CN110279854A - A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof - Google Patents

A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof Download PDF

Info

Publication number
CN110279854A
CN110279854A CN201910654291.9A CN201910654291A CN110279854A CN 110279854 A CN110279854 A CN 110279854A CN 201910654291 A CN201910654291 A CN 201910654291A CN 110279854 A CN110279854 A CN 110279854A
Authority
CN
China
Prior art keywords
micropterus salmoides
dna vaccine
mcp
virus dna
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910654291.9A
Other languages
Chinese (zh)
Inventor
李蔚
易婉盈
周天鸿
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jinan University
University of Jinan
Original Assignee
Jinan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jinan University filed Critical Jinan University
Priority to CN201910654291.9A priority Critical patent/CN110279854A/en
Publication of CN110279854A publication Critical patent/CN110279854A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/00034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention discloses a kind of Micropterus salmoides virus DNA vaccines and the preparation method and application thereof.The DNA vaccination is the recombinant plasmid containing Micropterus salmoides virus MCP genetic fragment；The nucleotide sequence of MCP genetic fragment is as shown in SEQ ID NO.1.DNA vaccination provided by the invention can significantly cause relevant specificity and nonspecific immune reaction in Micropterus salmoides body; good protecting effect is played to the poison of attacking of LMBV; immune protective rate can provide fundamental basis and scientific basis for later period Micropterus salmoides virus DNA vaccine in production application and industrialization production up to 63%.

