CN105754959A - NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof - Google Patents

NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof Download PDF

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CN105754959A
CN105754959A CN201610141600.9A CN201610141600A CN105754959A CN 105754959 A CN105754959 A CN 105754959A CN 201610141600 A CN201610141600 A CN 201610141600A CN 105754959 A CN105754959 A CN 105754959A
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dhav
gene
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马秀丽
宋敏训
黄兵
李玉峰
于可响
刘存霞
胡峰
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Poultry Research Institute Shandong Academy of Agricultural Sciences
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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes.The recombinant virus is recorded as rLS-1VP1-2A-3VP1 and is obtained inserting serially connected DHAV-1 and DHAV-3 VP1 genes into an NDV vector and carrying out saving.The NDV (Lasota strain) is used as the vector for the recombinant virus, the serially connected DHAV-1 and DHAV-3 VP1 genes are inserted into the NDV (Lasota strain) to obtain a vector, and the DNV recombinant virus co-expressing DHAV-1 and DHAV-3 VP1 genes is obtained by determining an optimal insertion site to insert the DHAV VIP1 gene; the recombinant virus is useful in preventing duck hepatitis A viruses (type 1 and type 3) and duck Newcastle disease and filling the current blank of DHAV-3 vaccines.

Description

Express NDV recombinant virus and the application thereof of DHAV-1 and DHAV-3 type VP1 gene
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of coexpression DHAV-1 type and The NDV recombinant virus of the VP1 gene of DHAV-3 type, and its preparation method is disclosed further.
Background technology
China is as the first in the world duck culturing big country, and popular duck hepatitis A frequently occurs, due to currently The prevention effect of the yolk antibody used instability, cause the prevention and control present situation of duck hepatitis A not allowed happy See, cause harm greatly not only to duck culturing industry, also China's food safety is caused certain hidden danger.
Duck hepatitis A is mainly by duck hepatitis A virus (HAV) 1 type (DHAV-1), 2 types (DHAV-2) and 3 types (DHAV-3) three kinds of viruses cause, the domestic epidemic isolates being currently known all with DHAV-1 and DHAV-3 is main.VP1 gene, as protected protein main for DHAV, encodes main antigen site And have in main type specificity and site, also become the preferred genes of DHAV research.Except this it Outward, currently also finding that Avian pneumo-encephalitis virus (NDV) there is also bigger harm to the cultivation of duck, it causes Characteristic of disease the most gradually strengthens.At present, duck group infects the existing more report of Avian pneumo-encephalitis virus case, its Sick rate is up to 20%-60%, and mortality rate is even as high as 90% up to 10%-15%, the infection rate of indivedual duck groups Above.Visible, for the safety of duck culturing industry, the not only preventing and treating of DHAV-1 and DHAV-3 virus Particularly significant, the immunoprophylaxis for Avian pneumo-encephalitis virus is the most imperative.
Prevention currently for duck hepatitis A virus (HAV) (DHAV) relies primarily on traditional vaccine, and traditional goes out Live vaccine not only production cost is high, and antibody produces the slowest, and culture experiment has proven to itself and discomfort Immunoprophylaxis in duckling.Existing DHAV-1 type Attenuate vaccine cannot be protected at all and be on the rise DHAV-3 virus, and DHAV-3 type Attenuate vaccine not yet gets the Green Light code, therefore, except for The Attenuate vaccine demand of DHAV-3 type virus urgently outside, for the demand also ten of duck hepatitis A virus (HAV) polyvalent vaccine Divide urgent.
Along with greatly developing of Protocols in Molecular Biology, reverse genetics manipulation technology has been developing progressively ripe, NDV is used as viral vector and the most increasingly comes into one's own with the research developing live recombined vaccines.Existing grind Study carefully it has proven convenient that NDV is to induce humoral immunization and cellular immunization as the advantage that carrier is maximum, can Propagation long-term expression antigen gene, will not integrate with host genome in vivo, and safety is wanted substantially It is better than DNA viral vector.GeJY etc. the most successfully construct the restructuring NDV expressing H5N1HA gene, The attack of anaphylactic type NDV and homology and allos HPAIV can be resisted simultaneously.Huang Z H etc. are also The restructuring NDV of the expression IBDV VP2 albumen built, all can effectively protect NDV virulent strain and infection The attack of property bursal disease virus highly virulent strain.The studies above all has proven to NDV can as vaccine carrier Stimulate body to produce stronger immunoreation, and NDV prevents and medical science in fowl diseases as vaccine carrier Practice has shown that its huge advantage and potentiality, there is extraordinary application prospect.
