CN103361356B - The preparation method of Marsupenaeus japonicus thymosin gene and Marsupenaeus japonicus Zadaxin thereof and application - Google Patents
The preparation method of Marsupenaeus japonicus thymosin gene and Marsupenaeus japonicus Zadaxin thereof and application Download PDFInfo
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Abstract
The preparation method of Marsupenaeus japonicus thymosin gene and Marsupenaeus japonicus Zadaxin thereof and application, relate to the nucleotide sequence of Marsupenaeus japonicus thymosin gene and the aminoacid sequence coded by it.Marsupenaeus japonicus thymosin gene comprises Marsupenaeus japonicus Zadaxin mjthm4 gene, Marsupenaeus japonicus Zadaxin mjthm3 gene and Marsupenaeus japonicus Zadaxin mjthm2 gene.The step of the extraction of a Marsupenaeus japonicus total serum IgE; The step of Marsupenaeus japonicus cDNA reverse transcription; The pcr amplification of a Marsupenaeus japonicus thymosin gene and the step of sequential analysis; The recombinant expressed step of a Marsupenaeus japonicus Zadaxin.Described Marsupenaeus japonicus Zadaxin can be used for preparing anti-prawn ' s virus medicine.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.
Description
Technical field
The present invention relates to the nucleotide sequence of Marsupenaeus japonicus (Marsupenaeusjaponicus) thymosin gene and the aminoacid sequence coded by it, especially relate to Marsupenaeus japonicus (Marsupenaeusjaponicus) Zadaxin albumen and preparation method thereof.
Background technology
Zadaxin is a kind of B cell growth factor secreted by thymic epithelial cells, and research finds, they are prevalent in Various Tissues cell.Difference according to iso-electric point (pI) is divided into Thymosin alpha, β, γ, wherein iso-electric point pI be less than 5.0 for alpha thymosin peptides, component that pI is positioned at 5.0 ~ 7.0 is called beta thymosin peptides, iso-electric point pI be greater than 7.0 be γ Zadaxin.So far, found that beta thymosin peptides has 15 kinds, except participating in except the regulation and control of Actin muscle in cell, beta thymosin peptides also participates in and the aspects such as the interaction of other albumen and the anti-infective protection of body.At present, Zadaxin, mainly as a kind of cellular immunization toughener, promotes lymphocyte maturation and enhancing body immunologic function.Research shows, beta thymosin peptides take part in immunomodulatory, tumor invasion and multiple physiology such as transfer, apoptosis, inflammatory reaction, vasculogenesis, coagulation process, wound healing, fetal development and neurodevelopment and regeneration etc. and pathologic process.Meanwhile, it also take part 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 seriously constrains the sound development of shrimp culture industry.Prawn belongs to invertebrates, lacks specific immunity system, but it has numerous immune factors, to resist the invasion and attack of cause of disease in external environment.Therefore, utilize the immune disease-resistance factor of prawn itself to strengthen its resistivity to disease, will the control to shrimp disease be contributed to.
Summary of the invention
First object of the present invention is to provide Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene sequence;
Second object of the present invention is to provide Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin protein sequence;
3rd object of the present invention is to provide the preparation method of Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin.
4th object of the present invention is to provide the application of Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin in the anti-prawn ' s virus medicine of preparation.
Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene of the present invention comprises Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene, Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm3 gene and Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm2 gene.
