CN104357460B - Restructuring duck enteritis virus, preparation method and application - Google Patents
Restructuring duck enteritis virus, preparation method and application Download PDFInfo
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Abstract
The present invention provides restructuring duck enteritis virus, preparation method and application, belongs to the vaccines arts of animal medicine.Avian influenza virus hemagglutinin gene of the present invention, its nucleotides sequence is classified as the 1437th to 3131 nucleotide of SEQ ID NO:1.The present invention recombinates duck enteritis virus, is to insert the expression cassette containing described avian influenza virus hemagglutinin gene in the spacer between LORF11 and the UL55 gene of duck enteritis virus genome.Avian influenza virus hemagglutinin gene of the present invention, as vaccine activity composition, can reach preferable cross-protection.The present invention recombinates duck enteritis virus, can express avian influenza virus hemagglutinin gene, viral steady, well-grown with stability and high efficiency; the live vaccine of preparation; higher to the Vaccine effectiveness of duck pestilence and bird flu, create higher avian influenza antibody, and the antibody duration is longer.
Description
Technical field
The invention belongs to the vaccines arts of animal medicine, be specifically related to recombinate duck enteritis virus, preparation method and
Application.
Background technology
Duck pestilence reported first sees earlier 1900s, and there is the report of this disease in the most of duck culturing in current whole world country, by
Duck plague virus (also known as duck enteritis virus) causes, and its M & M is up to 100%, and duck plague virus can be duck
Trigeminal ganglion forms persistent infection, and duck pestilence attenuated live vaccines is widely used in the immunoprophylaxis of duck pestilence, can effectively stop morbidity.
Bird flu (H5N1) is a kind of Infectious Diseases of duck and chicken, is not only current threat whole world domestic fowl farming cultivation peace
One of complete most important infectious disease, and the fatality rate to the mankind is high, constitutes a serious threat mankind's public health security.
The method based on Pathogen Purification is taked in prevention and control measure to domestic fowl farming bird flu, Countries and area, such as European Union's great majority
Country, achieves preferable effect.It is comprehensive that the prevention and control of domestic fowl farming bird flu (H5N1) are taked based on compulsory immunization by China
The anti-measure processed of property, effectively controls this disease propagation impetus in China poultry.Aquatic bird based on duck is the important of bird flu
Carry and store host, in the propagation and mutation process of bird flu (H5N1) virus, play key player, therefore, the fowl of duck
The anti-system of influenza (H5N1) is the key of these disease prevention and control.China uses totivirus inactivation epidemic disease at present to poultry compulsory immunization
Seedling, although this vaccine has the advantages such as safety is good, but also shows weak point following aspects in practical application,
One, inactivated vaccine to carry out large-scale Virus culture aborning, there is the biological safety hidden danger of diffusion virus.Two, its
Immunity produces relatively slow, and forms lump at injection site, and the application for production cycle shorter fast-growing type poultry is limited
System.Three, though inactivated vaccine can produce efficient humoral immunization, but cellular immunity is not enough, it is impossible to virus thoroughly cleared out of
Body, consequently, it is possible to form inapparent infection.Especially inactivated vaccine is poor to the immunogenicity of duck, and immune effect is the best.
Summary of the invention
It is an object of the invention to provide avian influenza virus hemagglutinin gene, this gene expression product becomes as vaccine activity
Point, preferable cross-protection can be reached.
It is a further object of the present invention to provide restructuring duck enteritis virus, bird flu virus can be expressed with stability and high efficiency
Hemagglutinin gene, viral steady, well-grown.
It is a further object of the present invention to provide the construction method of described restructuring duck enteritis virus, the method is simple,
Efficiency is high.
Another object of the present invention is to provide the live vaccine with described restructuring duck enteritis virus as active component, should
After live vaccination, higher to the Vaccine effectiveness of duck pestilence and bird flu, create higher avian influenza antibody, and antibody duration
Longer.
The purpose of the present invention adopts the following technical scheme that realization.
The present invention provides avian influenza virus hemagglutinin gene, its nucleotides sequence be classified as the 1437th of SEQ ID NO:1 to
3131 nucleotide.
The present invention also provide for recombinate duck enteritis virus, duck enteritis virus genome LORF11 and
Spacer between UL55 gene inserts the expression cassette containing avian influenza virus hemagglutinin gene described in claim 1.
Preferably in technical scheme, the promoter in described expression cassette is pMCMV IE promoter.
Preferably in technical scheme, described expression cassette substituted for spacer the 263rd between UL55 and LORF11 gene to
The nucleotide fragments of 291.
The present invention also provides for the construction method of described restructuring duck enteritis virus, uses methods of homologous recombination to contain
The expression cassette of described avian influenza virus hemagglutinin gene inserts LORF11 and the UL55 gene of duck enteritis virus genome
Between spacer.
In the present invention, the expression cassette containing described avian influenza virus hemagglutinin gene inserts duck enteritis virus
Method comprises the steps:
(1) to be replaced is determined in the spacer between UL55 and the LORF11 gene of duck enteritis virus genome
Section, expands the nucleotide fragments of fragment upstream and downstream to be replaced respectively, obtains upstream homology arm and downstream homology arm；To express
Described in the expression cassette of green fluorescent protein and expression claim 1, the expression cassette of gene inserts upstream homology arm respectively and downstream is same
Between source arm, respectively obtain homologous recombination fragment A and homologous recombination fragment B；
(2) genomic DNA of duck enteritis virus and homologous recombination fragment A carry out homologous recombination, select in ultraviolet
The recombinant virus of green fluorescence is sent, it is thus achieved that recombinant virus C under light；
(3) homologous recombination fragment B carries out homologous recombination with the genomic DNA of recombinant virus C, under ultraviolet light can not select
Send the recombinant virus of green fluorescence, obtain carrying the restructuring duck viral enteritis of avian influenza virus hemagglutinin gene expression cassette
Virus.