Summary of the invention
To this end, the technical problem to be solved be to provide a kind of coexpression DHAV-1 type and The NDV recombinant virus of the VP1 gene of DHAV-3 type, is effective to current duck hepatitis A virus (HAV) and duck is new The prevention of city epidemic disease.
The NDV recombinant virus of expression DHAV-1 and DHAV-3 type VP1 gene of the present invention It is designated as rLS-1VP1-2A-3VP1, is will to insert after DHAV-1 type and DHAV-3 type VP1 gene tandem Enter to NDV be carrier, through rescue obtain recombinant virus.
The invention also discloses the NDV weight of described expression DHAV-1 and DHAV-3 type VP1 gene The method of papova, comprises the steps:
(1) SOE-PCR method is used DHAV-1 type and DHAV-3 type VP1 gene to be carried out even Connect, it is thus achieved that DHAV 1VP1-2A-3VP1 recombination；
(2) DHAV 1VP1-2A-3VP1 gene is connected with LaSota strain linearized vector, and Product will be connected convert to Stbl2 competent cell, the weight of construction expression DHAV-1VP1-2A-3VP1 Group NDV viral vector, filters out positive colony；
(3) save and obtain recombinant virus rLS-1VP1-2A-3VP1.
Further, in described step (1), described acquisition DHAV 1VP1-2A-3VP1 recombinates base The step of cause specifically includes:
(1a) respectively with DHAV-1 and DHAV-3 as material, RT-PCR method is utilized to expand respectively Obtain DHAV-1VP1-2A gene and DHAV-3VP1 gene；And reclaim with the two groups of PCR obtained Product is template, utilizes the amplification of SOE-PCR method to obtain recombination DHAV-1VP1-2A-3VP1；
(1b) PCR primer that will reclaim, is connected to pMD18-T-Vector, through EcoR I/Not I Double digestion is identified, identifies correct positive recombinant.
More excellent, in described step (1a),
Described DHAV-1 carries out the primer P1 that PCR amplification is DHAV-1VP1-2A gene:
Forward primer: 5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
Downstream primer: 5'-CTGATTGGAATCACCTTGATCTGTAGTAAT-3 ',
The clip size of its amplified production DHAV-1VP1-2A is 1652bp；Amplified production The nucleotide sequence of DHAV-1VP1-2A is as shown in SEQ ID NO:1；
Described DHAV-3 carries out the primer P2 that PCR amplification is DHAV-3VP1 gene:
Forward primer: 5'-ATTACTACAGATCAAGGTGATTCCAATCAGC-3 ',
Downstream primer: 5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NOTI),
The clip size of amplified production DHAV-3VP1 is 746bp；Amplified production DHAV-3VP1 Nucleotide sequence as shown in SEQ ID NO:2；
Described DHAV-1VP1-2A gene and DHAV-3VP1 gene carry out SOE-PCR amplification and obtain The primer P3 obtaining recombination DHAV-1VP1-2A-3VP1 gene is:
Forward primer: 5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
Downstream primer: 5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NotI),
The clip size of amplified production DHAV-1VP1-2A-3VP1 is 2368bp, amplified production The nucleotide sequence of DHAV-1VP1-2A-3VP1 is as shown in SEQ ID NO:3.
Further, described step (2) specifically includes:
(2a) with recombiant plasmid pLS-RFP as template, the primer of design is utilized, at plasmid pLS-RFP RFP ORF two ends by Inverse PCR amplification obtain containing LaSota full-length cDNA linear Change carrier；
(2b) the DHAV 1VP1-2A-3VP1 gene that step (1) obtains is carried out PCR amplification, The gene end obtained contains the 15bp sequence spreading identical with LaSota linearized vector end；
(2c) after QIAEXII Gel Extraction Kit reclaims, In-is passed throughPCR DHAV 1VP1-2A-3VP1 gene is connected by Cloning Kit with LaSota linearized vector；
(2d) the connection product obtained is converted to Stbl2 competent cell, cultivate 24h, profit for 30 DEG C Carry out PCR with the primer of design to identify and DNA nucleotide sequence qualification, filter out positive colony, Named pLS-1VP1-2A-3VP1.
