CN108384763A - A kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and its preparation method and application - Google Patents
A kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and its preparation method and application Download PDFInfo
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- CN108384763A CN108384763A CN201810209600.7A CN201810209600A CN108384763A CN 108384763 A CN108384763 A CN 108384763A CN 201810209600 A CN201810209600 A CN 201810209600A CN 108384763 A CN108384763 A CN 108384763A
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Abstract
The present invention provides a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and its preparation method and application, for manufactured attenuated viral strains after infectious spleen and kidney necrosis virus strain missing ORF074 genes.The present invention also provides a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and its preparation method and application with selection markers, ORF074 genes, and the manufactured attenuated viral strains after riddled basins are inserted into the position of gene delection homologous recombination have been lacked for infectious spleen and kidney necrosis virus.Infectious spleen and kidney necrosis virus ORF074 gene-deleted strains provided by the invention are a kind of recombination engineered vaccines having knocked out ORF074 genes, virulence is weaker, substantially reduce the fish morbidity lethality being immunized, significant immune effect can reach using immersion immunity, it need not inject, application value is very big.
Description
Technical field
The present invention relates to aquaculture control and prevention of disease fields, and in particular to a kind of infectious spleen and kidney necrosis virus ORF074 bases
Because of gene-deleted strain and its preparation method and application.
Background technology
Mandarin fish also known as mandarin fish, because the advantages that growth is fast, best in quality and delicious flavour becomes the main fresh water name spy in China
One of excellent cultured fishes.By infectious spleen and kidney necrosis virus (infectious spleen and kidney necrosis
Virus, ISKNV) caused by mandarin fish iridescent virus disease be mandarin fish cultivation main epidemic disease, cause the mandarin fish death rate close to 100%, become system
The about main bottleneck of mandarin fish aquaculture development.
Infectious spleen and kidney necrosis virus (ISKNV) is the representative species of enlargement Tobamovirus, and the group nucleic acid sequence of full genome is
After measured, overall length 111362bp, G+C content are 54.78%, including 124 potential open reading frame (ORF), coding length
Degree amino acid from 40 to 1208.By sequence analysis, it is main capsid protein to deduce ORF006L at present；ORF19R、
ORF46L, ORF27L and ORF63L are related with DNA replication dna, modification and processing；ORF24, ORF28L, ORF34R, ORF64L and
ORF87R is related with transcription and nucleotide metabolism；ORF111L and ORF048R is related with host's interaction；ORF12、ORF65、
ORF66, ORF99 and ORF111L encode ring finger protein.
ISKNV vaccines currently used for prevention and control infectious spleen and kidney necrosis virus are mainly cell inactivation vaccine, cell
Inactivated vaccine has many advantages, such as that safety is good, immunogenicity is strong, protective rate is high, the R＆D cycle is short, becomes most common vaccine kind
Class, but inactivated vaccine can only cause humoral immunity, type of immune response is more single, immune to hold time shorter, needs repeatedly to connect
Kind and inoculum concentration it is big the shortcomings of.Present many documents are it has been reported that gene-deletion attenuated live vaccine can effectively and safely rise
To the effect of protection, attenuated live vaccine can induce humoral immunity and cellular immunity, and type of immune response is more comprehensive, when maintenance is immunized
Between it is longer, the advantages that inoculation times are few and inoculum concentration is small as current ISKNV vaccines the new hot spot of research.And with ISKNV
ORF074 gene-deleted strains do not have been reported that as the research of gene-deletion attenuated live vaccine.
Invention content
In view of this, the present invention provides, a kind of good immune effect, easy to use, application is strong, need not injection infectiousness
Spleen and kidney necrosis virus vaccine and its preparation method and application.It is knocked out in the genome of wildness infectious spleen and kidney necrosis virus
It is viral (ISKNV △ ORF074) to construct ORF074 gene delections for ORF074 genes.Compared to the immunization ways of traditional vaccine, leaching
It is the sharpest edges of gene-deleted vaccine that bubble is immune, this is the great innovation of fishes virus vaccine immunity mode, is had wide
Application space.
