CN1837368A - Gene recombine plasmid of pralichthys olivaceus 'whole fish' lysozyme - Google Patents
Gene recombine plasmid of pralichthys olivaceus 'whole fish' lysozyme Download PDFInfo
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- CN1837368A CN1837368A CN 200510046100 CN200510046100A CN1837368A CN 1837368 A CN1837368 A CN 1837368A CN 200510046100 CN200510046100 CN 200510046100 CN 200510046100 A CN200510046100 A CN 200510046100A CN 1837368 A CN1837368 A CN 1837368A
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Abstract
The invention discloses a Bothide 'whole fish' lisozyma gene recombination plasmid, which is characterized by the following: possessing SEQ ID NO.1 base sequence in the sequence list; strengthening the fish immune ability and resistant ability for Bothide pathogeny; improving the product; cloning the fish itself gene to guarantee the safety; applying in the aquaculture for gene-transfer fish.
Description
Technical field
The present invention relates to marine organisms pralichthys olivaceus ' whole fish ' lysozyme gene, the specifically clone of lefteye flounder c type lysozyme gene and the structure of expression plasmid, especially a kind of pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid.
Background technology
N,O-Diacetylmuramidase (Lysozyme) is a kind of lytic enzyme that acts on microorganism wall specially, because of it has bacteriolysis, so the called after N,O-Diacetylmuramidase.N,O-Diacetylmuramidase is as one of non-specific immunity (innate immunity) material of fish, plays an important role in the fish body is resisted the forefront defense mechanisms of infectious pathogenic bacterium.Existing known, N,O-Diacetylmuramidase be a kind ofly be antibiosis, by lymph corpuscle excretory solvability enzyme. and mainly act on gram-positive microorganism (G ' bacterium) and some specific bacterium; One of its main mode is the mucopolysaccharide in the dissolution of bacteria cell wall.In the fish body, N,O-Diacetylmuramidase mainly is present in blood, muscle and the Lymphoid tissue; Represent the lysozyme activity of certain non-specific immunity level, even more important in mammalian body in fish body internal ratio, because the specific immunity of fish (acquired immunity) system imperfection also.Abroad about the existing further investigation of fish N,O-Diacetylmuramidase, make the lysozyme gene donor as more rainbow trouts of selection lysozyme such as BGrinde, in the fish-egg of the less kind of lysozyme content (as Atlantic salmon and cod), inject lysozyme gene, improve the immune state of these fish from heredity.
Experiment showed, that N,O-Diacetylmuramidase has participated in the nonspecific defense process of serum, netcher in 1973 and Sia4cki in 1987 etc. find in Pangasiidae fish and cyprinid fish that respectively the content of N,O-Diacetylmuramidase and activity all significantly improve after immunity.N,O-Diacetylmuramidase also can react on the cell walls of bacterium or other cause of diseases with chitinase one, more effectively attacks cause of disease.
Summary of the invention
The object of the present invention is to provide a kind of security good, can strengthen the pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid of Paralichthys olivaceus to the cause of disease resistivity.N,O-Diacetylmuramidase derives from Paralichthys olivaceus, and name is called the pralichthys olivaceus ' whole fish ' lysozyme recombinant plasmid.
To achieve these goals, the technical solution used in the present invention is as follows:
A kind of pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid has SEQ ID NO.1 base sequence in the sequence table.
The proteins encoded of described pralichthys olivaceus ' whole fish ' lysozyme gene has the aminoacid sequence shown in the SEQ ID No.2.
The present invention has cloned the Paralichthys olivaceus lysozyme gene, is connected to then on the expression vector opAFP.
The purpose of gene of the present invention, purposes, meaning are as follows: this gene can strengthen the immunological competence of fish, molten mattress enzyme recombinant plasmid plasmid changed in the Paralichthys olivaceus (transgenosis lefteye flounder) also can strengthen its immunizing power, strengthen the resistivity of Paralichthys olivaceus, thereby improve output cause of disease.Simultaneously, consider that from the biological safety angle this plasmid is cloned the gene from fish self fully, helps genetically engineered fish and is applied in aquaculture.
Description of drawings
Fig. 1 is a relevant electrophoretogram in the building process of recombinant expression plasmid opAFP-ly; Wherein, A shows that length that RT-PCR amplifies is about the Lysozyme gene of 486bp; B shows that length that primer A, B screening and cloning opAFP-ly amplify is about the These positive bands of 692bp; C shows that the PstI enzyme cuts positive colony opAFP-ly, and what endonuclease bamhi length was about 1.8kb (Fig. 1 C) is the positive colony of forward for inserting fragment; M1, M2 are DNA size markers, and M1 is λ DNA/HindIII, and M2 is 100bp DNAladder plus;
Fig. 2 is the full fish gene of the linearizing element mode chart after EcoR I enzyme is cut; Wherein, A shows opAFPpromoter; B shows opAFP 5 ' untranslated sequence; C shows lysozyme gene; D shows opAFP 3 ' sequence;
Fig. 3 is the technological line of construction of recombinant plasmid.