The present invention also provides for the live vaccine with described restructuring duck enteritis virus as active component.
The original strain of the restructuring duck enteritis virus expressing avian flu virus hemagglutinin (HA) gene can be duck
Viral enteritis virus Embryo Gallus domesticus low virulent strain or other duck enteritis virus low virulent strains or engineered through artificial gene
Strain and virulent strain.
The present invention, with the described restructuring duck enteritis virus (the DEV-H5 ULwf strain) live vaccine as active component, uses
The duck, goose and the immunoprophylaxis of the Anseriformes birds infectious disease such as Muscovy duck and wild duck that cause in duck plague virus and bird flu virus and chicken
The immunoprophylaxis of bird flu.
Live vaccine of the present invention, is using described restructuring duck enteritis virus DEV-H5 ULwf strain as kind of a poison, with reference to duck
The production technology of viral enteritis live vaccine produces.Immunological adjuvant and vaccine excipients can be added in live vaccine of the present invention
Deng, those skilled in the art can easily produce.
The expression product of the avian influenza virus hemagglutinin gene that present invention process optimizes, as vaccine activity composition, can reach
Preferably cross-protection.
The present invention passes through to insert avian influenza virus hemagglutinin gene expression cassette in duck enteritis virus genome,
Obtain restructuring duck enteritis virus.This restructuring duck enteritis virus energy stability and high efficiency expresses bird flu virus blood clotting
Plain gene, viral steady, well-grown.
The present invention recombinates, and the construction method of duck enteritis virus is simple, efficiency is high.
The present invention live vaccine with described restructuring duck enteritis virus as active component, after inoculation chicken and duck, safety
Property good, the highest to the Vaccine effectiveness of duck pestilence and bird flu, especially create higher and that the duration is longer bird flu resist
Body.
Accompanying drawing explanation
The structure diagram of Fig. 1, GFP gene transfer vector.
Fig. 2, GFP recombinate duck enteritis virus rDEV-GFP plaque fluorescence photo.
Fig. 3, avian flu virus hemagglutinin (HA) expression casette schematic diagram.
Fig. 4, the indirect immunofluorescence figure of recombinant virus DEV-H5 ULwf strain expression HA.
Fig. 5, recombinant virus DEV-H5 ULwf strain growth characteristics curve chart, wherein DEV C-KCE is that duck viral enteritis is sick
The abbreviation of poison Embryo Gallus domesticus low virulent strain C-KCE strain.
Vaccine effectiveness figure to duck pestilence after Fig. 6, recombinant virus DEV-H5 ULwf strain immunity commodity duck.
Anti-avian influenza virus (H5) the HI antibody that the induction of Fig. 7, recombinant virus DEV-H5 ULwf strain immunity commercial chicken produces
Titer level figure.
Detailed description of the invention
Further illustrate the present invention below by way of specific embodiment, but scope and spirit of the present invention are not limited to listed reality
Execute example.
Recombinant bacterium pHA2 corr, Berlin, Germany Free University give, and is disclosed in document:Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant.Schumacher D, Tischer BK, Fuchs W, Osterrieder N.J Virol. 2000 Dec;74 (23):11088-98。
Duck enteritis virus Embryo Gallus domesticus low virulent strain C-KCE strain (is abbreviated as duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain), public
Open document: Li, Y., B. Huang, et al. (2009). " Molecular characterization of the
genome of duck enteritis virus." Virology 391(2): 151-161。
Embodiment 1, the structure of GFP metastasis transplanting physique grain
1. obtain GFP expression cassette
GFP expression cassette is the expression cassette of green fluorescent protein (green fluorescent protein), sequence such as SEQ
Shown in ID NO:2.GFP expression cassette contains pHCMV promoter, green fluorescence protein gene GFP and terminator sequence.With recombinant bacterium
The DNA of pHA2 corr is template, with Primer GFP-UL55 F and Primer GFP-UL55 R as primer, uses grads PCR
Method amplification GFP expression cassette.
Primer GFP-UL55 F(SEQ ID NO:4): 5 '-TATCCCGGGTTAACCGGGCTGCATCCGAT-3 '；
Primer GFP-UL55 R(SEQ ID NO:5): 5 '-ATACCCGGGCGAAGTTATGCGGCCATTTA-3 '.
The reaction system of grads PCR method is: 10 × PCR Buffer 5 μ L, dNTPs (2.5 mM each) 4 μ L,
Mg2+(25 mM) 3 μ L, each 2 μ L, ExTaq enzyme 0. 5 μ L of upstream and downstream primer (10 pmol/ μ L), DNA profiling 2 μ L, dd
H2O 31.5 μ L(PCR reagent is from Takara company).The reaction condition of grads PCR method: 94 DEG C of denaturation 3 min；94℃
Degeneration 30 sec, 52 DEG C of annealing 40sec, 72 DEG C extend 1min40 sec, circulate 10 times；Then 94 DEG C of degeneration 30 sec, 60
DEG C annealing 40sec, 72 DEG C extend 1min40 sec, circulate 27 times；Last 72 DEG C extend 10 min.Reaction takes PCR after terminating
Product carries out agarose gel electrophoresis, and amplification obtains the fragment that length is about 1500bp, standby after recovery.This sheet is found through order-checking
Segment length is 1536bp, and sequence is as shown in SEQ ID NO:3.