Description

A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of Micropterus salmoides virus DNA vaccine and its preparation side Method and application.
Background technique
Micropterus salmoides (Micropterus salmoides) is one of Important Economic fish of China's freshwater aquiculture, close several Year, Chinese market was increasing the demand of Micropterus salmoides, and national Culture Techniques of Micropterws Salmoides increased than 2016 within 2017 31.57%, growth rate is first of cultured freshwater fish.
Culture Techniques of Micropterws Salmoides scale is increasing, and frequency is also got in the outburst of the disease of Micropterus salmoides especially viral disease It is numerous.Micropterus salmoides virus (Largermouth bass virus, LMBV) belongs to one of Iridoviridae Ranavirus member, right Micropterus salmoides has high pathogenic, can cause the high mortality of Micropterus salmoides.Currently, viral for LMBV both at home and abroad having Effect treatment skill there is no report, based on the civil precautionary measures for mostly using conservative, such as science raising, sterilize in time etc..Cause This, the economic loss for caused by LMBV is not stopped loss effectively yet.
Therefore, there is an urgent need to develop it is a kind of can effective preventive inoculation vaccine that LMBV is broken out, seek new treatment skill.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of Micropterus salmoides viral DNA Vaccine.
Another object of the present invention is to provide the preparation methods of above-mentioned Micropterus salmoides virus DNA vaccine.
A further object of the present invention is to provide the applications of above-mentioned Micropterus salmoides virus DNA vaccine.
The purpose of the invention is achieved by the following technical solution:
A kind of Micropterus salmoides virus DNA vaccine, to contain viral (LMBV) main capsid protein (MCP) the gene piece of Micropterus salmoides The recombinant plasmid of section；The nucleotide sequence of MCP genetic fragment is as shown in SEQ ID NO.1.
The MCP genetic fragment can be obtained by artificial synthesized, or from Micropterus salmoides virus LMBV-C Genomic PCR Amplification obtains.
The Micropterus salmoides virus LMBV-C genome is the genome for extracting Micropterus salmoides virus LMBV-C and obtaining DNA。
The preparation method of above-mentioned Micropterus salmoides virus DNA vaccine includes the following steps: Micropterus salmoides viral (LMBV) is main Capsid protein (MCP) genetic fragment is inserted into carrier for expression of eukaryon, obtains Micropterus salmoides virus DNA vaccine.
The carrier for expression of eukaryon is preferably the eukaryon expression plasmid with Flag label.
The eukaryon expression plasmid is preferably pCDNA3.1 (+).
Application of the above-mentioned DNA vaccination in cultured fishes Micropterus salmoides prevention and cure of viruses.
In the application, DNA vaccination is administered by pectoral fin base portion injection system, and administration concentration is preferably 30~50 μ G/ tail, more preferably 40 μ g/ tails.
The present invention has the following advantages and effects with respect to the prior art:
DNA vaccination prepared by the present invention for Micropterus salmoides disease can significantly cause relevant specificity and non-spy in fish body Specific immunological reaction, plays good protecting effect to the poison of attacking of LMBV, immune protective rate can be later period DNA vaccination up to 63% It provides fundamental basis in production application and industrialization production and scientific basis.
Detailed description of the invention
Fig. 1 is to detect MCP gene expression dose result figure using RT-qPCR and Western Blot experimental method；In figure, A is RT-qPCR testing result figure, and B is Western Blot testing result figure.
Fig. 2 is serum neutralize antibody titers qualification result figure.
Fig. 3 is leukocyte differential count result figure；In figure, A is the testing result figure of neutrophil cell, and B is monocyte Testing result figure, C are the testing result figure of lymphocyte.
Fig. 4 is the expression analysis result figure of gene involved in immunity；In figure, A is the detection knot of interleukin-1 ' beta ' in liver Fruit figure, B are the testing result figure of interleukin-1 ' beta ' in spleen, and C is the testing result figure of interleukin-1 ' beta ' in head-kidney, D For the testing result figure of interleukin 8 in liver, E is the testing result figure of interleukin 8 in spleen, and F is in head-kidney The testing result figure of interleukin 8, G are the testing result figure of tumor necrosis factor-alpha in liver, and H is that tumour is bad in spleen The testing result figure of necrosis factor-α, I are the testing result figure of tumor necrosis factor-alpha in head-kidney, and J is antiviral gene in liver Testing result figure, K be spleen in antiviral gene testing result figure, L be head-kidney in antiviral gene testing result figure.
Fig. 5 is challenge viral dosage result figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.
The preparation of 1 DNA vaccination of embodiment
(1) recombinant plasmid of building expression LMBV-MCP albumen:
According to the MCP gene order (GenBank:FR682503.1) of LMBV on NCBI, specific primer is designed and synthesized Right, primer is synthesized by Huada gene company, and the sequence of primer is as shown in table 1:
The primer sequence of 1 PCR of table acquisition MCP genetic fragment
Target gene Forward primer MCP-F1 (5 ' -3 ') Reverse primer MCP-R1 (5 ' -3 ')
MCP CGGGATCCGCCACCATGTCTTCTGTTACGG CGGAATTCCAGGATGGGGAAACCCATGG
Micropterus salmoides virus LMBV-C is taken (to be presented by Zhongshan University He Jianguo laboratory, and in document " Chen Zhensheng mono- Separation, identification and specificity analysis [D] master thesis of strain Micropterus salmoides viral (LMBV), Zhongshan University are public in 2014 " It opens).It is taken out with viral DNA extracts kit (OMEGA, Viral DNA Kit) referring to the method in " molecular cloning texts guide " Genomic DNA is mentioned, is expanded as template by round pcr, MCP full length gene is obtained.
