CN111499720B - Macrobrachium rosenbergii thymosin beta 4 gene, protein, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of molecular biological gene engineering, relates to a nucleotide sequence of Macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta 4 gene, an encoded amino acid sequence and a nucleotide sequence after codon optimization, and particularly relates to a recombinant expression preparation method of Macrobrachium rosenbergii thymosin beta 4 protein. The recombinant expression of the MrThy beta 4 protein can overcome the problem of low yield in the purification and separation process, further research the function of the protein and lay a foundation for the wide application of the protein in the research fields of biology, medicine, agriculture and the like.

Description

Macrobrachium rosenbergii thymosin β4 gene, protein and preparation method and application thereof

technical field

The invention belongs to the field of molecular biology genetic engineering, and relates to a nucleotide sequence and an encoded amino acid sequence of a thymosin β4 gene of Macrobrachium rosenbergii, as well as a codon-optimized nucleotide sequence, in particular to a macrobrachium rosenbergii (Macrobrachium rosenbergii) rosenbergii) Recombinant expression preparation method of thymosin β4 protein.

Background technique

Thymosin widely exists in vertebrates and invertebrates, and can be divided into three categories: Tα, Tβ, Tγ, etc. according to the difference of isoelectric point. The functional domain of thymosin is a thymosin (THY) domain, and β-thymosin proteins in invertebrates usually have multiple domains. Thymosin is currently involved in various biological functions of the body, including regulation of actin and remodeling of the cytoskeleton. In addition, β-thymosin can also play an important role in physiological activities such as cell motility, endocytosis, immune regulation and cascade reactions, tumor diagnosis and treatment, development and wound healing. At present, thymosin from vertebrates has been developed as a clinical drug for the treatment of tumors, viral hepatitis and other diseases. The thymosin protein is highly conserved in the evolutionary process, and the thymosin protein isolated from shrimp also has important biological activities.

Macrobrachium rosenbergii is one of the main farmed freshwater shrimp species in my country. Its strong adaptability, fast growth rate and wide breeding range have brought huge benefits to farmers. However, shrimps are invertebrates and lack the adaptive immune system, mainly relying on innate immune defense. The strong adaptability and anti-stress ability of Macrobrachium rosenbergii are related to its strong innate immune system. Thymosin protein is an effector molecule of shrimp innate immunity and plays an important role in the process of disease resistance. Therefore, the specific immune disease resistance factor of shrimp itself is developed, its functional activity is studied, and its resistance to disease is strengthened. However, the process of directly separating thymosin from shrimp is complicated, with high cost and low yield, and it is difficult to achieve stable and high-efficiency expression of thymosin β4 protein by traditional prokaryotic expression and recombinant methods. Therefore, the scheme of the present invention combines synthetic biology and bioengineering technology to transform the thymosin β4 gene and achieve high-efficiency expression in Escherichia coli.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a recombinant expression preparation method and application of the thymosin β4 protein of Macrobrachium rosenbergii. By recombinantly expressing the MrThyβ4 protein, the problem of low yield in the purification and separation process can be overcome.

The technical scheme of the present invention is as follows:

A M. rosenbergii thymosin β4 gene, the M. rosenbergii thymosin β4 gene is named MrThyβ4, and the sequence is shown in SED ID NO: 1.

A thymosin β4 gene of Macrobrachium rosenbergii, the sequence of which is shown in SED ID NO: 2.

A thymosin β4 protein of Macrobrachium rosenbergii, the sequence of which is shown in SED ID NO: 3.

A preparation method of Macrobrachium rosenbergii thymosin β4 protein, comprising the following steps:

The codon-optimized sequence of MrThyβ4, SEQ ID No. 2, was directly linked to the pET30a vector, transformed into E. coli BL21 competent cells, and single clones were selected for shaking, induced by IPTG for expression, and collected for SDS-PAGE protein detection. Bacteria were lysed by sonication, purified and renatured.

