CN108728419A - Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method - Google Patents
Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method Download PDFInfo
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- CN108728419A CN108728419A CN201810580569.8A CN201810580569A CN108728419A CN 108728419 A CN108728419 A CN 108728419A CN 201810580569 A CN201810580569 A CN 201810580569A CN 108728419 A CN108728419 A CN 108728419A
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Abstract
The present invention relates to expression aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and its construction method, the newcastle disease virus strain rAI4-penton, its preserving number CGMCC No：15492.Its construction method is the reverse genetic manipulation platform using established newcastle disease attenuated IBDVs, the penton gene orders of aviadenovirus are inserted into AI4 pnca gene group overall length transcription vectors pNDV/rAI4, recombinant Newcastle disease virus full length cDNA clone pNDV/rAI4-penton is obtained, the recombinant virus rAI4-penton successful expression penton albumen obtained through transfection.Recombinant virus rAI4-penton has higher reproductive titer in chicken embryo, and continuous passage, which is appointed, can stablize penton albumen, be suitable for the large-scale production of vaccine, can be used for manufacturing vaccine.
Description
Technical field
The present invention relates to using Reverse Genetics, one plant of rescue expresses aviadenovirus by carrier of newcastle disease virus
The recombinant virus rAI4-penton of penton albumen, for developing vaccine.
Background technology
It is cDNA that reverse Genetics Technique, which is by rna virus cdna group reverse transcription, then cDNA and various auxilin phases
Interaction is assembled into process [Neumann G, the Kawaoka Y.A decade after the generation of infectious RNA
of a negative-sense RNA virus from cloned cDNA-what have we learned？[J].The
Journal of general virology,2002,83(Pt 11):2635-62.], this process is otherwise known as " viral
Rescue ".It, can be in the level of DNA since the RNA virus of reverse Genetics Technique final " rescue " derives from cDNA clone
On various external manual operations are carried out to the genome of RNA virus, it is slotting such as to carry out gene mutation, gene knockout (missing), gene
Enter, the transformations such as gene substitution and gene complementation, solves and rna virus cdna group is difficult to operate this problem, it is greatly convenient
People carry out the research of the function, pathogenesis and virosis Control srtategy of each gene of virus, while grinding for new generation vaccine
System and exploitation provide new thinking [Huang Z, Elankumaran S, Panda A, et al.Recombinant
Newcastle disease virus as a vaccine vector.Poultry Science,2003,82:899-
906.]。
Newcastle disease (Newcastle disease, ND) be by newcastle disease virus (Newcastle disease virus,
NDV the poultry such as main infection chicken, dove, quail, turkey caused by) and a kind of acute, septic of wild birds, high degree in contact
Infectious disease.The genome of newcastle disease virus is non-segmented negative, sub-thread strand RNA, 3 '-leader-NP-P- of genome pattern
M-F-HN-L-trailer-5 ' encodes 6 kinds of structural proteins successively：Nucleocapsid protein (Nucleocapsid protein, NP),
Phosphoprotein (Phosphoprotein, P), stromatin (Matrix protein, M), fusion protein (Fusion protein,
F), hemagglutinin-neuraminidase albumen (Heamagglutinin-Neuraminidase protein, HN) and high molecular weight protein
(Large protein,L).Wherein F protein has the function of inducing cell fusion, destroys cell, and F protein is different NDV virulence
The main reason for strong and weak, and the cracking site of F protein play important function [peeters B P, Deleeuw, Koch G,
et al. Rescue of Newcastle disease virus from cloned cDNA:evidence that
cleavability of the fusion protein is a major determinant for virulence[J]
.Journal of virology,1999,73(6):5001-9.]。
Aviadenovirus (Fowl adenovirus, FAV) belongs to Adenoviridae Aviadenovirus, the nucleocapsid of aviadenovirus
It is made of hexon (hexon), penton (penton) and filament proteins (fiber).According to the difference of group specific antigen point
For 3 groups：I group I fowl adenovirus include from the isolated each serotype strain of different birds, have just as group specificity it is anti-
Original can be divided into five genotype of A-E according to RFLP technologies, can be divided into 12 serotypes according to serum cross-neutralization reaction
(serotype 1-7,8a, 8b, 9-11), representative strains are CELO virus (CELOV) [Meulemans G, ouvreur
B, Decaesstecker M,et al.Phylogenetic analysis of fowl adenoviruses[J C]
.Avian pathology:journal of the WVPA,2004,33(2):164-70.].II group I fowl adenovirus includes turkey
Hemorrhagic Enteritis Virus (HEV), the pheasant marble lienopathla (MSDV) and birds splenomegaly are viral (AASV)；III group I fowl adenovirus packet
Include egg drop syndrome (EDSV).Wherein I group of 4 type aviadenovirus (FAdV4) of serum can cause inclusion body hepatitis and hydropericardium
Syndrome (HPS) [Kim J N, Byun S H, Kim M J, et al.Outbreaks of hydropericardium
syndrome and molecular characterization of Korean fowl adenoviral isolates
[J].Avian diseases,2008,52(3):526-30.]。
Countries in the world mainly use oil-emulsion bacterin to prevent HPS [Du D, Zhang P, Li X, et at present
al.Cell-culture derived fowl adenovirus serotype 4inactivated vaccine
provides complete protection for virus infection on SPF chickens[J]
.Virusdisease,2017,28(2):182-8.], but with the vaccine of this approach production there are of high cost, it is inconvenient for use, and
Not exclusively there is the risk of diffusion virus in the presence of inactivation, while attenuated live vaccines have genetic recombination, the anti-strong risk of virulence again,
Therefore the novel genes engineered vaccine such as genetic vaccine, subunit vaccine, nucleic acid vaccine becomes the heat of vaccine research in recent years
Point.
