CN1569899A - Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses - Google Patents
Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses Download PDFInfo
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Abstract
The invention relates to a recombinant nerve putrescence virus protein vaccine, the nucleic acid sequence coding the recombinant protein, and the use of the recombinant protein in preparing fish use vaccine product for nerve putrescence viruses resistance. Wherein the cDNA of the capsid nucleocapsid for the grouper nerve putrescence viruses is utilized to obtain the polypeptide recombinant proteins through DNA recombination process and pronucleus expression method.
Description
Technical field
The present invention relates to the nucleotide sequence of a kind of nervous necrosis virus recombinant protein vaccine, this recombinant protein of encoding, and this recombinant protein the preparation fish with the application in the anti-nervous necrosis virus vaccine product.
Background technology
Cabrilla is one of famous and precious seafood fish renowned in the world, stablizes, costs an arm and a leg in the selling market.The cabrilla cultivation density increases in recent years, and disease is serious day by day.Wherein, (viral nervous necrosis is one of the great disease of serious harm fry VNN) to viral nervous necrosis, can cause great financial loss, even makes to grow seedlings to produce and be forced to stop.Dayawan Aquatic Products Experimental Center, Guangdong Prov. began to carry out the test of Epinephelus coioide artificial breeding in 1999, break out viral nervous necrosis between nursery stages in 2000, during only first seedling of May was produced, dead Epinephelus coioide prelarva reached 6,000,000 tails, and direct economic loss reaches more than 200 ten thousand yuan.This disease takes place once more between the 2001-2002 nursery stage, and the fry mortality ratio reaches more than 90%.
Fish viral nervous necrosis (viral nervous necrosis, VNN) be to infect and a kind of transmissible disease of causing by the kind that the β promise da virus of promise da virus section (Nodaviridae) belongs to (Betanodaviruses), the young postlarva of seawater fish is easily infected, infect mortality ratio up to more than 90%, even 100%.At present, except that Africa, other each continents all have seawater fish to be subjected to the relevant report of this virus infection.Infected fish are many, kind surplus the seawater fish of the ill toxicity nervous necrosis of having reported reaches 40.Promise da virus section comprises two genus: β promise da virus belongs to and α promise da virus belongs to (alphanodaviruses), and β promise da virus owner will infect fish, and α promise da virus owner wants infected insect.The promise da virus be a class individuality tiny, be icosahedron, no togavirus, viral genome is made up of the positive chain RNA of two strands, the 5 ' end of two RNA all has a cap sequence, 3 ' end does not have poly (A) tail.Small pieces RNA2 size is generally 1.3-1.4kb, the precursor protein of main capsid protein (MCP) that encode; Big segment RNA1 size is 3.0-3.2kb, and proteins encoded A, albumin A are the composition units of the RNA polymerase (RdRp) of viral dependenc RNA.
Phenomenons such as that swimming often appears in the fish that suffers from nervous necrosis is inharmonious, spirrillum swimming, blind, body colour are unusual, appetite decline, poor growth.Also there is not specific medicament to treat to this virus disease at present.Reducing and prevent the generation of VNN, at first is to select health, virus-free parent population, fish-egg, fry to grow seedlings to produce and culture; Next is to strengthen management, and carries out every disinfection; In addition, development cost is low, the high antiviral vaccine of tiring will be one of this sick most effectual way of control.Numerous research datas show, though being classes, fish hang down the vertebrates of waiting, but it still has more perfect immunity system, and the head-kidney of Teleostei, kidney, spleen, thymus gland, gastral Lymphoid tissue, blood and lymph are major organs and the tissues that produces immunne response.More substantial evidence, fish have similar mammiferous T lymphocyte and bone-marrow-derived lymphocyte.Therefore, in theory, it is feasible that the application vaccine carries out immunoprophylaxis to fishes infectious disease.
Summary of the invention
The purpose of this invention is to provide a kind of nervous necrosis virus recombinant protein vaccine and encoding sequence thereof, and this recombinant protein vaccine the preparation fish with the application in the anti-nervous necrosis virus vaccine product.
The present invention has cloned the cDNA of Epinephelus coioide nervous necrosis virus master capsid protein gene by reverse transcription-polymerase chain reaction (RT-PCR).Obtained to contain the recombinant protein of nervous necrosis virus master capsid protein gene encoded polypeptides by DNA recombinant technology and prokaryotic expression method.Through evidence, this recombinant protein is used for immune fish, can effectively improve the resistibility of fish body to nervous necrosis virus, is a kind of recombinant protein vaccine of anti-nervous necrosis virus, can be used for preparing the recombinant protein vaccine goods of the anti-nervous necrosis virus of fish.
Nervous necrosis virus recombinant protein vaccine of the present invention is to contain ordered list sequence＜400〉recombinant protein of 2 aminoacid sequence (i.e. Chong Zu nervous necrosis virus master capsid protein gene encoded polypeptides).Normally contain ordered list sequence＜400 by what prokaryotic expression method obtained〉fusion rotein of aminoacid sequence shown in 2; Particularly have sequence table sequence＜400〉fusion rotein of aminoacid sequence shown in 5.
The present invention also provides the polynucleotide of the above-mentioned recombinant protein vaccine of coding.These polynucleotide are to contain ordered list sequence＜400〉1 nucleotide sequence or its complementary sequence (sequence table sequence＜400〉3); Particularly sequence table sequence＜400〉4 nucleotide sequence or its complementary sequence (sequence table sequence＜400〉6).
The present invention also provides a kind of recombinant expression vector, and it contains the polynucleotide of above-mentioned code book invention recombinant protein vaccine.This expression vector can be to cut the nucleotide sequence of site insertion sequence table sequence＜400〉1 and the expression plasmid that is built at the multienzyme of prokaryotic expression carrier (for example pET32a (+)).
The present invention also provides a kind of host cell (recombinant bacterial strain) that contains above-mentioned recombinant expression vector.It can be by obtaining above-mentioned recombinant expression vector transformed host cell (for example intestinal bacteria).
Through evidence,, can effectively improve the ability of fish body opposing nervous necrosis virus, the outburst of prevention nervous necrosis viral disease with above-mentioned recombinant protein vaccine immunity fish of the present invention.Therefore, this recombinant protein vaccine can be used for preparing fish with anti-nervous necrosis virus vaccine product.
Recombinant protein vaccine of the present invention one of can be by the following method preparation:
According to the nucleotide sequence of described nervous necrosis virus master capsid protein gene (sequence table sequence＜400〉1 polynucleotide, GenBank sequence number: AF534998) design a pair of Auele Specific Primer, send out with reverse transcription-polymerase chain and should (RT-PCR) amplify required nervous necrosis virus master capsid protein gene sequence (be sequence table sequence＜400〉1 polynucleotide).Then, with this nucleotide sequence expression vector (for example pET32a) of recombinating, and the carrier transformed host cell after will recombinating (for example intestinal bacteria), carry out recombinant expressedly, extract expression product.
2. according to the nucleotide sequence of described nervous necrosis virus master capsid protein gene (sequence table sequence＜400〉1 polynucleotide), use the chemical process synthesizing ribonucleotide sequence, and add restriction enzyme site at 5 ' and 3 ' end; Then, with this nucleotide sequence expression vector (for example pET32a) of recombinating, and the carrier transformed host cell after will recombinating (for example intestinal bacteria), carry out recombinant expressedly, extract expression product.
