CN105801707A - Oral vaccine for treating grass carp hemorrage as well as preparation and application thereof - Google Patents
Oral vaccine for treating grass carp hemorrage as well as preparation and application thereof Download PDFInfo
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- CN105801707A CN105801707A CN201610213530.3A CN201610213530A CN105801707A CN 105801707 A CN105801707 A CN 105801707A CN 201610213530 A CN201610213530 A CN 201610213530A CN 105801707 A CN105801707 A CN 105801707A
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- fusion protein
- nucleotide sequence
- cotc
- bacillus subtilis
- seqidno
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- 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
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
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- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the technical field of biology and particularly relates to an oral vaccine for treating grass carp hemorrage as well as preparation and an application thereof. The invention provides a fusion protein firstly. The fusion protein comprises bacillus subtilis spore capsid protein CotC and grass carp reovirus VP4 protein. The oral vaccine for treating grass carp hemorrhage is prepared from the fusion protein, can be colonized successfully in the intestinal tract of a fish body and has good immune effect, and the death rate of fish bodies after challenge infection is reduced by 30%-45% compared with a negative group.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of hemorrhagic disease of grass carp oral vaccine and preparation and application thereof.
Background technology
Hemorrhagic disease of grass carp (GrassCarpHemorrhage) is a kind of by GCRV (Grasscarpreovirus, the viral infectious of the harm Ctenopharyngodon idellus cultivation GCRV) caused, mainly cause each organ of ill Grass Carp Juveniles, organize hyperemia in various degree, bleeding, and hence obtain one's name.Within 1970, find first, hereafter in succession popular in each culture zones of provinces, municipalities and autonomous regions such as China Hubei, Hunan, Guangdong, Guangxi, Jiangsu, Zhejiang, Anhui, Fujian, Shanghai, Sichuan, also can infect the multiple fingerlings such as Mylopharyngodon piceus, Carassius auratus, pseudorasbora parva.Owing to hemorrhagic disease of grass carp harm fish species is many, Epidemic Scope is wide, and morbidity season length (annual late June is to being Major Epidemic season by the end of September), aquaculture, up to 85%, is brought significant damage and economic loss by mortality rate.
GCRV is subordinate to Aquareovirus (Aquareoviridae, AQRV), is the first fishes virus of China's separation, is also the strain that in Aquareovirus, pathogenicity is the strongest.GCRV virus belongs to the C group of AQRV, and for the spheroplast of icosahedral symmetry, ripe viral diameters is 70-80nm, has double capsid, and without cyst membrane, genome is made up of 11 dsRNA.Mainly through respiratory tract infection host, there is hyperinfection and lethal.The Grass Carp Juveniles (4-5 monthly age) of GCRV about main harm 10cm, entering fish body by the fish gill, first 3 days is incubation period, starts morbidity after the 4th day, infect the tissues such as Liver of Ctenopharyngodon Idellus, kidney, intestinal, muscle, substantially congested, bleeding occurred after 5-7 days.
At present, immunity inoculation is still the preventing and treating maximally effective approach of this viral disease, including inactivated vaccine, attenuated vaccine, subunit vaccine, carrier bacterin, DNA vaccination etc..Protease inhibitor, RNA perturbation technique, immunostimulant also obtain certain progress and application.Such as, China's Pearl River Fishery Research Institute of Aquatic Science Research Institute has been realized commercialization by the attenuated live vaccines of many cultures, attenuation development and has been used widely, and makes hemorrhagic disease of grass carp be under control to a certain extent.In recent years, preparation subunit vaccine and nucleic acid vaccine are that integrated control hemorrhagic disease of grass carp provides new method.Subunit vaccine has titre height, the advantages such as morning, persistent period length occurs in antibody, has the immunogenic character of thymus dependent.Nucleic acid vaccine also referred to as gene vaccine, transformation convenience, guard time length strong with its antigenicity, preparation and convenient transportation and be increasingly subject to people's attention.Tribute Cheng Liang etc. also construct the recombinant baculovirus BmNPV-IIVP6 with GCHV structure protein VP6 gene and be seeded to five age silkworm larva or pupa, then by five age silkworm larva or the lyophilized powder prepared of pupa lyophilization obtain oral vaccine with fish meal Homogeneous phase mixing.
