CN109735567A - A kind of the recombinant adenoviral vector building and adenovirus packing method of African swine fever EP153R and P54 gene co-expressing - Google Patents
A kind of the recombinant adenoviral vector building and adenovirus packing method of African swine fever EP153R and P54 gene co-expressing Download PDFInfo
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Abstract
The present invention provides a kind of building of the recombinant adenoviral vector of African swine fever EP153R and P54 gene co-expressing and adenovirus packing methods, belong to gene engineering technology field.A kind of EP153R gene and P54 gene overexpression adenovirus vector introduce CTLA4, EP153R, P54 gene based on pShuttle-CMV carrier for expression of eukaryon at multiple cloning sites.The present invention also provides the recombined adhenovirus of coexpression EP153R, P54 gene, adenovirus packaging process is realized using the pAD-Shuttle-CMV-CTLA4-EP153R-P54 carrier that building obtains, obtain the adenovirus for capableing of direct infection animal or eukaryocyte, to realize the purpose that EP153R and P54 gene co-express in eukaryocyte, realize that the adenovirus vaccine of EP153R and P54 gene co-expressing is laid a good foundation for research.
Description
Technical field
The invention belongs to gene engineering technology fields, specifically relate to a kind of African swine fever virus CTLA4, EP153R, P54 base
Because of coexpression recombinant adenoviral vector, construction method and adenovirus packing method.
Background technique
African swine fever (african swine fever, ASF) is one kind of the pig as caused by African swine fever virus (ASFV)
Acute, hot, high degree in contact communicable disease.It is characterized in that the course of disease is short, case fatality rate is high, clinical symptoms are similar with pathological change
In acute swine fever, high fever, dermohemia, miscarriage, oedema and internal organs bleeding are showed.ASFV is positive two that a kind of diameter is 200nm
Decahedron virus, virus contain 70~100nm of diameter DNA core, and periphery is the icosahedral capsid of 172~191nm of diameter
With the cyst membrane containing lipoid.The unimolecule threadiness double-stranded DNA that ASFV genome is closed for terminal covalent, 170~190kb of size (with
Strain it is different and variant), end interactive connection, have inverted terminal repeats.Genome can encode 150~200 kinds
Protein, many genes for encoding these protein have been cloned and have expressed.
CTLA4 (cytotoxic T-lymphocyte associated protein 4) gene (NC_010457.5)
That is cytotoxic T cell related antigen -4: contactin, mainly CD4+ the and CD8+T cell of activation with
And the B cell surface expression of activation.CTLA-4 can be with the B7 molecule on CD28 molecule competitive binding T cell, and its affinity is
10~50 times of CD28, therefore be the major surfaces binding molecule of immune response negative regulator, it can be used as the target of antigen presenting cell
To molecule, strong humoral and cellular immune response is induced.
EP153R gene (GI:22220439) overall length 474bp, coding generate the transmembrane protein of 158 base compositions, with
There is significant homology in the N-terminal region of CD44 molecule, comprising N- glycosylation site, phosphorylation site, acylation site,
Central transmembrane region, c type animal agglutinate structure domain, cell attachment sequence column.EP153R gene mainly early stage virus infection with evening
Phase, it was reported that EP153R gene can induce or keep the combination of virus CD2 homologue and its corresponding receptor；The C of EP153R gene
Type animal agglutinate structure domain is inhibited to MHC-I class antigen presentation；The presence of EP153R gene makes virus infection or star
The transcriptional activity that shape spore rhzomorph handles P53 albumen in Vero cell reduces, this is the c type that first description has anti-apoptotic characteristic
Zoo-agglutinin.The recombinant adenoviral vector of building expression EP153R gene can preferably study gene function, non-to research and develop
Continent swine fever candidate vaccine provides technical support.
P54 albumen is the I type transmembrane structure albumen of ASFV, is encoded by E183L (GI:290782550) gene, in virus
It plays an important role in reproduction process, participates in absorption and intrusion of the virus to cell, Apoptosis of Host Cells can be caused, become in virus
Shape and infection early stage play an important role.P54 albumen produces disulfide bond, when ASFV transfects cell, forms coating precursor, leads to
It crosses targeting positioning endoplasmic reticulum and carries out protein expression, damaging cells, P54 antibody can neutralize P54 albumen, and part blocks ASFV's invades
Enter, mitigates the damage of host cell.
