CN107759684B - Recombinant periplaneta americana thymosin protein PaTHY1 and expression method thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses a recombinant periplaneta americana thymosin protein PatHY1 and an expression method thereof, wherein the method comprises the following steps: (1) extracting RNA from periplaneta americana tissues and carrying out reverse transcription to obtain cDNA; (2) synthesizing a primer: (3) obtaining a periplaneta americana thymosin protein coding gene fragment by using the total cDNA as a template through PCR; (4) cloning the coding gene into a prokaryotic expression vector, transforming host bacteria, culturing the obtained recombinant engineering bacteria, and then inducing the recombinant engineering bacteria to express a target protein; (5) and purifying the target protein by adopting an affinity chromatography method so as to obtain the recombinant periplaneta americana thymosin protein PatHY 1. The recombinant protein is obtained by gene engineering for the first time, solves the problems of extremely low content of thymosin in complex chemical components of the periplaneta americana and difficulty in purification, and lays a foundation for the research on pharmacodynamic functions of the thymosin.

Description

Recombinant periplaneta americana thymosin protein PaTHY1 and expression method thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a recombinant periplaneta americana thymosin protein PatHY1 and an expression method thereof.

Background

Thymosin (THY) is a lymphocyte growth factor produced primarily by the thymus, and is ubiquitous in a variety of tissue cells. Originally discovered by Goldstein and White in 1966 for the first time from fetal bovine thymus protein extracts, it was a group of small polypeptides containing over 40 fractions and hence named thymosins. Thymosins can be classified into α, β, γ 3 families according to their isoelectric Points (PI), among which those with PI ranging from 5.0 to 7.0 are called β -family thymosins (T β), which are highly conserved in structure, consist of 40 to 44 amino acids, have a molecular mass of about 5.0ku, and are widely found in vertebrates and invertebrates. The beta thymosin family belongs to a family of polar protein molecules, originally thought of as a thymic hormone, with properties that affect angiogenesis and the induction of apoptosis to varying degrees. To date, 15 forms of beta thymosin have been discovered, with 3 predominant forms of beta thymosin in humans, thymosin beta 4, thymosin beta 10 and thymosin beta 15. Wherein, the thymosin beta 4 is distributed most widely and has the most content, which accounts for 70 to 80 percent of the total amount of the beta thymosin. They play an important role in maintaining the dynamic balance of the agonist protein, and various physiological and pathological processes such as tumor onset and metastasis, cell apoptosis, inflammation, angiogenesis, wound healing and the like.

In recent years, the biological function of thymosins has been of great interest, and it plays an important role in many physiological and pathological activities. However, current research on beta-family thymosins has focused primarily on vertebrates. In invertebrates, the presence of thymosins has also been found, but very few have been studied. The insect THY is a multifunctional molecule, has the functions of wound repair and tissue regeneration, and is found to regulate the physiological function and morphological structure of the insect by combining actin in the research of the silkworm THY, and the sequence of the insect THY has high conservation. Only 18 research data related to the protein can be found in NCBI, mainly in fruit fly, Aedes aegypti, domestic silkworm and other species, and the homology with T beta is relatively high, and the beta-family thymosin is found to have antibacterial capacity in oyster and sea urchin research. In invertebrates, these proteins usually have multiple THY domains compared to T β 4, and are therefore also known as polymeric thymosins, which are structurally more complex than vertebrates and whose function is to be studied further.

The new rehabilitation liquid developed by the periplaneta americana alcohol extract has very good effects on wound repair and wound healing, and also has the functions of diminishing inflammation, resisting bacteria, enhancing the immunity of the organism and the like, which is very similar to the research result of thymosin. We found in the Periplaneta americana transcriptome study that there were 4 transcripts encoding thymosin beta-4 protein family domains. However, the thymosin has extremely low content in the complex chemical components of the periplaneta americana and is difficult to purify, so the PaTHY1 protein is obtained by constructing a pET-28a (+) -THY prokaryotic expression vector and purifying a prokaryotic expression product, and a foundation is laid for the research of the pharmacodynamic function of the periplaneta americana thymosin.

Disclosure of Invention

The invention aims to provide a periplaneta americana thymosin protein PaTHY1 through genetic engineering, and an expression method of the periplaneta americana thymosin protein PaTHY1, solves the problems of extremely low content and difficult purification of thymosin in periplaneta americana extract, and lays a foundation for the research of the function of the periplaneta americana thymosin.

