CN104357460A - Recombinant duck viral enteritis virus, preparation method and applications - Google Patents
Recombinant duck viral enteritis virus, preparation method and applications Download PDFInfo
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Abstract
The invention provides a recombinant duck viral enteritis virus, a preparation method and applications, belonging to the field of animal medicine vaccines. The avian influenza virus haemagglutinin gene is 1437th to 3131th nucleotide with the nucleotide sequence of SEQ ID NO:1. According to the recombinant duck viral enteritis virus, an expression cassette containing the avian influenza virus haemagglutinin gene is inserted in a spacer region between LORF11 and UL55 genes of duck viral enteritis virus genome. As a vaccine active ingredient, the avian influenza virus haemagglutinin gene can achieve better cross protection effect. The recombinant duck viral enteritis virus can stably and efficiently express the avian influenza virus haemagglutinin gene, virus is stable and grows well, the prepared live vaccine has higher protective efficacy on duck plague and avian influenza, higher avian influenza antibody is generated, and the duration period of the antibody is long.
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 plague reported first sees earlier 1900s, current whole world great majority support the report that there is this disease in duck country, caused by duck plague virus (also known as duck enteritis virus), its M & M can reach 100%, duck plague virus can form persistent infection at the gasserian ganglion of duck, duck plague attenuated live vaccines is widely used in the immunoprophylaxis of duck plague, effectively can stop morbidity.
Bird flu (H5N1) is a kind of Infectious Diseases of duck and chicken, is not only one of most important transmissible disease of current threat whole world aviculture cultivation safety, and high to the lethality rate of the mankind, constitutes a serious threat to mankind's public health security.To the prevention and control measure of aviculture bird flu, the method based on Pathogen Purification is taked in Countries and area, as European Union's most countries, achieves good effect.The comprehensive anti-measure processed based on compulsory immunization is taked in the prevention and control of China to aviculture bird flu (H5N1), effectively controls the propagation impetus of this disease in China poultry.Aquatic bird based on duck is that the important of bird flu is carried and store host
,in the propagation and mutation process of bird flu (H5N1) virus, play key player, therefore, the anti-system of the bird flu (H5N1) of duck is the key of these sick prevention and control.China at present to poultry compulsory immunization adopt be inactivated virus vaccine, although this vaccine has the advantages such as security is good, but also show in practical application following several in weak point, one, inactivated vaccine will carry out large-scale virus culture aborning, there is the biological safety hidden danger of diffusion virus.Two, its immunizing power produces comparatively slow, and forms lump at injection site, and for the production cycle, the application of shorter fast-growing type poultry is restricted.Three, though inactivated vaccine can produce efficient humoral immunization, cellular immunity is not enough, virus thoroughly cannot be cleared out of body, thus may form inapparent infection.Especially the immunogenicity of inactivated vaccine to duck is poor, and immune effect is not good.
Summary of the invention
The object of this invention is to provide avian influenza virus hemagglutinin gene, this gene expression product, as vaccine activity composition, can reach good cross-protection.
Another object of the present invention is to provide restructuring duck enteritis virus, can express avian influenza virus hemagglutinin gene, viral steady, well-grown by stability and high efficiency.
Another object of the present invention is to provide the construction process of described restructuring duck enteritis virus, and the method is simple, efficiency is high.
The living vaccine that it is activeconstituents that another object of the present invention is to provide with described restructuring duck enteritis virus, after this live vaccination, higher to the Vaccine effectiveness of duck plague and bird flu, create higher avian influenza antibody, and the antibody extended period is longer.
Object of the present invention adopts following technical scheme to realize.
The invention provides avian influenza virus hemagglutinin gene, its nucleotides sequence is classified as the 1437 to 3131 Nucleotide of SEQ ID NO:1.
The present invention also provides restructuring duck enteritis virus, inserts the expression cassette containing avian influenza virus hemagglutinin gene described in claim 1 in the transcribed spacer between genomic LORF11 and the UL55 gene of duck enteritis virus.
In preferred technical scheme, the promotor in described expression cassette is pMCMV IE promotor.
In preferred technical scheme, described expression cassette substituted for the nucleotide fragments of the 263 to 291, transcribed spacer between UL55 and LORF11 gene.
