CN104120110A - Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain - Google Patents
Duck plague virus recombinant vaccine strain expressing enhanced green fluorescent protein gene, constructing method thereof and applications of the recombinant vaccine strain Download PDFInfo
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Abstract
The invention discloses a duck plague virus recombinant vaccine strain expressing an enhanced green fluorescent protein (EGFP) gene, a constructing method thereof and applications of the recombinant vaccine strain. In particular, by utilization of a recombinant clone technology, a gene fragment CMV-EGFP containing an enhanced green fluorescent protein (EGFP) and a CMV promoter sequence replaces a US10 gene of a duck plague virus, and a recombinant EGFP duck plague virus lacking the US10 gene with a CMV-EGFP expression cassette being inserted into the corresponding position is constructed. The recombinant virus can stably express the EGFP gene. The duck plague virus recombinant vaccine strain is named as rDEVUS10-EGFP. The microbial accession number is CGMCC No.8660. The invention also relates to constructing methods of duck plague virus recombinant vaccine strains capable of stably expressing other poultry pathogeny exogenous genes, and applications of the recombinant vaccine strains in preparation of vaccines preventing duck plague and other poultry infectious diseases.
Description
Technical field
The present invention relates to recombinant viral vaccine strain and construction process thereof and application, relate in particular to a kind of restructuring duck plague virus strain rDEVUS10-EGFP and construction process and application that can stable expression of exogenous gene, the invention belongs to biological medicine technology field.
Background technology
Duck plague claims again duck viral enteritis (Duck viral enteritis, DVE), that the para-infectious one of the multiple Anseriformes fowl such as duck, goose that caused by duck plague virus (claiming again duck enteritis virus (Duck enteritis virus, DEV)) is acute, hot, contagious disease.Its principal feature is popular extensive, propagates rapidly, and sickness rate and mortality ratio are high, and the equal susceptible of the duck of different days, and provisions duck industry causes tremendous economic loss, is one of important epidemic disease of the foster duck industry of harm.
The natural infection of duck plague virus only limits to anseriform Anatidae member (duck, goose, swan etc.).Contagium is mainly disease duck, sick goose, with malicious poultry and wildfowl.Water is this sick natural propagation medium, and hematophagous bug may be the potential communication media of this disease.Infecting this sick rehabilitation fowl likely becomes carrier, and periodicity toxin expelling.Duck plague virus is the same with other simplexvirus, DEV hide and activate can cause this disease domestic and migrate aquatic bird in break out.The infection of virus and kind, age and sex are irrelevant, and the younger duck of duck infection rate that grows up is high.But the infection of discovered in recent years DEV is towards the future development that becomes younger, and the infection rate of goose constantly increases, and M & M is many more than 80%.Duck plague virus only has a serotype, and immunization is the effective means of this disease of control.The use of prevention duck plague is attenuated vaccine more widely both at home and abroad at present, and this type of vaccine has good immune effect, immune protective efficiency produces fast feature, carries out immunization can effectively prevent this disease before arriving season occurred frequently to duck group.In the time that epidemic situation occurs, in the beginning initial stage should be immediately for duck group, do not occur that the duck of symptom carries out urgent immunity inoculation, can prevent the further diffusion of epidemic disease, for controlling epidemic situation, reduce financial loss and there is unusual effect.But, along with the development of livestock and poultry intensive culture industry, univalent vaccine has demonstrated its certain drawback, and repeatedly the prevention and control measure of immunity is wasted time and energy, multivalence combined vaccines just becomes the most popular control and prevention of disease product of aquaculture, thereby carries out poultry diease multivalence combined vaccines and become 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. have reported by the most of ORF sequences of DEV VAC strain weakening strain DEV Clone-03 (Liu et al., 2009).Sequential analysis shows the genomic D of the being configured as type of DEV VAC genome (Li et al., 2009), and genome structure is: UL-IRS-US-TRS.Comprise altogether 78 opening code-reading frames (ORF), wherein have 65 ORF to be positioned at UL district, 11 ORF are positioned at US district, and other 2 ORF (ICP4 and IE180) lay respectively at HeTRS district of IRS district (Wu et al., 2012).