Further, the transformation steps are as follows: take the competent cells E.coli BL21 and put them in ice and let them melt; add recombinant plasmids to the competent cells, mix the contents gently, and let stand in an ice bath for 3 min; centrifuge the cells The tubes were placed in a 42°C water bath for 90s, quickly transferred to an ice bath, cooled for 2-3 minutes, and do not shake; add 900 μL of sterile LB medium to each centrifuge tube, mix well, and place on a shaker at 37°C for 45 minutes. to revive the bacteria; put the recovered bacteria in a centrifuge at 1000 rpm for 3 min, aspirate 850 μL of the supernatant, mix evenly and add competent cells to LB solid medium containing the corresponding antibiotics. Gently spread the cells evenly with a sterile elbow glass rod, place at room temperature until the liquid is absorbed, invert the plate, and incubate at 37°C for 12-16h.

Further, the inducing expression step is as follows: selecting Escherichia coli containing the recombinant plasmid, adding it to 250mL LB liquid medium, and culturing with shaking at 37°C until the bacterial liquid OD600=0.4-0.6; taking the above bacterial liquid as a control and preserving seeds, the remaining bacteria Add an appropriate amount of IPTG to the solution to the final concentration of 1 mM, and continue to culture at 37 °C for 2 to 3 hours; centrifuge the bacterial solution induced by IPTG at 8000 rpm, 4 °C for 10 min, carefully pour off the supernatant, and collect the bacterial pellet; About 20 mL of sterilized water was used to resuspend the cells, put them in -80°C for freezing, and freeze and thaw them repeatedly for 1-2 times; take the resuspended cells after freezing and thawing, and ultrasonically crush them under ice bath conditions. Gradually become clear, put the broken bacterial liquid at 4°C, 8000rpm, centrifuge for 10min, put the supernatant into a new 50mL centrifuge tube, and store the supernatant and pellet at -20°C; Bacterial liquid, after crushing, supernatant and precipitate were added with appropriate amount of loading buffer, mixed and centrifuged, boiled at 99.9 °C for 5 min, and analyzed by SDS-PAGE electrophoresis to determine the expression form of the recombinant protein.

Further, the crushing conditions are: crushing for 3s, interval for 2s, and crushing for 30min.

Further, the purification and renaturation step is as follows: the semipermeable membrane is slightly boiled in boiling water for 5-7 minutes, after processing, the purified recombinant protein is added to it, and after sealing, the semipermeable membrane is placed in the dialysis renaturation solution for 12 hours. Replace the renaturation solution once, take the renatured recombinant protein into a clean centrifuge tube, centrifuge at 5000 rpm for 5 min, and store the supernatant at -20°C.

Further, the urea concentration in the renaturing solution is 6M, 4M, 3M, 2M, 1M, and 0M in turn.

The present invention also provides the application of the method for preparing the above-mentioned M. rosenbergii thymosin β4 protein in the field of pharmacy.

The first object of the present invention is to provide the thymosin β4 gene sequence of Macrobrachium rosenbergii;

The second object of the present invention is to provide the thymosin β4 protein sequence of Macrobrachium rosenbergii;

The third object of the present invention is to provide a codon-optimized nucleotide sequence of Macrobrachium rosenbergii thymosin β4;

The fourth object of the present invention is to provide a method for recombinant expression and renaturation of thymosin β4 protein of Macrobrachium rosenbergii.

The molecular type of the macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta4 gene is a cDNA sequence: 501 bp in length, and the type is double-stranded, linear, nucleic acid, and the sequence of the macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta4 gene MrThybeta4 passed PCR Amplified and sequenced. PCR-specific primers were as follows: F-5'-ATGAGTACCGAAACCGCCC-3', R-5'-TTAGGCGTTCTTCTCCTGC-3'. specific sequence

As shown below, denoted as SEQ ID No.1:

ATGAGTACCGAAACCGCCCTCAAGGATCTCCCCAAGGTCGACCCCACCCTCAAGGGCCAGCTGGAAGGATTCACCCCCGACAAACTCAAGAAGACCGACACAGAGGAGAAGACCATCTTGCCTTCCAAAGAGGACGTGGAAAGTGAGAAGCTTCGGAACGAACACCTGGAGAACATCAGCAAATTCCCGAGTGGGAGGCTGAAACGCACCTCTACTTCGGAGAAAATTGTCCTCCCCTCTAGCGCAGATGTGGAAGCCGAGAAGAAAGAAAAAGCCCACCTACAGGCTGTGGAGGGCTTCAATGCAGCCAATTTGAAGCATGCAAACACGAAAGAGAAAATTGTGTTGCCGGCCAAAGAAGATATCGAGAAAGAAAAGGGTCAGCAGGCGCTGTTCCAAGGAATCGAAGGATTCAACCAATCTAATCTTAAGAAGACTGAAACACAGGAGAAGAACCCTCTCCCAACTAAGGAGATAATCGAGCAGGAGAAGAACGCCTAA