Hay spore, as oral type vaccine carrier, has the advantage that a lot of conventional vaccine carrier is incomparable: it belongs to gram positive bacteria, does not produce toxin, biological safety is high, excellent stability, is one of conventional probiotic bacteria, has been used as the functional food of people, animal feed additive etc..In addition, hay spore has the immunological characteristic of uniqueness, oral spore can be identified by gut-associated lymphoid tissue at animal intestinal, play immunoadjuvant function, the lymphoid tissue on activation intestinal mucosa, increase secreting type (SIgA) antibody-secreting, improve Immune discrimination ability, and induce T, bone-marrow-derived lymphocyte and Factor of Macrophage, activate systemic immune system by lymphocyte recirculation, thus the non-specific and specific immune system of enhancing body.
Bacillus subtilis has perfect excretory system, by, in foreign recombinant proteins direct secretion to culture fluid, greatly reducing the purification procedures of expression product, can be applied to the expression of many recombiant proteins.Bacillus subtilis spore production cost is low, and growth is quickly, low to nutritional requirement, is suitable to modern industry fermenting and producing.The recombinant spore vaccine produced, stability is high, convenient storage, transport, it is possible to use as food additive.Therefore bacillus subtilis has been used widely as probiotic bacteria animal husbandry and aquatic products industry, especially as additive, not only overcome antibiotic to the harmful effect of other microorganism species in digestive tract, and avoid the toxic and side effects of antibiotic life-time service, Drug resistance and drug resistance, and the growth of poultry and Fish can be promoted, strengthen animal disease resistance ability, improve the digestion of feedstuff, absorption rate and conversion ratio, improve aquaculture yield.Owing to hay spore is as the had unique advantage of vaccine carrier, it is believed that be the most promising potential carrier for oral vaccine delivery the development starting to be applied to pathogenic microorganism vaccine.
Existing inactivated vaccine, attenuated live vaccines make hemorrhagic disease of grass carp be under control to a certain extent, but in the wild application in its one by one injection system still have certain limitation, fish body be will also result in damage.Simultaneously inactivated vaccine, attenuated live vaccines there is also the safety risks of virulence reversion.By comparison, it is convenient that oral vaccine has enforcement, not by the restriction of time, place and fish body size, to advantages such as fish not damageds.
Although bacillus subtilis has good microorganism vaccine researching value, but in genetic engineering operating process, low-conversion and genetic instability limit its extensive use in industrial fermentation produces.The structure of escherichia coli/B. subtilis shuttle expression plasmid and application technology can improve bacillus subtilis and change into power, simplifies genetic engineering operating procedure, is conducive to bacillus subtilis as the application of vaccine carrier.
Summary of the invention
In order to overcome problem existing in prior art, it is an object of the invention to provide a kind of hemorrhagic disease of grass carp oral vaccine and preparation and application thereof.
To achieve these goals and other relevant purposes, the present invention adopts the following technical scheme that
A first aspect of the present invention, it is provided that a kind of fusion protein, containing bacillus subtilis spore capsid protein CotC and GCRV VP4 albumen.
Preferably, the aminoacid sequence of described bacillus subtilis spore capsid protein CotC is corresponding to the aminoacid sequence coded by nucleotide sequence as shown in SEQIDNO.2.
It is highly preferred that the aminoacid sequence of described bacillus subtilis spore capsid protein CotC is such as shown in SEQIDNO.1, particularly as follows:
MGYYKKYKEEYYTVKKTYYKKYYEYDKKDYDCDYDKKYDDYDKKYYDHDKKDYDYVVEYKKHKKHY。
It is further preferred that the coding nucleotide sequence of described bacillus subtilis spore capsid protein CotC is such as shown in SEQIDNO.2, particularly as follows:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTAC。
Preferably, the aminoacid sequence of described GCRV VP4 albumen is corresponding to the aminoacid sequence coded by nucleotide sequence as shown in SEQIDNO.4.