And currently, in the prior art without the recombinant adenoviral vector of CTLA4, EP153R, P54 gene co-expressing.
Summary of the invention
In order to overcome drawbacks described above, the purpose of the present invention is to provide a kind of African swine fever virus EP153R and P54 gene
Recombined adhenovirus, construction method and the adenovirus packing method of coexpression establish technical foundation for research African swine fever antibody.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of African swine fever virus EP153R and P54 gene co-expressing recombinant adenoviral vector pAD-
Shuttle-CMV-CTLA4-EP153R-P54, based on pShuttle-CMV carrier for expression of eukaryon, at multiple cloning sites
Introduce CTLA4, EP153R, P54 gene；The CTLA4 gene has the nucleotides sequence as shown in Seq ID No.1 in sequence table
Column, the EP153R gene have the nucleotide sequence as shown in Seq ID No.2 in sequence table, and the P54 gene has such as
Nucleotide sequence shown in Seq ID No.3 in sequence table.The multiple cloning sites are the restriction enzyme site of KpnI, HindIII.
The present invention also provides the buildings of a kind of African swine fever virus EP153R and P54 gene co-expressing recombinant adenoviral vector
Method, comprising the following steps:
1) EP153R gene (GI:22220439), P54 gene (GI:290782550) that the inquiry website NCBI is included, lead to
Artificial synthesized genetic fragment after obtaining optimization is crossed, and corresponding in EP153R gene, the addition of P54 gene order both ends respectively
Restriction enzyme site and homologous region；Corresponding restriction enzyme site and homologous region are added in CTLA4 gene order both ends, and CTLA4 template is that pig is thin
Born of the same parents system；
2) double digestion is carried out to pShuttle-CMV carrier with restriction enzyme KpnI and HindIII, is linearized
PShuttle-CMV carrier；
3) the linearisation pShuttle-CMV carrier for obtaining the step 2) carries out homologous with CTLA4-EP153R, P54
Recombination, is obtained pShuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid, is surveyed using CMV-F and SV40-R
Sequence；
4) the pShuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid for obtaining the step 3) carries out instantaneous
Expression, label protein verify protein expression；
5) the pShuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid for obtaining the step 3) carries out PmeI
Digestion is transformed into BJ5183 Escherichia coli and is recombinated, and obtains coexpression adenovirus vector pAD-Shuttle-CMV-CTLA4-
EP153R-P54。
The present invention also provides a kind of recombined adhenovirus packing methods, by pAD-Shuttle-CMV-CTLA4-EP153R-
P54 co-expresses adenovirus vector and carries out single endonuclease digestion with PacI, and the plasmid after linearisation is for transfecting；293A cell is transfected, is realized
Recombined adhenovirus packaging.Specifically it is prepared by the following steps to obtain:
1) pAD-Shuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid is subjected to single endonuclease digestion with PacI, linearly
Plasmid after change is for transfecting；293A cell is transfected using Lip3000 transfection reagent；
2) more to cell detachment after transfecting, cell and supernatant are collected, as P1 is for adenovirus；
3) P1 observes cell state for adenovirus infection 293A cell.
The invention further relates to the recombined adhenovirus that recombined adhenovirus packing method above-mentioned is prepared, and comprising aforementioned
Recombined adhenovirus vaccine.
The present invention provides a kind of EP153R gene overexpression adenovirus vectors
PAD-Shuttle-CMV-CTLA4-EP153R-P54, based on pShuttle-CMV carrier for expression of eukaryon,
CTLA4, EP153R, P54 gene are introduced at multiple cloning sites.The carrier carries CMV strong promoter, and table can be crossed in eukaryocyte
The gene carried up to it, while CTLA4 can enhance immune response, be screened, and finally obtain and be capable of direct infection eukaryocyte
Adenovirus is further research based on table altogether to realize the purpose that EP153R, P54 gene co-express in eukaryocyte
It lays a solid foundation up to EP153R, P54 gene recombinant adenovirus vector vaccine.