The present invention also provides the construction process of described restructuring duck enteritis virus, adopts methods of homologous recombination that the expression cassette containing described avian influenza virus hemagglutinin gene is inserted the transcribed spacer between genomic LORF11 and the UL55 gene of duck enteritis virus.
In the present invention, the method that the expression cassette containing described avian influenza virus hemagglutinin gene inserts duck enteritis virus comprises the steps:
(1) determine fragment to be replaced in the transcribed spacer between duck enteritis virus genomic UL55 and LORF11 gene, the nucleotide fragments of the fragment upstream and downstream to be replaced that increases respectively, obtains upstream homology arm and downstream homology arm; The expression cassette of gene described in the expression cassette of expressing green fluorescent protein and expression claim 1 is inserted between upstream homology arm and downstream homology arm respectively, obtains homologous recombination Segment A and homologous recombination fragment B respectively;
(2) genomic dna of duck enteritis virus and homologous recombination Segment A carry out homologous recombination, select the recombinant virus sending green fluorescence under ultraviolet light, obtain recombinant virus C;
(3) genomic dna of homologous recombination fragment B and recombinant virus C carries out homologous recombination, selects the recombinant virus that can not send green fluorescence under ultraviolet light, obtains the restructuring duck enteritis virus carrying avian influenza virus hemagglutinin gene expression cassette.
The living vaccine that it is activeconstituents that the present invention also provides with described restructuring duck enteritis virus.
The original strain of restructuring duck enteritis virus of expressing avian flu virus hemagglutinin (HA) gene can be duck enteritis virus chicken embryo low virulent strain or other duck enteritis virus low virulent strains or through the engineered strain of artificial gene and virulent strain.
The living vaccine that the present invention is activeconstituents with described restructuring duck enteritis virus (DEV-H5 ULwf strain), the immunoprophylaxis of the duck caused for duck plague virus and avian influenza virus, goose and kind immunoprophylaxis of the Anseriformes such as duck and wild duck bird transmissible disease and the bird flu of chicken.
Living vaccine of the present invention is using described restructuring duck enteritis virus DEV-H5 ULwf strain as kind of a poison, and the production technique with reference to duck viral enteritis living vaccine is produced.Can add immunological adjuvant and vaccine excipients etc. in living vaccine of the present invention, those skilled in the art can easily produce.
The present invention passes through the expression product of the avian influenza virus hemagglutinin gene optimized as vaccine activity composition, can reach good cross-protection.
The present invention, by inserting avian influenza virus hemagglutinin gene expression cassette in duck enteritis virus genome, obtains restructuring duck enteritis virus.This restructuring duck enteritis virus energy stability and high efficiency expresses avian influenza virus hemagglutinin gene, viral steady, well-grown.
The present invention recombinates, and the construction process of duck enteritis virus is simple, efficiency is high.
The living vaccine that the present invention is activeconstituents with described restructuring duck enteritis virus, after inoculating chicken and duck, security is good, all very high to the Vaccine effectiveness of duck plague and bird flu, especially creates higher and that the extended period is longer avian influenza antibody.
Accompanying drawing explanation
The structure diagram of Fig. 1, GFP gene transfer vector.
Fig. 2, GFP restructuring duck enteritis virus rDEV-GFP plaque fluorescence photo.
Fig. 3, avian flu virus hemagglutinin (HA) expression casette schematic diagram.
The indirect immunofluorescence figure of Fig. 4, recombinant virus DEV-H5 ULwf strain expression HA.
Fig. 5, recombinant virus DEV-H5 ULwf strain growth characteristics graphic representation, wherein DEV C-KCE is the abbreviation of duck enteritis virus chicken embryo low virulent strain C-KCE strain.
Vaccine effectiveness figure to duck plague after Fig. 6, recombinant virus DEV-H5 ULwf strain immunity commodity duck.
Anti-avian influenza virus (H5) the HI antibody titer level view that Fig. 7, the induction of recombinant virus DEV-H5 ULwf strain immunity commercial chicken produce.
Embodiment
Further illustrate the present invention below by way of specific embodiment, but scope and spirit of the present invention are not limited to listed embodiment.
Recombinant bacterium pHA2 corr; Berlin, Germany Free University is so kind as to give; be 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 chicken embryo low virulent strain C-KCE strain (being abbreviated as duck plague chicken embryo low virulent strain C-KCE strain), open source literature: Li, Y., B. Huang, et al. (2009). " Molecular characterization of the genome of duck enteritis virus. "
virology391 (2): 151-161.