It is highly preferred that the aminoacid sequence of described GCRV VP4 albumen is such as shown in SEQIDNO.3, particularly as follows:
MSAIWEPETVSAAGNYYLWPTVIGDASMTSDWGTISTSLANGRLRVAPLDLTHALHKGNVVEPIVPSDVLGNASPEEMRSALPADVLTAFKAKLTTVASVVGRALNPNDSAHAPSSGTVLGPLAIENKAQSKPKPVSDLWIAAXRGVNLFVASPSASLQTGVPVMGDSSVLTSLTGGATTALNTGDMGTPSLEATAKRAARVAGGLLRQRVMDKLTRYWPLR。
It is further preferred that the coding nucleotide sequence of described GCRV VP4 albumen is such as shown in SEQIDNO.4, particularly as follows:
TTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTC。
Preferably, containing, for example the aminoacid sequence coded by nucleotide sequence shown in SEQIDNO.5 in the aminoacid sequence of described fusion protein.
It is further preferred that containing, for example the nucleotide sequence shown in SEQIDNO.5 in the coding nucleotide sequence of described fusion protein.Nucleotide sequence as shown in SEQIDNO.5 is specifically:
TCTATAATAATATTTGGGAAATATTCATTCTAATTGGCTCGAGGTCGACTGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACGGATCCTTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTCGAGCTCTAGCACTTTGCCACTCATTTTTTGCGTTAGCAA。
A second aspect of the present invention, it is provided that a kind of method preparing foregoing fusion albumen, comprises the steps:
(1) genes of interest is obtained；
(2) construction recombination plasmid: step (1) gained genes of interest and vector plasmid are adopted identical restricted enzyme cutting, is then attached, it is thus achieved that recombiant plasmid；
(3) step (2) gained recombiant plasmid is imported competent cell, convert；
(4) screening positive clone, and by correct recombinant plasmid transformed host's competent cell；
(5) abduction delivering fusion protein.
Preferably, in step (1), the nucleotide sequence of described genes of interest contains the coding nucleotide sequence of bacillus subtilis spore capsid protein CotC and the coding nucleotide sequence of GCRV VP4 albumen.
Preferably, in step (1), the coding nucleotide sequence of described bacillus subtilis spore capsid protein CotC is such as shown in SEQIDNO.2.The coding nucleotide sequence of described GCRV VP4 albumen is such as shown in SEQIDNO.4.
It is further preferred that in step (1), the nucleotide sequence of described genes of interest is such as shown in SEQIDNO.6, particularly as follows:
GTCGACTGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACGGATCCTTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTCGAGCTC。
Preferably, in step (2), described vector plasmid is PEB03 plasmid.
Described PEB03 plasmid is existing plasmid in prior art.Such as it is referred to document " productionofn-acetyl-d-neuraminicacidbyuseisofanefficien tsporesurfacedisplaysystem ".
Preferably, in step (2), described restricted enzyme is SalI, SacI.
Preferably, in step (3), described competent cell is bacillus subtilis WB600 competent cell.
Preferably, in step (4), described host's competent cell is bacillus subtilis WB600 competent cell.
Preferably, in step (5), described induction adopts the induction of DSM spore culture medium.
A third aspect of the present invention, it is provided that foregoing fusion albumen purposes in preparing hemorrhagic disease of grass carp oral vaccine.
A fourth aspect of the present invention, it is provided that a kind of hemorrhagic disease of grass carp oral vaccine, containing foregoing fusion albumen.
Preferably, described hemorrhagic disease of grass carp oral vaccine is possibly together with vaccine carrier, and described vaccine carrier selects bacillus subtilis spore.
It is further preferred that described hemorrhagic disease of grass carp oral vaccine is possibly together with cod-liver oil and feedstuff.
Compared with prior art, there is advantages that
The fusion protein that the present invention is obtained by extensive and deep research, prepares into hemorrhagic disease of grass carp oral vaccine, it is possible to success field planting in fish body intestinal, and immune effect is good, it is possible to after fish body counteracting toxic substances is infected, more negative group of mortality rate reduces by 40%～50%.
Accompanying drawing explanation
The qualification of the fragment of Fig. 1: CotC order, the amplification of VP4 coding sequence fragment and CotC-VP4 genes of interest fragment, illustrates fragment and the VP4 coding sequence fragment successful connection of CotC order.
Fig. 2: SDS-PAGE analysis fusioning protein PEBO3-CotC-VP4 expression.
Fig. 3: Westernblotting analysis fusioning protein PEBO3-CotC-VP4 expression.
