CN104312982A - Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore - Google Patents
Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore Download PDFInfo
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Abstract
The invention discloses a duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein (EGFP) genes and a constructing method and application of the duck plague virus recombinant vaccine strain rDEVTK-EGFP. The microorganism preservation number of the vaccine strain rDEVTK-EGFP is CGMCC No. 9456. According to the duck plague virus recombinant vaccine strain rDEVTK-EGFP, the constructing method and the application, a recombinant clone technology is adopted; a gene segment sCMV-EGFP containing enhanced green fluorescent proteins (EGFP) and sCMV promoter sequences is inserted into a duck plague virus TK gene region; recombinant EGFP duck plague viruses with an sCMV-EGFP expression cassette inserted into the corresponding position of a TK gene is constructed; the EGFP genes are stably expressed by the recombinant viruses. The invention further relates to a method for constructing the recombinant duck plague virus vaccine strain for stably expressing other poultry pathogeny exogenous genes and the application of the recombinant duck plague virus vaccine strain to preparation of vaccines for preventing duck plagues and other poultry infectious diseases.
Description
Technical field
The present invention relates to recombinant viral vaccine strain, particularly relate to a kind of can the restructuring duck plague virus strain rDEV TK-EGFP of stable expression of exogenous gene and construction process thereof and application, the invention belongs to biomedicine field.
Background technology
Duck plague is also known as duck viral enteritis (Duck viral enteritis, DVE), by duck plague virus (duck, the goose that cause also known as duck enteritis virus (Duck enteritis virus, DEV) and the para-infectious one of multiple Anseriformes fowl is acute, hot, contagious disease.Its principal feature is extensively popular, propagates rapidly, sickness rate and mortality ratio high, the equal susceptible of duck of different days, provisions duck industry causes tremendous economic lose, is one of important epidemic disease of the foster duck industry of harm.
The natural infection of duck plague virus is only limitted to anseriform Anatidae member (duck, goose, swan etc.).Contagium is sick duck, sick goose, the poultry being with poison and wildfowl mainly.Water is the natural propagation medium of this disease, and hematophagous bug may be the potential communication media of this disease.The rehabilitation fowl infecting this disease likely becomes carrier, and periodicity toxin expelling.Duck plague virus is the same with other simplexvirus, and hiding and activating of DEV can cause this disease to break out with migrating in aquatic bird domestic.The infection of virus and kind, age and sex have nothing to do, and the younger duck of duck infection rate that grows up is high.But the infection of discovered in recent years DEV is towards becoming younger future development, and the infection rate of goose constantly increases, and M & M is many more than 80%.Duck plague virus only has a serotype.Immunization is the most effective means of this disease of control.Prevention duck plague uses both at home and abroad is at present attenuated vaccine more widely, and this type of vaccine has good immune effect, immune protective efficiency produces fast feature, carries out immunization to before season arrives occurred frequently duck group and can effectively prevent this disease.When epidemic situation occurs, the beginning initial stage should immediately for duck group in do not occur that the duck of symptom carries out urgent immunity inoculation, the further diffusion of epidemic disease can be prevented, for control epidemic situation, reduce financial loss there is unusual effect.But, along with the development of livestock and poultry intensive culture industry, univalent vaccine has demonstrated its certain drawback, and the prevention and control measure of repeatedly immunity is wasted time and energy, multivalence combined vaccines just becomes the most popular control and prevention of disease product of aquaculture, thus carries out poultry diease multivalence combined vaccines and becomes inevitable choice.
DEV is simplexvirus, and its genome is bifilar linear DNA, and size is about 168Kb, is made up of covalently bound two regions, comprises long distinct zones (UL) and short distinct zones (US).The whole genome sequence of the reported first DEV VAC strains such as Li in 2009, in the same year, Liu etc. report by the most of ORF sequence of DEV VAC strain weakening strain DEV Clone-03 (Liu et al., 2009).Sequential analysis shows that DEV VAC is genomic and is configured as D type genome (Li et al., 2009), and genome structure is: UL-IRS-US-TRS.Comprise 78 opening code-reading frames (ORF) altogether, wherein have 65 ORF to be positioned at UL district, 11 ORF are positioned at US district, and other 2 ORF (ICP4 and IE180) then lay respectively at IRS district and TRS district (Wu et al., 2012).
