CN107760716A - The preparation method of GCRV S11 gene eucaryon expression recombinant plasmids and its application as nucleic acid vaccine - Google Patents

The preparation method of GCRV S11 gene eucaryon expression recombinant plasmids and its application as nucleic acid vaccine Download PDF

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CN107760716A
CN107760716A CN201711022072.6A CN201711022072A CN107760716A CN 107760716 A CN107760716 A CN 107760716A CN 201711022072 A CN201711022072 A CN 201711022072A CN 107760716 A CN107760716 A CN 107760716A
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gcrv
nucleic acid
fish body
gene
grass carp
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裴超
高岩
孔祥会
孙效迎
李莉
吕爱军
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Henan Normal University
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Henan Normal University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

Application the invention discloses a kind of preparation method of GCRV S11 gene eucaryon expression recombinant plasmids and its as nucleic acid vaccine, belongs to genetic engineering and molecular immunology technical field.Technical scheme main points are：Extract virus genome RNA, reverse transcription is cDNA, amplify corresponding DNA sequence dna, the DNA sequence dna is building up to pcDNA 3.1 (+) plasmid and converts bacillus coli DH 5 alpha, filter out the positive colony bacterium containing recombinant plasmid and by positive bacteria mass propgation, extract the recombinant plasmid S11 pcDNA3.1 contained in thalline.Using the recombinant plasmid as nucleic acid vaccine intramuscular injection grass carp, the nucleic acid vaccine enters muscle cell and the VP35 albumen of GCRV is expressed in muscle cell, so as to stimulate fish body immune cell propagation, the expression of up-regulation Antiviral related gene and stimulate fish body to produce antiviral antibody, the ability of the anti-GCRV infection of grass carp is effectively improved, and then for preventing the hemorrhagic disease of grass carp caused by the virus in aquaculture.

Description

The preparation method of GCRV S11 gene eucaryon expression recombinant plasmids and its Application as nucleic acid vaccine
Technical field
The invention belongs to genetic engineering and molecular immunology technical field, and in particular to a kind of GCRV S11 The preparation method of gene eucaryon expression recombinant plasmid and its application as nucleic acid vaccine.
Background technology
GCRV（Grass carp reovirus, GCRV）An age is infected to two age Grass carps' fries, causes fish Body body surface and fin ray end black, and belly enlargement, expophthalmos, lower jaw, fin base are congested, oral cavity, muscle, visceral hemorrhage, referred to as grass carp Hemorrhage.Since GCRV is since the 1980s is found separation, it is found that the viral prevalence is in extensive range, There is prevalence on the ground such as Guangdong and Guangxi Provinces, Hubei and Hunan Provinces, Anhui, Jiangxi, Guizhou, Henan, and fashionable colors length, 6-10 months can be popular, and summer is high Warm season easy outbreak of epidemic, up to 90% death rate can be caused.And grass carp is China's freshwater aquiculture yield highest fish, grass Hemorrhagic disease of grass carp caused by fish reovirus causes massive losses to the grass carp aquaculture industry in China.
GCRV belongs to Reoviridae（Reoviridae）, Aquareovirus （Aquareovirus）, it is diplornavirus, viral genome includes 11 RNA fragments, is followed successively by S1- from big to small S11, a gene is included in each RNA fragments, contains two genes on individual segments.3 kinds of genotype are had now been found that GCRV, be referred to as I types, II types and type III GCRV, I type virus and be found in the eighties in last century, find Time is earliest, correlative study more horn of plenty, represents strain as GCRV-873, and the type virus has 7 capsid proteins, is VP1- respectively VP7, wherein VP7 are main outer capsid proteins, in addition also 5 non-capsid proteins.In recent years, II types and type III GCRV Also it is found to separate in succession, II types represent strain as GCRV-GD108, GCRV-HZ08 etc., and epidemiological study shows, mesh It is preceding it is generally popular be II type GCRV, and virulence is better than I types, and the gene of II types virus, which is formed, is different from I types, and its capsid protein removes Outside VP1-VP6, VP35, VP35 that the also one VP56 and S11 gene segment for being similar to adenovirus spike protein encodes Contain the zinc finger binding motif similar with I type VP7 albumen（CxxC-n16-HxC zinc-binding motif）, therefore quilt It is considered the major capsid protein of II types virus, but there is huge for VP35 amino acid sequence and VP7 amino acid sequence Difference, both similarities only have about 16%, therefore predict that its antigenicity also differs.
