CN112522286A - EP402R, CP204L and E183L co-expression recombinant adenovirus and construction and application thereof - Google Patents
EP402R, CP204L and E183L co-expression recombinant adenovirus and construction and application thereof Download PDFInfo
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Abstract
The invention provides an EP402R, CP204L and E183L co-expression recombinant adenovirus and construction and application thereof, belonging to the technical field of genetic engineering, wherein the gene is inserted into a pShuttle-CMV eukaryotic expression vector and can be used for constructing a recombinant adenovirus for expressing African swine fever virus EP402R, CP204L and E183L proteins, and the recombinant adenovirus can directly infect eukaryotic cells, so that the aim of co-expression of the EP402R, CP204L and E183L genes in the eukaryotic cells is fulfilled, and a foundation is laid for further research of a recombinant adenovirus vector vaccine based on co-expression of EP402R, CP204L and E183L genes.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a co-expression recombinant adenovirus of EP402R, CP204L and E183L, and construction and application thereof.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease in pigs caused by African Swine Fever Virus (ASFV). It is characterized by short course of disease, high fatality rate, clinical symptoms and pathological changes similar to acute swine fever, and high fever, cutaneous congestion, abortion, edema and organ bleeding. CD2v is the only characteristic viral protein in the outer membrane proteins of ASFV, encoded by ORF EP402R gene, also known as pEP 402R. p30 is an early membrane protein expressed by ASFV and is encoded by ORF CP204L gene. The P54 protein is a type I transmembrane structural protein of ASFV, is coded by E183L gene, plays an important role in the replication process of virus, participates in the adsorption and invasion of the virus to cells, can cause apoptosis of host cells, and plays an important role in the early stage of virus deformation and infection. However, there is currently no recombinant adenoviral vaccine against EP402R, CP204L and E183L and no feasible method for preparing the recombinant adenoviral vaccine.
Disclosure of Invention
The invention aims to provide a co-expression recombinant adenovirus of EP402R, CP204L and E183L, and construction and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a gene co-expressed by EP402R, CP204L and E183L, wherein the gene is obtained by sequentially connecting an EGFP gene, a first connecting gene, an EP402R gene, a second connecting gene, a CP204L gene, a second connecting gene and an E183L gene in series; the nucleotide sequence of the EP402R gene is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the CP204L gene is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the E183L gene is shown as SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the first connecting gene is shown as SEQ ID NO: 11 is shown in the figure; the nucleotide sequence of the second connecting gene is shown as SEQ ID NO: shown at 12.
The invention also provides a recombinant vector containing the gene in the scheme, wherein the recombinant vector takes a pShuttle-CMV vector as an original vector; the insertion site of the gene is between KpnI and HindIII.
The invention also provides a recombinant adenovirus vector, which is inserted into the recombinant vector of the scheme, and the insertion site is between the first site and the second site of the PmeI enzyme cutting site sequence; the nucleotide sequence of the first site is shown as SEQ ID NO: 13 is shown in the figure; the nucleotide sequence of the second site is shown as SEQ ID NO: as shown at 14.
The invention also provides a construction method of the recombinant adenovirus vector in the scheme, which comprises the following steps:
and (3) carrying out enzyme digestion on the recombinant vector in the scheme by using a restriction enzyme PmeI to obtain a linearized plasmid, transforming the linearized plasmid into escherichia coli containing a pAdEasy-1 plasmid, and recombining to obtain the recombinant adenovirus vector.
The invention also provides a recombinant adenovirus containing the recombinant adenovirus vector in the scheme or the recombinant adenovirus vector obtained by the construction method.
The invention also provides a construction method of the recombinant adenovirus in the scheme, which comprises the following steps: and carrying out enzyme digestion linearization on the recombinant adenovirus vector through PacI to obtain a linearized plasmid, and transfecting HEK293A cells with the linearized plasmid to culture to obtain the recombinant adenovirus.