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene is DNA, sequence signature: length is 501bp, type is nucleic acid, chain is double-strand, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene is as follows, is designated as SEQIDNo.1:
1ATGAGCGCCGAAACTCCCCTCAAGGACTTGCCCAAGGTTGACCCCACCCTCAAGGGACAG
61CTCGAGGGATTCTCCGCCGTAAACCTTAAGAAGATCGAGACGGAGGAAAAGATCCACCTG
121CCAAACAAGGAGGACGTGGCACAAGAGAAGCAACACATTGAACATCTACAGAACATCAGC
181GAGTTTCGCAGCGAAAGACTCAAACGAACGTCAACCTCTGAGAAGTTGGTCCTCCCCACT
241AGTCAAGACGTGGAAGCAGAGAAGAAAGCACAGGCCCATCTGCAGGCTGTCGAAGGCTTC
301AATGCTGCACAACTCAAGCATGCCAATACCCAAGAAAAAATTGTTTTACCTGCTAAGGAA
361GATATTGAGAATGAGAAGGGTCAGCAGGCACTCCGCCAGGGTATTGAGGGCTTTGACCAT
421ACCGCTCTGAAGAAGGCTCAGACGGCAGAGAAGAATACCCTTCCAACTAAGGAAATGATT
481GAGGAAGAGAAGAAGGCCTAA；
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm3 gene is DNA, sequence signature: length is 387bp, type is nucleic acid, chain is double-strand, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm3 gene is as follows, is designated as SEQIDNo.2:
1ATGAGCGCCGAAACTCCCCTCAAGGACTTGCCCAAGGTTGACCCCACCCTCAAGGGACAG
61CTCGAGGGATTCTCCGCCGTAAACCTTAAGAAGATCGAGACGGAGGAAAAGATCCACCTG
121CCAAACAAGGAGGACGTGGAAGCAGAGAAGAAAGCACAGGCCCATCTGCAGGCTGTCGAA
181GGCTTCAATGCTGCACAACTCAAGCATGCCAATACCCAAGAAAAAATTGTTTTACCTGCT
241AAGGAAGATATTGAGAATGAGAAGGGTCAGCAGGCACTCCGCCAGGGTATTGAGGGCTTT
301GACCATACCGCTCTGAAGAAGGCTCAGACGGCAGAGAAGAATACCCTTCCAACTAAGGAA
361ATGATTGAGGAAGAGAAGAAGGCCTAA；
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm2 gene is DNA, sequence signature: length is 273bp, type is nucleic acid, chain is double-strand, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm2 gene is as follows, is designated as SEQIDNo.3:
1ATGAGCGCCGAAACTCCCCTCAAGGACTTGCCCAAGGTTGACCCCACCCTCAAGGGACAG
61CTCGAGGGATTCTCCGCCGTAAACCTTAAGAAGATCGAGACGGAGGAAAAGATCCACCTG
121CCAAACAAGGAGGATATTGAGAATGAGAAGGGTCAGCAGGCACTCCGCCAGGGTATTGAG
181GGCTTTGACCATACCGCTCTGAAGAAGGCTCAGACGGCAGAGAAGAATACCCTTCCAACT
241AAGGAAATGATTGAGGAAGAGAAGAAGGCCTAA。
The nucleotide sequence of Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene of the present invention can adopt following methods to obtain: the RNA extracting Marsupenaeus japonicus Marsupenaeusjaponicus, and RNA reverse transcription is become cDNA, be template further with cDNA, SEQIDNo.1 is obtained, the sequence of SEQIDNo.2 and SEQIDNo.3 through pcr amplification.Relevant nucleotide sequence obtains the polypeptide (SEQIDNo.4, SEQIDNo.5 and SEQIDNo.6) of genes encoding by prediction, then carries out Prokaryotic expression vector construction and obtains recombinant expression protein.
Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin of the present invention is Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4, Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm3 and Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm2.
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4 is protein, sequence signature: length is 166aa, type is amino acid, and the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4 is as follows, is designated as SEQIDNo.4:
1MSAETPLKDLPKVDPTLKGQLEGFSAVNLKKIETEEKIHLPNKEDVAQEKQHIEHLQNIS
61EFRSERLKRTSTSEKLVLPTSQDVEAEKKAQAHLQAVEGFNAAQLKHANTQEKIVLPAKE
121DIENEKGQQALRQGIEGFDHTALKKAQTAEKNTLPTKEMIEEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm3 is protein, sequence signature: length is 128aa, type is amino acid, and the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm3 is as follows, is designated as SEQIDNo.5:
1MSAETPLKDLPKVDPTLKGQLEGFSAVNLKKIETEEKIHLPNKEDVEAEKKAQAHLQAVE
61GFNAAQLKHANTQEKIVLPAKEDIENEKGQQALRQGIEGFDHTALKKAQTAEKNTLPTKE
121MIEEEKKA；
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm2 is protein, sequence signature: length is 90aa, type is amino acid, and the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm2 is as follows, is designated as SEQIDNo.6:
1MSAETPLKDLPKVDPTLKGQLEGFSAVNLKKIETEEKIHLPNKEDIENEKGQQALRQGIE
61GFDHTALKKAQTAEKNTLPTKEMIEEEKKA。
These albumen have the polypeptide of aminoacid sequence shown in SEQIDNo.4, SEQIDNo.5 and SEQIDNo.6; Or formed through the replacement of one or more amino-acid residue, disappearance or interpolation by SEQIDNo.4, SEQIDNo.5 and SEQIDNo.6 aminoacid sequence, and there is the polypeptide with the aminoacid sequence identity function shown in SEQIDNo.4, SEQIDNo.5 and SEQIDNo.6.