Embodiment
A kind of pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid has following feature:
(1) sequence signature:
Length: 38716 base pairs; Type: Nucleotide; Chain: strand; Topological framework: linearity
(2) molecule type: DNA
(3) suppose: not
(4) antisense: not
(5) initial source: Paralichthys olivaceus (Paralichthys olivaceus).
：GGATCCCCCAGAATGAGCTGGAACATGTTGCGGGGAGAGGGAAGTCTGGGTCAGCCTGCTTGGCCTGCTGCCACCGTGACCCGACCTCAGATAAGCGGAGGAAAATGGATGGATGGATTGAATCACAGAATGTTTCTGAAGACAGATATCACCTTCGCTTCAAAGAGGTGCGCACCTGGGCAGGCACCCACACAGCCACACAAATGGCATATGAATCAACCAAGAAGACGGTTGGAACTGGTCAAAACCTTCACTATACCATGTGTGACAGTTGTTTGTCACAGTGTATAAAAGACAGGGACTTAGAGACAGAGCTCTGAGCAGCTATGAGATTGTAGTTTGGCCAGGATGCGCTTAAGACCTTTGTGATGAAAAGTTATCAAATTCGTGAGTTTTCATGGAAGAACCTTGACGTGGCGTGGTGGCCATTTTGCGTCATTCGGCATGGAAAAGGAAGTCGTTATAACTCCCAGGTACATTATCTTATCTACACAAAATGTCTAATGCATGATACTACTTAAAGCCTGAGCATATTTCAAGGCCAGCACTTTTCAATAACTCATAGGCCACCTGCTGGCAAAAGGAAATGCCACATTTTATACTTTTATTTACTCCTAGACAGTTGACCTGATCAGTCTCAAATTTGGTAAGGATAGCCTTAAGACAATGAAGATGCTTCATCAGGAATATTGTGAGTTGTCGTTGAACGTTGTTGCCGTGGCAACGCATCATTCGCCATGAAAAAGAAGCTGATGGTTCAGTGGCTTGGGATGCTCAAAAAGTCATGGAACTTTGTACATGTGTCATAATTGATGGGAAGTTGTATGGGTTTTTGGCTTGCTTGTTATAAATTGTCTCCATAGCGCCCCCTACAATATTTCAAAAGAGCAGCCCCAGTGCTACGTACATGTATGAAACTTAGTAGCCAGATGTACCATATAGAGACTTACAAAAAGGTATCTTGGCCATGCTCTCAACCGTACTGGAAGTCGGCCATTTTGATTTTTGCATAATTTTTCAATAGATTTTTGCACATTTGTAATCGCTATACTTTAACGAACTCCTCCAAGGAACTTTGTCTAATCAATTTCAAATTTTGTCAGTACAATCTCAGTACTACAGTACCAAATCTACAGTTCTGCATCTCGTAGCTGCTCAGAGGTCTGTCTCTAAGTCCCTGTCTTTTATACACTGTGACAAACAACTGTCACACATGGTATAGTGAAGGTTTTGACCAGTTCCAACCGTCTTGTTGGTTGATTCATATGCCATTCGTGTGGCTGTGTGGGTGCCTACCCAGATGCGCACCTCTTTGAAGCGAATGTGATATCTGTCTTCATAAACATTCTGTTATTAGCAAGTTCATATGAGAATGAAGGCTGTATGCAAACAGGTGCACAGTCTGTTTCTAAGCATCATGGAAAAGTACAAGCAATTTGCACAAATCATTCTGTATTTTTCCAATAGCTAACAATGTCACCGGGACATTGTGCTATTGGATAGAAGAGACCAGCTGATCTAGACAGTTGATATCATGATCAACAGCCCCAAACAACAAGTGTGCATGCGCGAGGAGTGATTGGCAGATGTATGAGAACTAAACCACTGACTGAACTTGCACTAGAGGCATCTATTTTGTCTTTTCTCATATGATGTTGGGATGGCACATGGGAGTTTTTCCCCTGTCTCAGCTTGCTTTTTACCCCAAATATTGTATATCTATTAGAACCGTTGTCACAGGGTTCAAATTAACGTTTTAGTTTAGTTTTGATCATGATATACACATTTTATCCGTAAAGCATGTGCATATACAGTAAGGGCTTGTTATTCGACAGCAAGAAGAAGAGGATATGTGTGCAGGCAGTCAGCTAATGCATGGATCACAAGTTATAGAATGCAAGCTTGTGATAGTTTGGACAAAAACAAGTTATACTTTACTTATAAGAATATAAAATTTCCATTGCAATTGGCATAAGGAGGTGTGACACAGTGACCTACTTTCAGGCCAATAGGAAACGGGATATGCCGGTTAAGTCCTCCCACATACTGTATATTAGATGCAGCACATGGACCTGTCCTGTCAGAAGTCTCAGCTACAGCTTTCACTTCGATCCAGATCTTTTCACTTCGATCTCCGATAATTAATTAATTAATTAATTATTAATTAATTAAGTCTCAGCCACCGTTGACGATT
AATCAGAGAAACATC