The structure of embodiment 1, GFP metastasis transplanting physique grain
1. obtain GFP expression cassette
GFP expression cassette is the expression cassette of green fluorescent protein (green fluorescent protein), and sequence is as shown in SEQ ID NO:2.GFP expression cassette contains pHCMV promotor, green fluorescence protein gene GFP and terminator sequence.With the DNA of recombinant bacterium pHA2 corr for template, with Primer GFP-UL55 F and Primer GFP-UL55 R for primer, adopt 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, Mg
2+(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 H
2o 31.5 μ L(PCR reagent is from Takara company).The reaction conditions of grads PCR method: 94 DEG C of denaturation 3 min; 94 DEG C of sex change 30 sec, 52 DEG C of annealing 40sec, 72 DEG C extend 1min40 sec, circulate 10 times; Then 94 DEG C of sex change 30 sec, 60 DEG C of annealing 40sec, 72 DEG C extend 1min40 sec, circulate 27 times; Last 72 DEG C extend 10 min.Get PCR primer after reaction terminates and carry out agarose gel electrophoresis, amplification obtains the fragment that length is about 1500bp, for subsequent use after reclaiming.Find that this fragment length is 1536bp through order-checking, sequence is as shown in SEQ ID NO:3.
2. obtain upstream and downstream homology arm
Spacer sequence between duck plague chicken embryo low virulent strain C-KCE strain LORF11 and UL55 gene is as shown in SEQ ID NO:3.The nucleotide fragments of the 263 to 291, this transcribed spacer is selected to be fragment to be replaced.The design of upstream and downstream homology arm: the sequence being 1011bp from the 263rd the supreme brigade commander's degree of Nucleotide of transcribed spacer is upstream homology arm H1, and the sequence being 1039bp from the 291st Nucleotide to downstream length of transcribed spacer is downstream homology arm H2.
With the genomic dna of duck plague chicken embryo low virulent strain C-KCE strain for template, increase upstream homology arm H1 and downstream homology arm H2 respectively.The primer of amplification upstream homology arm is Primer UL55 H1 F and Primer UL55 H1 R, and the primer of amplification downstream homology 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 5 μ L, dNTPs (2.5 mM each) 4 μ L, Mg
2+(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 H
2o 31.5 μ L(PCR reagent is from Takara company).PCR reaction conditions: 94 DEG C of denaturation 3 min; 94 DEG C of sex change 30 sec, 52 DEG C of annealing 40sec, 72 DEG C extend 1min10 sec, circulate 10 times; Then 94 DEG C of sex change 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, get PCR primer sepharose, obtain the amplified fragments that length is about 1000-1100bp, reclaim upstream and downstream homology arm pcr amplified fragment respectively.
By the pcr amplified fragment of upstream homology arm H1 and pUC19 plasmid, application Sac I and Sma I restriction enzyme carry out double digestion (cleavage reagent is from TaKaRa company) respectively.The enzyme system of cutting is: Sac I 1 μ L, Sma I 1 μ L, and 10 × T Buffer 1 μ L, 100 × BSA 0.2 μ L, DNA 1 μ g, adds water and supply volume to 20 μ L.Endonuclease reaction, 37 DEG C of effects 1 hour, carries out electrophoresis recovery after reaction terminates.Apply T4 ligase enzyme (TaKaRa company) again to connect.Linked system is: T4 ligase 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 spent the night at 4 DEG C, obtains the recombinant plasmid pUC19-H1 (DEV UL55) containing upstream homology arm, then transformed competence colibacillus cell DH5 α.
By the pcr amplified fragment of downstream homology arm H2 and recombinant plasmid pUC19-H1 (DEV UL55), application Sma I and Hind III restriction enzyme carry out double digestion respectively, and cleavage reagent is from TaKaRa company.The enzyme system of cutting is: Hind III 1 μ L, Sma I 1 μ L, 10 × T Buffer 1 μ L, 100 × BSA, and 0.2 μ L, DNA 1 μ g, adds water and supply volume to 20 μ L.Enzyme cuts system 37 DEG C of effects 1 hour, carries out electrophoresis recovery after reaction terminates.Apply T4 ligase enzyme again to connect, connect reagent from TaKaRa company.Linked system is: T4 ligase enzyme 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.Linked system is spent the night at 4 DEG C, obtains the plasmid pUC19-H1-H2 (DEV UL55) containing upstream and downstream homology arm, then transformed competence colibacillus cell DH5 α.