2. obtain upstream and downstream homology arm
Spacer sequence such as SEQ ID NO:3 between duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain LORF11 and UL55 gene
Shown in.The nucleotide fragments selecting the 263rd to 291, this spacer is fragment to be replaced.The design of upstream and downstream homology arm: from
The sequence of the 263rd the nucleotide a length of 1011bp of supreme trip of spacer is upstream homology arm H1, from the 291st of spacer the
Nucleotide to the sequence that downstream length is 1039bp is downstream homology arm H2.
With the genomic DNA of duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain as template, amplification upstream homology arm H1 and downstream respectively
Homology arm H2.The primer of amplification upstream homology arm is Primer UL55 H1 F and Primer UL55 H1 R, and amplification downstream is same
The primer of source arm is Primer UL55 H2 F and Primer UL55 H2 R.
Primer UL55 H1 F(SEQ ID NO:6): 5 '-CTAGAGCTCCGCTAATAAATAGTAACGCC-3 '；
Primer UL55 H1 R(SEQ ID NO:7): 5 '-TATCCCGGGTATTACCCAAATACCCTGTT-3 '；
Primer UL55 H2 F(SEQ ID NO:8): 5 '-ATACCCGGGAATTGTCCTAGCTTGTTCAG-3 '；
Primer UL55 H2 R(SEQ ID NO:9): 5 '-CGGAAGCTTTCTCGTGTCAGTTAAAGGTA-3 '.
Upstream homology arm H1 is identical with condition with the PCR reaction system of downstream homology arm H2.PCR reaction system is: 10 ×
PCR Buffer 5μL、dNTPs(2.5 mM each) 4μL、Mg2+(25 mM) 3 μ L, upstream and downstream primer (10 pmol/ μ L)
Each 2 μ L, ExTaq enzyme 0. 5 μ L, DNA profiling 2 μ L, dd H2O 31.5 μ L(PCR reagent is from Takara company).PCR is anti-
Answer condition: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 52 DEG C of annealing 40sec, 72 DEG C extend 1min10 sec, follow
Ring 10 times；Then 94 DEG C of degeneration 30 sec, 60 DEG C of annealing 40sec, 72 DEG C extend 1min10 sec, circulate 27 times；Last 72
DEG C extend 10 min.
After PCR reaction terminates, take PCR primer agarose gel, obtain the amplified fragments of length about 1000-1100bp, point
Hui Shou upstream and downstream homology arm pcr amplified fragment.
By the pcr amplified fragment of upstream homology arm H1 and pUC19 plasmid, application Sac I and Sma I restriction enzyme respectively
Enzyme carries out double digestion (cleavage reagent is from TaKaRa company).Enzyme action system is: Sac I 1 μ L, Sma I 1 μ L, 10 × T
Buffer 1 μ L, 100 × BSA 0.2 μ L, DNA 1 μ g, adds water and supplies volume to 20 μ L.Endonuclease reaction acts on 1 at 37 DEG C
Hour, reaction carries out electrophoresis recovery after terminating.Reapply T4 ligase (TaKaRa company) to connect.Linked system is: T4 connects
Enzyme 1 μ L, 10 × Buffer 1 μ L, pUC19 plasmid enzyme restriction product 2 μ L(200ng), upstream homology arm H1 digestion products 5 μ L
(500ng), add water and supply volume to 10 μ L.Linked system is at 4 DEG C overnight, it is thus achieved that containing the recombiant plasmid of upstream homology arm
PUC19-H1 (DEV UL55), then transformed competence colibacillus cell DH5 α.
By pcr amplified fragment and the recombiant plasmid pUC19-H1 (DEV UL55) of downstream homology arm H2, apply Sma respectively
I Yu Hind III restricted enzyme carries out double digestion, and cleavage reagent is from TaKaRa company.Enzyme action system is: Hind III
1 μ L, Sma I 1 μ L, 10 × T Buffer 1 μ L, 100 × BSA, 0.2 μ L, DNA 1 μ g, add water and supply volume to 20
μL.Enzyme action system acts on 1 hour at 37 DEG C, and reaction carries out electrophoresis recovery after terminating.Reapply T4 ligase to connect, connect reagent
From TaKaRa company.Linked system is: T4 ligase 1 μ L, 10 × Buffer 1 μ L, pUC19-H1 (DEV UL55)
Digestion products 2 μ L(200ng), upstream homology arm H2 digestion products 5 μ L(500ng), add water and supply volume to 10 μ L.Connector
Tie up to 4 DEG C overnight, it is thus achieved that the plasmid pUC19-H1-H2 (DEV UL55) containing upstream and downstream homology arm, then transformed competence colibacillus cell
DH5α。
3. build GFP gene transfer vector
Sma I restricted enzyme is applied to carry out enzyme respectively GFP expression cassette and pUC19-H1-H2 (DEV UL55) plasmid
Cutting, cleavage reagent is from TaKaRa company.Enzyme action system is: Sma I 1 μ L, 10 × T Buffer 2 μ L, 100 × BSA
0.2 μ L, DNA 1 μ g, adds water and supplies volume to 20 μ L.Enzyme action system 37 DEG C acts on 1 hour, and reaction carries out electrophoresis after terminating
Reclaim.Reapply T4 ligase to be attached.Linked system is: T4 ligase 1 μ L, 10 × Buffer 1 μ L, pUC19-
H1-H2 (DEV UL55) digestion products 2 μ L(200ng), GFP expression cassette digestion products 5 μ L(500ng), add water and supply volume
To 10 μ L.At 4 DEG C overnight, connect product is GFP gene transfer vector pDEV-GFP (UL55) (Fig. 1) to linked system.By GFP base
Because of transfer vector pDEV-GFP (UL55) transformed competence colibacillus cell DH5 α, it is thus achieved that the positive recombinant bacterium containing plasmid.Finally by base
The transfer vector pDEV-GFP (UL55) obtained because of sequence verification is correct clone.