The most important feature of vector virus can be exactly used to develop rapidly for newly sending out infectious disease highly pathogenic
Recombinant vaccine, newcastle disease virus do not recombinate, mass expressing external base in addition to having this feature with homologous virus
Because the serious decrease of self-replication will not be caused, cause it is weak after do not have to humans and animals pathogenic, a kind of safety barrier can be used as
[Bukreyev A, Skiadopoulos M H,Murphy B R,et al.Nonsegmented negative-strand
viruses as vaccine vectors[J]. Journal of Virology,2006,80(21):10293-10306.]。
It is inserted into the recombinant virus after foreign gene in NDV genomes, can replicate in animal body and stablizes constantly expression external source egg
In vain, it uses this recombinant virus as vaccine, body can be induced to generate the Double immune protection for foreign protein and newcastle disease
[Veits J,Wiesner D,Fuchs W,et al.Newcastle disease virus expressing
H5hemagglutinin gene protects chickens against Newcastle disease and avian
influenza[J].ProcNatlAcadSci U S A,2006,103(21):8197-8202.Schroer D,Veits J,
Grund C,et al.Vaccination with Newcastle disease virus vectored vaccine
protects chickens against highly pathogenic H7avian influenza virus[J].Avian
Dis,2009,53(2):190-197.], [Lamb, R.A., Kolakofsky, D., the et al. (2001) such as Lamb
.Paramyxoviridae:the viruses and their replication,p.1305-1340.In D. M.Knipe
And P.M.Howley, Fields virology.Lippincott Williams&Wilkins Philadephia, Pa.] it grinds
Study carefully and find that the transcription of paramyxovirus is in polar effect, its transcription product is more closer to the gene at the ends 3', is transcribed closer to the ends 5'
The amount of product is lower.From the point of view of current research, when foreign gene is inserted between P and M genes, expression quantity is higher, is external source
The first choice that gene is inserted into.The F protein for the VII d hypotype NDV strains JS5/05 that Hu Zenglei in 2011 etc. detaches this laboratory cracks
Full-length clone pNDV/AI4 is successfully constructed after site mutation, is obtained through reverse genetic manipulation and causes weak AI4, is had excellent numerous
Performance is grown, the potency of allantoic fluid can reach 10log2, be suitable as carrier [Hu Z, the Hu S, Meng of expression alien gene
C,et al.Generation of a genotype VII Newcastle disease virus vaccine
candidate with high yield in embryonated chicken eggs[J].Avian diseases,2011,
55(3):391-7.]。
The protective antigens of aviadenovirus includes：Hexon (hexon), penton (penton) and filament proteins
(fiber), there is presently no using newcastle disease virus as carrier, express aviadenovirus foreign gene to build the elder generation of bivalent vaccine
Example.
Penton albumen is the important structural proteins of aviadenovirus, participates in the mistake of Virus entry cell and virion assembling
Journey.Some researches show that the antibody of anti-penton albumen to have significant virus neutralization, and 90% protection can be provided for animal
Rate [Shah M S, Ashraf A, Rahman M, et al.A subunit vaccine against
hydropericardium syndrome using adenovirus penton capsid protein[J].Vaccine,
2012,30(50):7153-6.]。
Invention content
It is an object of the invention to express the recombinant virus of aviadenovirus penton albumen using NDV as carrier, for making
Make vaccine.
The present invention expresses the recombinant virus rAI4-penton of aviadenovirus penton genes, is protected on March 20th, 2018
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No：15492.
The present invention discloses the construction method of the expression aviadenovirus penton Protein reconstitution Strain rAI4-penton：
Using the full-length genome transcription vector pNDV/rAI4 of genotype Ⅶ NDV attenuated IBDVs AI4 as skeleton, by aviadenovirus
Penton genes are inserted between P, M gene of AI4 pnca gene group overall length transcription vectors pNDV/rAI4, through reverse genetics skill
Art obtains recombinant virus rAI4-penton.
The present invention utilizes established newcastle disease virus AI4 plants of reverse genetic manipulation platform, by the Ministry of Agriculture of Yangzhou University
The penton genes of one plant of FAdV4 of livestock and poultry infectious disease emphasis open laboratory separation are inserted into NDV overall length transcription vectors pNDV/
Between P, M gene of rAI4, recombinant virus rAI4-penton is obtained, recombinant virus rAI4-penton all has compared with Gao Fan
Performance is grown, the large-scale production of vaccine is suitble to.
The method of the present invention specifically includes following steps：
1. the structure of full-length clone pNDV/rAI4-penton
1) structure of interstitial granules pNAI4-blunt (I-BstZ17 I of Age) in
Product is connected into blunt carriers, structure by design primer primer1 and primer6 using pNDV/rAI4 as template PCR
It builds to obtain plasmid pNAI4-blunt (I-BstZ17 I of Age).
Primer1：5'-AACTCTCACAACCGGTGC-3'(SEQ ID NO.1)
Primer6：5'-CTGAGACGAGGTGTATACATTGACTG-3'(SEQ ID NO.6)
2) clone of aviadenovirus penton genes
According to the full length sequence of the 1 plant of aviadenovirus strain CH/JSXZ/2015 delivered in GenBank, pass through Jin Sirui public affairs
Department's synthesis penton genes, the design primer NAI4-P3 and NAI4-P4 amplification gene coding regions penton (SEQ ID NO.9) are led to
The method for crossing homologous recombination is connected into plasmid pNAI4-blunt (I-BstZ17 I of Age), and the correct plasmid of sequencing result is named
For pNAI4-penton-blunt (I-BstZ17 I of Age).
NAI4-P3:5’-AAAAAATACGGGTAGAAGCCACCATGTGGGGGTTGCAGCCGC-3’(SEQ ID NO.3)
NAI4-P4:5’-GATTCCGTGTGACGCCTACTGCAAGGTCGCGGAAC-3’(SEQ ID NO.4)
3) acquisition of full-length clone pNDV/rAI4-penton
Plasmid pNAI4-penton-Blunt (I-BstZ17 I of Age) and plasmid pNDV/rAI4 passes through Age I and BstZ17 I
Double digestion, through agarose gel electrophoresis recycling pNAI4-penton-Blunt (I-BstZ17 I of Age) 3428bp band and
The big band of pNDV/rAI4, is then attached, and constructs recombination NDV full-length genomes pNDV/rAI4-penton, specific structure
Construction method is shown in Fig. 1
2. the rescue that pNDV/rAI4-penton plants of recombinant virus
By overall length plasmid pNDV/rAI4-penton and tri- helper plasmid cotransfection BSR- of pCI-NP, pCI-P and pCI-L
T7/5 cells are inoculated with 9~11 age in days SPF chicken embryos after 60h, recombinant virus rAI4-penton are obtained after a blind passage generation.
The present invention is inserted into the protective antigens hexon of different aviadenovirus using different newcastle disease virus as carrier respectively
(hexon), penton (penton) and filament proteins (fiber), it is final to be only inserted into obtained recombinant Newcastle disease virus
The recombinant virus of penton genes can stimulate animal to generate the neutralizing antibody for aviadenovirus.
For the present invention using pNDV/rAI4 as skeleton, structure obtains to express the recombinant Newcastle disease of aviadenovirus penton albumen
Strain rAI4-penton successfully stimulates animal to produce the antibody for newcastle disease virus and aviadenovirus, can be used for answering
To the prevalence of current newcastle disease and hydropericardium.