Because I type GCRVs discovery time is early, gene functional research is goed deep into, therefore most of grass carp at present The vaccine of reovirus is prepared both for I types virus, includes the GCRV inactivated vaccine of early stage, in recent years The subunit vaccine and nucleic acid vaccine prepared using I types virus VP 4, VP6, VP7.And currently for vaccine prepared by II types virus Species is few, mainly there is subunit vaccine and nucleic acid vaccine for II type virus VP 4 proteins, and I types virus and II types are viral This notable difference be present on protein structure composition, identical guarantor can not be played to II type viruses for the vaccine of I types virus Shield property, therefore more available antigen sites are found for II types virus, it is very necessary to develop a greater variety of vaccines 's.It is this kind of viral major capsid protein that the VP35 albumen of II type virus S11 sections coding, which is predicted, up to the present, also Not on the relevant report by the use of the GFP as nucleic acid vaccine, therefore this patent is provided with the gene constructed nucleic acid epidemic disease The method of seedling, and demonstrate the practical application effect of nucleic acid vaccine.
The content of the invention
Present invention solves the technical problem that it there is provided a kind of GCRV S11 gene eucaryon expression recombinant plasmids Preparation method, recombinant plasmid S11-pcDNA3.1 can effectively prevent caused by GCRV as nucleic acid vaccine Hemorrhagic disease of grass carp.
The present invention is to solve above-mentioned technical problem to adopt the following technical scheme that, GCRV S11 gene eucaryon tables Up to the preparation method of recombinant plasmid, it is characterised in that concretely comprise the following steps：
（1）Designed for expanding the specific primer of GCRV S11 target gene, wherein GCRV The base sequence of S11 target gene is as shown in sequence table SEQ ID No.1, primer sequence S11F：5’- TGTGGATCCACCAATTATCGGTAAGTATGGAA-3 ', S11R：5’-CTGCTCGAGTGGTATGGA ATCAGTCATTACTG - 3 ', underscore is restriction enzyme site；
（2）Culture GCRV simultaneously extracts reovirus genes of grass carps group RNA；
（3）Reverse transcription is carried out to reovirus genes of grass carps group RNA using the pair of primers of design and obtains cDNA, then with cDNA Enter performing PCR with primer similar to the above for template to expand；
（4）The DNA fragmentation that PCR expands to obtain carries out double digestion with BamH I and Xho I, pcDNA-3.1 plasmids also with it is above-mentioned Identical restriction enzyme carries out double digestion, by the DNA fragmentation after digestion and plasmid fragments T4DNA ligase is connected Connect；
（5）Connection product converts bacillus coli DH 5 alpha, is applied on the culture medium containing ampicillin and is cultivated, picking colony Clone, PCR detections filter out the positive colony bacterium containing recombinant plasmid；
（6）Positive colony bacterium is continued to expand culture, collects thalline, extracts plasmid therein i.e. GCRV restructuring matter Grain S11-pcDNA3.1.
The application of GCRV S11 gene eucaryon expression recombinant plasmids of the present invention, it is characterised in that：Institute State GCRV recombinant plasmid S11-pcDNA3.1 and exhale the lonely disease of intestines for improving grass carp infection grass carp as nucleic acid vaccine The survival rate of fish body after poison.
The application of GCRV S11 gene eucaryon expression recombinant plasmids of the present invention, it is characterised in that：Will After nucleic acid vaccine is according to 0.5 μ g nucleic acid vaccines/g fish bodies weight injection grass carp muscle, the nucleic acid vaccine enter muscle cell and GCRV VP35 albumen is expressed in muscle cell, by stimulating fish body immune cell propagation, induction fish body antiviral Gene upregulation expresses and stimulated fish body specific antibody to produce to improve the ability of the anti-GCRV infection of grass carp, enters And it is used to prevent the hemorrhagic disease of grass carp caused by GCRV in aquaculture, wherein GCRV VP35 eggs White amino acid sequence is as shown in sequence table SEQ ID No.4.