Utilize DNAWorks 2.4 online software, carry out codon optimization to the nucleotide sequence of MrThyβ4, select Escherichia coli expression system, to the sequence after codon optimization of MrThyβ4 sequence, mark SEQ ID No.2:

ATGAGCACCGAAACCGCACTGAAAGATCTGCCGAAAGTGGACCCGACCCTGAAAGGCCAGCTGGAAGGCTTCACCCCGGACAAACTGAAAAAGACCGACACCGAAGAAAAAACCATTCTGCCGAGCAAAGAAGATGTTGAAAGCGAAAAACTGCGTAATGAACATCTGGAAAATATTAGCAAATTTCCGAGCGGTCGTCTGAAACGTACCAGCACCAGCGAAAAAATTGTTCTGCCGAGCAGCGCAGATGTTGAAGCAGAAAAAAAAGAAAAAGCACATCTGCAGGCAGTTGAAGGTTTTAATGCAGCAAATCTGAAACATGCAAATACCAAAGAAAAAATTGTTCTGCCGGCAAAAGAAGATATTGAAAAAGAAAAAGGTCAGCAGGCACTGTTTCAGGGTATTGAAGGTTTTAATCAGAGCAATCTGAAAAAAACCGAAACCCAGGAAAAAAATCCGCTGCCGACCAAAGAAATTATTGAACAGGAAAAAAATGCATAA

Described MrThyβ4 molecular type is protein, sequence feature: length 166aa, type is amino acid, sequence information is as follows, marked as SEQ ID No.3:

MSTETALKDLPKVDPTLKGQLEGFTPDKLKKTDTEEKTILPSKEDVESEKLRNEHLENISKFPSGRLKRTSTSEKIVLPSSADVEAEKKEKAHLQAVEGFNAANLKHANTKEKIVLPAKEDIEKEKGQQALFQGIEGFNQSNLKKTETQEKNPLPTKEIIEQEKNA

The preparation method of the MrThyβ4 thymosin peptide of Macrobrachium rosenbergii according to the present invention comprises the following steps:

Synthesis of a codon-optimized gene of Macrobrachium rosenbergii MrThyβ4;

A recombinant expression step of Macrobrachium rosenbergii MrThyβ4;

Refolding and antibacterial steps of the MrThyβ4 thymosin recombinant protein.

In the present invention, the sequence of MrThyβ4 codon-optimized SEQ ID No. 2 is directly connected to the pET30a vector, transformed into Escherichia coli BL21 competent cells, single clones are selected to be shaken, the expression is induced by IPTG, and the cells are collected for SDS-PAGE. For detection, ultrasonically disrupted and lysed bacteria, purified the target protein with Ni ²⁺ affinity chromatography column, renatured by dialysis, and verified the antibacterial activity. Through the method of the present invention, the problem of low yield in the purification and separation process can be overcome, the concentration of the produced thymosin recombinant protein can reach mg/ml level, and the renatured thymosin has obvious bacteriostatic activity (as shown in Figure 2), which can be used as a Potential antibacterial peptide drugs, applied in research fields such as biology, medicine and agriculture.

Description of drawings

Figure 1. SDS-PAGE electrophoresis detection of MrThyβ4 recombinant protein. M represents protein marker, 1 represents bacterial protein without IPTG induction, 2 represents bacterial protein after IPTG induction, and 3 represents MrThyβ4 recombinant protein purified by Ni ²⁺ .

Figure 2. Identification of the antibacterial activity of MrThyβ4 recombinant protein against Aeromonas hydrophila.

