CN102335424B - II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using IX protein - Google Patents
II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using IX protein Download PDFInfo
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Abstract
The invention discloses an II type adenovirus live vector recombinant vaccine of a dog for showing a lyssa virus protective antigen by using an IX protein and relates to a recombinant vaccine for respectively showing the lyssa virus protective antigen or an epitope by using characteristics of different structure proteins of the II type adenovirus of the dog. A lyssa virus protective antigen gene or a nucleotide sequence for expressing the epitope is inserted in a hydrophobic locus of the structure protein gene of the II type adenovirus of the dog by using the IX protein, so that an exogenous antigen or epitope is subjected to fusion expression with the structure proteins of the II type adenovirus of the dog on the surface of the adenovirus. After toxic substances are eliminated from the recombinant vaccine, the closed isolated feeding for a test animal is carried out and the food searching and pathogenic conditions of the test animal are observed and recorded. The result shows that the dog subjected to oral administration and intramuscular injection of any recombinant vaccine can resist attack of virulent strain and has a survival rate of over 90 percent.
Description
Technical field:
The present invention relates to utilize the characteristic of dog II type adenovirus different structure albumen, distinguish the recombiant vaccine of displaying protective antigen of rabies virus or epitope, can be used for the development and application of novel animal rabies vaccine.
Background technology:
Rabies are a kind of important infectious diseases common to human beings and animalss that the apyetous encephalomyelitis is principal character of take that caused by rabies virus, and after infecting humans and animals, once morbidity, mortality rate is almost 100%.China is rabic multiple ground, has every year thousands of people to die from rabies, is mainly to be caused by viruliferous lyssodexis.Although the people is safe and effective with commercially available vaccine at present, the existence with malicious animal has still formed safely direct threat to the human life in a large number.Particularly in recent years, die from rabic number and be the trend risen year by year.Therefore, reducing or control the storage collection of rabies virus in domestic animal is effectively to control the important channel that human rabies occurs.
Vaccine for the animal rabies prevention is mainly inactivated vaccine and attenuated vaccine at present, and the two is to the antirabic effect of playing positive important.But the inactivated vaccine immunity cycle is short, and vaccination ways is single, is unsuitable for the field immunity of animal, and also do not realize production domesticization in China, import inactivated vaccine complicated process of preparation, the cost costliness, relatively be applicable to developed country.Attenuated vaccine has the danger of virulence reversion in the Sensitivity animal body, can not fundamentally eliminate rabies, therefore, is difficult to be promoted in China.
Along with the continuous research to the rabies virus molecular biological characteristic, proved that glycoprotein is the unique rabies virus structural protein that can induce body to produce neutralizing antibody.Many laboratorys have all been carried out and take the research of the new semple type rabies vaccines such as subunit vaccine that glycoprotein is protective antigen, genetic engineering subunit vaccine, DNA vaccination in the world.Although the generation that these vaccines can the inducing specific neutralizing antibody, and there is certain immune protective efficiency, due to less immunogenic, do not obtain practical application.The live vector recombinant vaccine research that poxvirus and human adenovirus type 5 be the vector expression rabies virus glucoprotein of take has obtained certain progress, and the immunity test in Europe and North America proves to have good immunogenicity.But, owing to having again immunologic rejection or safety issue, and obtain official approval, do not use.For improving the actual application value of recombinant adenovirus vaccine, at present, the existing research of the displaying vaccine that the human adenovirus type 5 of take is carrier, there is not yet report at home and abroad and take the displaying vaccine technologies that dog II type adenovirus is carrier.
The recombiant vaccine that the dog II type adenovirus of take is carrier, to the Canis animals susceptible, also can effectively copy in the livestock animals bodies such as cat, cattle and pig, and inherited character is stable, and safety is good, in the research of new semple type rabies vaccine, has larger potentiality to be exploited.The dog II type adenovirus live vector recombinant vaccine of the expressing glycoprotein of rabies virus that this laboratory builds, obtained national inventing patent, and completed the bio-safety evaluation in the industrial experimentation stage, proves and have good immune effect and safety.On this basis, for further improving immune efficacy and the actual application value of dog II type adenovirus recombiant vaccine, the invention provides a series of recombiant vaccinies with dog II type adenovirus structural protein presenting and expressing protective antigen of rabies virus or epitope.
Summary of the invention:
The technical problem to be solved in the present invention is to provide the dog II type adenovirus live vector recombinant vaccine that utilizes IX albumen displaying protective antigen of rabies virus.
Technical scheme of the present invention is that to take dog II type adenovirus be carrier; insert the nucleotide sequence of protective antigen of rabies virus gene or antigen expressed epi-position in the hydrophobicity site of its structural protein gene, make exogenous antigen or epi-position at adenovirus surface and its structural protein amalgamation and expression.