ATGAGGACTCTGGTGGTTCTGCTCCTCGTGGCTGTGGCC AACGCTCGAGTCTACGAACGCTGTGAATGGGCCCGACTGCTGAGAAA TCAGGGGATGGATGGTTACCGTGGCATCAGTCTGGCTAACTGGGTTTG TCTGAGTGAGTGGGAGTCACGCTACAACACCAGAGCCACCAACCACA ACACTGATGGATCCACTGACTACGGCATCTTCCAGATCAACAGTCGCT GGTGGTGTAACGACAGCCAGACGCCCACTTCTAACGCCTGTAACATC AGATGCAGTGAGCTTCTGACTGATGATGTCATTGTGGCGATCAAATGT GCCAAAAGAGTGGTCCGGGATCCAAACGGGATCGGAGCCTGGGTGG CGTGGCGTCAGCACTGTCAGGGCCAGGACCTGTCGTCCTATCTGGCA GGATGTGGTCTGTAAAAGCAACAT
AATCTCTAGAGGATCCCCAACTGAACATGTCAAAACCTGTGGAGACTGTTGAGATTTGATGTTCTGAAAAGATAAAGCCTATAAATAAAATGTTGCCCAAATTTCCTGCCTGATGTTTTTCTTTGTCTTTGCTACATGGCTTTGCTGCTCGGATCGGCTCACTCTGTGTATGCCACGTTCACTTTGTACTCTCCTTCTCACGGTAGGTTTATTATTTTTAGATGTGCAGTTAGTTTCTGTGAAATAACACACCACACACTGATATTGTCTGTGCATTGACTTGGTGAGTGCACATTGTTTTTGATCTTGACATATTTATATTTGATTGATCAGGTGAACTGTGTGAATCTAAAGTGCTCCATACAGATGTTCTGCATTGAAAATATTCTCATTTTATTAGTGGAAGTGAGTGTATGCCACATCCAATCAATTTCAGCAAACACCCCAGTATGATTTAATGCAAAAAAATGAAGGTATCAAACACGCATTACTACTTTGCAGTTAAATATTTAACATTTATTCCAACACGAAAAAAAGCAGTAAATAACACTTTGACAAACACGTCAGGACATCTTATTTTTGTCACCCTCACAGGCAATTTAGTATAATATATTATATATATATATATATCATATAATAATATTCAGTATAATATATATATATATATCATATTATAATATTCAGTATAATATAAAACACAAACACATATATGTATAATATAATATAACATTTTTATTTATTGAGATGCCTCTATGGACCGTGTTATAAGAAGTAAAGATCAGGAGAAGTAAACATGAAGTGTAATTATGAATACTGATGTTAAATTAAGCTATGATGAGTTTTCACTGTTAATTTACCATCTCAATTAAATGTTGATGCCTCCATGACCAAGTTAAGCAGATGAGACTGAGACAACTGTAGAAGACAAGATGTTCACTTTGCTGAATATAGCTGGCTTGACAGTTATCTATGACTCTATAAATATATATATATTTTTTTTTTTATAAAATGATTTATTTATAACTATATATCCATTTCTCAGACAGGTGCTTCATATCCCTCACTCCCGTAGCTGTCCATGCTGGATCTGTCCCCGTTGTTTTTAAAAAGCTAAATAAGTTATTAACATGACTGCATCCAGCGAGCCAAACCTGTCTGGTGTACAGCTACCAGAGAAGCTT
In sequence, the part of band underscore is represented the coding region, and its sequence is:
ATGAGGACTCTGGTGGTTCTGCTCCTCGTGGCTGTGGCCAACGCT CGAGTCTACGAACGCTGTGAATGGGCCCGACTGCTGAGAAATCAGGG GATGGATGGTTACCGTGGCATCAGTCTGGCTAACTGGGTTTGTCTGAG TGAGTGGGAGTCACGCTACAACACCAGAGCCACCAACCACAACACT GATGGATCCACTGACTACGGCATCTTCCAGATCAACAGTCGCTGGTGG TGTAACGACAGCCAGACGCCCACTTCTAACGCCTGTAACATCAGATGC AGTGAGCTTCTGACTGATGATGTCATTGTGGCGATCAAATGTGCCAAA AGAGTGGTCCGGGATCCAAACGGGATCGGAGCCTGGGTGGCGTGGCG TCAGCACTGTCAGGGCCAGGACCTGTCGTCCTATCTGGCAGGATGTG GTBe total to 426bp, the position on former sequence is: 2230-2655.Its encoded protein matter sequence is: MRTLVVLLLVAVANARVYERCEWARLLRNQGMDGYRGISLANWVCLSEWESRYNTR ATNHNTDGSTDYGIFQINSRWWCNDSQTPTSNACNIRCSELLTDDVIVAIKCAKRV VRDPNGIGAWVAWRQHCQGQDLSSYLAGCG, totally 142 bases.Two double underlines partly are the primer of RT-PCR, and the part between two is the product of RT-PCR, base pair 2195-2690,496bp altogether.