3. according to sequence table sequence＜400〉5 aminoacid sequence, obtain required recombinant protein vaccine by chemical process is synthetic.
Recombinant protein vaccine of the present invention and nucleotide sequence thereof also have following purposes:
1. the application in aquaculture: this recombinant protein vaccine can be prepared into vaccine product, by injection, make an addition to method such as feed, is used for immune fish, the generation of prevention nervous necrosis viral disease.
2. this recombinant protein vaccine can immune animal, the preparation polyclonal antibody.This antibody can in and nervous necrosis virus, be used for prevention and treatment fish nervous necrosis viral disease.Also can detect virus infection situation in the fish body tissue by immunohistochemical method.
3. can be that antigen is set up the ELISA detection method with this recombinant protein vaccine, be used for detecting the anti-nervous necrosis virus antibody titer of fish body blood, judge that with this fish body is subjected to this virus and feels right situation.
4. according to sequence table sequence＜400〉1 or the different sections design of the polynucleotide of its complementary sequence Auele Specific Primer, can directly detect the infection conditions that other aquatic animal is subjected to nervous necrosis virus by RT-PCR.Also can be with sequence＜400 of RT-PCR amplification〉1 or the part segment of the polynucleotide of its complementary sequence carry out mark, use technology such as Southern blot, Northern blot, gene chip, microarray, in situ hybridization, original position PCR again, detect animal and be subjected to the nervous necrosis virus infection conditions.
Description of drawings
Fig. 1 is tiltedly with the pcr amplification result of lithosporic nervous necrosis virus master capsid protein gene; Wherein: the 1.PCR product; The M.DNA standard molecular weight.
Fig. 2 is the Sac I+Hind III double digestion qualification result of recombinant plasmid; Wherein: the M.DNA standard molecular weight; The double digestion of (1.pET32a+) expression vector; 2-3.pET32a-MCP the double digestion of recombinant expression vector.
Fig. 3 is a recombinant plasmid PCR qualification result; Wherein: the 1.DNA standard molecular weight; The 2-4PCR amplified production.
Fig. 4 is the SDS-PAGE and the Western-blot analytical results of total protein behind the reorganization bacterium BL21/pET32a-MCP abduction delivering; Wherein: the Western-blot of 1-3. reorganization bacterium total protein analyzes (arrow shows the recombinant protein band of expression); 4-6. the SDS-PAGE of reorganization bacterium total protein analyzes: 4. the total protein of bacterium BL21/pET32a-MCP behind the IPTG abduction delivering of recombinating; 5. negative control; 6. protein standard molecular weight.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment one: Epinephelus coioide nervous necrosis virus master capsid protein gene cDNA obtains
The inventor has designed a pair of Auele Specific Primer according to the Epinephelus coioide nervous necrosis virus genome sequence of having measured, and the main capsid protein gene of this virus is used to increase.With Epinephelus coioide nervous necrosis virus RNA is template,, its reverse transcription is become the first chain cDNA, go out double-stranded cDNA with primer amplified.
1. the first chain cDNA's is synthetic
With Epinephelus coioide nervous necrosis virus RNA (GenBank sequence number: AF534998) be template.With RNA template sex change 5min in 95 ℃ of water-baths, take out earlier, place standby rapidly on ice.Get the 0.2ml centrifuge tube, add following reagent successively:
5×buffer 2μl
DTT 1μl
10mmol/l?dNTP 1μl
20pmol/l 6bp random primer 1 μ l
Rnase inhibitor 0.2 μ l
M-MLV ThermoScript II 0.5 μ l
RNA template 1 μ l
Add the DEPC treating water to cumulative volume 10 μ l, 37 ℃ of water bath with thermostatic control reaction 50-60min, 95 ℃ of water-bath 5min termination reactions ,-20 ℃ of preservations.
Described Epinephelus coioide nervous necrosis virus RNA also can obtain by the following method: get the cerebral tissue of the Epinephelus coioide of suffering from the nervous necrosis viral disease, extract RNA with Trizol Blue (biotech firm is widely collected in Shanghai).
2. primer design
Initiation site AUG according to the viral main capsid protein gene of coding (sequence table sequence＜400〉1 polynucleotide) designs a pair of primer to termination site UAA, and primer sequence is as follows:
Upstream primer F1:5 '-TCGAGCTCATGGTACGCAAAGGT-3 '
Downstream primer R1:5 '-GCCCAAGCTTTAGTTTTCCGAGTC-3 '
The about 1017bp of primer spacing, the wherein TC of 5 ' of F1 and R1 end and the GCCC protectiveness base that is adding, and added Sac I and Hind III restriction endonuclease recognition sequence respectively.Primer can be synthetic by last sea base health biotech company.
3.PCR amplification
The amplified reaction cumulative volume is 50 μ l, wherein the template first chain cDNA (the GenBank sequence number: AF534998) 1 μ l, 100mmol/LdNTPs 1 μ l, each 1 μ l of the F1 of 20pmol/L, R1 primer, 10 * PCR buffer (contains Mg
2+) 5 μ l, pfu enzyme 0.5 μ l.Loop parameter is: 95 ℃ of 5min, and 94 ℃ of 40s, 55 ℃ of 40s, 72 ℃ of 120s, 35 circulations are extended 10min for back 72 ℃.Amplified production is identified (Fig. 1) by 0.8% agarose gel electrophoresis, and as can be seen from Fig. 1, the PCR product conforms to the purpose cDNA molecular weight size of expectation, shows to have amplified purpose cDNA.This cDNA is the gene cDNA of Epinephelus coioide nervous necrosis virus master capsid protein, and its nucleotide sequence is as sequence table sequence＜400〉shown in 1.
Embodiment two: the structure of Epinephelus coioide nervous necrosis virus master capsid protein gene recombinant expression plasmid pET32a-MCP
1.cDNA with being connected of expression vector
With Sac I and Hind III cDNA and the prokaryotic expression plasmid pET32a (+) that embodiment one obtains carried out double digestion respectively, reclaim respectively with agarose gel again.The product that reclaims carries out ligation, obtains containing the recombinant expression vector of nervous necrosis virus master capsid protein encoding sequence (sequence table sequence＜400〉1 nucleotide sequence), called after pET32a-MCP.In fact this plasmid pET32a-MCP contains ordered list sequence＜400〉4 nucleotide sequence.
The ligation system is as follows:
cDNA 2μl
pET32a 6μl
10 * connection damping fluid, 1 μ l
T
4Dna ligase 1 μ l
Above reagent is added the 0.5ml centrifuge tube successively, mix, 4 ℃ of reactions are spent the night.
