CN111549040A - Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method - Google Patents

Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method Download PDF

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CN111549040A
CN111549040A CN202010543868.1A CN202010543868A CN111549040A CN 111549040 A CN111549040 A CN 111549040A CN 202010543868 A CN202010543868 A CN 202010543868A CN 111549040 A CN111549040 A CN 111549040A
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recombinant adenovirus
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周远成
邝声耀
阴文奇
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Livestock Bioengineering Co ltd
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Abstract

The invention discloses a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method thereof, wherein the recombinant adenovirus can synchronously express African swine fever p72 and B602L proteins after infecting cells, and B602L is expressed in series with p72 through self-cleavage 2A signal peptide. The recombinant adenovirus vector capable of expressing the p72 and B602L proteins is constructed, the recombinant adenovirus capable of expressing the African swine fever virus p72 and B602L proteins is obtained after the vector and an adenovirus skeleton system are used for cotransfecting cells, and after the recombinant adenovirus is used for immunizing a test animal, the test animal can be stimulated to generate specific antibodies, so that new test data are provided for the development of an African swine fever vaccine.

Description

Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method.
Background
African Swine Fever Virus (ASFV) is a nucleoplasm large DNA virus and is the only arbovirus, can infect domestic pigs and wild pigs, and has stronger infectivity and pathogenicity. The clinical symptoms of swine infection African swine fever are similar to swine fever, swine erysipelas and other diseases, the severity of the symptoms is different from the virulence, infection dosage and infection route of ASFV, and the symptoms can be divided into acute infection, subacute infection, invisible infection and the like according to the severity of the clinical symptoms. Acute infections are characterized mainly by anorexia, hyperpyrexia, leukopenia, bleeding of the skin and internal organs, with a high mortality rate, some of which can reach 100%. Subacute infections are characterized by transient thrombocytopenia and cytopenia, with observable foci of bleeding that can lead to respiratory changes, abortion and death, with a lower mortality rate than acute infections. At present, no specific vaccine and therapeutic drug aiming at African swine fever exist, so that the forced catching and killing mode is adopted at home and abroad to treat the sick swine herd.
The African swine fever is introduced into China in 2018, and the epidemic situation reported nationwide at present exceeds 150. The outbreak of African swine fever causes huge economic loss to the domestic pig breeding industry. In the time of more than one year of the African swine fever in China, the stock volume of breeding pigs in China is reduced from about 4000 million before the epidemic situation appears to about 1900 million of the lowest peak, and according to the statistics of incompleteness, the direct and indirect economic losses caused by the African swine fever can exceed 1 trillion yuan. After the outbreak of the African swine fever, the research on the African swine fever vaccine including subunit vaccine, polypeptide vaccine, gene deletion vaccine and the like is carried out by a plurality of domestic units, but no effective vaccine is available at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a nucleotide sequence for coding African swine fever virus p72 and B602L proteins is shown as SEQ ID NO: 1 is shown.
A recombinant adenovirus vector for expressing p72 and B602L proteins of african swine fever virus, comprising SEQ ID NO: 1.
The construction method of the recombinant adenovirus vector for expressing the proteins p72 and B602L of the African swine fever virus comprises the following steps: artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector to obtain the vector.
A recombinant adenovirus expressing p72, B602L protein of african swine fever virus, comprising SEQ ID NO: 1 or the recombinant adenovirus vector.
The construction method of the recombinant adenovirus for expressing the proteins of African swine fever virus p72 and B602L comprises the following steps:
(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector;
(2) and (2) co-transfecting the vector constructed in the step (1) and a pBHGloxdel E13cre vector into 293A cells, and then culturing to obtain the recombinant vector.
The recombinant adenovirus can be used for preparing African swine fever vaccines.
