CN102240399B

CN102240399B - Application of siniperca chuatsi ISKNV (Infectious Spleen and Kidney Necrosis Virus) ORF093 protein

Info

Publication number: CN102240399B
Application number: CN 201110191339
Authority: CN
Inventors: 付小哲; 吴淑勤; 李宁求; 石存斌; 潘厚军
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2011-07-09
Filing date: 2011-07-09
Publication date: 2013-06-19
Anticipated expiration: 2031-07-09
Also published as: CN102240399A

Abstract

The invention discloses application of a siniperca chuatsi ISKNV (Infectious Spleen and Kidney Necrosis Virus) ORF093 protein in preparing a vaccine or a medicament for preventing a siniperca chuatsi ISKNV disease. The siniperca chuatsi ISKNV ORF093 protein plays a better immunoprophylaxis role on a siniperca chuatsi iridescent virus disease, has protection ratio exceeding 45 percent and can be developed into a new vaccine or a new medicament for resisting the siniperca chuatsi ISKNV disease.

Description

The application of mandarin fish infectious spleen and kidney necrosis virus (ISKNV) ORF093 albumen

Technical field

The invention belongs to gene engineering technology field, be specifically related to the application of mandarin fish infectious spleen and kidney necrosis virus (ISKNV) ORF093 albumen.

Background technology

Mandarin fish ( Siniperca chuatsi) be one of important fresh-water fishes Special Breed kind of China, by infectious spleen and kidney necrosis virus (Infectious spleen and kidney necrosis virus, ISKNV) the Siniperca chuatsi iridescent virus disease that causes is the main epidemic disease of Siniperca chuatsi cultivation, cause the mandarin fish mortality rate near 100%, become the Main Bottleneck of restriction mandarin fish aquaculture development.For a long time, never have effective medicine for the control of mandarin fish virosis, and the disease preventing and treating drawback such as antibiotic, chemical medicine is numerous, as cause of disease drug resistance, environmental pollution, food-safety problem etc.Therefore as the disease control measure that meet environmental friendliness and the strategy of sustainable development, the biological preparation such as vaccine, antiserum are just becoming the standard production standard of international modern culture fishery and the forward position hot fields of research and development.Therefore, development has strategic importance with development mandarin fish infectious spleen and kidney necrosis virus disease vaccine and treatment preparation to control Siniperca chuatsi iridescent virus disease, and seeking new vaccine candidate antigen and antiviral serum is one of emphasis of current Siniperca chuatsi iridescent virus disease research.

Infectious spleen and kidney necrosis virus (ISKNV) is the representative species that Iridoviridae enlargement cell belongs to, be the entozoic tool cyst membrane of Cytoplasm icosahedron viruses, diameter is 150nm, contain double-stranded DNA, genome is by 111,362 base compositions comprise 125 prediction opening code-reading frame ORF(open reading frames), Siniperca chuatsi is its main infection host.Control at present viral disease and mainly contain two kinds of means: vaccination and use antiviral drugs.Aspect mandarin fish iridescent virus disease vaccine research, the employing deactivation tissue milk vaccines such as Pan Houjun are prevented and treated it, and are better in indoor and field experiment effect, but due to the vaccine material source in sick mandarin fish viscera tissue, be subjected to season and quantitative restriction, apply certain difficulty.Be not subjected to the restriction of above-mentioned condition due to recombinant vaccine, become a desirable method of development mandarin fish ISKNV vaccine.2004, Zhang Min etc. have carried out ISKNV master's capsid protein (Major capsid protein, MCP) prokaryotic expression, adopt the method for the immune marking to determine that tentatively restructuring MCP albumen has identical antigenic characteristic with ISKNV MCP albumen, for the development of ISKNV recombinant subunit vaccine provides thinking.2009, Fu Xiaozhe etc. carried out preliminary study to the immunogenicity of restructuring MCP albumen, found that restructuring MCP albumen has immunogenicity preferably, can excite mandarin fish specific immunity and nonspecific immune response.Aspect other iridescent virus disease vaccine developments; the research worker of Japan is developed anti-red-sea bream iridovirus (RSIV) cell inactivated vaccine; immune protective rate more than 75%, is the unique vaccine of successfully commercially producing and using the fishes virus disease of Asia.The Japanology personnel also are studied RSIV nucleic acid vaccine (ORF380R, ORF 569R), and result shows that recombinant dna vaccine can produce the immunoprotection of 45 ~ 60% left and right.Park etc. have built oplegnathus fasciatus irido virus (RBIV) nucleic acid vaccine according to ORF055, and result shows that immune snapper serum has certain neutralization to virus on the BF-2 cell, but there is no the immunoprotection data.There is no correlational study aspect the antiviral drugs such as antiserum.