Fig. 4: immunofluorescence analysis fusion protein PEBO3-CotC-VP4 expression.
Fig. 5: PCR detection recombinant spore colonisation in grass carp intestinal.
Fig. 6: ELISA measures Grass Carp Serum specific antibody IgM level.
Fig. 7: real-timeqPCR detection Ctenopharyngodon idellus spleen, kidney relevant cell factor level.
Fig. 8: the statistics of cumulative mortality after variable concentrations GCRV counteracting toxic substances.
Detailed description of the invention
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, rather than in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, generally conventionally condition, or according to each manufacturer it is proposed that condition.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment, material, record according to those skilled in the art's grasp to prior art and the present invention, it is also possible to use similar with the method described in the embodiment of the present invention, equipment, material or that be equal to any method of prior art, equipment and material to realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all adopt the conventional molecular biology of the art, biochemistry, chromatin Structure and analysis, analytical chemistry, cell are cultivated, the routine techniques of recombinant DNA technology and association area.These technology existing improving in existing document illustrates, specifically can referring to the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001；Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, john wiley & sons, NewYork, 1987andperiodicupdates；TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego；Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998；METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999；And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc..
Terminological interpretation involved in the present invention:
Bpbasepair base pair
BSAbovineserumalbumin bovine serum albumin
CDNAcomplementaryDNA complementary DNA
CFUColony-FormingUnits colony-forming units
Cy3CyanineDyes fluorometric reagent
DEPCDiethypyrocarbonate pyrocarbonic acid diethyl ester
E.coliEscherichiacoli escherichia coli
EDTAethylenediaminetetraaceticacid ethylenediaminetetraacetic acid
ELISAenzymelinkedimmunosorbentassay enzyme-linked immunosorbent assay
HRPHorseradishPeroxidase horseradish peroxidase
LBlysogenybrothLB culture medium
LD50medianlethaldose median lethal dose(LD 50)
ODopticaldensity optical density
ORFopenreadingframe open reading frame
PAGEpolyacrylamidegelelectrophoresis polyacrylamide gel electrophoresis
PCRPolymeraseChainReaction polymerase chain reaction
PMSFphenylmethanesulfonylfluoride Phenylmethanesulfonyl fluoride
SDSsodiumdodecylsulfate sodium lauryl sulphate
WBWesternblotting western blotting
The preparation of embodiment 1 hemorrhagic disease of grass carp oral vaccine
(1) structure of bacillus subtilis/bacillus coli shuttle expression system
Extract bacillus subtilis WB600 genomic DNA, after the fragment of CotC order is amplified from Bacillus subtilis genes group DNA with carrier pBluescriptIISK (+) recombinate, it is thus achieved that recombiant plasmid pBluescriptIISK (+)-CotC；Again VP4 coding sequence fragment is connected to recombiant plasmid pBluescriptIISK (+)-CotC, it is thus achieved that recombiant plasmid pBluescriptIISK (+)-CotC-VP4；By overall for CotC-VP4 genes of interest fragment from recombiant plasmid pBluescriptIISK (+)-CotC-VP4 cuts, be connected to PEB03 and obtain recombiant plasmid PEB03-CotC-VP4.
Recombinate with PEBO3 after the fragment of CotC order being amplified from Bacillus subtilis genes group DNA, it is thus achieved that recombiant plasmid PEBO3-CotC is as comparison simultaneously.Shuttle expression plasmid PEB03-CotC-VP4 and PEBO3-CotC is proceeded to abduction delivering in bacillus subtilis WB600 by electrotransformation respectively.
