CN111499720B

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

Info

Publication number: CN111499720B
Application number: CN202010237012.1A
Authority: CN
Inventors: 杨辉; 熊浩然; 付立霞; 张莹莹
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-06-14
Anticipated expiration: 2040-03-30
Also published as: CN111499720A

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 beta 4 gene, protein, preparation method and application thereof

Technical Field

The invention belongs to the field of molecular biological gene engineering, relates to a nucleotide sequence of a 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 a Macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta 4 protein.

Background

Thymus peptides are widely present in vertebrates and invertebrates and can be classified into 3 types such as T alpha, T beta and T gamma according to their isoelectric points. The functional domain of thymosin is a thymosin (thy) domain, and β -thymosin proteins in invertebrates typically have multiple domains. Currently, thymosin is involved in a variety of biological functions in the body, including regulation of actin and cytoskeleton remodeling. In addition, the beta-thymosin protein can play an important role in physiological activities such as cell movement, endocytosis, immunoregulation and cascade reaction, tumor diagnosis and treatment, development, wound healing and the like. At present, the thymosin of vertebrates is developed into clinical medicines for treating diseases such as tumor, viral hepatitis and the like, the thymosin protein is highly conserved in the evolution process, and the thymosin protein separated from the shrimp body also has important biological activity.

Macrobrachium rosenbergii is one of the main freshwater shrimp varieties cultivated in China. The method has the advantages of strong adaptability, high growth speed and wide culture range, and brings huge benefits to farmers. However, shrimps belong to invertebrates and lack the adaptive immune system, mainly aiming at innate immune defense. The strong adaptability and anti-stress capability of macrobrachium rosenbergii are related to the strong innate immune system. The 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-resistant factor of the shrimps is developed, the functional activity of the specific immune disease-resistant factor is researched, and the disease resistance of the specific immune disease-resistant factor is enhanced. However, the process for directly separating the thymosin from the shrimp body is complex, the cost is high, the yield is low, and the stable and efficient expression of the thymosin beta 4 protein is difficult to realize by the traditional prokaryotic expression recombinant method. Therefore, the scheme of the invention combines synthetic biology and biological engineering technology to transform the thymosin beta 4 gene and realize high-efficiency expression in escherichia coli.

Disclosure of Invention

In order to solve the technical problems, the invention provides a recombinant expression preparation method and application of the macrobrachium rosenbergii thymosin beta 4 protein, and the problems of low yield in the purification and separation process can be solved by recombinant expression of the MrThy beta 4 protein.

The technical scheme of the invention is as follows:

the macrobrachium rosenbergii thymosin beta 4 gene is named as MrThy beta 4, and has a sequence shown as SED ID NO: 1 is shown.

The macrobrachium rosenbergii thymosin beta 4 gene has a sequence shown in SED ID NO: 2, respectively.

The macrobrachium rosenbergii thymosin beta 4 protein has a sequence shown in SED ID NO: 3, respectively.

A preparation method of macrobrachium rosenbergii thymosin beta 4 protein comprises 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.

Further, the conversion steps are as follows: putting competent cells E.coli BL21 in ice until they are thawed; 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; and (3) 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 bent glass rod, placing the mixture at room temperature until the liquid is absorbed, inverting the plate, and culturing for 12-16h at 37 ℃.

Further, the induction expression step is as follows: selecting escherichia coli containing recombinant plasmids, adding the escherichia coli into 250mL of LB liquid culture medium, and performing shake culture at 37 ℃ until bacterial liquid OD600 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 pre-induction bacterial liquid and the post-induction bacterial liquid, adding a proper amount of loading buffer solution into the crushed supernatant and the precipitate respectively, uniformly mixing and centrifuging, boiling at 99.9 ℃ for denaturation for 5min, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis to determine the expression form of the recombinant protein.

Further, the crushing conditions are as follows: crushing for 3s at intervals of 2s for 30 min.

Further, 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.

Furthermore, the concentration of urea in the renaturation solution is 6M, 4M, 3M, 2M, 1M and 0M in sequence.

The invention also provides the application of the preparation method of the macrobrachium rosenbergii thymosin beta 4 protein in the field of pharmacy.

The first purpose of the invention is to provide the thymulin beta 4 gene sequence of Macrobrachium rosenbergii (Macrobrachium rosenbergii);

the second purpose of the invention is to provide a Macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta 4 protein sequence;

the third purpose of the invention is to provide a nucleotide sequence of Macrobrachium rosenbergii (Macrobrachium rosenbergii) after the codon optimization of thymosin beta 4;

the fourth purpose of the invention is to provide a recombinant expression and renaturation method of the Macrobrachium rosenbergii (Macrobrachium rosenbergii) thymosin beta 4 protein.