More excellent, in described step (2a):
Described Inverse PCR amplification obtains drawing of the linearized vector step containing LaSota full-length cDNA Thing is:
Forward primer: 5'-GGTGGCTACAACTATCAACTAAACT-3 ',
Downstream primer: 5'-GTGTGTAACTACCGTGTACTAAGC-3 '；
Amplified production clip size is 18.524kb；The nucleotide sequence of its amplified production such as SEQ ID NO: Shown in 4；
In described step (2b), described PCR amplification DHAV 1VP1-2A-3VP1 gene end contains The 15bp sequence spreading primer identical with LaSota linearized vector end is had to be:
Forward primer: 5'-atagttgtagccaccATGGGTGATTCTAACCAGTTG-3 ',
Downstream primer: 5'-acggtagttacacacCTAAATCTCCAGATGGAGCTC-3 '；
The clip size of amplified production is 2382bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:5；
In described step (2d), the PCR of described recombiant plasmid pLS-1VP1-2A-3VP1 identifies step Rapid primer is:
Forward primer: 5'-GCTCCTAAGCAAGTTAGATGC-3 ',
Downstream primer: 5'-CCCAACTTGAAAGATGAATCC-3 '；
Amplified production clip size is 3048bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:6.
Further, rescue side to recombinant virus rLS-1VP1-2A-3VP1 in described step (3) Method specifically includes:
(3a) the recombined vaccinia virus MVA/T7 expressing T7 polymerase is inoculated in length and has 90% monolayer In 24 orifice plates of HEp-2 cell, after hatching 1h, by 1.0 μ g pLS-1VP1-2A-3VP1,0.5 μ g PTM-NP, 0.25 μ g pTM-P and 0.05 μ g pTM-L helper plasmid cotransfection to HEp-2 cell, After transfection 6h, will the cells rinsed with PBS of transfection, and add containing 2%FBS and antibiotic DMEM culture medium；To transfection 72h, multigelation transfectional cell 3 times, the restructuring of results rescue Virus；
(3b) recombinant virus of results rescue is seeded to 9 age in days SPF Embryo Gallus domesticus, gathers in the crops Blood coagulation test For positive allantoic fluid, after filtering poxvirus, continuous passage on 9 age in days SPF Embryo Gallus domesticus, Obtain recombinant virus, and named rLS-1VP1-2A-3VP1, in-80 DEG C of preservations.
Further, the step that described recombinant virus is identified also is included after described step (3) (4), specifically include:
(4a) by E5 for recombinant virus rLS-1VP1-2A-3VP1 and parent recombinate poison rLS-RFP divide Not Gan Ran CEF cell monolayer, every 24h collect sample, each time point has two independently to repeat reality Test, detect TCID50, represent the titer of virus with Log10TCID50/ml, and draw virus Growth curve；
(4b) E5 generation restructuring poison rLS-1VP1-2A-3VP1 and parent's poison LaSota strain are felt respectively Dye CEF cell monolayer, carries out IFA and detects DHAV-1VP1 and DHAV-3VP1 gene respectively Express；
(4c) E5 generation restructuring poison rLS-1VP1-2A-3VP1 is tested duck through eye dripping Nasal immunization, often Plumage 1ml, takes a blood sample after 10 days after primary immune response；After the serum collected is inactivated 30min in 56 DEG C, enter 2 times of serial dilutions of row, respectively with equivalent 200ELD50DHAV-1 and DHAV-3 virus carry out And test, by Reed-Muench method calculate this restructuring poison anti-DHAV-1 of immune duck serum and DHAV-3 antibody mean titre.
The invention also discloses expression DHAV-1 and DHAV-3 type VP1 that said method prepares The NDV recombinant virus of gene.
The invention also discloses described recombinant virus for preparing prevention and treatment duck hepatitis A virus (HAV) and duck The purposes of newcastle disease vaccine.
