04-03 华南农业大学 Gene segment for coding porcine interferon-gamma and application of gene segment
CN107056941A (en) * 2017-05-09 2017-08-18 杨凌博德越生物科技有限公司 A kind of specific recognition goat IFN γ method for preparing monoclonal antibody

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HTTPS://WWW.ABBKINE.COM: "Rat IFN-gamma protein Cat#PRP1214", HTTPS://WWW.ABBKINE.COM, pages 1 *
MEETUL KUMAR ET AL.: "Optimization of conditions for expression of recombinant interferon-c in E.coli", MOL BIOL REP., vol. 41 *
REIN DUKEMA ET AL.: "Cloning , expression, and purification of rat IFN-γ", METHODS IN ENZYMOLOGY, vol. 119, pages 453 - 464 *
胡大强;: "蛋白转导结构域修饰小鼠干扰素-γ的研制", 中国药房, no. 1, pages 47 - 49 *

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