CN113150103A - Saccharomyces cerevisiae expression recombinant fish thioredoxin and preparation method thereof - Google Patents

Saccharomyces cerevisiae expression recombinant fish thioredoxin and preparation method thereof Download PDF

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CN113150103A
CN113150103A CN202110354989.6A CN202110354989A CN113150103A CN 113150103 A CN113150103 A CN 113150103A CN 202110354989 A CN202110354989 A CN 202110354989A CN 113150103 A CN113150103 A CN 113150103A
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rftrx
saccharomyces cerevisiae
pyes2
thioredoxin
alpha
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赵俊
刘洁
徐文俊
夏兵兵
周炜
张勇
夏洁
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Wuhu Yingtefeier Biological Products Industry Research Institute 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The invention belongs to the technical field of biology, and particularly relates to recombinant fish thioredoxin and a preparation method thereof, which comprise the following steps: (1) constructing a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rFTRX-His; (2) preparing and transforming pYES2/CT-MF alpha-rFTRX-His engineering bacteria; (3) shake flask culture, induced expression purification and verification of saccharomyces cerevisiae engineering bacteria containing pYES2/CT-MF alpha-rFTRX-His. The FTRX gene in the engineering bacteria prepared by the invention has the preference characteristic of saccharomyces cerevisiae, contains saccharomyces cerevisiae signal peptide, can ensure that the FTRX is secreted and expressed, greatly improves the yield of expression products, and has simple acquisition process and lower cost compared with an escherichia coli expression system and other beneficial bacteria secretion expression systems.

Description

Saccharomyces cerevisiae expression recombinant fish thioredoxin and preparation method thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of saccharomyces cerevisiae expression recombinant fish thioredoxin.
Background
Thioredoxin (TRX) is a highly conserved low molecular weight protein, an acidic small molecule protein soluble in cells, stable to heat, and having redox activity, with a molecular weight of about 12 kDa. Thioredoxin has two structural types, thioredoxin-1 and thioredoxin-2. A first structural type, mainly present in the cytoplasm and nucleus; whereas the second structural type, thioredoxin-2, is present only in mitochondria.
TRX is widely distributed in plants, bacteria, yeasts and animals, and forms a thioredoxin system (TRX system) together with Thioredoxin Reductase (TR) and reduced Nicotinyl Adenine Dinucleotide Phosphate (NADPH), so that the TRX system is an effective protein reduction system. TRX contains dithio-sulfydryl and disulfide bond in the conserved activation region, and the redox activity of TRX makes thioredoxin have the functions of maintaining stable redox state in vivo, regulating cell growth, inhibiting apoptosis, regulating gene transcription influence protein content in animal body, regulating immunity and the like.
Verification and functional identification of thioredoxin and thioredoxin reductase in large yellow croaker (Chenmennau, Zhejiang ocean university) indicate that: large yellow croaker (Larimichthys crocea) is a seawater cage culture fish which is unique in China and largest in scale in China, and is also the most popular seawater fish in domestic markets. However, with the continuous expansion of the culture scale and density of large yellow croakers, various diseases continuously outbreak in the culture process, wherein vibriosis is common, and vibrio parahaemolyticus is one of the pathogens of the diseases. Thioredoxin (Trx), Thioredoxin reductase (TrxR) and reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) form an effective protein reduction system, namely a Thioredoxin system, can regulate the processes of cell proliferation and survival, and plays an important role in the regulation of organism redox and antioxidant defense.
Disclosure of Invention
The invention provides a method for preparing recombinant fish thioredoxin, which selects a saccharomyces cerevisiae eukaryotic expression system to perform exogenous expression on the fish thioredoxin and comprises the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rFTRX-His specifically comprises the following steps:
artificial optimization of fish thioredoxin gene to obtain plasmid pMD 19-T-rFTRX-His;
carrying out double enzyme digestion on the plasmid pYES2/CT-MF alpha and the rFTRX-His fragment recovered from the gel to obtain a positive clone pYES2/CT-MF alpha-rFTRX-His;
(2) the preparation and transformation of pYES2/CT-MF alpha-rFTRX-His engineering bacteria specifically comprise the following steps:
preparing a common solution and a culture medium for a saccharomyces cerevisiae expression system;
respectively transforming the pYES2/CT-MF alpha-rFTRX-His and the pYES2/CT-MF alpha plasmids obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clone INVSC1(pYES2/CT-MF alpha-rFTRX-His) through culture of a culture medium, PCR amplification and screening;
(3) the shake flask culture, induced expression purification and verification of the saccharomyces cerevisiae engineering bacteria containing pYES2/CT-MF alpha-rFTRX-His specifically comprise the following steps:
culturing and centrifuging the positive clone INVSC1(pYES2/CT-MF alpha-rFTRX-His) obtained in the step (2), collecting supernatant, performing column chromatography to obtain purified supernatant protein liquid, and observing specific bands through SDS-PAGE electrophoresis and Westernblot identification of a mouse anti-His monoclonal antibody, namely the saccharomyces cerevisiae expression recombinant fish thioredoxin obtained by the invention.