Recombinant virus of the present invention selects NDV (Lasota strain) to be carrier, by DHAV-1 type and It is inserted into NDV (Lasota strain) carrier after the VP1 gene tandem of DHAV-3 type, utilizes and express T7 The recombined vaccinia virus MVA/T7 of polymerase and helper plasmid, save out coexpression DHAV-1 type and The NDV recombinant virus of the VP1 gene of DHAV-3 type, can be used for duck hepatitis A virus (HAV) (1 type, 3 types) With the prevention of duck newcastle, make up the blank of current DHAV-3 vaccine.Meanwhile, the present invention is obtained The application of multiple vaccines can reduce production of vaccine cost, simplify immune programme for children, reduce immunological stress, Overcome the interference phenomenon produced between different virus Attenuate vaccine, promote the sound development of duck culturing industry.
Accompanying drawing explanation
In order to make present disclosure be more likely to be clearly understood, below according to the concrete reality of the present invention Executing example and combine accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is the amplification electrophoretogram for DHAV-1VP1-2A-3VP1, and wherein M is DNA Marker DL15000,1 be the amplification of DHAV-1VP1-2A, 2 be DHAV-3VP1 amplification, 3 is the amplification of DHAV-1VP1-2A-3VP1；
Fig. 2 is that the PCR of recombiant plasmid pLS-1VP1-2A-3VP1 identifies electrophoretogram, and wherein M is The result of DNA Marker DL15000,1 be the result of vector plasmid pLS-RFP, 2 for restructuring matter The result of grain pLS-1VP1-2A-3VP1；
Fig. 3 is the RT-PCR electrophoretogram of the 1VP1-2A-3VP1 gene of recombinant virus, and wherein M is DNA Marker DL15000,1 be recombinant virus rLS-1VP1-3VP1,2 for recombinant virus rLS-RFP；
Fig. 4 is the growth curve of recombinant virus rLS-1VP1-2A-3VP1；
Fig. 5 is indirect immunofluorescene assay DHAV 1VP1 and the expression of results of DHAV 3VP1 albumen (× 200 times), wherein 1 is mouse-anti DHAV-1 serum and restructuring poison rLS-1VP1-2A-3VP1；2 For mouse-anti DHAV-3 serum and restructuring poison rLS-1VP1-2A-3VP1；3 is mouse-anti DHAV-1 serum With LaSota；4 is mouse-anti DHAV-3 serum and LaSota.
Detailed description of the invention
Embodiment 1DHAV-1 type, the connection of VP1 gene of DHAV-3 type
The DHV DHAV-1VP1-2A gene included with reference to GenBank and DHAV-3VP1 Gene nucleotide series, respectively with DHAV-1 and DHAV-3 as material, utilizes conventional RT-PCR side Method expands respectively and obtains DHAV-1VP1-2A gene and DHAV-3VP1 gene.According to description, Utilize Trizol reagent to extract DHAV-1 and DHAV-3 viral RNA, utilize following 1VP1-2A-F/1VP1-2A-SOE-R, 3VP1-SOE-F/3VP1-R primer expands respectively and obtains DHAV-1VP1-2A and DHAV-3VP1 gene.
Described DHAV-1 carries out the primer P1 that PCR amplification is DHAV-1VP1-2A gene:
1VP1-2A-F:5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
1VP1-2A-SOE-R:5'-CTGATTGGAATCACCTTGATCTGTAGTAAT-3 '；
The clip size of its amplified production DHAV-1VP1-2A is 1652bp；Amplified production The nucleotide sequence of DHAV-1VP1-2A is as shown in SEQ ID NO:1.
Described DHAV-3 carries out the primer P2 that PCR amplification is DHAV-3VP1 gene:
3VP1-SOE-F:5'-ATTACTACAGATCAAGGTGATTCCAATCAGC-3 ',
3VP1-R:5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NOTI).
The clip size of amplified production DHAV-3VP1 is 746bp；Amplified production DHAV-3VP1 Nucleotide sequence as shown in SEQ ID NO:2.
After gel reclaims, with two groups of PCR recovery product obtained above as template, with 1VP1-2A-F It is the amplification (knot that primer carries out the fragment of fusion gene DHAV 1VP1-2A-3VP1 mesh with 3VP1-R Fruit sees Fig. 1), utilize PCR method to merge and obtain recombination DHAV-1VP1-2A-3VP1.