Detailed description of the invention
Fig. 1 is pShuttle-CMV-CTLA4-EP153R-P54 plasmid enzyme restriction proof diagram in embodiment 3；1:1 plasmid
SalI+XhoI；2:2 plasmid SalI+XhoI；M1:DL2000DNA Marker；M2:1KB DNA Marker；
Fig. 2 is to turn HA label protein WB for 24 hours in embodiment 4 pShuttle-CMV-CTLA4-EP153R-P54 plasmid wink to test
Card figure；1:pShuttle-CMV-CTLA4-EP153R-P54 plasmid；2:pShuttle-EGFP-CTLA4-EP153R-P54 matter
Grain；3:293T blank control；M: albumen Marker；
Fig. 3 is pAD-Shuttle-CMV-CTLA4-EP153R-P54PacI digestion verification figure in embodiment 5；1:1 matter
Grain；2:2 plasmid；III digest of M1: λ-Hind；M2:1kb DNA Marker；
Fig. 4 is ADV infection cell 48h HA label protein WB proof diagram in embodiment 7；
1:ADV infects ST cell；2:ADV infects 293A；3:ST cell；4:293A；M: albumen Marker；
Fig. 5 is pShuttle-CTLA4-EP153R-P54 plasmid construct figure；
Fig. 6 is pADShuttle-CMV-CTLA4-EP153R-P54 plasmid construct figure.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
In the following example, test method without specific conditions, usually routinely condition, such as " fine works molecular biosciences
Learn experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the chief editor such as J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates Beijing: section
Learn publishing house, 2004) described in method carry out.
The present invention provides the construction methods of a kind of EP153R and P54 gene co-expressing adenovirus vector, including following step
It is rapid:
1) EP153R gene (GI:22220439), P54 gene (GI:290782550) that the inquiry website NCBI is included, lead to
Artificial synthesized genetic fragment after obtaining optimization is crossed, CTLA4 template is pig cell system, and phase is added at the both ends of 3 segments respectively
The restriction enzyme site and homologous sequence answered (restriction enzyme site and homologous sequence are embodied in SEQ ID No.4-11).
2) the step 1) genetic fragment and pShuttle-CMV carrier through KpnI, HindIII double digestion and are recycled, is passed through
Homologous recombination is crossed to convert to obtain recombinant plasmid pShuttle-CMV-CTLA4-EP153R-P54, using CMV-F and SV40-R into
Row sequencing.
3) the pShuttle-CMV-CTLA4-EP153R-P54 plasmid for obtaining the step 2) with use restriction enzyme
Enzyme PmeI digestion；Linearization plasmid is transformed into BJ5183 Escherichia coli, obtains pAD-Shuttle-CMV- by recombination
CTLA4-EP153R-P54 recombinant plasmid.
4) pAD-Shuttle-CMV-CTLA4-EP153R-P54 recombinant plasmid transfects 293A by PacI linearization for enzyme restriction
Cell obtains recombined adhenovirus.
The optimum synthesis of 1 gene of embodiment
Inquiry the website NCBI include EP153R gene (GI:22220439), P54 gene (GI:290782550) and
Derived from the CTLA4 gene (NC_010457.5) of pig cell system, by artificial synthesized, acquisition optimization (refers to and carries out to nucleotide sequence
Optimization) after gene, sequence such as SEQ ID No.1-3.