The invention discloses an amino acid sequence of a recombinant periplaneta americana thymosin protein PaTHY1 shown in SEQ ID No. 1.

On the other hand, the invention also discloses an expression method of the recombinant periplaneta americana thymosin protein PatHY1, which comprises the following steps:

(1) extracting RNA from periplaneta americana tissues and carrying out reverse transcription to obtain cDNA;

(2) synthesizing a primer:

Primer F CGCGAATTCATGTCGGCCCCAGTC

Primer R CCGCTCGAGTTATGCTTTCTTCTCTTCATCG

(3) obtaining a periplaneta americana thymosin protein coding gene fragment by using the total cDNA as a template through PCR;

(4) cloning the coding gene into a prokaryotic expression vector, transforming host bacteria, culturing the obtained recombinant engineering bacteria, and then inducing the recombinant engineering bacteria to express a target protein;

(5) and purifying the target protein by adopting an affinity chromatography method so as to obtain the recombinant periplaneta americana thymosin protein PatHY 1.

In the expression method, the nucleotide sequence of the periplaneta americana thymosin protein coding gene fragment in the step (3) is shown as SEQ ID No. 2.

In the expression method, the PCR reaction conditions in the step (3) are 94 ℃, pre-denaturation for 4min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 50s, extension at 72 ℃ for 20s-30s, 35 cycles, and extension at 72 ℃ for 10 min.

In the expression method, the expression vector in the step (4) is PET-28 a.

In the expression method, the host bacterium in the step (4) is escherichia coli BL 21.

In the expression method, the inducer for inducing in the step (4) is IPTG, the concentration is 0.5mmol/L, the induction temperature is 37 ℃, and the induction time is 6 h.

Compared with the prior art, the invention has the following beneficial effects: the invention extracts total RNA from the periplaneta americana tissues, amplifies the coding gene of the periplaneta americana thymosin protein PaTHY1, induces and expresses the recombinant periplaneta americana thymosin protein in escherichia coli through a gene engineering means, solves the problems of extremely low content and difficult purification of thymosin in the periplaneta americana extract, and lays a foundation for the research of the pharmacodynamic function of the periplaneta americana thymosin.

Drawings

FIG. 1 is an electrophoresis diagram of the extraction of Periplaneta americana RNA;

FIG. 2 shows PCR amplification products of thymosin genes THY1 and THY 2;

FIG. 3PCR identification of recombinant (PET-THY1) bacterial liquid;

FIG. 4 expression of proteins by PET-THY1 at different induction times, where a: no-load PET-28(a +) (BL21) was induced (6 h); b: no-load PET-28(a +) (BL21) induction (6 h); c: PET-THY1 without inducer (6 h); d, e, f, g, h: respectively shows that PET-THY1 induces 0, 2, 4, 6 and 8 (h);

FIG. 5 expression of proteins of PET-THY1 at different inducer concentrations, wherein a, b, c, d, e, f: respectively representing the concentrations of the PET-THY1 inducer to be 0, 0.25, 0.5, 0.75, 1.0 and 1.2 mmol/L;

FIG. 6 gel diagram of purified PatHY1 protein;

FIG. 7 Western blot of PatHY1 protein.

Detailed Description

Example 1

1. Test materials

1.1 Periplaneta americana samples

The American cockroach sample is provided by Sichuan good doctors to climb West Chang breeding base of western pharmaceutical industry Limited liability company.

1.2 plasmid vectors and recipient strains

The PET28a vector for prokaryotic gene expression is stored in the laboratory. The recipient strain Escherichia coli DH5a, prokaryotic expression cell BL21(DE3) from Boo Wei Xin Co.

1.3 restriction enzymes and other enzymes

Restriction enzymes (XhoI, EcoR I), T4DNA Ligase (DNA Ligase), Taq enzyme and the like are available from Genistein (Takara) Bio Inc.

1.4 Instrument and consumables

Ultraviolet spectrophotometer, PCR amplification instrument (Biometra, Germany), vertical pressure steam sterilization pot (Shanghai Bispi industries, Ltd.), protein vertical electrophoresis tank, transfer electrophoresis tank, shaking table, oscillator, ultrasonic crusher, and protein purification instrument.