The molecular type of the thymus peptide beta 4 gene of the Macrobrachium rosenbergii (Macrobrachium rosenbergii) is a cDNA sequence, the length is 501bp, the type is double-chain, linear and nucleic acid, and the sequence of the thymus peptide beta 4 gene MrThy beta 4 of the Macrobrachium rosenbergii (Macrobrachium rosenbergii) is obtained by PCR amplification and sequencing. The PCR specific primers were as follows: F-5'-ATGAGTACCGAAACCGCCC-3' and R-5'-TTAGGCGTTCTTCTCCTGC-3'. Specific sequence

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

ATGAGTACCGAAACCGCCCTCAAGGATCTCCCCAAGGTCGACCCCACCCTCAAGGGCCAGCTGGAAGGATTCACCCCCGACAAACTCAAGAAGACCGACACAGAGGAGAAGACCATCTTGCCTTCCAAAGAGGACGTGGAAAGTGAGAAGCTTCGGAACGAACACCTGGAGAACATCAGCAAATTCCCGAGTGGGAGGCTGAAACGCACCTCTACTTCGGAGAAAATTGTCCTCCCCTCTAGCGCAGATGTGGAAGCCGAGAAGAAAGAAAAAGCCCACCTACAGGCTGTGGAGGGCTTCAATGCAGCCAATTTGAAGCATGCAAACACGAAAGAGAAAATTGTGTTGCCGGCCAAAGAAGATATCGAGAAAGAAAAGGGTCAGCAGGCGCTGTTCCAAGGAATCGAAGGATTCAACCAATCTAATCTTAAGAAGACTGAAACACAGGAGAAGAACCCTCTCCCAACTAAGGAGATAATCGAGCAGGAGAAGAACGCCTAA

using DNAworks 2.4 online software to perform codon optimization on the nucleotide sequence of MrThy beta 4, selecting an escherichia coli expression system, and marking the sequence of the MrThy beta 4 after codon optimization as SEQ ID No.2:

ATGAGCACCGAAACCGCACTGAAAGATCTGCCGAAAGTGGACCCGACCCTGAAAGGCCAGCTGGAAGGCTTCACCCCGGACAAACTGAAAAAGACCGACACCGAAGAAAAAACCATTCTGCCGAGCAAAGAAGATGTTGAAAGCGAAAAACTGCGTAATGAACATCTGGAAAATATTAGCAAATTTCCGAGCGGTCGTCTGAAACGTACCAGCACCAGCGAAAAAATTGTTCTGCCGAGCAGCGCAGATGTTGAAGCAGAAAAAAAAGAAAAAGCACATCTGCAGGCAGTTGAAGGTTTTAATGCAGCAAATCTGAAACATGCAAATACCAAAGAAAAAATTGTTCTGCCGGCAAAAGAAGATATTGAAAAAGAAAAAGGTCAGCAGGCACTGTTTCAGGGTATTGAAGGTTTTAATCAGAGCAATCTGAAAAAAACCGAAACCCAGGAAAAAAATCCGCTGCCGACCAAAGAAATTATTGAACAGGAAAAAAATGCATAA

the MrThy beta 4 molecular type is protein, and the sequence characteristics are as follows: length 166aa, type amino acid, sequence information as follows, labeled SEQ ID No.3:

MSTETALKDLPKVDPTLKGQLEGFTPDKLKKTDTEEKTILPSKEDVESEKLRNEHLENISKFPSGRLKRTSTSEKIVLPSSADVEAEKKEKAHLQAVEGFNAANLKHANTKEKIVLPAKEDIEKEKGQQALFQGIEGFNQSNLKKTETQEKNPLPTKEIIEQEKNA

the preparation method of the Macrobrachium rosenbergii MrThy beta 4 thymosin disclosed by the invention comprises the following steps of:

synthesizing a Macrobrachium rosenbergii Rosenbergii MrThy beta 4 codon optimization gene;

a Macrobrachium rosenbergii Rosenbergii MrThy beta 4 recombinant expression step;

and (3) renaturation and antibiosis of the MrThy beta 4 thymosin recombinant protein.

The invention directly connects the sequence SEQ ID No.2 optimized by MrThy beta 4 codon into pET30a vector, through transforming into escherichia coli BL21 competent cell, selecting single clone to shake bacteria, IPTG induced expression, collecting bacteria to detect SDS-PAGE protein, ultrasonic breaking and cracking bacteria, using Ni to detect SDS-PAGE protein²⁺And (3) purifying the target protein by using an affinity chromatography column, dialyzing and renaturing, and verifying the antibacterial activity. The method of the invention can overcome the problems of low yield in the purification and separation process, the concentration of the produced thymosin recombinant protein can reach the level of mg/ml, and the renatured thymosin has obvious bacteriostatic activity (as shown in figure 2), so that the thymosin recombinant protein can be used for treating the diseases of high yield, high yield and the likeThe antibacterial peptide is used as a potential antibacterial peptide drug and is applied to the research fields of biology, medicine, agriculture and the like.