Further; the dog II type adenovirus vaccine strain of take is carrier; on its structural protein hexonmer, fine prominent or IX albumen; utilize the original paper of transcribing of these structural protein genes self; amalgamation and expression protective antigen of rabies virus or epitope; it is showed on dog II type adenovirus particles surface, thereby reach the purpose that strengthens the recombiant vaccine immune efficacy.Immunity test proves, this series recombiant vaccine all can be induced the neutralizing antibody for rabies virus and hepatitis infectiosa canis virus that produces high titre, and can resist the attack of rabies virus standard virulent strain and hepatitis infectiosa canis virus virulent strain.
At first, base deletion is not occurring and is not affecting under the prerequisite that structural protein transcribe, hexonmer in the full genome of dog II type adenovirus, fine prominent or IX protein gene are being transformed:
1, lap over PCR is respectively in 4 sites of the full genome hexonmer of dog II type adenovirus gene hypervariable region, the reading frame of transcribing by hexonmer is inserted respectively in rabies virus glucoprotein and epitope, makes itself and hexonmer at adenovirus particles outer surface amalgamation and expression;
2, introduce the nucleotide sequence (containing Xma I restriction enzyme site) of one section synthetic at the full genome 28106bp of dog II type adenovirus place, at Xma I restriction enzyme site place, the transcribe reading frame prominent by fibre inserted in rabies virus glucoprotein and epitope, and itself and fibre are dashed forward at adenovirus particles outer surface amalgamation and expression;
3, utilize the restriction enzyme A se I site in dog II type adenovirus IX protein gene, insert rabies virus glucoprotein film outskirt gene and SV40 virus poly A transcription stop signals at IX albumen tanscription termination codon place, make IX albumen and Surface Display of Foreign Epitopes utilize the same reading frame of transcribing at adenovirus particles outer surface amalgamation and expression.
Secondly, protective antigen of rabies virus or epitope can be transcribed with canine recombinant II type adenovirus structural protein, on the virion surface, produce a plurality of copies, and its immune efficacy is better than the recombiant vaccine of single copy; The 3rd, the mode of the propagation of recombiant vaccine, amplification culture and immunity inoculation is all consistent with vector virus vaccine strain dog II type adenovirus; The 4th, after the animal inoculation recombiant vaccine, infect and the immunity of rabies virus infection obtain for hepatitis infectiosa canis virus simultaneously, and can resist the attack of normal fatal dose virulent strain.
The building process of the canine recombinant II type adenovirus of displaying protective antigen of rabies virus:
That 1, utilizes laboratory in earlier stage to build obtains the large fragment containing the hexonmer gene containing two single restriction enzyme sites in the full geneome plasmid pPOLYII-CAV2 of dog II type adenovirus, and it is cloned into to the pET-28a carrier, obtains recombiant plasmid pET-LH.In recycling pET-LH, two single restriction enzyme sites of large fragment obtain the small fragment containing the hexonmer gene, and it is cloned into to the pET-28a carrier, obtain recombiant plasmid pET-SH.To in rabies virus, with epitope, insert respectively 4 different loci (respectively at the complete genomic 17378bp of dog II type adenovirus, 17450bp, 17623bp and 18084bp place) in six adjacent body hypervariable regions by overlapping PCR, and the PCR product cloning is entered to the pMD18-T carrier, carry out sequencing.After order-checking is correct, utilize two single restriction enzyme sites in hexonmer that improved hexonmer gene clone is entered to pET-SH.Finally, according to above-mentioned route back clone, obtain the full genome of canine recombinant II type adenovirus.
2, utilize restriction enzyme site Sac I and the Kpn I in pBluescriptII KS (+/-), insert artificial synthesized sequence Linker SK (successively containing Sal I, Xma I, Spe I and many restriction enzyme sites of Pac I), obtain recombiant plasmid pBS-SKI.Press the fiber protein gene sequence, synthetic dog II type adenovirus whole genome sequence 28050bp～28106bp section, and insert in Linker SK between Sal I～Xma I, utilize PCR to obtain 30182bp～29358bp sequence in the full genome of dog II type adenovirus, two fragments are inserted in Linker SK between Spe I～Pac I, obtained recombiant plasmid pBS-SKII.Again by rabies virus and epitope by the reading frame of transcribing of fiber protein gene, insert in the Linker SK of pBS-SKII between Xma I and Spe I site, obtain improved fiber protein gene.Finally, by restriction enzyme site Sal I and the Pac I at two ends in Linker SK, the fiber protein gene of transformation is inserted in pPOLYII-CAV2, obtain the full genome of canine recombinant II type adenovirus.