1.5 media and solutions

1.5.1 liquid LB Medium

100ml of formula: dissolving tryptone 1.0g, yeast extract 0.5g, and NaCl1.0g in 90mlddH solution₂Adjusting pH to 7.2-7.5 in O, diluting to 100ml, sterilizing, and storing at 4 deg.C.

1.5.2 solid LBA Medium (containing kanamycin)

100ml of formula: 1.5g of agar powder is weighed and then poured into a conical flask. Further, 1.0g of tryptone, 0.5g of yeast extract and 1.0g of NaCl1.0g were weighed and dissolved in 80ml of dH20, pH was adjusted to 7.2 to 7.5, and the volume was adjusted to 100 ml. Then poured into a conical flask filled with agar powder. Sterilizing, cooling to 40-50 deg.C, adding kanamycin (kana) to 50ug/ml, mixing, pouring into culture dish, cooling to obtain flat plate (about 20 ml/dish), and wrapping with plastic wrap at 4 deg.C.

1.5.3 PCR primers with restriction sites (restriction site sequence underlined)

TABLE 1 primers with restriction sites for prokaryotic expression

Primer name	Sequence (5 '- - - -3')
		Primer F	CGCGAATTCATGTCGGCCCCAGTC
Primer R	CCGCTCGAGTTATGCTTTCTTCTCTTCATCG

2. Experimental methods

2.1 extraction and reverse transcription of Periplaneta americana RNA

The experiment adopts a tissue total RNA extraction kit of Chengdu Fuji biotechnology limited company to extract total RNA according to the instruction, and takes 1 microliter of RNA sample to carry out agarose gel electrophoresis detection. Then, the first strand cDNA was synthesized by reverse transcription using a kit for reverse transcription of DNA-free from Beijing Quanji Biotechnology Ltd.

2.2 design of primers and PCR reaction conditions

The gene sequence was obtained by transcriptome analysis, primers were designed based on the gene sequence and the characteristics of the multiple cloning site region of the PET-28 expression vector, and primers were designed using Primer Premier5 software.

Performing PCR reaction by using the first strand cDNA product, wherein the reaction system is as follows: 50 microliter system

Reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 50s, extension at 72 ℃ for 20s-30s, 35 cycles, final extension at 72 ℃ for 10min, and forever at 4 ℃. The PCR product was stored at-20 ℃.

2.3 recovery and purification of PCR products

The PCR product was subjected to Gel-green staining in 1% agarose Gel electrophoresis, the Gel was excised under an ultraviolet lamp, and the DNA fragment was recovered using a Gel Extraction Kit according to the instructions.

2.4 construction of prokaryotic expression vectors

2.4.1 restriction and ligation

The PCR amplification product of the periplaneta americana thymosin gene THY1 and the PET-28a plasmid are subjected to double enzyme digestion by using Xho1 and EcoR1, and a 50 microliter system is as follows:

	PCR product	PET-28a plasmid
			Substrate DNA	43 microliter (1 microgram)	43 microliter (0.5-1 microgram)
10 Xrecommended buffer	5 microliter	5 microliter
			Xho1	1 microliter	1 microliter
EcoR1	1 microliter	1 microliter

Reacting at 37 deg.C for 1h, inactivating enzyme at 65 deg.C for 20min, and recovering product by sodium acetate precipitation. The purified PCR digestion product and the plasmid digestion product are connected by T4DNA Ligase. The reaction system was as follows (10. mu.l system):

the reaction was carried out at 16 ℃ for 12 hours. Escherichia coli BL21(DE3) competent cells are transformed, and positive recombinants are screened by bacterial liquid PCR, sequencing and enzyme digestion and named as PET-THY 1.

2.4.2 transformation and double restriction enzyme identification of recombinant PET-THY1 plasmid

A single colony of Escherichia coli BL21(DE3) containing the recombinant plasmid was picked up by a sterile pipette tip and cultured with 500. mu.l of LB liquid medium (containing kanamycin) at 37 ℃ for 3 hours by shaking, and then PCR was carried out. The primer is a universal primer of PET-28 a.