Further preferably, the fish body immunocyte is monocyte, neutrophil cell and lymphocyte, the fish body Antiviral gene is IFN I, MHC I, IgM and TLR22.
The present invention has the advantages that compared with prior art：
1st, GCRV S11 target genes used in this nucleic acid vaccine, into fish body muscle cell after can express Go out the VP35 albumen of GCRV, there is good immunogenicity, the specificity and non-specificity that can induce fish body are exempted from Epidemic disease response；
2nd, this nucleic acid vaccine can induce fish body and produce specific antibody, can effectively antagonize the infection of GCRV, exempt from Epidemic disease protective efficacy reaches 70.4%；
3rd, this nucleic acid vaccine is not pathogenic, will not cause pathologic reaction, while preparation method is simple, without adding assistant during use Agent, available for large-scale production.
Brief description of the drawings
Fig. 1 is S11-pcDNA3.1 plasmid double digestion proof diagrams, and the fragment of 1kb sizes is GCRV S11 Target gene；
Fig. 2 is the expression in the grass carp nephrocyte that detection recombinant plasmid is cultivated in vitro, and wherein swimming lane 1 is Transfected Recombinant Plasmid Cell, swimming lane 2 are the cells of unloaded pcDNA3.1 plasmid transfections, and swimming lane 3 is the cell of untransfected；
Fig. 3 is the expression that nucleic acid vaccine immunity injects fish body musculature amplifying nucleic acid vaccine in different time sections after fish body；
Fig. 4 is red blood cell number and immunocyte number change total in fish body blood after nucleic acid vaccine immunity injection fish body, is injected The fish body of PBS is as a control group；
Fig. 5 is that nucleic acid immunization injects after fish body fish body lymphocyte, monocyte and neutrophil cell in different time sections Percentage changes；
Fig. 6 be nucleic acid vaccine immunity inject antiviral gene IFN I after fish body in immune organ head-kidney and spleen, MHC I, IgM and TLR22 relative expression quantity change；
Fig. 7 is the change of specific antiviral antibody content in fish body serum after immunoprotection fish body；
Fig. 8 is challenge viral dosage result, using the experimental group of vaccine, using empty plasmid control group, be not used vaccine control The comparison diagram of group fish body survival rate.
Embodiment
The above of the present invention is described in further details by the following examples, but this should not be interpreted as to this The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair Bright scope.
Embodiment 1
The preparation of GCRV S11 target gene nucleic acid vaccines
1st, GCRV recombinant plasmid S11-pcDNA3.1 structure
GCRV is isolated from the cultivating pool of Xinxiang City, Henan Province one, takes the grass carp harslet for suffering from hemorrhagic disease of grass carp, adds After the homogenate of PBS mixed grinding, take homogenate 8000g to centrifuge, take supernatant to be filtered with the filter in 0.22 μm of aperture, filtered fluid It is inoculated into the grass carp nephrocyte of in vitro culture（CIK）In, cell is collected in culture after one week, the grass carp containing propagation exhales intestines in cell Lonely virus.Cell and the total serum IgE of virus are extracted, RT-PCR detections show that the GCRV is that II type grass carps exhale the lonely disease of intestines Poison.Using reovirus genes of grass carps group RNA as template, with primer S11F：5’- TGTGGATCCACCAATTATCGGTAAGTATGGAA-3 ' and primer S11R：5’- CTGCTCGAGTGGTATGGAATCAGTCATTA CTG -3 ' carry out reverse transcription PCR, and process of reverse-transcription is：The μ L of RNA templates 8 are taken, Each 1 μ L of primer, 94 DEG C of 5min, are immediately placed on ice, afterwards RNasin Ribonuclease Inhibitor 0.5 μ L, dNTP Mixture（10mM each）1 μ L, 5 × RT Buffer, 3.5 μ L, AMV Reverse Transcriptase 1 μ L, RNase free dH2The μ L of O 3.5, reaction condition are 37 DEG C of 1h, 75 DEG C of 10min, obtain target gene cDNA.Then 1 μ L cDNA are taken Enter performing PCR amplification for template：95 DEG C of pre-degeneration 5min；95 DEG C of 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 1min, 34 circulate； 72 DEG C extend 10min eventually.PCR primer carries out double digestion with BamH I and Xho I after purification, reclaims 1kb fragment.pcDNA- 3.1 plasmids carry out double digestion with enzyme same as described above, reclaim 5.4kb fragment, by two recovery fragment T4DNA connections Enzyme connects, and connection product conversion bacillus coli DH 5 alpha, is coated on the LB solid mediums containing ampicillin and cultivates 12h, The monoclonal bacterium colony grown on picking culture medium, it is inoculated with the LB liquid medium of the benzyl containing ammonia and expands culture, PCR detects each expansion The bacterium colony of culture, choose positive bacteria sample presentation therein and detect plasmid dna sequence therein, be with verifying purpose gene fragment amplification It is no correct and whether be correctly connected with plasmid.After the correct positive bacteria of sequence verification expands culture, plasmid therein is extracted i.e. GCRV recombinant plasmid S11-pcDNA3.1.