CTLA4 sequence Seq ID No.1
GCTAGAGATCTGGTACCGCCACCATGCACGTGGCCCAACCTGCAGTA GTGCTGGCCAACAGCCGGGG
TGTTGCCAGCTTTGTGTGTGAGTATGGGTC TGCAGGCAAAGCTGCCGAGGTCCGGGTGACAGTGCTGCGGCGGGC
CGGC AGCCAGATGACTGAAGTCTGTGCCGCGACATATACTGTGGAGGATGAGTT GACCTTCCTTGATGACTCTA
CATGCACTGGCACCTCCACCGAAAACAAAG TGAACCTCACCATCCAAGGGCTGAGAGCCGTGGACACTGGGCTCT
ACATC TGCAAGGTGGAGCTCCTGTACCCACCACCCTACTATGTGGGTATGGGCAA CGGGACCCAGATTTATGTC
ATTGATCCAGAACCATGCCCAGATTCTGATA GTACTGATTACAAAGACGATGACGATAAG
EP153R sequence Seq ID No.2
AAAGACGATGACGATAAGACCGGTTTCAGCAACAAGAAGTACATCG GCCTGATCAACAAGAAGGAGG
GCCTGAAGAAGAAGATCGATGACTACAG CATCCTGATCATCGGCATCCTGATCGGCACCAACATCCTGAGCCTGA
TCAT CAACATCATCGGCGAGATCAATAAGCCTATCTGCTACCAGAATGATGACA AGATCTTCTACTGCCCCAAG
GACTGGGTGGGCTACAACAACGTGTGTTAC TACTTCGGCAACGAGGAGAAGAACTACAACAATGCCAGCAACTAC
TGCA AGCAGTTGAACAGCACACTGACAAACAATAACACCATCCTGGTGAACCTG ACGAAGACCCTGAACCTGAC
CAAGACCTACAACCACGAGAGCAACTACT GGGTGAACTACAGCCTGATCAAGAACGAGAGCGTGCTGCTGCGCGA
CAG CGGCTACTACAAGAAGCAGAAGCACGTGAGCCTGCTGTACATCTGCAGCA
AGGCTAGCGAGCAGAAACTCATCTCTGAA
P54 sequence Seq ID No.3
gtcgacATGTCTTCAAGAAAGAAAAAAGCTGCTGCTATTGAGGAGGAAG ATATACAGTTTATAAATC
CTTATCAAGATCAGCAGTGGGTAGAAGTCACT CCACAACCAGGTACCTCTAAACCAGCTGGAGCGACTACAGCAA
GTGTAGG CAAGCCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAA CCAGTTACGGACAACCCA
GTTACGGACAGACTAGTCATGGCAACTGGCGG GCCGGCGGCCGCACCTGCGGCCGCGAGTGCTCCTGCTCATCCG
GCTGAGC CTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCT ATTGAAAATTTACGACA
AAGAAACACCTATACGCATAAAGACCTAGAAA ACTCCTTGctcgag
The modification and amplification of 2 segment of embodiment
1) design of primers is shown in Table 1:
1 design of primers of table
2) CTLA4, EP153R, P54 segment PCR
CTLA4:CTLA4-KpnI-F+CTLA4-R+CTLA4-Flag-R
EP153R:EP153R-Flag-F+EP153R-Myc-R
P54:P54-Myc-F+P54-HA-R+HA-R
CTLA4-EP153R-P54:CTLA4-KpnI-F+EP153R-Myc-R+HA-R (template CTLA4+EP153R+
P54)
That is, amplification CTLA4 gene (PCR product 1) Shi Caiyong CTLA4-KpnI-F+CTLA4-R+CTLA4-Flag-R
Primer combination, DNA profiling are Seq IDNo.1；Expand EP153R gene (PCR product 2) Shi Caiyong EP153R-Flag-F+
The primer of EP153R-Myc-R combines, and DNA profiling is Seq IDNo.2；Expand P54 (PCR product 3) Shi Caiyong P54-Myc-F+
The primer of P54-HA-R+HA-R combines, and DNA profiling is Seq IDNo.3.When expanding CTLA4-EP153R-P54 (PCR product 4),
It is combined using the primer of CTLA4-KpnI-F+EP153R-Myc-R+HA-R, and DNA profiling is the mixed of PCR product 1,2,3 at this time
Close object.
Reaction system such as table 2 by PCR:
2 PCR reaction system of table
| Reaction solution ingredient | Volume |
| GXL Primer | 1μL |
| 5×GXL Buffer | 10μL |
| dNTP Mix | 4μL |
| F(10μM) | 2μL |
| R(10μM) | 2μL |
| Template | 1μL |
| ddH2O | 30μL |
| It amounts to | 50μL |
Wherein F is forward primer, and R is reverse primer.
PCR reaction condition such as table 3:
3 PCR reaction condition of table
PCR uses 1% agarose gel electrophoresis to detect digestion purpose band size, blend compounds QIAquick Gel Extraction Kit after reaction
Recovery product.