Description of the drawings
Fig. 1 recombinant plasmid pNDV/rAI4-penton forming types figures.
Fig. 2 recombinant plasmid pNDV/rAI4-fiber2 forming types figures.
Fig. 3 recombinant virus cDNA RT-PCR identify electrophoretogram.M:1000bp DNA ladder marker, 1:rAI4-
Penton amplified fragments, 2:RAI4-fiber2 amplified fragments.
Fig. 4 recombinant plasmids infect the western blot testing results of foreign protein after DF1 cells.1：Marker, 2：
RAI4-penton, 3：RAI4-fiber2,4：AI4.
The HI antibody levels of anti-NDV viruses after Fig. 5 is immune.
Fig. 6 immune chicken serum moderate resistance aviadenovirus neutralization titers.
Interstitial granules pAI4-2FHN-blunt (I-Afl II of Age) forming types figure in Fig. 7.
Fig. 8 recombinant plasmid pNDV/rAI4-2FHN-hexon forming types figures.
Fig. 9 recombinant virus foreign gene RT-PCR results.M:1000bp DNA ladder marker, 1:rAI4-2FHN-
Hexon amplified fragments.
Figure 10 immunoblotting assay exogenous protein expression situations.1：Marker, 2：RAI4-2FHN-hexon, 3： rAI4-
2FHN-penton, 4：rAI4-2FHN-fiber2.
The HI antibody levels of anti-vector virus after Figure 11 is immune.
Anti- aviadenovirus neutralizing antibody is horizontal after Figure 12 is immune
The expression aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton that the present invention is built in
On March 20th, 2018 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Court of Beijing
The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica), Classification And Nomenclature is：Avian paramyxoviruses I types；Preserving number
CGMCC No.15492。
Specific implementation mode
Embodiment 1：Using pNDV/rAI4 as carrier
Step 1：It is total length expressed to express aviadenovirus penton Protein reconstitution newcastle disease vaccine Candidate Strains rAI4-penton
The structure of clone
Total length expressed carrier pNDV/rAI4 (Hu Z, Hu S, Meng C, the et al.Generation of of AI4 plants of NDV
a Genotype VII Newcastle Disease Virus Vaccine Candidate with HighYield in
Embryonated Chicken Eggs.Avian Diseases 2011,55(3):1759-1769) by the Ministry of Agriculture of Yangzhou University
Livestock and poultry infectious disease emphasis open laboratory structure preserves.Blunt carriers：Purchased from Transgen companies；
Dephospharylation(BAP)Kit：Purchased from precious bioengineering (Dalian) Co., Ltd；AMV reverse transcriptase, High
Fidelity DNA polymerase, T4DNA ligases, Agarose Gel DNA ExtractionKit are public purchased from Roche
Department；Transfection reagent SuperFect and plasmid extraction kit (QIAprep Spin MiniPrep Kit) are produced for QIAGEN companies
Product；Remaining conventional reagent is that domestic analysis is pure.
1, the structure of middle interstitial granules pNAI4-blunt (I-BstZ17 I of Age)
According to the primers primer1 and primer6 of plasmid pNDV/rAI4, for expanding plasmid pNDV/rAI4
Sequence between restriction enzyme site Age I (2879)-BstZ17 I (4705).Primer sequence is as follows：
Primer1：5'-AACTCTCACAACCGGTGC-3'(SEQ ID NO.1)
Primer6：5'-CTGAGACGAGGTGTATACATTGACTG-3'(SEQ ID NO.6)
Thickened portion is restriction enzyme site sequence in primer sequence, and italicized item is homologous sequence, and the above primer is given birth to by Shanghai
Object Engineering Co., Ltd synthesizes.
PCR is reacted using plasmid pNDV/rAI4 as the following system of template configuration：
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 2min, carry out
15 cycles；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 2min carry out 15 cycles；72 DEG C extension 10min, 4 DEG C
It preserves.
The band that 1852bp is cut after agarose gel electrophoresis preserves after recycling pcr products in 4 degrees Celsius.
It is connect with blunt carriers after PCR product recovery purifying, is transformed into DH5 α competence, the plasmid Primer1 of extraction
PCR verifications are carried out with Primer6, positive plasmid is named as pNAI4-blunt (I-BstZ17 I of Age).
2, the clone of aviadenovirus penton genes
Aviadenovirus strain CH/JSXZ/2015 (GenBank are synthesized by Jin Sirui companies:KU569296.1)
Penton genes, according to penton gene order design primer NAI4-P3 and NAI4-P4, the volume for expanding penton genes
Code area.It is homologous heavy for carrying out according to primers Primer1, Primer2, Primer5, Primer6 of pNDV/rAI4
Group reaction.
In primer sequence, thickened portion is restriction enzyme site sequence, and lower stroke of dotted line is kozak sequences, can improve external source base
The expression of cause, downslide solid line are GE the and GS sequences of newcastle disease virus, and italicized item is the homologous sequence on primer, primer
By Nanjing spun gold, auspicious company synthesizes.Using Primer1 and Primer2 as 2879 (Age I) of primer amplification pNDV/rAI4 extremely
Segment between 3114 (Pac I) is primer amplification with the ORF segments of penton genes using NAI4-P3 and NAI4-P4,
Primer5 and Primer6 is 3114 (Pac I) of primer amplification pNDV/rAI4 to the piece between 4705 (BstZ17 I)
Plasmid pNAI4-blunt (I-BstZ17 I of Age) is exposed homology arm by section using Age I and BstZ17 I progress double digestions,
Carrier segments are recycled, above 3 PCR fragments, which are reacted with carrier segments using homologous sequence progress homologous recombination, can build to obtain
Plasmid pNAI4-penton-blunt (I-BstZ17 I of Age)；By plasmid pNAI4-penton-blunt (I-BstZ17 I of Age)
It is connected into overall length plasmid pNDV/rAI4 by Age I and I double digestions of BstZ17, recombinant virus overall length plasmid pNDV/ can be obtained
rAI4-penton。
PCR reaction systems：
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15
A cycle；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15 cycles；72 DEG C of extension 10min, 4 DEG C of guarantors
It deposits.
To NAI4-P3 and NAI4-P4, the segment of primer amplification is sequenced in this, obtains the gene sequence of penton albumen
Row, i.e. sequence shown in SEQ ID NO.7：
By PCR product into row agarose gel electrophoresis, the purpose band of 265bp, 1794bp and 1603bp can be respectively obtained,
Will purpose band with glue reclaim reagent recycle after be positioned over 4 DEG C it is spare.