Detailed ways

Example 1

1. Transformation of MrThyβ4-pET30A recombinant plasmid:

The competent cells E.coli BL21 were taken and placed in ice and thawed. Add the recombinant plasmid to the competent cells, mix the contents gently, and let stand in an ice bath for 3 min. Place the centrifuge tube in a 42°C water bath for 90s, quickly transfer it to an ice bath, and cool for 2-3min without shaking. Add 900 μL of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, place it on a shaker at 37° C. for 45 min (15 rpm/min), and recover the cells. Place the recovered bacterial solution in a centrifuge at 1000 rpm for 3 min, aspirate and discard 850 μL of the supernatant, and after mixing, aspirate the competent cells and add them to the LB solid medium containing the corresponding antibiotics. Use a sterile elbow glass rod. Gently spread the cells evenly, place at room temperature until the liquid is absorbed, invert the plate, and incubate at 37°C for 12-16h.

2. Induction expression of MrThyβ4-pET30A protein:

Select the Escherichia coli containing the recombinant plasmid, add it to 250mL LB liquid medium (containing antibiotics, Kana resistance), shake and cultivate at 37°C until the bacterial liquid OD600=0.4-0.6; Add an appropriate amount of IPTG (1M) to the bacterial solution to a final concentration of 1 mM, and continue to culture at 37°C for 2-3 hours; centrifuge the bacterial solution induced by IPTG at 8000 rpm, 4°C for 10 min, carefully pour off the supernatant, and collect the bacterial pellet; Add about 20 mL of sterilized water to the above-mentioned precipitation to resuspend the cells, put them in -80°C for freezing, and freeze and thaw 1-2 times repeatedly; The conditions are: break for 3s, interval for 2s, break for 30min; after the break, the bacterial liquid gradually becomes clear, put the broken bacterial liquid at 4 ℃, 8000rpm, centrifuge for 10min, and put the supernatant into a new 50mL centrifuge tube , store the supernatant and the precipitate at -20°C; take the pre- and post-induction bacterial solutions, add an appropriate amount of loading buffer to the supernatant and the precipitate after crushing, mix and centrifuge, boil at 99.9°C for 5 min, and conduct SDS-PAGE electrophoresis analysis. Determine the expression form of the recombinant protein.

3. Purification and renaturation of MrThyβ4-pET30A recombinant protein:

To separate and purify the MrThyβ4-pET30A recombinant protein: dissolve the sonicated precipitate with 8M urea, and then load the sample on a Ni ²⁺ ion affinity chromatography column at a flow rate of 10 times the column volume/hour, and collect the flow-through liquid. The column was washed with 15 column volumes of Binding Buffer (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 0.5 M NaCl, 8 M urea) to remove impurities. Elution was performed with 5 mL of Elution Buffer (20 mM Tris-HCl pH 7.9, 500 mM imidazole, 0.5 M NaCl, 8 M urea), and the eluted protein was collected.

Refolding the MrThyβ4-pET30A recombinant protein: slightly boil the semipermeable membrane in boiling water for 5-7 minutes, add the purified recombinant protein to it after processing, and place the semipermeable membrane in the dialysis renaturation solution after sealing. Replace the renaturation solution once every 12h (the urea concentration in the renaturation solution is 6M, 4M, 3M, 2M, 1M, 0M in sequence), take the recombinant protein after renaturation in a clean centrifuge tube, centrifuge at 5000rpm for 5min, take the supernatant and set it Store at -20°C.

4. Detection of antibacterial activity of MrThyβ4-pET30A recombinant protein:

The inhibitory activity of the recombinant protein MrThyβ4 against Aeromonas hydrophila was determined by the zone of inhibition method. The specific steps are as follows: suck the bacterial solution preserved in the laboratory and inoculate it in 5 mL of sterile LB liquid medium, respectively. Cultivate at a constant temperature for several hours; when the bacterial liquid reaches the logarithmic growth stage (ie, OD=0.6-0.8), dilute the bacterial liquid by 10 times and draw 100 μL of the bacterial liquid to evenly spread it on the anti-anti-LB plate. Place the small disc of filter paper gently on the surface of the plate, draw 15 μL of recombinant protein MrThyβ4 stock solution and drop it vertically into the center of the disc, do not shake it left and right; place it in a 30°C constant temperature incubator and incubate overnight to observe whether a bacteriostatic zone is formed. As shown in Figure 2, compared with the intermediate control, the recombinant protein MrThyβ4 was able to significantly kill Aeromonas hydrophila, resulting in a significant inhibition zone. Aeromonas hydrophila is a major pathogenic bacteria in aquatic animals, which endangers the health of aquaculture animals, and the extensive use of antibiotics is prone to produce drug-resistant bacteria. Therefore, the specific activity of recombinant protein MrThyβ4 in killing Aeromonas hydrophila has a certain prospect for its application in aquaculture as an antimicrobial peptide drug.