2nd, recombinant plasmid S11-pcDNA3.1 functional verification
The S11-pcDNA3.1 recombinant plasmids of preparation send company to carry out sequencing, and measurement result shows that target gene sequence is inserted Angle of striking is correct, and target sequence is in itself without frameshit or deletion mutation, and the recombinant plasmid structure of structure is correct, and plasmid construct is just True property has also carried out double digestion checking, and carrying out double digestion using BamH I and Xho I can be by the base of GCRV S11 mesh Because being cut from recombinant plasmid（Fig. 1）.
The expression checking in cell is cultivated in vitro：Grass carp nephrocyte CIK is subjected to passage training in 6 porocyte culture plates Support, after cells grow up to the individual layer, take 4 μ g recombinant plasmids and 4 μ L liposome Lipofectamine 2000（Invitrogen） Mixing, it is added thereto after standing 20min in the culture cell conditioned medium in a hole, the cell training containing transfection liquid is suctioned out after transfecting 6h Supernatant is supported, changes normal cell culture medium into, is scraped the cell on culture plate with cell scraper after continuing 48h, collected after centrifugation Cell.4 μ g pcDNA-3.1 empty carriers plasmid is also transfected to the culture cell in a hole by same operation in addition, as control, The cell not transfected is resuspended with 30 μ L PBSs after this three groups of cells are collected as negative control, adds loading buffer Liquid, SDS-PAGE electrophoresis is carried out after boiling water bath 8min, then the albumen after electrophoresis is transferred on NC films, after BSA close membranes plus Enter VP35 albumen primary antibody and be incubated 1.5h, film is cleaned 3 times with TBST, the secondary antibody for then adding alkali phosphatase enzyme mark is incubated 1h, clearly After washing three times, substrate colour developing is added.As a result show that recombinant plasmid successful expression in CIK cell has gone out GCRV VP35 albumen（Fig. 2）, the amino acid sequence of the VP35 albumen is as shown in sequence table SEQ ID No.4.
Expression checking in grass carp fish body：Amount intramuscular injection grass carp by recombinant plasmid according to 0.5 μ g/g fish body weight Fry, the 1st after injection, 20mg musculatures are respectively taken within 7,14,21,28,35,42,49 days, add RIPA lysates and cracked, Then take the total protein after 100 μ g cracking to carry out SDS-PAGE electrophoresis, equally go on NC films, with the primary antibody and enzyme of VP35 albumen Mark secondary antibody and carry out Western blot detections, as a result show, can in grass carp muscle cell amplifying nucleic acid vaccine after the 7th day The VP35 albumen of successful expression virus（Fig. 3）, the amino acid sequence of the VP35 albumen is as shown in sequence table SEQ ID No.4.