The acquisition of embodiment 2, GFP restructuring duck enteritis virus and qualification
Take 9～10 age in days SPF Embryo Gallus domesticus, conventionally prepare and cultivate primary and second generation chick embryo fibroblast
(Primary chicken embryo fibroblasts, CEF).Take duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain by infection multiplicity
For 0.05(Multiplicity of infection, MOI) inoculation monolayer chick embryo fibroblast, cultivate about 72 h, treat 95%
Cell occurs collecting cell during pathological changes, conventionally extracts viral DNA in sick cell, carries out DNA through gel scanner
Quantitatively, DNA concentration is adjusted to 0.1～0.2 g/ L.From recombinant bacterium, transfer vector pDEV-GFP is extracted with alkaline lysis
(UL55) DNA, application Eppendorf Biophotometer instrument carries out quantitatively (or sepharose electrophoresis measurement method is quantitative),
Adjusting concentration is 700 ng/ L.Transfect according to conventional calcium phosphate infection protocol, by the DNA of 5 g pDEV-GFP (UL55)
Carry out homologous recombination with the primary CEF of DNA cotransfection of 2 g duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strains, absorb next day and cultivate
Liquid, adds the methylcellulose medium (Beijing Lv Yuan) containing 10% hyclone and covers, and continues to cultivate 24-48 h, at wavelength is
Restructuring duck enteritis virus plaque (Fig. 2) sending green fluorescence, picking green fluorescence is found under 488nm ultraviolet excitation
The plaque fresh CEF of inoculation, so repeat several circulation, until viral purification success, it is thus achieved that GFP recombinates duck viral enteritis
Virus, named rDEV-GFP strain.
Embodiment 3, avian flu virus hemagglutinin (HA) expression casette and the structure of transfer vector thereof
1. obtain avian flu virus hemagglutinin (HA) gene
Hemagglutinin (HA) gene order of H5N1 type bird flu will published on GenBank since 2010, adopts
Carry out Multiple sequence alignments with MEGA software and obtain consensus sequence.The base in each site of consensus sequence takes out present HA
Four kinds of base (A, C, G, T) medium frequencys maximums of the same site of sequence.Remove in consensus sequence HA1 Yu HA2 fragment it
Between basic amino acid cracking site, referring next to the H5N1 type avian flu virus hemagglutinin of the most popular 2.3.2.1 pedigree
(HA) gene order (GenBank:AB700635.1; JN986881.1; JN986882.1; JN646713.1;
JN646716.1; HQ020376.1; CY098758.1; JF975561.1;), optimize design and obtain avian influenza of the present invention
Poison hemagglutinin (HA) gene, sequence is the 1437th to 3131 nucleotide in SEQ ID No:1.HA expression casette
PMCMV IE promoter is the of the mouse cytomegalovirus complete genome sequence (GenBank:GU305914.1) published
The complementary series of the nucleotide sequence of 184336 to 182946.By above-mentioned pMCMV IE promoter, avian flu virus hemagglutinin
HA gene and termination signal sequence constitute avian flu virus hemagglutinin (HA) expression casette (SEQ ID No:1), and structure is shown
It is intended to as shown in Figure 3.Avian flu virus hemagglutinin (HA) expression casette obtains through synthetic.
With avian flu virus hemagglutinin (HA) the expression casette DNA of synthetic as template, with Primer HA
Cassette F and Primer HA cassette R primer, use grads PCR method amplification avian flu virus hemagglutinin (HA)
Expression casette.
Primer HA cassette F(SEQ ID NO:10): 5 '-TATCCCGGGAACTCCGCCCGTTTTATGAC-
3’；
Primer HA cassette R(SEQ ID NO:11): 5 '-ATACCCGGGTTGTCGACTCTAGAGGATCC-
3’。
The reaction system of grads PCR method is: 10 × PCR Buffer 5 μ L, dNTPs (2.5 mM each) 4 μ L,
Mg2+(25 mM) 3 μ L, each 2 μ L, ExTaq enzyme 0. 5 μ L of upstream and downstream primer (10 pmol/ μ L), DNA profiling 2 μ L, dd H2O
31.5 μ L(PCR reagent are from Takara company).Response procedures: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 48 DEG C
Annealing 40sec, 72 DEG C extend 3min40 sec, circulate 10 times；Then 94 DEG C of degeneration 30 sec, 58 DEG C of annealing 40sec, 72
DEG C extend 3min40 sec, circulate 27 times；Last 72 DEG C extend 10 min.
After reaction terminates, taking PCR primer and carry out agarose gel electrophoresis, expanding fragment length is 3357bp, reclaims amplification
Fragment.Sma I restricted enzyme is applied to carry out enzyme respectively amplified fragments and pUC19-H1-H2 (DEV UL55) plasmid DNA
Cutting, application T4 ligase is attached, it is thus achieved that avian flu virus hemagglutinin (HA) gene transfer vector pDEV-H5 (UL55).