And (3) PCR system:

respectively taking 5ul of two positive recombinant plasmid bacterial liquids, inoculating 5ml of LB (Kana) liquid culture medium, and carrying out shake culture at 37 ℃ overnight; extracting plasmid, sequencing and double enzyme digestion identification. After correct identification, ligation product 5ul (100. mu.g/ml) was added to 33ul of BL21 competent cells and gently mixed (experimental group), intact PET-28(a +)3ul (135. mu.g/ml) was added to 33ul of BL21 competent cells and gently mixed (control group). And (4) coating a plate, selecting the single clone, performing sequencing identification and preserving strains.

2.5 sequence analysis

Sequence similarity analysis was performed using BLASTN and BLASTX in the National Center for Biotechnology Information (NCBI); protein properties were analyzed with ScanSite PI/PW; using single P4.0 software to search signal peptide; TMHMM software predicts protein transmembrane structure and the domain is analyzed online using the software Motif Scan (http:// myhits. isb. ch/cgi-bin/Motif _ Scan).

2.6 inducible expression

Researches find that the concentration of a bacterial liquid, the concentration of an IPTG inducer and the induction temperature are key factors influencing the soluble expression of the recombinant protein, and the solubility of the soluble protein can be increased by timely reducing the conditions.

(1) Selecting the optimal time;

(a) inoculating 10 μ l of the strain containing recombinant plasmid obtained by screening to 5mL LB liquid medium (containing Kana), and performing shake culture at 37 deg.C and 200rpm for 16 h;

(b) according to the following steps: 100, transferring the bacterial liquid into 5mL LB liquid culture medium containing 50mg/L Kana, and carrying out shaking culture at 37 ℃ until the absorbance is between 0.4 and 0.6;

(c) adding isopropyl thiogalactoside (IPTG) to make the final concentration be 0.5mmol/L, inducing and culturing at 37 ℃ and 200rpm for 0h, 4h, 6h, 8h and 10h respectively, and designing a negative control for PET-28(a +) airborne bacteria culture;

(d) at 4 ℃, 12,000rpm for 10min, and discarding the supernatant;

(e) then adding 3mL of PBS (phosphate buffer solution) suspension bacteria into every 100mg of thalli (wet weight), crushing cells by using an ultrasonic crusher, setting crushing parameters to be 34-40% of output power, 3s of ultrasonic pulse and 3s of pause pulse, and setting the crushing time to be about 15-30 min;

(f) taking supernatant at 4 deg.C and 12,000rpm for 10 min;

(g) and (6) detecting.

(2) Selection of optimum concentration of IPTG

The steps are consistent with the above, after the above optimum temperature is selected, the concentration of IPTG is changed to 0, 0.25, 0.5, 0.75, 1 and 1.2mmol/L, and other conditions are unchanged, so that the optimum concentration is obtained.

2.7 deformation discontinuous Polyacrylamide gel electrophoresis (SDS-PAGE)

The glass plate was assembled on a holder and formulated into a separation gel and a concentrated gel according to the following formulation.

Components	12% separation gel (ml)	5% concentrated glue (ml)
			30% acrylamide	2	0.415
dd H2O	1.75	1.455
			4 Xconcentrated gel buffer (containing SDS)	------	0.625
4 XSEPARATION GEL BUFFER (containing SDS)	1.25	--------
			10% ammonium persulfate	0.025	0.0125
TEMED	0.0025	0.00125

Before loading the protein sample, adding 5X SDS-PAGE loading buffer, boiling at 100 ℃ for 10min, and centrifuging at 10000 rpm for 10 min. Protein samples and marker were added to the wells using a microsyringe. And (3) applying a voltage of 80V for 30-40min to enable the strip to reach the interface of the concentrated gel and the separation gel, adjusting the voltage to 120V for 40-50min until bromophenol blue just reaches the bottom of the separation gel, stripping the polyacrylamide gel from the glass gel plate, marking, placing the glass gel plate in a plastic container, adding Coomassie brilliant blue staining solution R-250 to cover the gel, and slowly shaking for 2-4 h. Discarding the dye solution, adding Coomassie brilliant blue decolorization solution, slowly shaking for 4-6h, changing the solution, continuing shaking until a blue clear band is obtained, namely a clean background, and taking a picture for recording.