Embodiment 2
Applications of the GCRV recombinant plasmid S11-pcDNA3.1 as nucleic acid vaccine
1st, the increase of GCRV S11 target gene nucleic acid vaccine energy obvious stimulation fish body immunocyte quantity
By healthy grass carp fry（Per tail about 20g）90 are randomly divided into 3 groups, every group 30, respectively as experimental group, empty plasmid Control group and negative control group, experimental group is per the μ g/g fish body weight of tail intramuscular injection nucleic acid vaccine 0.5, empty plasmid control group note The pcDNA-3.1 empty carrier plasmids of equivalent are penetrated, negative control group injects isometric PBS, 25 DEG C of raisings.After injection 1st, 7,14,21,28,35,42,49 day, 3 are respectively taken in 3 groups of fishes daily, tail vein takes blood, with volume ratio 1:After 200 dilutions Immunocyte number is counted using Neubauer tallies, and is observed with Giemsa staining, counts single in 100 immunocytes Nucleus, neutrophil cell and each accounting example of lymphocyte.As a result show, experimental group immunocyte quantity is immune 7th day Significantly raise and reach peak value (9.58 ± 0.72) × 107/mL（P<0.05）, still pole is significantly higher than two control groups within the 14th day（P <0.01）（Fig. 4 b）；Mean constant of red blood cell experimental group and control group are without significant difference（Fig. 4 a）；Experimental group neutrophil leucocyte ratio exists Dramatically increase and reach peak value (24.13 ± 2.38) % within 7th day（P<0.05）, percentage of lymphocyte after immunoprotection 14 days it is aobvious Writing increases and reaches peak value (93.30 ± 4.71) %（P<0.05）, still it is significantly higher than control group within the 21st day（P<0.05）（Fig. 5）.
2nd, GCRV S11 target gene nucleic acid vaccine can significantly improve the expression of fish body antiviral gene
The 1st after injection, the grass carp fish body of 7,14,21,28,35,42,49 days, respectively take 3, take head-kidney and spleen tissue, extract RNA, with the method for fluorescence quantitative RT-RCR, detection wherein antiviral gene IFN I, MHC I, IgM and TLR22 expression, knot Fruit shows expression quantity all significantly up-regulations after immunoprotection relative to control group of 4 immunogenes of experimental group（P<0.05）.Head IFN I reach extreme value for the 14th day and 21 days in immunoprotection and raise 12 times, 18 times respectively in kidney and spleen（P<0.01）；Head-kidney and MHC I reach extreme value on the 21st day in immunoprotection and raise 50 times and 30 times respectively in spleen（P<0.05）；TLR22 is in head-kidney and spleen Dirty middle immunoprotection raises 6 times on the 14th day（P<0.05）With 125 times（P<0.01）.IgM is in head-kidney and spleen after immunoprotection The the 35th and 28 day phase reached extreme value, raised 12 times and 17 times respectively（P<0.05）（Fig. 5）.
3rd, GCRV S11 target gene nucleic acid vaccine energy obvious stimulation fish body produces the antibody of protectiveness
After the fish body blood clotting layering of the experimental group taken in 1 and control group, the serum on upper strata is taken, with ELISA method The content of detection wherein antiviral antibody, as a result shows, vaccinates specific antibody content in the fish body of group and starts after 7 days Dramatically increase and continue to increase, be still significantly higher than control group at the 6th week, reach highest, about 500 in the 21st day antibody content（P <0.05）（Fig. 6）.
4th, GCRV S11 target gene nucleic acid vaccine has aobvious for hemorrhage caused by GCRV The protective effect of work
It is same that healthy grass carp 90 is divided into 3 groups, every group 30, respectively as experimental group and control group, experimental group injection weight Group subunit vaccine, empty carrier plasmid control group injection equivalent pcDNA-3.1, negative control group injection PBS.Injection 21 After it, the GCRV that 10 LD50 are injected to every tail fish of experimental group and control group is infected, after infecting 14 days Count the death toll and survival number of two groups of fishes, calculate the immune protective rate RPS, RPS of vaccine=（The 1- immune groups death rate/control group The death rate）×100%.The nucleic acid vaccine drawn according to experimental result is for the immunoprotection efficiency of GCRV 70.4%（Fig. 7）, grass carp can be significantly improved by illustrating the GCRV recombinant plasmid S11-pcDNA3.1 as nucleic acid vaccine The ability of anti-GCRV infection, and then effectively prevent hemorrhagic disease of grass carp caused by GCRV.