3, utilize two single restriction enzyme sites in pPOLYII-CAV2 to obtain the large fragment containing the IX protein gene, it is cloned into to pBluescriptII KS (+/-) carrier, obtain recombiant plasmid pBS-LIX.In recycling pBS-LIX, two single restriction enzyme sites of large fragment obtain the small fragment containing the IX protein gene, and it is cloned into to the pEGFP-C1 carrier, obtain recombiant plasmid pEGFP-SIX.Then, at IX albumen termination codon place, utilize the restriction enzyme site of himself to insert rabies virus glucoprotein film outskirt gene and SV40 poly A transcription terminator, realize IX albumen and glycoprotein film outskirt amalgamation and expression.Finally, according to above-mentioned route, the back clone obtains the full genome of canine recombinant II type adenovirus.
4, alkaline lysis prepares various canine recombinant II type adenoviral gene group plasmids in a large number, respectively through Asc I and the free full postgenome of Pme I double digestion, by liposome transfection 5 μ g genomic DNAs in dog kidney continuous cell line MDCK, go down to posterity every other day 1 time, until typical hepatitis infectiosa canis virus pathological changes (thyrsiform) occurs.
The dog II type adenovirus live vector recombinant vaccine immunogenicity of displaying protective antigen of rabies virus
1, utilize mdck cell to prepare in a large number recombiant vaccine, and measure the TCID of vaccine
50(all be not less than 10
6.5TCID
50/ 0.1ml).The domesticated dog of getting without the rabies immune history is experimental animal, respectively the injection of both sides vastus medialis meat and oral vaccination.
2, when immunity every dog intramuscular injection 1ml or oral 2ml recombiant vaccine, immunity once, blood sampling separation of serum weekly before immunity and after immunity.
3, adopt fluorescent antibody rabies virus neutralization test (FAVN) method; detect immune dog serum NAT; result shows; the average neutralizing antibody level that recombiant vaccine is induced is after immunity the 3rd week the time; more than all reaching international minimal protection level (0.5IU/ml), within 4th～5 weeks, reach the highest (4.62～6.43IU/ml).By hemagglutination inhibition test, detect test dog body inner gland virus antibody titer, result shows, the adenovirus antibody level that recombiant vaccine is induced is the highest the 4th week the time after immunity, is 1: 2
10.
The immanoprotection action of the dog II type adenovirus live vector recombinant vaccine of displaying protective antigen of rabies virus
After immunity 6 weeks, each test group animal is carried out to counteracting toxic substances, use respectively 100LD for every group
50The strong malicious CVS-24 of rabies virus standard and the strong poison of hepatitis infectiosa canis virus carry out counteracting toxic substances.After counteracting toxic substances, experimental animal is sealed to isolated rearing, do not contacted each other, observed and record searching for food and incidence of experimental animal every day, observed 40 days.The result demonstration, the oral and dog any recombiant vaccine of intramuscular injection all can be resisted the attack of strong poison, and survival rate is more than 90%.
Specific embodiments:
Below in conjunction with embodiment, the present invention is described further:
Embodiment 1: with the complete genomic clone of recombiant vaccine of hexonmer displaying protective antigen of rabies virus
Eco52I and SnaB I double digestion pPOLYII-CAV2, reclaim the 12516bp fragment through agarose gel electrophoresis, is cloned into the pET-28a carrier, obtains recombiant plasmid pET-LH.Use again Nde I and Mlu I double digestion pET-LH, through agarose gel electrophoresis, reclaim the 4325bp fragment, be cloned into the pET-28a carrier, obtain recombiant plasmid pET-SH.By overlapping PCR will be in rabies virus and epitope insert respectively 4 different loci in six adjacent bodies, and by the PCR product cloning to the pMD18-T carrier, carry out sequencing.After order-checking is correct, utilize two natural restriction enzyme sites of hexonmer Kpn I and Dra I that improved hexonmer gene clone is entered to pET-SH.Finally, according to above-mentioned route, the back clone obtains 4 kinds of full geneome plasmid pPolyII-CAV2/HVR1-GAP of canine recombinant II type adenovirus, pPolyII-CAV2/HVR2-GAP, pPolyII-CAV2/HVR3-GAP and pPolyII-CAV2/HVR4-GAP.