Concrete:
The extraction of 1.1 bacillus subtilis WB600 genomic DNAs
1)-80 DEG C of refrigerators take out bacillus subtilis WB600 bacterial strain, draw LB flat board, 37 DEG C of overnight incubation；
2) picking monoclonal colony inoculation is in 5mlLB culture medium, 37 DEG C of shaking table overnight incubation；
3) take 1ml bacterium solution in 1:100 ratio and proceed in 100mlLB culture medium, 37 DEG C of shaking table overnight incubation；
4) 4 DEG C of centrifugal collections antibacterial (weighing about 0.5g), use the EDTA liquid containing 0.5% lysozyme to blow outstanding thalline, and 37 DEG C stand 30 minutes；
5) adding 10%SDS to final concentration 0.5%, blow even, 65 DEG C of water-baths stand 30 minutes；
6) add and step 5) in the isopyknic phenol of 10%SDS used, chloroform, isoamyl alcohol mixed liquor (phenol: chloroform: isoamyl alcohol volume ratio is 25:24:1) extracting twice, each ice bath 15 minutes, centrifugal 5 minutes of 12000g, abandon supernatant；
7) genomic DNA, room temperature 30min are precipitated with 0.6 times of volume isopropanol；Centrifugal 10 minutes of 12000g, abandons supernatant；
8) precipitating by 70% washing with alcohol, dry, DNA is dissolved in 500 μ lTE buffer, adds RNase to final concentration 100 μ g/ml, and 65 DEG C of water-baths stand 30 minutes；Repeat this step twice；
9) being dissolved in by DNA in 100 μ lTE buffer, take 5 μ l samples and carry out agarose gel electrophoresis qualification ,-20 DEG C of all the other samples save backup.
1.2 recombiant plasmid pBluescriptIISK (+) structure of-CotC
According to spore capsid CotC coded sequence and carrier pBluescriptIISK (+) multiple clone site, design specific gene amplimer, after the fragment of CotC order is expanded from Bacillus subtilis genes group DNA with carrier pBluescriptIISK (+) recombinate, acquisition recombiant plasmid pBluescriptIISK (+)-CotC, introduce multiple clone site.Technique for gene engineering replicates operation in bacterial strain DH5 α escherichia coli, and errorless rear routine preservation is identified in recombiant plasmid order-checking.
The nucleotide sequence of the fragment of described CotC order is such as shown in SEQIDNO.2, particularly as follows:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTAC。
Primer is as follows:
Forward primer P1:5 '-CATGTCGACTGTAGGATAAATCGTT-3 ' (SEQIDNO.7), underscore is SalI restriction enzyme site；
Downstream primer P2:5 '-CGGGGATCCGTAGTGTTTTTTATGC-3 ' (SEQIDNO.8), underscore is BamHI restriction enzyme site.
1.3 recombiant plasmid pBluescriptIISK (+) structure of-CotC-VP4
According to albumen conserved domain ReovirusM2 (Accession:cl05506) of GCRV-HZ08 strain S6 gene (GenBank:GQ896337.1) sequential coding in GenBank, expand VP4 gene with Primerpremier5.0 software design specific gene amplimer.
The nucleotide sequence of described VP4 coding sequence fragment is such as shown in SEQIDNO.4, particularly as follows:
TTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTC。
Forward primer P1:5 '-CGCGGGATCCTTTGGCAATATTCGCAACATTACC-3 ' (SEQIDNO.9), underscore is BamHI restriction enzyme site；
Downstream primer P2:5 '-CGTGAGCTCGACGGAGGAGGCCAGTATCGA-3 ' (SEQIDNO.10), underscore is SacI restriction enzyme site.
Pcr amplification goes out GCRVVP4 coding sequence fragment ORF (701bp), be connected to above-mentioned recombiant plasmid pBluescriptIISK (+)-CotC.Technique for gene engineering replicates operation in bacterial strain DH5 α escherichia coli, and errorless rear routine preservation is identified in recombiant plasmid order-checking.