The invention also provides application of the recombinant adenovirus obtained by the scheme or the construction method in preparation of African swine fever vaccines.
The invention also provides an African swine fever vaccine which comprises an immunologic adjuvant and an immunogen and is characterized in that the immunogen is the recombinant adenovirus obtained by the scheme or the construction method.
The invention provides a gene co-expressed by EP402R, CP204L and E183L, wherein the gene is inserted into a pShuttle-CMV eukaryotic expression vector and can be used for constructing recombinant adenovirus for expressing African swine fever virus EP402R, CP204L and E183L proteins, the recombinant adenovirus can directly infect eukaryotic cells, so that the aim of co-expression of the genes EP402R, CP204L and E183L in the eukaryotic cells is fulfilled, and a foundation is laid for further research of a recombinant adenovirus vector vaccine based on co-expressed genes EP402R, CP204L and E183L.
Drawings
FIG. 1 is a map of a recombinant plasmid of the present invention;
FIG. 2 is a map of a recombinant adenovirus vector of the present invention;
FIG. 3 is a PacI single-restriction linearization diagram and a BamHI single-restriction linearization diagram of a recombinant adenovirus vector pAD-Shuttle-CMV-EGFP-P2A-EP402R-CP204L-E183L-HA, detected by 1% agarose gel electrophoresis, and the PacI single-restriction linearization diagram electrophoresis shows two bands, one band is 4.5kb and the other band is about 33.8 kb; the BamHI enzyme digestion linearized map electrophoresis shows two bands, one is 26.6kb and the other is about 11.7 kb;
FIG. 4 is a graph showing the WB validation of HA tag protein 48h after ADV infection of 293A cells;
FIG. 5 is a graph of CPE of 72h cells of ADV infected 293A cells;
FIG. 6 is a 48h fluorescence expression profile of ADV infected 293A cells.
Detailed Description
The gene is obtained by connecting an EGFP gene, a first connecting gene, an EP402R gene, a second connecting gene, a CP204L gene, a second connecting gene and an E183L gene in series in sequence; the nucleotide sequence of the EP402R gene is shown as SEQ ID NO: 1, specifically: atgataatacttatttttttaatattttctaacatagttttaagtattgattattgggttagttttaataaaacaataattttagatagtaatattactaatgataataatgatataaatggagtatcatggaatttttttaataattcttttaatacactagctacatgtggaaaagcaggtaacttttgtgaatgttctaattatagtacatcaatatataatataacaaataattgtagcttaactatttttcctcataatgatgtatttgatacaacatatcaagtagtatggaatcaaataattaattatacaataaaattattaacacctgctactcccccaaatatcacatataattgtactaattttttaataacatgtaaaaaaaataatggaacaaacactaatatatatttaaatataaatgatacttttgttaaatatactaatgaaagtatacttgaatataactggaataatagtaacattaacaattttacagctacatgtataattaataatacaattagtacatctaatgaaacaacacttataaattgtacttatttaacattgtcatctaactatttttatactttttttaaattatattatattccattaagcatcataattgggataacaataagtattcttcttatatccatcataacttttttatctttacgaaaaagaaaaaaacatgttgaagaaatagaaagtccaccacctgaatctaatgaagaagaacaatgtcagcatgatgacaccacttccatacatgaaccatctcccagagaaccattacttcctaagccttacagtcgttatcagtataatacacctatttactacatgcgtccctcaacacaaccactcaacccatttcccttacctaaaccgtgtcctccacccaaaccatgtccgccacccaaaccatgtcctccacctaaaccatgtccttcagctgaatcctattctccacccaaaccactacctagtatcccgctactacccaatatcccgccattatctacccaaaatatttcgcttattcacgtagatagaattatttaa, respectively; the nucleotide sequence of the CP204L gene is shown as SEQ ID NO: 2, specifically: atggattttattttaaatatatccatgaaaatggaggtcatcttcaaaacggatttaagatcatcttcacaagttgtgtttcatgcgggtagcctgtataattggttttctgttgagattatcaatagcggtagaattgttacgaccgctataaaaacattgcttagtactgttaagtatgatattgtgaaatctgctcgtatatatgcagggcaagggtatactgaacatcaggctcaagaagaatggaatatgattctgcatgtgctgtttgaagaggagacggaatcctcagcatcttcggagaacattcatgaaaaaaatgataatgaaaccaatgaatgcacatcctcctttgaaacgttgtttgagcaagagccctcatcggaggtacctaaagactccaagctgtatatgcttgcacaaaagactgtgcaacatattgaacaatatggaaaggcacctgattttaacaaggttattagagcacataattttattcaaaccatttatggaacccctctaaaggaagaagaaaaagaggtggtaagactcatggttattaaacttttaaaaaaaataagcttttatctcacctacatttaa, respectively; the nucleotide sequence of the E183L gene is shown as SEQ ID NO: 3, specifically: atggattctgaattttttcaaccggtttatccgcggcattatggtgagtgtttgtcaccagtcactacaccaagcttcttctccacacatatgtatactattctcattgctatcgtggtcttagtcatcattatcatcgttctaatctatctattctcttcaagaaagaaaaaagctgctgctattgaggaggaagatatacagttataaatccttatcaagatcagcagtgggtagaagtcactccacaaccaggtacctctaaaccagctggagcgactacagcaagtgtaggcaagccagtcacgggcagaccggcaacaaacagaccagcaacaaacaaaccagttacggacaacccagttacggacagactagtcatggcaactggcgggccggcggccgcacctgcggccgcgagtgctcctgctcatccggctgagccttacacgacagtcactactcagaacactgcttcacaaacaatgtcggctattgaaaatttacgacaaagaaacacctatacgcataaagacctagaaaactccttgtaa, respectively; the nucleotide sequence of the first connecting gene is shown as SEQ ID NO: shown at 11, specifically AGTACTGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGGTCGAC; the nucleotide sequence of the second connecting gene is shown as SEQ ID NO: 12, specifically: GGCGCCCCC are provided.
In the present invention, the EP402R gene, CP204L gene and E183L gene are obtained by artificial synthesis, and the method of artificial synthesis is not particularly limited in the present invention, and may be any method that is conventional in the art.
The invention also provides a recombinant vector containing the gene in the scheme, wherein the recombinant vector takes a pShuttle-CMV vector as an original vector; the insertion site of the gene is between KpnI and HindIII. FIG. 1 is a map of a recombinant plasmid of the present invention.
In the present invention, the method for constructing the recombinant vector preferably comprises the steps of:
artificially synthesizing the gene, and carrying out homologous recombination on the gene and a linearized plasmid of the pShuttle-CMV vector to obtain a recombinant vector. The method of homologous recombination in the present invention is not particularly limited, and may be performed by a method conventional in the art.
The invention also provides a recombinant adenovirus vector, which is inserted with the gene linearized by the recombinant vector in the scheme, and the insertion site is between the first site and the second site of the PmeI enzyme cutting site sequence; the nucleotide sequence of the first site is shown as SEQ ID NO: 13, specifically: GGTTT; the nucleotide sequence of the second site is shown as SEQ ID NO: 14, specifically: AAACC. FIG. 2 is a map of a recombinant adenovirus vector of the present invention.
The invention also provides a construction method of the recombinant adenovirus vector in the scheme, which comprises the following steps:
and (3) carrying out enzyme digestion on the recombinant vector in the scheme by using a restriction enzyme PmeI to obtain a linearized plasmid, transforming the linearized plasmid into BJ5183 escherichia coli containing a pAdEasy-1 plasmid, and carrying out recombination to obtain the recombinant adenovirus vector.
The invention also provides a recombinant adenovirus containing the recombinant adenovirus vector in the scheme or the recombinant adenovirus vector obtained by the construction method.