2. the amplification of recombinant expression vector
(1) .CaCl
2Legal system is equipped with competence cell liquid or dull and stereotyped TG1 cell (intestinal bacteria) 37 ℃ of activation in the LA liquid nutrient medium of preserving are spent the night, and be inoculated in once more in 1% ratio and be cultured to logarithmic phase (the OD value is about about 0.6) in the LA liquid nutrient medium next day; Ice bath 30 minutes, centrifugal 3 minutes of 4 ℃, 6000rpm are abandoned supernatant, and every 1.5ml bacterium liquid adds the CaCl of the 0.1mol/L of 200 μ l precoolings
2Resuspended, centrifugal 3 minutes of 4 ℃, 6000rpm are abandoned supernatant, add the CaCl of the 0.1mol/L of 200 μ l precoolings for the second time
2Resuspended, centrifugal 3 minutes of 4 ℃, 6000rpm are abandoned supernatant, add the CaCl of 100 μ l once more
2Solution is blown and beaten suspension gently, puts 4 ℃, uses in 24 hours after 30 minutes or glycerol adding to final concentration is that 10% back is frozen in-80 ℃, uses in two months.
(2). transform to get and connect product (recombinant expression vector pET32a-MCP) 5 μ l and mix, ice bath 42 ℃ of thermal shocks 90 seconds after 30 minutes, rapid ice bath 5 minutes with competent cell 100 μ l.Add SOC liquid nutrient medium 800 μ l, 37 ℃ of slow joltings of 120-180rpm were cultivated 1 hour or 37 ℃ of water-baths were cultivated 1 hour.Get 100 μ l and evenly be coated with the LA flat board, treat that liquid is absorbed fully by flat board after, be inverted to cultivate 12-16 hour in 37 ℃.
3. the evaluation of recombinant expression vector pET32a-MCP
Double digestion is identified and is used alkaline lysis method of extracting plasmid DNA, carry out double digestion with Sac I+Hind III restriction enzyme, get an amount of endonuclease reaction liquid and DNA Marker and carry out agarose gel electrophoresis (Fig. 2) jointly, the long 5.9kb of pET32a (+) carrier, recombinant plasmid pET32a-MCP is behind Sac I and Hind III double digestion, obtaining size, to be about two segments of 1kb and 6kb consistent with expected results, shows on purpose cDNA sequence (as sequence table sequence＜400〉1 shown in) pET32a (+) expression vector of recombinating.
PCR identifies that with alkaline lysis method of extracting plasmid DNA be template, carries out pcr amplification.The condition of pcr amplification and reaction system are carried out according to 3 method among the embodiment one.After reaction finishes, get and carry out agarose gel electrophoresis (Fig. 3) in right amount, as seen from the figure, the PCR product conforms to the purpose cDNA molecular weight size of expectation, shows that recombinant plasmid pET32a-MCP contains purpose cDNA sequence (as sequence table sequence＜400〉1 shown in).
4. the sequencing of recombinant expression vector pET32a-MCP and analysis
Extract to cut with plasmid extraction kit (go up sea base health bio-engineering corporation) and identify and the plasmid DNA of PCR evaluation, utilize the T7 universal primer at pET32a (+) carrier two ends to carry out forward and reverse order-checking (sequencing reaction is finished by last sea base health bio-engineering corporation) through enzyme.Sequencing result is analyzed with biosoftwares such as DNAstar, BLAST.Confirm by analysis on cDNA sequence (as sequence table sequence＜400〉1 shown in) pET32a (+) expression vector of recombinating of Epinephelus coioide nervous necrosis virus master capsid protein gene.
Embodiment three: the expression of recombinant expression vector pET32a-MCP in intestinal bacteria
With the plasmid DNA of recombinant expression vector pET32a-MCP, transformed into escherichia coli BL21 competent cell, next day, picking transformed the single bacterium colony that grows on the flat board, was the protokaryon reorganization bacterium that contains the pET32a-MCP plasmid DNA, called after BL21/pET32a-MCP.Picking transforms the single bacterium colony 37 ℃ of activation in the LA liquid nutrient medium that grow on the flat board and spends the night, and transfer in fresh LA liquid nutrient medium in according to 1% ratio next day, is cultured to OD
600When being about 0.8 left and right sides, adding 100mmol/L IPTG is 1mmol/L to final concentration, and continuation was cultivated 2-3 hour.Collect thalline, analyze (Fig. 4) through SDS-PAGE, the reorganization bacterium has more the band that a molecular weight is about 55.3kD than empty carrier bacterium, and content is more.Further identify (Fig. 4) through Western-blot, this band can specific and anti-wolf perch nervous necrosis virus monoclonal antibody react, and has confirmed that molecular weight is about the albumen that 55.3kD is with and is the recombinant protein that contains nervous necrosis virus master capsid protein gene encoded polypeptides.Its aminoacid sequence is as sequence table sequence＜400〉shown in 5.
Embodiment four: the preparation of Epinephelus coioide nervous necrosis virus recombinant protein vaccine and as the application of vaccine
Induce reorganization bacterium BL21/pET32a-MCP to express, centrifugal collection thalline adds bacterium lysis buffer (50mmol/lTris.Cl, pH8.0; 2mmol/l EDTA) resuspended.Adding 50mg/ml N,O-Diacetylmuramidase stock solution to final concentration is 100 μ g/ml, stirs under the room temperature after 15-20 minute, and ultrasonication is to solution thickness no longer on the ice bath.12000g, 4 ℃ centrifugal 15 minutes, collecting precipitation.Add and contain 0.1%Triton X-100 lavation buffer solution (50mmol/l Tri.Cl, pH8.0; 2mmol/l EDTA) precipitation is dissolved fully, 12000g, centrifugal 15 minutes, abandons supernatant by 4 ℃.In precipitation, add 1 * PBS and make dissolving fully, 12000g, 4 ℃, centrifugal 15 minutes, abandon supernatant, washing precipitation is once as stated above to use 1 * PBS again, centrifugal collecting precipitation is recombinant protein (its aminoacid sequence is as sequence table sequence＜400〉5 shown in) more than 60% in the precipitation.Add 1 * PBS and make it to dissolve fully in the gained precipitation, be recombinant protein vaccine solution, get 10 μ l and carry out the SDS-PAGE analysis, all the other-20 ℃ frozen standby.
Can be with direct abdominal injection of recombinant protein vaccine solution or intramuscular injection immunity fish body, perhaps in recombinant protein vaccine solution, add vegetables oil and be prepared into water-in-oil emulsion injecting immune fish body, dosage is pressed per kilogram fish body weight injection 1-10mg recombinant protein vaccine, every injection in 7-15 days once, inject 2-3 time altogether.Immunity back relative survival rate can reach 40% or more than, relatively protection ratio reach 55.6% or more than.
Embodiment five: the immanoprotection action of nervous necrosis virus recombinant protein vaccine
Get healthy tiltedly band lithosporic _ fish 36 tails (mean body weight 11.25 ± 0.37g/ tail), be divided into 2 groups (A1, A2), the recombinant protein vaccine solution of A1 abdominal injection 0.1ml (containing 50-100 μ g recombinant protein vaccine approximately), the negative bacterium liquid of the same dosage of A2 abdominal injection.After the first immunisation 15 days, carry out the immunity second time, method and dosage are with for the first time.Immunity is for the third time carried out in immunity for the second time after 7 days, method and dosage are with for the first time.Immunity was for the third time attacked poison after 7 days, the viral liquid that will prepare by the amount of 300 μ l/ tails in the dorsal fin both sides muscle injection fish body of close the afterbody of fish, every side injection 150 μ l.The result is as shown in table 1.