Hsv gene group comprises a large amount of dispensable genes that copies, and comprises TK, gC, gG, gK, US1, US2, US10, UL41, UL42, US7 and US8 etc.Foreign gene is inserted or is substituted the nonessential gene of simplexvirus in correlative study, 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, Ma Likeshi virus vector and herpes turkey virus carrier etc. as the report of carrier.Along with the continuous intensification to duck plague virus research, be subject to paying close attention to more widely taking DEV as virus live vector, DEV is that as the advantage of carrier this virus host range is narrower, to the equal no pathogenicity of chicken, turkey, dove and Mammals, therefore can not impact people and the healthy and safe of other animals in these instantaneous propagation of non-natural reservoir (of bird flu viruses) respiratory tract, this is significant to exploitation DEV virus live vector vaccine.At present, smooth for DEV virus vector progress, Wang utilizes BAC technology that H5N1 HA Gene of H 9 Subtype AIV is inserted in duck plague virus gC gene, success construction expression is expressed the restructuring duck plague virus (Wang et al., 2011) of H5N1 HA Gene of H 9 Subtype AIV.Liu etc. insert H5N1 HA Gene of H 9 Subtype AIV in duck plague virus UL41 gene and between US7 and US8 gene; build two strains and express the restructuring duck plague virus of H5N1 HA Gene of H 9 Subtype AIV; immune duck group all can produce immunoprotection (Liu et al., 2011) to DEV and H5N1 subtype avian influenza virus afterwards.But the recombinant virus that utilizes at present the nonessential gene US10 of DEV site to carry out foreign gene builds and application is not reported.
Summary of the invention
One of technical problem to be solved by this invention is to provide the method for the duck plague virus recombinant vaccine strain of a kind 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 that has expression enhanced green fluorescence protein (EGFP) gene that described method prepares;
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 of preparation prevention duck plague and other bird transmissible disease.
Technical problem to be solved by this invention realizes by following technological approaches:
The method of the duck plague recombinant virus of a kind of construction expression enhanced green fluorescence protein of the present invention (EGFP) gene, it is characterized in that replacing with the gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence the US10 gene of duck plague virus, build the duck plague recombinant virus that obtains disappearance US10 gene and insert CMV-EGFP expression cassette in its corresponding position.
In method of the present invention, preferred, the nucleotide sequence of the described gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence is as shown in SEQ ID NO:2.
In method of the present invention, preferred, the nucleotide sequence of the US10 gene of disappearance is as shown in SEQ ID NO:1.
In method of the present invention, preferred, described CMV promotor derives from the plasmid that contains CMV promotor, more preferably pEGPF-N1 plasmid.
In method of the present invention, preferred, comprise the following steps:
(1) recombinant transfer vector of the gene fragment CMV-EGFP that structure comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence, wherein be connected with respectively in described gene fragment CMV-EGFP upstream and downstream the flanking sequence that is positioned at duck plague virus US10 gene both sides that amplification obtains, the nucleotide sequence of the described gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence is as shown in SEQ ID NO:2;
(2) the complete genome group DNA of extraction duck plague virus;
(3) utilize the recombinant transfer vector of acquisition in step (1) and the complete genome group DNA cotransfection of the middle duck plague virus obtaining of step (2) inferior to chick embryo fibroblast, screen the virus of expressing green fluorescent protein by fluorescent microscope, pass through again viral plaque purification, obtain the duck plague recombinant virus of single stably express EGFP.
In method of the present invention, preferred, the primer of flanking sequence that is positioned at duck plague virus US10 gene both sides in step (1) for increasing is as follows:
VL1
upstream: 5 '-ATCGATTGACGATGAGCGATCGGAAT-3 '
VL2
downstream: 5 '-CCTAGGGTTGCGCGTTGTGTATAAGT-3 '
VR1
upstream: 5 '-ACGCGTGACTCTGACTGATACTCTAC-3 '
VR2
downstream: 5 '-ATGCATCTAATCGGTTATTTGCTGCT-3 '.
Further, the invention allows for the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene preparing according to the method described in above any one.
Wherein, in a preferred embodiment of the present invention, obtain the duck plague recombinant virus of a kind of stably express enhanced green fluorescence protein (EGFP) gene, called after rDEVUS10-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 December 27th, 2013, and its microbial preservation number is: CGMCC No.8660.
In described duck plague recombinant virus rDEVUS10-EGFP strain, recombinant transfer vector EGFP gene two ends have added Xho I and Not I restriction endonuclease sites, can EGFP be replaced with to other foreign genes by this site, as avian infectious bronchitis virus N gene or NDV HN chimeric gene etc., with duck plague virus cotransfection chick embryo fibroblast CEF, to obtain the duck plague recombinant virus of other foreign genes of stably express (as avian infectious bronchitis virus N gene or NDV HN chimeric gene etc.).
