CN111269306B - Art V1 recombinant protein and preparation method and application thereof - Google Patents
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Abstract
The invention provides an Art V1 recombinant protein, a preparation method and an application thereof, wherein a coding gene of the Art V1 recombinant protein comprises a nucleic acid sequence shown as SEQ ID NO. 1. The coding gene of the Art V1 recombinant protein is subjected to codon optimization, is artificially synthesized and then is subcloned to a eukaryotic vector to obtain a recombinant vector, and is prepared by adopting an eukaryotic expression system for expression, so that the efficient expression of the Art V1 recombinant protein in the eukaryotic expression system is favorably realized.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to an Art V1 recombinant protein, a preparation method and application thereof, and particularly relates to an Art V1 recombinant protein as a main component allergen of folium artemisiae argyi, a preparation method and application thereof.
Background
In recent years, with the change of life style and the increase of work pressure, the incidence rate of allergic diseases is increasing year by year, and the allergic diseases become one of important diseases affecting the life quality of people. About 3 hundred million people suffer from allergic bronchial asthma, 5 hundred million people suffer from allergic rhinitis, and 3-5 hundred million people suffer from food allergy every year worldwide. Allergic diseases seriously affect physical and mental health, and therefore, are particularly important for diagnosis and prevention of allergic diseases.
Mugwort is an important allergen causing type I anaphylaxis, can cause symptoms such as allergic rhinitis and asthma, and mainly comprises four allergen components of Art V1, art V2, art V3 and Art V4, wherein the most important allergen component is Art V1.Art V1 is a secretory protein containing two IgE antibody recognition regions, and the recombinant expression Art V1 has wide application prospect in the diagnosis and desensitization treatment of artemisia argyi allergy.
CN 106729698A discloses that NY-ESO-1 has the function as a molecular adjuvant for enhancing the immunoreaction of Art v1 (wtArt) allergen, but does not disclose how to obtain the Art v1 allergen.
Therefore, the research on an economical and effective method for preparing the Art V1 component allergen has important significance in the fields of diagnosis and treatment of allergic diseases.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior Art, the invention provides the Art V1 recombinant protein and the preparation method and the application thereof, the coding gene of the Art V1 recombinant protein is subjected to codon optimization and is prepared by adopting a eukaryotic expression system, so that the yield is high, the cost is low, and the application prospect is wide.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a nucleic acid molecule encoding an Art V1 recombinant protein, said nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO. 1;
SEQ ID NO:1：
ATGACCAGACTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCAGCAGCAGAGCCGCCGGCAGCAAGCTGTGCGAGAAGACCAGCAAGACCTACAGCGGCAAGTGCGACAACAAGAAGTGCGACAAGAAGTGCATCGAGTGGGAGAAGGCCCAGCACGGCGCCTGCCACAAGAGAGAGGCCGGCAAGGAGAGCTGCTTCTGCTACTTCGACTGCAGCAAGAGCCCCCCCGGCGCCACCCCCGCCCCCCCCGGCGCCGCCCCCCCCCCCGCCGCCGGCGGCAGCCCCAGCCCCCCCGCCGACGGCGGCAGCCCCCCCCCCCCCGCCGACGGCGGCAGCCCCCCCGTGGACGGCGGCAGCCCCCCCCCCCCCAGCACCCAC。
according to the invention, according to the mRNA sequence of the main component allergen Art V1 of mugwort, the original signal peptide of the Art V1 gene is replaced by an Azurocidin preproprotein signal peptide sequence, and codon optimization is carried out, wherein the codon optimization is mainly carried out by adjusting GC content, mRNA secondary structure and the like, and an online codon optimization tool (https:// www.vectorbuilder.cn/tool/codon-optimization. Html) is utilized to obtain the nucleic acid molecule for coding the Art V1 recombinant protein, the nucleic acid molecule is artificially synthesized and then is subcloned to a eukaryotic vector to obtain a recombinant vector, and the efficient expression of the Art V1 recombinant protein in the eukaryotic expression system is favorably realized.
In a second aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule as described in the first aspect.
In the invention, the recombinant vector has multiple cloning sites and is a molecular biological vector capable of regulating and controlling the expression of exogenous genes.