Table 1 recombinant protein vaccine immunity Epinephelus coioide test-results
Group name individual death number accumulation hipocratic face is to the survival rate % relative protection ratio % of non-sickness rate % that falls ill
The sum rate % that dies is dead individual
A1 18 3 16.7 40 1 22.2 55.6
A2 18 5 27.8 - 4 50 -
Sequence table
＜110〉Zhongshan University
＜120〉a kind of nervous necrosis virus recombinant protein vaccine and encoding sequence and purposes
<160>6
<210>1
<211>1017
<212>cDNA
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus)
<220>
<221>CDS
<222>(1)...(1017)
<400>1
atg?gta?cgc?aaa?ggt?gag?aag?aaa?ttg?gca?aaa?ccc?gcg?acc?acc?aag 48
Met?Val?Arg?Lys?Gly?Glu?Lys?Lys?Leu?Ala?Arg?Pro?Ala?Thr?Thr?Arg
1 5 10 15
gcc?gcg?aat?ccg?caa?ccc?cgc?cga?cgt?gct?aac?aat?cgt?cgg?cgt?agt 96
Ala?Ala?Asn?Pro?Gln?Pro?Arg?Arg?Arg?Ala?Asn?Asn?Arg?Arg?Arg?Ser
20 25 30
aat?cgc?act?gac?gca?cct?gtg?tct?aag?gcc?tcg?act?gtg?act?gga?ttt 144
Asn?Arg?Thr?Asp?Ala?Pro?Val?Ser?Lys?Ala?Ser?Thr?Val?Thr?Gly?Phe
35 40 45
gga?cgt?ggg?acc?aat?gac?gtc?cat?ctc?tca?ggt?atg?tcg?aga?atc?tcc 192
Gly?Arg?Gly?Thr?Asn?Asp?Val?His?Leu?Ser?Gly?Met?Ser?Arg?Ile?Ser
50 55 60
cag?gcc?gtc?ctc?cca?gcc?ggg?aca?gga?act?gac?gga?tac?gtt?gtc?gtt 240
Gln?Ala?Val?Leu?Pro?Ala?Gly?Thr?Gly?Thr?Asp?Gly?Tyr?Val?Val?Val
65 70 75 80
gac?gca?acc?atc?gtc?ccc?gac?ctc?ctg?cca?cga?ctg?gga?cac?gct?gct 288
Asp?Ala?Thr?Ile?Val?Pro?Asp?Leu?Leu?Pro?Arg?Leu?Gly?His?Ala?Ala
85 90 95
aga?atc?ttc?cag?cga?tac?gct?gtt?gaa?aca?ctg?gag?ttt?gaa?att?cag 336
Arg?Ile?Phe?Gln?Arg?Tyr?Ala?Val?Glu?Thr?Leu?Glu?Phe?Glu?Ile?Gln
100 105 110
cca?atg?tgc?ccc?gca?aac?acg?ggc?ggt?ggt?tac?gtt?gct?ggc?ttc?ctg 384
Pro?Met?Cys?Pro?Ala?Asn?Thr?Gly?Gly?Gly?Tyr?Val?Ala?Gly?Phe?Leu
115 120 125
cct?gat?cca?act?gac?aac?gac?cac?acc?ttc?gac?gcg?ctt?caa?gca?act 422
Pro?Asp?Pro?Thr?Asp?Asn?Asn?His?Thr?Phe?Asp?Ala?Leu?Gln?Ala?Thr
130 135 140
cgt?ggt?gca?gtc?gtt?gcc?aaa?tgg?tgg?gaa?agc?aga?aca?gtc?cga?cct 480
Arg?Gly?Ala?Val?Val?Ala?Lys?Trp?Trp?Glu?Ser?Arg?Thr?Val?Arg?Pro
145 150 155 160
cag?tac?acc?cgc?acg?ctc?ctc?tgg?acc?tcg?tcg?gga?aag?gag?cag?cgt 528
Gln?Tyr?Thr?Arg?Thr?Leu?Leu?Trp?Thr?Ser?Ser?Gly?Lys?Glu?Gln?Arg
165 170 175
ctc?acg?tca?cct?ggt?cgg?ctg?ata?ctc?ctg?tgt?gtc?ggc?aac?aac?act 576
Leu?Thr?Ser?Pro?Gly?Arg?Leu?Ile?Leu?Leu?Cys?Val?Gly?Asn?Asn?Thr
180 185 190
gat?gtg?gtc?aac?gtg?tca?gtg?ctg?tgt?cgc?tgg?agt?gtt?cga?ctg?agc 624
Asp?Val?Val?Asn?Val?Ser?Val?Leu?Cys?Arg?Trp?Ser?Val?Arg?Leu?Ser
195 200 205
gtt?cca?tct?ctt?gag?aca?cct?gaa?gag?acc?acc?gct?ccc?atc?atg?aca 672
Val?Pro?Ser?Leu?Glu?Thr?Pro?Glu?Glu?Thr?Thr?Ala?Pro?Ile?Met?The
210 215 220
caa?ggt?tcc?ctg?tac?aac?gat?tcc?ctt?tcc?aca?aat?gac?ttc?aag?tcc 720
Gln?Gly?Ser?Leu?Tyr?Asn?Asp?Ser?Leu?Ser?Thr?Asn?Asp?Phe?Lys?Ser
225 230 235 240
atc?ctc?cta?gga?tcc?aca?cca?ctg?gat?att?gcc?cct?gat?gga?gca?gtc 768
Ile?Leu?Leu?Gly?Ser?Thr?Pro?Leu?Asp?Ile?Ala?Pro?Asp?Gly?Ala?Val
245 250 255
ttc?cag?ctg?gac?cgt?ccg?ctg?tcc?att?gac?tac?agc?ctt?gga?act?gga 816
Phe?Gln?Leu?Asp?Arg?Pro?Leu?Ser?Ile?Asp?Tyr?Ser?Leu?Gly?Thr?Gly
260 265 270
gat?gtt?gac?cgt?gct?gtt?tat?tgg?cac?ctc?aag?aag?ttt?gct?gga?aat 864
Asp?Val?Asp?Arg?Ala?Val?Tyr?Trp?His?Leu?Lys?Lys?Phe?Ala?Gly?Ash
275 280 285
gct?ggc?aca?cct?gca?ggc?tgg?ttt?cgc?tgg?ggc?atc?tgg?gac?aac?ttc 912
Ala?Gly?Thr?Pro?Ala?Gly?Trp?Phe?Arg?Trp?Gly?Ile?Trp?Asp?Asn?Phe
290 295 300
aac?aag?acg?ttc?aca?gat?ggc?gtt?gcc?tac?tac?tct?gat?gag?cag?ccc 960
Asn?Lys?Thr?Phe?Thr?Asp?Gly?Val?Ala?Tyr?Tyr?Ser?Asp?Glu?Gln?Pro
305 310 315 320
cgt?caa?atc?ctg?ctg?cct?gtt?ggc?act?gtc?tgc?acc?agg?gtt?gac?tcg 1008
Arg?Gln?Ile?Leu?Leu?Pro?Val?Gly?Thr?Val?Cys?Thr?Arg?Val?Asp?Ser
325 330 335
gaa?aac?taa 1017
Glu?Asn?***
<210>2
<211>338
<212>PRT
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus)
<400>2
Met?Val?Arg?Lys?Gly?Glu?Lys?Lys?Leu?Ala?Arg?Pro?Ala?Thr?Thr?Arg
1 5 10 15
Ala?Ala?Asn?Pro?Gln?Pro?Arg?Arg?Arg?Ala?Asn?Asn?Arg?Arg?Arg?Ser
20 25 30
Asn?Arg?Thr?Asp?Ala?Pro?Val?Ser?Lys?Ala?Ser?Thr?Val?Thr?Gly?Phe
35 40 45
Gly?Arg?Gly?Thr?Asn?Asp?Val?His?Leu?Ser?Gly?Met?Ser?Arg?Ile?Ser