3 pShuttle-CMV carrier digestion of embodiment and homologous recombination
1) pShuttle-CMV carrier is subjected to digestion, digestion system such as table 4.
4 digestion system of table
| Reaction solution ingredient | Volume |
| Plasmid (350 μ g/ μ L) | 20μL |
| 10×Buffer | 5μL |
| KpnI | 2μL |
| HindIII | 2μL |
| ddH2O | 31μL |
| It amounts to | 50μL |
Sample-adding mixes and is placed on 37 DEG C of digestion 6h, detects digestion purpose item with 1% agarose gel electrophoresis after reaction
Band size, blend compounds QIAquick Gel Extraction Kit recycle linearization plasmid.
2) carrier and segment homologous recombination:
The reaction system of table 5 carrier and segment homologous recombination
Wherein CTLA4-EP153-P54 is PCR product 4 above-mentioned.
37 DEG C of reaction 35min convert DH5 α competent escherichia coli cell, kalamycin resistance LB plate screening, picking
Plasmid enzyme restriction identification, positive plasmid sequencing identification (Fig. 1) are extracted in monoclonal PCR identification, positive bacterium colony amplification；
Sequencing primer is Seq ID No.12:CMV-F CGCAAATGGGCGGTAGGCGTG
Seq ID No.13:SV40-R GAAATTTGTGATGCTATTGC
All sequences herein are 5 ' -3 '.
It is pShuttle-CMV-CTLA4-EP153R-P54 that correct plasmid, which is sequenced, can be used for subsequent experimental.
4 293T cell transient expression of embodiment and its verifying
1) 293T cell transfecting
Cell prepares: by 1 × 106A 293T cell is uniformly layered in every hole of 6 orifice plates, is used after cell is adherent within second day
In cell transfecting；
Plasmid prepares: by 2.5 μ g pShuttle-CMV-CTLA4-EP153R-P54 plasmids and 7.5 μ L Lip3000 lipids
Body is mixed with 500 μ L plasma-free DMEM mediums respectively, stands 4min, and plasmid mixed liquor and Lip3000 mixed liquor are mixed, quiet
Set 10min；
Mixed liquor is added dropwise in culture medium, is gently shaken up, 37 DEG C of cultures.
Collect cell afterwards for 24 hours.
2) WB detects protein expression
Protein extraction: PBS is cleaned 2 times, and 250 μ l cell pyrolysis liquids are added and (press down containing protease inhibitors and phosphoprotein phosphatase
Preparation), cell is blown off in digestion, and concussion mixes, and ice bath 25min, 12000rpm are centrifuged 15min, is drawn 200 μ l supernatants and is added
40 μ l 6 × SDS Buffer, boil 5min；
Albumen PAGE gel electrophoresis: 30 μ l protein samples, electrophoresis, transferring film, closing is added in every hole；
The detection of FLAG tag antibody: 4 DEG C of FLAG primary antibody overnight incubations, TBST cleaning, source of mouse secondary antibody are incubated at room temperature 1h, TBST
Cleaning, developer solution colour developing；Testing result is as shown in Fig. 2, EP153R and P54 gene co-expressing can be realized in 293T cell.
The recombination of 5 pAD-Shuttle-CMV-CTLA4-EP153R-P54 adenovirus vector of embodiment
1) pShuttle-CMV-CTLA4-EP153R-P54 plasmid PmeI linearization for enzyme restriction (Fig. 3), gel recycling；
2) linearized vector is transformed into BJ5183 Escherichia coli, blocks the screening of that resistant panel, bacterium colony PCR identification, positive bacteria
Amplification is fallen, plasmid is extracted.
3) PacI digestion verification, and endonuclease bamhi is recycled using ethanol precipitation, for transfecting cell packaging adenovirus.
6 adenovirus of embodiment packaging
1) 293A plating cells: 2 × 105 293T cells are uniformly spread with 12 orifice plates, convergence degree reaches 30% and is used for
Transfection；
2) plasmid prepare: by 1.5 μ g linearization plasmids and 4 μ L Lip3000 liposomes respectively with 250 μ L serum-free DMEM
Culture medium mixes, and stands 4min, and plasmid mixed liquor and Lip3000 mixed liquor are mixed, and stands 10min；Mixed liquor is added dropwise
Enter into culture medium, gently shakes up, 37 DEG C of cultures.