The structure of 1.4 recombinant shuttle plasmid PEB03-CotC-VP4 and electricity convert to bacillus subtilis WB600
Adopt SalI, SacI restriction enzyme site by overall for CotC-VP4 genes of interest fragment from recombiant plasmid pBluescriptIISK (+)-CotC-VP4 cuts, be connected to PEB03 and obtain recombinant shuttle plasmid PEB03-CotC-VP4.New recombiant plasmid electricity is converted to bacillus subtilis WB600, specifically comprises the following steps that
1) bacterium WB600 is met in 3mlLB culture medium, 37 DEG C, 250rpm, overnight incubation；
2) 2.6ml bacterium solution is proceeded to 40ml electricity and converts in A culture medium, 37 DEG C, 250rpm, shakes to OD value 0.8-0.9；
3) bacterium solution ice-water bath 10min；5,000g, 4 DEG C of centrifugal 5min；
4) convert B culture medium with the electricity of 50ml pre-cooling and blow outstanding thalline；5,000g, 4 DEG C of centrifugal 5min；Repeat this step 4 time；
5) thalline is blown be suspended from 1ml electricity convert B culture medium, subpackage 10 is managed, each EP pipe subpackage 100 μ l；
6) by 100 μ lWB600 competent cells add 5 μ l (about 1 μ g) recombiant plasmid PEB03-CotC-VP4,2min is stood on ice；
7) being drawn in the electric shock cup of pre-cooling (1mm) by 100 μ lWB600 competent cells, adjust electricity conversion instrument parameter: 2.0kV, 1mm, 25 μ F, 200 Ω, 4.5-5.0ms, electric shock is once；
8) electric shock terminate rear sucking-off electric shock cup in competent cell be transferred in new EP pipe, immediately add 1ml electricity convert C culture medium, 37 DEG C, 150rpm, recovery 3h, be coated with LB flat board (Spectinomycin resistance), in 37 DEG C of incubators cultivate 20h；
9) picking monoclonal is to (Spectinomycin resistance) in LB culture medium, 37 DEG C, 250rpm, overnight incubation；
10) positive colony is identified in bacterium solution PCR and order-checking.
Bacterium solution PCR identifies as it is shown in figure 1, fragment and the VP4 coding sequence fragment successful connection of CotC order is described.
Order-checking is identified, fragment and the VP4 coding sequence fragment successful connection of CotC order are also described.
The nucleotide sequence of CotC-VP4 genes of interest as shown in (SEQIDNO.6), particularly as follows:
GTCGACTGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACGGATCCTTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTCGAGCTC。
The nucleotide sequence of encoding fusion protein is containing, for example the nucleotide sequence shown in SEQIDNO.5.Nucleotide sequence as shown in SEQIDNO.5 is specifically:
TCTATAATAATATTTGGGAAATATTCATTCTAATTGGCTCGAGGTCGACTGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTGATTTGTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAGTATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACGGATCCTTTGGCAATATTCGCAACATTACCGACTTCTCAATGTCTGCAATTTGGGAACCAGAGACAGTCAGCGCGGCAGGCAATTACTATCTATGGCCGACCGTAATCGGTGATGCATCAATGACATCAGATTGGGGGACAATTAGCACATCCCTAGCTAATGGCAGACTCCGTGTCGCACCTCTGGACCTGACTCATGCACTCCACAAAGGCAATGTCGTGGAGCCGATCGTACCATCTGATGTGCTAGGTAATGCCTCACCAGAGGAAATGCGTTCCGCGTTGCCAGCAGATGTGCTGACAGCTTTCAAAGCCAAGCTCACAACAGTGGCTTCCGTAGTCGGCCGTGCCTTAAATCCCAACGACAGTGCGCATGCACCATCATCCGGCACCGTCCTCGGCCCGCTTGCAATCGAAAACAAGGCCCAATCAAAACCTAAACCCGTATCAGATCTGTGGATAGCCGCTYGTCGTGGTGTGAATCTATTCGTAGCCTCTCCAAGCGCGAGTCTGCAAACAGGTGTGCCGGTCATGGGGGACAGCAGCGTGTTGACAAGCTTGACGGGTGGTGCAACTACCGCGTTGAATACTGGTGACATGGGTACCCCAAGCTTGGAGGCCACAGCGAAACGGGCAGCTAGAGTTGCTGGAGGACTGCTACGACAGAGAGTCATGGACAAACTAACTCGATACTGGCCCCTCCGTCGAGCTCTAGCACTTTGCCACTCATTTTTTGCGTTAGCAA。
(2) detection of bacillus subtilis amalgamation and expression GCRVVP4 (fusion protein)
Recombinant bacterium cultivates 0h, 6h, 12h, 24h (Fig. 2 in DSM spore culture medium, 1-4 swimming lane) sampling process, and the spore supernatant of separation and Culture 24h and capsid protein (Fig. 2,5,6 swimming lane) row SDS-PAGE, observes fusion protein expression in bacillus subtilis and the location in spore thereof.Full bacterium precipitation (Fig. 3 that induction 24h is left and taken, 1 swimming lane) and ultrasonication separate spore supernatant protein and capsid protein (Fig. 3,2,3 swimming lane) after carrying out SDS-PAGE, proceed Westernblotting, detection fusion albumen expression in bacillus subtilis and in the location of spore capsid.After taking the liquid-solid fixed process of spore of induction 24h, react with the IgG of big mouse-anti VP4 serum (matched group hatches normal rabbit serum), goat anti rat Cy3 labelling successively, fluorescence microscopy Microscopic observation fusion protein is at spore capsid expression (Fig. 4, spore form under A white light, B experimental group is notable fluorescence as seen, and C matched group has no fluorescence).