The recombinant adenovirus vector and the recombinant adenovirus expressing the African swine fever virus p72 and B602L proteins and the construction method thereof provided by the invention have the following beneficial effects:
the invention constructs a recombinant adenovirus vector capable of simultaneously expressing proteins of African swine fever virus p72 and B602L, obtains the recombinant adenovirus capable of expressing proteins of African swine fever virus p72 and B602L after the vector and an adenovirus skeleton system are used for cotransfecting cells, can stimulate a test animal to generate a specific antibody after the test animal is immunized by using the adenovirus, and provides new test data for the development of an African swine fever vaccine.
Drawings
FIG. 1 is a diagram showing the construction of the vector pDC316-p 72-B602L.
FIG. 2 is a graph showing the result of fluorescent plaque formation by the prepared recombinant adenovirus.
Detailed Description
EXAMPLE 1 construction of recombinant adenovirus vectors
The amino acid sequence for expressing African swine fever virus p72 protein is shown in SEQ ID NO: 2, the amino acid sequence of the protein expressing African swine fever virus B602L is shown in SEQ ID NO: 3, histidine tag HHHHHHHHHHHHHH is added at the tail end of the p72 protein, and T2A self-cutting signal peptide is added between the p72 protein added with the histidine tag HHHHHHHHHHHHHHHHHHHHHHHHHHHH and the B602L protein, wherein the amino acid sequence of T2A is shown in SEQ ID NO: 4.
according to the amino acid sequence, a nucleotide sequence capable of expressing p72 and B602L proteins is artificially designed, and the nucleotide sequence is shown in SEQ ID NO: 1, synthesizing the designed nucleotide sequence by Nanjing Kingsry Biotechnology Limited, inserting the synthesized sequence between Nhe I and Hind III enzyme cutting sites of a pDC316-mCMV-EGFP vector, and naming the constructed vector as pDC316-p72-B602L, wherein the structural diagram is shown in figure 1.
EXAMPLE 2 preparation of recombinant adenovirus
Passage of 293A cells to 12-well plates following conventional protocol, and use of transfection reagents the next day after passage: (
Figure BDA0002539981100000031
HD, available from Promega corporation) was transfected with pDC316-p72-B602L plasmid and pBHGloxdel E13cre plasmid using 6ul of transfection reagent, pDC316-p72-B602L plasmid and pBHGloxdel E13cre plasmid each at 1 ug. After transfection, the cells were cultured at 37 ℃ in a 5% carbon dioxide incubator. The cell status was observed daily. Expression of green fluorescent protein was observed 12 hours after transfection, followed by 7 days of observation of green fluorescent plaques (results figure 2). After fluorescent spots appear, the cells are cultured for 48 hours, and are repeatedly frozen and thawed three times at minus 80 ℃, centrifuged at 12000rpm, and stored at minus 80 ℃ to be counted as ADV P72/B602L P1 generation virus.
The 293A cells are passaged into a 96-well plate according to the conventional method, ADV P72/B602L P1 virus substitute stored at the temperature of-80 ℃ is diluted by 10 times of gradient by serum-free DMEM cell culture solution, and 10 times of dilution is taken1-108Dilutions were seeded in culture wells containing 293A cell suspension, 100ul virus dilution was added to each well, and 24 wells were diluted in each gradient. Adding virus diluent, culturing at 37 deg.C in 5% carbon dioxide incubator, and observing every day. Taking out after green fluorescent spot appearsInoculating 293A with the lowest dilution single fluorescent spot hole virus solution for amplification culture, harvesting the virus when the cytopathic effect reaches 80%, repeatedly freezing and thawing at-80 ℃ for 1 time, centrifuging at 12000rpm for 5min, taking the supernatant, subpackaging and storing at-80 ℃, and marking as ADV P72/B602L P3 generation virus. Inoculating 293A cells with the ADV P72/B602L P3 generation virus according to a conventional method, continuously culturing and propagating to the ADV P72/B602L P5 generation virus, and storing all harvested viruses at-80 ℃.