Therefore, further, the invention allows for the application of described duck plague recombinant virus in the restructuring duck plague vaccine of preparation prevention duck plague.And described duck plague recombinant virus prevents other bird transmissible disease (as infectious bronchitis in preparation, newcastle disease etc.) recombinant multivalent vaccine in application, comprise the expression cassette of enhanced green fluorescence protein (EGFP) gene is replaced with to the viral gene that causes other bird transmissible diseases, wherein, other described bird transmissible disease comprises the viral infectious of Anseriformes bird and chicken, described Anseriformes bird comprises duck and goose, preferably, the described viral gene that causes other bird transmissible diseases comprises duck hepatitis virus VP1 gene, chicken infectious bronchitis N and S gene and F gene of NDV strain.
Low virulent strain range of application of the present invention is wider, for example, can be applicable to be prepared into the multivalence combined vaccines (live seedling or inactivated vaccine) of prevention duck plague or its duck plague and his bird cause of disease etc.
In a specific embodiment of the present invention, relate to a kind of for preventing the bivalent vaccine of duck plague and duck viral hepatitis, the duck plague recombinant virus that contains stably express duck hepatitis virus VP1 gene, the described duck plague recombinant virus that contains stably express duck hepatitis virus VP1 gene builds and obtains by the following method: be duck hepatitis virus VP1 gene by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, obtain final product.
In a specific embodiment of the present invention, relate to a kind of for preventing the bivalent vaccine of duck plague and chicken infectious bronchitis, the duck plague recombinant virus that contains stably express chicken infectious bronchitis N or S gene, the described duck plague recombinant virus that contains stably express chicken infectious bronchitis N or S gene builds and obtains by the following method: be chicken infectious bronchitis N or S gene by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, obtain.
In a specific embodiment of the present invention, relate to a kind of for preventing the bivalent vaccine of duck plague and newcastle disease, the duck plague recombinant virus that contains stably express F gene of NDV strain, the described duck plague recombinant virus that contains stably express F gene of NDV strain builds and obtains by the following method: be F gene of NDV strain by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, obtain final product.
Brief description of the drawings
Fig. 1 is the present invention's CMV promotor and enhanced green fluorescence protein (EGFP) gene order (shown in SEQ ID NO:2) schematic diagram that duck plague virus pnca gene group US10 disappearance region inserts of recombinating, wherein, dash area is EGFP sequence, and underscore part is CMV promoter sequence;
Fig. 2 is the carrier collection of illustrative plates of transfer vector pUS10-EGFP.
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative completely, and they are only used for the present invention to be specifically described, and not should be understood to limitation of the present invention.
The structure of embodiment 1 transfer vector
According to DEV viral genome US10 gene order flanking sequence (the GenBank number of logging in is EF524095), application Oligo6.0 software design 2 is to primer, and primer sequence is as follows:
VL1 (upstream): 5 '-ATCGATTGACGATGAGCGATCGGAAT-3 '
VL2 (downstream): 5 '-CCTAGGGTTGCGCGTTGTGTATAAGT-3 '
VR1 (upstream): 5 '-ACGCGTGACTCTGACTGATACTCTAC-3 '
VR2 (downstream): 5 '-ATGCATCTAATCGGTTATTTGCTGCT-3 '
First apply primer VL1 (upstream) and VL2 (downstream) the left homology arm that increases, with primer VR1 (upstream) and VR2 (downstream) the right homology arm that increases, left homology arm (Cla I+Bln I) and right homology arm (Mlu I+Ava III) are inserted respectively in the pT-EGFP carrier of the EGFP expression cassette (CMV-EGFP) with CMV promotor being built by this research department, build transfer vector pUS10-EGFP (carrier collection of illustrative plates as shown in Figure 2), the nucleotide sequence of the gene fragment (CMV-EGFP) that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence is as shown in SEQ ID NO:2.
The genomic extraction of embodiment 2 duck plague virus
DEV Clone-03 cell toxicant is inoculated in to (preparation reference " philosophy and technique of the vitro culture " operation (Xue Qingshan of chick embryo fibroblast in the 5mL cell bottle that is paved with CEF individual layer with 0.001 MOI, 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 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 the extracting of 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 precipitations and spend the night, 4 DEG C of centrifugal 15min, add appropriate amount of deionized water lytic virus genomic dna after air-dry, agarose gel electrophoresis detects genomic integrity, be stored in-20 DEG C for subsequent use.