The invention also provides a construction method of the recombinant adenovirus in the scheme, which comprises the following steps: and carrying out PacI enzyme digestion linearization on the recombinant adenovirus vector to obtain a linearized plasmid, and transfecting 293A cells with the linearized plasmid to culture to obtain the recombinant adenovirus.
The invention also provides application of the recombinant adenovirus in the scheme in preparation of an African swine fever vaccine.
The invention also provides an African swine fever vaccine which comprises the recombinant adenovirus in the scheme.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the experimental procedures without specifying the specific conditions were generally carried out by the methods described in "molecular biology laboratory Manual of Fine text" (edited by F.M. Osber, R.E. Kingston, J.G. Sedman, et al, Mashimi, Shujiong, Beijing: scientific Press, 2004).
Example 1
1) The optimized and connected gene fragment is obtained by artificially synthesizing an EP402R gene (GI:41902828), a CP204L gene (GI:41902863) and an E183L gene (GI:41902895) which are recorded in an NCBI website. .
1. Modification and amplification of fragments
1) Primer design
2) PCR of EGFP, EGFP-P2A-EP402R-CP204L-E183L fragment
EGFP：EGFP-F(KpnI)+EGFP-R(+P2A)
EP402R-CP 204L-E183L: CD2V-P30-P54-F + CD2V-P30-P54-R (the template is PUC57-CD2V-P30-P54)
EGFP-P2A-EP402R-CP204L-E183L：EGFP-F(KpnI)+HA-R(HindIII)
The PCR reaction system is as follows:
| components of reaction solution | Volume of |
| GXL Primer | 1μL |
| 5×GXL Buffer | 10μL |
| dNTP Mix | 4μL |
| F(10μM) | 2μL |
| R(10μM) | 2μL |
| Form panel | 1μL |
| ddH2O | 30μL |
| Total of | 50μL |
The PCR reaction conditions were as follows:
after the PCR reaction is finished, detecting the size of a band of a target enzyme digestion by using 1% agarose gel electrophoresis, and recovering a product by using a gel recovery kit.
Restriction enzyme digestion and homologous recombination of pShuttle-CMV vector
1) The pShuttle-CMV vector was digested in the following manner
| Components of reaction solution | Volume of |
| Plasmid (350 ng/. mu.L) | 20μL |
| 10×Buffer | 5μL |
| KpnI | 2μL |
| HindIII | 2μL |
| ddH2O | 31μL |
| Total of | 50μL |
Adding the sample, uniformly mixing, placing at 37 ℃ for enzyme digestion for 6h, detecting the size of a band of the enzyme digestion target by using 1% agarose gel electrophoresis after the reaction is finished, and recovering the linearized plasmid by using a gel recovery kit.
2) Homologous recombination of vector and fragment:
reacting at 37 ℃ for 35min, transforming DH5 alpha escherichia coli competent cells, screening kanamycin-resistant LB plates, selecting monoclonal PCR identification, amplifying and extracting plasmid enzyme digestion identification by positive colony amplification, and sequencing and identifying positive plasmids;
the sequencing primer is SEQ ID NO. 9: CMV-F CGCAAATGGGCGGTAGGCGTG;
SEQ ID NO.10：SV40-R GAAATTTGTGATGCTATTGC。
the plasmid with the correct sequencing is pShuttle-CMV-EGFP-P2A-EP402R-CP204L-E183L-HA, and can be used for subsequent experiments.
3.293T cell transient expression and validation thereof
1) Transfection of 293T cells
Cell preparation: will be 1 × 106293T cells are uniformly paved in each hole of a 6-hole plate, and used for cell transfection after the cells adhere to the wall on the next day;
plasmid preparation: respectively mixing 2.5 μ g plasmid and 7.5 μ L Lipofectamine3000 liposome with 500 μ L serum-free DMEM medium, standing for 4min, mixing the plasmid mixed solution and Lipofectamine3000 mixed solution, and standing for 10 min;
the mixture was added dropwise to the medium, shaken gently and cultured at 37 ℃.
Cells were harvested after 24 h.