Preferably, the empty vector of the recombinant vector comprises pcDNA3.1/myc-His A.
In a third aspect, the present invention provides a eukaryotic expression system transfected with a nucleic acid molecule as described in the first aspect and/or a recombinant vector as described in the second aspect.
Preferably, the eukaryotic expression system comprises eukaryotic cells that can express the exogenous gene.
Preferably, the eukaryotic cell comprises a mammalian cell.
Preferably, the mammalian cells comprise HEK293F cells.
In a fourth aspect, the present invention provides an Art V1 recombinant protein, wherein the Art V1 recombinant protein is expressed using the eukaryotic expression system according to the third aspect.
Preferably, the Art V1 recombinant protein comprises an amino acid sequence shown as SEQ ID NO. 2;
SEQ ID NO:2：
AGSKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYFDCSKSPPGATPAPPGAAPPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPSTH。
in a fifth aspect, the present invention provides a method for producing the Art V1 recombinant protein according to the fourth aspect, the method comprising:
(1) Inserting a nucleic acid molecule for coding the Art V1 recombinant protein into a eukaryotic vector to obtain a recombinant vector;
(2) Transforming the recombinant vector into a eukaryotic expression system, and carrying out induced culture;
(3) Collecting the culture supernatant of the eukaryotic expression system, and purifying to obtain the Art V1 recombinant protein.
Preferably, the nucleic acid molecule of step (1) is as shown in SEQ ID NO 1.
Preferably, the eukaryotic vector of step (1) comprises pcDNA3.1/myc-His A.
Preferably, the nucleic acid molecule of step (1) is inserted into the HindIII and XbaI restriction sites of a eukaryotic vector.
Preferably, the eukaryotic expression system of step (2) comprises eukaryotic cells.
Preferably, the eukaryotic cell comprises a mammalian cell.
Preferably, the mammalian cells comprise HEK293F cells.
Preferably, the transformation method of step (2) comprises chemical transfection and/or physical transfection.
Preferably, the method of purification in step (3) comprises affinity chromatography.
Preferably, the affinity chromatography is performed using a nickel column.
As a preferred embodiment, the present invention provides a method for producing an Art V1 recombinant protein according to the fourth aspect, the method comprising:
(1) Inserting nucleic acid molecule SEQ ID NO 1 of coding Art V1 recombinant protein into HindIII and XbaI restriction enzyme cutting sites of eukaryotic vector pcDNA3.1/myc-His A to obtain a recombinant vector;
(2) Transforming the recombinant vector into HEK293F cells, and performing induction culture;
(3) Collecting culture supernatant of the HEK293F cells, and performing nickel column affinity chromatography to obtain the Art V1 recombinant protein.
In a sixth aspect, the present invention provides a nucleic acid molecule according to the first aspect, a recombinant vector according to the second aspect, a eukaryotic expression system according to the third aspect, or an Art V1 recombinant protein according to the fourth aspect, for use in the preparation of a diagnostic agent and/or a therapeutic agent for allergic diseases.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention replaces the original signal peptide of the Art V1 gene with an Azurocidin preproprotein signal peptide sequence, and carries out codon optimization to obtain a nucleic acid molecule which is suitable for a eukaryotic expression system and encodes the Art V1 recombinant protein;
(2) The invention subclones the nucleic acid molecule into a eukaryotic vector after artificial synthesis to obtain a recombinant vector, and then transfects the recombinant vector into a eukaryotic expression system, thereby realizing the high-efficiency expression of the Art V1 recombinant protein, obtaining 10.5mg of Artemisia argyi Art V1 recombinant protein by 1L induction expression amount, and the purity of the Artemisia argyi Art V1 recombinant protein is as high as more than 90%;
(3) The preparation method of the invention has the advantages of simple operation, low cost and high yield, and has wide application prospect in the field of diagnosis and treatment of allergic diseases.