Overlapping PCR primer sequence
HV(forward)：
5’-ATATCAGGGGTACCCTAGACAGAG-3’；
HV(reverse)：
5’-GGTGTGTATTTAAAGGCATCAGGC-3’；
HVR 1-GAP(forward)：
5’-CTCGGATCCTGGAGCCCTATTGACATACTAGGTTCAGCTGTCACAAACAATACCTACCA-3’；
HVR1-GAP(reverse)：
5’-TGAACCTAGTATGTCAATAGGGCTCCAGGATCCGAGCACAGTTATGGCACCTGCAGAGG-3’；
HVR2-GAP(forward)：
5’-CTCGGATCCTGGAGCCCTATTGACATACTAGGTTCAGAGCTGGGGGATGCGTCTGGCCG-3’；
HVR2-GAP(reverse)：
5’-TGAACCTAGTATGTCAATAGGGCTCCAGGATCCGAGATCGACCCAGCTTTCAGGGCCCA-3’；
HVR3-GAP(forward)：
5’-CTCGGATCCTGGAGCCCTATTGACATACTAGGTTCAGCCATGCTATACACTGAAGATGT-3’；
HVR3-GAP(reverse)：
5’-TGAACCTAGTATGTCAATAGGGCTCCAGGATCCGAGGGTGGTCACCTTATAAAATCTGG-3’；
HVR4-GAP(forward)：
5’-CTCGGATCCTGGAGCCCTATTGACATACTAGGTTCATTTCAGGCAGAAAATACCAATGT-3’；
HVR4-GAP(reverse)：
5’-TGAACCTAGTATGTCAATAGGGCTCCAGGATCCGAGAACCTTCATGGTGGTCATGTTTG-3’。
Wherein, while at the full genome 17378bp of dog II type adenovirus place, inserting exogenous gene, with primer HV and HVR1-GAP, finally obtain the full geneome plasmid pPolyII-CAV2/HVR1-GAP of canine recombinant II type adenovirus; While inserting at the 17450bp place, with primer HV and HVR2-GAP, finally obtain the full geneome plasmid pPolyII-CAV2/HVR2-GAP of canine recombinant II type adenovirus; While inserting at the 17623bp place, with primer HV and HVR3-GAP, finally obtain the full geneome plasmid pPolyII-CAV2/HVR3-GAP of canine recombinant II type adenovirus; While inserting at the 18084bp place, with primer HV and HVR4-GAP, finally obtain the full geneome plasmid pPolyII-CAV2/HVR4-GAP of canine recombinant II type adenovirus.
In rabies virus glucoprotein and the epitope DNA sequence
5’-TGAACCTAGTATGTCAATAGGGCTCCAGGATCCGAG-3’。
The PCR reaction condition
Take at the full genome 17378bp of dog II type adenovirus place insertion exogenous gene is example, plasmid pET-SH is template, HVR (reverse) and HVR1-GAP (forward) are primer, under the effect of TaKaRa Ex Taq archaeal dna polymerase, 95 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 59 ℃ of annealing 45s, 72 ℃ are extended 1min, and totally 30 circulations obtain left side, mutational site fragment (2L).Take pET-SH as template, HVR1-GAP (reverse) and HV (forward) are primer, under the effect of TaKaRa Ex Taq archaeal dna polymerase, 95 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 30 circulations, 72 ℃ are extended 10min amplification right side, mutational site fragment (2R).Pcr amplification product reclaims purification through agarose gel electrophoresis.Then, carry out second step PCR, get respectively 25-50ng 2L and the 2R fragment fully mixes as template, take HV (forward) and HV (reverse) is primer, under the effect of TaKaRa Ex Taq archaeal dna polymerase, 95 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 30 circulations, amplification obtains HVR1-GAP sudden change fragment.Amplified production reclaims purification through agarose gel electrophoresis, is connected to pMD18-T Vector, is checked order.When inserting, other positions in the same way, utilize primer separately to carry out overlapping pcr amplification.
Embodiment 2: with the complete genomic clone of recombiant vaccine of the prominent displaying protective antigen of rabies virus of fibre
Utilize restriction enzyme site Sac I and Kpn I in pBluescriptII KS (+/-), insert artificial synthesized sequence Linker SK (successively containing Sal I, Xma I, Spe I and many restriction enzyme sites of Pac I), obtain recombiant plasmid pBS-SKI.Press the fiber protein gene sequence, synthetic dog II type adenovirus whole genome sequence 28050bp～28106bp section, and insert in Linker SK between Sal I～Xma I, utilize PCR to obtain 30182bp～29358bp sequence in the full genome of dog II type adenovirus, two fragments are inserted in Linker SK between Spe I～Pac I, obtained recombiant plasmid pBS-SKII.Again by rabies virus and epitope by the reading frame of transcribing of fiber protein gene, insert in the Linker SK of pBS-SKII between Xma I and Spe I site, obtain improved fiber protein gene.Finally, by restriction enzyme site Sal I and the Pac I at two ends in Linker SK, the fiber protein gene of transformation is inserted in pPOLYII-CAV2, obtain the full geneome plasmid pPOLYII-CAV2/Fiber-GAP of canine recombinant II type adenovirus.
