CN113651879A

CN113651879A - Preparation method and application of TRIM21 full-length protein

Info

Publication number: CN113651879A
Application number: CN202110951053.1A
Authority: CN
Inventors: 徐娅; 易汪雪; 于倩; 王营; 殷婷子; 廖怡辉; 李梦茜
Original assignee: Cusabio Biotech Co ltd
Current assignee: Cusabio Biotech Co ltd
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-11-16

Abstract

The invention discloses a preparation method and application of TRIM21 full-length protein, wherein the coding gene of TRIM21 protein is subjected to codon optimization to obtain the TRIM21 protein coding gene shown as SEQ ID NO.1, so that the in-vitro expression efficiency of TRIM21 protein is obviously improved, and a yeast system is used for in-vitro expression to obtain the high-purity TRIM21 full-length protein with high binding activity with ligand protein SKP 2.

Description

Preparation method and application of TRIM21 full-length protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of TRIM21 full-length protein.

Background

Human TRIM21 protein contains 475 amino acids and is composed of multiple domains. The gene comprises a RING domain, a B-box domain, a coil-coil domain, a C-terminal PRYSPRY domain and the like. The RING domain is the basis of TRIM21 as ubiquitin ligase participating in cell metabolism, and the PRYSPRY domain can be specifically combined with the IgG Fc segment CH2-CH3 domain. TRIM21 recognizes virus surface antibodies attached to cells, TRIM21 recognizes and binds these antibodies, labels the antibody-virus complex as "garbage" and delivers it to proteasomes in cells for degradation, and based on this principle, the Trim-away technology has been invented, which can distinguish different proteins even two different variants of the same protein by using the significant specificity of antibodies, and has important significance in most of the research on diseases caused by protein variants, and TRIM21, which is the core of the technology, can be regarded as an ideal molecular biological tool for intracellular protein targeted knock-out.

TRIM21 has important functions in immunity, such as neutralizing viral infection and regulating inflammatory signaling pathways. In addition, TRIM21 may be involved in cell proliferation and apoptosis in cancer biology. Various research results show that TRIM21 may have tumor inhibiting effect, and the maintenance of TRIM21 expression is related to better prognosis of tumor patients. TRIM21 plays an important role in the process of resisting viruses in host cells as an important signal molecule in innate immunity, TRIM21 is used as a biomarker molecule to indicate certain diseases, and the exploration of the direction possibly has important significance for the diagnosis and prognosis of other certain diseases. However, how to realize the in vitro high-efficiency expression of the TRIM21 protein and maintain the binding activity of the TRIM21 protein is a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a preparation method and application of TRIM21 full-length protein, wherein codon optimization is carried out on a coding gene of TRIM21 protein to obviously improve the in-vitro expression efficiency of TRIM21 protein, and a yeast system is used for in-vitro expression, so that the high-purity TRIM21 full-length protein with high binding activity with ligand protein SKP2 is obtained.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a codon-optimized TRIM21 protein coding gene, wherein the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1.

Furthermore, the amino acid sequence of TRIM21 protein encoded by the encoding gene is shown in a sequence table SEQ ID NO. 2.

The invention also provides an expression vector, which comprises the codon-optimized TRIM21 protein coding gene.

Further, the expression vector is selected from a PET vector or a Pichia expression vector.

Further, the expression vector is a pPIC3.5K vector.

The invention also provides a cell, which comprises the expression vector.

Further, the cell is selected from escherichia coli, yeast or eukaryotic cells.

Further, the cell is a yeast cell.

The invention also provides a preparation method of the TRIM21 full-length protein, which comprises the following steps: connecting the coding gene shown as SEQ ID NO.1 with an expression vector, transferring the coding gene into a receptor cell, inducing the expression of the coding gene, cracking the cell, collecting and purifying to obtain the TRIM21 protein.

Further, the method further comprises: and (3) detecting the binding activity of the TRIM21 protein and the ligand SKP2 protein by using an ELISA detection method.