Hsv gene group comprises and a large amount of copies dispensable gene, comprises TK, gC, gG, gK, US1, US2, US10, UL41, UL42, US7 and US8 etc.In correlative study, foreign gene inserted or substitute the nonessential gene of simplexvirus, construction of recombinant virus vaccine, this is considered to a kind of good live recombined vaccines virus vector, at present about simplexvirus comprises pseudoabies carrier, infectious laryngotracheitis virus carrier, horsepower kirschner virus vector and herpes turkey virus carrier etc. as the report of carrier.Along with the continuous intensification of duck plague virus research, be that virus live vector is subject to paying close attention to more widely with DEV, as the advantage of carrier, DEV is that this virus host range is narrower, to the equal no pathogenicity of chicken, turkey, dove and Mammals, can not impact non-host animals and the healthy and safe of people.Duck plague virus can drive the expression of foreign gene as transient copying in chicken body non-natural host; therefore utilize duck plague virus as the protective antigen of vector expression chicken source cause of disease; be the new approaches of exploitation DEV virus live vector vaccine, there is important application prospect.At present, smooth for DEV virus vector progress, Wang utilizes BAC technology to be inserted into by H5N1 HA Gene of H 9 Subtype AIV in duck plague virus gC gene, success construction expression expresses the restructuring duck plague virus (Wang et al., 2011) of H5N1 HA Gene of H 9 Subtype AIV.H5N1 HA Gene of H 9 Subtype AIV inserts in duck plague virus UL41 gene and between US7 and US8 gene by Liu etc.; build the restructuring duck plague virus that H5N1 HA Gene of H 9 Subtype AIV is expressed in two strains; immune duck group all can produce immunoprotection (Liu et al., 2011) to DEV and H5N1 subtype avian influenza virus afterwards.But the recombinant virus utilizing DEV nonessential gene TK site to carry out foreign gene at present builds and application is not reported.
Summary of the invention
One of technical problem to be solved by this invention is to provide a kind of method of duck plague virus recombinant vaccine strain of construction expression enhanced green fluorescence protein (EGFP) gene;
Two of technical problem to be solved by this invention is to provide a kind of duck plague virus recombinant vaccine strain of expression enhanced green fluorescence protein (EGFP) gene having described method to prepare;
Three of technical problem to be solved by this invention is to provide the application of described duck plague virus recombinant vaccine strain in the restructuring duck plague vaccine of preparation prevention duck plague.And the application of described duck plague recombinant virus in the recombiant vaccine preparing prevention duck plague and other bird transmissible disease.
Technical problem to be solved by this invention is realized by following technique means:
The method of the duck plague recombinant virus of a kind of construction expression enhanced green fluorescence protein (EGFP) gene of the present invention, it is characterized in that inserting in TK gene 3 ' end HpaI site the gene fragment sCMV-EGFP comprising sCMV promotor and enhanced green fluorescence protein (EGFP) gene order, build the duck plague recombinant virus obtaining and insert sCMV-EGFP expression cassette in TK gene corresponding position.
In the present invention, preferably, the method for the duck plague recombinant virus of construction expression enhanced green fluorescence protein (EGFP) gene comprises the following steps:
(1) pcr amplification obtains the flanking sequence of duck plague virus genome TK and both sides thereof, and the eukaryotic promoter sCMV promotor that can identify for virus and EGFP gene insert 3 ' end HpaI site of TK gene, build the recombinant transfer vector obtained containing EGFP expression cassette, called after pTK-EGFP.
(2) the complete genome group DNA of duck plague virus is extracted;
(3) utilize the duck plague virus genomic dna cotransfection obtained in the recombinant transfer vector pTK-EGFP and step (2) obtained in step (1) secondary to chick embryo fibroblast CEF, by the virus of fluorescent microscope screening expressing green fluorescent protein, again through virus plaques purifying, obtain the duck plague recombinant virus rDEV TK-EGFP of single stably express EGFP.
Wherein, preferably, the middle recombinant transfer vector pTK-EGFP of step (1) builds by the following method and obtains:
(1) according to DEV viral genome TK gene order and flanking sequence thereof, application Oligo 6.0 software design 1 pair of primer, primer sequence is as follows:
T1 (upstream): 5 '-GACGTGTTGGCATCGGTTC-3 '
T4 (downstream): 5 '-AAACAAATAGGGAGTAGCGAAGG-3 '
(2) apply described primer amplification DEV TK gene and flanking sequence thereof, and be cloned in pMD18-T Simple carrier, build plasmid pTK; The Hpa I site in the gene fragment sCMV-EGFP insertion vector pTK of sCMV promotor and enhanced green fluorescence protein (EGFP) gene order will be comprised, build transfer vector pTK-EGFP, preferably, described sCMV promotor derives from pCS2+ plasmid, preferably, add Hind III and Stu I restriction endonuclease sites at EGFP gene two ends, by this site, EGFP is replaced with other foreign genes.
In the present invention, preferably, the described nucleotide sequence comprising the gene fragment sCMV-EGFP of sCMV promotor and enhanced green fluorescence protein (EGFP) gene order is as shown in SEQ ID NO:2 (Fig. 2), and the nucleotides sequence of the DEV TK gene that amplification obtains and flanking sequence thereof is classified as shown in SEQ ID NO:1 (Fig. 1).
Further, the invention allows for the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene prepared according to described method.