2.8 amplification culture of bacterial solution and Collection of cells

10 microliter of the glycerol strain stored was inoculated into 10mL of LB medium containing kanamycin, shaken at 37 ℃ and 200 ℃ overnight, 6mL of the overnight-cultured bacterial solution was inoculated into 600mL of LB medium containing kanamycin, shaken at 37 ℃ and 200rpm until the OD value of the bacterial solution was 0.4-0.8, approximately 3-4 hours, and then added with an inducer and shaken at 37 ℃ for 6 hours.

2.9 protein purification

Recombinant proteins His-tagged proteins were purified using a nickel column, and the protein sample and loading buffer were first filtered through a 0.45 μm filter. The tube was washed with ddH2O and pooling buffer, respectively, and the column was mounted on the machine, setting the flow rate at 1mL/min and the maximum column pressure at 0.4 MPa. A buffer and a junction containing 20mmol/L sodium phosphate, 0.25mmol/L NaCl, and 10mmol/L imidazole was pumped from the A pump to the equilibration column to UV detection level. And (3) loading the cell disruption supernatant, collecting the effluent sample, repeating the loading once, and eluting nonspecific binding protein by using a binding buffer solution until reaching the reference line of an ultraviolet detection area. And (5) suspending the system, changing the pump B into gradient sample injection elution, and collecting samples according to the ultraviolet detection peak value. The samples were analyzed by SDS-PAGE.

2.10Western blot identification

2.10.1 transfer film (Wet transfer)

1. Marking a PVDF membrane by using a sign pen, and soaking in methanol;

2. soaking filter paper and sponge by using a rotating membrane buffer solution at 1 x;

3. the film transfer interlayer is manufactured in this order: black gel holder, sponge, filter paper, glue, PVDF membrane, filter paper, sponge, white gel holder, closed clamp;

4. the black surface of the clip is opposite to the black surface of the groove;

5. adding a refrigerator, fully adding a membrane-rotating buffer solution, and running for 90min at 300mA in a refrigerating chamber;

6. after the rotation is finished, sealing the container with 5% milk for 1 h.

2.10.2 protein detection

1. Washing with TBST for three times (5 min/time), adding primary antibody, and incubating at 37 deg.C for 1-2 hr;

2. washing with TBST for three times (5 min/time), washing nasal discharge, adding 2 antibody, and incubating at 37 deg.C for 1 hr;

3. washing with TBST for three times (5 min/time);

mixing the solutions A and B of the BeyoECL Plus in equal volume, uniformly dripping the mixture on a PVDF membrane, and contacting the PVDF membrane for 2 min;

5. and developing and fixing.

3. Results of the experiment

3.1 cloning and identification of THY1 Gene

The result of the total RNA extraction kit extracted Periplaneta americana RNA is shown in figure 1, the prokaryotic expression primer is designed according to the gene sequence obtained by transcriptome analysis, and the PCR result is shown in figure 2. As can be seen from the figure, the PCR product has three distinct bands after agarose gel electrophoresis, the size of the three bands is consistent with the predicted result, the expression quantity of two of the three bands is relatively high, and the two bands have main functions and are named as THY1 and THY2, the two bands are almost derived from the same gene, and only the sequences are different due to different splicing modes of introns, so that the functions are possibly different. The length of the coding region of the gene THY1 obtained by sequencing, identification and analysis is 507bp, 169 amino acids are coded, the length of the coding region of the THY2 is 393bp, and 131 amino acids are coded. Different splicing of the RNA can result in different thymosin peptides, thus forming the polymeric thymosin. The base sequence of THY1 is shown in SEQ ID No. 2.

3.1 determination of the recombinants PET-THY1

Cloning the amplified THY1 coding region sequence to a PET-28(a +) expression vector, transforming to escherichia coli BL21(DH3), selecting a single clone for screening and identifying, taking a recombinant bacterium solution as a template, detecting by a PCR method, wherein the size of the recombinant bacterium solution is consistent with that of the expected recombinant bacterium solution as shown in figure 3, and then sequencing to further confirm that the cloned DNA insert is THY 1. And the recombinant plasmid was designated PET-THY 1.