Plasmid pNAI4-Blunt (I-BstZ17 I of Age) is subjected to double digestion with Age I and BstZ17 I, recycles 3982bp items
Band is tried with the counterband tape of 265bp, 1794bp and 1603bp by In Fusion Advantage PCR Cloning Kit
Agent box is attached, and is transformed into DH5 α competent cells, and the plasmid of extraction send Nanjing spun gold auspicious company's sequencing, and sequencing result is correct
Plasmid be named as pNAI4-penton-blunt (I-BstZ17 I of Age).
3, the structure of full-length clone pNDV/rAI4-penton
Plasmid pNAI4-penton-blunt (I-BstZ17 I of Age) and plasmid pNDV/rAI4 passes through Age I and BstZ17 I
Double digestion, through agarose gel electrophoresis recycling pNAI4-penton-blunt (I-BstZ17 I of Age) 3428bp band and
The big band of pNDV/rAI4, is then attached, and is transformed into DH5 α competent cells, identifies that positive plasmid is named as
pNDV/rAI4-penton.(see Fig. 1)
Step 2：It is total length expressed to express aviadenovirus fiber2 Protein reconstitution newcastle disease vaccine Candidate Strains rAI4-fiber2
The structure of clone
1, the clone of aviadenovirus fiber2 genes
The fiber2 genes of aviadenovirus strain CH/JSXZ/2015, GenBank sequence numbers are synthesized by Jin Sirui companies
For KU569296.1, according to fiber2 gene order design primer NAI4-F3 and NAI4-F4, for expanding fiber2 genes
Code area.It is homologous for carrying out according to primers Primer1, Primer2, Primer5, Primer6 of pNDV/rAI4
Recombining reaction.
In primer sequence, thickened portion is restriction enzyme site sequence, and lower stroke of dotted line is kozak sequences, can improve external source base
The expression of cause, downslide solid line are GE the and GS sequences of newcastle disease virus, and primer is synthesized by Shanghai bioengineering Co., Ltd.
PCR reaction systems：
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15
A cycle；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15 cycles；72 DEG C of extension 10min, 4 DEG C of guarantors
It deposits.
To NAI4-F3 and NAI4-F4, the segment of primer amplification is sequenced in this, obtains the gene sequence of fiber2 albumen
Row, i.e. sequence shown in SEQ ID NO.10：
Plasmid pNAI4-blunt (I-BstZ17 I of Age) is subjected to double digestion with Age I and BstZ17 I, exposes homology arm,
Fiber2 genes are connected into carrier, by what is obtained by recycling 3982bp bands as the method in step 1 by homologous recombination
Plasmid order-checking, the correct plasmid of sequencing result are named as pNAI4-fiber2-blunt (I-BstZ17 I of Age).
2, the structure of full-length clone pNDV/rAI4-fiber2
Plasmid pNAI4-fiber2-blunt (I-BstZ17 I of Age) and plasmid pNDV/rAI4 passes through Age I and BstZ17 I
Double digestion, through agarose gel electrophoresis recycling pNAI4-fiber2-blunt (I-BstZ17 I of Age) 3428bp band and
The big band of pNDV/rAI4, is then attached, and is transformed into DH5 α competent cells, identifies that positive plasmid is named as
PNDV/rAI4-fiber2 (see Fig. 2).
Step 3：The rescue that rAI4-penton plants and rAI4-fiber2 plants of recombinant virus
The BSR-T7/5 cells of expression t7 rna polymerase can be stablized：By Harbin Veterinary Medicine Inst., China Academy of Agriculture
Step chigo researcher give.CEF cells are voluntarily prepared according to a conventional method by laboratory.
Eukaryon expression plasmid pCI-NP, pCI-P, pCI-L (Hu Shunlin, Yanmei ZHANG, the goose's Newcastle diseases such as Sun Qing, Liu Yuliang
Foundation [J] microbiologies of virus rescue are notified to .2007,34 (3))：By the Ministry of Agriculture of Yangzhou University livestock and poultry pestology weight
Point open laboratory structure preserves.Wherein, eukaryon expression plasmid pCI-NP, pCI-P of expression NP and P genes are also disclosed in (Liu
Beautiful good (2005) generate ZJ1 plants of goose's Newcastle diseases of infectivity from cDNA clone)；Express the eukaryon expression plasmid pCI- of L genes
L is also disclosed in (foundation and its application of Hu Shunlin (2007) Newcastle disease virus from goose Reverse Genetics platforms).
SPF hatching eggs are purchased from Beijing Cimmeria company, in livestock and poultry pestology emphasis open laboratory of the Ministry of Agriculture of Yangzhou University
Hatching.
1, the rescue of recombinant virus
Plasmid (overall length plasmid, pCI-NP, pCI-P and pCI-L) used is all made of when transfection
QIAprepSpinMiniPrepKit is extracted.By 3 helper plasmids of pCI-NP, pCI-P and pCI-L respectively with recombination NDV genes
Group full length cDNA clone pNDV/ rAI4-penton and pNDV/rAI4-fiber2 cotransfection BSR-T7/5 cells, after 18-24h
Final concentration of 10%SPF embryo allantoic liquids are added, after transfection sample freeze thawing 1 time, will be harvested after 96h and be inoculated with 9~11 age in days SPF chickens
Embryo collects chick embryo allantoic liquid, by OIE standard test hemagglutinative titers, obtains the recombinant Newcastle disease vaccine of expression aviadenovirus albumen
Candidate Strain rAI4-penton and rAI4-fiber2.The blood clotting characteristic of two plants of recombinant viruses can be by the polyclonal antibody for NDV
Inhibit.
After rAI4-penton plants pass on 5 times in chicken embryo, surveyed HA potency is 9log2, compared with maternal strain AI4 under
1 titre has dropped, and rAI4-fiber2 plants of HA potency of recombinant virus are 7log2, 2 titres are had dropped compared with AI4 plants,
Fiber2 genes are inserted into the more obvious duplication for inhibiting newcastle disease virus.
2, RT-PCR verifies recombinant virus of being rescued
Hemagglutination test (HA test) (hemagglutination, HA) and erythrocyte agglutination Inhibition test
(hemagglutination inhibition, HI) detection is that positive allantoic fluid is collected after SPF chicken embryos uploaded for 4 generations
The total serum IgE of allantoic fluid extracting virus is reacted at after cDNA for RT-PCR with 6nt random primed reverse transcriptions.