In one particular embodiment of the present invention, a kind of duck plague recombinant virus of stably express enhanced green fluorescence protein (EGFP) gene, called after rDEVTK-EGFP strain, Classification And Nomenclature is Duck Anatid Herpesvirus, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, preservation date is on July 8th, 2014, and its microbial preservation number is: CGMCC No.9456.
Further, the invention allows for described duck plague recombinant virus in preparation prevention duck plague or the application in the restructuring duck plague vaccine preparing prevention duck plague and other bird transmissible disease, comprise gene enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of described expression enhanced green fluorescence protein (EGFP) gene being replaced with the virus causing other bird transmissible diseases, preferably, other described bird transmissible disease comprises causing and comprises duck, the Anseriformes bird of goose and the viral infectious of chicken.
Preferably, the described gene of the virus of other bird transmissible diseases that causes comprises Avian Influenza Virus HA Gene, NDV HN chimeric gene.
In a preferred embodiment of the present invention, utilize the recombinant transfer vector built that EGFP gene is replaced with other genes as Avian Influenza Virus HA Gene or NDV HN chimeric gene etc., express EGFP with TK genetically deficient to recombinate duck plague virus genome cotransfection chick embryo fibroblast CEF, obtain the duck plague recombinant virus of other foreign genes of stably express (Avian Influenza Virus HA Gene or NDV HN chimeric gene etc.).
In one particular embodiment of the present invention, disclose a kind of for preventing the bivalent vaccine of duck plague and bird flu, it is characterized in that the duck plague recombinant virus containing stably express Avian Influenza Virus HA Gene, the described duck plague recombinant virus containing stably express Avian Influenza Virus HA Gene builds by the following method and obtains: enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of described expression enhanced green fluorescence protein (EGFP) gene is replaced with Avian Influenza Virus HA Gene, to obtain final product.
In another specific embodiment of the present invention, disclose a kind of for preventing the bivalent vaccine of duck plague and newcastle disease, it is characterized in that the duck plague recombinant virus containing stably express NDV HN chimeric gene, the described duck plague recombinant virus containing stably express NDV HN chimeric gene builds by the following method and obtains: enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of described expression enhanced green fluorescence protein (EGFP) gene is replaced with NDV HN chimeric gene, to obtain final product.
Low virulent strain range of application of the present invention is wider, such as, can be applicable to the multivalence combined vaccines (live seedling or inactivated vaccine) etc. being prepared into prevention duck plague or other bird cause of diseases.
Accompanying drawing explanation
Fig. 1 is that the present invention recombinates the TK nucleotide sequence of duck plague virus pnca gene group TK gene insertion site and disappearance;
Fig. 2 is that the present invention recombinates the sCMV promotor and the gene order of enhanced green fluorescence protein (EGFP) that duck plague virus pnca gene group TK gene internal inserts;
Wherein underlined sequences is the gene order of sCMV promotor, and the sequence shown in grey font is the gene order of enhanced green fluorescence protein (EGFP).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The structure of embodiment 1 transfer vector
According to DEV viral genome TK gene order and flanking sequence (the GenBank number of logging in is AY963569) thereof, application Oligo 6.0 software design 1 pair of primer, primer sequence is as follows:
T1 (upstream): 5 '-GACGTGTTGGCATCGGTTC-3 '
T4 (downstream): 5 '-AAACAAATAGGGAGTAGCGAAGG-3 '
First apply primer T1 (upstream) and T4 (downstream) to increase DEV TK gene and flanking sequence thereof (shown in SEQ ID NO:1), and be cloned in pMD18-T Simple carrier, structure plasmid pTK; By with the Hpa I site (shown in Fig. 1) in EGFP expression cassette (shown in SEQ ID NO:2) the insertion vector pTK of sCMV promotor, build transfer vector pTK-EGFP.Wherein, described sCMV promotor derives from pCS2+ plasmid, and adds Hind III and Stu I restriction endonuclease sites when increasing at EGFP gene two ends, by this site, EGFP is replaced with other foreign genes.
The genomic extraction of embodiment 2 duck plague virus
DEV Clone-03 cell toxicant is inoculated in 0.001 MOI and is paved with (preparation reference " philosophy and technique of the vitro culture " operation (Xue Qingshan of chick embryo fibroblast in the 5mL cell bottle of CEF individual layer, 2001)), 37 DEG C of absorption 2h, discard virus liquid, change DMEM cell maintenance medium (containing 2%FBS), after cytopathy reaches 80% ~ 90%, discard cell maintenance medium, add cell dissociation buffer (1860 μ L STE; 100 μ L 10%SDS; 40 μ L Proteinase K 20mg/mL), 37 DEG C of digestion are spent the night, and add the extracting of equal-volume phenol once, and equal-volume phenol chloroform (1:1) extracting once, adds equal-volume chloroform once; Add 1/10 volume NaAC (3M, pH5.2), 2.5 times of volume dehydrated alcohols, be placed in-20 DEG C of precipitates overnight, 4 DEG C of centrifugal 15min, after air-dry, add appropriate amount of deionized water lytic virus genomic dna, agarose gel electrophoresis detects genomic integrity, be stored in-20 DEG C for subsequent use.
