CN103361356A - Marsupenaeus japonicus thymulin genes, and preparation method and applications of marsupenaeus japonicus thymulins - Google Patents

Marsupenaeus japonicus thymulin genes, and preparation method and applications of marsupenaeus japonicus thymulins Download PDF

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CN103361356A
CN103361356A CN2013103395732A CN201310339573A CN103361356A CN 103361356 A CN103361356 A CN 103361356A CN 2013103395732 A CN2013103395732 A CN 2013103395732A CN 201310339573 A CN201310339573 A CN 201310339573A CN 103361356 A CN103361356 A CN 103361356A
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marsupenaeus japonicus
zadaxin
marsupenaeus
japonicus
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CN103361356B (en
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阮灵伟
施泓
徐洵
马金友
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Third Institute of Oceanography SOA
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Abstract

The invention relates to marsupenaeus japonicus thymulin genes, and a preparation method and applications of marsupenaeus japonicus thymulins, and relates to nucleotide sequences of the marsupenaeus japonicus thymulin genes, and amino acid sequences encoded by the nucleotide sequences. The marsupenaeus japonicus thymulin genes comprise a marsupenaeus japonicus thymulin mjthm4 gene, a marsupenaeus japonicus thymulin mjthm3 gene and a marsupenaeus japonicus thymulin mjthm2 gene. The preparation method comprises a step of extracting total RNA of marsupenaeus japonicus; a step of performing inverse transcription for marsupenaeus japonicus cDNA; a step of performing PCR amplification and sequence analysis for the marsupenaeus japonicus thymulin genes; and a step of recombining and expressing the marsupenaeus japonicus thymulins. The marsupenaeus japonicus thymulins can be used for preparing anti-prawn-virus medicines. Through homologous recombination and expression of the three thymulins, deficiencies, namely complex and tedious processes, low yield, etc. of separation and purification are overcame, thus laying the foundations of applications of the proteins in the research fields of biology, food, medicine, agriculture, and the like.

Description

The preparation method of Marsupenaeus japonicus thymosin gene and Marsupenaeus japonicus Zadaxin thereof and application
Technical field
The nucleotide sequence that the present invention relates to Marsupenaeus japonicus (Marsupenaeus japonicus) thymosin gene with and coded aminoacid sequence, especially relate to Marsupenaeus japonicus (Marsupenaeus japonicus) Zadaxin albumen and preparation method thereof.
Background technology
Zadaxin is a kind of B cell growth factor by thymic epithelial cells's secretion, the research discovery, and they are prevalent in the Various Tissues cell.Difference according to iso-electric point (pI) is divided into Thymosin alpha, β, γ, wherein iso-electric point pI less than 5.0 be alpha thymosin peptides, pI is positioned at component of 5.0～7.0 and is called beta thymosin peptides, iso-electric point pI greater than 7.0 be the γ Zadaxin.So far, found that beta thymosin peptides has 15 kinds, except the regulation and control of participation Actin muscle in cell, beta thymosin peptides also participates in and the interaction of other albumen and the aspects such as anti-infective protection of body.At present, Zadaxin promotes lymphocyte maturation and enhancing body immunologic function mainly as a kind of cellular immunization toughener.Studies show that, beta thymosin peptides has participated in multiple physiology such as immunomodulatory, tumor invasion and transfer, apoptosis, inflammatory reaction, vasculogenesis, coagulation process, wound healing, fetal development and neurodevelopment and regeneration etc. and pathologic process.Simultaneously, it has also participated in the transduction of cell surface and nucleus inner recipient signal.
Prawn is one of Important Economic animal of culture fishery, and day by day serious disease problem has seriously restricted the sound development of shrimp culture industry.Prawn belongs to invertebrates, lacks specific immunity system, but it has numerous immune factors, with the invasion and attack of cause of disease in the opposing external environment.Therefore, utilize the immune disease-resistance factor of prawn itself to strengthen it to the resistivity of disease, will help the control to shrimp disease.
Summary of the invention
First purpose of the present invention provides Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene sequence;
Second purpose of the present invention provides Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin protein sequence;
The 3rd purpose of the present invention provides the preparation method of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin.