2) WB detection of protein expression
Protein extraction: washing with PBS for 2 times, adding 250 μ l cell lysate (containing protease inhibitor and protein phosphatase inhibitor), digesting and blowing off cells, shaking and mixing, ice-cooling for 25min, centrifuging at 12000rpm for 15min, sucking 60 μ l supernatant, adding 20 μ l 4 xSDS Buffer, and boiling for 5 min;
protein PAGE gel electrophoresis: adding 10 mul of protein sample into each hole, performing electrophoresis, transferring a membrane, and sealing;
detection of HA-tagged antibody: incubating HA primary antibody at 4 ℃ overnight, washing with TBST, incubating rabbit source secondary antibody at room temperature for 1h, washing with TBST, and developing with a developing solution.
pAD-Shuttle-CMV-EGFP-P2A-EP402R-CP204L-E183L-HA adenovirus vector recombination
1) Carrying out enzyme digestion linearization on the plasmid PmeI of pShuttle-CMV-EGFP-P2A-EP402R-CP204L-E183L-HA, and recovering gel;
2) the linearized vector is transformed into BJ5183 escherichia coli containing pAdEasy-1 plasmid, screening on a kana resistance plate, identifying colony PCR, amplifying positive colonies, and extracting plasmid.
3) PacI enzyme digestion verification is carried out, and an enzyme digestion fragment is recovered by using ethanol precipitation and is used for transfecting cells to package adenovirus. The results of the identification are shown in FIG. 3. As can be seen from FIG. 3, the 1kb DNA ladder with TaKaRa as M, and the electrophoresis results of the products cleaved with PacI and BamHI in lanes 1 and 2, respectively. As can be seen in FIG. 3, the PacI cleavage yielded about 4.5kb and a larger band, presumably 33.8 kb; digestion with BamHI gave two relatively close bands, 1 of which was approximately 11.7kb above 10kb, and the other band was slightly larger, presumably 26.6kb in size. Thus, the recombinant adenovirus plasmid is correctly constructed.
5. Adenovirus packaging
1) Plating 293A cells: 2 x 10 to5Uniformly spreading 293A cells in a 12-well plate, and using the cell with the confluence degree reaching 30% for transfection;
2) plasmid preparation: respectively mixing 1.5 μ g linearized plasmid and 4 μ L Lipofectamine3000 liposome with 250 μ L serum-free DMEM medium, standing for 4min, mixing the plasmid mixed solution and Lipofectamine3000 mixed solution, and standing for 10 min; the mixture was added dropwise to the medium, shaken gently and cultured at 37 ℃.
3) Observing the cell state for 48h, after 5d, leading the cells to die, paying attention to fluid infusion, collecting the cells and the supernatant, freezing and thawing for 2 times at minus 80 ℃, shaking to release adenovirus, and centrifuging to remove precipitate;
4) mixing the adenovirus supernatant with 10% culture medium 1:1, inoculating in 6cm dish, observing cell state, and collecting virus after most of cells die;
5) repeatedly infecting, inoculating in T75 flask, inoculating in T175 flask, and collecting toxin;
6) concentrating and purifying the adenovirus.
6. Verification of adenovirus expression
1) Cell preparation: will be 5X 105HEK293A were spread evenly in 12-well plates with 80% confluency for adenovirus infection;
2) HEK293A cells were infected with 100. mu.L of unpurified virus supernatant, and Cytopathic (CPE) status was observed at 72h and expression status of green fluorescent protein was observed at 48h, as shown in FIGS. 5 and 6. The HEK293A cells inoculated with the unpurified adenovirus supernatant were visibly rounded and shed (FIG. 5) by observation with a common light microscope; while a clear green fluorescence expression was visible by fluorescence microscopy (fig. 6).