sequence listing

<110> Yangzhou University

<120> Macrobrachium rosenbergii thymosin β4 gene, protein and preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 501

<212> DNA

<213> Artificial Sequence

<400> 1

atgagtaccg aaaccgccct caaggatctc cccaaggtcg accccaccct caagggccag 60

ctggaaggat tcacccccga caaactcaag aagaccgaca cagaggagaa gaccatcttg 120

ccttccaaag aggacgtgga aagtgagaag cttcggaacg aacacctgga gaacatcagc 180

aaattcccga gtgggaggct gaaacgcacc tctacttcgg agaaaattgt cctcccctct 240

agcgcagatg tggaagccga gaagaaagaa aaagcccacc tacaggctgt ggagggcttc 300

aatgcagcca atttgaagca tgcaaacacg aaagagaaaa ttgtgttgcc ggccaaagaa 360

gatatcgaga aagaaaaggg tcagcaggcg ctgttccaag gaatcgaagg attcaaccaa 420

tctaatctta agaagactga aacacaggag aagaaccctc tcccaactaa ggagataatc 480

gagcaggaga agaacgccta a 501

<210> 2

<211> 501

<212> DNA

<213> Artificial Sequence

<400> 2

atgagcaccg aaaccgcact gaaagatctg ccgaaagtgg acccgaccct gaaaggccag 60

ctggaaggct tcaccccgga caaactgaaa aagaccgaca ccgaagaaaa aaccattctg 120

ccgagcaaag aagatgttga aagcgaaaaa ctgcgtaatg aacatctgga aaatattagc 180

aaatttccga gcggtcgtct gaaacgtacc agcaccagcg aaaaaattgt tctgccgagc 240

agcgcagatg ttgaagcaga aaaaaaagaa aaagcacatc tgcaggcagt tgaaggtttt 300

aatgcagcaa atctgaaaca tgcaaatacc aaagaaaaaa ttgttctgcc ggcaaaagaa 360

gatattgaaa aagaaaaagg tcagcaggca ctgtttcagg gtattgaagg ttttaatcag 420

agcaatctga aaaaaaccga aacccaggaa aaaaatccgc tgccgaccaa agaaattatt 480

gaacaggaaa aaaatgcata a 501

<210> 3

<211> 166

<212> PRT

<213> Artificial Sequence

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Met Ser Thr Glu Thr Ala Leu Lys Asp Leu Pro Lys Val Asp Pro Thr

1 5 10 15

Leu Lys Gly Gln Leu Glu Gly Phe Thr Pro Asp Lys Leu Lys Lys Thr

20 25 30

Asp Thr Glu Glu Lys Thr Ile Leu Pro Ser Lys Glu Asp Val Glu Ser

35 40 45

Glu Lys Leu Arg Asn Glu His Leu Glu Asn Ile Ser Lys Phe Pro Ser

50 55 60

Gly Arg Leu Lys Arg Thr Ser Thr Ser Glu Lys Ile Val Leu Pro Ser

65 70 75 80

Ser Ala Asp Val Glu Ala Glu Lys Lys Glu Lys Ala His Leu Gln Ala

85 90 95

Val Glu Gly Phe Asn Ala Ala Asn Leu Lys His Ala Asn Thr Lys Glu

100 105 110

Lys Ile Val Leu Pro Ala Lys Glu Asp Ile Glu Lys Glu Lys Gly Gln

115 120 125

Gln Ala Leu Phe Gln Gly Ile Glu Gly Phe Asn Gln Ser Asn Leu Lys

130 135 140

Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Thr Lys Glu Ile Ile

145 150 155 160

Glu Gln Glu Lys Asn Ala

165

Claims (9)

1. The macrobrachium rosenbergii thymosin beta 4 gene is characterized by having a sequence shown as SED ID NO: 2, respectively.