First aspect present invention provides a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains, is infectious spleen
Kidney necrosis virus strain lacks manufactured attenuated viral strains after ORF074 genes.
Preferably, the ORF074 gene orders are and SEQ ID NO：Sequence shown in 1 have at least 90%, at least
95%, the nucleotide sequence of at least 98%, at least 99% or 100% homology.
Second aspect, the infectious spleen and kidney necrosis virus ORF074 gene delections with selection markers that the present invention provides a kind of
Strain has lacked ORF074 genes for infectious spleen and kidney necrosis virus, and has been inserted respectively by homologous recombination at the position of gene delection
Enter manufactured attenuated viral strains after the first screening-gene and the second screening-gene.
Preferably, the ORF074 gene orders be with sequence shown in SEQ ID NO.1 have at least 90%, at least
95%, the nucleotide sequence of at least 98%, at least 99% or 100% homology.
Preferably, first screening-gene and the second screening-gene are different, and it is red glimmering to be separately selected from insertion
Aequorin and resistance screening gene.
It is further preferred that the red fluorescent protein gene is DsRed2 protein gene.
It is further preferred that the resistance screening gene is puromycin-resistant screening-gene.
The third aspect, the present invention provides a kind of structure sides of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains
Method, including：It detaches and identifies infectious spleen and kidney necrosis virus strain；It is by the method for homologous recombination that infectious spleen renal necrosis is sick again
The ORF074 genes of strain are lacked, and infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are obtained；
Preferably, the ORF074 gene orders be with sequence shown in SEQ ID NO.1 have at least 90%, at least
95%, the nucleotide sequence of at least 98%, at least 99% or 100% homology.
Fourth aspect, the infectious spleen and kidney necrosis virus ORF074 gene delections with selection markers that the present invention provides a kind of
The construction method of strain, including：It detaches and identifies infectious spleen and kidney necrosis virus strain；Again by the method for homologous recombination by infectiousness
The ORF074 genes of spleen and kidney necrosis virus strain are lacked, and are inserted into the first screening-gene and respectively at the position of gene delection
Two screening-genes obtain infectious spleen and kidney necrosis virus ORF074 gene-deleted strains.
Preferably, the ORF074 gene orders be with sequence shown in SEQ ID NO.1 have at least 90%, at least
95%, the nucleotide sequence of at least 98%, at least 99% or 100% homology.
Preferably, first screening-gene and the second screening-gene are different, and it is red glimmering to be separately selected from insertion
Aequorin and resistance screening gene.
It is further preferred that the red fluorescent protein gene is DsRed2 protein gene.
It is further preferred that the resistance screening gene is puromycin-resistant screening-gene.
Preferably, the construction method specifically includes：
A) screening-gene is transformed
First screening-gene is cloned into the multiple cloning sites of Puc19 carriers, obtains the first Puc19 recombinant vectors；Again will
Second screening-gene is cloned into the multiple cloning sites of the first Puc19 recombinant vectors, obtains the 2nd Puc19 recombinant vectors；
B) structure of transfer vector
Recombinate the structure of ORF074 gene delection Baculovirus transfer vectors：Using ISKNV pnca gene group DNA as template, pass through PCR
Obtain the underarm gene of the upper arm gene and ORF074 of ORF074；By the underarm base of the upper arm gene of gained ORF074 and ORF074
Cause is cloned into the 2nd Puc19 recombinant vectors prepared by step a), obtains in the 3rd Puc19 recombinant vectors, gained third
In Puc19 recombinant vectors, the first screening-gene, the second screening-gene are located at the upper arm gene of ORF074 and the underarm base of ORF074
Because between；
C) homologous recombination
Mandarin fish cell is transfected using the ORF074 recombinant transfer vectors built, ISKNV virus liquids are added after transfection and attack
Poison collects the cell of morbidity, obtains the virus liquid that the ORF074 of △ containing ISKNV are mixed with wild type ISKNV；Using limiting dilution assay
Purified virus liquid obtains ISKNV △ ORF074 recombinant viruses；
It is further preferred that the step c) obtains ISKNV △ ORF074 recombinations using limiting dilution assay purifying 5-10 generations
Virus.