Embodiment 3 transfection
Day 1: prepare cell
Be laid in 5mL cell bottle by CEF passage in advance, 37 DEG C of 2% constant incubator is cultivated.
Day 2: transfection
(1). before transfection, 3-4h changes cell culture fluid
(2). rotaring redyeing system: A liquid: 18 μ L 2M CaCl2,10 μ g DNA (transfer vector and viral genome ratio are 3:1), add deionized water and supply volume to 150 μ L.B liquid: 150 μ L 2 × Hepes Buffered Saline (HBS)
(3). with pipettor, A liquid is dropwise added B liquid, while adding A liquid, utilize another pipettor to be slowly blown into air in B liquid, this process should complete within 1-2min.
(4). A B mixed solution is placed in incubated at room 30min.
(5). mixed solution is added in cell culture fluid.
(6). cell is placed in 37 DEG C of 2% constant incubator and cultivates continuation cultivation.
Day 3: change cell culture fluid and cell is suffered a shock
(1). change cell culture fluid.
(2). with 1 × PBS fine laundering cell 2 times.
(3). utilize DMSO to suffer a shock to cell.
1) 15%DMSO is prepared with 1 × PBS.
2) liquid of being suffered a shock by 2mL DMSO adds in the cell after fine laundering.
3) incubated at room 2.5min.
4) discard DMSO and add fresh cell culture fluid.
(4). cell is placed in 37 DEG C of 5%CO2 constant incubators and cultivates continuation cultivation.
1) 15%DMSO shock liquid is prepared with 1 × PBS.
2) liquid of being suffered a shock by 2mL DMSO adds in the cell after fine laundering.
3) incubated at room 2.5min.
4) discard DMSO shock liquid and add fresh cell culture fluid.
Cell is placed in 37 DEG C, and 5%CO2 constant incubator continues to cultivate in cultivating.Every day observe to there is cytopathy (CPE), whether fluorescence microscopy Microscopic observation has the CPE of expressing green fluorescent protein, after CPE reaches 80% ~ 90% results virus, multigelation 3 times, as plaque purification kind poison, be stored in-70 DEG C for subsequent use.
The screening of embodiment 4 recombinant virus and qualification
The virus liquid serum-free DMEM containing recombinant virus of results is done 10
-1~ 10
-4dilution, by well-grown CEF cell monolayer PBS fine laundering 3 times, add viral dilution liquid, 37 DEG C hatch 2h after, virus liquid is abandoned in suction, adds DMEM substratum (containing 1% low melting-point agarose, 10%FBS), after room temperature places 30min culture medium solidifying, move in 37 DEG C of 5%CO2 thermostat containers and continue to cultivate.After plaque occurs, the screening of recombinant virus plaque is carried out in the expression of fluorescence microscopy Microscopic observation green fluorescent protein, treat that plaque rises to suitable size, the plaque of picking express fluorescent protein is in serum-free DMEM, after multigelation three times, be again inoculated in CEF cell and proceed plaque purification.Repeat above step, take turns plaque purification through 3, obtain pure recombinant virus.By the recombinant virus multigelation three times of purifying, get 200 μ L virus liquids and extract recombinant virus genomes, method is as follows: get 200 μ L virus liquids, adds 5 μ L Proteinase Ks (20mg/mL) and 20 μ L 10%SDS, is placed in 56 DEG C of water-baths and digests 2h; Equal-volume phenol chloroform (1:1) extracting once, adds equal-volume chloroform once; Add 1/10 volume NaAC (3M, pH5.2) and 2.5 times of volume dehydrated alcohols, after being placed in-20 DEG C of 15min, 4 DEG C, centrifugal 15min collects viral genome, after adding appropriate amount of deionized water dissolving, with this recombinant virus genomes for template, utilize primer T1+T2 to carry out PCR preliminary evaluation to recombinant virus, PCR primer, after 0.9% agarose gel electrophoresis qualification, is cut glue and is reclaimed object fragment, be cloned into pMD18-T carrier, after Screening and Identification, positive plasmid checks order, and shown in result with SEQ ID NO:2, sequence is consistent, and explanation successfully constructs.
The mensuration of embodiment 5 recombinant virus stability
1, growth stability (duplicating dynamics/one step growth)
Previously prepared good CEF passage is laid in 12 porocyte culture plates, calculates cell quantity.Grow up to after individual layer until cell, recombinant virus is inoculated in well-grown CEF individual layer with 0.001 MOI, hatch 2h, discard virus liquid for 37 DEG C, add DMEM cell maintenance medium (containing 2%FBS) and be placed in 37 DEG C of 5%CO2 constant incubators cultivations.Respectively at results virus after 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, each time point does 3 parallel repetitions, multigelation three times, and-20 DEG C of storages are for subsequent use.