Further, in the step (1), the artificial optimization of the fish thioredoxin gene is performed to obtain the plasmid pMD19-T-rFTRX-His, and the specific steps are as follows:
according to the amino acid sequence of the fish thioredoxin and the preference of saccharomyces cerevisiae to codons, a saccharomyces cerevisiae preferred recombinant fish thioredoxin gene is artificially designed, and the amino acid sequence of the recombinant fish thioredoxin is shown in a sequence table 1;
the recombinant fish thioredoxin gene is inserted between Hind III enzyme cutting site and Xba I enzyme cutting site of pMD19-T Simple Vector in the sequence of FTRX-His, Hind III enzyme cutting site is arranged at the 5 'end, Xba I enzyme cutting site is arranged at the 3' end, and plasmid pMD19-T-rFTRX-His is obtained.
Further, in the step (1), double enzyme digestion is performed on plasmid pYES2/CT-MF α and rFTRX-His fragments recovered from the above gel, so as to obtain positive clone pYES2/CT-MF α -rFTRX-His, and the specific steps are as follows:
designing and synthesizing a forward primer rFTRX-His F and a reverse primer rFTRX-His R according to the sequence of the rFTRX-His gene, amplifying the rFTRX-His gene in a pMD19-T-rFTRX-His plasmid through PCR, and performing gel recovery according to the instruction of a gel recovery kit;
the purchased plasmid pYES2/CT-MF alpha and the rFTRX-His fragment recovered from the gel are subjected to double enzyme digestion by QuickCut Not I and QuickCut Xba I respectively, the rFTRX-His gene fragment and the pYES2/CT-MF alpha fragment are recovered by gel digestion respectively, then T4DNA ligase is used for connection to obtain recombinant plasmid, the recombinant plasmid is transformed into escherichia coli (DH 5), positive clones are selected on an LB plate culture medium containing ampicillin, and the positive clones are identified by bacterial liquid PCR (forward primer: FTRRX-His F; reverse primer: rFTRX-His R) and double enzyme digestion (QuickCut Not I and QuickCut Xba I) to select the positive clones pYES2/CT-MF alpha-rFTRX-His.
Further, in the step (2), a common solution and a culture medium for a saccharomyces cerevisiae expression system are prepared, and the specific method comprises the following steps:
YPD medium: dissolving peptone 20g, yeast extract 10g, and glucose 20g (20 g agar powder is added when preparing solid culture medium) in 800ml water, diluting to 1L, and autoclaving at 121 deg.C for 20 min;
SC-U liquid medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 900ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of 20% glucose solution for filtration sterilization, mixing uniformly, and storing at 4 ℃ for later use;
SC-U plate culture medium, 6.70g yeast nitrogen-free extract, 0.15g compound amino acid, 20g agar powder, 900ml deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml 20% glucose solution for filtration sterilization, mixing, and storing at 4 ℃ for later use;
SC-U induction medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 800ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of filter-sterilized 20% glucose solution and 100ml of filter-sterilized 20% galactose solution, mixing uniformly, and storing at 4 ℃ for later use.