Up to the present, also do not have to occur with the research report of ISKNV ORF093 gene as vaccine precursor and antiserum prepd thereof.

Summary of the invention

The invention provides the application of mandarin fish infectious spleen and kidney necrosis virus (ISKNV) ORF093 albumen in preparation prevention mandarin fish infectious spleen and kidney necrosis virus disease vaccine and medicine.

The technical solution adopted in the present invention is as follows:

The application of mandarin fish infectious spleen and kidney necrosis virus (ISKNV) ORF093 albumen in preparation prevention mandarin fish infectious spleen and kidney necrosis virus disease vaccine or medicine.

Preferably, the aminoacid sequence of described (ISKNV) ORF093 albumen is as shown in SEQ ID NO:2.

The present invention has following beneficial effect:

Mandarin fish ISKNV ORF093 albumen has immunoprophylactic effect preferably to the mandarin fish iridescent virus disease, and the antiserum that is prepared by the ORF093 recombiant protein also has neutralization preferably to the mandarin fish iridescent virus disease.The ORF093 recombinant dna vaccine immunity Siniperca chuatsi of preparation is after 30 days, and protective rate surpasses 45%, and the rabbit anti-serum that is prepared by the ORF093 recombiant protein also has stronger neutralization to ISKNV, and neutralizer injection Siniperca chuatsi is after 28 days, and relative survival rate surpasses 56%.Therefore, mandarin fish ISKNV ORF093 albumen can be developed becomes the sick novel vaccine of anti-Siniperca chuatsi infectious spleen and kidney necrosis virus or novel drugs.

Description of drawings

Fig. 1 is the pcr amplification figure of ISKNV ORF093 gene, and wherein M is maker, and 1 is ISKNV ORF093 gene;

Fig. 2 is the expression figure that SDS-PAGE analyzes ISKNV ORF093 recombination fusion protein, and wherein 1 is albumen marker, and 2 is pET32a (+) zero load, and 3 is the abduction delivering supernatant, and 4 is thalline before abduction delivering, and 5,6 is bacterial protein after abduction delivering;

Fig. 3 is the purification figure that SDS-PAGE analyzes ISKNV ORF093 recombination fusion protein, and wherein 1 is albumen marker, and 2 is pET32a (+) zero load, and 3 is the recombiant protein after purification, and 4 for expressing the recombiant protein inclusion body;

Fig. 4 is the western-bloting analysis chart of recombiant plasmid pET32a-093 expression product, and wherein 1 is pET32a (+) zero load, and 2 is the ORF093 recombiant protein, and 3 for dying in advance albumen marker;

Fig. 5 is that immunodotting detects ISKNV figure, and wherein 1 is the mandarin fish brain cell, and 2 is the ISKNV of cell culture.

The specific embodiment

The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.Experiment material used in following embodiment if no special instructions, is to buy from routine biochemistry reagent company and obtains.% in following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1

The prokaryotic expression of 1 mandarin fish ISKNV ORF093 recombiant protein in escherichia coli

1.1 according to infectious spleen and kidney necrosis virus in GenBank (ISKNV) [NC_003494] ORF093 sequence (SEQ ID NO:1) design primer, and introduce restriction enzyme site (following underlining).Primer sequence is as follows:

P1:[5`-CG GAATTCATGAATAAAACGC-3'] (SEQ ID NO:3), introduce EcoR I restriction enzyme site;

P2:[5'-CGG AAGCTTTTAAACATGGCGTC-3'] (SEQ ID NO:4), introduce Hind III restriction enzyme site.