Linker SK sequence
Sense sequence：
5’-CGTCGACAGTCCCGGGAGTGGCGCCAGTGGGCCCAGTACTAGTGCCTTAATTAAGGTAC-3’；
Antisense sequence：
5’-CTTAATTAAGGCACTAGTACTGGGCCCACTGGCGCCACTCCCGGGACTGTCGACGAGCT-3’。
The full genome 28050bp of synthetic dog II type adenovirus～28106bp sector sequence
Sense sequence：
5’-TCGACGGTGCCCCCAGCAGAAGTATCGACTGCATGCTAATTATTAACAAACCAAAAC-3’；
Antisense sequence：
5’-CCGGGTTTTGGTTTGTTAATAATTAGCATGCAGTCGATACTTCTGCTGGGGGCACCG-3’。
The PCR primer sequence
Forward：5’-TGATCACCGCAACGGTGAATG-3’
Reverse：5’-TGAGTATGATGATGCTCTTGCAC-3’
The PCR reaction condition
Under the effect of TaKaRa Ex Taq archaeal dna polymerase, 95 ℃ of denaturation 5min, 94 ℃ of degeneration 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 30 circulations, 72 ℃ are extended 10min.Amplified production reclaims purification through agarose gel electrophoresis, is connected to pMD18-T Vector, is checked order.
Embodiment 3: with the complete genomic clone of recombiant vaccine of IX albumen displaying protective antigen of rabies virus
EcoR I and Cla I double digestion pPOLYII-CAV2, agarose gel electrophoresis reclaims the 3610bp fragment, is cloned into pBluescriptII KS (+/-), obtains recombiant plasmid pBS-LIX.Mlu I and EcoR I double digestion pBS-LIX, agarose gel electrophoresis reclaims the 340bp fragment, is cloned into pEGFP-C1, obtains recombiant plasmid pEGFP-SIX.At IX albumen termination codon place, utilize single restriction enzyme site Ase I to insert rabies virus glucoprotein film outskirt gene (G ') and SV40 poly A transcription stop signals sequence, realize IX albumen and glycoprotein film outskirt amalgamation and expression.Finally, according to above-mentioned route, the back clone obtains the full geneome plasmid pPOLYII-CAV2/IX-G ' of canine recombinant II type adenovirus.
Embodiment 4: the packing of recombiant vaccine and evaluation
1, the packing of recombiant vaccine
Get dog two type adenoviral plasmid pPolyII-CAV2/HVR1-GAP, pPolyII-CAV2/HVR2-GAP, pPolyII-CAV2/HVR3-GAP, the pPolyII-CAV2/HVR4-GAP of transformation, each 5 μ g of pPOLYII-CAV2/Fiber-GAP and pPOLYII-CAV2/IX-G ', discharge respectively the full genome of restructuring with Asc I and Pme I double digestion, utilize Lipofectamine
TM2000 transfections to mdck cell is, until pathological changes appears in cell.
The enzyme action condition
Restriction enzyme A sc I and each 10U of Pme I, 10 * Buffer, 5 μ l, plasmid 5 μ g, add water and mend to 50 μ l.After 37 ℃ of water-bath 1h, chloroform: isoamyl alcohol (24: 1) extracting 1 time, 2 times of volume dehydrated alcohol precipitations, the water dissolution precipitation is heated up in a steamer in sterilizing four, and-20 ℃ save backup.
2, the evaluation of recombiant vaccine
The recombiant vaccine genome identification
Get the monolayer mdck cell that has been the thyrsiform pathological changes in the 50ml culture bottle, the tipping culture medium, with PBS washing 2 times, add the freshly prepared cell pyrolysis liquid of 800 μ l (0.6%SDS, 10mM EDTA, 100 μ g/ml E.C. 3.4.21.64s), 37 ℃ of incubation 1h, add 200 μ l 5M NaCl, after mixing gently, ice-water bath 1h.Entire mixture is moved into to centrifuge tube, in 4 ℃, the centrifugal 40min of 12000rpm, supernatant moves into and to add a centrifuge tube, and with equal-volume phenol: chloroform extracting 2 times adds final concentration 0.25M sodium acetate and 2 times of volume dehydrated alcohol, the centrifugal 10min of 12000rpm, DNA precipitation is with 70% washing with alcohol 1 time, aseptic air-dry after, be dissolved in 50 μ l deionized waters.Take genome as template, the pcr amplification exogenous gene, order-checking is identified.
Identify and use the PCR primer sequence
1. CAV2/HVR 1-GAP, CAV2/HVR2-GAP, CAV2/HVR3-GAP and CAV2/HVR4-GAP identify and use primer sequence
Forward：5’-GGTGTGTATTTAAAGGCATCAGGC-3’；
Reverse：5’-ATATCAGGGGTACCCTAGACAGAG-3’。
2. CAV2/Fiber-GAP identifies and uses primer sequence
Forward：5’-AGCAGAAGTATCGACTGCAT-3’；
Reverse：5’-TGAGTATGATGATGCTCTTGCAC-3’。
3. CAV2/IX-G ' identifies and uses primer sequence
Forward：5’-AGCTGAAGATCCAGGTGGCT-3’；
Reverse：5’-ACGCTGGACACGGTATTATC-3’。
The PCR reaction condition
The same.