2. The macrobrachium rosenbergii thymosin beta 4 protein is characterized by having a sequence shown as SED ID NO: 3, respectively.

3. A preparation method of macrobrachium rosenbergii thymosin beta 4 protein is characterized by comprising the following steps:

the sequence SEQ ID No.2 optimized by MrThy beta 4 codon is directly connected into a pET30a vector, is transformed into escherichia coli BL21 competent cells, a single clone is selected for shake bacteria, IPTG induction expression is carried out, thalli are collected for SDS-PAGE protein detection, bacteria are cracked by ultrasonic disruption, and the renaturation is purified.

4. The method for preparing the thymosin beta 4 protein of the macrobrachium rosenbergii according to claim 3, wherein the converting step comprises: placing competent cells E, coli BL21 in ice, and waiting for the cells to melt; adding the recombinant plasmid into the competent cells, gently mixing the contents uniformly, and standing in an ice bath for 3 min; placing the centrifuge tube in 42 deg.C water bath for 90s, rapidly transferring into ice bath, cooling for 2-3min without shaking; adding 900 μ L of sterile LB culture medium into each centrifuge tube, mixing, and shake culturing at 37 deg.C for 45min to recover thallus; placing the recovered bacterial liquid in a centrifuge, centrifuging for 3min at 1000rpm, sucking 850 mu L of supernatant, uniformly mixing, sucking competent cells, adding the competent cells to an LB solid culture medium containing corresponding antibiotics, slightly and uniformly spreading the cells by using a sterile elbow glass rod, placing the mixture at room temperature until the liquid is absorbed, inverting the plate, and culturing for 12-16h at 37 ℃.

5. The method for preparing the thymosin beta 4 protein of macrobrachium rosenbergii according to claim 3, wherein the inducing expression step is: selecting escherichia coli containing recombinant plasmids, adding the escherichia coli into 250mL of LB liquid culture medium, and performing shake culture at 37 ℃ until the OD600 of a bacterial liquid is 0.4-0.6; taking the bacterial liquid as a reference, preserving the seeds, adding a proper amount of IPTG into the residual bacterial liquid to the final concentration of 1mM, and continuously culturing for 2-3 h at 37 ℃; centrifuging the IPTG-induced bacterial liquid at 8000rpm and 4 ℃ for 10min, carefully pouring off the supernatant, and collecting thalli precipitates; adding about 20mL of sterilized water into the precipitate to resuspend the thalli, putting the thalli into a freezing chamber at the temperature of minus 80 ℃, and repeatedly freezing and thawing for 1 to 2 times; taking the frozen and thawed heavy suspension thalli, carrying out ultrasonic crushing under an ice bath condition, gradually clarifying the bacterial liquid after the crushing is finished, placing the crushed bacterial liquid at 4 ℃, 8000rpm, centrifuging for 10min, placing the supernatant into a new 50mL centrifuge tube, and placing the supernatant and the precipitate at-20 ℃ for preservation; taking the inducing pre-and post-bacteria liquid, crushing, adding proper amount of sample buffer solution into the crushed supernatant and precipitate, mixing, centrifuging, boiling at 99.9 deg.c to denature for 5min, and SDS-PAGE analyzing to determine the expression form of the recombinant protein.

6. The method for preparing the thymosin beta 4 protein of macrobrachium rosenbergii according to claim 5, wherein the crushing condition is: crushing for 3s at intervals of 2s for 30 min.

7. The method for preparing the thymosin beta 4 protein of macrobrachium rosenbergii according to claim 3, wherein the purification and renaturation steps are as follows: boiling the semipermeable membrane in boiling water for 5-7min, adding purified recombinant protein, sealing, placing the semipermeable membrane in dialysis renaturation solution, replacing disposable renaturation solution for 12 hr, centrifuging the renaturated recombinant protein in clean centrifuge tube at 5000rpm for 5min, collecting supernatant, and storing at-20 deg.C.

8. The method for preparing the thymosin beta 4 protein of macrobrachium rosenbergii according to claim 7, wherein the concentration of urea in the renaturation solution is 6M, 4M, 3M, 2M, 1M and 0M in sequence.

9. The use of the Macrobrachium rosenbergii thymosin beta 4 protein of claim 2 in the preparation of a medicament for inhibiting Aeromonas hydrophila.

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