1.2 carry out pcr amplification from the mandarin fish ISKNV genome of preserving as template take this laboratory, 25 μ L PCR reaction systems comprise: 10 * buffer(contains Mg2+) 2.5 μ L, 4 * dNTP(10mmol/L) 0.5 μ L, each 0.5 μ L of upstream and downstream primer (20 μ mol/L), Taq archaeal dna polymerase (5U/ μ L) 0.3 μ L, DNA 1.0 μ L, aseptic double-distilled water is supplied.PCR reaction condition: 94 ℃ of denaturation 4min; 94 ℃ of degeneration 45S, 50 ℃ of renaturation 45S, 72 ℃ are extended 30S, 30 circulations; 72 ℃ are extended 10min.The PCR product is after 1% agarose gel electrophoresis detects, and result shows, bright band occurs in the 900bp left and right, and clip size and the expection 927bp (see figure 1) that substantially conforms to is carried out glue with the purpose product and reclaimed purification, delivers to order-checking company and checks order, and sequence is seen SEQ ID NO:1.

1.3 with the ISKNV ORF093 PCR product after purification and the prokaryotic expression plasmid pET32a(+ of extraction), carry out double digestion with Hind III and EcoR I respectively.PET32a(+) enzyme action system is: 10 * NEB Buffer, 5 μ L, 100 * BSA, 0.5 μ L, EcoR I (20U/ μ L) 1 μ L, Hind III (20U/ μ L) 1 μ L, pET32a(+) 15 μ L(7 μ g/ μ L), the sterilization distilled water is supplied 50 μ L.ORF093 PCR product enzyme action system is: 10 * NEB Buffer, 5 μ L, 100 * BSA, 0.5 μ L, EcoR I (20U/ μ L) 1 μ L, Hind III (20U/ μ L) 1 μ L, ORF09340 μ L(2.5 μ g/ μ L), the sterilization distilled water is supplied 50 μ L.The enzyme action product is carried out glue reclaim purification.

1.4 the PCR product after purification is connected with pET32a+) 16 ℃ of connections of plasmid spend the night, linked system is: pET32a (+) (9ng/ μ L) 3.0 μ L, ORF093 5.0 μ L(7ng/ μ L), T4 DNA ligase (5U/ μ L) 1.0 μ L, 2 * Rapid Ligation Buffer, 1.0 μ L.

1.5 coupled reaction liquid 5 μ L are added 100 μ L DH5 α competent cell (Takara, Japan) in, the rotating centrifugal pipe is with mixing, after putting ice bath 30min gently, competent cell is placed in 42 ℃ of water-bath heat shock 90S, then forwards fast cooling 3min in ice bath to.Add the LB culture medium of 900 μ L sterilizations in each centrifuge tube, mixing is placed on 37 ℃ of shaking table shaken cultivation 45min(rotating speed 200rpm/min again).Draw the competent cell coating LB dull and stereotyped (containing amp 50 μ g/mL) that 100 μ L have transformed, 37 ℃ just putting cultivate 30min after, be inverted and cultivate 16h.

1.6 the picking white colony is inoculated in 3mL and adds Amp(final concentration 50 μ g/mL) the LB fluid medium in, 37 ℃ of 200rpm overnight incubation.Draw 1mL bacterium liquid and change in the 1.5mL centrifuge tube, deliver to the order-checking of order-checking company, identify whether recombinant expression carrier pET32a-ORF093 correctly builds, and sequence is seen SEQ ID NO:1.

1.7 draw to contain and identify that correct pET32a-ORF093 DH5 α bacterium liquid 30 μ L are added in the LB fluid medium that 3 mL contain Amp (final concentration 50 μ g/mL), 37 ℃, 200rpm overnight incubation.Adopt plasmid extraction kit (OMEGA, USA) to extract recombiant plasmid pET32a-ORF093.Get 2 μ L recombiant plasmid and add in 100 μ L BL21 (DE3) competent cells (Takara, Japan) and transform, the subsequent experimental method is seen step 1.5.

1.8 picking white colony, being inoculated in 3ml and adding Amp(final concentration 50 μ g/ mL) in the LB culture medium, 37 ℃ of concussions are spent the night, and the inoculum concentration by 1% again is inoculated in and contains that to continue to be cultured to the OD value in the Amp culture medium be 0.6, adding the IPTG(final concentration is 1mmol/L), 37 ℃ of abduction delivering 4h.