The preparation method of Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin of the present invention, comprises the following steps:
The step of the extraction of a Marsupenaeus japonicus Marsupenaeusjaponicus total serum IgE;
The step of Marsupenaeus japonicus MarsupenaeusjaponicuscDNA reverse transcription;
The pcr amplification of a Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene and the step of sequential analysis;
The recombinant expressed step of a Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin.
Described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin can be used for preparing anti-prawn ' s virus medicine.
The present invention is by extracting the total serum IgE of Marsupenaeus japonicus Marsupenaeusjaponicus, pass through reverse transcription, by primer amplification and identify 3 thymosin genes mjthm4, mjthm3 and mjthm2, recombinant prokaryotic expression vector is constructed by pGEX-4T-2, expression of recombinant proteins is carried out, for the practical application of this albumen provides the foundation in e. coli bl21.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 for research material, compares research to the gene expression difference of normal shrimp and disease-resistant shrimp with Marsupenaeus japonicus Marsupenaeusjaponicus, and screening obtains three kinds of thymosin genes.Research finds that virus stimulates their transcriptional expression levels early stage obviously to raise, on average reach more than 3 times levels, wherein lower at disease-resistant shrimp transcription expression level and reach conspicuous level for two, illustrate that the infection of Marsupenaeus japonicus Zadaxin and prawn ' s virus exists close contacting, it may have important antiviral using value and pharmaceutical use.
Accompanying drawing explanation
Fig. 1 is the changing conditions (phosphoric acid buffer (PBS) is experiment contrast group, and WSSVinjection is WSSV infected group) of Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene mjthm4 its transcriptional level after WSSV infects.In FIG, 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 experiment contrast group, and WSSV infects for WSSV infected group) of Marsupenaeus japonicus Marsupenaeusjaponicus 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 experiment contrast group, and WSSV infects for WSSV infected group) of Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene mjthm2 its transcriptional level after WSSV infects.In figure 3, a is phosphoric acid buffer (PBS) contrast, and b is that WSSV infects.
Fig. 4 is Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene mjthm3 in the situation (not infecting WSSV shrimp is the common prawn group not infecting WSSV, and Anti-infection to WSSV shrimp is the Anti-infection to WSSV prawn group not infecting WSSV) of common prawn and Anti-infection to WSSV prawn transcription level.In the diagram, a is not for infect WSSV shrimp, and b is Anti-infection to WSSV shrimp.
Fig. 5 is Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene mjthm2 in the situation (not infecting WSSV shrimp is the common prawn group not infecting WSSV, and Anti-infection to WSSV shrimp is the Anti-infection to WSSV prawn group not infecting WSSV) of common prawn and Anti-infection to WSSV prawn transcription level.In Figure 5, a is not for infect WSSV shrimp, and b is Anti-infection to WSSV shrimp.
Fig. 6 is the SDS-PAGE collection of illustrative plates of the recombinant expressed purifying of Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4 and Mjthm3.1: recombinant vectors pGEX-4T-2-Mjthm3 at the non-abduction delivering of bacterial strain BL21; 2: recombinant vectors pGEX-4T-2-Mjthm4 at the non-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, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition as Molecular Cloning: A Laboratory room handbook (1, Pehanorm Brooker, Russell's (work), Huang Peitang (translating), " Molecular Cloning: A Laboratory guide ", Science Press, 2002, the third edition .) described in experiment condition, or according to the condition that reagent or instrument manufacturer facility business advise.
For achieving the above object, the present invention adopts following technical measures, and its concrete steps are:
1. the extraction of Marsupenaeus japonicus Marsupenaeusjaponicus total serum IgE
Get 100mg Marsupenaeus japonicus Marsupenaeusjaponicus hepatic tissue, add liquid nitrogen and clay into power, add 1mLTRIzol(Invitrogen company) mixing after proceed to 1.5mL centrifuge tube, room temperature leaves standstill 5min lysing cell; Add 0.2mL chloroform, thermal agitation 15s, room temperature leaves standstill 10min; 4 DEG C, the centrifugal 15min of 12000g; Be transferred to by supernatant liquid one new in RNase centrifuge tube, add 0.5mL Virahol, mixing, room temperature places 8min; 4 DEG C, the centrifugal 10min of 12000g; Discard liquid, add 1mL75% ethanol rinse; 4 DEG C, the centrifugal 5min of 7500g; Discard liquid, be dissolved in the water of appropriate RNase-free after precipitating drying a little; Total serum IgE is measured at OD with micro-ultraviolet spectrophotometer
230, OD
260and OD
280absorbancy, judge the concentration of total serum IgE and purity.