Its concrete preparation process is as follows:
The separation of 1 lefteye flounder c type lysozyme gene
A.RNA extracts: utilize lefteye flounder peripheral blood (back blood drawing) mononuclearcell at 15 monthly ages to extract the source as RNA.With cell according to being not less than 1: 10 (weight g: volume ml) add TRIZOL reagent (TRIROL
TMTest kit is available from LIFE TECHNOLOGY company), after milling with grinding rod on ice, temperature was bathed 5 minutes under the room temperature, the ratio adding chloroform that adds 0.2 milliliter chloroform then according to every milliliter of TRIZOL, thermal agitation 15 seconds, room temperature was placed 3 minutes then, and 4 ℃ of 12000g centrifugal 15 minutes then; The water intaking phase, the ratio that adds 0.5 milliliter of Virahol according to every milliliter of TRIZOL reagent adds Virahol, and the bath of room temperature temperature is 10 minutes behind the mixing, and 4 ℃ of 12000g centrifugal 10 minutes then; Abandon supernatant, the ethanol with 75% (every milliliter of TRIZOL with 1 milliliter 75% ethanol) washing precipitation, 4 ℃ of 7500g centrifugal 5 minutes then, abandon supernatant, and the natural sedimentation drying precipitates with the water dissolution of no Rnase;
B.RT-PCR design of primers:, design a pair of RT-PCR primer: P15 ' gtagctgcagctgtggagac, P2 5 ' aatcaccggagatgtttcag according to lefteye flounder (Paralichthys olivaceus) c type N,O-Diacetylmuramidase cDNA sequence; P1 5 ' end band Pst I restriction enzyme site;
C.RT-PCR (the RT-PCR test kit is available from Promega company): do not have at 10 μ l and to add 2 μ gRNA and 1 μ g primer in the sterilized water of Rnase, 70 ℃ of water-baths 5 minutes are placed on cooled on ice then immediately.Add reagent according to following order then:
M-MLV 5 * Reaction Buffer 5 μ l, dATP 10mM 1.25 μ l, dCTP 10mM1.25 μ l, dGTP 10mM 1.25 μ l, dTTP 10mM 1.25 μ l, rRNasinRibonuclease Inhibitor 25units, M-MLV RT 200units; Sterilized water to the 25 μ l that adds no Rnase, PCR is in 42 ℃ of water-baths 60 minutes behind the mixing gently then; The PCR reaction parameter is 95 ℃ of sex change 30s, 54 ℃ of annealing 30s, and 72 ℃ are extended 45s; Circulate 30 times, 72 ℃ are extended 10min.
2 make up " full fish " element genes
A.RT-PCR product purification: use test kit glue to reclaim (in a small amount) test kit (GelExtraction Mini Kit (Watson Biotechnologies) Shanghai China Shun biotechnology company limited, W5211) purified product.