3) 48h observes luciferase expression, and cell has more death after 5d, pays attention to fluid infusion, collects cell and supernatant, -80 DEG C of jellies
Melt 2 times, concussion release adenovirus, centrifugation goes to precipitate；
4) adenovirus supernatant and 10% culture medium 1:1 are mixed, is inoculated in 6cm dish disk, observe cell state, to
Poison is received after the most of death of cell；
5) superinfection is inoculated in T75 culture bottle, is inoculated in T175 culture bottle, receives poison；
6) adenovirus is concentrated and purified.
The verifying of 7 gland virus expression of embodiment
1) cell prepares: by 5 × 105A 293A and ST (porcine kidney cell) is uniformly layered in 12 orifice plates, and convergence degree reaches
80% for transfecting；
2) taking the unpurified viral supernatants infection of 100 μ L, infection cell, 48h observe fluorescence respectively；
3) WB label protein detects, and testing result is as shown in Figure 4, it is known that being packaged into adenovirus postoperative infection cell can realize
EP153R and P54 gene co-expressing.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Yangzhou University
<120>a kind of building of the recombinant adenoviral vector of African swine fever EP153R and P54 gene co-expressing and adenovirus are packed
Method
<130> xhx2018011801
<141> 2019-01-18
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 425
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gctagagatc tggtaccgcc accatgcacg tggcccaacc tgcagtagtg ctggccaaca 60
gccggggtgt tgccagcttt gtgtgtgagt atgggtctgc aggcaaagct gccgaggtcc 120
gggtgacagt gctgcggcgg gccggcagcc agatgactga agtctgtgcc gcgacatata 180
ctgtggagga tgagttgacc ttccttgatg actctacatg cactggcacc tccaccgaaa 240
acaaagtgaa cctcaccatc caagggctga gagccgtgga cactgggctc tacatctgca 300
aggtggagct cctgtaccca ccaccctact atgtgggtat gggcaacggg acccagattt 360
atgtcattga tccagaacca tgcccagatt ctgatagtac tgattacaaa gacgatgacg 420
ataag 425
<210> 2
<211> 522
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaagacgatg acgataagac cggtttcagc aacaagaagt acatcggcct gatcaacaag 60
aaggagggcc tgaagaagaa gatcgatgac tacagcatcc tgatcatcgg catcctgatc 120
ggcaccaaca tcctgagcct gatcatcaac atcatcggcg agatcaataa gcctatctgc 180
taccagaatg atgacaagat cttctactgc cccaaggact gggtgggcta caacaacgtg 240
tgttactact tcggcaacga ggagaagaac tacaacaatg ccagcaacta ctgcaagcag 300
ttgaacagca cactgacaaa caataacacc atcctggtga acctgacgaa gaccctgaac 360
ctgaccaaga cctacaacca cgagagcaac tactgggtga actacagcct gatcaagaac 420
gagagcgtgc tgctgcgcga cagcggctac tacaagaagc agaagcacgt gagcctgctg 480
tacatctgca gcaaggctag cgagcagaaa ctcatctctg aa 522
<210> 3
<211> 411
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtcgacatgt cttcaagaaa gaaaaaagct gctgctattg aggaggaaga tatacagttt 60
ataaatcctt atcaagatca gcagtgggta gaagtcactc cacaaccagg tacctctaaa 120
ccagctggag cgactacagc aagtgtaggc aagccagtca cgggcagacc ggcaacaaac 180
agaccagcaa caaacaaacc agttacggac aacccagtta cggacagact agtcatggca 240
actggcgggc cggcggccgc acctgcggcc gcgagtgctc ctgctcatcc ggctgagcct 300
tacacgacag tcactactca gaacactgct tcacaaacaa tgtcggctat tgaaaattta 360
cgacaaagaa acacctatac gcataaagac ctagaaaact ccttgctcga g 411
<210> 4
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gctagagatc tggtaccgcc accatgcacg tggcccaacc t 41
<210> 5
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atcagaatct gggcatggtt ctggatcaat gacataaatc tggg 44
<210> 6
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cttatcgtca tcgtctttgt aatcagtact atcagaatct gggcatgg 48
<210> 7
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aaagacgatg acgataagac cggtttcagc aacaagaagt aca 43
<210> 8
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ttcagagatg agtttctgct cgctagcctt gctgcagatg tacag 45
<210> 9
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aagaggatct ggtcgacatg tcttcaagaa aga 33