It is further preferred that the step c) obtains ISKNV △ ORF074 bases using limiting dilution assay purifying 5-10 generations
Because gene-deleted strain specifically includes：
By the ORF074 of △ containing ISKNV of gained and wild type ISKNV hybrid virus liquid multigelation 3-5 times, membrane filtration
Degerming dilutes the mandarin fish cell of postoperative infection health, selects while expressing the first screening-gene and the second screening-gene after the onset
Cell dilutes the mandarin fish cell of postoperative infection health, continues culture, observation；
After the onset of the cell of equal cultures, the cell of the first screening-gene and the second screening-gene is selected while being expressed, is diluted
The mandarin fish cell of postoperative infection health continues culture, observation；Repeatedly, 5-10 generations are purified using the method for limiting dilution, obtained
The ISKNV △ ORF074 gene-deleted strains of purifying.
5th aspect, the present invention provides a kind of infectious spleen and kidney necrosis virus ORF074 gene delections recombinant virus epidemic diseases
Seedling, including the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains described in first aspect or the band screening described in second aspect
The infectious spleen and kidney necrosis virus ORF074 gene-deleted strains of label.
Preferably, the infectious spleen and kidney necrosis virus ORF074 gene delection recombinant viral vaccines further include pharmaceutically connecing
Diagnosticum, supporting agent, excipient or the diluent received.
6th aspect, the present invention provides the infectious spleen and kidney necrosis virus ORF074 genes described in a kind of first aspect to lack
It loses the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers described in strain or second aspect and presses following one kind
Or a variety of methods are applied：
(1) the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are individually applied；
(2) by the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and one or more of vaccine use in conjunction；
(3) the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are thrown to cultivation water by the way of impregnating
Body is applied；
(4) the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are thrown to cultivation water by the way of feeding
Body is applied.
7th aspect, the present invention provides the infectious spleen and kidney necrosis virus ORF074 genes described in a kind of first aspect to lack
Lose infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers described in strain or second aspect prepare diagnosis,
The reagent for preventing, treating enlargement cell virus disease or the application in drug.
Preferably, the enlargement cell virus includes mandarin fish infectious spleen and kidney necrosis virus, red-sea bream iridovirus, oplegnathus fasciatus
One in irido virus, jewfish irido virus, rheum officinale fish virus, Taiwan grouper irido virus, Epinephelus coioides irido virus
Kind is a variety of.
Advantageous effect of the present invention：
The present invention constructs infectious spleen and kidney necrosis virus ORF074 gene-deleted strains, and strain by being had after purification
The recombination engineered vaccine of immunogenicity.
Recombination engineered vaccine provided by the invention is a kind of attenuated vaccine with good immunogenicity, can be induced
Fish preferably generate immune response, have the effect of immune.In addition, recombination engineered vaccine can be induced by immune fish
Effectively generate specific antibody for a long time.Secondly, other kinds of vaccine is compared, recombination engineered vaccine has immunization rate
High advantage can reach better immune effect in production application.
Importantly, recombination engineered vaccine is because having certain virology activity, it is more convenient on immunization ways,
The method that immersion immunity may be used makes virus enter in fish body from the gill or alimentary canal, plays immunogenicity, and induction fish generate
Immune response.Great amount of cost can be saved in production application.Simultaneously as the recombination engineered vaccine that the present invention is built knocks out
ORF074 genes, so virulence dies down, the fish that reducing causes to be immunized fall ill dead probability, preferably exempt to reach
Epidemic disease effect.