Example 3 ADV p72/B602L Mini preparation and purification
293A cells were passaged to a T175 cell flask by a conventional method, after the cells had grown to a dense monolayer, the cell culture solution was removed, 0.5ml of ADV P72/B602L P3 virus was inoculated, and adsorbed in a cell incubator at 37 ℃ for 30 minutes, and then 30ml of a DMEM (purchased from GIBCO) culture solution of 2% calf serum (purchased from GIBCO) was added to the inoculated cell flask. Culturing at 37 deg.C in 5% carbon dioxide incubator. After more than 90% of cells have cytopathic effect, harvesting virus liquid, centrifuging at 12000rpm for 10 minutes, taking supernatant, adding into a 100KD ultrafiltration centrifuge tube (purchased from MERCK company), centrifuging at 5000rpm to 1/10 of the original volume, adding 0.1M phosphate buffer to the original volume, centrifuging at 5000rpm to 1/100 of the original volume, and finally, according to the ratio of 5: adding glycerol at the ratio of 1, subpackaging and storing at-80 ℃.
Example 4 determination of viral content of ADV p72/B602L different generations and of purified viruses
293A cells were passaged to a 96-well plate according to a conventional method, and cultured in a 5% carbon dioxide incubator at 37 ℃. ADVp72/B602L P4, P5 virus and purified virus were diluted 10-fold gradient to 10 with 2% serum DMEM10After dilution, passaged 293A cells were seeded, and 8 wells were seeded at each dilution. After inoculation, the mixture was cultured in a 5% carbon dioxide incubator at 37 ℃ for 6 days. Placing the cultured 96-well plate under an inverted fluorescence microscope to observe whether cytopathic effect and green fluorescent spot appear in each dilution hole, recording the number of the green fluorescent spots and cytopathic holes in each dilution hole, and calculating TCID of ADV P72/B602L P4, ADV P72/B602L P5 generation and ADV P72/B602L purified virus according to the Reed-Muench method50Are respectively 109.5TCID50/ml、1010.2TCID50/ml、1011.5TCID50/ml。
Example 5 measurement of the humoral immune Effect of ADV p72/B602L immunized piglets
1. Immunization test for piglets
Purchasing 15 weaned piglets of 4 weeks old, randomly dividing the 15 piglets into 3 groups, taking purified ADV p72/B602L purified virus, diluting with serum-free DMEM, and injecting 108TCID50、109TCID50ADV p72/B602L purified virus and DMEM were injected intramuscularly in the neck at 2 ml/head, labeled as groups 1, 2, and 3, and serum collected before, 2 weeks after, and 4 weeks after immunization was assayed for serum antibodies.
2. Serum antibody assay
P72 protein polypeptide FPENSHNIQTAGKQDC was selected, synthesized by tsinggis biotechnology ltd, and the carbon terminal C was labeled with BSA protein. The BSA synthesized and conjugated polypeptides (99% pure) were dissolved in sterile water for injection to 1mg/ml and then diluted to 4ug/ml using 0.05M pH9.6 carbonate buffer. Taking an enzyme label plate, adding 100ul of diluted polypeptide solution into each hole, and coating for 12-16 hours at 2-8 ℃; removing the coating solution containing polypeptide, adding 0.1ml blocking solution (0.15MPBS, 0.05% Tween 20, 3% BSA, pH 7.4) per well, and blocking at 37 deg.C for 3 hr; remove blocking solution and wash 5 times with PBST (0.15MPBS, 0.05% tween 20, pH 7.4); diluting the collected serum by 100 times, adding 100ul of diluted serum into each hole, setting up a diluent control hole, and incubating at room temperature for 60 min; serum was removed and washed 5 times with PBST; adding 100ul of HRP-labeled goat anti-pig IgG diluted 1:5000 to each well, incubating for 30min at room temperature, and washing 5 times with PBST; adding 100ul TMB developing solution, incubating at room temperature for 15min, adding 50ul 2M sulfuric acid to terminate reaction, and measuring OD450 light absorption value to determine the wells and the negative control well A450nmIf the result is more than 2.1, the result is positive and the measurement result is shown in Table 1.