The virus liquid that different time points is gathered in the crops is done 10
-1~ 10
-7doubly dilution.Virus stock solution used and serum-free DMEM cell culture fluid are fully mixed with the ratio of 1:10.Draw 100 μ L viral dilution liquid to be added in the EP pipe that 900 μ L serum-free DMEM cell culture fluids are housed, repeat above step until viral dilution to 10
-7doubly, be inoculated in by viral dilution liquid and be paved with in 96 porocyte culture plates of CEF individual layer in advance, each extent of dilution 8 repetition, hatches 2h, discards virus liquid for 37 DEG C, and every hole adds the DMEM cell maintenance medium (containing 2%FBS) of 100 μ L.The virus liquid in 3 different holes of each time point results carries out replicate(determination) according to above method.Observation of cell pathology situation is started, Continuous Observation, result of determination after 7d after 12h.The TCID of virus is calculated according to Karber method
50.Add up the TCID of all time point virus liquids
50replicate(determination) is carried out to the growth rhythm of parental virus DEV Clone-03 simultaneously, be inoculated in well-grown CEF individual layer with 0.001 MOI, respectively at results virus after 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h after inoculation, each time point does three parallel repetitions.After measured, the recombinant virus titre that rDEV TK-EGFP gathers in the crops at 72h is the highest, is 10
6.8tCID
50/ mL, along with the increase of infection time, virus copy the damage of cell increasing, number of viable cells reduces gradually, causes copying of virus to be suppressed due to the death of host cell, virus titer 72h reach the highest after decline gradually.Parental virus DEV Clone-03 reaches the highest in the titre that 72h gathers in the crops, and is 10
7.9tCID
50/ mL, along with the increase of infection time, its titre declines gradually.The visible rDEV TK-EGFP recombinant virus viral titer comparatively wild poison of parent slightly declines, but its titre still can reach 10
6.8tCID
50/ mL.
2, recombinant virus genetic stability measures
Recombinant virus is inoculated in the continuous passage of CEF cell.Poison of recombinating inoculates CEF cell with 0.001MOI, and 37 DEG C, 5%CO2 hatches 2h, discards virus liquid, adds DMEM Growth of Cells maintenance medium (containing 2%FBS).The expression of fluorescence microscopy Microscopic observation green fluorescent protein, gathers in the crops virus when CPE reaches 80% ~ 90%.In continuous passage to 25 generation, in every 5 generations, are identified.The expression of fluorescence microscopy Microscopic observation green fluorescent protein, and extract virus genom DNA and carry out PCR qualification and sequencing, result shows, recombinant virus all can keep genetic stability through continuous passage.
The duck plague recombinant virus of a kind of stably express enhanced green fluorescence protein (EGFP) gene of the present invention, called after rDEVTK-EGFP strain, Classification And Nomenclature is Duck Anatid herpesvirus, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, preservation date is on July 8th, 2014, and its microbial preservation number is: CGMCC No.9456.
Foreign gene is inserted duck plague virus TK gene internal by the present invention, with EGFP gene for reporter gene, causes the disappearance of TK gene while reporter gene inserts, and the TK of construction expression green fluorescent protein lacks duck plague recombinant virus.In order to verify that the present invention inserts the possibility of other foreign genes and the validity of expressing protein as reform patterns virus at TK gene internal, stably express fowl multiple epidemic disease cause of disease immunogen gene is in succession constructed by the method for construction of recombinant virus of the present invention, as Avian Influenza Virus HA Gene, NDV HN chimeric gene, and different recombinant virus is all excited as antigen immune above-mentioned Hosts animal the antibody producing corresponding cause of disease immunogen protein, the method of the restructuring duck plague virus strain that other foreign genes of stably express utilizing the present invention to obtain are described and the restructuring duck plague virus vaccine strain building other bird cause of disease foreign genes of stably express, the recombiant vaccine of prevention duck plague and other bird transmissible diseases can be prepared, there is widespread use be worth.The recombinant virus obtained for confirmation the present invention and construction process are in preparation prevention duck plague and the application in the restructuring duck plague vaccine of other bird transmissible disease, application of the present invention is set forth further by following examples and experimental example, but it should be understood that, following examples and experimental example are illustrational object, and do not mean that limit the scope of the invention and spirit.