Recombination engineered vaccine preparation method provided by the invention is simple：On the basis of ISKNV wildness Strain,
Using DsRed2 red fluorescent proteins gene and puromycin-resistant screening-gene as selection markers, technique for gene engineering is utilized
The recombinant transfer vector containing ORF074 genetic recombination arms is built, makes recombinant transfer vector and wild-type virus using rotaring dyeing technology
Homologous recombination is carried out in mandarin fish cell, knocks out the ORF074 genes in wild type, and the virus for obtaining ISKNV △ ORF074 is outstanding
Liquid, then ISKNV △ ORF074 Strain is purified by limiting dilution assay, finally utilize cell culture technology to expand culture missing disease
Recombination engineered vaccine is made in strain, purification.
Description of the drawings
Fig. 1 is ORF074 recombinant transfer vector Puc19-RP collection of illustrative plates；
Fig. 2 is the qualification result of ORF074 recombinant transfer vectors Puc19-RP, wherein each swimming lane is respectively from left to right
1：1kb marker, 2：The double digestion qualification result of DsRed2 gene-puro genes, 3：The double digestion identification knot of ORF074 upper arm
Fruit, 4：The double digestion qualification result of ORF074 underarms；
Fig. 3 is the mandarin fish cell that IKSNV △ ORF074 infection is observed under the inverted fluorescence microscope that amplification factor is 100 times
Red light-emitting situation；
Fig. 4 is that PCR identifies that the one of recombinant virus ORF074 genes expands as a result, each swimming lane is respectively from left to right：Swimming lane
1：ISKNV MCP negative controls, swimming lane 2：ISKNV MCP positive controls, swimming lane 3：ORF074 knocks out strain MCP amplifications, swimming
Road 4：DS2000marker, swimming lane 5：ISKNV ORF074 negative controls, swimming lane 6：ISKNV ORF074 positive controls, swimming lane 7：
ORF074 knocks out strain testing result；
Fig. 5 is that PCR identifies that the two of recombinant virus ORF074 genes expands result；Each swimming lane is respectively from left to right：Swimming lane
1：DS2000marker, swimming lane 2：ISKNV ORF074 negative controls, swimming lane 3：ISKNV ORF074 positive controls, swimming lane 4：
ORF074 knocks out strain testing result；
Fig. 6 is that ORF074 gene delection recombinant viruses attack malicious survival experiment result figure, is shown compared with wild-type virus,
Gene delection recombinant virus is attacked toxicity and is substantially reduced；
Fig. 7 is that ORF074 gene delection recombinant viruses attack malicious protective rate experimental result picture, shows ORF074 gene delection weights
Group virus makes mandarin fish generate preferable immunocompetence to wild-type virus after attacking poison.
Specific implementation mode
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to normal condition.In the embodiment of the present invention unless otherwise noted, agents useful for same and consumptive material are
Commercial goods.
An embodiment of the present invention provides a kind of infectious spleen and kidney necrosis virus ORF074 gene delection recombinant viral vaccines
Preparation method includes the following steps：
1. building the pUC19-Red carriers with DsRed2 genes
Using pDsRed-Monomer-N1Vector, separately design：
Left and right primer：
RFP-F：ATAGTAATCAATTACGGGGT (SEQ ID NO.2),
RFP-R：TGATGAGTTTGGACAAACCA (SEQ ID NO.3),
Over-lap PCR (Overlap) primer：
RFP-OL-F：CAGATCCGCTAGCGCTCGCCACCATGGACAACACCG (SEQ ID NO.4),
RFP-OL-R:GTTGTCCATGGTGGCGAGCGCTAGCGGATCTGACGG (SEQ ID NO.5),
Multiple cloning sites sequence (the 591bp to 671bp in original DsRed2 carriers is deleted using Overlap technologies
Base sequence, convenient for connecting into new recombinant transfer vector).The first time amplification of Overlap PCR amplifies respectively
Two sections of sequences of DsRed2 carrier base serial numbers 9-591bp and 671-1590bp.Use the Kod Fx polymerases of TOYOBO companies
System, PCR system are specially：
| Reagent | Dosage (totally 50 μ L systems) unit：μL |
| Kod Fx DNA heat-stabilised poly synthase | 1 |
| 2×Kod Buffer | 25 |
| Sterile water | 10 |
| dNTPs | 10 |
| RFP-F/RFP-OL-R | 1.5 |
| RFP-OL-F/RFP-R | 1.5 |
| DsRed2 templates | 1 |
95 DEG C, time 30s of denaturation temperature, 50 DEG C, time 30s of annealing temperature, 62 DEG C, extension of time 30s of elongating temperature, into
30 cycles of row.