2. Marsupenaeus japonicus MarsupenaeusjaponicuscDNA reverse transcription
Prepare cDNA inverse transcription reaction liquid at 0.5mL centrifuge tube, comprise 2 μ g total serum IgE, 1 μ L6mer random primer (200ng), 1 μ LdNTP(10mM), with the H of DEPC process
2cumulative volume is mended to 13 μ L by O; Reaction system 65 DEG C process 5min, puts into ice bath 1min immediately; Slightly centrifugal, add following component: 4 μ L5 × First-StrandBuffer, 1 μ LDTT(0.1M), 1 μ LRNaseInhibitor(40U/ μ L) (TaKaRa company) and 1 μ LSuperScript
tMiIIreversetranscriptase(200U/ μ L) (Invitrogen company); Reaction system flicks mixing, slightly centrifugal; 25 DEG C of 5min, 50 DEG C of incubation 60min, 70 DEG C of process 15min termination reactions.
3. the pcr amplification of Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene and sequential analysis
With Marsupenaeus japonicus MarsupenaeusjaponicuscDNA for template, increased respectively by primer and obtain the open reading frame of thymosin gene mjthm4, mjthm3 and mjthm2.
Containing 1 μ L template in the reaction system of 25 μ L, the positive anti-primer of 1 μ L (20 μMs), each 2.5mM of 2 μ LdNTP(), 5 μ L5 × PrimerSTAR
tMbuffer, 0.5 μ LPrimerSTAR
tMhSDNAPolymerase(2.5U/ μ L) (TaKARa company), use H
2cumulative volume is mended to 25 μ L by O.PCR reaction conditions is: 98 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 35s(30cycles); 4 DEG C of preservations.Be connected with carrier T after PCR primer is purified, be transformed in competent escherichia coli cell Top10, choose after bacterium colony PCR and have the positive colony of DNA fragmentation to check order.
Derive the aminoacid sequence of Mjthm4, Mjthm3 and Mjrhm2 according to the nucleotide sequence of amplification acquisition, respectively containing 166,128 and 90 amino acid whose polypeptide, its aminoacid sequence refers to SEQIDNo.4, SEQIDNo.5, SEQIDNo.6.
4. Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin response that WSSV is infected
From WSSV falls ill culturing pool, obtain the Marsupenaeus japonicus of survival as disease-resistant shrimp, the Marsupenaeus japonicus simultaneously choosing a collection of health carries out WSSV infectable infection.Get the Marsupenaeus japonicus hepatopancreas liquid nitrogen cryopreservation of disease-resistant shrimp and WSSV infection different time points (0h, 6h, 12h, 24h, 48h), extract total serum IgE, remove genomic dna, reverse transcription is cDNA, determine that WSSV infects the rear transcriptional expression amount of different phase thymosin gene and the transcriptional expression amount of disease-resistant shrimp thymosin gene by real-time quantitative PCR, find that three thymosin genes of clone infect at WSSV and obviously raise (Fig. 1 ~ 3) in early days, infer that it is relevant to the infection of WSSV; In disease-resistant shrimp, thymosin gene mjthm3, mjthm2 downward amount almost reaches half (Figure 4 and 5), these two genes may take part in early days to WSSV infect defence or by WSSV infect utilize.This gene and albumen can by as antiviral or medicinal applications.
5. the expression of Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene
By the primer amplification of the plasmid containing mjthm4, mjthm3 and mjthm2 gene with band restriction enzyme site, then carry out enzyme with corresponding restriction enzyme to cut, glue reclaims mjthm4, mjthm3 and mjthm2 gene fragment, plasmid pGEX-4T-2 is also carried out enzyme with identical enzyme simultaneously and cuts.Will both 16 DEG C of connection 6h.Connect product conversion in competent escherichia coli cell DH5 α, extract plasmid, sequence verification.