TCID
50Mensuration
By 1 * 10
7Mdck cell imports 96 porocyte culture plates into, every hole inner cell approximately 1 * 10
6, supplemented medium to 100 μ l.Next day, access virus sample to be measured.In 1～11 hole, carry out 10
-1～10
-11Dilution, A～H classifies same concentration as and repeats.After 7 days, calculate the TCID of recombiant vaccine to be measured with the Karber method
50.
Immunity identification (Western blotting)
Get containing rabies virus SRV
9The cell pyrolysis liquid 50 μ l of strain, add equivalent 2 * SDS-PAGE sample-loading buffer, and boiling water bath 10min, be cooled to room temperature, of short durationly carries out the SDS-PAGE electrophoresis after centrifugal.Electrophoresis is complete, by protein sample electrotransfer on glue to nitrocellulose filter, respectively with the multi-resistance of the anti-recombiant vaccine of dog and positive and negative control multi-resistance, the anti-reaction of the anti-dog two of HR labelling rabbit, finally with DAB/H
2O
2Whether colour developing, occur and whether the location determination recombiant vaccine expresses Rabies Virus Antigen according to colour developing band.
Virulence is identified
Get 60 of the negative dogs of 4～5 monthly age hepatitis infectiosa canis viruses and Antibody against rabies virus, by inoculation recombiant vaccine difference, be divided into 6 groups, 10 every group, respectively with containing 10
8TCID
50With 10
7TCID
505 dogs of each intramuscular injection of recombiant vaccine cell culture fluid.After injection, observe the performances such as diet, the mental status of injection dog, take temperature, counting quantity of leucocyte and other clinical manifestation etc., and dissected the injection dog after 4 weeks, observe the pathological change of dog body, and carry out the tissue slice observation of the main organs such as liver.
Repeated pruning
1 * 10
6Mdck cell imports 25cm into
2In Tissue Culture Flask, after cell grows up to monolayer, press 0.5% access recombiant vaccine of culture volume in culture bottle, within after inoculation 3～4 days, receive poison, to various recombiant vaccinies difference continuous passage 40 times, labelling is also preserved each culture fluid, simultaneously every 5 generations, extract the adenoviral gene group in infection cell, size, direction and the position of by PCR and DNA sequencing, identifying exogenous gene.
Result
1. all correctly inserted protective antigen of rabies virus in each recombiant vaccine genome, transcriptional orientation is consistent with the viral genome transcriptional orientation;
2.0.1ml the TCID of each recombiant vaccine
50Without significant difference, be 10
6.5～10
7
3. each recombiant vaccine has all been expressed the rabies virus glucoprotein neutralizing epitope, antibody can with the rabies virus glucoprotein specific binding;
4. the virulence testing result shows, the injection dog does not occur that diet and spiritual expression etc. are abnormal, and body temperature is (37.5～38.5 ℃) within normal range.In body, numeration of leukocyte shows, total white blood cells (7.5～16.0 * 10
9/ L) all, within normal range, the clinical manifestations such as blue eye or anorexia do not appear in the injection dog in differential counting.Dissection has no the Main Tissues organ and ANOMALOUS VARIATIONS occurs, and the tissue slice of the tissue slice such as recombiant vaccine injection dog lung, liver and kidney and healthy dogs is without obvious difference.
5. the repeated pruning result shows, each recombiant vaccine went down to posterity through 40 generations, and its genome (size, direction and the position that comprise exogenous gene) all remains unchanged, and exogenous gene sequence does not all change.
Embodiment 5: with the immune effect of the dog II type adenovirus live vector recombinant vaccine of hexonmer displaying protective antigen of rabies virus
Laboratory animal
Rabies virus and adenovirus antibody feminine gender 4～5 the monthly age 80 of dogs, be divided at random 8 groups, 10 every group.
Animal immune
Respectively with TCID
50Be 10
6.5Recombiant vaccine CAV2/HVR1-GAP, CAV2/HVR2-GAP, CAV2/HVR3-GAP and CAV2/HVR4-GAP 8 groups of dogs are carried out to immunity.20 dogs of every kind of each immunity of recombiant vaccine, wherein intramuscular injection is 10, the 1ml/ bar; Oral 10, the 2ml/ bar.
Immune detection
Gather respectively weekly a small amount of venous blood before immunity and after immunity, separation of serum, detect tested rabies virucidin level with FAVN.
Challenge test
After immunity 6 weeks, randomly draw 5 dogs for every group, with 100LD
50Rabies standard virulent strain CVS-24 inoculation; All the other dogs are with 100LD
50The strong malicious mixture inoculation of dog I type and II type adenovirus.Quarantine each dog to inoculating latter 40 days, and itemized record are respectively organized morbidity and the death condition of dog.