Product carried out 1% agarose electrophoresis, cut purpose segment blob of viscose, S1 liquid (Shanghai China Shun biotechnology company limited in the test kit that the back adding triploid of weighing amasss, W5211), 50 ℃ of water-baths 10 minutes are melted blob of viscose fully, add and the isopyknic Virahol of S1 liquid, 50 ℃ of water-baths 1 minute, with liquid be transferred to adsorption column (Shanghai China Shun's biotechnology company limited, W5211) in, centrifugal 30 seconds of 12000rpm, abandon from fluid, add the W1 liquid Shanghai China Shun biotechnology company limited in the 500 microlitre test kits, W5211), centrifugal 15 seconds of 12000rpm, abandon from fluid, add 500 microlitre W1 liquid, room temperature left standstill 1 minute, centrifugal 15 seconds of 12000rpm, abandon from fluid centrifugal 1 minute of 12000rpm; Adsorption column is transferred in the 1.5 milliliters clean centrifuge tube, and (Shanghai China Shun biotechnology company limited, W5211) in the post center, room temperature was placed 1 minute, centrifugal 1 minute of 12000rpm, subzero 20 ℃ of preservations to add T1 liquid in the 30 microlitre test kits.
B. reclaim being connected of product and carrier: reclaim product and PMD-T carrier at T
4Room temperature connects 3 hours under the effect of dna ligase (takara); The ligation system is as follows: reclaim product 3 μ l; PMD-T carrier 1 μ l; 10 * connection damping fluid, 1 μ l; Sterilized water 5 μ l; Cumulative volume 10 μ l.
C. connect the conversion of product: add 10 microlitres and connect among product to the 100 microlitre competent cell DH-5 α, placed on ice 35 minutes, 42 ℃ of water-baths 45 seconds, put on ice 2 minutes, add 500 microlitre LB liquid nutrient mediums (peptone: 1% yeast powder 0.5%NaCl 0.5%), cultivated 50 minutes for 37 ℃, be laid on the LB flat board that contains penbritin that adds 10 μ l IPTG (isopropyl-) and 50 μ l X-gal (5-bromo-4 chloro-3-indoles-β-D-galactosides) 37 ℃ of grow overnight.
D. the detection of converted product: the picking single bacterium colony of white (positive colony) is inoculated in the LB liquid nutrient medium that contains 50 μ l/ml penbritins, and 37 ℃ of following 220rpm shaking culture are spent the night; 5000rpm collected thalline in 5 minutes, and PCR method detects determines positive colony PMD-T-ly, and delivered to rich inferior order-checking.
The a small amount of of e.PMD-T-ly is extracted: the picking single bacterium colony of white (positive colony) is inoculated in 2.5 milliliters of LB liquid nutrient mediums that contain 50 μ l/ml penbritins, and 37 ℃ of following 220rpm shaking culture are spent the night; 5000rpm 5 minutes collects thalline, abandons supernatant, adds 200 microlitre solution 1, and the thermal agitation mixing adds 200 microlitre solution 2, mixing gently, and room temperature was placed 5 minutes, added 200 microlitre solution 3, placed on ice behind the mixing 5 minutes; 12000rpm is centrifugal 5 minutes then; Shift supernatant liquid to 1.5 milliliters clean centrifuge tube, add 600 microlitre Virahols, placed on ice behind the mixing 5 minutes; 12000rpm is centrifugal 5 minutes then; Clean Virahol suction, add 300 microlitres, 70% ethanol, centrifugal 5 minutes of vibration back 12000rpm, the ethanol suction is clean, be dissolved in the 40 microlitre sterilized waters after air-dry.
Wherein: solution 1:50mMol/L glucose, 25mMol/LTrisCl (PH8.0), 10mMol/LEDTA (PH8.0);
Solution 2:0.2MolNaOH, 1%SDS; Solution 3:5Mol/L;
The double digestion of f.PMD-T-ly 2 and the single endonuclease digestion of opAFP-V: with Sma I and 37 ℃ of double digestion PMD-T-ly of HindII (Sangon company) (extracting product in a small amount) 2 hours, with 37 ℃ of single endonuclease digestion expression vector opAFP-V of Hpa I (Sangon company) 2 hours, its reaction system was as follows respectively:
The double digestion reaction system of PMD-T-ly 2: Buffer Y
+1 μ l, DDW 4 μ l, SmaI 1 μ l, HindII 1 μ l, dna profiling 3 μ l, the reaction system of totally 10 μ l.The single endonuclease digestion reaction system of opAFP-V: Buffer Y
+1 μ l, DDW 5 μ l, HpaI 1 μ l, dna profiling 3 μ l, the reaction system of totally 10 μ l.
G. enzyme is cut (the same 2-a of method) behind the product purification, with ly gene fragment and linearizing expression vector opAFP-V at T
4Terminal connection 3 hours is put down in dna ligase (takara) effect room temperature down, and transforms 100 μ l competence DH-5 α, is laid on the LB flat board that contains penbritin that adds 10 μ l IPTG and 50 μ l X-gal 37 ℃ of following overnight incubation.