Embodiment 3 transfections
Day1: prepare cell
By CEF passage and be laid in 5mL cell bottle, 37 DEG C of 2% constant incubator cultivated in advance.
Day2: transfection
(1) before transfection, 3-4h changes cell culture fluid.
(2) rotaring redyeing system: A liquid: 18 μ L2M 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 L2 × Hepes Buffered Saline (HBS).
(3) with pipettor, A liquid is dropwise added to B liquid, utilize another pipettor to be slowly blown into air in adding A liquid in B liquid, this process should complete within 1-2min.
(4) A B mixed solution is placed in to incubated at room 30min.
(5) mixed solution is added in cell culture fluid.
(6) cell is placed in to 37 DEG C of 2% constant incubator and cultivates continuation cultivation.
Day3: 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) with 1 × PBS preparation 15%DMSO.
2) 2mL DMSO shock liquid is added 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 to 37 DEG C of 5%CO2 constant incubators and cultivates continuation cultivation.
1) with 1 × PBS preparation 15%DMSO shock liquid.
2) 2mL DMSO shock liquid is added 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.Observe to there is cytopathy (CPE) every day, and whether fluorescence microscopy Microscopic observation has the CPE of expressing green fluorescent protein, results virus after CPE reaches 80%～90%, multigelation 3 times, as the kind poison of plaque purification, be stored in-70 DEG C for subsequent use.
Screening and the qualification of embodiment 4 recombinant viruses
The virus liquid that contains recombinant virus of results is done to 10 with serum-free DMEM
-1～10
-4doubly dilution, by PBS fine laundering 3 times of well-grown CEF cell monolayer, add viral dilution liquid, hatch after 2h for 37 DEG C, virus liquid is abandoned in suction, adds DMEM substratum (containing 1% low melting-point agarose, 10%FBS), room temperature is placed after 30min culture medium solidifying, moves in 37 DEG C of 5%CO2 thermostat containers and continues to cultivate.After plaque occurs, the screening of recombinant virus plaque is carried out in expression at 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 of purifying three times, to get 200 μ L virus liquids and extract recombinant virus genomes, method is as follows: get 200 μ L virus liquids, add 5 μ L Proteinase Ks (20mg/mL) and 20 μ L10%SDS, be placed in 56 DEG C of water-baths and digest 2h, equal-volume phenol chloroform (1:1) extracting once, adds the extracting of equal-volume chloroform once, add 1/10 volume NaAC (3M, pH5.2) and 2.5 times of volume dehydrated alcohols, be placed in after-20 DEG C of 15min, 4 DEG C, centrifugal 15min collects viral genome, after adding appropriate amount of deionized water to dissolve, taking this recombinant virus genomes as template, utilize primer VL1+VR2 to carry out PCR preliminary evaluation to recombinant virus, PCR product is after 0.9% agarose gel electrophoresis qualification, cut glue and reclaim object fragment, be cloned into pMD18-T carrier, after Screening and Identification, positive plasmid checks order, result is consistent with sequence shown in SEQ ID NO:2, illustrate that the EGFP expression cassette that contains CMV promoter sequence has inserted in viral genome, recombinant virus successfully constructs.
The mensuration of embodiment 5 recombinant virus stability
1, growth stability (duplicating dynamics/one step growth)
By previously prepared good CEF passage and be laid in 12 porocyte culture plates, calculate cell quantity.After 24h, recombinant virus is inoculated in to well-grown CEF individual layer with 0.001 MOI, hatches 2h for 37 DEG C, discard virus liquid, add DMEM cell maintenance medium (containing 2%FBS) to be placed in 37 DEG C of 5%CO
2constant incubator is cultivated.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 of different time points results is done to 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 and 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, viral dilution liquid is inoculated in the 96 porocyte culture plates that are paved with in advance CEF individual layer, 8 repetitions of each extent of dilution, hatch 2h for 37 DEG C, discard virus liquid, 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.After 12h, start observation of cell pathology situation, Continuous Observation, result of determination after 7d.Calculate viral TCID according to Karber method
50.Add up the TCID50 of all time point virus liquids, the growth rhythm of parental virus DEV is carried out to replicate(determination) 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, rDEVUS10-EGFP is the highest in the recombinant virus titre of 72h results, is 10
6.1tCID
50/ mL, along with the increase of infection time, virus copy the damage of cell increasingly, viable cell number reduces gradually, causes copying because the death of host cell is suppressed of virus, virus titer reaches after the highest and declines gradually at 72h.Parental virus DEV reaches the highest in the titre of 72h results, is 10
7.9tCID
50/ mL, along with the increase of infection time, its titre declines gradually.Visible rDEVUS10-EGFP recombinant virus viral titer slightly declines compared with the wild poison of parent, but its titre still can reach 10
6.1tCID
50/ mL.