3. build GFP gene transfer vector
GFP expression cassette and pUC19-H1-H2 (DEV UL55) plasmid are applied respectively Sma I restriction enzyme to carry out enzyme and cut, cleavage reagent is from TaKaRa company.The enzyme system of cutting is: Sma I 1 μ L, 10 × T Buffer 2 μ L, and 100 × BSA 0.2 μ L, DNA 1 μ g, adds water and supply volume to 20 μ L.Enzyme cuts system 37 DEG C effect 1 hour, carries out electrophoresis recovery after reaction terminates.Apply T4 ligase enzyme again to connect.Linked system is: T4 ligase enzyme 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.Linked system is spent the night at 4 DEG C, and connecting product is GFP gene transfer vector pDEV-GFP (UL55) (Fig. 1).By GFP gene transfer vector pDEV-GFP (UL55) transformed competence colibacillus cell DH5 α, obtain the positive recombinant bacterium containing plasmid.Verify that the transfer vector pDEV-GFP (UL55) obtained is correct clone finally by gene sequencing.
The acquisition of embodiment 2, GFP restructuring duck enteritis virus and qualification
Get 9 ~ 10 age in days SPF chicken embryos, conventionally prepare and cultivate primary and secondary to chick embryo fibroblast (Primary chicken embryo fibroblasts, CEF).Getting duck plague chicken embryo low virulent strain C-KCE strain is 0.05(Multiplicity of infection by infection multiplicity, MOI) individual layer chick embryo fibroblast is inoculated, cultivate about 72 h, cell is collected when pathology appears in 95% cell, conventionally extract viral DNA in sick cell, DNA is carried out quantitatively, adjustment DNA concentration to 0.1 ~ 0.2 μ g/ μ L through gel scanner.From recombinant bacterium, extract the DNA of transfer vector pDEV-GFP (UL55) with alkaline lysis, application Eppendorf Biophotometer instrument carries out quantitatively (or agarose electrophoresis method of masurement is quantitative), and adjustment concentration is 700 ng/ μ L.Conveniently calcium phosphate transfection method carries out transfection, the primary CEF of DNA cotransfection of the DNA of 5 μ g pDEV-GFP (UL55) and 2 μ g duck plague chicken embryo low virulent strain C-KCE strains is carried out homologous recombination, next day absorbs nutrient solution, the methylcellulose medium (Beijing Lv Yuan) added containing 10% foetal calf serum covers, continue to cultivate 24-48 h, the restructuring duck enteritis virus plaque (Fig. 2) sending green fluorescence is found under wavelength is 488nm ultraviolet excitation, the plaque of picking green fluorescence inoculates fresh CEF, so repeat several circulation, until viral purification success, obtain GFP restructuring duck enteritis virus, called after rDEV-GFP strain.
The structure of embodiment 3, avian flu virus hemagglutinin (HA) expression casette and transfer vector thereof
1. obtain avian flu virus hemagglutinin (HA) gene
By hemagglutinin (HA) gene order of H5N1 type bird flu published on GenBank since 2010, adopt MEGA software to carry out Multiple sequence alignments and obtain consensus sequence.Four kinds of base (A, C, G, T) medium frequencys that the base in each site of consensus sequence takes out the same site of present HA sequence are maximum.Remove the basic aminoacids cracking site between HA1 and HA2 fragment in consensus sequence, then with reference to H5N1 type avian flu virus hemagglutinin (HA) gene order (GenBank:AB700635.1 of recently popular 2.3.2.1 pedigree; JN986881.1; JN986882.1; JN646713.1; JN646716.1; HQ020376.1; CY098758.1; JF975561.1; ), optimization design obtains avian flu virus hemagglutinin of the present invention (HA) gene, and sequence is the 1437th to 3131 Nucleotide in SEQ ID No:1.The pMCMV IE promotor of HA expression casette is the complementary sequence of the nucleotide sequence of the 184336 to 182946 of the mouse cytomegalovirus complete genome sequence (GenBank:GU305914.1) published.Constitute avian flu virus hemagglutinin (HA) expression casette (SEQ ID No:1) by above-mentioned pMCMV IE promotor, avian flu virus hemagglutinin HA gene and termination signal sequence, structural representation as shown in Figure 3.Avian flu virus hemagglutinin (HA) expression casette obtains through synthetic.