50 55 60
Gln?Ala?Val?Leu?Pro?Ala?Gly?Thr?Gly?Thr?Asp?Gly?Tyr?Val?Val?Val
65 70 75 80
Asp?Ala?Thr?Ile?Val?Pro?Asp?Leu?Leu?Pro?Arg?Leu?Gly?His?Ala?Ala
85 90 95
Arg?Ile?Phe?Gln?Arg?Tyr?Ala?Val?Glu?Thr?Leu?Glu?Phe?Glu?Ile?Gln
100 105 110
Pro?Met?Cys?Pro?Ala?Asn?Thr?Gly?Gly?Gly?Tyr?Val?Ala?Gly?Phe?Leu
115 120 125
Pro?Asp?Pro?Thr?Asp?Asn?Asn?His?Thr?Phe?Asp?Ala?Leu?Gln?Ala?Thr
130 135 140
Arg?Gly?Ala?Val?Val?Ala?Lys?Trp?Trp?Glu?Ser?Arg?Thr?Val?Arg?Pro
145 150 155 160
Gln?Tyr?Thr?Arg?Thr?Leu?Leu?Trp?Thr?Ser?Ser?Gly?Lys?Glu?Gln?Arg
165 170 175
Leu?Thr?Ser?Pro?Gly?Arg?Leu?Ile?Leu?Leu?Cys?Val?Gly?Asn?Asn?Thr
180 185 190
Asp?Val?Val?Asn?Val?Ser?Val?Leu?Cys?Arg?Trp?Ser?Val?Arg?Leu?Ser
195 200 205
Val?Pro?Ser?Leu?Glu?Thr?Pro?Glu?Glu?Thr?Thr?Ala?Pro?Ile?Met?The
210 215 220
Gln?Gly?Ser?Leu?Tyr?Asn?Asp?Ser?Leu?Ser?Thr?Asn?Asp?Phe?Lys?Ser
225 230 235 240
Ile?Leu?Leu?Gly?Ser?Thr?Pro?Leu?Asp?Ile?Ala?Pro?Asp?Gly?Ala?Val
245 250 255
Phe?Gln?Leu?Asp?Arg?Pro?Leu?Ser?Ile?Asp?Tyr?Ser?Leu?Gly?Thr?Gly
260 265 270
Asp?Val?Asp?Arg?Ala?Val?Tyr?Trp?His?Leu?Lys?Lys?Phe?Ala?Gly?Asn
275 280 285
Ala?Gly?Thr?Pro?Ala?Gly?Trp?Phe?Arg?Trp?Gly?Ile?Trp?Asp?Asn?Phe
290 295 300
Asn?Lys?Thr?Phe?Thr?Asp?Gly?Val?Ala?Tyr?Tyr?Ser?Asp?Glu?Gln?Pro
305 310 315 320
Arg?Gln?Ile?Leu?Leu?Pro?Val?Gly?Thr?Val?Cys?Thr?Arg?Val?Asp?Ser
325 330 335
Glu?Asn?***
<210>3
<211>1017
<212>cDNA
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus)
<400>3
ttagttttcc?gagtcaaccc?tggtgcagac?agtgccaaca?ggcagcagga?tttgacgggg 60
ctgctcatca?gagtagtagg?caacgccatc?tgtgaacgtc?ttgttgaagt?tgtcccagat 120
gccccagcga?aaccagcctg?caggtgtgcc?agcatttcca?gcaaacttct?tgaggtgcca 180
ataaacagca?cggtcaacat?ctccagttcc?aaggctgtag?tcaatggaca?gcggacggtc 240
cagctggaag?actgctccat?caggggcaat?atccagtggt?gtggatccta?ggaggatgga 300
cttgaagtca?tttgtggaaa?gggaatcgtt?gtacagggaa?ccttgtgtca?tgatgggagc 360
ggtggtctct?tcaggtgtct?caagagatgg?aacgctcagt?cgaacactcc?agcgacacag 420
cactgacacg?ttgaccacat?cagtgttgtt?gccgacacac?aggagtatca?gccgaccagg 480
tgacgtgaga?cgctgctcct?ttcccgacga?ggtccagagg?agcgtgcggg?tgtactgagg 540
tcggactgtt?ctgctttccc?accatttggc?aacgactgca?ccacgagttg?cttgaagcgc 600
gtcgaaggtg?tggtcgttgt?cagttggatc?aggcaggaag?ccagcaacgt?aaccaccgcc 660
cgtgtttgcg?gggcacattg?gctgaatttc?aaactccagt?gtttcaacag?cgtatcgctg 720
gaagattcta?gcagcgtgtc?ccagtcgtgg?caggaggtcg?gggacgatgg?ttgcgtcaac 780
gacaacgtat?ccgtcagttc?ctgtcccggc?tgggaggacg?gcctgggaga?ttctcgacat 840
acctgagaga?tggacgtcat?tggtcccacg?tccaaatcca?gtcacagtcg?aggccttaga 900
cacaggtgcg?tcagtgcgat?tactacgccg?acgattgtta?gcacgtcggc?ggggttgcgg 960
attcgcggcc?ttggtggtcg?cgggttttgc?caatttcttc?tcacctttgc?gtaccat 1017
<210>4
<211>1524
<212>cDNA
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus) and pET32a (+) expression vector
<220>
<221>CDS
<222>(1)...(1524)
<400>4
atg?agc?gat?aaa?att?att?cac?ctg?act?gac?gac?agt?ttt?gac?acg?gat 48
Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?The?Asp
-170 -165 -160
gta?ctc?aaa?gcg?gac?ggg?gcg?atc?ctc?gtc?gat?ttc?tgg?gca?gag?tgg 96
Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp
-155 -150 -145
tgc?ggt?ccg?tgc?aaa?atg?atc?gcc?ccg?att?ctg?gat?gaa?atc?gct?gac 144
Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp
-140 -135 -130
gaa?tat?cag?ggc?aaa?ctg?acc?gtt?gca?aaa?ctg?aac?atc?gat?caa?aac 192
Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn
-125 -120 -115 -110
cct?ggc?act?gcg?ccg?aaa?tat?ggc?atc?cgt?ggt?atc?ccg?act?ctg?ctg 240
Pro?Gly?Thr?Ala?Pro?Asp?Tyr?Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu
-105 -100 -95 -90
ctg?ttc?aaa?aac?ggt?gaa?gtg?gcg?gca?acc?aaa?gtg?ggt?gca?ctg?tct 288
Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser
-85 -80 -75
aaa?ggt?cag?ttg?aaa?gag?ttc?ctc?gac?gct?aac?ctg?gcc?ggt?tct?ggt 336
Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp?Ala?Asn?Leu?Ala?Gly?Ser?Gly
-70 -65 -60
tct?ggc?cat?atg?cac?cat?cat?cat?cat?cat?tct?tct?ggt?ctg?gtg?cca 384
Ser?Gly?His?Met?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
-55 -50 -45
cgc?ggt?tct?ggt?atg?aaa?gaa?acc?gct?gct?gct?aaa?ttc?gaa?cgc?cag 432