Further, in the step (2), the specific steps for obtaining the positive clone INVSC1(pYES2/CT-MF α -rFTRX-His) are as follows:
adding 10 mu l of pYES2/CT-MF alpha-rFTRX-His plasmid into 80 mu l of Saccharomyces cerevisiae INVSCl competent cells, blowing and sucking to mix the plasmids evenly, then transferring the cells into a precooled electric shock cup, carrying out ice bath for 5min, adjusting a Bio-Rad electric converter to a fungus level, selecting PIC, placing the electric shock cup on the Bio-Rad electric converter for electric shock, simultaneously quickly adding 500 mu l of precooled 1M sorbitol solution into the electric shock cup, mixing the solution evenly, coating an SC-U plate culture medium, carrying out inversion culture at constant temperature of 30 ℃ until a monoclonal colony grows out, preparing the Saccharomyces cerevisiae transformant containing pYES2/CT-MF alpha-rFTRX-His in the monoclonal colony, preparing bacterial liquid for PCR amplification, and screening to obtain the INVSC1/pYES2/CT-MF alpha-rFTRX-His positive clone.
Further, in the step (3), the purified supernatant protein solution is obtained by culturing, centrifuging, collecting supernatant and performing column chromatography, and the specific steps are as follows:
the positive clone INVSc1(pYES 2/CT-MF. alpha. -rFTRX-His) was picked up in 30ml of liquid SC-U medium, shake-cultured overnight at 30 ℃ and OD was measured600nmLight absorption value, inoculating appropriate amount of culture solution into 100ml SC-U induction culture medium, shake culturing overnight, and measuring OD600nmTo 0.4;
centrifuging at 4 ℃ and 1500g for 5min, collecting thalli, suspending the thalli by using 1-2 ml of SC-U induction culture medium, re-inoculating 100ml of SC-U induction culture medium, placing in 30 ℃ for shake culture for 96h, centrifuging at 4 ℃ and 15000g for 5min, collecting thalli and supernatant, and filtering the supernatant subjected to induced expression by a 0.22 mu m filter membrane;
self-packed column using chemical Sepharose TM Fast Flow Nickel ion chelate affinity chromatography packing and washing Ni with 3 column volumes of purified water2+Chelating affinity chromatography column, balancing 2-3 column volumes with Binding Buffer (pH 8.0 PBS); detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value are stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min; then, the mixture is passed through a chromatographic column by using a Binding Buffer, and the foreign protein which is not combined with the chromatographic column is washed off until the OD is reached280nmStabilizing;
eluting by an Elution Buffer (pH 8.0PBS contacting 100-300 mM Imidazole) with gradient Imidazole concentration, and collecting protein corresponding to an Elution peak, namely the saccharomyces cerevisiae expression recombinant fish thioredoxin obtained by the invention.
Furthermore, the SC-U liquid culture medium and the SC-U induction culture medium are respectively added with 2% of raffinose and 2% of galactose.
Compared with the prior art, the invention has the beneficial effects that:
1. the fish TRX gene in the engineering bacteria prepared by the invention has the preference type characteristic of saccharomyces cerevisiae, contains saccharomyces cerevisiae signal peptide, can make fish thioredoxin secrete and express, greatly improves the yield of an expression product, and has simple acquisition process and lower cost compared with an escherichia coli expression system and other beneficial bacteria secretion and expression systems.
2. At present, no research report for obtaining the recombinant fish thioredoxin through a saccharomyces cerevisiae expression system exists, so that the preparation method has very important application value.
Description of the drawings:
FIG. 1 plasmid pYES2/CT-MF α map;
description of sequence listing:
sequence table 1 amino acid sequence of recombinant fish thioredoxin of the invention
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
The specific implementation mode is as follows:
the invention is further illustrated by the following examples:
example 1: construction of Saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rFTRX-His
(1) Artificial optimization of fish thioredoxin genes
According to the fish thioredoxin amino acid sequence and the codon preference of saccharomyces cerevisiae, a saccharomyces cerevisiae preferred fish thioredoxin gene is artificially designed, and the recombinant fish thioredoxin amino acid sequence is shown in a sequence table 1.
The gene sequence is inserted between HindIII enzyme cutting site and XbaI enzyme cutting site of pMD19-T Simple Vector in the sequence of FTRX-His, the 5 'end of the gene sequence is connected with HindIII enzyme cutting site, the 3' end of the gene sequence is connected with XbaI enzyme cutting site, and thus, the plasmid pMD19-T-rFTRX-His is obtained.
(2) Construction of Saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rFTRX-His
A forward primer rFTRX-His F and a reverse primer rFTRX-His R are synthesized according to the sequence design of the rFTRX-His gene, and the rFTRX-His gene in a pMD19-T-rFTRX-His plasmid is amplified through PCR.