6 × DNA electrophoresis Loading Buffer of 10 μ L are added in PCR product, and mixing rear electrophoresis is identified and carries out glue recycling.
It carries out second using the recovery product that first time expands as template later to expand, second of amplification system is：
Using Touchdown PCR, 95 DEG C of denaturation temperature, first round annealing temperature declines 10 cycles from 55 DEG C -46 DEG C,
It is denaturalized 30s, anneal 30s, 62 DEG C of elongating temperature, extension of time 1 minute.25 cycles are carried out using 50 DEG C as annealing temperature again,
Denaturation, annealing, extension of time are constant.
By Overlap PCR obtain by DsRed2 base sequences 9bp to 590bp and 672bp to
6 × DNA electrophoresis Loading Buffer of 10 μ L are added in the PCR product that 1590bp is connected, and the identification of mixing rear electrophoresis is gone forward side by side
Row glue recycles.Recovery product using TAKARA Taq enzyme carry out tailings reactions, 72 DEG C 20 minutes.
Using the pmd 19T carrier systems of TAKARA, it is attached reaction, converts DH5 α competent escherichia coli cells,
Coated plate after cultivating 12-16h, chooses bacterium sequencing, clones the DsRed2 Expression elements of 1509bp sizes.
The primer RFP-K-F and RFP-B-R respectively with I restriction enzyme digestion sites of Kpn I and BamH are designed, is utilized
Kod Fx polymerase systems as before are carried as template with being connected into the T of DsRed feux rouges albumen of transformation, pass through PCR amplification
Obtain the transformation feux rouges albumen with I restriction enzyme digestion sites of Kpn I and BamH.We utilize Kpn I and BamH I later
Restriction endonuclease enzyme carries out endonuclease reaction to PCR product obtained in the previous step and Puc19 carriers respectively, and digestion system is such as
Under：
| Reagent | Dosage (totally 50 μ L systems) |
| I restriction enzyme enzymes of Kpn | 2μL |
| I restriction enzyme enzymes of BamH | 2μL |
| 10 × digestion buffer | 5μL |
| PCR product | 2μg |
| Sterile water | up to 50μL |
37 DEG C of 12h of endonuclease reaction, are used in combination T4DNA ligases to connect the Expression element in pUC19 carriers, are built into band
The pUC19-Red carriers of DsRed2.
2. building the pUC19-Red carriers of puro genes
Puro genes are inserted into pUC19-Red carriers, following overlap primers are synthesized：
It is utilized respectively rp-ol-1/rp-ol-2, rp-ol-3/rp-ol-4, rp-ol-5/rp-ol-6 primer P and goes out segment
Overlap1, overlap2, overlap3, PCR system are as follows：
| Unit：μL | |
| Template | 1 |
| Primer forward | 1.5 |
| Primer reverse | 1.5 |
| Kod Fx | 1 |
| Kod Buffer | 25 |
| ddH2O | 20 |
95 DEG C, time 30s of denaturation temperature, 55 DEG C, time 30s of annealing temperature, 68 DEG C, extension of time 1min of elongating temperature,
Carry out 30 cycles.
1% agarose gel electrophoresis identification, the Gel provided using E.D.Z.A companies are provided after the completion of PCR
Extraction Kit carry out glue recycling, obtain glue recovery product overlap1, overlap2, overlap3.