3) And (3) detecting WB tag protein. HA antibody, GFP antibody and GAPDH antibody were mixed at an optimum ratio and used for Western-Blot detection, and the results are shown in FIG. 4. M is protein Marker (Thermo Fisher #26616), lane 1 is total adenovirus-infected cell protein, and lane 2 is total control cell protein. A band of about 29kD is seen in lane 1, which is a GFP-P2A tagged protein; the multiple bands of 100kD are EGFP-P2A-EP402R-CP204L-E183L-HA, EP402R-CP204L-E183L-HA fusion protein and possible post-translational modification bands; while no band at the above position was detected in lane 2, a band similar to that in lane 1 was detected only in the vicinity of 35kD, which is the GAPDH reference.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yangzhou university
<120> EP402R, CP204L and E183L co-expression recombinant adenovirus, construction and application
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1083
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgataatac ttattttttt aatattttct aacatagttt taagtattga ttattgggtt 60
agttttaata aaacaataat tttagatagt aatattacta atgataataa tgatataaat 120
ggagtatcat ggaatttttt taataattct tttaatacac tagctacatg tggaaaagca 180
ggtaactttt gtgaatgttc taattatagt acatcaatat ataatataac aaataattgt 240
agcttaacta tttttcctca taatgatgta tttgatacaa catatcaagt agtatggaat 300
caaataatta attatacaat aaaattatta acacctgcta ctcccccaaa tatcacatat 360
aattgtacta attttttaat aacatgtaaa aaaaataatg gaacaaacac taatatatat 420
ttaaatataa atgatacttt tgttaaatat actaatgaaa gtatacttga atataactgg 480
aataatagta acattaacaa ttttacagct acatgtataa ttaataatac aattagtaca 540
tctaatgaaa caacacttat aaattgtact tatttaacat tgtcatctaa ctatttttat 600
acttttttta aattatatta tattccatta agcatcataa ttgggataac aataagtatt 660
cttcttatat ccatcataac ttttttatct ttacgaaaaa gaaaaaaaca tgttgaagaa 720
atagaaagtc caccacctga atctaatgaa gaagaacaat gtcagcatga tgacaccact 780
tccatacatg aaccatctcc cagagaacca ttacttccta agccttacag tcgttatcag 840
tataatacac ctatttacta catgcgtccc tcaacacaac cactcaaccc atttccctta 900
cctaaaccgt gtcctccacc caaaccatgt ccgccaccca aaccatgtcc tccacctaaa 960
ccatgtcctt cagctgaatc ctattctcca cccaaaccac tacctagtat cccgctacta 1020
cccaatatcc cgccattatc tacccaaaat atttcgctta ttcacgtaga tagaattatt 1080
taa 1083
<210> 2
<211> 606
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggatttta ttttaaatat atccatgaaa atggaggtca tcttcaaaac ggatttaaga 60
tcatcttcac aagttgtgtt tcatgcgggt agcctgtata attggttttc tgttgagatt 120
atcaatagcg gtagaattgt tacgaccgct ataaaaacat tgcttagtac tgttaagtat 180
gatattgtga aatctgctcg tatatatgca gggcaagggt atactgaaca tcaggctcaa 240
gaagaatgga atatgattct gcatgtgctg tttgaagagg agacggaatc ctcagcatct 300
tcggagaaca ttcatgaaaa aaatgataat gaaaccaatg aatgcacatc ctcctttgaa 360
acgttgtttg agcaagagcc ctcatcggag gtacctaaag actccaagct gtatatgctt 420
gcacaaaaga ctgtgcaaca tattgaacaa tatggaaagg cacctgattt taacaaggtt 480
attagagcac ataattttat tcaaaccatt tatggaaccc ctctaaagga agaagaaaaa 540
gaggtggtaa gactcatggt tattaaactt ttaaaaaaaa taagctttta tctcacctac 600
atttaa 606
<210> 3
<211> 554
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggattctg aattttttca accggtttat ccgcggcatt atggtgagtg tttgtcacca 60
gtcactacac caagcttctt ctccacacat atgtatacta ttctcattgc tatcgtggtc 120
ttagtcatca ttatcatcgt tctaatctat ctattctctt caagaaagaa aaaagctgct 180
gctattgagg aggaagatat acagttataa atccttatca agatcagcag tgggtagaag 240
tcactccaca accaggtacc tctaaaccag ctggagcgac tacagcaagt gtaggcaagc 300
cagtcacggg cagaccggca acaaacagac cagcaacaaa caaaccagtt acggacaacc 360
cagttacgga cagactagtc atggcaactg gcgggccggc ggccgcacct gcggccgcga 420
gtgctcctgc tcatccggct gagccttaca cgacagtcac tactcagaac actgcttcac 480
aaacaatgtc ggctattgaa aatttacgac aaagaaacac ctatacgcat aaagacctag 540
aaaactcctt gtaa 554
<210> 4
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctagagatc tggtaccgcc accatggtga gcaagggcg 39