Result
1. can't check the specific antibody of anti-hepatitis infectiosa canis virus and rabies virus in the preimmune serum of all tested dogs;
2. after all dog immunity 2～3 weeks, all can detect Anti-adenovirus antibody in its serum, during to 6 weeks, antibody still maintains higher level;
3. after all tested dog immunity 3～4 weeks the time, in its serum, all can detect the rabies neutralizing antibody, the antibody Fluctuation is consistent with adenovirus antibody;
4. tested dog can be resisted the attack of dog I type and the strong poison of II type adenovirus, and survival rate is 95%;
5. tested dog can be resisted the attack of the strong poison of rabies, and survival rate is 90%;
Conclusion
The different recombiant vaccinies of 4 strains all can induce body to produce the specificity neutralizing antibody for hepatitis infectiosa canis virus and rabies virus, and hepatitis infectiosa canis virus and rabies virus infection are had to protective effect.
Embodiment 6: with the immune effect of the dog II type adenovirus live vector recombinant vaccine of the prominent displaying protective antigen of rabies virus of fibre
Laboratory animal
Rabies virus and adenovirus antibody feminine gender 4～5 the monthly age 20 of dogs, be divided at random 2 groups, 10 every group.
Animal immune
Respectively with TCID
50Be 10
6.5Recombiant vaccine CAV2/Fiber-GAP 2 groups of dogs are carried out to immunity.Wherein intramuscular injection is 10, the 1ml/ bar; Oral 10, the 2ml/ bar.
Immune detection
Gather respectively weekly a small amount of venous blood before immunity and after immunity, separation of serum, detect tested rabies virucidin level with FAVN.
Challenge test
After immunity 6 weeks, randomly draw 5 dogs for every group, with 100LD
50Rabies standard virulent strain CVS-24 inoculation; All the other dogs are with 100LD
50The strong malicious mixture inoculation of dog I type and II type adenovirus.Quarantine each dog to inoculating latter 40 days, and itemized record are respectively organized morbidity and the death condition of dog.
Result
1. can't check the specific antibody of anti-hepatitis infectiosa canis virus and rabies virus in the preimmune serum of all tested dogs;
2. after all dog immunity 2～3 weeks, all can detect Anti-adenovirus antibody in its serum, during to 6 weeks, antibody still maintains higher level;
3. after all tested dog immunity 3～4 weeks the time, in its serum, all can detect the rabies neutralizing antibody, the antibody Fluctuation is consistent with adenovirus antibody;
4. tested dog can be resisted the attack of dog I type and the strong poison of II type adenovirus, and survival rate is 95%;
5. tested dog can be resisted the attack of the strong poison of rabies, and survival rate is 90%;
Conclusion
Recombiant vaccine CAV2/Fiber-GAP can induce body to produce the specificity neutralizing antibody for hepatitis infectiosa canis virus and rabies virus, and hepatitis infectiosa canis virus and rabies virus infection are had to protective effect.
Embodiment 7: with the immune effect of the dog II type adenovirus live vector recombinant vaccine of IX albumen displaying protective antigen of rabies virus
Laboratory animal
Rabies virus and adenovirus antibody feminine gender 4～5 the monthly age 20 of dogs, be divided at random 2 groups, 10 every group.
Animal immune
Respectively with TCID
50Be 10
6.5Recombiant vaccine CAV2/IX-G ' 2 groups of dogs are carried out to immunity.Wherein intramuscular injection is 10, the 1ml/ bar; Oral 10, the 2ml/ bar.
Immune detection
Gather respectively weekly a small amount of venous blood before immunity and after immunity, separation of serum, detect tested rabies virucidin level with FAVN.
Challenge test
After immunity 6 weeks, randomly draw 5 dogs for every group, with 100LD
50Rabies standard virulent strain CVS-24 inoculation; All the other dogs are with 100LD
50The strong malicious mixture inoculation of dog I type and II type adenovirus.Quarantine each dog to inoculating latter 40 days, and itemized record are respectively organized morbidity and the death condition of dog.
Result
1. can't check the specific antibody of anti-hepatitis infectiosa canis virus and rabies virus in the preimmune serum of all tested dogs;
2. after all dog immunity 2～3 weeks, all can detect Anti-adenovirus antibody in its serum, during to 6 weeks, antibody still maintains higher level;
3. after all tested dog immunity 3～4 weeks the time, in its serum, all can detect the rabies neutralizing antibody, the antibody Fluctuation is consistent with adenovirus antibody;
4. tested dog can be resisted the attack of dog I type and the strong poison of II type adenovirus, and survival rate is 95%;
5. tested dog can be resisted the attack of the strong poison of rabies, and survival rate is 90%;
Conclusion
Recombiant vaccine CAV2/IX-G ' can induce body to produce the specificity neutralizing antibody for hepatitis infectiosa canis virus and rabies virus, and hepatitis infectiosa canis virus and rabies virus infection are had to protective effect.