The 4th purpose of the present invention provides the application of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin in the anti-prawn ' s virus medicine of preparation.
Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene of the present invention comprises Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene, Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene and Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene.
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene is DNA, sequence signature: length is 501bp, type is nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene is as follows, is designated as SEQ ID No.1:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGACGTGGC ACAAGAGAAG CAACACATTG AACATCTACA GAACATCAGC
181 GAGTTTCGCA GCGAAAGACT CAAACGAACG TCAACCTCTG AGAAGTTGGT CCTCCCCACT
241 AGTCAAGACG TGGAAGCAGA GAAGAAAGCA CAGGCCCATC TGCAGGCTGT CGAAGGCTTC
301 AATGCTGCAC AACTCAAGCA TGCCAATACC CAAGAAAAAA TTGTTTTACC TGCTAAGGAA
361 GATATTGAGA ATGAGAAGGG TCAGCAGGCA CTCCGCCAGG GTATTGAGGG CTTTGACCAT
421 ACCGCTCTGA AGAAGGCTCA GACGGCAGAG AAGAATACCC TTCCAACTAA GGAAATGATT
481 GAGGAAGAGA AGAAGGCCTA A；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene is DNA, sequence signature: length is 387bp, type is nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene is as follows, is designated as SEQ ID No.2:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGACGTGGA AGCAGAGAAG AAAGCACAGG CCCATCTGCA GGCTGTCGAA
181 GGCTTCAATG CTGCACAACT CAAGCATGCC AATACCCAAG AAAAAATTGT TTTACCTGCT
241 AAGGAAGATA TTGAGAATGA GAAGGGTCAG CAGGCACTCC GCCAGGGTAT TGAGGGCTTT
301 GACCATACCG CTCTGAAGAA GGCTCAGACG GCAGAGAAGA ATACCCTTCC AACTAAGGAA
361 ATGATTGAGG AAGAGAAGAA GGCCTAA；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene is DNA, sequence signature: length is 273bp, type is nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene is as follows, is designated as SEQ ID No.3:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGATATTGA GAATGAGAAG GGTCAGCAGG CACTCCGCCA GGGTATTGAG
181 GGCTTTGACC ATACCGCTCT GAAGAAGGCT CAGACGGCAG AGAAGAATAC CCTTCCAACT
241 AAGGAAATGA TTGAGGAAGA GAAGAAGGCC TAA。
The nucleotide sequence of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene of the present invention can adopt following methods to obtain: the RNA that extracts Marsupenaeus japonicus Marsupenaeus japonicus, and the RNA reverse transcription become cDNA, further take cDNA as template, obtained SEQ ID No.1 through pcr amplification, the sequence of SEQ ID No.2 and SEQ ID No.3.Relevant nucleotide sequence obtains the polypeptide (SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6) of genes encoding by prediction, then carry out Prokaryotic expression vector construction and obtain recombinant expression protein.
Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin of the present invention is Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4, Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 and Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2.
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4 is protein, sequence signature: length is 166aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4 is as follows, is designated as SEQ ID No.4:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDVAQEK QHIEHLQNIS
61 EFRSERLKRT STSEKLVLPT SQDVEAEKKA QAHLQAVEGF NAAQLKHANT QEKIVLPAKE
121 DIENEKGQQA LRQGIEGFDH TALKKAQTAE KNTLPTKEMI EEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 is protein, sequence signature: length is 128aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 is as follows, is designated as SEQ ID No.5:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDVEAEK KAQAHLQAVE
61 GFNAAQLKHA NTQEKIVLPA KEDIENEKGQ QALRQGIEGF DHTALKKAQT AEKNTLPTKE
121 MIEEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2 is protein, sequence signature: length is 90aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2 is as follows, is designated as SEQ ID No.6:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDIENEK GQQALRQGIE
61 GFDHTALKKA QTAEKNTLPT KEMIEEEKKA。
These albumen have the polypeptide of aminoacid sequence shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6; Or replacement, disappearance or the interpolation through one or more amino-acid residues of SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 aminoacid sequence formed, and has polypeptide with the aminoacid sequence identity function shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6.