The structure of the recombinant virus of embodiment 6 stably express Avian Influenza Virus HA Gene and immune efficacy evaluation
1, the structure of the recombinant virus of stably express Avian Influenza Virus HA Gene
The plasmid carrying Avian Influenza Virus HA Gene is built by Harbin Veterinary Medicine Inst., China Academy of Agriculture and preserves, and application Oligo 6.0 software design HA gene primer, upstream primer introduces Hind III site, and downstream primer introduces Stu I site.Utilize restriction enzyme Hind III and Stu I process pTK-EGFP transfer vector, knock out EGFP gene, plasmid backbone is still with TK flanking sequence and the eukaryotic promoter sCMV promotor that can supply virus identification, the pretreated Avian Influenza Virus HA Gene with Hind III and Stu I sticky end is inserted in pTK skeleton, builds the transfer vector pTK-HA containing HA expression cassette.Particularly, utilize Hind III and Stu I to carry out enzyme to pTK-EGFP plasmid and HA fragment and cut process, reaction system (30 μ L): dH
2o:17 μ L, 10 × HBuffer:3 μ L, Xho I and Not I: each 1 μ L, plasmid 8 μ L, after 37 DEG C of water-bath 2h, cut result through 0.9% agarose gel electrophoresis qualification enzyme.Digestion products, after agarose gel electrophoresis, adopts DNA gel to reclaim test kit (Omega) purifying and reclaims target DNA fragment.Carrier after being cut by enzyme and the link of HA fragment, reaction system is: 10 × T
4dNA Ligase Buffer:1 μ L, T
4dNA ligase: 1 μ L, carrier 1.5 μ L, object fragment 6.5 μ L, 16 DEG C of connections are spent the night.Link product conversion intestinal bacteria TG 1, qualification is screening positive clone also, extracts positive plasmid also through cloning and sequencing, ensures that sequence is errorless.
Being inoculated in 0.001 MOI by rDEV TK-EGFP recombinant virus is paved with in the 5mL cell bottle of CEF individual layer, hatch 2h for 37 DEG C, discard virus liquid, change DMEM cell maintenance medium (containing 2%FBS), after cytopathy reaches 80% ~ 90%, discard cell maintenance medium, add cell dissociation buffer (1860 μ L STE; 100 μ L10%SDS; 40 μ L Proteinase K 20mg/mL), 37 DEG C of digestion are spent the night, and add the extracting of equal-volume phenol once, and equal-volume phenol chloroform (1:1) extracting once, adds equal-volume chloroform once; Add 1/10 volume NaAC (3M, pH5.2), 2.5 times of volume dehydrated alcohols, are placed in-20 DEG C of precipitates overnight, 4 DEG C of centrifugal 15min, add appropriate amount of deionized water and dissolve rDEV TK-EGFP genomic dna after air-dry.
Adopt transfection reagent, by transfer vector pTK-HA and rDEVTK-EGFP genomic dna cotransfection to CEF cell, method is with embodiment 3, obtain duck plague recombinant fowl influenza HA recombinant virus rDEVTK-HA, the screening of recombinant virus and purification process, with embodiment 4, then utilize primer T1+T2 to identify to the qualification of recombinant virus.Recombinant virus is inoculated well-grown CEF individual layer simultaneously, 6h after infection, 12h, 24h, 48h, 72h is harvested cell respectively, discard supernatant, PBS fine laundering three times, add RIPA, be placed on ice after cracking 20min, scrape with cell and cell is scraped, add 1/4 volume 5 × SDS sample-loading buffer, boiling water bath boils 10min, place 5min on ice, carry out the SDS-PAGE electrophoresis of protein, after conveniently Western blot method transferring film filter paper and nitrocellulose membrane (NC film) are cut into gel onesize, according to filter paper, NC film, gel, the order of filter paper is stacked together successively, emptying bubble, (NC film side is positioned at anode to be placed in half-dried electroporation, gel side is positioned at negative electrode), 40mA, 300V transfer printing 90min, take out NC film, transferring effect is determined through ponceau dyeing, successful for transfer printing NC film is placed in the PBS containing 5% skimming milk, 37 DEG C of closed 1h, PBST washs 3 times, each 10min, with chicken anti-avian influenza HA antibody (1:300) for primary antibodie, 37 DEG C hatch 2h after, PBST washs 3 times, each 10min, be two to resist with the anti-chicken IgG-HRP (1:5000) of rabbit, 37 DEG C hatch 2h after, PBST washs 3 times, each 10min, DAB colour developing, after object fragment colour developing to be detected, termination reaction.Result shows, and the expression of Avian Influenza Virus HA Gene can be detected after recombinant virus rDEVTK-HA infects CEF cell 48h, after 72h, the expression amount of HA gene is more, and inoculates the CEF cell of parent's strain and CEF cell does not detect corresponding albumen.
Embodiment has also carried out the mensuration of growth stability and genetic stability according to the method for embodiment 5 to recombinant virus rDEVTK-HA, finds that recombinant virus rDEVTK-HA can keep genetic stability through continuous passage, and continuous expression avian influenza virus HA protein.
2, the immune efficacy evaluation after recombinant virus rDEVTK-HA inoculation duck
2.1 test materials
2.1.1rDEVTK-HA strain recombinant virus is built by this research team and in CEF cell proliferation, and the strong poison of wild duck plague preserves qualification by Harbin Veterinary Medicine Inst., China Academy of Agriculture.