Further, when the TRIM21 protein was purified, a target protein peak was detected by Coomassie Brilliant blue method or SDS-PAGE.

The invention also provides application of the TRIM21 protein prepared by the method in preparation of antiviral drugs.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a preparation method of TRIM21 full-length protein aiming at the blank of the existing TRIM21 protein in vitro expression, which is characterized in that codon optimization is carried out on the known coding gene of the TRIM21 protein, and in vitro expression is carried out by utilizing a yeast system, so that the expression efficiency of the TRIM21 protein in the in vitro expression is obviously improved, the target protein is expressed and purified, the elution condition is optimized, the purity of the target protein is obviously improved, and simultaneously, the high binding activity of the target protein and the ligand protein SKP2 is kept. The method is simple to operate and low in cost, and the TRIM21 full-length protein prepared by the method can be used as a molecular marker of diseases such as tumors and the like or as an antiviral drug, and has extremely high production and application values.

Drawings

FIG. 1 shows the results of agarose gel electrophoresis detection of gene amplification in example 1 of the present invention;

FIG. 2 is a map of recombinant plasmid HU-TRIM21-pPIC3.5K constructed in example 1 of the present invention;

FIG. 3 shows the SDS-PAGE results of in vitro expression and purification of the original TRIM21 protein encoding gene before optimization in example 1;

FIG. 4 shows the SDS-PAGE results of the in vitro expression and purification of the codon-optimized TRIM21 protein-encoding gene in example 1 of the present invention;

FIG. 5 shows SDS-PAGE detection results of the recombinant protein obtained after elution optimization in example 1 of the present invention;

FIG. 6 shows the results of examining the binding activity of TRIM21 protein to SKP2 in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example provides a method for expressing the full-length recombinant protein TRIM21 in a yeast system.

1. Experimental Material

Ultraviolet instrument (WD-9403F, six Beijing);

a desk-top high-speed centrifuge (H1650-W, Hunan instrument);

a constant temperature water bath (DK-S22, Shanghai sperm macro);

a constant temperature incubator (GNP-P270, Shanghai sperm macro);

clean bench (SW-CJ-1FD, Antai, Suzhou);

PCR amplification instrument (DL9700, Beijing Donglingchang);

constant temperature shaking table (TS-100C, Shanghai Tian Xia);

-80 ℃ refrigerator (DW-86L628, Qingdao Haier Special appliances Co., Ltd.);

restriction enzymes were purchased from Fermentas;

methanol, agar powder, sodium chloride and imidazole are purchased from national medicine group chemical reagent limited.

2. Vector construction

(1) Codon optimization

The NCBI accession number of the original nucleotide sequence of the TRIM21 protein is NM-003141.4, wherein the coding region sequence is 1425bp, and the nucleotide sequence is shown as SEQ ID NO. 3. Aiming at the codon preference of an expression system, the nucleotide sequence is subjected to codon optimization, the nucleotide sequence of the TRIM21 protein coding gene after codon optimization is shown as a sequence table SEQ ID NO.1, and the corresponding amino acid sequence is shown as a sequence table SEQ ID NO. 2.

(2) Gene amplification

The original coding gene (SEQ ID NO.3) and the optimized TRIM21 coding gene (SEQ ID NO.1) are subjected to gene synthesis by Wuhan Kingkurui bioengineering Co., Ltd. And corresponding PCR amplification primers are designed, wherein F-1 and R-1 amplify a sequence shown in SEQ ID NO.1, F-2 and R-2 amplify a sequence shown in SEQ ID NO.3, and the primer sequences are as follows:

taking the synthesized coding gene as a template to carry out PCR amplification reaction, wherein the reaction system is as follows:

reagent	Volume (μ L)
		Form panel	4
Upstream primer (F)	2
		Downstream primer (R)	2
10×Buffer	20
		dNTPs	16
pFu polymerase	2
		Adding water to	154

The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; 30 cycles of denaturation at 94 ℃ for 30sec, annealing at 52 ℃ for 45sec, and elongation at 72 ℃ for 90 sec; further extension for 10min at 72 ℃; keeping the temperature at 15 ℃ for 5 min.