TABLE 1 results of antibody assay after immunization with ADV p72/B602L
Figure BDA0002539981100000061
As can be seen from Table 1, the antibodies in the immune group were all positive, and ADV p72/B602L 109TCID50Group height above 108TCID50Group, thereby indicating that ADV p72/B602L can induce the piglet to generate specific humoral immune response.
Sequence listing
<110> animal bioengineering Co., Ltd
<120> recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method
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<213> Artificial Sequence (Artificial Sequence)
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atggcctccg gcggcgcctt ctgcctgatc gccaatgacg gcaaggctga caagattatc 60
ctggcccagg acctgctgaa cagcaggatc agcaatatca agaacgtcaa taagagctac 120
gggaagccgg accccgagcc aaccctgagc cagatcgagg agacccacct ggtgcacttc 180
aacgcccatt tcaagcctta cgtgcccgtg gggtttgagt acaacaaggt gcgcccccac 240
accggaaccc ccaccctggg caataagctc accttcggga tcccacagta cggagacttc 300
ttccacgaca tggtggggca tcacatcctg ggagcctgcc acagcagctg gcaggacgcc 360
cccatccagg ggacatccca gatgggagcc cacggccagc tgcagacctt tccaagaaat 420
gggtacgact gggacaatca gaccccgctg gagggcgccg tgtataccct ggtggaccca 480
ttcggccgcc ccatcgtgcc tgggaccaag aacgcctacc ggaacctcgt ctactactgt 540
gagtaccccg gcgagaggct gtacgagaat gtcaggttcg atgtgaacgg gaatagcctg 600
gacgagtact ccagcgacgt gaccaccctg gtcaggaagt tttgtatccc tggcgacaaa 660
atgacaggct acaagcatct ggtgggacag gaggtgtccg tggagggaac cagcgggcct 720
ctgctgtgta atatccacga cctgcacaag ccacaccagt ctaagccgat cctgaccgat 780
gagaatgata cccagcgcac ctgctctcac acaaacccca agtttctgtc tcagcacttc 840
cctgagaata gccacaacat ccagaccgcc gggaagcagg acatcacccc catcaccgac 900
gccacctacc tggacatccg gaggaacgtg cactactcgt gcaacggccc tcagacccca 960
aagtactacc agcccccact ggctctgtgg atcaagctgc ggttctggtt caacgagaat 1020
gtgaatctgg ccattccctc tgtgagcatc cccttcgggg agcgcttcat caccataaag 1080
ctggcctccc agaaggacct ggtgaatgag ttccccggcc tgttcgtccg gcagagcaga 1140
ttcatcgccg gccgcccctc aagacgaaac attcgcttca aaccctggtt catccctggt 1200
gtgatcaatg agatcagcct gaccaacaac gagctgtata tcaacaacct gttcgtgact 1260
cctgagatcc ataatctctt cgtgaagcgg gtgcgatttt ccctcatccg ggtgcacaag 1320
acccaggtga cccacaccaa taacaaccac cacgacgaga agctgatgag cgccctgaag 1380
tggcctatcg aatacatgtt catcggcctg aagcccacct ggaacatctc cgaccagaac 1440
ccccatcagc accgggactg gcataagttc ggccacgtgg tgaacgccat catgcagcca 1500
acccaccacg ccgagattag cttccaggac agagataccg ccctgcctga cgcctgctcc 1560
agcatttccg acatcagtcc tgtgacctac cctatcaccc tgcccatcat caagaatatt 1620
agcgtgaccg cccacggaat taacctgatt gacaaattcc ccagcaaatt ctgctccagc 1680
tacatcccct tccactacgg aggcaacgcc atcaagaccc ctgacgatcc cggcgccatg 1740
atgattactt tcgccctgaa gcccagggag gagtaccagc cctctggcca catcaacgtg 1800