3.2 protein expression and purification

The recombinant plasmid PET-THY1 strain is predicted to obtain target protein with molecular mass of about 23Kda after IPTG induction, and the target protein consists of mature peptide part of 19Kda target gene and about 4Kda label protein on PET-28(a +) carrier. The supernatant after ultrasonication and centrifugation was subjected to SDS-PAGE, and it was found that an expression band was observed at 25Kda as compared with the control, as shown in FIG. 4. The prediction and the actual size are different, and researches show that the occurrence of the situation is caused by a plurality of reasons, and the research is not clear, the deviation is probably caused by the fact that the migration speed of the protein in SDS-PAGE is slowed due to the action of basic amino acid in His-tag, and the difference of only a few Kda is generally generated, but the future research is not influenced. FIG. 4, which shows the expression level of the PatHY1 protein at different induction times, shows that the protein expression level is relatively high at 6h of induction. The amino acid sequence of the target protein is shown as SEQ ID No. 1.

After the expression time is determined, the concentration of IPTG is further optimized, and the result is shown in figure 5, and the figure shows that the concentration of IPTG has little influence on the expression of protein, because IPTG has certain toxicity, and the final concentration of IPTG is selected to be 0.5mmol/L comprehensively.

In conclusion, we select the optimum time of 6h and the optimum IPTG concentration of 0.5mmol to perform mass induction expression and purification on the protein, and the results are shown in FIG. 6.

3.3Western blot results

In order to further verify whether the purified protein is the target protein, a Western blot experiment is further performed, the experimental result is shown in fig. 7, and the result shows that the protein is the required target protein.

SEQUENCE LISTING

<110> Sichuan good doctor Panxi pharmaceutical industry Limited responsibility company

<120> recombinant periplaneta americana thymosin protein PatHY1 and expression method thereof

<130> 2016

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 204

<212> PRT

<213> Periplaneta americana

<400> 1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Met Ser Ala Pro Val Ser Pro Gln Leu Lys Asp Leu

35 40 45

Pro Lys Val Asn Leu Asp Leu Lys Ser Glu Leu Glu Gly Phe Lys Thr

50 55 60

Val Asn Met Lys Lys Ala Glu Thr His Glu Lys Asn Val Leu Pro Thr

65 70 75 80

Ala Glu Asp Val Lys Gln Glu Arg Gln His Ser Glu Leu Ile Gln Gly

85 90 95

Val Glu Ser Phe Lys Pro Glu Arg Leu Lys Arg Thr Asn Thr Gln Glu

100 105 110

Lys Ile Val Leu Pro Asn Ala Gln Asp Val Ala Thr Glu Lys Thr Gln

115 120 125

Lys Ala Leu Leu Gln Gly Val Glu Ala Phe Asp Thr Gly Lys Leu Lys

130 135 140

His Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Asp Lys Asp Ala Ile

145 150 155 160

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

165 170 175

Asp Pro Arg Lys Leu Lys His Thr Glu Thr Gln Glu Lys Asn Pro Leu

180 185 190

Pro Thr Lys Glu Ala Ile Asp Glu Glu Lys Lys Ala

195 200

<210> 2

<211> 507

<212> DNA

<213> Periplaneta americana

<400> 2

atgtcggccc cagtcagccc acagttgaag gacctgccca aggtgaacct agacctgaaa 60

agcgaattgg aaggcttcaa aactgtaaat atgaagaagg ctgaaaccca cgaaaaaaat 120

gttttaccca cagcagaaga tgtgaaacaa gagcggcaac acagcgagct tattcaaggt 180

gtcgagagct ttaaacccga gaggttaaag cggaccaaca ctcaagaaaa aattgttcta 240

ccaaacgcac aagatgttgc caccgaaaaa actcagaagg ctcttcttca gggtgtagaa 300

gctttcgaca cggggaaact gaaacacaca gaaactcagg aaaaaaatcc ccttccagac 360

aaagatgcca ttgagcaaga aaggggaaag cagaacctga ttgctggaat tgaaaatttt 420

gatcccagaa agttgaagca tactgaaacc caagaaaaga atcctctgcc aacaaaagaa 480

gccatcgatg aagagaagaa agcataa 507

Claims (2)

1. A recombinant Periplaneta americana thymosin protein PaTHY1 is characterized in that the amino acid sequence of the thymosin protein PaTHY1 is shown as SEQ ID No. 1.

2. A gene for coding recombinant periplaneta americana thymosin protein PaTHY1 is characterized in that the nucleotide sequence of the gene for coding the periplaneta americana thymosin protein PaTHY1 is shown as SEQ ID No. 2.

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