With the avian flu virus hemagglutinin of synthetic (HA) expression casette DNA for template, with Primer HA cassette F and Primer HA cassette R primer, adopt 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, Mg
2+(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 H
2o 31.5 μ L(PCR reagent is from Takara company).Response procedures: 94 DEG C of denaturation 3 min; 94 DEG C of sex change 30 sec, 48 DEG C of annealing 40sec, 72 DEG C extend 3min40 sec, circulate 10 times; Then 94 DEG C of sex change 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, get PCR primer and carry out agarose gel electrophoresis, expanding fragment length is 3357bp, reclaims amplified fragments.Amplified fragments and pUC19-H1-H2 (DEV UL55) plasmid DNA are applied respectively Sma I restriction enzyme to carry out enzyme and cut, application T4 ligase enzyme connects, and obtains avian flu virus hemagglutinin (HA) gene transfer vector pDEV-H5 (UL55).Verify that the transfer vector pDEV-H5 (UL55) obtained is correct clone finally by gene sequencing.By pDEV-H5 (UL55) transformed competence colibacillus cell DH5 α, obtain reconstitution cell.
The acquisition of embodiment 4, avian flu virus hemagglutinin (HA) gene recombination duck enteritis virus and qualification
Conventionally prepare and cultivate primary and secondary to CEF, after cell grows up to individual layer, getting GFP gene recombination duck enteritis virus rDEV-GFP strain is 0.05 inoculating cell by infection multiplicity, cultivate about 72 h, cell is collected when pathology appears in 95% cell, conventionally extract viral DNA in sick cell, adjustment DNA concentration to 0.1 ~ 0.2 μ g/ μ L.From reconstitution cell, avian flu virus hemagglutinin HA gene transfer vector pDEV-H5 (UL55) is extracted, adjustment DNA concentration to 700 ng/ μ L with alkaline lysis.Conveniently calcium phosphate transfection method carries out transfection, and the primary CEF of DNA cotransfection of the DNA of 5 μ g pDEV-H5 (UL55) and 2 μ g GFP gene recombination duck enteritis virus rDEV-GFP strains is carried out homologous recombination.Next day absorbs nutrient solution, add and cover containing 10% foetal calf serum methylcellulose medium, after continuing to cultivate 24-48 h, the restructuring duck enteritis virus plaque not sending green fluorescence is found under wavelength is 488nm ultraviolet excitation, the plaque that picking does not send green fluorescence inoculates fresh CEF, so repeat several circulation, until viral purification success, obtain avian flu virus hemagglutinin (HA) gene recombination 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 amplification HA expression cassette, and sequence verification result shows that institute's 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) detects the situation that HA gene is expressed in recombinant virus DEV-H5 ULwf strain strain.96 orifice plates are adopted to cultivate CEF, treat that cell grows up to individual layer, get recombinant virus DEV-H5 ULwf strain, by 50 ~ 100, every hole plaque forming unit (Plaque Forming Unit, PFU) amount inoculation, cultivate 48 ~ 72 hours, apply after 2% paraformaldehyde is fixed process to culture, first the mouse resource monoclonal antibody (Jin Sirui) adding H5N1 avian influenza hemagglutinin HA reacts, then anti-(the Alexa488 of sheep anti mouse two of band green fluorescent label is added, Invitrogen) react, under wavelength is 488nm ultraviolet excitation, observes plaque whether sends green fluorescence, set duck plague chicken embryo low virulent strain C-KCE strain as contrast simultaneously.Result shows that the plaque of recombinant virus DEV-H5 ULwf strain can detect the plaque (Fig. 4) sending fluorescence, and the plaque of duck plague chicken embryo low virulent strain C-KCE strain does not send green fluorescence, the HA expression casette energy high expression HA antigen inserted in DEV-H5 ULwf strain is described.