Penton and fiber-2 is expanded with primer Primer1 and AI4a, length is respectively 2044bp and 1906bp, by PCR
Product recycling and sequencing result, show that foreign gene is successfully plugged by reverse genetics in newcastle Disease poisonous carrier.(see figure
3)
Primer1:5’-AACTCTCACAACCGGTGC-3’(SEQ ID NO.1)
AI4a：5'-CCTGGATGAGTCCATTTTGGTG-3'(SEQ ID NO.11)
Step 4：The Identification of Biological Characteristics of recombinant virus
1, the measurement of MDT
The chicken embryo median lethal time (mean death time, MDT) measures：Recombinant virus is divided with sterile saline
It Zuo not be 10 times (10-6、10-7……10-10) gradient dilution, each dilution is inoculated with 5 piece of 9~11 age in days SPF chicken embryo, per embryo
0.1mL, while the chicken embryo of 5 pieces of inoculation physiological saline is set as a contrast.37 DEG C of incubations, discard chicken embryo dead in for 24 hours, later
Every 12h observations once until 120h, the MDT of virus is calculated by OIE standard methods.
As a result：Recombinant virus shows recombinant virus rAI4- in inoculated into chick embryo no death in 120h of 5 dilutions
The MDT values of penton and rAI4-fiber2 are more than 120h.
2, Westernblot is tested
By recombinant virus rAI4-penton, rAI4-fiber2 with the dose inoculation DF1 cells of MOI=1, after infection for 24 hours
Lytic cell collects plasmosin and cracks the chicken embryo fibroblasts of uninfecting virus as blank control simultaneously.Treated sample
Product carry out SDS-PAGE electrophoresis, and albumen is transferred on the pvdf membrane of the apertures 0.22um, 5% skimmed milk closing, with the anti-fowl glands of Ji Yuan
For viral polyvalent antibody as primary antibody, the rabbit anti-chicken IgG of HRP labels is that secondary antibody carries out antigen-antibody binding reaction.With ECL luminescence methods
Colour developing acquires signal with gel imaging system.
As a result：Successful expression goes out penton albumen and fiber2 respectively by recombinant virus rAI4-penton and rAI4-fiber2
Albumen (see Fig. 4).
Step 5：The immuning effect test of vaccine carrier strain
1 age in days SPF chickens are grouped at random, 10/group, carry out isolated rearing, carrier bacterin is inoculated with respectively through collunarium eye droppings
RAI4- penton and rAI4-fiber2, dosage 106EID50/ only, while AI4 inoculation groups are set as blank control, PBS connects
Kind group is used as negative control.3 weeks acquisition venous blood, detection turn for the serum of NDV and aviadenovirus after every experiment chicken immune
Change horizontal.
As a result：Vaccinated flock does not show any clinical symptoms, illustrates that recombinant virus is not pathogenic to chicken, can make safely
With.Animal can be stimulated to generate for newcastle disease virus after recombinant virus rAI4-penton and rAI4-fiber2 inoculation animal
Antibody, HI potency is in 4log2(refer to Fig. 5) above；RAI4-penton Strain can effectively stimulate generation anti-in SPF chickens
The antibody of aviadenovirus；But rAI4-fiber2 Strain does not detect that the neutralization for aviadenovirus resists after animal is immunized
Body is suspected since the insertion of fiber2 genes causes the reproductive performance of recombinant virus rAI4-fiber2 to decline, leads to its immunogene
Property reduce (referring to Fig. 6).
Embodiment two：Using pNDV/rAI4-T4FHN as carrier
The preserving number of plasmid pNDV/rAI4-T4FHN is CGMCC No：12987, by Chinese patent application
201710596136.7 open.
Step 1：Express aviadenovirus hexon Protein reconstitution newcastle disease vaccine Candidate Strain rAI4-2FHN-hexon overall length tables
The structure of Dyclonine
1, the structure of middle interstitial granules pAI4-2FHN-blunt (I-BstZ17 I of Age)
It is as follows with reference to pNDV/rAI4-T4FHN design primers：
Thickened portion is restriction enzyme site sequence in primer sequence, and italicized item is overlap, and primer transfers to the auspicious public affairs of spun gold
Department's synthesis.
PCR reactions are carried out by template of pNDV/rAI4-T4FHN：
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C extend 4min, carry out
15 cycles；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 4min carry out 15 cycles；72 DEG C extension 10min, 4 DEG C
It preserves.
The band of 3371bp is cut after electrophoresis, is connect, is transformed into Blunt Zero carriers after PCR product recovery purifying
The plasmid of DH5 α competence, extraction carries out PCR verifications with Primer1 and FHa, and positive plasmid is named as pAI4-2FHN-blunt
(Age Ⅰ-AflⅡ).Build the visible Fig. 7 of flow.
2, the clone of aviadenovirus hexon genes
The hexon genes of aviadenovirus strain CH/JSXZ/2015, GenBank Serial No. are synthesized by Jin Sirui companies
KU569296.1, according to hexon gene order design primer NAI4-H3 and NAI4-H4, the coding for expanding hexon genes
Area.It is homologous heavy for carrying out according to primers Primer1, Primer2, Primer5, FHa of pNDV/rAI4-T4FHN
Group reaction.
In primer sequence, thickened portion is restriction enzyme site sequence, and lower stroke of dotted line is kozak sequences, can improve external source base
The expression of cause, downslide solid line are GE the and GS sequences of newcastle disease virus, and primer is synthesized by Shanghai bioengineering Co., Ltd.
PCR reaction systems：
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15
A cycle；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 15 cycles；72 DEG C of extension 10min, 4 DEG C of guarantors
It deposits.
To NAI4-H3 and NAI4-H4, the segment of primer amplification is sequenced in this, obtains the gene order of hexon albumen,
That is sequence shown in SEQ ID NO.16：
Plasmid pAI4-2FHN-blunt (I-Afl II of Age) is subjected to double digestion with Age I and Afl II, exposes homology arm,
Hexon genes are connected into carrier, by what is obtained by recycling 3982bp bands as the method in step 1 by homologous recombination
Plasmid order-checking, the correct plasmid of sequencing result are named as pNAI4-2FHN-hexon-blunt (I-Afl II of Age).
2, the structure of full-length clone pNDV/rAI4-2FHN-hexon
Plasmid pNAI4-2FHN-hexon-blunt (I-Afl II of Age) and plasmid pAI4-2FHN (I-FspA I of Age) passes through
II double digestion of Pac I and Afl is attached reaction after recycling target fragment, and the plasmid of successful connection is named as pNAI4-2FHN-
hexon (AgeⅠ-FspAⅠ)。
By pNDV/rAI4 and pNAI4-2FHN-hexon (I-FspA I of Age) by I double digestion of Pac I and FspA, mesh is recycled
Segment after be attached reaction, connection result is accredited as positive plasmid and is named as pNDV/rAI4-2FHN-hexon, positive
Plasmid is stored in -70 DEG C.Build the visible Fig. 8 of flow.