Described DHAV-1VP1-2A gene and DHAV-3VP1 gene carry out PCR amplification and merge acquisition The primer P3 of recombination DHAV-1VP1-2A-3VP1 gene is:
1VP1-2A-F:5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
3VP1-R:5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NotI)；
The clip size of amplified production DHAV-1VP1-2A-3VP1 is 2368bp, amplified production The nucleotide sequence of DHAV-1VP1-2A-3VP1 is as shown in SEQ ID NO:3.
The PCR primer that will reclaim, is connected to pMD18-T-Vector, through EcoR I/Not I double digestion Identify, identify that correct positive recombinant pT-1VP1-2A-3VP1 serves sea English Weihe River victory base trade limited Company checks order.
Embodiment 2 expresses the structure of DHAV-1VP1-2A-3VP1 recombinant Newcastle disease virus
With the recombiant plasmid pLS-RFP containing LaSota full-length cDNA, (P gene and F gene interleave Have RFP reporter gene) it is template, utilize primer LS-P-up and LS-F-down of designed, designed to exist The two ends of the RFP ORF of plasmid pLS-RFP are obtained containing LaSota total length by Inverse PCR amplification The linearized vector of cDNA:
LS-P-up:5'-GGTGGCTACAACTATCAACTAAACT-3 ',
LS-F-down:5'-GTGTGTAACTACCGTGTACTAAGC-3 '；
Amplified production clip size is 18.524kb；The nucleotide sequence of its amplified production such as SEQ ID NO: Shown in 4.
Specific primer plant-VP1-F and plant-VP1-R is utilized to expand DHAV 1VP1-2A-3VP1 Gene, the gene end obtained contains the 15bp mutually identical with LaSota linearized vector end and extends sequence Row:
Plant-VP1-F:5'-atagttgtagccaccATGGGTGATTCTAACCAGTTG-3 ',
Plant-VP1-R:5'-acggtagttacacacCTAAATCTCCAGATGGAGCTC-3 '；
The clip size of amplified production is 2382bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:5.
After QIAEXII Gel Extraction Kit reclaims, pass through In-PCR Cloning Kit DHAV 1VP1-2A-3VP1 gene is connected with LaSota linearized vector；And connection product is turned Change Stbl2 competent cell, cultivate 24h, carried out by primer LS-J-up, LS-J-down for 30 DEG C PCR identifies (result is shown in Fig. 2), filters out positive colony, named pLS-1VP1-2A-3VP1；
LS-J-up:5'-GCTCCTAAGCAAGTTAGATGC-3 ',
LS-J-down:5'-CCCAACTTGAAAGATGAATCC-3 '；
Amplified production clip size is 3048bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:6.
The rescue of embodiment 3 recombinant virus rLS-1VP1-2A-3VP1
The recombined vaccinia virus MVA/T7 (MOI=3) expressing T7 polymerase is inoculated in length and has 90% In 24 orifice plates of monolayer HEp-2 cell, after hatching 1h, according to LipofectamineTM2000 (Invitrogen) description is by 1.0 μ g pLS-1VP1-2A-3VP1,0.5 μ g pTM-NP, 0.25 μ g PTM-P and 0.05 μ g pTM-L helper plasmid cotransfection HEp-2 cell.After transfection 6h, will turn The cell PBS of dye washes once, adds containing 2%FBS and the DMEM culture medium of antibiotic. After transfection 72h, 3 transfectional cells of multigelation, the recombinant virus of results rescue, and inoculated 9 age in days SPF Embryo Gallus domesticus, gather in the crops allantoic fluid, by the allantoic fluid 0.22um of blood clotting (HA) test positive Filter be filtered to remove poxvirus, and continuous passage on 9 age in days SPF Embryo Gallus domesticus, the virus of results It is saved in-80 DEG C, by named for recombinant virus rLS-1VP1-2A-3VP1.Recombinant virus is SPF chicken The front 4 generation HA result of the tests of embryo continuous passage are respectively 4log2,6log2,8log2,8log2.
The RT-PCR of embodiment 4 recombinant virus identifies
By rLS-1VP1-2A-3VP1 9 age in days SPF Embryo Gallus domesticus continuous passage 5 times, use TRIzol Method extracts chick embryo allantoic liquid recombinant virus RNA, with plant-VP1-F and plant-VP1-R as primer, Utilizing RT-PCR method to amplify the purpose product (result is shown in Fig. 3) of 2345bp, check order knot further Fruit display, the expection site of recombinant virus genomes has been correctly inserted into DHAV 1VP-2A-3VP1 gene.