agccgggcta gagagttcta catcagctgg gacaccgact acgtgggatc catcacaacc 1860
gccgacctgg tggtgagcgc ttccgccatc aacttcctgc tgctgcaaaa tgggtctgcc 1920
gtgctgagat actccaccca ccaccaccat caccacgaag gcaggggctc actgctgacc 1980
tgcggggacg tggaggagaa cccaggcccc gccgagttta acatcgacga gctgctcaag 2040
aacgtgctgg aggacccatc aactgagatc tccgaggaga ccctgaagca gctctaccag 2100
cgcaccaacc catacaagca gtttaagaac gatagccggg tcgccttctg ttctttcact 2160
aatctgaggg agcagtatat ccggagactg attatgacca gctttatcgg ctacgtgttc 2220
aaggccctgc aggagtggat gccttcttac agcaagccca cccacaccac caaaaccctg 2280
ctgagcgagc tgatcaccct ggtggacacc ctgaaacagg agaccaatga cgtcccaagc 2340
gagagcgtgg tcaacacaat cctgagcatc gccgacagtt gtaagacaca gacccagaag 2400
tccaaggagg ctaagaccac catcgacagc ttcctgcgcg agcacttcgt gttcgacccc 2460
aacctccacg ctcagagtgc ctacacctgc gccgacacca acgtcgacac ctgcgcaagc 2520
atgtgcgctg acacaaatgt ggacacctgc gcctccatgt gcgcagacac caatgtcgac 2580
acctgtgcca gcacctgcac ctccactgaa tacacagacc tggctgaccc cgagcgcatc 2640
cctctccaca tcatgcagaa gaccctgaac gtgcccaacg agctgcaggc cgacatcgac 2700
gccatcaccc agacccctca gggatatcgg gccgccgccc acatcctgca gaacattgag 2760
ctccaccagt ccattaagca catgctggag aaccccaggg ctttcaaacc catcctgttt 2820
aacactaaga tcacgagata cctgagccag cacatccccc cccaggatac tttctataaa 2880
tggaactact acattgagga taactatgag gagctgaggg ccgccaccga atccatctac 2940
ccagagaagc ccgacctgga gtttgccttt atcatctacg acgtcgtgga ctcgagcaac 3000
cagcagaagg tggacgagtt ttactacaaa tataaggacc agatctttag cgaggtgtcc 3060
tccatccagc tggggaactg gaccctcctg ggctccttca aggccaaccg cgaaaggtat 3120
aactacttca accagaacaa cgaaatcatc aagagaatcc tggaccgcca cgaggaggac 3180
ctgaaaatcg gcaaggagat cctgcgcaac accatctacc ataagaaggc caaaaacatc 3240
caggagaccg ggcccgacgc ccccggcctg tccatctaca acagcacctt ccacactgat 3300
agcggcatta agggcctgct gagctttaag gagctgaaga atctggagaa ggccagcggc 3360
aacatcaaga aggcccgaga atacgacttc atcgacgact gtgaggagaa gatcaagcag 3420
ctgctgagca aggagaatct gacccccgac gaggagagcg agctgatcaa gaccaagaag 3480
cagctggaca acgccctgga gatgctgaac gtgcctgacg ataccatcag agtggatatg 3540
tgggtgaata acaataacaa gctggagaag gagatcctgt acaccaaggc cgagctgtaa 3600
<210>2
<211>652
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Ala Ser Gly Gly Ala Phe Cys Leu Ile Ala Asn Asp Gly Lys Ala
1 5 10 15
Asp Lys Ile Ile Leu Ala Gln Asp Leu Leu Asn Ser Arg Ile Ser Asn
20 25 30
Ile Lys Asn Val Asn Lys Ser Tyr Gly Lys Pro Asp Pro Glu Pro Thr
35 40 45
Leu Ser Gln Ile Glu Glu Thr His Leu Val His Phe Asn Ala His Phe
50 55 60
Lys Pro Tyr Val Pro Val Gly Phe Glu Tyr Asn Lys Val Arg Pro His
65 70 75 80
Thr Gly Thr Pro Thr Leu Gly Asn Lys Leu Thr Phe Gly Ile Pro Gln
8590 95
Tyr Gly Asp Phe Phe His Asp Met Val Gly His His Ile Leu Gly Ala