Step 3：The rescue of recombinant virus rAI4-2FHN-hexon
The BSR-T7/5 cells of expression t7 rna polymerase can be stablized：By Harbin Veterinary Medicine Inst., China Academy of Agriculture
Step chigo researcher give.CEF cells are voluntarily prepared according to a conventional method by laboratory.
Eukaryon expression plasmid pCI-NP, pCI-P, pCI-L (Hu Shunlin, Yanmei ZHANG, the goose's Newcastle diseases such as Sun Qing, Liu Yuliang
Foundation [J] microbiologies of virus rescue are notified to .2007,34 (3))：By the Ministry of Agriculture of Yangzhou University livestock and poultry pestology weight
Point open laboratory structure preserves.Wherein, eukaryon expression plasmid pCI-NP, pCI-P of expression NP and P genes are also disclosed in (Liu
Beautiful good (2005) generate ZJ1 plants of goose's Newcastle diseases of infectivity from cDNA clone)；Express the eukaryon expression plasmid pCI- of L genes
L is also disclosed in (foundation and its application of Hu Shunlin (2007) Newcastle disease virus from goose Reverse Genetics platforms).
SPF hatching eggs are purchased from Beijing Cimmeria company, in livestock and poultry pestology emphasis open laboratory of the Ministry of Agriculture of Yangzhou University
Hatching.
1, the rescue of recombinant virus
Plasmid (full-length clone, pCI-NP, pCI-P and pCI-L) used is all made of when transfection
QIAprepSpinMiniPrepKit is extracted.By 3 helper plasmids of pCI-NP, pCI-P and pCI-L respectively with recombination NDV genes
Full length cDNA clone rAI4-2FHN-hexon cotransfection BSR-T7/5 cells are organized, final concentration of 10%SPF is added after 18-24h
Embryo allantoic liquid after transfection sample freeze thawing 1 time, will harvest and be inoculated with 9~11 age in days SPF chicken embryos after 96h, collect chick embryo allantoic liquid,
By OIE standard test hemagglutinative titers, the recombinant Newcastle disease vaccine candidate strain rAI4-2FHN- of expression aviadenovirus albumen is obtained
hexon。
2, RT-PCR verifies recombinant virus of being rescued
Hemagglutination test (HA test) (hemagglutination, HA) and erythrocyte agglutination Inhibition test
(hemagglutination inhibition, HI) detection is that positive allantoic fluid is collected after SPF chicken embryos uploaded for 4 generations
The total serum IgE of allantoic fluid extracting virus is reacted at after cDNA for RT-PCR with 6nt random primed reverse transcriptions.
Hexon, length 2852bp are expanded with primer NAI4-H3 and NAI4-H4, PCR product is recycled into simultaneously sequencing result,
Show that foreign gene is successfully plugged by reverse genetics in newcastle Disease poisonous carrier.(see Fig. 9)
Step 3：The Identification of Biological Characteristics of recombinant virus
1, the measurement of MDT
The chicken embryo median lethal time (mean death time, MDT) measures：Recombinant virus is divided with sterile saline
It Zuo not be 10 times (10-6、10-7……10-10) gradient dilution, each dilution is inoculated with 5 piece of 9~11 age in days SPF chicken embryo, per embryo
0.1mL, while the chicken embryo of 5 pieces of inoculation physiological saline is set as a contrast.37 DEG C of incubations, discard chicken embryo dead in for 24 hours, later
Every 12h observations once until 120h, the MDT of virus is calculated by OIE standard methods.
As a result：Recombinant virus shows recombinant virus rAI4- in inoculated into chick embryo no death in 120h of 5 dilutions
The MDT values of 2FHN-hexon are more than 120h.
2, Westernblot is tested
By recombinant virus rAI4-2FHN-hexon with the dose inoculation DF1 cells of MOI=1, lytic cell after infection for 24 hours
It collects plasmosin and cracks the chicken embryo fibroblasts of uninfecting virus as blank control simultaneously.Treated, and sample carries out
Albumen is transferred on the pvdf membrane of the apertures 0.22um by SDS-PAGE electrophoresis, the closing of 5% skimmed milk, more with the anti-aviadenovirus of Ji Yuan
For antiserum as primary antibody, the rabbit anti-chicken IgG of HRP labels is that secondary antibody carries out antigen-antibody binding reaction.It is developed the color with ECL luminescence methods,
Signal is acquired with gel imaging system.
As a result：Three plants of recombinant viruses are constructed altogether with identical construction strategy：rAI4-2FHN-hexon,rAI4-2FHN-
penton, rAI4-2FHN-fiber2.Hexon, penton and fiber2 base are inserted as carrier using AI4-T4FHN respectively
Cause, recombinant virus rAI4-2FHN-hexon do not give expression to hexon albumen, but recombinant virus rAI4-2FHN-penton and
RAI4-2FHN-fiber2 successful expressions have gone out penton albumen and fiber2 albumen, and hexon mrna lengths are 2814bp,
Penton genes and fiber2 mrna lengths are respectively 1578bp and 1440bp, may be too big due to hexon genes, are had exceeded
The upper limit of newcastle Disease poisonous carrier expression alien gene (see Figure 10).
Step 5：The immuning effect test of vaccine carrier strain
1 age in days SPF chickens are grouped at random, 10/group, carry out isolated rearing, carrier bacterin is inoculated with respectively through collunarium eye droppings
RAI4-2FHN-hexon, rAI4-2FHN-penton, rAI4-2FHN-hexon dosage are 106EID50/ only, while setting PBS and connecing
Kind group is as a contrast.3 weeks acquisition venous blood, detection are horizontal for the seroconversion of aviadenovirus after every experiment chicken immune.
As a result：Vaccinated flock does not show any clinical symptoms, illustrates that recombinant virus is not pathogenic to chicken, can make safely
With.The HI antibody for carrier newcastle disease virus AI4-T4FHN is detected after recombinant virus inoculation animal, shows recombinant virus
It can carry out effectively replicating (referring to Figure 11) in animal body.