2, recombinant virus genetic stability is measured
Recombinant virus is inoculated in to the continuous passage of CEF cell.To recombinate poison with 0.001MOI inoculation CEF cell, and 37 DEG C, 5%CO2 is hatched 2h, discards virus liquid, adds DMEM Growth of Cells maintenance medium (containing 2%FBS).The expression of fluorescence microscopy Microscopic observation green fluorescent protein, results virus in the time that 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 present invention replaces the US10 gene of duck plague virus with the gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence, taking EGFP gene as reporter gene, when reporter gene inserts, cause the disappearance completely of US10 gene, build the US10 disappearance duck plague recombinant virus that obtains stably express enhanced green fluorescence protein.
The duck plague recombinant virus of a kind of stably express enhanced green fluorescence protein of the present invention (EGFP) gene, called after rDEVUS10-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 December 27th, 2013, and its microbial preservation number is: CGMCC No.8660.
In order to verify that the present invention inserts the possibility of other foreign genes and the validity of expressing protein as restructuring model virus in the corresponding disappearance of US10 position, in succession build the multiple epidemic disease cause of disease of stably express fowl immunogen gene as duck hepatitis virus VP1 gene by the method for construction of recombinant virus of the present invention, infectious bronchitis virus N-gene and S gene, F gene of NDV strain, and all excite different recombinant viruses as the above-mentioned Hosts animal of antigen immune and produce the antibody of corresponding cause of disease immunogen protein, the restructuring duck plague virus strain of other foreign genes of stably express and the method for the restructuring duck plague virus vaccine strain of other bird cause of disease foreign genes of structure stably express of utilizing the present invention to obtain are described, can prepare the recombiant vaccine of prevention duck plague and other bird transmissible diseases, having widespread use is worth.For recombinant virus and the application of construction process in the restructuring duck plague vaccine of preparation prevention duck plague and other bird transmissible disease of confirmation the present invention acquisition, further set forth application of the present invention 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.
Structure and the immune efficacy evaluation of the duck plague recombinant virus of embodiment 6 stably express duck hepatitis virus VP1 genes
1, the structure of the recombinant virus of stably express duck hepatitis virus VP1 gene
Carry duck hepatitis virus Du/CH/LBJ/090809 strain (Genbank accession number, JF828997) plasmid of VP1 gene is by Chinese Academy of Agricultural Sciences's Harbin animal doctor's research and establishment preservation, application Oligo6.0 software design VP1 gene primer, upstream primer is introduced Xho I site, and downstream primer inserts Not I site.Utilize restriction enzyme Xho I and Not I to process pUS10-EGFP transfer vector, knock out EGFP gene, plasmid skeleton is still with US10 flanking sequence and the eukaryotic promoter CMV promotor that can identify for virus, the pretreated duck hepatitis virus VP1 gene with Xho I and Not I sticky end is inserted in pUS10 skeleton, build the transfer vector pUS10-VP1 that contains VP1 expression cassette.Particularly, utilize Xho I and Not I to carry out enzyme to pUS10-EGFP plasmid and VP1 fragment and cut processing, reaction system (30 μ L): dH
2o:17 μ L, 10 × H Buffer: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.Enzyme is cut product after agarose gel electrophoresis, adopts DNA gel to reclaim test kit (Omega) purifying and reclaims target DNA fragment.Carrier after enzyme is cut and the link of VP1 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 transforms e. coli tg1, and qualification screening positive clone extract positive plasmid and through cloning and sequencing, ensure that sequence is errorless.
RDEVUS10-EGFP recombinant virus is inoculated in 0.001 MOI in the 5mL cell bottle that is paved with 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 the extracting of 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 precipitations and spend the night, and 4 DEG C of centrifugal 15min add after air-dry appropriate amount of deionized water to dissolve rDEVUS10-EGFP genomic dna.