100 105 110
Cys His Ser Ser Trp Gln Asp Ala Pro Ile Gln Gly Thr Ser Gln Met
115 120 125
Gly Ala His Gly Gln Leu Gln Thr Phe Pro Arg Asn Gly Tyr Asp Trp
130 135 140
Asp Asn Gln Thr Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro
145 150 155 160
Phe Gly Arg Pro Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu
165 170 175
Val Tyr Tyr Cys Glu Tyr Pro Gly Glu Arg Leu Tyr Glu Asn Val Arg
180 185 190
Phe Asp Val Asn Gly Asn Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr
195 200 205
Thr Leu Val Arg Lys Phe Cys Ile Pro Gly Asp Lys Met Thr Gly Tyr
210 215 220
Lys His Leu Val Gly Gln Glu Val Ser Val Glu Gly Thr Ser Gly Pro
225 230 235 240
Leu Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro
245 250255
Ile Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asn
260 265 270
Pro Lys Phe Leu Ser Gln His Phe Pro Glu Asn Ser His Asn Ile Gln
275 280 285
Thr Ala Gly Lys Gln Asp Ile Thr Pro Ile Thr Asp Ala Thr Tyr Leu
290 295 300
Asp Ile Arg Arg Asn Val His Tyr Ser Cys Asn Gly Pro Gln Thr Pro
305 310 315 320
Lys Tyr Tyr Gln Pro Pro Leu Ala Leu Trp Ile Lys Leu Arg Phe Trp
325 330 335
Phe Asn Glu Asn Val Asn Leu Ala Ile Pro Ser Val Ser Ile Pro Phe
340 345 350
Gly Glu Arg Phe Ile Thr Ile Lys Leu Ala Ser Gln Lys Asp Leu Val
355 360 365
Asn Glu Phe Pro Gly Leu Phe Val Arg Gln Ser Arg Phe Ile Ala Gly
370 375 380
Arg Pro Ser Arg Arg Asn Ile Arg Phe Lys Pro Trp Phe Ile Pro Gly
385 390 395 400
Val Ile Asn Glu Ile Ser Leu Thr Asn Asn Glu Leu Tyr Ile Asn Asn
405 410415
Leu Phe Val Thr Pro Glu Ile His Asn Leu Phe Val Lys Arg Val Arg
420 425 430
Phe Ser Leu Ile Arg Val His Lys Thr Gln Val Thr His Thr Asn Asn
435 440 445
Asn His His Asp Glu Lys Leu Met Ser Ala Leu Lys Trp Pro Ile Glu
450 455 460
Tyr Met Phe Ile Gly Leu Lys Pro Thr Trp Asn Ile Ser Asp Gln Asn
465 470 475 480
Pro His Gln His Arg Asp Trp His Lys Phe Gly His Val Val Asn Ala
485 490 495
Ile Met Gln Pro Thr His His Ala Glu Ile Ser Phe Gln Asp Arg Asp
500 505 510
Thr Ala Leu Pro Asp Ala Cys Ser Ser Ile Ser Asp Ile Ser Pro Val
515 520 525
Thr Tyr Pro Ile Thr Leu Pro Ile Ile Lys Asn Ile Ser Val Thr Ala
530 535 540
His Gly Ile Asn Leu Ile Asp Lys Phe Pro Ser Lys Phe Cys Ser Ser
545 550 555 560
Tyr Ile Pro Phe His Tyr Gly Gly Asn Ala Ile Lys Thr Pro Asp Asp
565 570 575
Pro Gly Ala Met Met Ile Thr Phe Ala Leu Lys Pro Arg Glu Glu Tyr
580 585 590
Gln Pro Ser Gly His Ile Asn Val Ser Arg Ala Arg Glu Phe Tyr Ile
595 600 605
Ser Trp Asp Thr Asp Tyr Val Gly Ser Ile Thr Thr Ala Asp Leu Val
610 615 620
Val Ser Ala Ser Ala Ile Asn Phe Leu Leu Leu Gln Asn Gly Ser Ala
625 630 635 640
Val Leu Arg Tyr Ser Thr His His His His His His
645 650
<210>3
<211>529
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Ala Glu Phe Asn Ile Asp Glu Leu Leu Lys Asn Val Leu Glu Asp Pro