H. the detection of converted product: the picking single bacterium colony of white (positive colony) is inoculated in the LB liquid nutrient medium that contains penbritin 37 ℃ of shaken overnight; 5000rpm collected thalline in 5 minutes, and PCR method detects determines positive colony opAFP-ly, order-checking (the same 2-d of method).Sequencing result shows that this plasmid construction is correct.
PCR detects primer A, B:, A5 ' atctcaacagtctccacaggt;
B5’tctgctgatgccagtcttact。
The PCR reaction parameter is 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, and 72 ℃ are extended 60s; Circulate 30 times, 72 ℃ are extended 10min.
The full fish of lefteye flounder
SEQUENCE?LISTING
＜110〉Institute of Oceanology of the Chinese Academy of Sciences
＜120〉the molten mattress enzyme gene recombination plasmid of a kind of lefteye flounder " full fish "
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>3871
<212>DNA
＜213〉Paralichthys olivaceus (Paralichthys olivaceus)
<220>
<221>CDS
<222>(2230)..(2655)
<223>
<400>1
ggatccccca?gaatgagctg?gaacatgttg?cggggagagg?gaagtctggg?tcagcctgct 60
tggcctgctg?ccaccgtgac?ccgacctcag?ataagcggag?gaaaatggat?ggatggattg 120
aatcacagaa?tgtttctgaa?gacagatatc?accttcgctt?caaagaggtg?cgcacctggg 180
caggcaccca?cacagccaca?caaatggcat?atgaatcaac?caagaagacg?gttggaactg 240
gtcaaaacct?tcactatacc?atgtgtgaca?gttgtttgtc?acagtgtata?aaagacaggg 300
acttagagac?agagctctga?gcagctatga?gattgtagtt?tggccaggat?gcgcttaaga 360
cctttgtgat?gaaaagttat?caaattcgtg?agttttcatg?gaagaacctt?gacgtggcgt 420
ggtggccatt?ttgcgtcatt?cggcatggaa?aaggaagtcg?ttataactcc?caggtacatt 480
atcttatcta?cacaaaatgt?ctaatgcatg?atactactta?aagcctgagc?atatttcaag 540
gccagcactt?ttcaataact?cataggccac?ctgctggcaa?aaggaaatgc?cacattttat 600
acttttattt?actcctagac?agttgacctg?atcagtctca?aatttggtaa?ggatagcctt 660
aagacaatga?agatgcttca?tcaggaatat?tgtgagttgt?cgttgaacgt?tgttgccgtg 720
gcaacgcatc?attcgccatg?aaaaagaagc?tgatggttca?gtggcttggg?atgctcaaaa 780
agtcatggaa?ctttgtacat?gtgtcataat?tgatgggaag?ttgtatgggt?ttttggcttg 840
cttgttataa?attgtctcca?tagcgccccc?tacaatattt?caaaagagca?gccccagtgc 900
tacgtacatg?tatgaaactt?agtagccaga?tgtaccatat?agagacttac?aaaaaggtat 960
cttggccatg?ctctcaaccg?tactggaagt?cggccatttt?gatttttgca?taatttttca 1020
atagattttt?gcacatttgt?aatcgctata?ctttaacgaa?ctcctccaag?gaactttgtc 1080
taatcaattt?caaattttgt?cagtacaatc?tcagtactac?agtaccaaat?ctacagttct 1140
gcatctcgta?gctgctcaga?ggtctgtctc?taagtccctg?tcttttatac?actgtgacaa 1200
acaactgtca?cacatggtat?agtgaaggtt?ttgaccagtt?ccaaccgtct?tgttggttga 1260
ttcatatgcc?attcgtgtgg?ctgtgtgggt?gcctacccag?atgcgcacct?ctttgaagcg 1320
aatgtgatat?ctgtcttcat?aaacattctg?ttattagcaa?gttcatatga?gaatgaaggc 1380
tgtatgcaaa?caggtgcaca?gtctgtttct?aagcatcatg?gaaaagtaca?agcaatttgc 1440
acaaatcatt?ctgtattttt?ccaatagcta?acaatgtcac?cgggacattg?tgctattgga 1500
tagaagagac?cagctgatct?agacagttga?tatcatgatc?aacagcccca?aacaacaagt 1560
gtgcatgcgc?gaggagtgat?tggcagatgt?atgagaacta?aaccactgac?tgaacttgca 1620