Above-mentioned glue recovery product overlap1, overlap2 is attached, overlap PCR systems are as follows：
| Unit：μL | |
| Overlap1 | 1 |
| Overlap2 | 1 |
| Primer rp-ol-1 | 1 |
| Primer rp-ol-4 | 1 |
| Kod Fx | 1 |
| Kod Buffer | 25 |
| ddH2O | 20 |
95 DEG C, time 30s of denaturation temperature, 50 DEG C, time 30s of annealing temperature, 68 DEG C, extension of time 1min of elongating temperature,
Carry out 30 cycles.
1% agarose gel electrophoresis identification, the Gel provided using E.D.Z.A companies are provided after the completion of PCR
The band of Extraction Ki recycling 1500bp or so, obtains glue recovery product overlap1/2.Similarly utilize overlap2,
Overlap3 is attached, and obtains overlap2/3.Overlap1/2, overlap2/3 are utilized into the side of overlap PCR again
Method connects to obtain overlap1/2/3.
Using Kpn1 and BamH1 restriction enzymes digestion puc19-red carriers and overlap1/2/3, digestion products are connected
It connects to obtain the recombinant transfer vector pUC19-RP being transformed, Vector map is as shown in Figure 1.
3. building ORF074 recombinant transfer vectors
The upper underarm of ORF074 is connected into carrier pUC19-RP：Use Invitrogen PureLink Viral DNA/
RNAKit extracts ISKNV DNA, is diluted to 50 μ g/mL and makees template.It is limited using band EcoR I, Kpn I and BamH I, Hind III
The primer of property endonuclease restriction enzyme site processed：
ORF074 upper arm-F：AAGCTTCTGGAACGCAAGCGGCAGTA (SEQ ID NO.12),
ORF074 upper arm-R：GGATCCTCAGACAGGTGTTTTGACCA (SEQ ID NO.13),
ORF074 underarms-F：GGTACCTAAAAGTGTTGCCGGCGTCT (SEQ ID NO.14),
ORF074 underarms-R：GAATTCAACAGTATGTCAAGCCGTGT(SEQ ID NO.15).
Upper arm (the I ISKNV of ORF074 are obtained by PCR using Kod Fx polymerase systems from the genome of ISKNV
68641bp to 69668bp in genome) and underarm (the 70683rd to 71760bp in ISKNV genomes), PCR reaction systems
It is as follows：
| Reagent | Dosage (totally 50 μ L systems) unit：μL |
| Kod Fx DNA heat-stabilised poly synthase | 1 |
| 2×Kod Buffer | 25 |
| Sterile water | 10 |
| dNTPs | 10 |
| ORF074 upper arm/underarm-F | 1.5 |
| ORF074 upper arm/underarm-R | 1.5 |
| ISKNV templates | 1 |
6 × DNA electrophoresis Loading Buffer of 10 μ L are added in PCR product, and mixing rear electrophoresis is identified and carries out glue recycling.
Recovery product uses EcoR I, Kpn I to be connected using T4 with after BamH I, III restriction endonuclease digestions of Hind recycling respectively
Enzyme connects on the Puc19-Red carriers that previous step is built, and constitutes ORF074 recombinant transfer vectors.Digestion system is as follows：
| Reagent | Dosage (totally 50 μ L systems) |
| I/BamH of EcoR, I restriction enzyme enzymes | 2μL |
| I/Hind of Kpn, III restriction enzyme enzymes | 2μL |
| 10 × digestion buffer | 5μL |
| PCR product | 2μg |
| Sterile water | up to 50μL |
Double digestion identification is carried out to recombinant transfer vector, the results are shown in Figure 2.
4. intracellular homologous recombination
Mandarin fish cell is transfected using the ORF074 recombinant transfer vectors built, transfection uses promega's
PromegaTMFuGENETMHD Transfection Reagent, 5 μ g of transfection amount, transfection 2 × 107A cell.