1 5 10 15
Ser Thr Glu Ile Ser Glu Glu Thr Leu Lys Gln Leu Tyr Gln Arg Thr
20 25 30
Asn Pro Tyr Lys Gln Phe Lys Asn Asp Ser Arg Val Ala Phe Cys Ser
35 40 45
Phe Thr Asn Leu Arg Glu Gln Tyr Ile Arg Arg Leu Ile Met Thr Ser
50 55 60
Phe Ile Gly Tyr Val Phe Lys Ala Leu Gln Glu Trp Met Pro Ser Tyr
65 70 75 80
Ser Lys Pro Thr His Thr Thr Lys Thr Leu Leu Ser Glu Leu Ile Thr
85 90 95
Leu Val Asp Thr Leu Lys Gln Glu Thr Asn Asp Val Pro Ser Glu Ser
100 105 110
Val Val Asn Thr Ile Leu Ser Ile Ala Asp Ser Cys Lys Thr Gln Thr
115 120 125
Gln Lys Ser Lys Glu Ala Lys Thr Thr Ile Asp Ser Phe Leu Arg Glu
130 135 140
His Phe Val Phe Asp Pro Asn Leu His Ala Gln Ser Ala Tyr Thr Cys
145 150 155 160
Ala Asp Thr Asn Val Asp Thr Cys Ala Ser Met Cys Ala Asp Thr Asn
165 170 175
Val Asp Thr Cys Ala Ser Met Cys Ala Asp Thr Asn Val Asp Thr Cys
180 185 190
Ala Ser Thr Cys Thr Ser Thr Glu Tyr Thr Asp Leu Ala Asp Pro Glu
195 200 205
Arg Ile Pro Leu His Ile Met Gln Lys Thr Leu Asn Val Pro Asn Glu
210 215 220
Leu Gln Ala Asp Ile Asp Ala Ile Thr Gln Thr Pro Gln Gly Tyr Arg
225 230 235 240
Ala Ala Ala His Ile Leu Gln Asn Ile Glu Leu His Gln Ser Ile Lys
245 250 255
His Met Leu Glu Asn Pro Arg Ala Phe Lys Pro Ile Leu Phe Asn Thr
260 265 270
Lys Ile Thr Arg Tyr Leu Ser Gln His Ile Pro Pro Gln Asp Thr Phe
275 280 285
Tyr Lys Trp Asn Tyr Tyr Ile Glu Asp Asn Tyr Glu Glu Leu Arg Ala
290 295 300
Ala Thr Glu Ser Ile Tyr Pro Glu Lys Pro Asp Leu Glu Phe Ala Phe
305 310 315 320
Ile Ile Tyr Asp Val Val Asp Ser Ser Asn Gln Gln Lys Val Asp Glu
325 330 335
Phe Tyr Tyr Lys Tyr Lys Asp Gln Ile Phe Ser Glu Val Ser Ser Ile
340 345 350
Gln Leu Gly Asn Trp Thr Leu Leu Gly Ser Phe Lys Ala Asn Arg Glu
355 360 365
Arg Tyr Asn Tyr Phe Asn Gln Asn Asn Glu Ile Ile Lys Arg Ile Leu
370375 380
Asp Arg His Glu Glu Asp Leu Lys Ile Gly Lys Glu Ile Leu Arg Asn
385 390 395 400
Thr Ile Tyr His Lys Lys Ala Lys Asn Ile Gln Glu Thr Gly Pro Asp
405 410 415
Ala Pro Gly Leu Ser Ile Tyr Asn Ser Thr Phe His Thr Asp Ser Gly
420 425 430
Ile Lys Gly Leu Leu Ser Phe Lys Glu Leu Lys Asn Leu Glu Lys Ala
435 440 445
Ser Gly Asn Ile Lys Lys Ala Arg Glu Tyr Asp Phe Ile Asp Asp Cys
450 455 460
Glu Glu Lys Ile Lys Gln Leu Leu Ser Lys Glu Asn Leu Thr Pro Asp
465 470 475 480
Glu Glu Ser Glu Leu Ile Lys Thr Lys Lys Gln Leu Asp Asn Ala Leu
485 490 495
Glu Met Leu Asn Val Pro Asp Asp Thr Ile Arg Val Asp Met Trp Val
500 505 510
Asn Asn Asn Asn Lys Leu Glu Lys Glu Ile Leu Tyr Thr Lys Ala Glu
515 520 525
Leu
<210>4
<211>18
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro

Claims (7)

1. A nucleotide sequence for coding African swine fever virus p72 and B602L proteins, wherein the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