The growth characteristics of embodiment 6, recombinant virus DEV-H5 ULwf strain
Get recombinant virus DEV-H5 ULwf strain and parent's poison duck plague chicken embryo low virulent strain C-KCE strain, carry out the detection of multistep processes growth kinetics.Virus infection plural number (multiplicity of infection by 0.01, MOI), two-strain is inoculated in respectively and grows up to the primary or secondary to CEF of individual layer, before infection with metainfective 6h, 12h, within 24h, 36h, 48h and 72h hour, detect in culture supernatant and the TCID that tires of cell-associated virus
50.Mensuration (the document 1:Wang J of virus titer in culture supernatant, 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, 2011 159 (1): 23-31.): the Supernatant samples getting 0.1mL at the time point of specifying, 375G gets supernatant in centrifugal 5 minutes and detects.The titer determination (detection method reference 1) of cell-associated virus: the supernatant of cells infected is absorbed at the time point of specifying, add 1mL nutritive medium after washing twice with PBS, cells infected is scraped 375G after 3 freeze thawing and within centrifugal 5 minutes, get supernatant and detect.The detection of virus titer and method of calculation (detection method reference 1): take 10 times of dilution methods, inoculation CEF adsorbs sucking-off virus liquid after 2 hours, adds fresh nutrient solution, cultivates observations after 7 days, all test repetitions 3 times, calculate TCID by Reed and Muench method
50.Result is as Fig. 5, can find out that recombinant virus DEV-H5 ULwf strain is consistent with parent's poison growth characteristics, avian flu virus hemagglutinin (HA) expression casette replaces the nucleotide fragments of the 263 to 291, transcribed spacer between LORF11 and UL55 gene, does not affect the growth of virus, breeding.
The stability of embodiment 7, recombinant virus DEV-H5 ULwf strain
Recombinant virus DEV-H5 ULwf strain after continuous passage 40 generation, is carried out the PCR detection of avian flu virus hemagglutinin HA expression casette, measured sequence, adopt indirect immunofluorescence assay to detect the expression of its HA simultaneously on CEF to the recombinant virus in the 40th generation.PCR detects by the identical method of embodiment 3, carries out electrophoresis, can observe the amplified fragments of length 3357bp, reclaim laggard row sequencing under ultra violet lamp after reaction terminates, result show with go down to posterity before completely the same.The method identical by embodiment 5 carries out indirect immunofluorescence assay, and detect the situation that HA is expressed in recombinant virus DEV-H5 ULwf strain, result shows that recombinant virus plaque energy 100% detects specificity fluorescent.Above result shows that recombinant virus DEV-H5 ULwf strain can stablize 40 generations of going down to posterity more than on CEF, and its foreign gene does not occur to lose or variation, illustrates that recombinant virus has good stability.
Embodiment 8, recombinant virus DEV-H5 ULwf strain are to the security of duck and immune efficacy
Get recombinant virus DEV-H5 ULwf strain kind poison, be inoculated in grow up to the primary or secondary to CEF of individual layer by 0.01 MOI, substratum is for containing 10% foetal calf serum, the EMEM (Biochrom company) of 100U/mL penicillin and 100U/mL Streptomycin sulphate.Within after infection 72h hour, receive poison, upper cleer and peaceful cell culture is carried out 3 freeze thawing together, then centrifugal 5 minutes of 375G, get supernatant liquor as recombinant virus DEV-H5 ULwf strain venom, rearmounted-70 DEG C of preservations of packing, and demarcate the TCID of virus
50.TCID
5010
-6.34/ 0.1mL.
Get recombinant virus DEV-H5 ULwf strain venom, by every plumage part 10
6tCID
50amount, the healthy commodity duck 10 of intramuscular inoculation 1 age in days, the duck of simultaneously setting 10 identical ages in days is only contrast, observes duck only spiritual, and diet and morbidity and death condition, to inoculating latter 14 days.Result show contrast duck only and inoculation duck only spirit is normal, diet is normal, without morbidity and dead, security 100%.
Get recombinant virus DEV-H5 ULwf strain venom, by every plumage part 10
6tCID
50amount, 4 ~ 5 weeks age of intramuscular inoculation healthy commodity duck 10,3 days after inoculation, 3 weeks and within 6 weeks, apply 1000MLD the strong malicious AV1221 strain virus liquid of duck plague virus (from Chinese animal doctor supervise standard malicious by force) carry out protest test.Result shows that recombinant virus DEV-H5 ULwf strain inoculation is 100%(Fig. 6 to the protection ratio of duck plague virus strong virus attack in latter 3 days, 3 weeks and 6 weeks).