Carry out the SDS-PAGE analysis 1.9 collect thalline.The separation gel of preparation 15% and 5% concentrated glue.Draw 20 μ l bacterium liquid, add equal-volume 2 * SDS-PAGE sample-loading buffer, 100 ℃ of water-bath 5min after mixing draw 20 μ l samples and add in the point sample hole, the 120V electrophoresis, stop electrophoresis when bromophenol blue indicator reaches bottom margin, gel is placed in the coomassie brilliant blue staining liquid of newly joining, dyeing 1h reclaims coomassie brilliant blue staining liquid, add under the destaining solution room temperature and shake decolouring, until background color is sloughed fully.Result is presented at approximately the 36KD place a specific expressed band, the (see figure 2) that conforms to the expection size, and its aminoacid sequence is seen SEQ ID NO:2.

1.10 fusion rotein is according to Bugbuster His Bind Purfification Kit(NOVAGEN, Canada) description carries out purification.

The bag filter 1.11 the protein solution that purification is good is packed into is dialysed with

dialysis solution

1,2,3,4,5 for 4 ℃ successively, and dialysis time is followed successively by 2h, 2h, 3h, 3h, 15h(spends the night).The good protein solution of dialysing again concentrates with PEG20000.The sample solution that sucking-off is dialysed, the centrifugal 10min of 12000rpm gets 10 μ l supernatants and carries out SDS-PAGE analysis (see figure 3), and method is seen step 1.9.

Dialysis solution 1:50 mM Tris-Cl (pH8.5), 4 mM Urea, 5mM EDTA,

5mM-mercaptoethanol, 20% glycerol;

Dialysis solution 2:50 mM Tris-Cl (pH8.5), 2 mM Urea, 2.5mM EDTA,

2.5mM-mercaptoethanol, 20% glycerol;

Dialysis solution 3:50 mM Tris-Cl (pH8.5), 1 mM Urea, 1mM EDTA,

1mM-mercaptoethanol, 20% glycerol;

Dialysis solution 4:50 mM Tris-Cl (pH8.5), 0.5 mM Urea, 5mM EDTA,

0.5mM-mercaptoethanol, 20% glycerol;

Dialysis solution 5:50 mM Tris-Cl (pH8.5), 20% glycerol.

The preparation of 2 anti-restructuring ORF093 fusion rotein polyclonal antibodies

2.1 the ORF093 recombiant protein concentration after renaturation is adjusted into 2mg/mL, fully emulsified with isopyknic Freund's complete adjuvant.

2.2 get 2 of New Zealand white rabbit, an injection ORF093 recombiant protein, an injection PBS(pH7.4).Before injection, get 1ml blood from ear vein and prepare serum, in contrast.

2.3 first immunisation adopts subcutaneous multi-point injection mode to carry out, every injection 2ml.Immunity was carried out booster immunization after 15 days, adopted incomplete Freunds adjuvant emulsifying during booster immunization, and immunizing dose is 2/3 of initial immunity.Later on every 7 days booster immunizations once, immunizing dose is 2/3 of initial immunity.Get blood 1ml by ear vein before each booster immunization, ELISA detects antibody titer.

2.4 when reaching required tiring wait tiring, cut off the blood-letting of rabbit carotid artery.The 50mL centrifuge tube is collected whole blood, and the standing 1h of room temperature after it solidifies, places 4 ℃ of refrigerator overnight.4 ℃ of centrifugal 10min of lower 5000rmp, getting supernatant is serum.Add 0.05% sodium azide in serum ,-20 ℃ of preservations after the tubule packing.

3 Diagnosis of Sghistosomiasis are scored and are analysed anti-restructuring ORF093 fusion rotein polyclonal antibody

3.1 the restructuring ORF093 fusion protein sample after purification is carried out the SDS-PAGE electrophoresis, and method is seen step 1.9.

3.2 after electrophoresis is complete, the protein delivery that separates on gel to nitrocellulose filter (NC film), is shifted 4h under the electric current of 80mA, transfer case is observed in Ponceaux dyeing.

3.3 with the potential binding site of the irrelevant albumen of confining liquid sealing that contains 5% defatted milk powder, the room temperature shaking table is hatched 1h.