Drawings
FIG. 1 is an SDS-PAGE of the recombinant protein Art V1, wherein lane 1 is the sample before purification, lane 2 is the wash harvest, lane 3 is the elution harvest, and lane 4 is the protein Marker;
FIG. 2 is a Western Blot analysis of Art V1 recombinant protein, wherein lane 1 shows purified Art V1-detected UniCAP mugwort negative serum, and lanes 2-4 show purified Art V1-detected UniCAP mugwort positive serum.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Cell and vector: coli DH 5. Alpha. Competent cells were purchased from Tiangen Biochemical technology (Beijing) Ltd, HEK293F cells were purchased from Thermo Fisher; pcDNA3.1/myc-His A vectors were purchased from Youbao.
Culture medium: SMM 293-TIS medium, sinofection-293 transfection reagent, SMS 293-SUPI from Beijing Yi Qiao Shen science and technology Co.
Biochemical engineeringReagent: pierce TM Coomassie (Bradford) Protein Assay Kit, fastdigest HindIII, fastdigest XbaI from Thermo Fisher; beyogold TM His-tag Purification Resin (reduction-resistant chelating type) and TMB color developing solution are purchased from Biyuntian biotechnology limited company; 4 × Loading buffer, 4-20% TEO gradient precast gel purchased from Shanghai Tianneng technology Ltd.
Example 1 design and Synthesis of Art V1 recombinant protein-encoding Gene
Taking the mRNA sequence (GenBank sequence accession number: AF493943, SEQ ID NO: 3) of main component allergen Art V1 of Artemisia argyi disclosed by GenBank as an optimization object, replacing the signal peptide of the original signal peptide of the Art V1 gene with Azurocidin preproprotein signal peptide sequence (GenBank sequence accession number: NM-001700, SEQ ID NO.
SEQ ID NO:3：
ATGGCAAAGTGTTCATATGTTTTCTGTGCGGTTCTTCTGATTTTCATAGTTGCTATCGGAGAAATGGAGGCCGCTGGTTCAAAGTTGTGTGAAAAGACAAGCAAGACGTATTCGGGTAAGTGCGACAACAAGAAATGTGACAAAAAGTGTATAGAGTGGGAGAAAGCGCAACATGGTGCTTGTCACAAGAGAGAAGCCGGCAAAGAAAGTTGCTTTTGCTACTTTGACTGTTCCAAATCGCCTCCTGGAGCAACACCAGCGCCTCCTGGTGCAGCTCCTCCCCCAGCTGCTGGCGGCTCTCCGTCACCTCCCGCTGATGGTGGCTCACCACCTCCTCCAGCTGATGGTGGATCTCCTCCTGTAGATGGTGGCTCTCCACCTCCTCCGTCCACTCACTAA；
SEQ ID NO:4：
ATGACCCGGCTGACAGTCCTGGCCCTGCTGGCTGGTCTGCTGGCGTCCTCGAGGGCC。
Example 2 expression of Art V1 recombinant protein
(1) After the coding gene (SEQ ID NO: 1) of the Art V1 recombinant protein was digested with restriction enzymes HindIII and XbaI, it was purified and recovered with TaKaRa Fragment Purification Kit; inserting the recovered coding gene into pcDNA3.1/myc-His A which is also subjected to enzyme digestion by restriction enzymes HindIII and XbaI to construct a recombinant vector;
(2) Dilution of pairs with preheated fresh SMM 293TIS MediumHEK293F cells in several growth phases to a cell density of 1X 10 6 cells/mL, 100mL of cell sap are added into a 500mL cell bottle, and the cell solution is placed in a shaking table for continuous culture for 3 hours;
(3) Diluting 50 mu g of recombinant vector by 150mM NaCl, uniformly mixing, standing for 5min, then adding 250 mu L of Sinofection-293 transfection reagent to ensure that the volume of the final transfection solution is 5mL, uniformly mixing, and standing for 10min;
(4) Adding the transfection solution dropwise into the cell culture solution, shaking, and returning to the shaker at 36.5 deg.C and 5% CO 2 Continuing the culture at 130rpm under the conditions;
(5) Adding 2mL of SMS 293-SUPI culture medium additive solution every day 24h after transfection;
(6) After transfection for 72h, collecting the culture medium, centrifuging at 10000rpm for 10min, and collecting the supernatant, wherein the supernatant contains Art V1 recombinant protein and the amino acid sequence is shown as SEQ ID NO. 2.