The reaction system of PCR amplification are as follows: 1 μ L of virus genom DNA template, primer (MCP forward primer/reverse primer) are each 2 μ L, PrimeSTART MAX archaeal dna polymerase (2 ×) 25 μ L, ddH2O complements to 50 μ L.
The reaction condition of PCR amplification are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s；58 DEG C of annealing 30s；72 DEG C of extensions 1.5min, 35 circulations；72 DEG C of extension 10min.
Pass through the artificial synthesized 3 × Flag tag template of Shanghai Sangon Biotech Company, then artificial synthesized band EcoRI restriction enzyme site respectively Forward primer and reverse primer with XbaI enzyme cutting site, the sequence of primer is as shown in table 2, carry out PCR amplification, after purification, Double digestion and recovery purifying are carried out to pCDNA3.1 (+) and 3 × Flag segment with restriction endonuclease EcoRI and XbaI, after by the two into Row connection, obtains pCDNA3.1 (+) plasmid for containing 3 × Flag label.Wherein, the sequence of 3 × Flag tag template is as follows:
5’-GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAA GTAA-3’。
By molecule clone technology, with restriction endonuclease BamHI and EcoRI to PCR product and above-mentioned 3 × Flag label PCDNA3.1 (+) plasmid recycles after carrying out double digestion, obtains the recombinant plasmid containing MCP genetic fragment by connection.
The primer sequence of 2 PCR of table acquisition 3 × Flag genetic fragment
Target gene 3 × Flag of forward primer F1 (5 ' -3 ') 3 × Flag of reverse primer R1 (5 ' -3 ')
3×Flag GGAATTCGACTACAAAGACCATG GCTCTAGATTACTTGTCATCGTCATCC
(2) recombinant plasmid built carries out expression analysis in cell:
The endotoxin-free recombinant plasmid that step (1) is obtained is conventional, and (lipofection, transfection reagent turn for efficient eukaryon Transfection reagent (GenStar), DNA: transfection reagent=1:3) transfection fish cell carp epidermoma cell line (EPC) it is (big by middle mountain The present of teacher He Jianguo laboratory is learned, in document " WANG Xiaohong, Weng Shaoping, He Jianguo's insect iridescent virus in vitro culture and its reason Change characteristic aquatic product journal, 2002,26 (4): disclosed in 363-366 "), after transfecting 48h, by RT-PCR to MCP gene expression It is identified, primer sequence is as shown in table 3, and testing result is as shown in Figure 1A, the results showed that recombinant plasmid is transcribed in cell MCP segment out；Wherein, the reaction system and reaction condition of RT-PCR is as follows: transfecting 1 μ L of cDNA sample, the primer of EPC, (MCP is just To primer/reverse primer) each 1 μ L, Premix Ex Taq archaeal dna polymerase (2 ×) 10 μ L, ddH2O complements to 20 μ L；95 DEG C pre- It is denaturalized 5min；95 DEG C of denaturation 30s；60 DEG C of annealing 30s；72 DEG C of extension 50s, 35 circulations；72 DEG C of extension 10min.With anti-Flag Antibody (SIGMA-Aldrich) referring to the Western Blot method in " molecular cloning texts guide " to MCP protein expression into Row identification, testing result are as shown in Figure 1B.
The primer sequence of table 3RT-PCR identification of M CP gene expression
RT-PCR the result shows that, recombinant plasmid in cell it is transcribed go out MCP segment；Western Blot result obtains, Recombinant plasmid can correctly express MCP destination protein.Therefore, recombinant plasmid is DNA vaccination, can be applied to later period immunity test.
Obtained recombinant plasmid is stored in -20 DEG C of refrigerators, it is immune for the later period.
2 DNA vaccination of embodiment is immunized living body Micropterus salmoides and verifies immune effect
The Micropterus salmoides (coming from sea bass farm at a gulp, Foshan city) of 180 tails health is randomly divided into 3 groups, every group 60 tails, respectively DNA vaccination group, unloaded group and reference group (PBS group).Micropterus salmoides is exempted from using pectoral fin base portion injection method Epidemic disease, DNA vaccination group: every tail fish injects 40 μ g of recombinant plasmid (being dissolved in the 100 sterile PBS of μ L)；Unloaded group: every tail fish injects zero load 40 μ g of plasmid (is dissolved in the 100 sterile PBS of μ L)；Reference group: every tail fish injects sterile 100 μ L of PBS.Respectively after immune the 1st, 7, the blood and tissue of every group of fish are collected within 14,21,28 days, leukocyte differential count, the identification of serum neutralize antibody titers are carried out and is immunized The expression analysis of related gene.
Every group takes 3 tail Micropterus salmoides at random, and tail vein blood is stored at room temperature 1h, 4 DEG C of standing 12h, 4000r/min centrifugations 10min takes upper serum, the measurement for antibody titer.Serum neutralize antibody titers qualification result is as shown in Fig. 2, DNA vaccination The serum neutralize antibody titers of group are extremely significant to be higher than control group, causes significant antibody response.
The above-mentioned every group 3 tail Micropterus salmoides taken at random, tail vein blood add anti-coagulants for Arneth's count.It is white Cell classification result is as shown in figure 3, neutrophil cell and the monocyte accounting in leucocyte significantly increase in fish body, lymph Cell accounts for significant decrease, causes certain inflammatory reaction.