Arg?Gly?Ser?Gly?Met?Lys?Glu?Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln
-40 -35 -30
cac?atg?gac?agc?cca?gat?ctg?ggt?acc?gac?gac?gac?gac?aag?gcc?atg 480
His?Met?Asp?Ser?Pro?Asp?Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Ala?Met
-25 -20 -15 -10
gct?gat?atc?gga?tcc?gaa?ttc?gag?ctc?atg?gta?cgc?aaa?ggt?gag?aag 528
Ala?Asp?Ile?Gly?Ser?Glu?Phe?Glu?Leu?Met?Val?Arg?Lys?Gly?Glu?Lys
-5 -1 1 5
aaa?ttg?gca?aaa?ccc?gcg?acc?acc?aag?gcc?gcg?aat?ccg?caa?ccc?cgc 576
Lys?Leu?Ala?Arg?Pro?Ala?Thr?Thr?Arg?Ala?Ala?Asn?Pro?Gln?Pro?Arg
10 15 20
cga?cgt?gct?aac?aat?cgt?cgg?cgt?agt?aat?cgc?act?gac?gca?cct?gtg 624
Arg?Arg?Ala?Asn?Asn?Arg?Arg?Arg?Ser?Asn?Arg?Thr?Asp?Ala?Pro?Val
25 30 35
tct?aag?gcc?tcg?act?gtg?act?gga?ttt?gga?cgt?ggg?acc?aat?gac?gtc 672
Ser?Lys?Ala?Ser?Thr?Val?Thr?Gly?Phe?Gly?Arg?Gly?Thr?Asn?Asp?Val
40 45 50 55
cat?ctc?tca?ggt?atg?tcg?aga?atc?tcc?cag?gcc?gtc?ctc?cca?gcc?ggg 720
His?Leu?Ser?Gly?Met?Ser?Arg?Ile?Ser?Gln?Ala?Val?Leu?Pro?Ala?Gly
60 65 70
aca?gga?act?gac?gga?tac?gtt?gtc?gtt?gac?gca?acc?atc?gtc?ccc?gac 768
Thr?Gly?Thr?Asp?Gly?Tyr?Val?Val?Val?Asp?Ala?Thr?Ile?Val?Pro?Asp
75 80 85
ctc?ctg?cca?cga?ctg?gga?cac?gct?gct?aga?atc?ttc?cag?cga?tac?gct 816
Leu?Leu?Pro?Arg?Leu?Gly?His?Ala?Ala?Arg?Ile?Phe?Gln?Arg?Tyr?Ala
90 95 100
gtt?gaa?aca?ctg?gag?ttt?gaa?att?cag?cca?atg?tgc?ccc?gca?aac?acg 864
Val?Glu?Thr?Leu?Glu?Phe?Glu?Ile?Gln?Pro?Met?Cys?Pro?Ala?Asn?Thr
105 110 115
ggc?ggt?ggt?tac?gtt?gct?ggc?ttc?ctg?cct?gat?cca?act?gac?aac?gac 912
Gly?Gly?Gly?Tyr?Val?Ala?Gly?Phe?Leu?Pro?Asp?Pro?Thr?Asp?Asn?Asn
120 125 130 135
cac?acc?ttc?gac?gcg?ctt?caa?gca?act?cgt?ggt?gca?gtc?gtt?gcc?aaa 960
His?Thr?Phe?Asp?Ala?Leu?Gln?Ala?Thr?Arg?Gly?Ala?Val?Val?Ala?Lys
140 145 150
tgg?tgg?gaa?agc?aga?aca?gtc?cga?cct?cag?tac?acc?cgc?acg?ctc?ctc 1008
Trp?Trp?Glu?Ser?Arg?Thr?Val?Arg?Pro?Gln?Tyr?Thr?Arg?Thr?Leu?Leu
155 160 165
tgg?acc?tcg?tcg?gga?aag?gag?cag?cgt?ctc?acg?tca?cct?ggt?cgg?ctg 1056
Trp?Thr?Ser?Ser?Gly?Lys?Glu?Gln?Arg?Leu?Thr?Ser?Pro?Gly?Arg?Leu
170 175 180
ata?ctc?ctg?tgt?gtc?ggc?aac?aac?act?gat?gtg?gtc?aac?gtg?tca?gtg 1104
Ile?Leu?Leu?Cys?Val?Gly?Asn?Asn?Thr?Asp?Val?Val?Asn?Val?Ser?Val
185 190 195
ctg?tgt?cgc?tgg?agt?gtt?cga?ctg?agc?gtt?cca?tct?ctt?gag?aca?cct 1152
Leu?Cys?Arg?Trp?Ser?Val?Arg?Leu?Ser?Val?Pro?Ser?Leu?Glu?Thr?Pro
200 205 210 215
gaa?gag?acc?acc?gct?ccc?atc?atg?aca?caa?ggt?tcc?ctg?tac?aac?gat 1200
Glu?Glu?Thr?Thr?Ala?Pro?Ile?Met?The?Gln?Gly?Ser?Leu?Tyr?Asn?Asp
220 225 230
tcc?ctt?tcc?aca?aat?gac?ttc?aag?tcc?atc?ctc?cta?gga?tcc?aca?cca 1248
Ser?Leu?Ser?Thr?Asn?Asp?Phe?Lys?Ser?Ile?Leu?Leu?Gly?Ser?Thr?Pro
235 240 245
ctg?gat?att?gcc?cct?gat?gga?gca?gtc?ttc?cag?ctg?gac?cgt?ccg?ctg 1296
Leu?Asp?Ile?Ala?Pro?Asp?Gly?Ala?Val?Phe?Gln?Leu?Asp?Arg?Pro?Leu
250 255 260
tcc?att?gac?tac?agc?ctt?gga?act?gga?gat?gtt?gac?cgt?gct?gtt?tat 1344
Ser?Ile?Asp?Tyr?Ser?Leu?Gly?Thr?Gly?Asp?Val?Asp?Arg?Ala?Val?Tyr
265 270 275
tgg?cac?ctc?aag?aag?ttt?gct?gga?aat?gct?ggc?aca?cct?gca?ggc?tgg 1392
Trp?His?Leu?Lys?Lys?Phe?Ala?Gly?Asn?Ala?Gly?Thr?Pro?Ala?Gly?Trp
280 285 290 295
ttt?cgc?tgg?ggc?atc?tgg?gac?aac?ttc?aac?aag?acg?ttc?aca?gat?ggc 1440
Phe?Arg?Trp?Gly?Ile?Trp?Asp?Asn?Phe?Asn?Lys?Thr?Phe?Thr?Asp?Gly
300 305 310
gtt?gcc?tac?tac?tct?gat?gag?cag?ccc?cgt?caa?atc?ctg?ctg?cct?gtt 1488
Val?Ala?Tyr?Tyr?Ser?Asp?Glu?Gln?Pro?Arg?Gln?Ile?Leu?Leu?Pro?Val
315 320 325
ggc?act?gtc?tgc?acc?agg?gtt?gac?tcg?gaa?aac?taa 1524
Gly?Thr?Val?Cys?Thr?Arg?Val?Asp?Ser?Glu?Asn?***
330 335
<210>5
<211>512
<212>PRT
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus) and pET32a (+) expression vector
<400>5
Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?The?Asp
-170 -165 -160
Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp
-155 -150 -145
Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp
-140 -135 -130
Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn
-125 -120 -115 -110
Pro?Gly?Thr?Ala?Pro?Asp?Tyr?Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu
-105 -100 -95 -90
Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser
-85 -80 -75
Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp?Ala?Asn?Leu?Ala?Gly?Ser?Gly