Example 3 purification of Art V1 recombinant protein
Protein purification was performed according to Beyogold from Biyuntian Biotechnology Ltd TM The method is carried out according to the instruction of His-tag Purification Resin (reduction-resistant chelating type), and comprises the following steps:
(1) Mixing 100mL of cell supernatant with 25mL of non-denatured lysate;
(2) 50% of 10mL of Beyogold TM His-tag Purification Resin (reduction chelate resistant type), centrifuging at 4 deg.C (1000 g × 10 s) to remove the storage solution, adding a column volume of non-denatured lysate into the gel, mixing to balance the gel, centrifuging at 4 deg.C (1000 g × 10 s) to remove the solution, repeating the balancing for 1-2 times, and removing the solution;
(3) Hybrid Beyogold TM His-tag purification resin (reduction-tolerant chelating type) and cell supernatant, slowly shaking in a side shaking table or a horizontal shaking table at 4 deg.C for 60min;
(4) Cell supernatants and Beyogold TM Putting mixture of His-tag Purification Resin (reduction-resistant chelating type) into appropriate empty column tube, opening the cover at the bottom of the Purification column, and allowing liquid in the column to flow out under gravity;
(5) Washing the column for 5 times, and adding 5mL of non-denaturing washing solution each time;
(6) Eluting the target protein for 2 times by using 5mL of non-denatured eluent each time to obtain the purified Art V1 recombinant protein.
Example 4 SDS-PAGE and protein concentration determination of the Art V1 recombinant protein
Respectively mixing 30 μ L of the collected samples with 10 μ L of Shanghai energy 4 × Loading buffer, and boiling in metal bath at 99 deg.C for 10min; after centrifugation at 10000rpm for 5min, 10. Mu.L of the supernatant was subjected to SDS-PAGE using 4-20% TEO gradient pre-gel, and the results were shown in FIG. 1, wherein lane 1 is a sample before purification, lane 2 is a wash pool, lane 3 is an elution pool, and lane 4 is a protein Marker.
It can be seen that the purified expression product has a distinct band at 10kDa, corresponding to the predicted molecular weight size of Art V1 (10 kDa), with a purity of >90% of the target protein.
By using Pierce TM The concentration of the purified Protein is measured by Coomassie (Bradford) Protein Assay Kit, and is 0.105mg/mL, and 100mL of cell expression can obtain about 1.05mg of high-purity Art V1 recombinant Protein.
Example 5 Western Blot analysis of Art V1 recombinant protein
Performing SDS-PAGE electrophoresis on 10 μ g of Art V1 recombinant protein after purification, electrically transferring to PVDF membrane, blocking BSA for 1h at room temperature, incubating mugwort allergen positive serum and negative serum for 1h at room temperature, respectively, washing with TBST for 3 times, each for 5min; then, incubation is carried out for 1h by using a mouse anti-human IgE secondary antibody coupled with HRP, TBST is washed for 3 times, each time is 5min, color development is carried out by using TMB color development solution, and the expression condition of the Art V1 recombinant protein is detected.
The results are shown in FIG. 2, wherein lane 1 is Art V1 mugwort allergen-negative serum, and lanes 2-4 are Art V1 mugwort allergen-positive serum, it can be seen that the Art V1 recombinant protein has reactivity with all of 3 cases of UniCAP mugwort-positive serum, but does not react with the negative serum, indicating that the Art V1 recombinant protein is successfully expressed in HEK293F cells.