The liver, spleen, head-kidney for taking each group Micropterus salmoides extract total serum IgE simultaneously using Trizol (Takara company) reagent Reverse transcription is carried out, cDNA is obtained, real-time fluorescence quantitative PCR is carried out to cDNA, wherein reaction system: SYBR Premix Ex 10 μ L of Taq II (2 ×), each 0.8 μ L, ROX Reference Dye II (50 ×), 0.4 μ L, cDNA template of primer (positive/negative primer) 2 μ L, ddH26 μ L of O, in total 20 μ L system；Reaction condition: 95 DEG C of initial denaturation 2min；95 DEG C of denaturation 10s, 60 DEG C of annealing 34s are followed Ring number 40 times.Solubility curve reaction condition: 95 DEG C of 15s；60 DEG C of 1min, 95 DEG C of 15s.To identify gene involved in immunity leucocyte The expression of -1 β of interleukin (IL-1 β), interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-α), antiviral gene (Mx). Primer sequence is shown in Table 4.The expression analysis result of gene involved in immunity is as shown in figure 4, gene involved in immunity in DNA vaccination group is in pole Significant up-regulation phenomenon, causes effective nonspecific immune reaction.
4 PCR of table identifies the primer sequence of gene involved in immunity expression in Micropterus salmoides
3 challenge test of embodiment
Micropterus salmoides after being immunized 30 days carries out the challenge test of LMBV, and the LMBV for attacking poison is LMBV-C virus Strain, attacking malicious concentration is 4.8 × 107TCID50/ mL, attacking toxic dose is 200 μ L/ tails.Day by day the death condition of fish body is observed, in time Death toll is recorded, the survival rate comparative analysis result of each group Micropterus salmoides is as shown in Figure 5.When fish body death tends towards stability, meter Calculate immune protective rate (Relative Percent Survival, RPS)=(the 1- immune group death rate/control group death rate).Knot Fruit show DNA vaccination to the immune protective rate of Micropterus salmoides up to 63%, it is extremely significant to be higher than control group.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.
<110>Ji'nan University
<120>a kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1392
<212> DNA
<213>Micropterus salmoides (Micropterus salmoides)
<220>
<223>MCP nucleotide sequencing
<400> 1
atgtcttctg ttacgggttc tggcatcact agcgggttca ttgatctcgc cacttatgac 60
agccttgaca aagcgctgta cggtggaaaa gatgcaacta cttatttcgt caaagagcat 120
tatcccgtgg gttggtttac caaactgcct acggctgcca caaaaacttc tggtacgcct 180
gctttcgggc agcacttttc cgtaggagtg cccaggtcgg gcgactatgt gctcaactct 240
tggctggtcc tcaagacccc ccagattaaa ctgctggcgg ccaaccagtt taacaatgac 300
ggtaccatca gatggaccaa aaatctcatg cacaacgttg tggagcacgc cgcactctcg 360
ttcaacgaga ttcaggccca gcagtttaac actgctttcc tggacgcctg gaacgagtac 420
accatgcccg aggccaagcg catcggctac tacaacatga ttggcaacac tagcgatctc 480
gtcaatcccg cccccgccac cggtcaagca ggagctaggg tcctgcccgc caaaaacctt 540
gtccttcctc tccccttctt tttcggcaga gacagcgggc tggccctgcc tacagtcacc 600
ctgccttaca acgaaattag aatcaccatc agcctgagat ccattcagga tctcctgatt 660
cttcagcaca agacgaccgg agaagtcaag cccatcgtgg ccacagatct ggaaggaggt 720
ctcccagaca cggtagaggc tcacgtctac atgactgtgg gtctggtgac tgccgccgag 780
cgtcaggcta tgagcagctc agtcagggac atggtggtgg agcagatgca gatggctccg 840
gtccacatgg tcaaccccaa gaacgccacc gtctttcacg cagacctgcg cttttcccac 900
gccgtcaaag cgctcatgtt tatggtgcaa aacgtcactc acaagtctgt gggttccaac 960
tacacttgcg tcactcctgt tgttggagcg ggtaacaccg tcctggagcc cgccctggcc 1020
gtcgatccgg tcaagagcgc cagtctggtg tacgaaaaca ctaccaggct tccagacatg 1080
agcgtagagt actattccct ggtgcagccc tggtactacg cacccgccat tcccatcagc 1140
actggccacc acctctactc ttacgccctg agcctcaacg atcctcaccc ttcagggtct 1200
accaatttcg gtcgcctgac caacgcaagc atcaacgtgt ctctgtctgc cgaggccgga 1260
actgccgccg gaggaggagg ggcagacaac tctggctaca aaaaccctca gaaatacgcc 1320
ctggtggtca tggccatcaa ccacaacatt atccgcatca tgaacggttc catgggtttc 1380
cccatcctgt aa 1392
<210> 2
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>PCR obtains the forward primer sequence of MCP genetic fragment
<400> 2
cgggatccgc caccatgtct tctgttacgg 30
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>PCR obtains the reverse primer sequences of MCP genetic fragment
<400> 3
cggaattcca ggatggggaa acccatgg 28
<210> 4
<211> 69
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>3 × Flag tag template sequences
<400> 4
gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat 60
gacaagtaa 69
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>PCR obtains the forward primer sequence of 3 × Flag genetic fragment
<400> 5
ggaattcgac tacaaagacc atg 23
<210> 6
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>PCR obtains the reverse primer sequences of 3 × Flag genetic fragment
<400> 6
gctctagatt acttgtcatc gtcatcc 27
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>the forward primer sequence of PCR identification of M CP gene expression
<400> 7
cgatcaagca ggagctaggg 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>reverse primer sequences of PCR identification of M CP gene expression
<400> 8
ggcagacaga gacacgttga 20
<210> 9
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>β-actin forward primer sequence
<400> 9
caggatgcag aaggagatca ca 22
<210> 10
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>β-actin reverse primer sequences
<400> 10
ctcctgcttg ctgatccaca t 21
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>IL-1 β forward primer sequence
<400> 11
tggtggaaaa cagcatggag c 21
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>IL-1 β reverse primer sequences
<400> 12
agggtgcacg tagttcgaca 20
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>IL-8 forward primer sequence
<400> 13
gagtttgagg agcctgggtg t 21
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>IL-8 reverse primer sequences
<400> 14
gggtccaggc aaacctcttg 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>TNF-α forward primer sequence
<400> 15
acttcgtcta cagccaggca 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>TNF-α reverse primer sequences
<400> 16
agtaacgcga gaccctgtgg 20
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Mx forward primer sequence
<400> 17
taaaatggct ggggtcgggg 20
<210> 18
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Mx reverse primer sequences
<400> 18
cattgcacgg aacgaccacc 20