After the reaction is finished, detecting the amplified product by 1% agarose gel electrophoresis, wherein the detection result of the optimized coding gene is shown in figure 1, and the fragment size is 1425 bp. Cutting the gel according to the size of the target segment, and placing the gel in a clean centrifuge tube. Placing the centrifuge tube in a refrigerator at-80 deg.C, taking out after 15min, dissolving at room temperature, mashing the gel with 1mL blue gun head, 12000r/min, 2 min. After centrifugation the supernatant was transferred to a new blank EP tube for use.

(3) Double digestion of vector and recovery of product

Taking a pPIC3.5K vector, and carrying out double enzyme digestion on the optimized pPIC3.5K vector, wherein a double enzyme digestion reaction system (150 mu L) is as follows:

reagent	Volume (μ L)
		Carrier	20
Buffer O	15
		BamHI	4
NotI	4
		Adding water to	150

The mixture is blown and sucked by a gun gently and mixed evenly, the mixture is cut by enzyme in water bath at 37 ℃ for more than 20h, then the double enzyme digestion products are recovered by a recovery kit (Tiangen Biochemical technology (Beijing) Co., Ltd.) according to the steps of the instruction, and the enzyme digestion products are detected by electrophoresis of 1 percent agarose gel.

(4) Ligation reaction

Respectively recovering the TRIM21 DNA fragments before and after optimization and the restriction enzyme products of the vector pPIC3.5K, and carrying out ligation reaction, wherein the reaction system (10 mu L) is as follows:

reagent	Volume (μ L)
		TRIM21 fragment	4
pPIC3.5K vector	1
		Buffer	1
Ligase	1
		Adding water to	10

And connecting for more than 2 hours at 22 ℃ to obtain a connection product, namely the recombinant plasmid.

(5) Transformation and Positive clone screening

The competent cells were removed from the freezer at-80 ℃ and the lid was opened and the recombinant plasmid (10. mu.L) was added; gently blowing and sucking, rotating the small gun head to fully and uniformly mix the recombinant plasmid and the competent cells, and carrying out heat shock for 90s at 42 ℃ for 1min on ice; add 700. mu.l of pre-warmed LB medium and shake at 37 ℃ for 158r/1.5 h.

Centrifuging at 6000r for 4min, sucking 700 μ l supernatant in a super clean bench, mixing the rest bacteria liquid, and spreading on a plate containing ampicillin; and (3) inverting the plate, and culturing in a constant-temperature incubator at 37 ℃ for 12-16 h to obtain bacterial colonies.

Screening positive clones: and (3) subpackaging 40 mu l of LB culture medium without antibiotics into PCR tubes, picking 6 single colonies of each clone into the PCR tubes, mixing uniformly, and placing in a shaker at 37 ℃ for 220r/2 h. Taking 2 mul of each tube as a template to carry out bacteria liquid PCR, wherein products and H are recovered by PCR₂O is used as a template of positive control and negative control respectively; and (3) carrying out agarose gel electrophoresis detection after the PCR reaction is finished, selecting the PCR tube with the size consistent with that of the target fragment as a positive clone, selecting the monoclonal bacterium liquid of 3 corresponding PCR tubes with positive results, inoculating the monoclonal bacterium liquid into 3mL LB culture medium containing ampicillin, culturing overnight, and preserving the seeds the next day. And (5) carrying out plasmid extraction on the instructions of the positive cloning case kit and sending the instructions to sequencing. The sequencing result is consistent with the target gene, and the positive clone is obtained without base mutation. Wherein the map of the recombinant plasmid HU-TRIM21-pPIC3.5K is shown in figure 2.