The preparation method of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin of the present invention may further comprise the steps:
The step of the extraction of a total RNA of Marsupenaeus japonicus Marsupenaeus japonicus;
The step of Marsupenaeus japonicus Marsupenaeus japonicus cDNA reverse transcription;
The pcr amplification of a Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene and the step of sequential analysis;
The recombinant expressed step of a Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin.
Described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin can be used for preparing anti-prawn ' s virus medicine.
The present invention is by extracting total RNA of Marsupenaeus japonicus Marsupenaeus japonicus, pass through reverse transcription, by primer amplification with identified 3 thymosin gene mjthm4, mjthm3 and mjthm2, made up recombinant prokaryotic expression vector by pGEX-4T-2, in e. coli bl21, carry out expression of recombinant proteins, for the practical application of this albumen provides the foundation.Express three kinds of Zadaxin by homologous recombination, can overcome that separation and purification process complexity is loaded down with trivial details, the deficiency such as yield poorly, for the widespread use of this albumen in research fields such as biology, food, medicine, agriculturals lays the foundation.
The present invention is take Marsupenaeus japonicus Marsupenaeus japonicus as research material, and the gene expression difference of normal shrimp and disease-resistant shrimp is compared research, and screening has obtained three kinds of thymosin genes.Research finds that virus stimulates early stage their transcriptional expression levels obviously to raise, on average reach level more than 3 times, wherein two in the downward modulation of disease-resistant shrimp transcription expression level and reach conspicuous level, there be close contacting in the infection that Marsupenaeus japonicus Zadaxin and prawn ' s virus are described, and it may have important antiviral using value and pharmaceutical use.
Description of drawings
Fig. 1 is the changing conditions (phosphoric acid buffer (PBS) is the experiment contrast group, and WSSV injection is the WSSV infected group) of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene mjthm4 its transcriptional level after WSSV infects.In Fig. 1, a is phosphoric acid buffer (PBS) contrast, and b is that WSSV infects.
Fig. 2 is the changing conditions (phosphoric acid buffer (PBS) contrast is the experiment contrast group, and WSSV infects and is the WSSV infected group) of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene mjthm3 its transcriptional level after WSSV infects.In Fig. 2, a is phosphoric acid buffer (PBS) contrast, and b is that WSSV infects.
Fig. 3 is the changing conditions (phosphoric acid buffer (PBS) contrast is the experiment contrast group, and WSSV infects and is the WSSV infected group) of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene mjthm2 its transcriptional level after WSSV infects.In Fig. 3, a is phosphoric acid buffer (PBS) contrast, and b is that WSSV infects.
Fig. 4 is Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene mjthm3 infects prawn transcription level at common prawn and anti-WSSV situation (do not infect the WSSV shrimp for not infecting the common prawn group of WSSV, anti-WSSV infects shrimp and infects the prawn group for the anti-WSSV that does not infect WSSV).In Fig. 4, a is not for infecting the WSSV shrimp, and b is that anti-WSSV infects shrimp.
Fig. 5 is Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene mjthm2 infects prawn transcription level at common prawn and anti-WSSV situation (do not infect the WSSV shrimp for not infecting the common prawn group of WSSV, anti-WSSV infects shrimp and infects the prawn group for the anti-WSSV that does not infect WSSV).In Fig. 5, a is not for infecting the WSSV shrimp, and b is that anti-WSSV infects shrimp.
Fig. 6 is the SDS-PAGE collection of illustrative plates of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4 and the recombinant expressed purifying of Mjthm3.1: recombinant vectors pGEX-4T-2-Mjthm3 is at the not abduction delivering of bacterial strain BL21; 2: recombinant vectors pGEX-4T-2-Mjthm4 is at the not abduction delivering of bacterial strain BL21; 3: recombinant vectors pGEX-4T-2-Mjthm3 is at the abduction delivering of bacterial strain BL21; 4: recombinant vectors pGEX-4T-2-Mjthm4 is at the abduction delivering of bacterial strain BL21; 5: through the target protein Mjthm3 of GST medium purification gained; 6: through the target protein Mjthm4 of GST medium purification gained; 7: molecular weight of albumen Marker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition such as molecular cloning laboratory manual (1, the Pehanorm Brooker, Russell's (work), Huang Peitang (translating), " molecular cloning experiment guide ", Science Press, 2002, the third edition .) described in experiment condition, or the condition of advising according to reagent or instrument production firm.