Claims (7)

1. a kind of Micropterus salmoides virus DNA vaccine, it is characterised in that: be the recombination containing Micropterus salmoides virus MCP genetic fragment Plasmid；The nucleotide sequence of MCP genetic fragment is as shown in SEQ ID NO.1.
2. Micropterus salmoides virus DNA vaccine according to claim 1, it is characterised in that: the MCP genetic fragment passes through It is artificial synthesized to obtain, or obtained from Micropterus salmoides virus LMBV-C genomic PCR amplification.
3. the preparation method of Micropterus salmoides virus DNA vaccine of any of claims 1 or 2, it is characterised in that include the following steps: Micropterus salmoides virus MCP genetic fragment is inserted into carrier for expression of eukaryon, Micropterus salmoides virus DNA vaccine is obtained.
4. the preparation method of Micropterus salmoides virus DNA vaccine according to claim 3, it is characterised in that: the eukaryon Expression vector is the eukaryon expression plasmid with Flag label.
5. the preparation method of Micropterus salmoides virus DNA vaccine according to claim 4, it is characterised in that: the eukaryon Expression plasmid is pCDNA3.1 (+).
6. application of the Micropterus salmoides virus DNA vaccine described in claim 1 in cultured fishes Micropterus salmoides prevention and cure of viruses.
7. application according to claim 7, it is characterised in that: by Micropterus salmoides virus DNA vaccine described in claim 1 It is administered by pectoral fin base portion injection system, administration concentration is 30~50 μ g/ tails.
CN201910654291.9A 2019-07-19 2019-07-19 A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof Pending CN110279854A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910654291.9A CN110279854A (en) 2019-07-19 2019-07-19 A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910654291.9A CN110279854A (en) 2019-07-19 2019-07-19 A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN110279854A true CN110279854A (en) 2019-09-27