2.1.2SPF duck is tested: from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
2.2 test method
By rDEVTK-HA strain recombinant virus with 0.1ml intramuscular inoculation 15 SPF duck in 4 week age, other 15 in contrast, inoculate latter 7 days wherein 10 immune ducks and 10 contrast ducks respectively intramuscular injection path attack the DEV virulent strain of lethal doses; Remaining 5 immune ducks and 5 contrast duck gather respectively determination of serum influenza virus blood clotting suppress valency.
2.3, test-results
2.3.1rDEVTK-HA strain recombinant virus is to the immunoprotection of the strong poison of DEV
With after recombinant virus immune duck 7 days, all immune group ducks all produced strong virus attack and protect completely, and 10 control group ducks are being attacked within latter 6 days of poison all dead (referring to table 1).
Table 1 rDEVTK-HA strain recombinant virus immune duck is to the Immunoprotection test result of DEV
2.3.2rDEVTK-HA strain recombinant virus immune serum is for the TPPA result of bird flu
With after recombinant virus immune duck 7 days, immune group duck all produced the antibody for avian influenza virus, and blood clotting suppresses valency measurement result in table 2.
After table 2 rDEVTK-HA strain recombinant virus immune duck, in serum, avian influenza virus blood clotting suppresses valency to measure
Test-results shows; can not only protect completely the strong poison generation of duck plague as vaccine with the recombinant virus that the present invention obtains using the duck plague recombinant fowl influenza virus HA gene that recombinant virus construction process builds; and the antibody that can produce for avian influenza virus, illustrate that the present invention has good using value.
The structure of the recombinant virus of embodiment 7 stably express newcastle disease HN gene and immune efficacy evaluation
1, the structure of the recombinant virus of stably express newcastle disease HN gene
According to construction of recombinant virus method of the present invention, particularly by the newcastle disease CK/CH/HLJ/1/06 strain virus HN gene design primer that embodiment 6 is cloned for this research team, upstream primer introduces Xho I site, and downstream primer inserts Not I site.Utilize restriction enzyme Xho I and Not I to build the cloning vector containing HN protein expression box, the fragment of application primer P6 and Pbupper amplification containing HN expression cassette, is inserted into the Nco I site of pTK carrier, builds transfer vector pTK-HN.According to embodiment 3 and 6, obtain duck plague recombination chicken newcastle disease virus HN Protein reconstitution virus rDEVTK-HN, the screening of recombinant virus and purification process are with embodiment 4, and the split product after recombinant virus-infected cell, through the qualification of Western blot method, can react with newcastle disease positive serum.Embodiment has also carried out growth stability and genetic stability mensuration according to the method for embodiment 5 to recombinant virus rDEVTK-HN, finds that recombinant virus rDEVTK-HN all can keep genetic stability through continuous passage, and continuous expression newcastle disease virus HN albumen.
2, the immune efficacy evaluation after recombinant virus rDEVTK-HN inoculation chicken
2.1 test materials
2.1.1rDEVTK-HN strain recombinant virus is built by this research team and in CEF cell proliferation, the strong malicious Beijing Strain of newcastle disease is preserved by this research team.
2.1.2SPF test chicken: from the Chinese Academy of Agricultural Sciences's Harbin veterinary institute Experimental Animal Center.
2.2 test method
By rDEVTK-HN strain recombinant virus respectively with 0.1ml intramuscular inoculation 10 15 age in days SPF chickens, other 5 in contrast, immune chicken and contrast chicken are attacked Virulent Newcastle Disease Virus Beijing Strain in latter 14 days and observe 14 days, statistics morbidity and death condition by immunity respectively.
2.3 test-results
After rDEVTK-HN strain recombinant virus inoculation chicken 14 days, immune group chicken all created the antibody for Avian pneumo-encephalitis virus, and strong virus attack protection ratio reaches 10/10, and control group chicken is then to attack latter 10 days of poison all dead.(referring to table 3).
Immunoprotection result to Virulent Newcastle Disease Virus after table 3 rDEVTK-HN strain recombinant virus immunity chicken
Test-results shows, the recombinant virus that the duck plague recombination chicken NDV HN chimeric gene built using recombinant virus construction process with the present invention obtains can produce the antibody for Avian pneumo-encephalitis virus as vaccine immune chicken, illustrates that the present invention has good using value.
Claims (10)
1. the method for the duck plague recombinant virus of construction expression enhanced green fluorescence protein (EGFP) gene, it is characterized in that inserting in TK gene 3 ' end HpaI site the gene fragment sCMV-EGFP comprising sCMV promotor and enhanced green fluorescence protein (EGFP) gene order, build the duck plague recombinant virus obtaining and insert sCMV-EGFP expression cassette in TK gene corresponding position.