ctagaggcat?ctattttgtc?ttttctcata?tgatgttggg?atggcacatg?ggagtttttc 1680
ccctgtctca?gcttgctttt?taccccaaat?attgtatatc?tattagaacc?gttgtcacag 1740
ggttcaaatt?aacgttttag?tttagttttg?atcatgatat?acacatttta?tccgtaaagc 1800
atgtgcatat?acagtaaggg?cttgttattc?gacagcaaga?agaagaggat?atgtgtgcag 1860
gcagtcagct?aatgcatgga?tcacaagtta?tagaatgcaa?gcttgtgata?gtttggacaa 1920
aaacaagtta?tactttactt?ataagaatat?aaaatttcca?ttgcaattgg?cataaggagg 1980
tgtgacacag?tgacctactt?tcaggccaat?aggaaacggg?atatgccggt?taagtcctcc 2040
cacatactgt?atattagatg?cagcacatgg?acctgtcctg?tcagaagtct?cagctacagc 2100
tttcacttcg?atccagatct?tttcacttcg?atctccgata?attaattaat?taattaatta 2160
ttaattaatt?aagtctcagc?caccgttgac?gattgtagct?gcagctgtgg?agacaatcag 2220
agaaacatc?atg?agg?act?ctg?gtg?gtt?ctg?ctc?ctc?gtg?gct?gtg?gcc?aac 2271
Met?Arg?Thr?Leu?Val?Val?Leu?Leu?Leu?Val?Ala?Val?Ala?Asn
1 5 10
gct?cga?gtc?tac?gaa?cgc?tgt?gaa?tgg?gcc?cga?ctg?ctg?aga?aat?cag 2319
Ala?Arg?Val?Tyr?Glu?Arg?Cys?Glu?Trp?Ala?Arg?Leu?Leu?Arg?Asn?Gln
15 20 25 30
ggg?atg?gat?ggt?tac?cgt?ggc?atc?agt?ctg?gct?aac?tgg?gtt?tgt?ctg 2367
Gly?Met?Asp?Gly?Tyr?Arg?Gly?Ile?Ser?Leu?Ala?Asn?Trp?Val?Cys?Leu
35 40 45
agt?gag?tgg?gag?tca?cgc?tac?aac?acc?aga?gcc?acc?aac?cac?aac?act 2415
Ser?Glu?Trp?Glu?Ser?Arg?Tyr?Asn?Thr?Arg?Ala?Thr?Asn?His?Asn?Thr
50 55 60
gat?gga?tcc?act?gac?tac?ggc?atc?ttc?cag?atc?aac?agt?cgc?tgg?tgg 2463
Asp?Gly?Ser?Thr?Asp?Tyr?Gly?Ile?Phe?Gln?Ile?Asn?Ser?Arg?Trp?Trp
65 70 75
tgt?aac?gac?agc?cag?acg?ccc?act?tct?aac?gcc?tgt?aac?atc?aga?tgc 2511
Cys?Asn?Asp?Ser?Gln?Thr?Pro?Thr?Ser?Asn?Ala?Cys?Asn?Ile?Arg?Cys
80 85 90
agt?gag?ctt?ctg?act?gat?gat?gtc?att?gtg?gcg?atc?aaa?tgt?gcc?aaa 2559
Ser?Glu?Leu?Leu?Thr?Asp?Asp?Val?Ile?Val?Ala?Ile?Lys?Cys?Ala?Lys
95 100 105 110
aga?gtg?gtc?cgg?gat?cca?aac?ggg?atc?gga?gcc?tgg?gtg?gcg?tgg?cgt 2607
Arg?Val?Val?Arg?Asp?Pro?Asn?Gly?Ile?Gly?Ala?Trp?Val?Ala?Trp?Arg
115 120 125
cag?cac?tgt?cag?ggc?cag?gac?ctg?tcg?tcc?tat?ctg?gca?gga?tgt?ggt 2655
Gln?His?Cys?Gln?Gly?Gln?Asp?Leu?Ser?Ser?Tyr?Leu?Ala?Gly?Cys?Gly
130 135 140
ctgtaaaagc?aacatctgaa?acatctccgg?tgattaatct?ctagaggatc?cccaactgaa 2715
catgtcaaaa?cctgtggaga?ctgttgagat?ttgatgttct?gaaaagataa?agcctataaa 2775
taaaatgttg?cccaaatttc?ctgcctgatg?tttttctttg?tctttgctac?atggctttgc 2835
tgctcggatc?ggctcactct?gtgtatgcca?cgttcacttt?gtactctcct?tctcacggta 2895
ggtttattat?ttttagatgt?gcagttagtt?tctgtgaaat?aacacaccac?acactgatat 2955
tgtctgtgca?ttgacttggt?gagtgcacat?tgtttttgat?cttgacatat?ttatatttga 3015
ttgatcaggt?gaactgtgtg?aatctaaagt?gctccataca?gatgttctgc?attgaaaata 3075
ttctcatttt?attagtggaa?gtgagtgtat?gccacatcca?atcaatttca?gcaaacaccc 3135
cagtatgatt?taatgcaaaa?aaatgaaggt?atcaaacacg?cattactact?ttgcagttaa 3195
atatttaaca?tttattccaa?cacgaaaaaa?agcagtaaat?aacactttga?caaacacgtc 3255
aggacatctt?atttttgtca?ccctcacagg?caatttagta?taatatatta?tatatatata 3315
tatatcatat?aataatattc?agtataatat?atatatatat?atcatattat?aatattcagt 3375
ataatataaa?acacaaacac?atatatgtat?aatataatat?aacattttta?tttattgaga 3435
tgcctctatg?gaccgtgtta?taagaagtaa?agatcaggag?aagtaaacat?gaagtgtaat 3495