(3) acquisition of the amplification culture of bacillus subtilis and spore
1) bacterium (Spectinomycin resistance) in 40mlLB culture medium, 37 DEG C of shaking table overnight incubation are connect according to 1:100 volume ratio；Proceeding in 4LDSM spore culture medium (without antibiotic) next day, 24h cultivated by 37 DEG C of shaking tables；
2) 12,000g centrifugal 10min, abandons supernatant and leaves and takes precipitation, adds lysozyme and repeatedly blows outstanding precipitation to final concentration 4mg/ml, and room temperature stands 30min；
3) wash spore with 1MNaCl, add PMSF to whole solubility 1mM simultaneously, fully mix；
4) 12,000g centrifugal 10min, abandons supernatant and leaves and takes precipitation, then add PMSF to whole solubility 1mM with 1MKCl washing spore simultaneously, fully mix；
5) deionized water wash spore three times, 12,000g centrifugal 10min, abandon supernatant and leave and take precipitation；
6) 40ml deionized water blows outstanding spore precipitation, and 65 DEG C of water-baths stand 1h to kill the bacillus subtilis brood body of residual；
7) take 3 μ l spore deionization dilutions, counted under microscope, spore is saved backup in-40 DEG C.
(4) Efficacy evaluation of oral vaccine
The preparation of 4.1 spores of bacillus subtilin vaccines and the immunity of fish body
The spore cod-liver oil of acquisition is coated rear and feedstuff Homogeneous phase mixing, namely obtains hemorrhagic disease of grass carp oral vaccine, spore amount about 109CFU/g feedstuff.Ctenopharyngodon idellus 150 tail, respectively oral vaccine group, negative control and positive controls (injection commercialization attenuated live vaccine), often organize 50 tail routine fish jars to raise, according to spores of bacillus subtilin on Growth of Grass Carps Ctenopharyngodon Idellus, immunity and the impact on breeding water body water quality, throw something and feed every day once, the about feedstuff of fish body weight 2%, throws something and feeds 6 weeks continuously.After immunity in 6 weeks terminates, three groups of Ctenopharyngodon idellus tail venous blood sampling respectively separating and collecting Grass Carp Serum, separate and preserve the tissue such as liver,spleen,kidney, intestinal ,-20 DEG C save backup.
4.2PCR method identifies grass carp intestinal endospore
After immunity 6 weeks, the LB solid culture plate of the fish intestinal lapping liquid coating in oral vaccine group grows a large amount of bacillus subtilis bacterium colony, picking monoclonal shakes bacterium, carry out bacterium solution PCR, PCR primer is through electroresis appraisal, obtain genes of interest band, spores of bacillus subtilin success field planting (Fig. 5) in intestinal is described.
4.3 indirect ELISAs measure Grass Carp Serum specific antibody level
With recombiant protein pET32a (+) coated elisa plate is overnight for-VP4 (1 μ g/ hole), Grass Carp Serum after immune 6 weeks is diluted according to 1:50, the goat-anti Ctenopharyngodon idellus IgM of HRP labelling is as two anti-(1:5000 dilution) detection immunity Grass Carp Serum antibody horizontal, oral vaccine group IgM level relatively matched group raises to some extent, but not as good as injection group notable (Fig. 6).
4.4.4 the detection of Ctenopharyngodon idellus spleen, the nephrocyte factor
Get the spleen of collection, nephridial tissue TRIzol method extracts total serum IgE also quantitatively.It is that after cDNA, 1:4 DEPC water dilutes, row real-timeqPCR detects relevant cell factor by 1 μ g total serum IgE reverse transcription.In result display Ctenopharyngodon idellus spleen, relevant cell factor injection group has notable rising, and oral vaccine group and negative group are without significant change；Kidney based intracellular cvtokine oral vaccine group and injection group all raise significantly, and wherein IgM, MyD88, TLR22 and negative group have significant difference (Fig. 7).