The transfer vector pDEV-H5 (UL55) obtained finally by gene sequencing checking is correct clone.By pDEV-H5 (UL55)
Transformed competence colibacillus cell DH5 α, it is thus achieved that reconstitution cell.
Embodiment 4, the acquisition of avian flu virus hemagglutinin (HA) gene recombinaton duck enteritis virus and qualification
Conventionally prepare and cultivate primary and second generation CEF, after cell grows up to monolayer, taking GFP gene recombinaton duck sick
Viral enteritis virus rDEV-GFP strain is 0.05 inoculating cell by infection multiplicity, cultivates about 72 h, treats that pathological changes occurs in 95% cell
Time collect cell, conventionally extract viral DNA in sick cell, adjust DNA concentration to 0.1～0.2 g/ L.Use alkali
Cracking process extracts avian flu virus hemagglutinin HA gene transfer vector pDEV-H5 (UL55) from reconstitution cell, adjusts DNA concentration
To 700 ng/ L.Transfect according to conventional calcium phosphate infection protocol, by the DNA and 2 g GFP of 5 g pDEV-H5 (UL55)
The primary CEF of DNA cotransfection of gene recombinaton duck enteritis virus rDEV-GFP strain carries out homologous recombination.Next day absorbs training
Nutrient solution, adds and covers containing 10% hyclone methylcellulose medium, after continuing to cultivate 24-48 h, is that 488nm is purple at wavelength
Outer light excites lower searching not send the restructuring duck enteritis virus plaque of green fluorescence, and picking does not send the erosion of green fluorescence
Speckle inoculates fresh CEF, so repeats several circulation, until viral purification success, it is thus achieved that avian flu virus hemagglutinin (HA) gene
Restructuring duck enteritis virus DEV-H5 ULwf strain.Extract the DNA of DEV-H5 ULwf strain, by embodiment 3 method, application
Primer HA cassette F and Primer HA cassette R carries out PCR and expands HA expression cassette, sequence verification result table
Bright institute insertion sequence is consistent with composition sequence.
Embodiment 5, restructuring duck enteritis virus DEV-H5 ULwf strain express the detection of HA gene
Application indirect immunofluorescence assay (Indirect Immunofluorescence test, IIF) detection restructuring disease
The situation of HA gene is expressed in poison DEV-H5 ULwf strain strain.Use 96 orifice plates to cultivate CEF, treat that cell grows up to monolayer, take recombinant virus
DEV-H5 ULwf strain, is inoculated by the amount of every hole 50～100 plaque forming units (Plaque Forming Unit, PFU),
Cultivate 48～72 hours, after culture is applied 2% paraformaldehyde be fixed process, be firstly added H5N1 avian influenza hemagglutinin
The mice resource monoclonal antibody (Jin Sirui) of HA reacts, and the sheep anti mouse two being subsequently adding band green fluorescent label resists
(Alexa488, Invitrogen) reacts, and observes whether plaque sends green glimmering under wavelength is 488nm ultraviolet excitation
Light, sets duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain as comparison simultaneously.Result shows the plaque energy of recombinant virus DEV-H5 ULwf strain
The plaque (Fig. 4) sending fluorescence detected, and the plaque of duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain does not send green fluorescence, explanation
The HA expression casette inserted in DEV-H5 ULwf strain can high efficient expression HA antigen.
Embodiment 6, the growth characteristics of recombinant virus DEV-H5 ULwf strain
Take recombinant virus DEV-H5 ULwf strain and parent's poison duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain, carry out multistep processes growth
Kinetic measurement.Viral infection multiplicity (multiplicity of infection, MOI) by 0.01, divides two-strain
It is not inoculated in and grows up to the primary of monolayer or second generation CEF, before infection with metainfective 6h, 12h, 24h, 36h, 48h and 72h hour
Detect in culture supernatant and titer TCID of cell-associated virus50.In culture supernatant virus titer mensuration (document 1:
Wang J, Osterrieder N. Generation of an infectious clone of duck enteritis
virus and generation of a vectored DEV expressing hemagglutinin of H5N1 avian
Influenza virus [J]. Virus Research, 2,011 159 (1): 23-31.): take at the time point specified
The Supernatant samples of 0.1mL, 375G is centrifuged 5 minutes and takes supernatant and detect.Titer determination (the detection method of cell-associated virus
List of references 1): at the time point specified, the supernatant of infection cell is absorbed, after washing twice with PBS, add 1mL nutritional solution, will
Infection cell scrapes 375G after 3 freeze thawing and is centrifuged 5 minutes and takes supernatant and detect.The detection of virus titer and computational methods
(detection method list of references 1): sucking-off virus liquid after taking 10 times of dilution methods, inoculation CEF to adsorb 2 hours, adds fresh training
Nutrient solution, observed result after cultivating 7 days, all tests are repeated 3 times, and calculate TCID by Reed and Muench method50.Result such as Fig. 5, can
Consistent with parent's poison growth characteristics to find out recombinant virus DEV-H5 ULwf strain, avian flu virus hemagglutinin (HA) gene expression
The nucleotide fragments of the 263rd to 291, spacer between LORF11 and UL55 gene replaced by box, do not affect virus growth,
Breeding.