3. Protein expression and purification

(1) Protein expression

The recombinant plasmid HU-TRIM21-pPIC3.5K is subjected to linearization reaction by ScaI enzyme, and is subjected to enzyme digestion overnight in water bath at 37 ℃ to obtain linearized recombinant plasmid, and the linearized recombinant plasmid is electrically transformed into yeast competent cells and coated on an RDB plate. Transformants were picked for PCR validation and single colonies were picked.

Inoculating the single colony into 10mL YPG liquid culture medium, shaking in a shaking table at 30 ℃ for 24 hours, collecting the bacteria, transferring the bacteria into 1L YPG liquid culture medium, and putting in the shaking table, shaking at 30 ℃ for 24 hours, and collecting the bacteria. The cells were then transferred to 1L YP medium (containing 1% methanol and 1mL of a sterilized antifoaming agent), supplemented with 1% methanol every 24 hours, cultured for three days, collected and mechanically disrupted, and the supernatant was collected and prepared for purification.

(2) Protein purification

Separating and purifying the intracellular supernatant by adopting a Ni-NTA column, which specifically comprises the following steps:

after the nickel column is pretreated, pretreating the intracellular supernatant to prepare a sample loading solution, and loading the sample on the pretreated nickel column; gradient elution is carried out by adopting 20mM imidazole buffer solution, 60mM imidazole buffer solution, 100mM imidazole buffer solution and 500mM imidazole buffer solution, and the target protein is obtained by elution. And detecting the target protein peak by adopting a Coomassie brilliant blue method and SDS-PAGE in the gradient elution process.

Wherein the in vitro expression and purification SDS-PAGE detection result of the original TRIM21 protein encoding gene before optimization is shown in FIG. 3, wherein lane 1 is the sample; lane 2 shows the permeate and lanes 3-6 show purified samples eluted with different gradient imidazole. The result of SDS-PAGE detection of in vitro expression and purification of the codon-optimized TRIM21 protein-encoding gene is shown in FIG. 4, wherein lane 1 is a control sample, i.e., the supernatant of yeast without transfer of the target plasmid; lane 2 as such; lanes 3-5 are purified samples eluted with 500mM, 100mM and 60mM imidazole in this order.

The result shows that when the gene encoding the original TRIM21 protein, which is not optimized, in the lane shown in fig. 3 is expressed in vitro, the target protein cannot be obtained after purification, that is, the gene encoding the original TRIM21 protein, which is not optimized, is not expressed basically, but the expression efficiency of the target protein is significantly improved after the encoding gene is optimized in the invention.

Further optimizing elution conditions, specifically: with CoCl₂Treating the column with the solution, pretreating intracellular supernatant to obtain a sample solution, and loading the sample solution onto the columnPretreating a nickel column; gradient elution is carried out by adopting 20mM imidazole buffer solution, 60mM imidazole buffer solution, 100mM imidazole buffer solution and 500mM imidazole buffer solution, and the target protein is obtained by elution. And detecting a target protein peak by adopting a Coomassie brilliant blue method and SDS-PAGE in the gradient elution process. The SDS-PAGE detection result of the finally obtained recombinant protein is shown in FIG. 5, wherein lane 1 is the purified sample after the elution conditions are optimized. The result shows that the invention finally obtains the target protein TRIM21 with high purity, wherein the purity is higher than 90%, by optimizing the elution conditions.

4. Detection of binding Activity of TRIM21 protein and SKP2

The method is characterized in that an ELISA detection method is adopted to detect the binding activity of the prepared TRIM21 protein and SKP2, and the specific steps are as follows:

diluting TRIM21 recombinant protein prepared by the method to 5 mu g/mL by using CB buffer, adding the diluted protein into an ELISA plate, and coating overnight at 4 ℃; after the coating solution was decanted, the cells were washed 3 times with PBS and blocked with 4% PBSM (PBS containing 4% skim milk) for 1 h; after washing 1 time with PBS, 100. mu.L of SKP2 protein (Cusabio, CSB-EP613392HU) diluted with 4% PBSM gradient was added to each well, wherein SKP2 protein was diluted with 18 gradients starting from 1000. mu.g/mL in 2-fold gradient and reacted at 37 ℃ for 1 h;