2. A recombinant adenoviral vector for expressing p72, B602L protein of african swine fever virus, comprising the nucleotide sequence of claim 1.
3. The method for constructing the recombinant adenovirus vector for expressing the African swine fever virus p72 and B602L proteins according to claim 2, which comprises the following steps: artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector to obtain the vector.
4. A recombinant adenovirus expressing p72, B602L protein of african swine fever virus, comprising the nucleotide sequence of claim 1 or the recombinant adenovirus vector of claim 3.
5. The method for constructing the recombinant adenovirus expressing the African swine fever virus p72 and B602L proteins according to claim 4, which comprises the following steps:
(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector;
(2) and (2) co-transfecting the vector constructed in the step (1) and a pBHGloxdel E13cre vector into 293A cells, and then culturing to obtain the recombinant vector.
6. The use of the recombinant adenovirus expressing the proteins p72 and B602L of african swine fever virus according to claim 4 in the preparation of an african swine fever vaccine.
7. An African swine fever vaccine, comprising the recombinant adenovirus expressing the p72 and B602L proteins of African swine fever virus of claim 4.
CN202010543868.1A 2020-06-15 2020-06-15 Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method Pending CN111549040A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363016A (en) * 2020-03-30 2020-07-03 中国农业大学 African swine fever immune antigen and preparation method and application thereof
CN112472801A (en) * 2020-12-22 2021-03-12 华南农业大学 DNA vaccine and subunit vaccine of African swine fever p30, p54, p72 and B602L, and preparation method and application thereof
CN112625095A (en) * 2021-01-13 2021-04-09 武汉科前生物股份有限公司 Porcine rotavirus recombinant protein, recombinant adenovirus expressing protein and application
CN113735943A (en) * 2021-05-13 2021-12-03 浙江海隆生物科技有限公司 Recombinant African swine fever virus p72 subunit protein, preparation method and application thereof
WO2022236977A1 (en) * 2021-05-13 2022-11-17 中国农业大学 African swine fever virus capsid protein p72, preparation method therefor, and application thereof
CN113755505A (en) * 2021-09-30 2021-12-07 绵阳市游仙区创新科技产业技术研究院 Vaccine for treating and/or preventing African swine fever virus and preparation method thereof
CN114107389A (en) * 2021-11-26 2022-03-01 中国农业科学院北京畜牧兽医研究所 Recombinant adenovirus expressing African swine fever virus B602L-B646L protein and construction method thereof

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Application publication date: 20200818