-70 -65 -60
Ser?Gly?His?Met?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
-55 -50 -45
Arg?Gly?Ser?Gly?Met?Lys?Glu?Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln
-40 -35 -30
His?Met?Asp?Ser?Pro?Asp?Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Ala?Met
-25 -20 -15 -10
Ala?Asp?Ile?Gly?Ser?Glu?Phe?Glu?Leu?Met?Val?Arg?Lys?Gly?Glu?Lys
-5 -1 1 5
Lys?Leu?Ala?Arg?Pro?Ala?Thr?Thr?Arg?Ala?Ala?Asn?Pro?Gln?Pro?Arg
10 15 20
Arg?Arg?Ala?Asn?Asn?Arg?Arg?Arg?Ser?Asn?Arg?Thr?Asp?Ala?Pro?Val
25 30 35
Ser?Lys?Ala?Ser?Thr?Val?Thr?Gly?Phe?Gly?Arg?Gly?Thr?Asn?Asp?Val
40 45 50 55
His?Leu?Ser?Gly?Met?Ser?Arg?Ile?Ser?Gln?Ala?Val?Leu?Pro?Ala?Gly
60 65 70
Thr?Gly?Thr?Asp?Gly?Tyr?Val?Val?Val?Asp?Ala?Thr?Ile?Val?Pro?Asp
75 80 85
Leu?Leu?Pro?Arg?Leu?Gly?His?Ala?Ala?Arg?Ile?Phe?Gln?Arg?Tyr?Ala
90 95 100
Val?Glu?Thr?Leu?Glu?Phe?Glu?Ile?Gln?Pro?Met?Cys?Pro?Ala?Asn?Thr
105 110 115
Gly?Gly?Gly?Tyr?Val?Ala?Gly?Phe?Leu?Pro?Asp?Pro?Thr?Asp?Asn?Asn
120 125 130 135
His?Thr?Phe?Asp?Ala?Leu?Gln?Ala?Thr?Arg?Gly?Ala?Val?Val?Ala?Lys
140 145 150
Trp?Trp?Glu?Ser?Arg?Thr?Val?Arg?Pro?Gln?Tyr?Thr?Arg?Thr?Leu?Leu
155 160 165
Trp?Thr?Ser?Ser?Gly?Lys?Glu?Gln?Arg?Leu?Thr?Ser?Pro?Gly?Arg?Leu
170 175 180
Ile?Leu?Leu?Cys?Val?Gly?Asn?Asn?Thr?Asp?Val?Val?Asn?Val?Ser?Val
185 190 195
Leu?Cys?Arg?Trp?Ser?Val?Arg?Leu?Ser?Val?Pro?Ser?Leu?Glu?Thr?Pro
200 205 210 215
Glu?Glu?Thr?Thr?Ala?Pro?Ile?Met?The?Gln?Gly?Ser?Leu?Tyr?Asn?Asp
220 225 230
Ser?Leu?Ser?Thr?Asn?Asp?Phe?Lys?Ser?Ile?Leu?Leu?Gly?Ser?Thr?Pro
235 240 245
Leu?Asp?Ile?Ala?Pro?Asp?Gly?Ala?Val?Phe?Gln?Leu?Asp?Arg?Pro?Leu
250 255 260
Ser?Ile?Asp?Tyr?Ser?Leu?Gly?Thr?Gly?Asp?Val?Asp?Arg?Ala?Val?Tyr
265 270 275
Trp?His?Leu?Lys?Lys?Phe?Ala?Gly?Asn?Ala?Gly?Thr?Pro?Ala?Gly?Trp
280 285 290 295
Phe?Arg?Trp?Gly?Ile?Trp?Asp?Asn?Phe?Asn?Lys?Thr?Phe?Thr?Asp?Gly
300 305 310
Val?Ala?Tyr?Tyr?Ser?Asp?Glu?Gln?Pro?Arg?Gln?Ile?Leu?Leu?Pro?Val
315 320 325
Gly?Thr?Val?Cys?Thr?Arg?Val?Asp?Ser?Glu?Asn?***
330 335
<210>6
<211>1524
<212>cDNA
＜213〉Epinephelus coioide nervous necrosis virus (Epinephelus coioids nervous necrosis virus) and pET32a (+) expression vector
<400>6
ttagttttcc?gagtcaaccc?tggtgcagac?agtgccaaca?ggcagcagga?tttgacgggg 60
ctgctcatca?gagtagtagg?caacgccatc?tgtgaacgtc?ttgttgaagt?tgtcccagat 120
gccccagcga?aaccagcctg?caggtgtgcc?agcatttcca?gcaaacttct?tgaggtgcca 180
ataaacagca?cggtcaacat?ctccagttcc?aaggctgtag?tcaatggaca?gcggacggtc 240
cagctggaag?actgctccat?caggggcaat?atccagtggt?gtggatccta?ggaggatgga 300
cttgaagtca?tttgtggaaa?gggaatcgtt?gtacagggaa?ccttgtgtca?tgatgggagc 360
ggtggtctct?tcaggtgtct?caagagatgg?aacgctcagt?cgaacactcc?agcgacacag 420
cactgacacg?ttgaccacat?cagtgttgtt?gccgacacac?aggagtatca?gccgaccagg 480
tgacgtgaga?cgctgctcct?ttcccgacga?ggtccagagg?agcgtgcggg?tgtactgagg 540
tcggactgtt?ctgctttccc?accatttggc?aacgactgca?ccacgagttg?cttgaagcgc 600
gtcgaaggtg?tggtcgttgt?cagttggatc?aggcaggaag?ccagcaacgt?aaccaccgcc 660
cgtgtttgcg?gggcacattg?gctgaatttc?aaactccagt?gtttcaacag?cgtatcgctg 720
gaagattcta?gcagcgtgtc?ccagtcgtgg?caggaggtcg?gggacgatgg?ttgcgtcaac 780
gacaacgtat?ccgtcagttc?ctgtcccggc?tgggaggacg?gcctgggaga?ttctcgacat 840
acctgagaga?tggacgtcat?tggtcccacg?tccaaatcca?gtcacagtcg?aggccttaga 900
cacaggtgcg?tcagtgcgat?tactacgccg?acgattgtta?gcacgtcggc?ggggttgcgg 960
attcgcggcc?ttggtggtcg?cgggttttgc?caatttcttc?tcacctttgc?gtaccatgag 1020
ctcgaattcg?gatccgatat?cagccatggc?cttgtcgtcg?tcgtcggtac?ccagatctgg 1080
gctgtccatg?tgctggcgtt?cgaatttagc?agcagcggtt?tctttcatac?cagaaccgcg 1140
tggcaccaga?ccagaagaat?gatgatgatg?atggtgcata?tggccagaac?cagaaccggc 1200
caggttagcg?tcgaggaact?ctttcaactg?acctttagac?agtgcaccca?ctttggttgc 1260
cgccacttca?ccgtttttga?acagcagcag?agtcgggata?ccacggatgc?catatttcgg 1320
cgcagtgcca?gggttttgat?cgatgttcag?ttttgcaacg?gtcagtttgc?cctgatattc 1380
gtcagcgatt?tcatccagaa?tcggggcgat?cattttgcac?ggaccgcacc?actctgccca 1440
gaaatcgacg?aggatcgccc?cgtccgcttt?gagtacatcc?gtgtcaaaac?tgtcgtcagt 1500
caggtgaata?attttatcgc?tcat 1524
Claims (10)
1. recombinant protein vaccine, it contains ordered list sequence＜400〉2 aminoacid sequence.