The reaction system is as follows:
Figure BDA0003003314060000071
PCR reaction procedure: pre-denatured at 95 ℃ 5 rain; 30s at 95 ℃, 25s at 58 ℃, 30s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min. The PCR amplification products were detected by 2.0% agarose gel electrophoresis and gel recovery was carried out according to the instructions of the Qiagen gel recovery kit.
The purchased plasmid pYES2/CT-MF α (plasmid map see FIG. 1) and the rFTRX-His fragment recovered from the above gel were double digested with QuickCut Not I and QuickCut Xba I, respectively.
The enzyme digestion reaction system is as follows:
Figure BDA0003003314060000072
and carrying out enzyme digestion reaction for 3h in a metal bath at 37 ℃, detecting the enzyme digestion product by 2% agarose gel electrophoresis, respectively cutting gel, recovering rFTRX-His gene fragment and pYES2/CT-MF alpha fragment, and then connecting by using T4DNA ligase.
The connection reaction system is as follows:
Figure BDA0003003314060000081
the reaction conditions are 16 ℃ and 14h, the recombinant plasmid is transformed into escherichia coli (DH5 alpha) by a conventional method (calcium chloride method), a positive clone is selected on an LB plate culture medium containing the ampicillin, and the positive clone is selected and identified by bacterial liquid PCR (forward primer: rFTRX-His F; reverse primer: rFTRX-His R) and double enzyme digestion (QuickCut Not I and QuickCut Xba I), and a positive clone pYES2/CT-MF alpha-rFTRX-His is selected.
Example 2: preparation and transformation of pYES2/CT-MF alpha-rFTRX-His engineering bacteria
(1) Preparation of common solution and culture medium for saccharomyces cerevisiae expression system
YPD medium: dissolving peptone 20g, yeast extract 10g, and glucose 20g (20 g agar powder is added when preparing solid culture medium) in 800ml water, diluting to 1L, and autoclaving at 121 deg.C for 20 min;
SC-U liquid medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 900ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of 20% glucose solution for filtration sterilization, mixing uniformly, and storing at 4 ℃ for later use;
SC-U plate culture medium, 6.70g yeast nitrogen-free extract, 0.15g compound amino acid, 20g agar powder, 900ml deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml 20% glucose solution for filtration sterilization, mixing, and storing at 4 ℃ for later use;
SC-U induction medium: 6.70g of yeast nitrogen-free extract, 0.15g of compound amino acid, 800ml of deionized water, autoclaving at 121 ℃ for 20min, cooling to 50 ℃, adding 100ml of filter-sterilized 20% glucose solution and 100ml of filter-sterilized 20% galactose solution, mixing uniformly, and storing at 4 ℃ for later use.
(2) pYES2/CT-MF alpha-rFTRX-His transformation saccharomyces cerevisiae
The plasmid pYES2/CT-MF alpha-rFTRX-His and pYES2/CT-MF alpha are respectively transformed into Saccharomyces cerevisiae INVSC1 competent cells by adopting an electric transformation method. Mu.l of pYES2/CT-MF α -rFTRX-His plasmid was added to 80. mu.l of s.cerevisiae INVSCl competent cells, well mixed by pipetting and then transferred to a pre-cooled cuvette. Ice-bath for 5min, and wiping the outer wall of the electric shock cup. The Bio-Rad electric converter was adjusted to the fungi range, PIC option, and the cuvette was placed on the Bio-Rad electric converter for electric shock. Add 500. mu.l of pre-chilled 1M sorbitol solution quickly to the cuvette, mix well and coat the SC-U plate. And (4) carrying out inverted culture at the constant temperature of 30 ℃ until a monoclonal antibody grows out. Grown in SC-U selection medium (containing ampicillin) were Saccharomyces cerevisiae transformants containing pYES2/CT-MF α -rFTRX-His and pYES2/CT-MF α plasmids, and the clones were screened for INVSC1/pYES2/CT-MF α -rFTRX-His positive clones by PCR (forward primer: rFTRX-His F; reverse primer: rFTRX-His R) from the broth.