RAI4-2FHN-penton recombinant viruses stimulation animal produces neutralizing antibody, but rAI4-2FHN-hexon and
RAI4-2FHN-fiber2 immune groups do not detect the neutralizing antibody for aviadenovirus, it is believed that due to recombinant virus
RAI4-2FHN-hexon does not give expression to hexon albumen, therefore does not have generation anti-for the neutralization of aviadenovirus after immune animal
Body；The antibody level that three weeks rAI4-2FHN-fiber2 stimulation animals generate after immune as seen from Figure 11 is minimum, therefore
The expression of fiber2 albumen reduces the replication capacity of vector virus, is not stimulated so as to cause rAI4-2FHN-fiber2 viruses
Animal generates the neutralizing antibody (referring to Figure 12) of anti-aviadenovirus.
The experimental results showed that being only inserted into the recombinant virus rAI4-penton and rAI4-2FHN-penton of penton genes
Animal can be stimulated to generate the neutralizing antibody for aviadenovirus, since vector virus used in rAI4-2FHN-penton is embedding
Newcastle disease virus AI4-T4FHN, F and HN albumen source is closed in 2 type of avian paramyxoviruses serum, with avian paramyxoviruses serum 1 type
Newcastle disease virus have serotype difference, the immunoprotection for newcastle disease virus can not be provided for animal；And recombinant virus
The newcastle epidemic disease antibody HI effects that itself F and HN albumen source of rAI4-penton generates after avian paramyxoviruses serum 1 type, immune animal
Valence is higher than 4log2, therefore can be used to prevent the infection of newcastle disease virus and aviadenovirus.
SEQUENCE LISTING
<110>Yangzhou University
<120>Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method
<130>
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
aactctcaca accggtgc 18
<210> 2
<211> 49
<212> DNA
<213>Artificial sequence
<400> 2
ctacccgtat tttttcttaa ttaacaggag cctgttatga gttgtgatg 49
<210> 3
<211> 42
<212> DNA
<213>Artificial sequence
<400> 3
aaaaaatacg ggtagaagcc accatgtggg ggttgcagcc gc 42
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence
<400> 4
gattccgtgt gacgcctact gcaaggtcgc ggaac 35
<210> 5
<211> 15
<212> DNA
<213>Artificial sequence
<400> 5
gcgtcacacg gaatc 15
<210> 6
<211> 26
<212> DNA
<213>Artificial sequence
<400> 6
ctgagacgag gtgtatacat tgactg 26
<210> 7
<211> 1578
<212> DNA
<213>Aviadenovirus
<400> 7
atgtgggggt tgcagccgcc gacgtcgatt ccgccgcctc ctccgccgac cgagttaacg 60
ccctcgacct atccggcgat ggtgaacggc tatccgcctc cggccgcgtc cgcgcagagc 120
tgtccctcta gcgacggtca gagcgagctg tatatgcccc ttcagcgggt gatggcccct 180
acggggggac ggaacagcat taagtatcgc gattacacgc cgtgtcgtaa caccaccaag 240
ctgttttacg tagacaacaa ggctagcgat atcgatacgt ataacaaaga cgccaaccat 300
agcaatttcc gcaccacggt gatccataac caggatctgg acgcggacac ggccgccacc 360
gagtccatcc agttggacaa ccgctcctgc tggggcggcg acctaaaaac agccgtgcgc 420
accaactgcc cgaacgtgag cagttttttc cagagtaaca gcgtgcgcgt gcgcatgatg 480
tggaagcgcg acccgccgac tagcacggct cctccgagcg cggtaggcag cggctattcg 540
gtgcccggcg cgcagtacaa gtggtacgac ctgacgatac ccgagggtaa ctacgcgctg 600
tgcgaactga tagacctgct caacgagggc atcgtgcagc tctacctgag cgaggggcgc 660
cagaacaacg tgcaaaaatc ggacatcggg gtcaagttcg acacgcgcaa cttcggcttg 720
ctccgcgacc ccgtgacggg actggtaact ccgggcacgt acgtgtacaa gggttaccac 780
cccgacatcg tgctgctgcc cggatgcgcg atcgacttta cgtacagccg cctgagcctg 840
ctcctgggca tagggaagcg cgagccctac tcgaaggggt tcgttattac ctacgaggat 900
ctgcagggag gggatatccc ggctctgctg gacctcgact ccgtcgacgt gaacgacgct 960
gacggtgaag tgatcgagct cgacaacgct gctccccttt tacatgacag cgcgggcgtg 1020
tcgtataacg tcatatacga ccaggtgacg ggtaaacccg tgacggtgta tcgatcgtgg 1080
atgttggctt acaacgtgcc taactcgccg gccaatcaga cgaccttgct gacggtgccc 1140
gatatggcgg gcgggatcgg ggcgatgtac acgtccctgc ccgatacctt tatcgcgcct 1200
accgggttca aggaagataa cacgaccaac ctttgcccgg tcgtcggcat gaacctgttc 1260
cccacctaca ataaagttta ttaccaggcg gcgtccacgt acgtgcagcg cctggaaaat 1320
tcctgccagt cggccacagc cgccttcaac cgctttcccg aaaacgagat tctgaagcaa 1380
gcgcccccca tgaatgtttc gtccgtgtgc gataaccaac ccgccgtcgt tcagcagggt 1440
gtgttgcctg tgaagacctc gctccccgga ctgcagcgcg tgctgatcac agacgaccag 1500
cgtcgtccga taccctacgt gtataagtct atcgcgacgg ttcagccgac cgttctgagt 1560
tccgcgacct tgcagtag 1578
<210> 8
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<212> DNA
<213>Artificial sequence
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aaaaaatacg ggtagaagcc accatgctcc gggcccctaa aag 43
<210> 9
<211> 33
<212> DNA
<213>Artificial sequence
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gattccgtgt gacgcttacg ggagggaggc cgc 33
<210> 10
<211> 1440
<212> DNA
<213>Aviadenovirus
<400> 10
atgctccggg cccctaaaag aagacattcc gaaaacggga agcccgagac cgaagcggga 60
ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120
gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180
ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240
atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300
ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360
gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420
gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480
accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540
gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600
accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgacagt 660
tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720
aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780
actaataccc tagcggtgac cgcgggcgct ttaaccgtgg tcggaggggg gagcgtctcc 840
acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgacc 900
gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960
gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020
ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080
caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140
gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200
tactatgaac catccatcgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260
aacccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggagagcga 1320
tacacccttc tatgctatag tctgcagtgc acgaacgcga gcatttttaa tccaaacaac 1380
agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcggcctc cctcccgtaa 1440
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence
<400> 11
cctggatgag tccattttgg tg 22
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence
<400> 12
atggactcat ccaggacaat c 21
<210> 13
<211> 31
<212> DNA
<213>Artificial sequence
<400> 13
gtaatttttt cttaagtctt ctacttgaca g 31
<210> 14
<211> 44
<212> DNA
<213>Artificial sequence
<400> 14
aaaaaatacg ggtagaagcc accatggcgg ccctcacgcc cgac 44
<210> 15
<211> 37
<212> DNA
<213>Artificial sequence
<400> 15
gattccgtgt gacgcttatt acacggcgtt gcctgtg 37
<210> 16
<211> 2814
<212> DNA
<213>Aviadenovirus
<400> 16
atggcggccc tcacgcccga cctgactacc gcgactccgc ggctccagta ttttcacatc 60
gcgggccccg ggacgcgcga atacctctct gaggacctcc aacagttcat ttccgccacc 120
ggaagctact ttgacttgaa aaacaagttc agacagacgg tcgtggcgcc cacccgaaat 180
gtcacgacag aaaaggctca acggctgcaa atccgctttt accccatcca aaccgacgac 240
acgtcgacgg gctaccgcgt gcggtacaac atcaatgtgg gcgacggttg ggtcctggac 300
atggggtcga cctatttcga catcaaggga atcctagacc gagggccgtc cttcaagccc 360
tactgcggca cggcttacaa cccgctggct cccaaggagt ccatgtttaa caactggtcg 420
gagacggcac ccgggcagaa cgtgtccgcc tccggtcagc tgtccaacgt ctataccaac 480
acgagcacct ccaaagacac gacggcggcg caggtgacga agatttccgg cgtcttcccc 540
aatcccaacc agggacccgg aagaaatcct ctgcgacggg tacaaaacgc caacaccggc 600
gtgctcggtc gcttcgccaa gtctcagtac aattacgctt acggtgccta cgtcaagccc 660
gtcgccgccg acggttccca gtccctcacg cagaccccct actggatcat ggataacacg 720
ggcaccaatt acctgggagc ggtggccgtc gaggactaca ccaacagcct ctcgtaccca 780
gataccatag tcgtgccgcc tcccgaggac tacgacgatt ataacatagg caccacgcgt 840
gcgctcaggc ccaactacat cgggttcagg gataacttca ttaacctgct gtatcacgac 900
tccggcgtgt gctcgggcac cctcaactcg gagcgttcgg gcatgaacgt ggtggtcgag 960
ctgcccgacc ggaataccga gctcagctac cagtacatgc tggccgacat gatgtcccgc 1020
catcactatt tcgccctgtg gaaccaggcc gtggaccagt acgaccccga ggtgcgagtc 1080
ttctccaatg acggttacga agaaggcgcg cccagctacg ccttcaaccc cgaagcggta 1140
ggcgcgggag aaggctacgg ccccgatctc agtcaaatta aactctacac caacaacacc 1200
gccgcgaacg acaaaaacac cgccgtggct aacgccacta ccaacttcta cttcggcacg 1260
gtaccctcct acgaaatcga tatcagcgct acccagaggc gcaactttat catggccaac 1320
atcgccgagt atctgcccga ccgttacaag tttagcatct ccggcttcga cgccaccagc 1380
gtcgcgccta ccacctacga gtacatgaac aagcgcgtcc ccctcaccaa cgtcgtcgac 1440
atgttcacga acgtgggtgc gcgttggtcc atcgaccaga tggacaacgt caaccccttc 1500
aaccaccaca gaaactgggg gctgaaatac cgctcccagc tgctgggaaa cagtcgctac 1560
gtcaacttcc acatccaagt gccccaaaaa ttcttcgcca tcaaaaacct gctgctgctc 1620
tccggctcgt acacctacga gtgggtgctg cgcaaagacc ccaacatgat cctccaatcc 1680
agtctgggca acgacctgcg cgccgacggc gccagcatca tctacaacga ggtgaacctc 1740
atggccaact tcatgcccat ggatcacaac accagtaacc agctcgagct gatgctgaga 1800
aacgccacca acgatcagac cttcgtggac tacctgggag ccaaaaacgc tctatactcg 1860
gtgcccgcgg gctccaccgc cctcaccatc aacattcccg ctcgcacctg ggaggggatg 1920
cgcgggtggt ccttcactcg catcaaggcg gccgagacgc ctcagctggg cgcccagtac 1980
gacgtcaact tcaagtactc gggcagcatc gcctactcag atggaggctt ctacctctcg 2040
cacaccttcc gtaacatgag catcctcttc gacacgtcca tcaactggcc gggcaacgac 2100
cggttgctca cgcctaacat gttcgagatc aagcgctcgg tggcgctcga caccgagggc 2160
ttcaccatga gccagtgcga catcaccaag gactggtacc tgatccagat ggccacgaac 2220
tacaacttcg tctataacgg ctatcgattc tggcccgatc gtcagtactt ccactacgac 2280
ttcctgcgaa atttcgaccc catgacgcgc cagggaccca acttcgcatt gcccggcctc 2340
ttcgacctcg tgtcttacac ccctaccacg gacaacagcg gacagcagcc tagtcaggaa 2400
gccgtgcgca acaactctgg gtttatcgcc ccccgctcct ggcccgtctg gagcgctcac 2460
cagggcgaga gctggcccgc caactggccg tacccgctct gcggtcagca ggccatccaa 2520
cccgcacagg tcctcagcta caagaagttc ctctgcgaca actacctgtg gaccatcccg 2580
ttcagttccg actttatgta catgggcgaa ctgacagatc tgggccagaa ccccatgtac 2640
accaacaact cgcacagcat ggtcatcaac ttcgagctcg accccatgga tgatcccact 2700
tacgtgtaca tgctctatgg cgtgttcgac accgttaggg tcaaccagcc cgaacgtaac 2760
gtgctagcta tggcttactt ccgtacgcct ttcgccacag gcaacgccgt gtaa 2814
Claims (2)
1. expressing recombinant Newcastle disease vaccine candidate the strain rAI4-penton, preserving number CGMCC of aviadenovirus penton albumen
No：15492.
2. expressing the recombinant Newcastle disease vaccine candidate strain rAI4-penton of aviadenovirus penton albumen described in claim 1
Construction method, it is characterised in that：Using newcastle disease attenuated IBDVs as carrier, aviadenovirus penton albumen is expressed.
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| CN110499296A (en) * | 2019-08-28 | 2019-11-26 | 扬州大学 | A kind of heat-resisting serum 8b type aviadenovirus recombinant vaccine Candidate Strain and its construction method |
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