3.4 abandon confining liquid, add immediately anti-restructuring ORF093 fusion rotein polyclonal antibody and new confining liquid (1:100), hatch 1h for 4 ℃.

3.5 shift confining liquid, with PBS rinsing 3 times, 10min/ time.Use at last the TBS rinsing 1 time, 10min.

Be diluted in (5% defatted milk powder, 50mM Tris-Cl, pH7.5 in the 10mL lysate 3.6 get the goat anti-rabbit igg (H+L) of 2 μ L Radix Cochleariae officinalis enzyme labellings, 150mM NaCl) (1:5000), film is soaked in this liquid, gets rid of bubble in bag, then airtight bag mouth, jog on shaking table, 37 ℃ of 1h.

3.7 remove horseradish peroxidase solution, TBS rinsing 3 times, 10min/ time.Film after rinsing moves into clean culture dish, adds substrate solution (DAB) and the H of 0.1mL/cm2 by membrane area ₂O ₂(the 10mL substrate solution adds 10 μ L 30%H to mixed liquor ₂O ₂Liquid uses after mixing immediately).

3.8 the room temperature jog examines the color producing reaction of protein band, in case the color depth of protein band reaches requirement (approximately 2-3min), i.e. cessation reaction is transferred to filter membrane in the culture dish of dress PBS in water rinsing a little.The result demonstration, antiserum can react with recombiant protein, and demonstrates the protein band (see figure 4) of a corresponding size.

4. the anti-restructuring of IDA ORF093 fusion rotein polyclonal antibody

4.1 the mandarin fish brain cell is passed bottle to 25cm ²Tissue Culture Flask, after forming until cell the monolayer converge, then the culture medium in the sucking-off culture bottle adds the approximately ISKNV virus filtrate of 1ml, 27 ℃ approximately 1h carry out viruses adsorption.Supernatant is infected in sucking-off, adds cell maintenance medium (L-15 adds 5% FBS), puts 27 ℃ of cell culture incubators and cultivates 5 days.

4.2 with the cell culture of ISKNV, after repeatedly freezing molten 3 times, 40000rpm/min is centrifugal, precipitation is also processed through same with TE buffer dissolving, the mandarin fish brain cell that does not infect.

4.3 the set point of 5 μ L dissolvings on nitrocellulose filter, is established the mandarin fish brain cell and is done negative control.60 ℃ of oven dry 1h, with the confining liquid sealing that contains 5% defatted milk powder, the room temperature shaking table is hatched 1h.Operation later on is all identical with immunoblotting assay, and method is seen step 3.5 ~ 3.8.Result shows, the cell sample place of ISKNV has obvious speckle to occur, and the mandarin fish brain cell contrast (see figure 5) that is speckless illustrate that restructuring ORF093 antiserum can react with ISKNV, and the ORF093 that recombinates has the antigenic characteristic similar to ISKNV ORF093.

5. mandarin fish ISKNV and anti-restructuring ORF093 fusion rotein polyclonal antibody neutralization test

5.1 experiment Siniperca chuatsi (available from three, lotus pool town, Fushan City, Guangdong Province plant), weight 40-50g supported for 2 weeks in inflation and flowing water culture pond temporarily.Therefrom random picking 20 tails, observe parasite, liver separation of bacterial, PCR detection ISKNV total negative through microscopy, confirms to be used for testing after health.27～31 ℃ of experimental session water temperatures, the forage fiss (Cirrhina molitorella) of throwing something and feeding every day changes the forage fiss of throwing something and feeding a time every 3 days into after counteracting toxic substances.

5.2 get Siniperca chuatsi spleen and head-kidney with typical mandarin fish virosis symptom, after PCR detects as the ISKNV positive, add the homogenate of PBS ice bath with the 1:10 mass volume ratio, 4 ℃ of 4 centrifugal 30min of 000r/min, get supernatant through 0.22 μ m filtering with microporous membrane degerming, add penicillin and streptomycin to 1000 UI/ml, preserve in 4 ℃ of refrigerators.Get viral filtrate and be inoculated on the TA culture medium, cultivation temperature is 30 ℃, and after 48h, on the observation culture medium, without bacterial growth, this viral filtrate is used for neutralization test.