Drawings

FIG. 1, SDS-PAGE electrophoretic detection of MrThy β 4 recombinant protein. M represents protein marker, 1 represents a bacterial protein not induced by IPTG, 2 represents a bacterial protein induced by IPTG, and 3 represents Ni²⁺Purified MrThy β 4 recombinant protein.

FIG. 2, identification of the bacteriostatic activity of MrThy beta 4 recombinant protein against Aeromonas hydrophila (Aeromonas hydrophylla).

Detailed Description

Example 1

Transformation of the MrThy β 4-pET30A recombinant plasmid:

coli BL21 was placed in ice and allowed to melt. The recombinant plasmid was added to the competent cells, the contents were gently mixed, and allowed to stand in an ice bath for 3 min. The tube was placed in a 42 ℃ water bath for 90s, transferred quickly to an ice bath, cooled for 2-3min without shaking. 900. mu.L of sterile LB medium (containing no antibiotics) was added to each tube, mixed well and placed on a shaker at 37 ℃ for shaking culture for 45min (15rpm/min) to resuscitate the cells. 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 ℃.

Inducible expression of MrThy β 4-pET30A protein:

selecting escherichia coli containing recombinant plasmids, adding the escherichia coli into 250mL of LB liquid medium (containing antibiotics and Kana resistance), and performing shake culture at 37 ℃ until the OD600 of a bacterial liquid is 0.4-0.6; taking a proper amount of the bacterial liquid as a reference, preserving the seeds, adding a proper amount of IPTG (1M) into the residual bacterial liquid to a 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 thallus to carry out ultrasonic crushing under the ice bath condition, wherein the crushing condition is as follows: crushing for 3s at intervals of 2s for 30 min; after the crushing is finished, the bacterial liquid becomes clear gradually, the crushed bacterial liquid is placed at 4 ℃, 8000rpm and centrifuged for 10min, the supernatant is placed into a new 50mL centrifuge tube, and the supernatant and the precipitate are stored at-20 ℃; 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.

Purification and renaturation of MrThy beta 4-pET30A recombinant protein:

the MrThy beta 4-pET30A recombinant protein is separated and purified: dissolving the precipitate after ultrasonic disruption with 8M urea, and then loading the sample with Ni²⁺And (3) collecting flow-through liquid by an ion affinity chromatography column at the flow rate of 10 times of the column volume per hour. The column was washed with 15 column volumes of Binding Buffer (20mM Tris-HCl pH 7.9,5mM imidazole, 0.5M NaCl, 8M Urea) to wash away the contaminating proteins. Eluted protein was collected by eluting with 5mL of Elution Buffer (20mM Tris-HCl pH 7.9,500mM imidazole, 0.5M NaCl, 8M urea).

Renaturation of the recombinant protein MrThy beta 4-pET 30A: 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 (urea concentration in renaturation solution is 6M, 4M, 3M, 2M, 1M and 0M in sequence) for 12h, placing renaturated recombinant protein in a clean centrifuge tube, centrifuging at 5000rpm for 5min, and collecting supernatant, and storing at-20 deg.C.

MrThy beta 4-pET30A recombinant protein antibacterial activity assay:

the inhibition activity of the recombinant protein MrThy beta 4 on Aeromonas hydrophila (Aeromonas hydrophila) is determined by a bacteriostatic circle method, which comprises the following specific steps: absorbing the bacterial liquid of the laboratory conservation, respectively inoculating the bacterial liquid into 5mL of sterile LB liquid culture medium, and culturing for several hours at the constant temperature of 37 ℃; when the bacterial liquid reaches the logarithmic growth period (namely OD is 0.6-0.8), diluting the bacterial liquid by 10 times, absorbing 100 mu L of bacterial liquid, uniformly coating the bacterial liquid on an anti-LB (Lunti-LB) flat plate, lightly placing a sterilized filter paper small disc on the surface of the flat plate, absorbing 15 mu L of recombinant protein MrThy beta 4 stock solution, and vertically dropping the stock solution into the center of the disc without left-right shaking; placing in a constant temperature incubator at 30 ℃, culturing overnight, and observing whether a bacteriostatic zone is formed. As shown in fig. 2, the recombinant protein MrThy β 4 was able to significantly kill aeromonas hydrophila, producing a significant zone of inhibition, compared to the intermediate control. Aeromonas hydrophila is a main pathogenic bacterium of aquatic animals, which is harmful to the health of aquatic animals, and the use of antibiotics in large quantities is easy to generate drug-resistant bacteria. Therefore, the recombinant protein MrThy beta 4 has certain prospect of being used as an antibacterial peptide medicament to be applied to aquaculture due to the specific activity of killing aeromonas hydrophila.

Sequence listing

<110> Yangzhou university

<120> Macrobrachium rosenbergii thymosin beta 4 gene, protein, preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 501

<212> DNA

<213> Artificial Sequence (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 (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 (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

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:

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.