Adopt transfection reagent, by transfer vector pUS10-VP1 and rDEVUS10-EGFP genomic dna cotransfection to CEF cell, method is with embodiment 3, obtain the duck plague recombinant virus rDEVUS10-VP1 of stably express duck hepatitis virus VP1 gene, the screening of recombinant virus and purification process be with embodiment 4, to the qualification of recombinant virus utilize primer DV1-U+DV1-L, VL1+DV1-U, DV1-L+VR2 tri-to primer respectively to identifying.Recombinant virus is inoculated to 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 cell is scraped with cell, 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 according to conventional Western blot method, transferring film filter paper and nitrocellulose membrane (NC film) are cut into gel onesize, in film transfering buffering liquid, soak 5min, 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, determine transfer printing effect through ponceau dyeing, successful transfer printing NC film is placed in to the PBS containing 5% skimming milk, 37 DEG C of sealing 1h, PBST washing 3 times, each 10min, taking the anti-duck hepatitis of rabbit VP1 antibody (1:100) as primary antibodie, hatch after 2h PBST washing 3 times, each 10min for 37 DEG C, taking goat-anti rabbit duck IgG-HRP (1:5000) as two anti-, hatch after 2h for 37 DEG C, PBST washing 3 times, each 10min, DAB colour developing, after object fragment colour developing to be detected, termination reaction.Result shows, the expression of duck hepatitis virus VP1 gene can be detected after recombinant virus rDEVUS10-VP1 infects CEF cell 48h, and after 72h, the expression amount of VP1 gene is more, and CEF cell and the CEF cell of inoculation parent strain do not detect corresponding albumen.
Embodiment has also carried out the mensuration of growth stability and genetic stability to recombinant virus rDEVUS10-VP1 according to the method for embodiment 5, find that recombinant virus rDEVUS10-VP1 can keep genetic stability through continuous passage, and continuous expression duck hepatitis virus VP1 albumen.
2, the immune efficacy evaluation after recombinant virus rDEVUS10-VP1 inoculation duck
2.1 test materials
2.1.1rDEVUS10-VP1 strain recombinant virus is built by this research team and in CEF cell proliferation, and the strong poison of wild duck plague is preserved qualification by Harbin Veterinary Medicine Inst., China Academy of Agriculture.
2.1.2SPF test duck: from the Chinese Academy of Agricultural Sciences's Harbin veterinary institute Experimental Animal Center.
2.2 test method
By rDEVUS10-VP1 strain recombinant virus with 15 30 age in days SPF ducks of 0.1ml intramuscular inoculation, other 15 in contrast, inoculate latter 7 days wherein 10 immune ducks and 10 contrast ducks respectively intramuscular injection paths attack the DEV virulent strain of lethal doses; Remaining 5 immune ducks and 5 contrast duck gathers respectively the neutralizing antibody of determination of serum duck hepatitis virus.
2.3, test-results
2.3.1rDEVUS10-VP1 the immunoprotection of strain recombinant virus to the strong poison of DEV
With after recombinant virus rDEVUS10-VP1 immune duck 7 days, all immune group ducks all produced protection completely to strong virus attack, and 10 control group ducks are being attacked within latter 6 days of poison all dead (referring to table 1).
The immunoprotection test-results of table 1 rDEVUS10-VP1 strain recombinant virus immune duck to DEV
2.3.2rDEVUS10-VP1 strain recombinant virus immune serum is in duck hepatitis virus and titration result
With after recombinant virus immune duck 7 days, immune group duck all produced the neutralizing antibody for duck hepatitis virus, and titration the results are shown in Table 2.
Serum duck hepatitis neutralizing antibody measurement result after table 2 rDEVUS10-VP1 strain recombinant virus immune duck
Test-results shows; the recombinant virus that the duck plague restructuring duck hepatitis virus VP1 gene building using recombinant virus construction process with the present invention obtains not only can produce protection completely to the strong poison of duck plague as vaccine; and can produce the neutralizing antibody for duck hepatitis virus, illustrate that the present invention has good using value.