Embodiment 7, the stability of recombinant virus DEV-H5 ULwf strain
Recombinant virus DEV-H5 ULwf strain after continuous passage 40 generation, is carried out fowl to the recombinant virus in the 40th generation on CEF
The PCR detection of influenza virus hemagglutinin HA expression casette, measures sequence, uses indirect immunofluorescence assay to detect its HA simultaneously
Expression.The method that embodiment 3 is identical is pressed in PCR detection, and reaction carries out electrophoresis after terminating, can observe under ultra violet lamp
To the amplified fragments of length 3357bp, reclaim laggard row sequencing, result show with pass on front completely the same.By embodiment 5
Identical method carries out indirect immunofluorescence assay, and the situation of HA is expressed in detection recombinant virus DEV-H5 ULwf strain, and result shows
Recombinant virus plaque energy 100% detects specificity fluorescent.Result above shows that recombinant virus DEV-H5 ULwf strain can be on CEF
Stably passing on 40 generations more than, its exogenous gene does not occur to lose or variation, illustrates that recombinant virus has preferable stability.
Embodiment 8, recombinant virus DEV-H5 ULwf strain are to the safety of duck and immune efficacy
Take recombinant virus DEV-H5 ULwf strain kind poison, be inoculated in by 0.01 MOI and grow up to the primary of monolayer or second generation CEF,
Culture medium is containing 10% hyclone, the EMEM (Biochrom company) of 100U/mL penicillin and 100U/mL streptomycin.In sense
72h hour receipts poison after dye, carries out 3 freeze thawing together by upper cleer and peaceful cell culture, and then 375G is centrifuged 5 minutes, takes supernatant
Liquid is as recombinant virus DEV-H5 ULwf strain venom, rearmounted-70 DEG C of preservations of subpackage, and demarcates the TCID of virus50。TCID50It is
10-6.34/0.1mL。
Take recombinant virus DEV-H5 ULwf strain venom, by every plumage part 106 TCID50Amount, intramuscular inoculation 1 age in days health commodity
Duck 10, sets the duck of 10 identical ages in days only as comparison simultaneously, observes duck only spirit, and diet and morbidity and death condition, to connecing
After Zhong 14 days.Result shows to compare duck, and only and inoculation duck spirit is normal, and diet is normal, without falling ill and dead, and safety 100%.
Take recombinant virus DEV-H5 ULwf strain venom, by every plumage part 106 TCID50Amount, intramuscular inoculation 4 ~ 5 week old health business
Product duck 10, the duck plague virus of 3 days after inoculation, 3 weeks and 6 weeks application 1000MLD poison AV1221 strain virus liquid by force is (from China
Veterinary supervise standard by force poison) carry out protest test.Result show recombinant virus DEV-H5 ULwf strain inoculate latter 3 days,
3 weeks and 6 weeks protective rates to duck plague virus strong virus attack are 100%(Fig. 6).
Embodiment 9, recombinant virus DEV-H5 ULwf strain are to the safety of chicken and immune efficacy
Take recombinant virus DEV-H5 ULwf strain venom, by every plumage part 106 TCID50Amount, intramuscular inoculation 4 ~ 5 week old health business
Product laying hen 10, observes chicken spirit, diet and morbidity and death condition；It addition, 1,2,3,4,5 and before inoculation and after inoculation
Blood sampling in 6 weeks separates serum, and (test kit is from Harbin dimension section biotechnology to detect its anti-avian influenza virus (H5) HI antibody titer
Development company).If duck pestilence Embryo Gallus domesticus low virulent strain C-KCE strain live vaccine immune group is comparison.Result shows, recombinant virus DEV-H5
The SPF chicken of ULwf strain immune group and C-KCE strain live vaccine immune group is all without morbidity and death, and spirit and diet are all normal, inoculation
Latter 2 weeks, DEV-H5 ULwf strain immune group bird flu (H5) HI titer meansigma methods reached 4log2, reach more than acceptance line, by 4 weeks
Time reach peak value 8.1log2(Fig. 7), C-KCE strain live vaccine immune group does not detects HI antibody.Antibody acceptance line is 4log2.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>restructuring duck enteritis virus, preparation method and should
<130> 20141110
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 3371
<212> DNA
<213> ARTIFICIAL
<220>
<223>avian flu virus hemagglutinin HA expression casette sequence
<400> 1
ctagtggatc ccccaactcc gcccgtttta tgactagaac caatagtttt taatgccaaa 60
tgcactgaaa tcccctaatt tgcaaagcca aacgccccct atgtgagtaa tacggggact 120
ttttacccaa tttcccaagc ggaaagcccc ctaatacact catatggcat atgaatcagc 180
acggtcatgc actctaatgg cggcccatag ggactttcca catagggggc gttcaccatt 240
tcccagcata ggggtggtga ctcaatggcc tttacccaag tacattgggt caatgggagg 300
taagccaatg ggtttttccc attactggca agcacactga gtcaaatggg actttccact 360
gggttttgcc caagtacatt gggtcaatgg gaggtgagcc aatgggaaaa acccattgct 420
gccaagtaca ctgactcaat agggactttc caatgggttt ttccattgtt ggcaagcata 480
taaggtcaat gtgggtgagt caatagggac tttccattgt attctgccca gtacataagg 540
tcaatagggg gtgaatcaac aggaaagtcc cattggagcc aagtacactg cgtcaatagg 600
gactttccat tgggttttgc ccagtacata aggtcaatag gggatgagtc aatgggaaaa 660
acccattgga gccaagtaca ctgactcaat agggactttc cattgggttt tgcccagtac 720