Example 3: shake flask culture, induced expression purification and verification of saccharomyces cerevisiae engineering bacteria containing pYES2/CT-MF alpha-rFTRX-His
Selecting positive clones INVSC1(pYES2/CT-MF alpha-rFTRX-His) and INVSC1(pYES2/CT-MF alpha) respectively in 30ml of liquid SC-U selection medium (containing 2% raffinose and ampicillin), shake-culturing at 30 deg.C overnight, and determining OD600nmLight absorption value, inoculating appropriate amount of culture solution into 100ml SC-U induction culture medium (containing 2% galactose and ampicillin), shake culturing overnight to make OD600 nmCentrifuging at 0.4 deg.C and 4 deg.C for 5min at 1500g, collecting thallus, suspending thallus with 1-2 ml SC-U induction culture medium (containing 2% galactose and ampicillin), inoculating 100ml SC-U induction culture medium (containing 2% galactose and ampicillin), shake culturing at 30 deg.C for 96 hr, centrifuging at 4 deg.C and 15000g for 5min, collecting thallus and supernatant, and filtering the supernatant with 0.22 μm filter membrane.
The column was self-packed using the GE Healthcare company chemical Sepharose TM Fast Flow Nickel ion chelate affinity chromatography packing, and the Ni was washed with 3 column volumes of purified water2+Chelating affinity chromatography column, and then Binding Buffer (pH 8)0PBS) to equilibrate 2-3 column volumes. And (3) detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value are both stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min. Then, the mixture is passed through a chromatographic column by using a Binding Buffer, and the foreign protein which is not combined with the chromatographic column is washed off until the OD is reached280nmAnd (4) stabilizing. Eluting with an Elution Buffer (pH 8.0PBS containing 100-300 mM imidazole) with gradient imidazole concentration, collecting protein corresponding to an Elution peak, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a protein sample in the purification process so as to analyze the purification effect.
In conclusion, the invention adopts the genetic engineering technology to express the recombinant fish thioredoxin, and finally obtains remarkable effect. The preparation method for expressing the recombinant fish thioredoxin related by the invention has the advantages of simple technology, low cost and practical application value to the breeding industry, provides a new idea for the development of feed additives in the breeding industry, has strong feasibility and greatly improves the working efficiency.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Sequence listing
<110> research institute of biological product industry, Inc. of Utafel, Utaki
<120> saccharomyces cerevisiae expression recombinant fish thioredoxin and preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 110
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Lys Ser Ser Ile Asp Leu Cys Ser Gln Glu Gly Phe Asp Lys Ala
1 5 10 15
Leu Ala Glu Ala Gly Asp Asn Leu Val Val Val Asp Phe Thr Ala Thr
20 25 30
Trp Cys Gly Pro Cys Gln Ser Ile Ala Pro Phe Tyr Lys Gly Leu Ser
35 40 45
Glu Asn Pro Ser Tyr Ala Lys Val Val Phe Leu Lys Val Asp Val Asp
50 55 60
Asp Ala Gln Asp Val Ala Gln Ser Cys Glu Ile Lys Cys Met Pro Thr
65 70 75 80
Phe His Phe Tyr Lys Asn Gly Lys Lys Leu Asp Asp Phe Ser Gly Ser
85 90 95
Asn Gln Ala Lys Leu Glu Glu Met Val Asn Gln Tyr Lys Asn
100 105 110

Claims (8)

1. The saccharomyces cerevisiae expression recombinant fish thioredoxin is characterized in that the amino acid sequence of the recombinant fish thioredoxin is shown in a sequence table 1.
2. The saccharomyces cerevisiae expression recombinant fish thioredoxin of claim 1, wherein the recombinant fish thioredoxin amino acid sequence is artificially designed according to the fish thioredoxin amino acid sequence and the preference of saccharomyces cerevisiae to codons.