<210> 10
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctagaaaact ccttgctcga gtacccctac gacgt 35
<210> 11
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tatcttatct agaagctttt aggcgtagtc gggcacgtcg taggggtact c 51
<210> 12
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cgcaaatggg cggtaggcgt g 21
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gaaatttgtg atgctattgc 20
Claims (7)
1. a kind of African swine fever virus EP153R and P54 gene co-expressing recombinant adenoviral vector, which is characterized in that with
Based on pShuttle-CMV carrier for expression of eukaryon, CTLA4, EP153R, P54 gene are introduced at multiple cloning sites to construct weight
Group adenovirus vector pAD-Shuttle-CMV-CTLA4-EP153R-P54；The CTLA4 gene, EP153R gene, P54 gene
It is respectively provided with the nucleotide sequence as shown in Seq ID No.1 in sequence table, Seq ID No.2, Seq ID No.3.
2. African swine fever virus EP153R according to claim 1 and P54 gene co-expressing recombinant adenoviral vector, special
Sign is that the multiple cloning sites are the restriction enzyme site of KpnI and HindIII.
3. the construction method of a kind of African swine fever virus EP153R and P54 gene co-expressing recombinant adenoviral vector, including it is following
Step:
1) it is directed to EP153R gene, P54 gene, by artificial synthesized, after obtaining optimization genetic fragment, and respectively in EP153R
Corresponding restriction enzyme site and homologous region are added in gene, P54 gene order both ends；Add corresponding enzyme in CTLA4 gene order both ends
Enzyme site and homologous region；
2) double digestion is carried out to pShuttle-CMV carrier with restriction enzyme KpnI and HindIII, is linearized
PShuttle-CMV carrier；
3) the linearisation pShuttle-CMV carrier for obtaining the step 2 and CTLA4-EP153R, P54 carry out homologous recombination,
PShuttle-CMV-CTLA4-EP153R- P54 eukaryon expression plasmid is obtained, is sequenced using CMV-F and SV40-R；
4) the pShuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid for obtaining the step 3) carries out instantaneous table
It reaches, label protein verifies protein expression；
5) the pShuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid for obtaining the step 3) carries out PmeI enzyme
It cuts, is transformed into BJ5183 Escherichia coli and is recombinated, obtain coexpression adenovirus vector pAD-Shuttle-CMV-CTLA4-
EP153R-P54。
4. a kind of recombined adhenovirus packing method, which is characterized in that by pAD-Shuttle-CMV- described in claim 1
CTLA4-EP153R-P54 co-expresses adenovirus vector and carries out single endonuclease digestion with PacI, and the plasmid after linearisation is for transfecting；Transfection
293A cell realizes recombined adhenovirus packaging.
5. a kind of recombined adhenovirus packing method according to claim 4, which is characterized in that be prepared by the following steps
It arrives:
1) pAD-Shuttle-CMV-CTLA4-EP153R-P54 eukaryon expression plasmid is subjected to single endonuclease digestion with PacI, after linearisation
Plasmid for transfecting；293A cell is transfected using Lip3000 transfection reagent；
2) more to cell detachment after transfecting, cell and supernatant are collected, as P1 is for adenovirus；
3) P1 observes cell state for adenovirus infection 293A cell.
6. the recombined adhenovirus that recombined adhenovirus packing method described in claim 4 or 5 is prepared.
7. including the vaccine of recombined adhenovirus as claimed in claim 6.
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| CN114377121A (en) * | 2022-01-11 | 2022-04-22 | 中国农业科学院兰州兽医研究所 | Recombinant African swine fever antigen 'cocktail' vaccine containing intramolecular adjuvant and application thereof |
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