Structure and the immune efficacy evaluation of the duck plague recombinant virus of embodiment 7 stably express chicken infectious bronchitis N and S gene
1, the structure of the recombinant virus of stably express chicken infectious bronchitis N and S gene
According to construction of recombinant virus method of the present invention, (this strain has been documented in document: in " qualification of the preparation of avian infectious bronchitis virus monoclonal antibody and B cell antigen epi-position thereof; Wang Fang etc.; Chinese Preventive Veterinary Medicine report; 4 phases in 2012 " in the chicken infectious bronchitis ck/CH/LDL/091022 strain of cloning for this research team particularly, now preserved by Chinese Academy of Sciences's Harbin veterinary institute) virus N-gene and S gene design primer, upstream primer is introduced Xho I site, and downstream primer is introduced Not I site.The transfer vector pUS10-N and the pUS10-S that utilize restriction enzyme Xho I and Not I to build respectively to contain N albumen and S protein expression box.According to embodiment 3 and 6, obtain the duck plague recombinant virus rDEVUS10-N of stably express chicken infectious bronchitis N gene and the duck plague recombinant virus rDEVUS10-S of stably express chicken infectious bronchitis S gene, the screening of recombinant virus and purification process are with embodiment 4, split product after 2 kinds of recombinant virus-infected cells, through the qualification of Western blot method, can react with chicken infectious bronchitis positive serum.Method according to embodiment 5 has been carried out growth stability and genetic stability mensuration to recombinant virus rDEVUS10-N and rDEVUS10-S, find that recombinant virus rDEVUS10-N and rDEVUS10-S all can keep genetic stability through continuous passage, and continuous expression avian infectious bronchitis virus N albumen and S albumen.
2, the immune efficacy evaluation after recombinant virus rDEVUS10-N and rDEVUS10-S inoculation chicken
2.1 test materials
2.1.1rDEVUS10-N built by this research team with rDEVUS10-S strain recombinant virus and in CEF cell proliferation, chicken infectious bronchitis antibody assay kit is purchased from IDEXX company.
2.1.2SPF test chicken: from the Chinese Academy of Agricultural Sciences's Harbin veterinary institute Experimental Animal Center.
2.2 test method
By rDEVUS10-N and rDEVUS10-S strain recombinant virus respectively with 10 15 age in days SPF chickens of 0.1ml intramuscular inoculation, other 5 in contrast, 2 groups of immunity chickens gather respectively determination of serum Infectious Bronchitis Antibody with contrast chicken, and method is carried out according to test kit ELISA method.
2.3, test-results
After rDEVUS10-N and rDEVUS10-S strain recombinant virus inoculation chicken 14 days, immune group chicken all produced Infectious Bronchitis Antibody, and 2 groups of immunity chicken antibody positive rates all reach 10/10, and control group chicken is all negative.(referring to table 3).
Serum antibody measurement result after table 3 rDEVUS10-N and rDEVUS10-S strain recombinant virus immunity chicken
Test-results shows, the recombinant virus that the duck plague recombination chicken infectious bronchitis virus N building using recombinant virus construction process with the present invention and S gene obtain can produce the antibody for infectious bronchitis virus as vaccine immune chicken, illustrates that the present invention has good using value.
Structure and the immune efficacy evaluation thereof of the duck plague recombinant virus of embodiment 8 stably express newcastle disease F genes
1, the structure of the recombinant virus of stably express newcastle disease F gene
According to construction of recombinant virus method of the present invention, by the newcastle disease CK/CH/HLJ/1/06 strain for the clone of this research team, (this strain has been documented in document: in " Monoclonal Antibodies To Newcastle Disease Virus preparation and part NP Protein Epitopes identification and analysis; Liu Peixin; 2011; Northeast Agricultural University's Ph D dissertation " particularly, now preserved by Chinese Academy of Sciences's Harbin veterinary institute) virus F gene design primer, upstream primer is introduced Xho I site, and downstream primer inserts Not I site.Utilize restriction enzyme Xho I and Not I to build the transfer vector pUS10-F that contains F protein expression box.According to embodiment 3 and 6, obtain the duck plague recombinant virus rDEVUS10-F of stably express newcastle disease F gene, 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 also according to the method for embodiment 5, recombinant virus rDEVUS10-F has been carried out to growth stability and genetic stability is measured, and finds that recombinant virus rDEVUS10-F all can keep genetic stability through continuous passage, and continuous expression newcastle disease virus F albumen.
2, the immune efficacy evaluation after recombinant virus rDEVUS10-F inoculation chicken
2.1 test materials
2.1.1rDEVUS10-F 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 rDEVUS10-F strain recombinant virus, respectively with 10 15 age in days SPF chickens of 0.1ml intramuscular inoculation, other 5 immunity is attacked Virulent Newcastle Disease Virus Beijing Strain by immune chicken with contrast chicken respectively in latter 14 days and is observed 14 days in contrast, and statistics is fallen ill and death condition.
2.3, test-results
After rDEVUS10-F strain recombinant virus inoculation chicken 14 days, immune group chicken all produced the antibody for newcastle disease, and strong virus attack protection ratio reaches 10/10, and control group chicken is to attack latter 10 days of poison all dead.(referring to table 4).