after washing 3 times with PBS and PBST respectively; mu.L of Sumo Monoclonal Antibody ((CSB-MA000132Mom) diluted 1: 1000 in 4% PBS was added to each well and reacted at 37 ℃ for 1 h;

after washing 3 times with PBS and PBST, 100. mu.L of Goat Anti-Mouse IgG (H + L) Antibody was added to each well; HRP conjugated (Cusabio, CSB-PA573747, diluted with 4% PBS at a ratio of 1: 10000) and incubated at 37 ℃ for 1 h;

washing with PBST and PBS three times, adding 100 μ L TMB substrate solution, reacting for 15min in dark, adding 25 μ L H₂SO₄(2mol/L) the reaction was terminated and OD was measured with a microplate reader_450nmThe value is obtained.

After reading the absorbance values, the EXCEL table was derived, and the results of absorbance measurements are shown in Table 1.

TABLE 1 TRIM21-SKP2 binding Activity assay results

Marking the corresponding groups, copying the concentration 1-concentration 18 data of the experimental group into Graphpad software, mapping, and reading EC₅₀Data, and finally a graph is derived, as shown in fig. 6.

The results show that TRIM21 recombinant protein prepared by the invention is coated by 5 mu g/mL, SKP2 protein (Cusabio, CSB-EP613392HU) is diluted by 18 gradients from 1000 mu g/mL in a 2-fold gradient way, and under the condition that a control group is normal, the EC of TRIM21 protein combined with SKP2 protein₅₀The value was 74.96-97.93. mu.g/mL. The structure of the TRIM21 protein prepared by in vitro expression and purification is similar to the structure of natural protein, and can be combined with SKP2 protein.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Wuhan Huamei bioengineering Co., Ltd

<120> preparation method and application of TRIM21 full-length protein

<160> 7

<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcttctg ctgctagatt gaccatgatg tgggaagaag tcacctgtcc aatctgcttg 60

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tcccaggtcg gtaaaggtgg tggttctgtt tgtccagtct gtagacagag attcctgctg 180

aagaacctga ggccaaacag acagttggct aacatggtca acaacctgaa agagatctcc 240

caagaggcca gagaaggtac tcaaggtgaa agatgtgctg tccacggtga gagattgcac 300

ttgttctgtg aaaaggacgg taaggccttg tgttgggttt gtgctcaatc cagaaagcac 360

agagatcacg ccatggttcc attggaagaa gctgctcaag agtaccaaga gaagttgcag 420

gttgctttgg gtgagctgag aagaaagcaa gagttggctg aaaagttgga ggtcgagatc 480

gctatcaaga gagccgactg gaagaaaacc gttgagactc aaaagtccag aatccacgct 540

gagttcgtcc agcagaagaa cttcttggtt gaagaggaac agagacagct gcaagagctg 600

gaaaaggatg agagagagca gttgagaatc ctgggtgaga aagaggctaa gttggctcaa 660

caatcccaag ccttgcaaga attgatctcc gagttggaca gaagatgtca ctcttccgct 720

ttggagttgt tgcaagaggt catcatcgtc ttggaaagat ccgagtcctg gaacttgaag 780

gacttggaca ttacttcccc agagttgaga tccgtttgtc acgttccagg tctgaagaaa 840

atgttgagaa cctgtgccgt ccacatcact ttggacccag atactgctaa cccatggttg 900

attttgtccg aggacagaag gcaggttaga ttgggtgata ctcagcagtc cattccaggt 960

aacgaagaga gattcgactc ttacccaatg gttttgggtg ctcaacactt ccactctggt 1020

aagcactact gggaagttga cgtcactggt aaagaagctt gggacttggg agtgtgtaga 1080

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caggttccac catgtcaggt tggtattttc ttggactacg aggccggtat ggtgtccttc 1260