The titration of embodiment 5 recombinant virus, pathogenic and growth kinetics detection
Recombinant virus titer and titration are respectively by HA, Embryo Gallus domesticus median infective dose (EID50) and tissue Median infective dose (TCID50) measures.Pathogenic analysis presses O.I.E standard respectively by average chicken Embryo lethal time (MDT), intracerbral pathogenicity index (ICPI) and intravenous pathogenic index (IVPI) etc. Pathogenicity is assessed.E5 is recombinated poison for recombinant virus rLS-1VP1-2A-3VP1 and parent RLS-RFP infects CEF cell monolayer (MOI=0.01) respectively, collects sample every 24h, each Time point has two independently to repeat experiment, detects TCID50, represents disease with Log10TCID50/ml The titer of poison, and draw the growth curve of virus, its growth kinetics in vitro and parental virus rLS-RFP Similar (result is shown in Fig. 4).
The EID50 in recombinant virus rLS-1VP1-2A-3VP1 the 5th generation is 107.2/0.1ml；MDT Be 0 more than 120h, ICPI and IVPI, according to OIE criterion (MDT >=90h, ICPI ＜ 0.5, IVPI=0), rLS-1VP1-2A-3VP1 is still low virulent strain, and maintains former recombinant vaccine strain rLS-RFP High titre growth adaptation and low pathogenic characteristic (result see table 1) to Embryo Gallus domesticus.
Table 1 recombinant Newcastle disease virus Identification of Biological Characteristics
Embodiment 6 indirect immunofluorescence assay (IFA)
By E5 generation restructuring poison rLS-1VP1-2A-3VP1 and parent's poison LaSota strain (MO=1.0) Infect CEF cell monolayer respectively, cultivate 24h for 37 DEG C, solid by acetone ethanol (3:2) room temperature of pre-cooling Determine 7min, with mouse-anti DHAV-1 hyper-immune serum and mouse-anti DHAV-3 hyper-immune serum be one resist, with FITC labelling sheep anti-mouse igg be two resist, carry out IFA detect respectively DHAV-1VP1 and The expression of DHAV-3VP1 gene.Result shows exempts from blood with mouse-anti DHAV-1 and mouse-anti DHAV-3 height It is clearly that an anti-detection rLS-1VP1-2A-3VP1 infection cell all becomes the fluorescence reaction that is positive；And with mouse-anti DHAV-1 and mouse-anti DHAV-3 hyper-immune serum are that an anti-detection LaSota infection cell does not observes sun Property reaction；Normal cell the most negative (result is shown in Fig. 5).
7 kinds of duck immunity tests of embodiment
By E5 generation restructuring poison rLS-1VP1-2A-3VP1 (viral titer 107.2EID50/0.1ml) through point Eye Nasal immunization test duck, every plumage 1ml, immunity is taken a blood sample after latter 10 days.By the serum of collection in 56 DEG C After inactivation 30min, carry out 2 times of serial dilutions, respectively with equivalent 200ELD50DHAV-1 and DHAV-3 virus mixes, 37 DEG C of effect 1h, often 5 pieces of duck embryos of group inoculation, 0.2mL/ embryo.Set simultaneously Virus control group and saline control group, 37 DEG C of Constant temperature hatchs observe 7d, by Reed-Muench method Calculate clear anti-DHAV-1 and the DHAV-3 antibody of this restructuring toxenia averagely to neutralize titer and be all higher than 1: 16。
Visible, recombinant virus of the present invention can be effectively used for prevention and treatment duck hepatitis A virus (HAV) and duck is new City epidemic disease vaccine.
Obviously, above-described embodiment is only for clearly demonstrating example, and not to embodiment party The restriction of formula.For those of ordinary skill in the field, the most also may be used To make other changes in different forms.Here without also all of embodiment being given With exhaustive.And the obvious change thus extended out or variation are still in the guarantor of the invention Protect among scope.