<210> 5
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tatcttatct agaagctttt aggcgtagtc gggcacgtcg taggggtact c 51
<210> 6
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atcctggacc ggtcgacatt gactactggg tgagctt 37
<210> 7
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tcgtaggggt actcgagcag gctgttctcc aggtcct 37
<210> 8
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggacgagc tgtacaagag tactgcaaca aacttc 36
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgcaaatggg cggtaggcgt g 21
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaaatttgtg atgctattgc 20
<210> 11
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agtactgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga 60
ccggtcgac 69
<210> 12
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggcgccccc 9
<210> 13
<211> 5
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggttt 5
<210> 14
<211> 5
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aaacc 5
Claims (8)
1. A gene co-expressed by EP402R, CP204L and E183L, wherein the gene is obtained by connecting an EGFP gene, a first connecting gene, an EP402R gene, a second connecting gene, a CP204L gene, a second connecting gene and an E183L gene in series in sequence; the nucleotide sequence of the EP402R gene is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the CP204L gene is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the E183L gene is shown as SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the first connecting gene is shown as SEQ ID NO: 11 is shown in the figure; the nucleotide sequence of the second connecting gene is shown as SEQ ID NO: shown at 12.
2. A recombinant vector containing the gene of claim 1, wherein the recombinant vector uses a pShuttle-CMV vector as an original vector; the insertion site of the gene is between KpnI and HindIII.
3. A recombinant adenovirus vector inserted with the recombinant vector of claim 2 at a position between the first site and the second site of the PmeI cleavage site sequence; the nucleotide sequence of the first site is shown as SEQ ID NO: 13 is shown in the figure; the nucleotide sequence of the second site is shown as SEQ ID NO: as shown at 14.
4. The method for constructing a recombinant adenovirus vector according to claim 3, comprising the steps of:
the recombinant vector of claim 2 is subjected to enzyme digestion by using a restriction enzyme PmeI to obtain a linearized plasmid, and the linearized plasmid is transformed into Escherichia coli containing a pAdEasy-1 plasmid, and is recombined to obtain the recombinant adenovirus vector.
5. A recombinant adenovirus comprising the recombinant adenovirus vector according to claim 3 or the recombinant adenovirus vector obtained by the construction method according to claim 4.
6. The method for constructing a recombinant adenovirus according to claim 5, comprising the steps of: and carrying out enzyme digestion linearization on the recombinant adenovirus vector through PacI to obtain a linearized plasmid, and transfecting HEK293A cells with the linearized plasmid to culture to obtain the recombinant adenovirus.
7. Use of the recombinant adenovirus according to claim 5 or the recombinant adenovirus obtained by the construction method according to claim 6 in the preparation of African swine fever vaccine.
8. An African swine fever vaccine, comprising an immunological adjuvant and an immunogen, wherein the immunogen is the recombinant adenovirus of claim 5 or the recombinant adenovirus obtained by the construction method of claim 6.
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| CN112592937A (en) * | 2020-12-29 | 2021-04-02 | 扬州大学 | D117L, F317L and EP364R co-expression recombinant adenovirus vector, recombinant adenovirus and construction and application |
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