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1 5 10 15

Pro Ile Cys Leu Asp Pro Phe Val Glu Pro Val Ser Ile Glu Cys Gly

20 25 30

His Ser Phe Cys Gln Glu Cys Ile Ser Gln Val Gly Lys Gly Gly Gly

35 40 45

Ser Val Cys Pro Val Cys Arg Gln Arg Phe Leu Leu Lys Asn Leu Arg

50 55 60

Pro Asn Arg Gln Leu Ala Asn Met Val Asn Asn Leu Lys Glu Ile Ser

65 70 75 80

Gln Glu Ala Arg Glu Gly Thr Gln Gly Glu Arg Cys Ala Val His Gly

85 90 95

Glu Arg Leu His Leu Phe Cys Glu Lys Asp Gly Lys Ala Leu Cys Trp

100 105 110

Val Cys Ala Gln Ser Arg Lys His Arg Asp His Ala Met Val Pro Leu

115 120 125

Glu Glu Ala Ala Gln Glu Tyr Gln Glu Lys Leu Gln Val Ala Leu Gly

130 135 140

Glu Leu Arg Arg Lys Gln Glu Leu Ala Glu Lys Leu Glu Val Glu Ile

145 150 155 160

Ala Ile Lys Arg Ala Asp Trp Lys Lys Thr Val Glu Thr Gln Lys Ser

165 170 175

Arg Ile His Ala Glu Phe Val Gln Gln Lys Asn Phe Leu Val Glu Glu

180 185 190

Glu Gln Arg Gln Leu Gln Glu Leu Glu Lys Asp Glu Arg Glu Gln Leu

195 200 205

Arg Ile Leu Gly Glu Lys Glu Ala Lys Leu Ala Gln Gln Ser Gln Ala

210 215 220

Leu Gln Glu Leu Ile Ser Glu Leu Asp Arg Arg Cys His Ser Ser Ala

225 230 235 240

Leu Glu Leu Leu Gln Glu Val Ile Ile Val Leu Glu Arg Ser Glu Ser

245 250 255

Trp Asn Leu Lys Asp Leu Asp Ile Thr Ser Pro Glu Leu Arg Ser Val

260 265 270

Cys His Val Pro Gly Leu Lys Lys Met Leu Arg Thr Cys Ala Val His

275 280 285

Ile Thr Leu Asp Pro Asp Thr Ala Asn Pro Trp Leu Ile Leu Ser Glu

290 295 300

Asp Arg Arg Gln Val Arg Leu Gly Asp Thr Gln Gln Ser Ile Pro Gly

305 310 315 320

Asn Glu Glu Arg Phe Asp Ser Tyr Pro Met Val Leu Gly Ala Gln His

325 330 335

Phe His Ser Gly Lys His Tyr Trp Glu Val Asp Val Thr Gly Lys Glu

340 345 350

Ala Trp Asp Leu Gly Val Cys Arg Asp Ser Val Arg Arg Lys Gly His

355 360 365

Phe Leu Leu Ser Ser Lys Ser Gly Phe Trp Thr Ile Trp Leu Trp Asn

370 375 380

Lys Gln Lys Tyr Glu Ala Gly Thr Tyr Pro Gln Thr Pro Leu His Leu

385 390 395 400

Gln Val Pro Pro Cys Gln Val Gly Ile Phe Leu Asp Tyr Glu Ala Gly

405 410 415

Met Val Ser Phe Tyr Asn Ile Thr Asp His Gly Ser Leu Ile Tyr Ser

420 425 430

Phe Ser Glu Cys Ala Phe Thr Gly Pro Leu Arg Pro Phe Phe Ser Pro

435 440 445

Gly Phe Asn Asp Gly Gly Lys Asn Thr Ala Pro Leu Thr Leu Cys Pro

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Leu Asn Ile Gly Ser Gln Gly Ser Thr Asp Tyr

465 470 475

<210> 3

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<213> human (Homo sapiens)