3. A preparation method of saccharomyces cerevisiae expression recombinant fish thioredoxin is characterized by comprising the following steps:
(1) the construction of a saccharomyces cerevisiae secretion expression vector pYES2/CT-MF alpha-rFTRX-His specifically comprises the following steps:
artificial optimization of fish thioredoxin gene to obtain plasmid pMD 19-T-rFTRX-His;
carrying out double enzyme digestion on the plasmid pYES2/CT-MF alpha and the rFTRX-His fragment recovered from the gel to obtain a positive clone pYES2/CT-MF alpha-rFTRX-His;
(2) the preparation and transformation of pYES2/CT-MF alpha-rFTRX-His engineering bacteria specifically comprise the following steps:
preparing a common solution and a culture medium for a saccharomyces cerevisiae expression system;
respectively transforming the pYES2/CT-MF alpha-rFTRX-His and the pYES2/CT-MF alpha plasmids obtained in the step (1) into Saccharomyces cerevisiae INVSC1 competent cells, and obtaining positive clone INVSC1(pYES2/CT-MF alpha-rFTRX-His) through culture of a culture medium, PCR amplification and screening;
(3) the shake flask culture, induced expression purification and verification of the saccharomyces cerevisiae engineering bacteria containing pYES2/CT-MF alpha-rFTRX-His specifically comprise the following steps:
culturing and centrifuging the positive clone INVSC1(pYES2/CT-MF alpha-rFTRX-His) obtained in the step (2), collecting supernatant, performing column chromatography to obtain purified supernatant protein liquid, and observing specific bands through SDS-PAGE electrophoresis and Westernblot identification of a mouse anti-His monoclonal antibody, namely the saccharomyces cerevisiae expression recombinant fish thioredoxin obtained by the invention.
4. The method for preparing recombinant fish thioredoxin expressed by saccharomyces cerevisiae according to claim 3, wherein in the step (1), the artificial optimization of fish thioredoxin gene is performed to obtain plasmid pMD19-T-rFTRX-His, and the specific steps are as follows:
according to the amino acid sequence of the fish thioredoxin and the preference of saccharomyces cerevisiae to codons, a saccharomyces cerevisiae preferred fish thioredoxin gene is artificially designed, and the amino acid sequence of the recombinant fish thioredoxin is shown in a sequence table 1;
the recombinant fish thioredoxin gene is inserted between Hind III enzyme cutting site and Xba I enzyme cutting site of pMD19-T Simple Vector in the sequence of FTRX-His, Hind III enzyme cutting site is arranged at the 5 'end, Xba I enzyme cutting site is arranged at the 3' end, and plasmid pMD19-T-rFTRX-His is obtained.
5. The method for preparing recombinant fish thioredoxin expressed by saccharomyces cerevisiae according to claim 3, wherein in the step (1), plasmid pYES2/CT-MF α and rFTRX-His fragment recovered from the above gel are subjected to double enzyme digestion to obtain positive clone pYES2/CT-MF α -rFTRX-His, and the specific steps are as follows:
designing and synthesizing a forward primer rFTRX-His F and a reverse primer rFTRX-His R according to the sequence of the rFTRX-His gene, amplifying the rFTRX-His gene in a pMD19-T-rFTRX-His plasmid through PCR, and performing gel recovery according to the instruction of a gel recovery kit;
the purchased plasmid pYES2/CT-MF alpha and the rFTRX-His fragment recovered from the gel are subjected to double enzyme digestion by QuickCut Not I and QuickCut Xba I respectively, the rFTRX-His gene fragment and the pYES2/CT-MF alpha fragment are recovered by gel digestion respectively, then T4DNA ligase is used for connection to obtain recombinant plasmid, the recombinant plasmid is transformed into escherichia coli (DH 5), positive clones are selected on an LB plate culture medium containing ampicillin, and the positive clones are identified by bacterial liquid PCR (forward primer: FTRRX-His F; reverse primer: rFTRX-His R) and double enzyme digestion (QuickCut Not I and QuickCut Xba I) to select the positive clones pYES2/CT-MF alpha-rFTRX-His.
6. The method for preparing recombinant fish thioredoxin expressed by saccharomyces cerevisiae according to claim 3, wherein the specific steps for obtaining positive clone INVSC1(pYES2/CT-MF α -rFTRX-His) in step (2) are as follows:
adding 10 mu l of pYES2/CT-MF alpha-rFTRX-His plasmid into 80 mu l of Saccharomyces cerevisiae INVSCl competent cells, blowing and sucking to mix the plasmids evenly, then transferring the cells into a precooled electric shock cup, carrying out ice bath for 5min, adjusting a Bio-Rad electric converter to a fungus level, selecting PIC, placing the electric shock cup on the Bio-Rad electric converter for electric shock, simultaneously quickly adding 500 mu l of precooled 1M sorbitol solution into the electric shock cup, mixing the solution evenly, coating an SC-U plate culture medium, carrying out inversion culture at constant temperature of 30 ℃ until a monoclonal colony grows out, preparing the Saccharomyces cerevisiae transformant containing pYES2/CT-MF alpha-rFTRX-His in the monoclonal colony, preparing bacterial liquid for PCR amplification, and screening to obtain the INVSC1/pYES2/CT-MF alpha-rFTRX-His positive clone.