By Plastid transformation BL21 correct for order-checking, after 37 DEG C of growth 15h, bacterium colony PCR verifies the exactness of Plastid transformation, selects and transforms correct bacterium colony, be inoculated into 5mL and contain in the LB substratum of 100IU penicillin, and 37 DEG C are shaken training to A
600when=0.5, add isopropylthio-β-D-galactoside (IPTG) to final concentration 0.1mM, 16 DEG C of induction 12 ~ 16h, are collected in the centrifuge tube of 200mL by bacterium liquid, 5000g centrifugation bacterial cell.By bacterial cell Eddy diffusion at 30mLPBS buffer A (140mMNaCl, 2.7mMKCl, 10mMNa
2hPO
412H
2o, 1.8mMKH
2pO
4, pH=7.4) in, ultrasonication becomes translucent to bacterium liquid, the centrifugal 20min of 18000g again, supernatant with in advance with the GST medium GlutathioneSepharose4FastFlow(GE company that PBS damping fluid balances) mix, 4 DEG C in conjunction with 4h, purge process illustrates according to purification kit carries out.The albumen of purifying through 12% SDS-PAGE electrophoretic analysis, its purity reaches more than 95% (see Fig. 6).
Claims (5)
1. Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene, is characterized in that for Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene;
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene is DNA, sequence signature: length is 501bp, type is nucleic acid, chain is double-strand, topological framework is linear, the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin mjthm4 gene is as follows, is designated as SEQIDNo.1:
1ATGAGCGCCGAAACTCCCCTCAAGGACTTGCCCAAGGTTGACCCCACCCTCAAGGGACAG
61CTCGAGGGATTCTCCGCCGTAAACCTTAAGAAGATCGAGACGGAGGAAAAGATCCACCTG
121CCAAACAAGGAGGACGTGGCACAAGAGAAGCAACACATTGAACATCTACAGAACATCAGC
181GAGTTTCGCAGCGAAAGACTCAAACGAACGTCAACCTCTGAGAAGTTGGTCCTCCCCACT
241AGTCAAGACGTGGAAGCAGAGAAGAAAGCACAGGCCCATCTGCAGGCTGTCGAAGGCTTC
301AATGCTGCACAACTCAAGCATGCCAATACCCAAGAAAAAATTGTTTTACCTGCTAAGGAA
361GATATTGAGAATGAGAAGGGTCAGCAGGCACTCCGCCAGGGTATTGAGGGCTTTGACCAT
421ACCGCTCTGAAGAAGGCTCAGACGGCAGAGAAGAATACCCTTCCAACTAAGGAAATGATT
481GAGGAAGAGAAGAAGGCCTAA。
2. Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene as claimed in claim 1, it is characterized in that its nucleotide sequence adopts following methods to obtain: the RNA extracting Marsupenaeus japonicus Marsupenaeusjaponicus, and RNA reverse transcription is become cDNA, take cDNA as template further, obtain the sequence of SEQIDNo.1 through pcr amplification; Relevant nucleotide sequence obtains the polypeptide SEQIDNo.4 of genes encoding by prediction, then carries out Prokaryotic expression vector construction and obtains recombinant expression protein.
3. Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin, is characterized in that for Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4;
The molecule type of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4 is protein, sequence signature: length is 166aa, type is amino acid, and the sequence of described Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin Mjthm4 is as follows, is designated as SEQIDNo.4:
1MSAETPLKDLPKVDPTLKGQLEGFSAVNLKKIETEEKIHLPNKEDVAQEKQHIEHLQNIS
61EFRSERLKRTSTSEKLVLPTSQDVEAEKKAQAHLQAVEGFNAAQLKHANTQEKIVLPAKE
121DIENEKGQQALRQGIEGFDHTALKKAQTAEKNTLPTKEMIEEEKKA。
4. Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin as claimed in claim 3, is characterized in that the polypeptide of albumen for aminoacid sequence shown in SEQIDNo.4.
5. the preparation method of Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin as claimed in claim 3, is characterized in that comprising the following steps:
The step of the extraction of a Marsupenaeus japonicus Marsupenaeusjaponicus total serum IgE;
The step of Marsupenaeus japonicus MarsupenaeusjaponicuscDNA reverse transcription;
The pcr amplification of a Marsupenaeus japonicus Marsupenaeusjaponicus thymosin gene and the step of sequential analysis;
The recombinant expressed step of a Marsupenaeus japonicus Marsupenaeusjaponicus Zadaxin.
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| CN103361356A (en) | 2013-10-23 |
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