<400> 3

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caggaggcca gagagggcac acagggggaa cggtgtgcag tgcatggaga gagacttcac 300

ctgttctgtg agaaagatgg gaaggccctt tgctgggtat gtgcccagtc tcggaaacac 360

cgtgaccacg ccatggtccc tcttgaggag gctgcacagg agtaccagga gaagctccag 420

gtggcattag gggaactgag aagaaagcag gagttggctg agaagttgga agtggaaatt 480

gcaataaaga gagcagactg gaagaaaaca gtggaaacac agaaatctag gattcacgca 540

gagtttgtgc agcaaaaaaa cttcctggtt gaagaagaac agaggcagct gcaggagctg 600

gagaaggatg agagggagca gctgagaatc ctgggggaga aagaggccaa gctggcccag 660

cagagccagg ccctacagga gttgatttcc gagctggaca gaagatgtca ctcttccgct 720

ttggagttgc tgcaagaggt tatcatcgtc ttggaaagat ccgagtcctg gaacttgaag 780

gacttggaca ttacttcccc agagttgaga tccgtttgtc acgttccagg tctgaagaaa 840

atgttgagaa cctgtgccgt ccacatcact ttggacccag atactgctaa cccttggttg 900

attttgtccg aggacagaag gcaggttaga ttgggtgaca ctcaacagtc cattccaggt 960

aacgaagaga gattcgactc ttacccaatg gttttgggtg ctcaacactt ccactctggt 1020

aagcactact gggaagttga cgtcactggt aaagaagctt gggacttggg agtctgtaga 1080

gactctgtta gaagaaaggg tcacttcctg ttgtcctcca agtctggttt ctggactatc 1140

tggttgtgga acaagcagaa gtacgaggct ggtacttacc cacaaactcc attgcacttg 1200

caggttccac catgtcaggt cggtattttc ttggactacg aagccggtat ggtgtccttc 1260

tacaacatta ctgaccacgg ttccctgatc tactccttct ctgaatgtgc tttcaccggt 1320

ccattgaggc catttttctc accaggtttc aacgacggtg gtaagaacac tgctccattg 1380

accttgtgtc cactgaacat tggttcccaa ggttccactg attac 1425

<210> 4

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcagggatcc gccaccatgc atcaccacca tcatcatatg gcttctgctg ctag 54

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgtagcggcc gcttagtaat cagtggaacc ttggg 35

<210> 6

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

actaattatt cgaaggatcc catcaccacc atcatcatat ggcttcagca gcacgc 56

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aggcgaatta attcgcggcc ttagtaatca gtggaacctt ggga 44

Claims

1. A codon-optimized TRIM21 protein coding gene is characterized in that the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1.

2. The encoding gene of claim 1, wherein the amino acid sequence of TRIM21 protein encoded by the encoding gene is shown in SEQ ID NO.2 of the sequence table.

3. An expression vector comprising the codon-optimized TRIM21 protein-encoding gene of claim 1.

4. The expression vector of claim 3, wherein the expression vector is selected from a PET vector or a Pichia expression vector.

5. A cell comprising the expression vector of claim 3 or 4.

6. The cell of claim 5, wherein the cell is selected from the group consisting of E.coli, yeast, and eukaryotic cells.

7. A method for preparing TRIM21 full-length protein, which comprises the following steps: connecting the coding gene shown as SEQ ID NO.1 with an expression vector, transferring the coding gene into a receptor cell, inducing the expression of the coding gene, cracking the cell, collecting and purifying to obtain the TRIM21 protein.

8. The method of manufacturing according to claim 7, further comprising: and (3) detecting the binding activity of the TRIM21 protein and the ligand SKP2 protein by using an ELISA detection method.

9. The method according to claim 7, wherein a peak of the target protein is detected by Coomassie Brilliant blue method or SDS-PAGE when the TRIM21 protein is purified.

10. Use of TRIM21 protein as defined in claim 7 for the preparation of an antiviral medicament.