7. The method for preparing recombinant fish thioredoxin expressed by Saccharomyces cerevisiae according to claim 3, wherein in the step (3),
the purified supernatant protein liquid is obtained by culturing, centrifuging, collecting supernatant and carrying out column chromatography, and the method comprises the following specific steps:
the positive clone INVSc1(pYES 2/CT-MF. alpha. -rFTRX-His) was picked up in 30ml of liquid SC-U medium, shake-cultured overnight at 30 ℃ and OD was measured600nmLight absorption value, inoculating appropriate amount of culture solution into 100ml SC-U induction culture medium, shake culturing overnight, and measuring OD600nmTo 0.4;
centrifuging at 4 ℃ and 1500g for 5min, collecting thalli, suspending the thalli by using 1-2 ml of SC-U induction culture medium, re-inoculating 100ml of SC-U induction culture medium, placing in 30 ℃ for shake culture for 96h, centrifuging at 4 ℃ and 15000g for 5min, collecting thalli and supernatant, and filtering the supernatant subjected to induced expression by a 0.22 mu m filter membrane;
self-packed column using chemical Sepharose TM Fast Flow Nickel ion chelate affinity chromatography packing and washing Ni with 3 column volumes of purified water2+Chelating affinity chromatography column, balancing 2-3 column volumes with Binding Buffer; detecting the conductivity value and the absorption value of 280nm wavelength on line, starting to sample after the conductivity value and the absorption value are stable, and setting the flow rate of the sample pumped through the chromatographic column to be 5-6 ml/min; then, the mixture is passed through a chromatographic column by using a Binding Buffer, and the foreign protein which is not combined with the chromatographic column is washed off until the OD is reached280nmStabilizing;
and eluting by using an Elution Buffer with gradient imidazole concentration, and collecting protein corresponding to an Elution peak, namely the saccharomyces cerevisiae expression recombinant fish thioredoxin obtained by the invention.
8. The method of claim 7, wherein the SC-U liquid culture medium and the SC-U induction culture medium are supplemented with 2% raffinose and 2% galactose, respectively.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146793A1 (en) * 1997-02-19 2002-10-10 Chiron S.P.A. , A Corporation Expression of heterologous proteins
US20110129872A1 (en) * 2009-12-01 2011-06-02 Mogam Biotechnology Research Institute Method for a production of a recombinant protein using yeast co-expression system
CN104480125A (en) * 2014-12-05 2015-04-01 中国水产科学研究院南海水产研究所 Trachinotus ovatus thioredoxin gene
US20170074858A1 (en) * 1998-10-13 2017-03-16 Peptide Biosciences, Inc. Stabilizied Bioactive Peptides and Methods of Identification, Synthesis and Use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020146793A1 (en) * 1997-02-19 2002-10-10 Chiron S.P.A. , A Corporation Expression of heterologous proteins
US20170074858A1 (en) * 1998-10-13 2017-03-16 Peptide Biosciences, Inc. Stabilizied Bioactive Peptides and Methods of Identification, Synthesis and Use
US20110129872A1 (en) * 2009-12-01 2011-06-02 Mogam Biotechnology Research Institute Method for a production of a recombinant protein using yeast co-expression system
CN104480125A (en) * 2014-12-05 2015-04-01 中国水产科学研究院南海水产研究所 Trachinotus ovatus thioredoxin gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG,W: ""Thioredoxin [Anabarilius grahami] GenBank: ROI47901.1"", 《GENBANK》 *
MENGNAN CHEN ET AL.: ""Cloning and functional characterization of thioredoxin genes from large yellow croaker Larimichthys crocea"", 《FISH AND SHELLFISH IMMUNOLOGY》 *
邹向晖 等: ""人硫氧还蛋白真核载体的构建及表达"", 《中国地方病学杂质》 *

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