Be divided at random 3 experimental grouies 5.3 will test Siniperca chuatsi, be respectively 093 group, positive controls, negative control group, every group of 30 tails, each group is established a parallel control group.The anti-restructuring of the rabbit for preparing ORF093 fusion rotein serum is mixed in the 1:1 ratio with viral filtrate, and 4 ℃ of placements are spent the night (about 12 hours), 093 group of fish body of lumbar injection next day; Positive controls lumbar injection virus filtrate; Negative control group lumbar injection PBS(pH7.4).The volume injected of each group is 200 μ L/ tails.Observed 28 days, and recorded incidence every day.Result shows (seeing Table 1); after 28 days; average accumulated mortality rate of each group of 093 group, positive controls, negative control group is respectively 43.3%, 100%, 0; the relative protection ratio of 093 group is 56.7%; the anti-restructuring ORF093 fusion rotein polyclonal antibody that preparation is described has neutralization preferably to ISKNV, can be used for preparing the pharmaceutical preparation of control mandarin fish iridescent virus disease.

Embodiment 2

1. the structure of ISKNV ORF093 carrier for expression of eukaryon

P3:[5 '-AT GGTACCA TGGTTTCGAGTGCACTGCT-3 '] (SEQ ID NO:5), introduce KpnThe I restriction enzyme site;

P4:[5 '-AT GAATTCTTAGGCCATAATGCCGCG-3 '] (SEQ ID NO:6), introduce EcoR I restriction enzyme site.

1.2 carry out pcr amplification take pET32a-ORF 093 plasmid as template, 25 μ L PCR reaction systems comprise: 10 * buffer(contains Mg2+) 2.5 μ L, 4 * dNTP(10mmol/L) 0.5 μ L, each 0.5 μ L of upstream and downstream primer (20 μ mol/L), Taq archaeal dna polymerase (5U/ μ L) 0.3 μ L, DNA 1.0 μ L, aseptic double-distilled water is supplied.PCR reaction condition: 94 ℃ of denaturation 4min; 94 ℃ of degeneration 45S, 68 ℃ of renaturation 45S, 72 ℃ are extended 30S, 30 circulations; 72 ℃ are extended 10min.The PCR product is after 1% agarose gel electrophoresis detects, and result shows, bright band occurs in the 900bp left and right, and clip size and the expection 927bp (see figure 1) that substantially conforms to is carried out glue with the purpose product and reclaimed purification, delivers to order-checking company and checks order.Sequence is seen SEQ ID NO:1.

1.3 with the ISKNV ORF093 PCR product after purification and the eukaryon expression plasmid pcDNA3.1+ of extraction, use respectively KpnI and EcoR I are carried out double digestion.The enzyme action system of pcDNA3.1+ is: 10 * NEB Buffer, 5 μ L, 100 * BSA, 0.5 μ L, EcoR I (20U/ μ L) 1 μ L, Hind III (20U/ μ L) 1 μ L, pcDNA3.1+ 20 μ L(5 μ g/ μ L), the sterilization distilled water is supplied 50 μ L.The enzyme action system of ORF093PCR product is: 10 * NEB Buffer, 5 μ L, 100 * BSA, 0.5 μ L, EcoR I (20U/ μ L) 1 μ L, Hind III (20U/ μ L) 1 μ L, ORF093 10 μ L(10 μ g/ μ L), the sterilization distilled water is supplied 50 μ L.The enzyme action product is carried out glue reclaim purification.

1.4 a ℃ connection that the PCR product after purification is connected with the pcDNA3.1+ plasmid is spent the night, linked system is: pcDNA3.1+ 5 μ L (10ng/ μ L), ORF093 8 μ L(10ng/ μ L), T4 DNA ligase (5U/ μ L) 2 μ L, 2 * Rapid Ligation Buffer, 2.0 μ L.