Embodiment 9, recombinant virus DEV-H5 ULwf strain are to the security of chicken and immune efficacy
Get recombinant virus DEV-H5 ULwf strain venom, by every plumage part 10
6tCID
50amount, 4 ~ 5 weeks age of intramuscular inoculation, healthy commodity egg 10, observed chicken spiritual, diet and morbidity and death condition; In addition, before inoculation and after inoculation, 1,2,3,4,5 and 6 week blood sampling separation of serum, detects its anti-avian influenza virus (H5) HI antibody titer (test kit is from Harbin Wei Ke biotechnology development company).If duck plague chicken embryo low virulent strain C-KCE strain living vaccine immune group is contrast.Result shows, the SPF chicken of recombinant virus DEV-H5 ULwf strain immune group and C-KCE strain living vaccine immune group all without morbidity with dead, spirit and diet are all normal, latter 2 weeks of inoculation, and DEV-H5 ULwf strain immune group bird flu (H5) HI mean value of tiring reaches 4log
2, reach more than acceptance line, reach peak value 8.1log to when 4 weeks
2(Fig. 7), C-KCE strain living vaccine immune group does not detect HI antibody.Antibody acceptance line is 4log2.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> recombinate 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 gggggtgagt 1020
caatgggttt ttcccattat tggcacgtac ataaggtcaa taggggtgag tcattgggtt 1080
tttccagcca atttaattaa aacgccatgt actttcccac cattgacgtc aatgggctat 1140
tgaaactaat gcaacgtgac ctttaaacgg tactttccca tagctgatta atgggaaagt 1200
accgttctcg agccaataca cgtcaatggg aagtgaaagg gcagccaaaa cgtaacaccg 1260
ccccggtttt cccctggaaa ttccatattg gcacgcattc tattggctga gctgcgttct 1320
acgtgggtat aagaggcgcg accagcgtcg gtaccgtcgc agtcttcggt ctgaccaccg 1380
tagaacgcag agctcctcgc tgcaggcggc cgctctagaa ctcgtcgatc gcagcgatgg 1440
agaaaatagt gcttcttttt gcaataatca gccttgttaa aagcgatcat atttgcattg 1500
gttatcatgc aaataactcg acagagcagg ttgacacaat aatggaaaag aacgtgactg 1560
ttacacatgc ccaagacata ctggaaaaga cacacaacgg taagctctgc gatctaaatg 1620
gagtgaagcc tctgatttta aaagattgta gtgtagcagg atggctcctc ggaaatccaa 1680
tgtgtgacga attcatcaat gtgccagaat ggtcttacat agtagagaag gccaatccag 1740
ccaatgacct ctgttaccca gggaatttca acgattatga agaattgaaa cacctattga 1800
gcaggataaa ccattttgag aaaatacaga tcatccccaa agattcttgg tcagatcatg 1860
aagcctcgtt gggggtgagt gcagcatgtc cataccaggg aaactcctcc ttcttcagaa 1920
atgtggtatg gcttatcaaa aaggacaatg catacccaac aataaagaaa agctacaata 1980
ataccaacca agaagatctc ttggtactgt gggggattca ccatcctaat gatgaggcag 2040
agcagacaag gctctatcaa aacccaacca cctatatttc cattgggaca tcaacactaa 2100
accagagatt ggtaccaaaa atagccacta ggtccaaaat aaacgggcaa agtggcagga 2160
tagatttctt ctggacaatt ttaaaaccga atgatgcaat ccacttcgag agtaatggaa 2220
atttcattgc tccagaatat gcatacaaaa ttgtcaagaa aggagactca acaattatga 2280
aaagtgaagt ggaatatggt aactgcaaca ccaggtgtca gactccaata ggggcgataa 2340
actccagtat gccattccac aacatacacc ctctcaccat cggagaatgt cccaaatatg 2400
tgaaatcaaa caaactagtc cttgcgactg ggctcagaaa tagtcctcaa ggagagacta 2460
gaggattgtt tggagctata gcaggtttta tagagggagg atggcaggga atggtagatg 2520
gttggtatgg gtaccaccac agcaatgagc aggggagtgg gtacgctgca gacaaagaat 2580
ctactcaaaa ggcaatagac ggggtcacca ataaggtcaa ctcgatcatt gacaaaatga 2640
acactcagtt tgaggccgta ggaagggaat ttaataactt agagaggaga atagagaatt 2700
tgaacaagaa gatggaagac ggattcctag atgtctggac ttataatgct gaacttctgg 2760
ttctcatgga aaatgagaga actctagact tccatgactc aaatgtcaag aacctttacg 2820
acaaggtcag actacagctt aaggataatg caaaagagct gggtaacggt tgtttcgagt 2880
tctatcacaa atgtgataat gaatgtatgg aaagtgtaag aaacggaacg tatgactacc 2940
cgcagtattc agaagaagca agattaaaaa gagaggaaat aagtggagta aaattggaat 3000
caataggaat ctaccaaata ctgtcaattt attcaacagt ggcgagttcc ctagtactgg 3060
caatcatgat ggctggtcta tctttatgga tgtgttccaa cgggtcgtta cagtgcagaa 3120
tttgcattta agcttgatct agagcggccg cggggatcca gacatgataa gatacattga 3180
tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 3240
tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 3300
ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt cggatcctct 3360