2. according to the described recombinant protein vaccine of claim 1, it is characterized in that it is to contain ordered list sequence＜400 by what prokaryotic expression method obtained〉fusion rotein of aminoacid sequence shown in 2.
3. according to the described recombinant protein vaccine of claim 2, it is characterized in that it has sequence table sequence＜400〉5 aminoacid sequence.
4. the polynucleotide sequence of coding claim 1,2 or 3 described recombinant protein vaccine.
5. according to the described polynucleotide sequence of claim 4, it is characterized in that it is sequence table sequence＜400〉4 nucleotide sequence or its complementary sequence.
6. recombinant expression vector, it contains claim 4 or 5 described polynucleotide sequences.
7. according to the described recombinant expression vector of claim 6, it is characterized in that it is to cut the nucleotide sequence of site insertion sequence table sequence＜400〉1 or its complementary sequence and the expression plasmid that is built at the multienzyme of prokaryotic expression carrier pET32a (+).
8. the host cell that contains claim 6 or 7 described expression vectors.
9. according to the described host cell of claim 8, it is characterized in that this host cell is intestinal bacteria.
The described recombinant protein vaccine of claim 1,2 or 3 the preparation fish with the application in the anti-nervous necrosis virus vaccine product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100271866A CN1569899A (en) | 2004-05-14 | 2004-05-14 | Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100271866A CN1569899A (en) | 2004-05-14 | 2004-05-14 | Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1569899A true CN1569899A (en) | 2005-01-26 |
Family
ID=34480913
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2004100271866A Pending CN1569899A (en) | 2004-05-14 | 2004-05-14 | Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1569899A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942496A (en) * | 2010-08-31 | 2011-01-12 | 中山大学 | Method for preparing virus analogs of nervous necrosis viruses |
| CN102367495A (en) * | 2011-12-09 | 2012-03-07 | 天津师范大学 | Specific primers and method used for detecting paralichthys olivaceus infected with beta nodavirus |
| CN104004068A (en) * | 2014-06-19 | 2014-08-27 | 天津师范大学 | Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof |
| CN104725503A (en) * | 2015-03-31 | 2015-06-24 | 华中农业大学 | Fugu rubripes nervous necrosis virus capsid protein egg yolk antibody and application thereof |
| CN106591328A (en) * | 2016-12-15 | 2017-04-26 | 斯澳生物科技（苏州）有限公司 | Preparation method and application of grouper nervous necrosis virus capsid protein |
| CN106831963A (en) * | 2016-06-29 | 2017-06-13 | 福建省水产研究所 | Grouper nervous necrosis virus Coat genes, in expression in escherichia coli method and application |
| CN108251356A (en) * | 2017-12-29 | 2018-07-06 | 中山大学 | A kind of fish-egg cleaning solution of anti-fish nervous necrosis virus |
| CN108904792A (en) * | 2018-07-19 | 2018-11-30 | 中山大学 | Using baculoviral as anti-nervous necrosis virus soaking vaccine of carrier and preparation method thereof |
| CN109355301A (en) * | 2018-08-22 | 2019-02-19 | 深圳大学 | A kind of nucleic acid sequence of the codon optimization of grouper recombination NNVcp, fusion expression vector, preparation method and itself |
| CN111777683A (en) * | 2019-04-03 | 2020-10-16 | 王大勇 | Fusion protein of nervous necrosis virus MCP and Edwardsiella ictaluri ompN1 and preparation method thereof |
-
2004
- 2004-05-14 CN CNA2004100271866A patent/CN1569899A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942496A (en) * | 2010-08-31 | 2011-01-12 | 中山大学 | Method for preparing virus analogs of nervous necrosis viruses |
| CN102367495A (en) * | 2011-12-09 | 2012-03-07 | 天津师范大学 | Specific primers and method used for detecting paralichthys olivaceus infected with beta nodavirus |
| CN104004068A (en) * | 2014-06-19 | 2014-08-27 | 天津师范大学 | Paralichthys olivaceus beta nodavirus capsid protein with immune protection function and preparing method thereof |
| CN104725503A (en) * | 2015-03-31 | 2015-06-24 | 华中农业大学 | Fugu rubripes nervous necrosis virus capsid protein egg yolk antibody and application thereof |
| CN104725503B (en) * | 2015-03-31 | 2018-08-17 | 华中农业大学 | A kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its application |
| CN106831963B (en) * | 2016-06-29 | 2019-10-11 | 福建省水产研究所 | Grouper nervous necrosis virus Coat gene, in expression in escherichia coli method and application |
| CN106831963A (en) * | 2016-06-29 | 2017-06-13 | 福建省水产研究所 | Grouper nervous necrosis virus Coat genes, in expression in escherichia coli method and application |
| CN106591328A (en) * | 2016-12-15 | 2017-04-26 | 斯澳生物科技（苏州）有限公司 | Preparation method and application of grouper nervous necrosis virus capsid protein |
| CN108251356A (en) * | 2017-12-29 | 2018-07-06 | 中山大学 | A kind of fish-egg cleaning solution of anti-fish nervous necrosis virus |
| CN108251356B (en) * | 2017-12-29 | 2021-06-22 | 中山大学 | Fish egg cleaning solution for resisting fish nervous necrosis virus |
| CN108904792A (en) * | 2018-07-19 | 2018-11-30 | 中山大学 | Using baculoviral as anti-nervous necrosis virus soaking vaccine of carrier and preparation method thereof |
| CN108904792B (en) * | 2018-07-19 | 2021-10-08 | 中山大学 | Anti-nervous necrosis virus soaking vaccine using baculovirus as carrier and preparation method thereof |
| CN109355301A (en) * | 2018-08-22 | 2019-02-19 | 深圳大学 | A kind of nucleic acid sequence of the codon optimization of grouper recombination NNVcp, fusion expression vector, preparation method and itself |
| CN111777683A (en) * | 2019-04-03 | 2020-10-16 | 王大勇 | Fusion protein of nervous necrosis virus MCP and Edwardsiella ictaluri ompN1 and preparation method thereof |
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