Embodiment above describes the general principle of the present invention, main features and advantages, the technical staff of the industry should Understand, the present invention is not limited to the above embodiments, the original for simply illustrating the present invention described in above-described embodiment and specification Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements are each fallen within In the scope of protection of the invention.
Sequence table
<110>He'nan Normal University
<120>The preparation method of GCRV S11 gene eucaryon expression recombinant plasmids and its answering as nucleic acid vaccine With
<130> 2017
<141> 2017-10-27
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 933
<212> DNA
<213>GCRV (Grass carp reovirus)
<400> 1
atggaaccag caaaaccatt gacgtttttg gatctcaccc gactcaatga ggcaatctgc 60
tctcgggctc ttttctttga taatgataac aactgctggt ctgtgtctcc aatcgccccg 120
aggcaagcca aattccatga ctcagttgtt tgtattcgat gtggagcgcc catcgacaaa 180
gtccatgcga tgtcaattcc accaccccct gtgcatggct gcatcccgat gctggggcat 240
agccaatggg aagatctgta tgagttggct gatgatatgg gtcgctgtat ttggtgggct 300
aaaaagcaac tgatcatctg gatggagggt atagtgaatc tgaaggctgg taaggtgtat 360
aatgataatg tgagtaaccg cagcgaatgg ccagatgagg tgtgggatga aacatgcaga 420
atcttctgta aatgggcaac gcaaaatcgt gtggcaagcc gttggataca gtcaccatca 480
cgtgtgtaca agtttctttg tgaccaggaa agtaagatga acattgatgc tctggagcta 540
tccaaccatc agatttttca ggcaccaccg aaatggccgg agctagctat ggctgtcccg 600
cattggtccc cgtccgtgca tgagatgcta aatggtcaca aaatggtgac gattgtgccc 660
cgcttgtcca tgccagtcat atttgacccc gccaacggtt acgtcgcccc aatctacacc 720
gcggccatga tcagcctccc gtcccaatgg tgggtgtcac aatacgtaaa agtccatgga 780
agccacatgg tgccacgttt atatggtgat gacgtcccaa ccttacgttc ccgcctgcga 840
aatgcttcta ccacaacctc ccactctcat acgcaactcc tgatgctgcc tgaagctcaa 900
tcgaccttca aacctgagat ccagggacag taa 933
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence (primer)
<400> 2
tgtggatcca ccaattatcg gtaagtatgg aa 32
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence (primer)
<400> 3
ctgctcgagt ggtatggaat cagtcattac tg 32
<210> 4
<211> 310
<212> PRT
<213>GCRV (Grass carp reovirus)
<400> 4
Met Glu Pro Ala Lys Pro Leu Thr Phe Leu Asp Leu Thr Arg Leu Asn
1 5 10 15
Glu Ala Ile Cys Ser Arg Ala Leu Phe Phe Asp Asn Asp Asn Asn Cys
20 25 30
Trp Ser Val Ser Pro Ile Ala Pro Arg Gln Ala Lys Phe His Asp Ser
35 40 45
Val Val Cys Ile Arg Cys Gly Ala Pro Ile Asp Lys Val His Ala Met
50 55 60
Ser Ile Pro Pro Pro Pro Val His Gly Cys Ile Pro Met Leu Gly His
65 70 75 80
Ser Gln Trp Glu Asp Leu Tyr Glu Leu Ala Asp Asp Met Gly Arg Cys
85 90 95
Ile Trp Trp Ala Lys Lys Gln Leu Ile Ile Trp Met Glu Gly Ile Val
100 105 110
Asn Leu Lys Ala Gly Lys Val Tyr Asn Asp Asn Val Ser Asn Arg Ser
115 120 125
Glu Trp Pro Asp Glu Val Trp Asp Glu Thr Cys Arg Ile Phe Cys Lys
130 135 140
Trp Ala Thr Gln Asn Arg Val Ala Ser Arg Trp Ile Gln Ser Pro Ser
145 150 155 160
Arg Val Tyr Lys Phe Leu Cys Asp Gln Glu Ser Lys Met Asn Ile Asp
165 170 175
Ala Leu Glu Leu Ser Asn His Gln Ile Phe Gln Ala Pro Pro Lys Trp
180 185 190
Pro Glu Leu Ala Met Ala Val Pro His Trp Ser Pro Ser Val His Glu
195 200 205
Met Leu Asn Gly His Lys Met Val Thr Ile Val Pro Arg Leu Ser Met
210 215 220
Pro Val Ile Phe Asp Pro Ala Asn Gly Tyr Val Ala Pro Ile Tyr Thr