ataaggtcaa tagggggtga gtcaacagga aagtcccatt ggagccaagt acattgagtc 780
aatagggact ttccaatggg ttttgcccag tacataaggt caatgggagg taagccaatg 840
ggtttttccc attactggca cgtatactga gtcattaggg actttccaat gggttttgcc 900
cagtacataa ggtcaatagg ggtgaatcaa caggaaagtc ccattggagc caagtacact 960
gagtcaatag ggactttcca ttgggttttg cccagtacaa aaggtcaata gggggtgagt1020
caatgggttt ttcccattat tggcacgtac ataaggtcaa taggggtgag tcattgggtt1080
tttccagcca atttaattaa aacgccatgt actttcccac cattgacgtc aatgggctat1140
tgaaactaat gcaacgtgac ctttaaacgg tactttccca tagctgatta atgggaaagt1200
accgttctcg agccaataca cgtcaatggg aagtgaaagg gcagccaaaa cgtaacaccg1260
ccccggtttt cccctggaaa ttccatattg gcacgcattc tattggctga gctgcgttct1320
acgtgggtat aagaggcgcg accagcgtcg gtaccgtcgc agtcttcggt ctgaccaccg1380
tagaacgcag agctcctcgc tgcaggcggc cgctctagaa ctcgtcgatc gcagcgatgg1440
agaaaatagt gcttcttttt gcaataatca gccttgttaa aagcgatcat atttgcattg1500
gttatcatgc aaataactcg acagagcagg ttgacacaat aatggaaaag aacgtgactg1560
ttacacatgc ccaagacata ctggaaaaga cacacaacgg taagctctgc gatctaaatg1620
gagtgaagcc tctgatttta aaagattgta gtgtagcagg atggctcctc ggaaatccaa1680
tgtgtgacga attcatcaat gtgccagaat ggtcttacat agtagagaag gccaatccag1740
ccaatgacct ctgttaccca gggaatttca acgattatga agaattgaaa cacctattga1800
gcaggataaa ccattttgag aaaatacaga tcatccccaa agattcttgg tcagatcatg1860
aagcctcgtt gggggtgagt gcagcatgtc cataccaggg aaactcctcc ttcttcagaa1920
atgtggtatg gcttatcaaa aaggacaatg catacccaac aataaagaaa agctacaata1980
ataccaacca agaagatctc ttggtactgt gggggattca ccatcctaat gatgaggcag2040
agcagacaag gctctatcaa aacccaacca cctatatttc cattgggaca tcaacactaa2100
accagagatt ggtaccaaaa atagccacta ggtccaaaat aaacgggcaa agtggcagga2160
tagatttctt ctggacaatt ttaaaaccga atgatgcaat ccacttcgag agtaatggaa2220
atttcattgc tccagaatat gcatacaaaa ttgtcaagaa aggagactca acaattatga2280
aaagtgaagt ggaatatggt aactgcaaca ccaggtgtca gactccaata ggggcgataa2340
actccagtat gccattccac aacatacacc ctctcaccat cggagaatgt cccaaatatg2400
tgaaatcaaa caaactagtc cttgcgactg ggctcagaaa tagtcctcaa ggagagacta2460
gaggattgtt tggagctata gcaggtttta tagagggagg atggcaggga atggtagatg2520
gttggtatgg gtaccaccac agcaatgagc aggggagtgg gtacgctgca gacaaagaat2580
ctactcaaaa ggcaatagac ggggtcacca ataaggtcaa ctcgatcatt gacaaaatga2640
acactcagtt tgaggccgta ggaagggaat ttaataactt agagaggaga atagagaatt2700
tgaacaagaa gatggaagac ggattcctag atgtctggac ttataatgct gaacttctgg2760
ttctcatgga aaatgagaga actctagact tccatgactc aaatgtcaag aacctttacg2820
acaaggtcag actacagctt aaggataatg caaaagagct gggtaacggt tgtttcgagt2880
tctatcacaa atgtgataat gaatgtatgg aaagtgtaag aaacggaacg tatgactacc2940
cgcagtattc agaagaagca agattaaaaa gagaggaaat aagtggagta aaattggaat3000
caataggaat ctaccaaata ctgtcaattt attcaacagt ggcgagttcc ctagtactgg3060
caatcatgat ggctggtcta tctttatgga tgtgttccaa cgggtcgtta cagtgcagaa3120
tttgcattta agcttgatct agagcggccg cggggatcca gacatgataa gatacattga3180
tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg3240
tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa3300
ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt cggatcctct3360
agagtcgaca a 3371
<210> 2
<211> 1535
<212> DNA
<213> ARTIFICIAL
<220>
<223>sequence of GFP expression casette
<400> 2
ttaaccgggc tgcatccgat gcaagtgtgt cgctgtcgag tttaaacatg catagttatt 60
aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 120
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 180
taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 240
agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 300
cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 360
tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 420
tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 480
gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 540
caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 600
aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc 660
caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 720
ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 780
ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 840
caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 900
gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 960
cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac1020
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg1080
gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa1140
gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct1200
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa1260
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat1320
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa1380
gtccggactc agatccaccg gatctagata actgatcata atcagccata ccacatttgt1440
agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat1500
gaatgcaatt gttgttgtta acttgtttat tgcag 1535
<210> 3
<211> 466
<212> DNA
<213>duck enteritis virus Embryo Gallus domesticus low virulent strain C-KCE
<400> 3
catttaatat accggtttta acttttgacg tctttcacat tacttaaata aaataaatat 60
gattaaacga atcagtctcg gctcttgact cttgttttga gttatagtgg ggaagcagac 120
tatgtaaaga cagtcgacgg actgccagtg aacgctgaac aagctaggac aattacgaga 180
ttgtctagct tgttcagcgt gttttattac ccaaataccc tgttagtagc ttgggtcttt 240
actttttgcg cggaacattg tccagatatc atctacgttt caatgaaatt ctccaaatgt 300
aaccaatgca actacaaata taccattaac acaatagtgt taactctacc tccaattcag 360