ISKNV virus liquids are added according to every milliliter of 1.30e+05 viral copy number after transfection for 24 hours.After attacking malicious 72h, receive
The cell for collecting morbidity, obtains the virus liquid that ISKNV △ ORF074 are mixed with wild type ISKNV.
5. purifying recombinant virus
By ISKNV △ ORF074 and wild type ISKNV hybrid virus liquid multigelation 3-5 times, 0.22 μm of filter membrane is used
Filtration sterilization, according still further to 1:The mandarin fish cell of 1000 ratio infection health.
72h or so observes fluorescence after the onset of starting under inverted fluorescence microscope, if red fluorescence is found, with 2ng/ μ l
Concentration be added puromycin (puromycin) kill except wherein wild type ISKNV infection cell.After for 24 hours, supernatant is siphoned away, and
After washing 3 times with PBS, fresh DMEM medium is added.The cell of remaining wild type ISKNV infection is fallen ill death entirely after about 48h, is received
Collect these cells, the membrane filtration degerming that multigelation is 3-5 times and 0.22 μm obtains the first generation through puromycin
(puromycin) the ISKNV △ ORF074 screened and wild type ISKNV hybrid virus liquid.This virus liquid is infected into health again
Mandarin fish cell, and repeat above screening step.The ISKNV △ ORF074 Strain of purifying is finally obtained after multiplicating.
The luminescence of cell situation of IKSNV △ ORF074 infection under inverted fluorescence microscope is as shown in Figure 3.
The purity of 6.PCR identification identification recombinant viruses
Viral DNA is extracted, and detection primer is separately designed in both sides/inside of ORF074 genes, carries out PCR identifications.PCR
Identification uses the rTaq systems of TAKARA companies, system as follows：
| Reagent | Dosage (totally 20 μ L systems) unit：μL |
| 2×rTaq | 10 |
| Sterile water | 7 |
| ORF074 detections-F | 1 |
| ORF074 detections-R | 1 |
| Recombinant virus genomes template | 1 |
95 DEG C of 30s of denaturation temperature, 55 DEG C of 30s of annealing temperature, 72 DEG C of 90s of elongating temperature, 25 cycles.
Use the product of an expansion as template later, identical system carries out two and expands, and identifies the purity of recombinant virus.Identification
As a result as shown in Figure 4, Figure 5.
7. mandarin fish live body challenge viral dosage
Mandarin fish live body challenge viral dosage is carried out with ISKNV △ ORF074 gene delection recombinant virus, average body is selected in experiment
The mandarin fish of weight 175g ± 30g is as sample.Experimental setup ISKNV △ ORF074 gene delection recombinant viruses experimental group, wild type
Viral group, DMEM control groups, sample number are respectively 43/20/20 tail.Experimental group is attacked with the concentration immersion of physics titre 9.0E3/mL
Malicious 3h, then changes water；Wild-type virus group attacks poison with the concentration same method of 8.0E5/mL, and control group is added in water body
20mLDMEM culture mediums.Cultivation 20 days, counts survival rate, the results are shown in Figure 6, ISKNV △ ORF074 gene delection weights daily
Survival rate 44.19% of the group Viral experiment group after cultivating 20 days, wild-type virus group is all dead in 11 days, shows
ISKNV △ ORF074 gene delection recombinant viruses are reduced compared to wild-type virus toxicity, and detailed data can see the table below：
The mandarin fish that above-mentioned recombinant virus experimental group is survived is attacked into malicious wild type strains with the concentration of 8.0E6/mL again, and is arranged
The control group of 20 tails cultivates 20 days, counts protective rate daily.The results are shown in Figure 7, the recombination of ISKNV △ ORF074 gene delections
Viral experiment group protective rate is 44.44%, and 20 tail mandarin fish of control group is immune without attenuated vaccine, after wild type strains attack poison
12 days in it is all dead, show that mandarin fish is immunized using ISKNV △ ORF074 gene delection recombinant virus, can obtain
Effective immunoprophylaxis effect, detailed data can see the table below：
In conclusion ISKNV △ ORF074 gene delections recombinant virus can be used as infection prevention spleen renal necrosis disease
Malicious attenuated vaccine ideal chose.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although ginseng
The present invention is explained in detail according to preferred embodiment, it will be understood by those of ordinary skill in the art that, it can be to the present invention's
Technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
Sequence table
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Claims (10)
1. a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains, which is characterized in that the infectious spleen renal necrosis disease
Malicious ORF074 gene-deleted strains are that infectious spleen and kidney necrosis virus lacks manufactured attenuated viral strains after ORF074 genes.