tatgaatact?gatgttaaat?taagctatga?tgagttttca?ctgttaattt?accatctcaa 3555
ttaaatgttg?atgcctccat?gaccaagtaa?agcagatgag?actgagacaa?ctgtagaaga 3615
caagatgttc?actttgctga?atatagctgg?cttgacagtt?atctatgact?ctataaatat 3675
atatatattt?ttttttttat?aaaatgattt?atttataact?atatatccat?ttctcagaca 3735
ggtgcttcat?atccctcact?cccgtagctg?tccatgctgg?atctgtcccc?gttgttttta 3795
aaaagctaaa?taagttatta?acatgactgc?atccagcgag?ccaaacctgt?ctggtgtaca 3855
gctaccagag?aagctt 3871
<210>2
<211>142
<212>PRT
＜213〉Paralichthys olivaceus (Paralichthys olivaceus)
<400>2
Met?Arg?Thr?Leu?Val?Val?Leu?Leu?Leu?Val?Ala?Val?Ala?Asn?Ala?Arg
1 5 10 15
Val?Tyr?Glu?Arg?Cys?Glu?Trp?Ala?Arg?Leu?Leu?Arg?Asn?Gln?Gly?Met
20 25 30
Asp?Gly?Tyr?Arg?Gly?Ile?Ser?Leu?Ala?Asn?Trp?Val?Cys?Leu?Ser?Glu
35 40 45
Trp?Glu?Ser?Arg?Tyr?Asn?Thr?Arg?Ala?Thr?Asn?His?Asn?Thr?Asp?Gly
50 55 60
Ser?Thr?Asp?Tyr?Gly?Ile?Phe?Gln?Ile?Asn?Ser?Arg?Trp?Trp?Cys?Asn
65 70 75 80
Asp?Ser?Gln?Thr?Pro?Thr?Ser?Asn?Ala?Cys?Asn?Ile?Arg?Cys?Ser?Glu
85 90 95
Leu?Leu?Thr?Asp?Asp?Val?Ile?Val?Ala?Ile?Lys?Cys?Ala?Lys?Arg?Val
100 105 110
Val?Arg?Asp?Pro?Asn?Gly?Ile?Gly?Ala?Trp?Val?Ala?Trp?Arg?Gln?His
115 120 125
Cys?Gln?Gly?Gln?Asp?Leu?Ser?Ser?Tyr?Leu?Ala?Gly?Cys?Gly
130 135 140
Claims (3)
1. a pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid is characterized in that: have the 2195-2690 base sequence in the SEQ ID NO.1 base sequence in the sequence table.
2. a pralichthys olivaceus ' whole fish ' lysozyme gene recombination plasmid is characterized in that: have SEQ ID NO.1 base sequence in the sequence table.
3. the proteins encoded of the described pralichthys olivaceus ' whole fish ' lysozyme gene of claim 1 is characterized in that: have the aminoacid sequence shown in the SEQ ID No.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510046100 CN1837368A (en) | 2005-03-25 | 2005-03-25 | Gene recombine plasmid of pralichthys olivaceus 'whole fish' lysozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510046100 CN1837368A (en) | 2005-03-25 | 2005-03-25 | Gene recombine plasmid of pralichthys olivaceus 'whole fish' lysozyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1837368A true CN1837368A (en) | 2006-09-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510046100 Pending CN1837368A (en) | 2005-03-25 | 2005-03-25 | Gene recombine plasmid of pralichthys olivaceus 'whole fish' lysozyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1837368A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952788A (en) * | 2012-09-11 | 2013-03-06 | 中国科学院海洋研究所 | Turbot C type lysozyme and building and application method thereof |
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2005
- 2005-03-25 CN CN 200510046100 patent/CN1837368A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952788A (en) * | 2012-09-11 | 2013-03-06 | 中国科学院海洋研究所 | Turbot C type lysozyme and building and application method thereof |
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