For achieving the above object, the present invention adopts following technical measures, and its concrete steps are:
1. the extraction of the total RNA of Marsupenaeus japonicus Marsupenaeus japonicus
Get 100mg Marsupenaeus japonicus Marsupenaeus japonicus hepatic tissue, add liquid nitrogen and clay into power, add 1mL TRIzol(Invitrogen company) change the 1.5mL centrifuge tube over to behind the mixing, room temperature leaves standstill the 5min lysing cell; Add the 0.2mL chloroform, thermal agitation 15s, room temperature leaves standstill 10min; 4 ℃, the centrifugal 15min of 12000g; Supernatant liquid is transferred to one new in the RNase centrifuge tube, adds the 0.5mL Virahol, mixing, room temperature is placed 8min; 4 ℃, the centrifugal 10min of 12000g; Discard liquid, add the rinsing of 1mL75% ethanol; 4 ℃, the centrifugal 5min of 7500g; Discard liquid, precipitate a little and be dissolved in the water of an amount of RNase-free after the drying; Measure total RNA at OD with micro-ultraviolet spectrophotometer 230, OD 260And OD 280Absorbancy, judge concentration and the purity of total RNA.
2. Marsupenaeus japonicus Marsupenaeus japonicus cDNA reverse transcription
Prepare the cDNA inverse transcription reaction liquid at the 0.5mL centrifuge tube, comprise the total RNA of 2 μ g, 1 μ L6mer random primer (200ng), 1 μ L dNTP(10mM), the H that processed with DEPC 2O mends cumulative volume to 13 μ L; 65 ℃ of processing of reaction system 5min puts into ice bath 1min immediately; Slightly centrifugal, add following component: 4 μ L5 * First-Strand Buffer, 1 μ L DTT(0.1M), 1 μ L RNase Inhibitor(40U/ μ L) (TaKaRa company) and 1 μ L SuperScript TMIIIreverse transcriptase(200U/ μ L) (Invitrogen company); Reaction system is flicked mixing, and is slightly centrifugal; 25 ℃ of 5min, 50 ℃ of incubation 60min process the 15min termination reaction for 70 ℃.
3. pcr amplification and the sequential analysis of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene
Take Marsupenaeus japonicus Marsupenaeus japonicus cDNA as template, increasing respectively by primer obtains the open reading frame of thymosin gene mjthm4, mjthm3 and mjthm2.
In the reaction system of 25 μ L, contain 1 μ L template, the positive anti-primer of 1 μ L (20 μ M), 2 each 2.5mM of μ L dNTP(), 5 μ L5 * PrimerSTAR TMBuffer, 0.5 μ L PrimerSTAR TMHS DNA Polymerase(2.5U/ μ L) (TaKARa company) uses H 2O mends cumulative volume to 25 μ L.The PCR reaction conditions is: 98 ℃ of 10s, 58 ℃ of 30s, 72 ℃ of 35s(30cycles); 4 ℃ of preservations.Be connected with the T carrier after the PCR product is purified, be transformed among the competent escherichia coli cell Top10, choose the positive colony order-checking that dna fragmentation is arranged behind the bacterium colony PCR.
Derive the aminoacid sequence of Mjthm4, Mjthm3 and Mjrhm2 according to the nucleotide sequence of amplification acquisition, contain respectively 166,128 and 90 amino acid whose polypeptide, its aminoacid sequence sees SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 for details.
4. Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin is to replying that WSSV infects
The Marsupenaeus japonicus that obtains survival from WSSV morbidity culturing pool is as disease-resistant shrimp, and the Marsupenaeus japonicus of choosing simultaneously a collection of health carries out the WSSV infectable infection.Get the Marsupenaeus japonicus hepatopancreas liquid nitrogen cryopreservation that disease-resistant shrimp and WSSV infect different time points (0h, 6h, 12h, 24h, 48h), extract total RNA, remove genomic dna, reverse transcription is cDNA, by real-time quantitative PCR determine WSSV infect after the transcriptional expression amount of the transcriptional expression amount of phase thymosin gene and disease-resistant shrimp thymosin gene simultaneously not, three thymosin genes finding the clone infect early stage obviously raise (Fig. 1～3) at WSSV, infer that its infection with WSSV is relevant; Thymosin gene mjthm3, mjthm2 downward modulation amount almost reach half (Figure 4 and 5) in disease-resistant shrimp, and these two genes may participate in utilizing to the defence of WSSV infection or by the WSSV infection in early days.This gene and albumen can be used as antiviral or medicinal application.