1.5 coupled reaction liquid 10 μ L are added 100 μ L DH5 α competent cell (Takara, Japan) in, the rotating centrifugal pipe is with mixing, after putting ice bath 30min gently, competent cell is placed in 42 ℃ of water-bath heat shock 90S, then forwards fast cooling 3min in ice bath to.Add the LB culture medium of 900 μ L sterilizations in each centrifuge tube, mixing is placed on 37 ℃ of shaking table shaken cultivation 45min(rotating speed 200rpm/min again).Draw the competent cell coating LB dull and stereotyped (containing amp 50 μ g/mL) that 100 μ L have transformed, 37 ℃ just putting cultivate 30min after, be inverted and cultivate 16h.

1.6 the picking white colony is inoculated in 3mL and adds Amp(final concentration 50 μ g/mL) the LB fluid medium in, 37 ℃ of 200rpm overnight incubation.Draw 1mL bacterium liquid and change in the 1.5mL centrifuge tube, deliver to the order-checking of order-checking company, identify whether recombinant expression carrier pcDNA093 correctly builds.Sequence is seen SEQ ID NO:1.

2. ISKNV ORF093 nucleic acid vaccine immunity protection test

2.1 embodiment 1 step 5.1 is seen in the preparation of experiment fish.

Be divided at random 2 experimental grouies 2.2 will test Siniperca chuatsi, be respectively 093 group, matched group, every group of 30 tails.Wherein 093 group of dorsal muscles is injected pcDNA093(20 μ g/ tail), the injection of matched group dorsal muscles Tris-Cl(50mmol/L, pH8.5).The volume injected of each group is 50 μ L/ tails.

2.3 the virus liquid preparation method is seen embodiment 1 step 5.2.

2.4 rear 30 days of immunity, dorsal muscles injecting virus liquid 100 μ L, Continuous Observation 15d, morning and evening every day observed and recorded is respectively organized the death condition of fish.Get the spleen of dead fish, the PCR detection that kidney carries out ISKNV, liver,spleen,kidney, brain are carried out antibacterial separate, with definite cause of the death, and calculate relative immunity protective rate (Relative percentage survival, RPS).Result shows (seeing Table 2), and the relative protection ratio of 093 group is 45.3%, illustrates that ORF093 albumen has immunogenicity preferably, can be used as the candidate antigens of preparation mandarin fish irido virus disease vaccine.

＜110〉China's Pearl River Fishery Research Institute of Aquatic Science Research Institute

＜120〉application of mandarin fish infectious spleen and kidney necrosis virus (ISKNV) ORF093 albumen

<130>

<160> 6

<170> PatentIn version 3.5

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＜213〉infectious spleen and kidney necrosis virus (Infectious spleen and kidney necrosis virus, ISKNV)

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acgtacctgc caaagataca ccgcggacct cgaccgcagc catcgagtac actgaggcca 120

caacgagtgc atgtcgatat ggtggcagaa gctaagagag tggcggctgt ggaccatgtc 180

gcactccaac accccaacta cgccaaggtg tctgtgcaca cagaagccag gccgactgtg 240

acactggacg tgctggccac tgtggacatt gagctgcctg gacgtaacgg catcctcctt 300

ggtgatcgcg ctacagtaca cggcggcatt cccgtgacat cgcatggaaa taaacccttt 360

acggatggcg tcaagtttgc cgacattgga caccagctca ctgcggggca tgaggccagt 420

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gtcatgccag gtagccgcat tacccccgtc ggcaccggcg ttgtgtacac tgctccagac 540

ggtgtgcgtc gagcctccca ccgatcttcg ctgggcgcgg ccaagtactt gtctgacaat 600

gccgttgcga cgggccacag tgcacccggc gtggtgagcc gcaacacggt gaagaggccg 660

gaccatgtca agacgtctat agccatgata gggcggacaa acacgtctag ggtagcggca 720

ccatcagaca gcaatcgcat cggacaagct acacagccgc atgtacgggc gtatacacag 780

caaccaacac gggccgacgc gcacaccatg cccacaaccg gctcaatgca cactgataca 840

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ttcgagggac gcggcattat ggcctaa 927

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Claims

1. the application of mandarin fish infectious spleen and kidney necrosis virus ORF093 albumen in preparation prevention mandarin fish infectious spleen and kidney necrosis virus disease vaccine or medicine, the aminoacid sequence of described mandarin fish infectious spleen and kidney necrosis virus ORF093 albumen is as shown in SEQ ID NO:2.