225 230 235 240
Ala Ala Met Ile Ser Leu Pro Ser Gln Trp Trp Val Ser Gln Tyr Val
245 250 255
Lys Val His Gly Ser His Met Val Pro Arg Leu Tyr Gly Asp Asp Val
260 265 270
Pro Thr Leu Arg Ser Arg Leu Arg Asn Ala Ser Thr Thr Thr Ser His
275 280 285
Ser His Thr Gln Leu Leu Met Leu Pro Glu Ala Gln Ser Thr Phe Lys
290 295 300
Pro Glu Ile Gln Gly Gln
305 310

Claims (4)

1. the preparation method of GCRV S11 gene eucaryon expression recombinant plasmids, it is characterised in that concretely comprise the following steps：
（1）Designed for expanding the specific primer of GCRV S11 target gene, wherein GCRV The base sequence of S11 target gene is as shown in sequence table SEQ ID No.1, primer sequence S11F：5’- TGTGGATCCACCAATTATCGGTAAGTATGGAA-3 ', S11R：5’-CTGCTCGAGTGGTATGGA ATCAGTCATTACTG - 3 ', underscore is restriction enzyme site；
（2）Culture GCRV simultaneously extracts reovirus genes of grass carps group RNA；
（3）Reverse transcription is carried out to reovirus genes of grass carps group RNA using the pair of primers of design and obtains cDNA, then with cDNA Enter performing PCR with primer similar to the above for template to expand；
（4）The DNA fragmentation that PCR expands to obtain carries out double digestion with BamH I and Xho I, pcDNA-3.1 plasmids also with it is above-mentioned Identical restriction enzyme carries out double digestion, by the DNA fragmentation after digestion and plasmid fragments T4DNA ligase is connected Connect；
（5）Connection product converts bacillus coli DH 5 alpha, is applied on the culture medium containing ampicillin and is cultivated, picking colony Clone, PCR detections filter out the positive colony bacterium containing recombinant plasmid；
（6）Positive colony bacterium is continued to expand culture, collects thalline, extracts plasmid therein i.e. GCRV restructuring matter Grain S11-pcDNA3.1.
2. the application of GCRV S11 gene eucaryon expression recombinant plasmids made from the method as described in claim 1, It is characterized in that：The GCRV recombinant plasmid S11-pcDNA3.1 is used to improve grass carp infection as nucleic acid vaccine The survival rate of fish body after GCRV.
3. the application of GCRV S11 gene eucaryon expression recombinant plasmids according to claim 2, its feature exist In：After nucleic acid vaccine is injected into grass carp muscle according to 0.5 μ g nucleic acid vaccines/g fish bodies weight, the nucleic acid vaccine enters muscle cell And GCRV VP35 albumen is expressed in muscle cell, by stimulating fish body immune cell propagation, induction fish body to resist Viral gene up-regulated expression and stimulation fish body specific antibody are produced to improve the energy of the anti-GCRV infection of grass carp Power, and then for preventing the hemorrhagic disease of grass carp caused by GCRV, wherein GCRV in aquaculture The amino acid sequence of VP35 albumen is as shown in sequence table SEQ ID No.4.
4. the application of GCRV S11 gene eucaryon expression recombinant plasmids according to claim 3, its feature exist In：The fish body immunocyte is monocyte, neutrophil cell and lymphocyte, and the fish body antiviral gene is IFN I, MHC I, IgM and TLR22.
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