Serum antibody measurement result after table 4 rDEVUS10-F strain recombinant virus immunity chicken
Test-results shows, can produce the antibody for Avian pneumo-encephalitis virus with the present invention using the recombinant virus of the duck plague recombination chicken NDV HN chimeric gene acquisition of recombinant virus construction process structure as vaccine immune chicken, illustrates that the present invention has good using value.
The foregoing is only preferred embodiment of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (10)
1. the method for the duck plague recombinant virus of a construction expression enhanced green fluorescence protein (EGFP) gene, it is characterized in that replacing with the gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence the US10 gene of duck plague virus, build the duck plague recombinant virus that obtains disappearance US10 gene and insert CMV-EGFP expression cassette in its corresponding position.
2. the method for claim 1, it is characterized in that the nucleotide sequence of the described gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence is as shown in SEQ ID NO:2, the nucleotide sequence of the US10 gene of disappearance is as shown in SEQ ID NO:1.
3. the method for claim 1, is characterized in that comprising the following steps:
(1) recombinant transfer vector of the gene fragment CMV-EGFP that structure comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence, wherein be connected with respectively in described gene fragment CMV-EGFP upstream and downstream the flanking sequence that is positioned at duck plague virus US10 gene both sides that amplification obtains, the nucleotide sequence of the described gene fragment CMV-EGFP that comprises enhanced green fluorescence protein (EGFP) and CMV promoter sequence is as shown in SEQ ID NO:2;
(2) the complete genome group DNA of extraction duck plague virus;
(3) utilize the recombinant transfer vector of acquisition in step (1) and the complete genome group DNA cotransfection of the middle duck plague virus obtaining of step (2) inferior to chick embryo fibroblast, screen the virus of expressing green fluorescent protein by fluorescent microscope, pass through again viral plaque purification, obtain the duck plague recombinant virus of single stably express EGFP.
4. method as claimed in claim 3, is characterized in that the primer of the flanking sequence that is positioned at duck plague virus US10 gene both sides in step (1) for increasing is as follows:
VL1
upstream: 5 '-ATCGATTGACGATGAGCGATCGGAAT-3 '
VL2
downstream: 5 '-CCTAGGGTTGCGCGTTGTGTATAAGT-3 '
VR1
upstream: 5 '-ACGCGTGACTCTGACTGATACTCTAC-3 '
VR2
downstream: 5 '-ATGCATCTAATCGGTTATTTGCTGCT-3 '.
5. the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene preparing according to the method described in claim 1-4 any one.
6. the duck plague recombinant virus of a stably express enhanced green fluorescence protein (EGFP) gene, it is characterized in that described duck plague recombinant virus, called after rDEVUS10-EGFP strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its microbial preservation number is: CGMCC No.8660.
7. the application of the duck plague recombinant virus described in claim 5 or 6 in the restructuring duck plague vaccine of preparation prevention duck plague, and in the application of preparing in the recombinant multivalent vaccine that prevents duck plague and other bird transmissible disease, comprise the expression cassette of 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 to the viral gene that causes other bird transmissible diseases, other described bird transmissible disease comprises the viral infectious of Anseriformes bird and chicken, described Anseriformes bird comprises duck and goose, preferably, the described viral gene that causes other bird transmissible diseases comprises duck hepatitis virus VP1 gene, chicken infectious bronchitis N and S gene and F gene of NDV strain.
8. one kind for preventing the bivalent vaccine of duck plague and duck viral hepatitis, it is characterized in that the duck plague recombinant virus that contains stably express duck hepatitis virus VP1 gene, the described duck plague recombinant virus that contains stably express duck hepatitis virus VP1 gene builds and obtains by the following method: be duck hepatitis virus VP1 gene by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, obtain final product.
9. one kind for preventing the bivalent vaccine of duck plague and chicken infectious bronchitis, it is characterized in that the duck plague recombinant virus that contains stably express chicken infectious bronchitis N or S gene, the described duck plague recombinant virus that contains stably express chicken infectious bronchitis N or S gene builds and obtains by the following method: be chicken infectious bronchitis N or S gene by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, 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 that contains stably express F gene of NDV strain, the described duck plague recombinant virus that contains stably express F gene of NDV strain builds and obtains by the following method: be F gene of NDV strain by enhanced green fluorescence protein (EGFP) Gene Replacement of the duck plague recombinant virus of expression enhanced green fluorescence protein (EGFP) gene described in claim 5 or 6, obtain final product.
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