2. a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers, which is characterized in that be infectiousness
Spleen and kidney necrosis virus lacks ORF074 genes, and is inserted into the first screening-gene respectively by homologous recombination at the position of gene delection
With manufactured attenuated viral strains after the second screening-gene.
3. the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers as claimed in claim 2, feature
It is, first screening-gene and the second screening-gene are different, and are separately selected from and are inserted into red fluorescent protein gene
And resistance screening gene.
4. a kind of construction method of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains, which is characterized in that including：Separation is simultaneously
Identify infectious spleen and kidney necrosis virus strain；Again by the method for homologous recombination by the ORF074 bases of infectious spleen and kidney necrosis virus strain
Because being lacked, infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are obtained.
5. a kind of construction method of the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers, feature exist
In, including：It detaches and identifies infectious spleen and kidney necrosis virus strain；It is by the method for homologous recombination that infectious spleen renal necrosis is sick again
The ORF074 genes of strain are lacked, and are inserted into the first screening-gene and the second screening base respectively at the position of gene delection
Cause obtains infectious spleen and kidney necrosis virus ORF074 gene-deleted strains.
6. a kind of infectious spleen and kidney necrosis virus ORF074 gene delection recombinant viral vaccines, which is characterized in that including such as right
It is required that infectious spleen and kidney necrosis virus ORF074 gene-deleted strains described in 1 or as claimed in claim 2 with selection markers
Infectious spleen and kidney necrosis virus ORF074 gene-deleted strains.
7. infectious spleen and kidney necrosis virus ORF074 gene delection recombinant viral vaccines as claimed in claim 6, feature exist
In the diagnosis that the infectious spleen and kidney necrosis virus ORF074 gene delection recombinant viral vaccines further include and pharmaceutically receive
Agent, supporting agent, excipient or diluent.
8. a kind of infectious spleen and kidney necrosis virus ORF074 gene-deleted strains as described in claim 1 or such as claim 2 institute
The infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers stated are answered by following one or more methods
With：
(1) the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains are individually applied；
(2) by infectious spleen and kidney necrosis virus ORF074 gene-deleted strains and one or more of vaccine use in conjunction；
(3) using impregnate by the way of by the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains be thrown to breeding water body into
Row application；
(4) by the way of feeding by the infectious spleen and kidney necrosis virus ORF074 gene-deleted strains be thrown to breeding water body into
Row application.
9. infectious spleen and kidney necrosis virus ORF074 gene-deleted strains as described in claim 1 are as claimed in claim 2
Infectious spleen and kidney necrosis virus ORF074 gene-deleted strains with selection markers are preparing diagnosis, are preventing, treating enlargement cytopathy
The reagent of malicious disease or the application in drug.
10. application as claimed in claim 9, which is characterized in that the enlargement cell virus includes that mandarin fish infectious spleen and kidney is bad
Dead virus, red-sea bream iridovirus, oplegnathus fasciatus irido virus, jewfish irido virus, rheum officinale fish virus, Taiwan grouper iris disease
It is one or more in poison, Epinephelus coioides irido virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810209600.7A CN108384763B (en) | 2018-03-14 | 2018-03-14 | Infectious spleen and kidney necrosis virus ORF074 gene deletion strain and preparation method and application thereof |
Applications Claiming Priority (1)
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