Family

ID=68023447

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910654291.9A Pending CN110279854A (en) 2019-07-19 2019-07-19 A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110279854A (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111073983A (en) * 2020-01-20 2020-04-28 中国水产科学研究院珠江水产研究所 SNP marker related to identification of northern subspecies and Florida subspecies of largemouth bass and application thereof
CN111135295A (en) * 2020-01-17 2020-05-12 浙江省淡水水产研究所 Iris virus disease inactivated vaccine for micropterus salmoides and preparation method thereof
CN112759629A (en) * 2020-12-29 2021-05-07 广东海大畜牧兽医研究院有限公司 ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof
CN113736747A (en) * 2021-07-22 2021-12-03 中国水产科学研究院珠江水产研究所 double-RNA virus, method for rapidly identifying double-RNA virus and application
CN114230660A (en) * 2022-02-25 2022-03-25 华南农业大学 Monoclonal antibody for resisting micropterus salmoides iridovirus LMBV and application thereof
CN114381551A (en) * 2021-12-21 2022-04-22 佛山科学技术学院 Real-time fluorescent RAA primer, probe and kit for detecting iridovirus of micropterus salmoides
CN114748617A (en) * 2022-03-24 2022-07-15 华南农业大学 Subunit vaccine of micropterus salmoides iridovirus as well as preparation method and application thereof
CN118389449A (en) * 2024-03-22 2024-07-26 中国水产科学研究院长江水产研究所 Lateolabrax japonicus virus attenuated strain and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006137724A (en) * 2004-11-15 2006-06-01 Meiji Seika Kaisha Ltd Dna vaccine against red sea bream iridovirus of marine fish
CN103041404A (en) * 2012-05-17 2013-04-17 上海七海复泰生物科技有限公司 Preparation method of fish iridovirus DNA (Deoxyribonucleic Acid) vaccine
CN104404057A (en) * 2014-12-03 2015-03-11 中国水产科学研究院长江水产研究所 Giant salamander iridescent virus vaccine, preparation method and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006137724A (en) * 2004-11-15 2006-06-01 Meiji Seika Kaisha Ltd Dna vaccine against red sea bream iridovirus of marine fish
CN103041404A (en) * 2012-05-17 2013-04-17 上海七海复泰生物科技有限公司 Preparation method of fish iridovirus DNA (Deoxyribonucleic Acid) vaccine
CN104404057A (en) * 2014-12-03 2015-03-11 中国水产科学研究院长江水产研究所 Giant salamander iridescent virus vaccine, preparation method and application

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BAI,J.J.ET AL.: "" GU256635.1 Largemouth bass ulcerative syndrome virus strain EPC060608-08 major capsid protein gene, complete cds"", 《GENBANK》 *
OHLEMEYER,S. ET AL.: ""FR682503 Largemouth bass virus MCP gene for major capsid protein"", 《GENBANK》 *
易婉盈: ""大口黑鲈病毒（LMBV）DNA疫苗的研制及其免疫效果的研究"", 《中国优秀硕士学位论文全文数据库》 *
曾宪辉等: ""大鲵虹彩病毒主衣壳蛋白 MCP 基因 DNA 疫苗的构建及其免疫效果"", 《中国水产科学》 *
苏友禄等: ""神经坏死病毒MCP重组疫苗对军曹鱼稚鱼的免疫保护"", 《生物技术通报》 *
马冬梅等: ""大口黑鲈溃疡综合征病毒 MCP 基因的原核表达及重组蛋白的免疫效果初步分析"", 《生物技术通报》 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111135295A (en) * 2020-01-17 2020-05-12 浙江省淡水水产研究所 Iris virus disease inactivated vaccine for micropterus salmoides and preparation method thereof
CN111135295B (en) * 2020-01-17 2023-08-25 浙江省淡水水产研究所 Inactivated vaccine for iridovirus disease of largemouth black bass and preparation method thereof
CN111073983A (en) * 2020-01-20 2020-04-28 中国水产科学研究院珠江水产研究所 SNP marker related to identification of northern subspecies and Florida subspecies of largemouth bass and application thereof
CN112759629A (en) * 2020-12-29 2021-05-07 广东海大畜牧兽医研究院有限公司 ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof
CN112759629B (en) * 2020-12-29 2021-10-26 广东海大畜牧兽医研究院有限公司 ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof
CN113736747A (en) * 2021-07-22 2021-12-03 中国水产科学研究院珠江水产研究所 double-RNA virus, method for rapidly identifying double-RNA virus and application
CN114381551A (en) * 2021-12-21 2022-04-22 佛山科学技术学院 Real-time fluorescent RAA primer, probe and kit for detecting iridovirus of micropterus salmoides
CN114381551B (en) * 2021-12-21 2024-02-02 佛山科学技术学院 Real-time fluorescence RAA primer, probe and kit for detecting largemouth black bass iridovirus
CN114230660A (en) * 2022-02-25 2022-03-25 华南农业大学 Monoclonal antibody for resisting micropterus salmoides iridovirus LMBV and application thereof
CN114748617A (en) * 2022-03-24 2022-07-15 华南农业大学 Subunit vaccine of micropterus salmoides iridovirus as well as preparation method and application thereof
CN114748617B (en) * 2022-03-24 2024-04-16 华南农业大学 Subunit vaccine of largemouth black bass iridovirus, and preparation method and application thereof
CN118389449A (en) * 2024-03-22 2024-07-26 中国水产科学研究院长江水产研究所 Lateolabrax japonicus virus attenuated strain and application thereof
CN118389449B (en) * 2024-03-22 2024-10-29 中国水产科学研究院长江水产研究所 Lateolabrax japonicus virus attenuated strain and application thereof