5. the expression of Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene
To contain the plasmid usefulness of mjthm4, mjthm3 and mjthm2 gene with the primer amplification of restriction enzyme site, then carrying out enzyme with corresponding restriction enzyme cuts, glue reclaims mjthm4, mjthm3 and mjthm2 gene fragment, simultaneously plasmid pGEX-4T-2 is also carried out enzyme with identical enzyme and cuts.With both 16 ℃ of connection 6h.Connect product and be transformed among the competent escherichia coli cell DH5 α, extract plasmid, sequence verification.
With the correct Plasmid Transformation BL21 of order-checking, the exactness of bacterium colony PCR checking Plasmid Transformation is selected and is transformed correct bacterium colony behind 37 ℃ of growth 15h, is inoculated in the LB substratum that 5mL contains 100IU penicillin, and 37 ℃ are shaken and train to A 600=0.5 o'clock, add isopropylthio-β-D-galactoside (IPTG) to final concentration 0.1mM, induce 12～16h for 16 ℃, bacterium liquid is collected in the centrifuge tube of 200mL 5000g centrifugation bacterial cell.With the bacterial cell Eddy diffusion at 30mL PBS buffer A (140mM NaCl, 2.7mM KCl, 10mM Na 2HPO 412H 2O, 1.8mM KH 2PO 4, pH=7.4) in, ultrasonication to bacterium liquid becomes translucent, the centrifugal 20min of 18000g again, supernatant and the GST medium Glutathione Sepharose4Fast Flow(GE company that uses in advance PBS damping fluid balance) mixing, 4 ℃ in conjunction with 4h, and purge process is carried out according to the purification kit explanation.The albumen of purifying is through 12% SDS-PAGE electrophoretic analysis, and its purity reaches (referring to Fig. 6) more than 95%.
Figure IDA00003627060900011
Figure IDA00003627060900021
Figure IDA00003627060900031
Figure IDA00003627060900051

Claims (10)

1. Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene is characterized in that comprising Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene, Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene and Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene.
2. Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene as claimed in claim 1, the molecule type that it is characterized in that described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene is DNA, sequence signature: length is 501bp, type is nucleic acid, chain is double-stranded, topological framework is linear, and the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm4 gene is as follows, is designated as SEQ ID No.1:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGACGTGGC ACAAGAGAAG CAACACATTG AACATCTACA GAACATCAGC
181 GAGTTTCGCA GCGAAAGACT CAAACGAACG TCAACCTCTG AGAAGTTGGT CCTCCCCACT
241 AGTCAAGACG TGGAAGCAGA GAAGAAAGCA CAGGCCCATC TGCAGGCTGT CGAAGGCTTC
301 AATGCTGCAC AACTCAAGCA TGCCAATACC CAAGAAAAAA TTGTTTTACC TGCTAAGGAA
361 GATATTGAGA ATGAGAAGGG TCAGCAGGCA CTCCGCCAGG GTATTGAGGG CTTTGACCAT
421 ACCGCTCTGA AGAAGGCTCA GACGGCAGAG AAGAATACCC TTCCAACTAA GGAAATGATT
481 GAGGAAGAGA AGAAGGCCTA A。
3. Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene as claimed in claim 1, the molecule type that it is characterized in that described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene is DNA, sequence signature: length is 387bp, type is nucleic acid, chain is double-stranded, topological framework is linear, and the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm3 gene is as follows, is designated as SEQ ID No.2:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGACGTGGA AGCAGAGAAG AAAGCACAGG CCCATCTGCA GGCTGTCGAA
181 GGCTTCAATG CTGCACAACT CAAGCATGCC AATACCCAAG AAAAAATTGT TTTACCTGCT
241 AAGGAAGATA TTGAGAATGA GAAGGGTCAG CAGGCACTCC GCCAGGGTAT TGAGGGCTTT
301 GACCATACCG CTCTGAAGAA GGCTCAGACG GCAGAGAAGA ATACCCTTCC AACTAAGGAA
361 ATGATTGAGG AAGAGAAGAA GGCCTAA。
4. Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene as claimed in claim 1, the molecule type that it is characterized in that described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene is DNA, sequence signature: length is 273bp, type is nucleic acid, chain is double-stranded, topological framework is linear, and the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin mjthm2 gene is as follows, is designated as SEQ ID No.3:
1 ATGAGCGCCG AAACTCCCCT CAAGGACTTG CCCAAGGTTG ACCCCACCCT CAAGGGACAG
61 CTCGAGGGAT TCTCCGCCGT AAACCTTAAG AAGATCGAGA CGGAGGAAAA GATCCACCTG
121 CCAAACAAGG AGGATATTGA GAATGAGAAG GGTCAGCAGG CACTCCGCCA GGGTATTGAG
181 GGCTTTGACC ATACCGCTCT GAAGAAGGCT CAGACGGCAG AGAAGAATAC CCTTCCAACT
241 AAGGAAATGA TTGAGGAAGA GAAGAAGGCC TAA。
5. Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene as claimed in claim 1, it is characterized in that its nucleotide sequence adopts following methods to obtain: the RNA that extracts Marsupenaeus japonicus Marsupenaeus japonicus, and the RNA reverse transcription become cDNA, further take cDNA as template, obtain SEQ ID No.1 through pcr amplification, the sequence of SEQ ID No.2 and SEQ ID No.3; Relevant nucleotide sequence obtains the polypeptide SEQ ID No.4 of genes encoding by prediction, then SEQ IDNo.5 and SEQ ID No.6 carry out Prokaryotic expression vector construction and obtain recombinant expression protein.
6. Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin is characterized in that comprising Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4, Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 and Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2.
7. Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin as claimed in claim 6, the molecule type that it is characterized in that described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4 is protein, sequence signature: length is 166aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm4 is as follows, is designated as SEQ ID No.4:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDVAQEK QHIEHLQNIS
61 EFRSERLKRT STSEKLVLPT SQDVEAEKKA QAHLQAVEGF NAAQLKHANT QEKIVLPAKE
121 DIENEKGQQA LRQGIEGFDH TALKKAQTAE KNTLPTKEMI EEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 is protein, sequence signature: length is 128aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm3 is as follows, is designated as SEQ ID No.5:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDVEAEK KAQAHLQAVE
61 GFNAAQLKHA NTQEKIVLPA KEDIENEKGQ QALRQGIEGF DHTALKKAQT AEKNTLPTKE
121 MIEEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2 is protein, sequence signature: length is 90aa, type is amino acid, the sequence of described Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin Mjthm2 is as follows, is designated as SEQ ID No.6:
1 MSAETPLKDL PKVDPTLKGQ LEGFSAVNLK KIETEEKIHL PNKEDIENEK GQQALRQGIE
61 GFDHTALKKA QTAEKNTLPT KEMIEEEKKA。
8. Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin as claimed in claim 7 is characterized in that described albumen has the polypeptide of aminoacid sequence shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6; Or replacement, disappearance or the interpolation through one or more amino-acid residues of SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 aminoacid sequence formed, and has polypeptide with the aminoacid sequence identity function shown in SEQ ID No.4, SEQ ID No.5 and the SEQ ID No.6.
9. the preparation method of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin as claimed in claim 6 is characterized in that may further comprise the steps:
The step of the extraction of a total RNA of Marsupenaeus japonicus Marsupenaeus japonicus;
The step of Marsupenaeus japonicus Marsupenaeus japonicus cDNA reverse transcription;
The pcr amplification of a Marsupenaeus japonicus Marsupenaeus japonicus thymosin gene and the step of sequential analysis;
The recombinant expressed step of a Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin.
10. the as claimed in claim 6 application of Marsupenaeus japonicus Marsupenaeus japonicus Zadaxin in the anti-prawn ' s virus medicine of preparation.
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