4.5 challenge infections
Above experimental result points out spores of bacillus subtilin in this experiment that fish body can be stimulated to produce a degree of specific immune response.Often organize Ctenopharyngodon idellus after taking the 20 above-mentioned immunity of tail respectively and inject 200ul about 103LD50GCRV-hunan1307 carries out counteracting toxic substances infection, and keeps constant 28 DEG C of water temperature with heating rod heating.Cumulative mortality, the i.e. immune protective efficiency of vaccine is added up after counteracting toxic substances.Ibid often organize and take 20 tails respectively with 104LD50GCRV carries out counteracting toxic substances infection.Result shows; negative group cumulative mortality is all up to 90%, and injection group all has notable protection, cumulative mortality 10% in twice challenge viral dosage; though oral vaccine group significantly also has certain protection not as good as injection group, cumulative mortality is 45% and 60% (Fig. 8) respectively.
4.6 statistical analysiss
SPSS16.0 software is for the statistical analysis of data in this research.Experimental result all represents with means standard deviation, and experimental group compares employing t-test and analyzes with matched group, and P value shows that less than 0.05 two groups of data have significant difference.
The above; it is only presently preferred embodiments of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the premise without departing from the inventive method, also can making some improvement and supplement, these improve and supplement and also should be regarded as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, the equivalent variations of a little change, modification and the differentiation made when available disclosed above technology contents, it is the Equivalent embodiments of the present invention；Meanwhile, all change of any equivalent variations, modification and differentiation above-described embodiment made according to the substantial technological of the present invention, all still fall within the scope of technical scheme.
Claims (12)
1. a fusion protein, it is characterised in that described fusion protein contains bacillus subtilis spore capsid protein CotC and GCRV VP4 albumen.
2. fusion protein according to claim 1, it is characterised in that the aminoacid sequence of described bacillus subtilis spore capsid protein CotC is corresponding to the aminoacid sequence coded by nucleotide sequence as shown in SEQIDNO.2.
3. fusion protein according to claim 1, it is characterised in that the coding nucleotide sequence of described bacillus subtilis spore capsid protein CotC is such as shown in SEQIDNO.2.
4. fusion protein according to claim 1, it is characterised in that the aminoacid sequence of described GCRV VP4 albumen is corresponding to the aminoacid sequence coded by nucleotide sequence as shown in SEQIDNO.4.
5. fusion protein according to claim 1, it is characterised in that the coding nucleotide sequence of described GCRV VP4 albumen is such as shown in SEQIDNO.4.
6. fusion protein according to claim 1, it is characterised in that containing, for example the aminoacid sequence coded by nucleotide sequence shown in SEQIDNO.5 in the aminoacid sequence of described fusion protein.
7. prepare a method for fusion protein as described in claim 1～6 any claim, comprise the steps:
(1) genes of interest is obtained；
(2) construction recombination plasmid: step (1) gained genes of interest and vector plasmid are adopted identical restricted enzyme cutting, is then attached, it is thus achieved that recombiant plasmid；
(3) step (2) gained recombiant plasmid is imported competent cell, convert；
(4) screening positive clone, and by correct recombinant plasmid transformed host's competent cell；
(5) abduction delivering fusion protein.
8. method according to claim 7, it is characterized in that, in step (1), the nucleotide sequence of described genes of interest contains the coding nucleotide sequence of bacillus subtilis spore capsid protein CotC and the coding nucleotide sequence of GCRV VP4 albumen.
9. method according to claim 7, it is characterised in that in step (2), described restricted enzyme is SalI, SacI.
10. method according to claim 7, it is characterised in that step (3) and in step (4), described host's competent cell is bacillus subtilis WB600 competent cell.
11. fusion protein purposes in preparing hemorrhagic disease of grass carp oral vaccine as described in claim 1～6 any claim.
12. a hemorrhagic disease of grass carp oral vaccine, containing, for example fusion protein described in claim 1～6 any claim.
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