agagtcgaca a 3371
<210> 2
<211> 1535
<212> DNA
<213> ARTIFICIAL
<220>
The sequence of <223> 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 agggcgacac 1020
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 1080
gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 1140
gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 1200
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 1260
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 1320
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 1380
gtccggactc agatccaccg gatctagata actgatcata atcagccata ccacatttgt 1440
agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat 1500
gaatgcaatt gttgttgtta acttgtttat tgcag 1535
<210> 3
<211> 466
<212> DNA
<213> duck enteritis virus chicken embryo 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 1437 to 3131 Nucleotide of SEQ ID NO:1.
2. restructuring duck enteritis virus, inserts the expression cassette containing gene described in claim 1 in the transcribed spacer between genomic LORF11 and the UL55 gene of duck enteritis virus.
3. to recombinate according to claim 2 duck enteritis virus, it is characterized in that the promotor in described expression cassette is pMCMV IE promotor.
4. to recombinate according to Claims 2 or 3 duck enteritis virus, it is characterized in that described expression cassette substituted for the nucleotide fragments of the 263 to 291, transcribed spacer between UL55 and LORF11 gene.
5. to recombinate described in claim 2 construction process of duck enteritis virus, adopt methods of homologous recombination by the transcribed spacer between genomic LORF11 and the UL55 gene of expression cassette insertion duck enteritis virus containing gene described in claim 1.
6. recombinate according to claim 5 the construction process of duck enteritis virus, the method that the expression cassette that it is characterized in that containing gene described in claim 1 inserts duck enteritis virus comprises the steps:
(1) determine fragment to be replaced in the transcribed spacer between duck enteritis virus genomic UL55 and LORF11 gene, the nucleotide fragments of the fragment upstream and downstream to be replaced that increases respectively, obtains upstream homology arm and downstream homology arm; The expression cassette of gene described in the expression cassette of expressing green fluorescent protein and expression claim 1 is inserted between upstream homology arm and downstream homology arm respectively, obtains homologous recombination Segment A and homologous recombination fragment B respectively;
(2) genomic dna of duck enteritis virus and homologous recombination Segment A carry out homologous recombination, select the recombinant virus sending green fluorescence under ultraviolet light, obtain recombinant virus C;
(3) genomic dna of homologous recombination fragment B and recombinant virus C carries out homologous recombination, selects the recombinant virus that can not send green fluorescence under ultraviolet light, obtains the restructuring duck enteritis virus carrying avian influenza virus hemagglutinin gene expression cassette.
7. with the living vaccine that duck enteritis virus of recombinating described in the 1-6 of claim is activeconstituents.
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| CN105002146A (en) * | 2015-08-11 | 2015-10-28 | 扬州大学 | RHVT (recombinant Herpesvirus of Turkey)-H9HA (H9 hemagglutinin) and construction method thereof |
| CN116656630A (en) * | 2023-07-27 | 2023-08-29 | 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 |
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| CN105002146A (en) * | 2015-08-11 | 2015-10-28 | 扬州大学 | RHVT (recombinant Herpesvirus of Turkey)-H9HA (H9 hemagglutinin) and construction method thereof |
| CN116656630A (en) * | 2023-07-27 | 2023-08-29 | 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 |
| 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 |
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