2. the method for claim 1, is characterized in that comprising the following steps:
(1) pcr amplification obtains the flanking sequence of duck plague virus genome TK and both sides thereof, and the eukaryotic promoter sCMV promotor that can identify for virus and EGFP gene insert 3 ' end HpaI site of TK gene, build the recombinant transfer vector obtained containing EGFP expression cassette, called after pTK-EGFP;
(2) the complete genome group DNA of duck plague virus is extracted;
(3) utilize the duck plague virus genomic dna cotransfection obtained in the recombinant transfer vector pTK-EGFP and step (2) obtained in step (1) secondary to chick embryo fibroblast CEF, by the virus of fluorescent microscope screening expressing green fluorescent protein, again through virus plaques purifying, obtain the duck plague recombinant virus rDEV TK-EGFP of single stably express EGFP.
3. method as claimed in claim 2, is characterized in that the middle recombinant transfer vector pTK-EGFP of step (1) builds by the following method and obtains:
(1) according to DEV viral genome TK gene order and flanking sequence thereof, application Oligo 6.0 software design 1 pair of primer, primer sequence is as follows:
T1 (upstream): 5 '-GACGTGTTGGCATCGGTTC-3 '
T4 (downstream): 5 '-AAACAAATAGGGAGTAGCGAAGG-3 '
(2) apply described primer amplification DEV TK gene and flanking sequence thereof, and be cloned in pMD18-T Simple carrier, build plasmid pTK; The Hpa I site in the gene fragment sCMV-EGFP insertion vector pTK of sCMV promotor and enhanced green fluorescence protein (EGFP) gene order will be comprised, build transfer vector pTK-EGFP, preferably, described sCMV promotor derives from pCS2+ plasmid, preferably, add Hind III and Stu I restriction endonuclease sites at EGFP gene two ends, by this site, EGFP is replaced with other foreign genes.
4. method as claimed in claim 3, it is characterized in that the described nucleotide sequence comprising the gene fragment sCMV-EGFP of sCMV promotor and enhanced green fluorescence protein (EGFP) gene order is as shown in SEQ ID NO:2, the nucleotides sequence of the DEV TK gene that amplification obtains and flanking sequence thereof is classified as shown in SEQ ID NO:1.
5. the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene prepared according to the method described in any one of claim 1-4.
6. the duck plague recombinant virus of stably express enhanced green fluorescence protein (EGFP) gene, it is characterized in that described duck plague recombinant virus, called after rDEVTK-EGFP strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its microbial preservation number is: CGMCC No.9456.
7. the duck plague recombinant virus described in claim 5 or 6 is in preparation prevention duck plague or the application in the restructuring duck plague vaccine preparing prevention duck plague and other bird transmissible disease, comprise gene enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6 being replaced with the virus causing other bird transmissible diseases, preferably, other described bird transmissible disease comprises causing and comprises duck, the Anseriformes bird of goose and the viral infectious of chicken.
8. application according to claim 7, the wherein said gene of the virus of other bird transmissible diseases that causes comprises Avian Influenza Virus HA Gene, NDV HN chimeric gene.
9. one kind for preventing the bivalent vaccine of duck plague and bird flu, it is characterized in that the duck plague recombinant virus containing stably express Avian Influenza Virus HA Gene, the described duck plague recombinant virus containing stably express Avian Influenza Virus HA Gene builds by the following method and obtains: enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6 is replaced with Avian Influenza Virus HA Gene, to obtain final product.
10. one kind for preventing the bivalent vaccine of duck plague and newcastle disease, it is characterized in that the duck plague recombinant virus containing stably express NDV HN chimeric gene, the described duck plague recombinant virus containing stably express NDV HN chimeric gene builds by the following method and obtains: enhanced green fluorescence protein (EGFP) gene of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6 is replaced with NDV HN chimeric gene, to obtain final product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385663A (en) * | 2015-11-26 | 2016-03-09 | 中国兽医药品监察所 | Establishment of duck enteritis virus gE and gI double gene deletion virus strain and application of establishment |
| CN110117612A (en) * | 2018-02-05 | 2019-08-13 | 南京农业大学 | A kind of Expressed in Xenopus Oocytes carrier pGH19-EGFP and application with green fluorescent protein tag |
| JP2019524154A (en) * | 2016-06-29 | 2019-09-05 | セバ・サンテ・アニマル | Duck enteritis virus and use thereof |
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- 2014-09-17 CN CN201410474947.6A patent/CN104312982A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385663A (en) * | 2015-11-26 | 2016-03-09 | 中国兽医药品监察所 | Establishment of duck enteritis virus gE and gI double gene deletion virus strain and application of establishment |
| JP2019524154A (en) * | 2016-06-29 | 2019-09-05 | セバ・サンテ・アニマル | Duck enteritis virus and use thereof |
| JP6990814B2 (en) | 2016-06-29 | 2022-01-12 | セバ・サンテ・アニマル | Duck plague virus and its use |
| CN110117612A (en) * | 2018-02-05 | 2019-08-13 | 南京农业大学 | A kind of Expressed in Xenopus Oocytes carrier pGH19-EGFP and application with green fluorescent protein tag |
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