In conclusion, the invention replaces the original signal peptide of the Art V1 gene with the Azurocidin preproprotein signal peptide sequence, and carries out codon optimization to obtain the nucleic acid molecule which is suitable for the eukaryotic expression system and used for coding the Art V1 recombinant protein, the nucleic acid molecule is artificially synthesized and then is subcloned into the eukaryotic vector to obtain the recombinant vector, and the recombinant vector is transfected into the eukaryotic expression system, thereby realizing the high-efficiency expression of the Art V1 recombinant protein, and the preparation method has the advantages of simple operation, low cost and high yield, and has wide application prospect in the fields of diagnosis and treatment of allergic diseases.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> blue Yi science and technology group, inc.; zhejiang Lanyi medicine Co., ltd
<120> Art V1 recombinant protein, preparation method and application thereof
<130> 20200309
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 381
<212> DNA
<213> Artificial Synthesis
<400> 1
atgaccagac tgaccgtgct ggccctgctg gccggcctgc tggccagcag cagagccgcc 60
ggcagcaagc tgtgcgagaa gaccagcaag acctacagcg gcaagtgcga caacaagaag 120
tgcgacaaga agtgcatcga gtgggagaag gcccagcacg gcgcctgcca caagagagag 180
gccggcaagg agagctgctt ctgctacttc gactgcagca agagcccccc cggcgccacc 240
cccgcccccc ccggcgccgc cccccccccc gccgccggcg gcagccccag cccccccgcc 300
gacggcggca gccccccccc ccccgccgac ggcggcagcc cccccgtgga cggcggcagc 360
cccccccccc ccagcaccca c 381
<210> 2
<211> 108
<212> PRT
<213> Artificial Synthesis
<400> 2
Ala Gly Ser Lys Leu Cys Glu Lys Thr Ser Lys Thr Tyr Ser Gly Lys
1 5 10 15
Cys Asp Asn Lys Lys Cys Asp Lys Lys Cys Ile Glu Trp Glu Lys Ala
20 25 30
Gln His Gly Ala Cys His Lys Arg Glu Ala Gly Lys Glu Ser Cys Phe
35 40 45
Cys Tyr Phe Asp Cys Ser Lys Ser Pro Pro Gly Ala Thr Pro Ala Pro
50 55 60
Pro Gly Ala Ala Pro Pro Pro Ala Ala Gly Gly Ser Pro Ser Pro Pro
65 70 75 80
Ala Asp Gly Gly Ser Pro Pro Pro Pro Ala Asp Gly Gly Ser Pro Pro
85 90 95
Val Asp Gly Gly Ser Pro Pro Pro Pro Ser Thr His
100 105
<210> 3
<211> 399
<212> DNA
<213> Artificial Synthesis
<400> 3
atggcaaagt gttcatatgt tttctgtgcg gttcttctga ttttcatagt tgctatcgga 60
gaaatggagg ccgctggttc aaagttgtgt gaaaagacaa gcaagacgta ttcgggtaag 120
tgcgacaaca agaaatgtga caaaaagtgt atagagtggg agaaagcgca acatggtgct 180
tgtcacaaga gagaagccgg caaagaaagt tgcttttgct actttgactg ttccaaatcg 240
cctcctggag caacaccagc gcctcctggt gcagctcctc ccccagctgc tggcggctct 300
ccgtcacctc ccgctgatgg tggctcacca cctcctccag ctgatggtgg atctcctcct 360
gtagatggtg gctctccacc tcctccgtcc actcactaa 399
<210> 4
<211> 57
<212> DNA
<213> Artificial Synthesis
<400> 4
atgacccggc tgacagtcct ggccctgctg gctggtctgc tggcgtcctc gagggcc 57
Claims (7)
1.A nucleic acid molecule for coding an Art V1 recombinant protein, which is characterized in that the nucleic acid molecule has a nucleic acid sequence shown as SEQ ID NO. 1.
2. A recombinant vector comprising the nucleic acid molecule of claim 1.
3. The recombinant vector according to claim 2, wherein the empty vector of the recombinant vector comprises pcDNA3.1/myc-His A.
4. A eukaryotic expression system transfected with a nucleic acid molecule according to claim 1 and/or a recombinant vector according to claim 2 or 3;
the eukaryotic expression system includes a eukaryotic cell.
5. The eukaryotic expression system of claim 4, wherein the eukaryotic cell comprises a mammalian cell.
6. The eukaryotic expression system of claim 5, wherein the mammalian cell comprises a HEK293F cell.
7. Use of a nucleic acid molecule according to claim 1, a recombinant vector according to claim 2 or 3, a eukaryotic expression system according to any one of claims 4 to 6 for the preparation of a diagnostic agent and/or a therapeutic agent for allergic diseases caused by Art V1.
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