Claims (10)

1. expressing a NDV recombinant virus for DHAV-1 and DHAV-3 type VP1 gene, it is special Levying and be, described recombinant virus is designated as rLS-1VP1-2A-3VP1, is by DHAV-1 type and DHAV-3 Being inserted into NDV after type VP1 gene tandem is carrier, through the recombinant virus that rescue obtains.
2. prepare expression DHAV-1 and DHAV-3 type VP1 gene described in claim 1 for one kind The method of NDV recombinant virus, it is characterised in that comprise the steps:
(1) SOE-PCR method is used DHAV-1 type and DHAV-3 type VP1 gene to be carried out even Connect, it is thus achieved that DHAV 1VP1-2A-3VP1 recombination；
(2) DHAV 1VP1-2A-3VP1 gene is connected with LaSota strain linearized vector, and Product will be connected convert to Stbl2 competent cell, the weight of construction expression DHAV-1VP1-2A-3VP1 Group NDV viral vector, filters out positive colony；
(3) save and obtain recombinant virus rLS-1VP1-2A-3VP1.
Preparation method the most according to claim 2, it is characterised in that in described step (1), The step of described acquisition DHAV 1VP1-2A-3VP1 recombination specifically includes:
(1a) respectively with DHAV-1 and DHAV-3 as material, RT-PCR method is utilized to expand respectively Obtain DHAV-1VP1-2A gene and DHAV-3 VP1 gene；And reclaim with the two groups of PCR obtained Product is template, utilizes SOE-PCR method to obtain recombination DHAV-1VP1-2A-3VP1；
(1b) PCR primer that will reclaim, is connected to pMD18-T-Vector, through EcoR I/Not I Double digestion is identified, identifies correct positive recombinant.
Preparation method the most according to claim 3, it is characterised in that in described step (1a),
Described DHAV-1 carries out the primer P1 that PCR amplification is DHAV-1VP1-2A gene:
Forward primer: 5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
Downstream primer: 5'-CTGATTGGAATCACCTTGATCTGTAGTAAT-3 ',
The clip size of its amplified production DHAV-1VP1-2A is 1652bp；Amplified production The nucleotide sequence of DHAV-1VP1-2A is as shown in SEQ ID NO:1；
Described DHAV-3 carries out the primer P2 that PCR amplification is DHAV-3 VP1 gene:
Forward primer: 5'-ATTACTACAGATCAAGGTGATTCCAATCAGC-3 ',
Downstream primer: 5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NOTI),
The clip size of amplified production DHAV-3 VP1 is 746bp；Amplified production DHAV-3 VP1 Nucleotide sequence as shown in SEQ ID NO:2；
Described DHAV-1VP1-2A gene and DHAV-3 VP1 gene carry out SOE-PCR amplification and obtain The primer P3 obtaining recombination DHAV-1VP1-2A-3VP1 gene is:
Forward primer: 5'-TGGAATTCGGTGATTCTAACCAGTTG-3 ' (BamHI),
Downstream primer: 5'-AATGCGGCCGCTTCAATYTCCARAT-3 ' (NotI),
The clip size of amplified production DHAV-1VP1-2A-3VP1 is 2368bp, amplified production The nucleotide sequence of DHAV-1VP1-2A-3VP1 is as shown in SEQ ID NO:3.
5. according to the arbitrary described preparation method of claim 2-4, it is characterised in that described step (2) Specifically include:
(2a) with recombiant plasmid pLS-RFP as template, the primer of design is utilized, at plasmid pLS-RFP RFP ORF two ends by Inverse PCR amplification obtain containing LaSota full-length cDNA linear Change carrier；
(2b) the DHAV 1VP1-2A-3VP1 gene that step (1) obtains is carried out PCR amplification, The gene end obtained contains the 15bp sequence spreading identical with LaSota linearized vector end；
(2c) after QIAEXII Gel Extraction Kit reclaims, pass throughPCR DHAV 1VP1-2A-3VP1 gene is connected by Cloning Kit with LaSota linearized vector；
(2d) the connection product obtained is converted to Stbl2 competent cell, cultivate 24h, profit for 30 DEG C Carry out PCR with the primer of design to identify and DNA nucleotide sequence qualification, filter out positive colony, Named pLS-1VP1-2A-3VP1.