atcagactag agcacttcca ggcggtactg ttcgatcttt actattaaaa aatcaatttc 420
ttatggtttt aataaaacgc tttattacat tgtagtgtaa caagac 466
<210> 4
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer GFP-UL55 F
<400> 4
tatcccgggt taaccgggct gcatccgat 29
<210> 5
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer GFP-UL55 R
<400> 5
atacccgggc gaagttatgc ggccattta 29
<210> 6
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer UL55 H1 F
<400> 6
ctagagctcc gctaataaat agtaacgcc 29
<210> 7
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer UL55 H1 R
<400> 7
tatcccgggt attacccaaa taccctgtt 29
<210> 8
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer UL55 H2 F
<400> 8
atacccggga attgtcctag cttgttcag 29
<210> 9
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer UL55 H2 R
<400> 9
cggaagcttt ctcgtgtcag ttaaaggta 29
<210> 10
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer HA cassette F
<400> 10
tatcccggga actccgcccg ttttatgac 29
<210> 11
<211> 29
<212> DNA
<213> ARTIFICIAL
<220>
<223> Primer HA cassette R
<400> 11
atacccgggt tgtcgactct agaggatcc 29
Claims (7)
1. avian influenza virus hemagglutinin gene, its nucleotides sequence is classified as the 1437th to 3131 nucleotide of SEQ ID NO:1.
2. restructuring duck enteritis virus, between LORF11 and the UL55 gene of duck enteritis virus genome between
Septal area inserts the expression cassette containing gene described in claim 1.
Recombinate the most according to claim 2 duck enteritis virus, it is characterised in that the promoter in described expression cassette is
PMCMV IE promoter.
4. according to duck enteritis virus of recombinating described in Claims 2 or 3, it is characterised in that described expression cassette substituted for UL55
And the nucleotide fragments of the 263rd to 291, the spacer between LORF11 gene.
5. recombinate described in claim 2 construction method of duck enteritis virus, use methods of homologous recombination to contain and has the right
Require that described in 1, the expression cassette of gene inserts the interval between LORF11 and the UL55 gene of duck enteritis virus genome
District.
Recombinate the most according to claim 5 the construction method of duck enteritis virus, it is characterised in that containing claim 1
The expression cassette of described gene inserts the method for duck enteritis virus and comprises the steps:
(1) fragment to be replaced in the spacer between UL55 and the LORF11 gene of duck enteritis virus genome is determined,
Expand the nucleotide fragments of fragment upstream and downstream to be replaced respectively, obtain upstream homology arm and downstream homology arm；To express green
Described in the expression cassette of color fluorescin and expression claim 1, the expression cassette of gene inserts upstream homology arm and downstream homology respectively
Between arm, respectively obtain homologous recombination fragment A and homologous recombination fragment B；
(2) genomic DNA of duck enteritis virus and homologous recombination fragment A carry out homologous recombination, select under ultraviolet light
Send the recombinant virus of green fluorescence, it is thus achieved that recombinant virus C；
(3) homologous recombination fragment B carries out homologous recombination with the genomic DNA of recombinant virus C, selects and can not send under ultraviolet light
The recombinant virus of green fluorescence, the restructuring duck viral enteritis obtaining carrying avian influenza virus hemagglutinin gene expression cassette is sick
Poison.
7. with the duck enteritis virus live vaccine as active component of recombinating described in any one of 2-3 of claim.
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| CN116656630B (en) * | 2023-07-27 | 2023-10-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant duck plague virus vaccine and construction method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559610A (en) * | 2010-12-08 | 2012-07-11 | 中国农业科学院哈尔滨兽医研究所 | Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof |
| CN103881981A (en) * | 2013-08-26 | 2014-06-25 | 华中农业大学 | Living-vector vaccine of H5N1 subtype of avian influenza virus and duck enteritis virus |
-
2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559610A (en) * | 2010-12-08 | 2012-07-11 | 中国农业科学院哈尔滨兽医研究所 | Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof |
| CN103881981A (en) * | 2013-08-26 | 2014-06-25 | 华中农业大学 | Living-vector vaccine of H5N1 subtype of avian influenza virus and duck enteritis virus |
Non-Patent Citations (4)
| Title |
|---|
| "A Duck Enteritis Virus-Vectored Bivalent Live Vaccine Provides Fast and Complete Protection against H5N1 Avian Influenza Virus Infection in Ducks";Jinxiong Liu 等;《Journal of Virology》;20110824;第85卷(第21期);第10989-10998页 * |
| "Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens";Jichun Wang;《Virology Journal》;20150813;第12卷(第126期);第1-14页 * |
| "GenBank Accession No：KP019932.1";Wang,J.等;《GenBank》;20150517;第1-2页 * |
| "Generation of an infectious clone of duck enteritis virus (DEV) and of a vectored DEV expressing hemagglutinin of H5N1 avian influenza virus";Jichun Wang 等;《Virus Research》;20110428;第159卷(第1期);第23-31页 * |
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