Similar Documents

Publication Publication Date Title
CN110279854A (en) A kind of Micropterus salmoides virus DNA vaccine and the preparation method and application thereof
Wang et al. Mass mortality caused by Cyprinid Herpesvirus 2 (CyHV-2) in Prussian carp (Carassius gibelio) in China
Yi et al. Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with largemouth bass virus (LMBV)
CN108251384B (en) Fish rhabdovirus attenuated vaccine strain
CN113736800B (en) Pigeon-derived Newcastle disease virus recombinant vaccine strain and construction method and application thereof
CN109652357B (en) Mycoplasma bovis mutant strain with growth defect under cell co-culture and application thereof
CN109321535A (en) A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain
Li et al. Establishment and characterization of a fin tissue cell line derived from silver pomfret, Pampus argenteus
CN106520710A (en) Preparation method and application of live vector vaccine for expressing duck Tembusu virus (DTMUV) prm and E protein recombinant Newcastle disease virus (NDV)
CN113416768B (en) Application of PRKRA gene as target in inhibiting replication of peste des petits ruminants virus
CN112899404B (en) Nested-RAA detection primer pair for mandarin fish frog iridovirus as well as application and detection method thereof
CN104152417B (en) Attenuated vaccine strain expressing GM-CSF (granulocyte-macrophage colony-stimulating factor) recombinant PRRSV (porcine reproductive and respiratory syndrome virus) as well as preparation method and application thereof
KR101958169B1 (en) Composiotion for Vaccine Containing VHSV Vaccine and adjuvant
CN109182277A (en) A kind of swamp eel rhabdovirus CrERV and RT-PCR detection primer and application
CN115779084B (en) Application of preparation for activating TUSC1 gene expression of pigs in preparation of anti-pseudorabies virus infection medicines of pigs
CN116286686A (en) Construction of double-gene deletion attenuated African swine fever virus strain and application of strain as vaccine
CN115820638A (en) Exogenous artificial miRNA for inhibiting replication of waterfowl-derived avian reovirus and application thereof
CN102363792A (en) Lentivirus method for preparing IGF2b transgenic fish
CN101972476A (en) DNA vaccine adjuvant using Micro RNA-155 and construction method thereof
CN101928727A (en) Newcastle disease virus Italien strain auxiliary expression plasmid systems, micro genome for detecting system and application thereof
CN110042083B (en) BHK-21 cell strain for stably expressing MAP3K8 protein and construction and application thereof
CN116059371B (en) Application of preparation for activating VASH gene expression of pig in preparation of medicine for resisting pseudorabies virus infection of pig
CN117965632B (en) Preparation method and application of functional enhancement type NK cells
CN117802097B (en) SgRNA for knocking out ISG15 gene, ISG15 gene-deleted bovine kidney cell and application thereof
CN103243106A (en) Epinephelus coioides interferon IFNgamma1 and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20190927

RJ01 Rejection of invention patent application after publication