Preparation method the most according to claim 5, it is characterised in that: in described step (2a),
Described Inverse PCR amplification obtains drawing of the linearized vector step containing LaSota full-length cDNA Thing is:
Forward primer: 5'-GGTGGCTACAACTATCAACTAAACT-3 ',
Downstream primer: 5'-GTGTGTAACTACCGTGTACTAAGC-3 '；
Amplified production clip size is 18.524kb；The nucleotide sequence of its amplified production such as SEQ ID NO: Shown in 4；
In described step (2b), described PCR amplification DHAV 1VP1-2A-3VP1 gene end contains The 15bp sequence spreading primer identical with LaSota linearized vector end is had to be:
Forward primer: 5'-atagttgtagccaccATGGGTGATTCTAACCAGTTG-3 ',
Downstream primer: 5'-acggtagttacacacCTAAATCTCCAGATGGAGCTC-3 '；
The clip size of amplified production is 2382bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:5；
In described step (2d), the PCR of described recombiant plasmid pLS-1VP1-2A-3VP1 identifies step Rapid primer is:
Forward primer: 5'-GCTCCTAAGCAAGTTAGATGC-3 ',
Downstream primer: 5'-CCCAACTTGAAAGATGAATCC-3 '.
Amplified production clip size is 3048bp；The nucleotide sequence of described amplified production such as SEQ ID Shown in NO:6.
7. according to the preparation method described in claim 5 or 6, it is characterised in that: described step (3) In the rescue method of recombinant virus rLS-1VP1-2A-3VP1 is specifically included:
(3a) the recombined vaccinia virus MVA/T7 expressing T7 polymerase is inoculated in length and has 90% monolayer In 24 orifice plates of HEp-2 cell, after hatching 1h, by 1.0 μ g pLS-1VP1-2A-3VP1,0.5 μ g PTM-NP, 0.25 μ g pTM-P and 0.05 μ g pTM-L helper plasmid cotransfection to HEp-2 cell, After transfection 6h, will the cells rinsed with PBS of transfection, and add containing 2%FBS and antibiotic DMEM culture medium；To transfection 72h, multigelation transfectional cell 3 times, the restructuring of results rescue Virus；
(3b) recombinant virus of results rescue is seeded to 9 age in days SPF Embryo Gallus domesticus, gathers in the crops Blood coagulation test For positive allantoic fluid, after filtering poxvirus, continuous passage on 9 age in days SPF Embryo Gallus domesticus, Obtain recombinant virus, and named rLS-1VP1-2A-3VP1, in-80 DEG C of preservations.
8. according to the arbitrary described preparation method of claim 2-7, it is characterised in that: described step (3) The most also include the step (4) that described recombinant virus is identified, specifically include:
(4a) by E5 for recombinant virus rLS-1VP1-2A-3VP1 and parent recombinate poison rLS-RFP divide Not Gan Ran CEF cell monolayer, every 24h collect sample, each time point has two independently to repeat reality Test, detect TCID50, represent the titer of virus with Log10TCID50/ml, and draw virus Growth curve；
(4b) E5 generation restructuring poison rLS-1VP1-2A-3VP1 and parent's poison LaSota strain are felt respectively Dye CEF cell monolayer, carries out IFA and detects DHAV-1VP1 and DHAV-3VP1 gene respectively Express；
(4c) E5 generation restructuring poison rLS-1VP1-2A-3VP1 is tested duck through eye dripping Nasal immunization, often Plumage 1ml, takes a blood sample after 10 days after primary immune response；After the serum collected is inactivated 30min in 56 DEG C, enter 2 times of serial dilutions of row, respectively with equivalent 200ELD50DHAV-1 and DHAV-3 virus carry out And test, by Reed-Muench method calculate this restructuring poison anti-DHAV-1 of immune duck serum and DHAV-3 antibody mean titre.
9. the arbitrary described method of claim 2-8 prepares expression DHAV-1 and DHAV-3 type The NDV recombinant virus of VP1 gene.
10. the recombinant virus described in claim 1 or 9 is used for preparing prevention and treatment duck hepatitis A virus (HAV) Purposes with duck newcastle disease vaccine.
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CN111518777A (en) * 2020-05-19 2020-08-11 扬州大学 Construction method of recombinant baculovirus expressing avian adenovirus serotype 4 spike protein F1

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