Attached: dog II type adenovirus structural protein gene sequence after transformation
1. dog II type adenovirus structural protein hexonmer gene order after transforming
Hexonmer gene order in recombiant vaccine CAV2/HVR1-GAP:
ATGGCAACCCCGTCGATGCTGCCACAATGGTCTTACATGCACATTGCTGGCCAGGACGCCGCCGAATACTTGTCTCCCGCCCTGGTTCAGTTTGCCCAAGCAACCAGTTCTTACTTTAAGTTGGACAACAAGTTCAGAAACCCCACTGTGGCCCCCACCCACGATGTGACCACTGAGAGGTCGCAGCGCTTGCAGCTGCGCTTTGTGCCAGTCATGCAAGAGGATGGCCAGTACACTTACAAAACCCGCTTCCAGCTTGCGGTGGGAGACAACAGGGTGCTGGACATGGCCAGTACTTACTTCGATATCAGGGGTACCCTAGACAGAGGCCCCTCCTTCAAGCCTTACAGCGGCACCGCCTACAATGCCCTCGCCCCCAAGGCCGGGGCTAACAACTGTCTTTTTAATGGACAGGGTGCCAATATTAACACTTTAGCCCAGGTGCCCTCTGCAGGT
ACTGTGGCTGTCACAAACAATACCTACCAGCCAGAGCCCCAGCTGGGCCCTGAAAGCTGGGTCGATGGCAGCCTAGCAGAGCTGGGGGATGCGTCTGGCCGTGCCCTTAAGGCTTCAACCCCGCGCATGCCTTGCTATGGTTCCTATGCTCCCCCCACCAACGAAAATGGAGGTACTGGTCCAGTGGAATCCAGATTTTATAAGGTGACCACCAACAATAACAATGAAGCAGATGCCATGCTATACACTGAAGATGTAAACCTGCAGGCCCCAGACACCCACCTGGTGCACCAAGTGCCAGAGGGTCAGGTTACAGGGGTGCAAGGGCTGGGCCAGCAGGCTGCGCCCAACAGGCCGAACTACATAGGCTTCAGGGACAACTTCATAGGCCTCATGTACTACAATAGTAATGGAAACCTAGGGGTGCTGGCGGGTCAGTCATCTCAGCTCAATGCCGTGGTGGACTTGCAAGACAGAAACACAGAGCTCTCTTACCAGCTGCTGCTGGATGCCCTCACAGACAGGTCCCGCTACTTTTCCATGTGGAACCAGGCTGTAGATAGCTATGACCAGGATGTTAGGATTATTGACAACCATGGCGTGGAAGATGATATGCCCAACTATTGCTACCCACTGAGCGGCATGGGGCCCCTAACAAACATGACCACCATGAAGGTTTTTCAGGCAGAAAATACCAATGTGGGGCCCATTCAAAAGATTGGTTTTGGAAATGTTGAGGCCATGGAAATCAACCTCAATGCCAACCTCTTCAAAAGCTTCCTTTACTCCAATGTGGCCTTATACTTGCCTGATGCCTTTAAATACACACCTGAAAACATTGTGGCCCCTGCCAATGTGAATACCTATGCTTACATGAATGTTAGATTACCCGCCGCCAACCTTATAGATACCTTTGTAAATATTGGCGCCAGATGGTCACCAGATGTAATGGACACTGTTAATCCTTTCAACCACCACAGAAATGCAGGACTCCGCTACCGTTCACAACTGCTTGGCAATGGCCGCTATTGCTCGTTCCATATTCAGGTCCCTCAAAAATTTTTTGCAATCAAAAATCTCCTCCTACTGCCTGGAACGTACACGTACGAGTGGTCTTTCAGAAAGGATGTAAACATGATCCTTCAAAGCAGCTTGGGCAATGACCTCCGAGTGGATGGGGCCACCATCAACATTCAGAGCATCAACCTATATGCAAGCTTTTTCCCAATGGCACACAACACTGCCTCCACTCTGGAAGCCATGCTGCGCAACGATGTAAATGACCAGTCCTTTGCAGACTACCTGTCTTCTGCCAACATGCTTTATCCCATCCCTGCCAACACTACTAACCTGCCAATCTCCATTCCCGCCAGAAACTGGGCGGGATTTAGAGGGTGGAGCTTTACCAGAATTAAGCAACGAGAAACTCCTGCCCTGGGCTCGCCTTATGATCCCTACTTCACTTATTCGGGCAGTATTCCATATCTGGATGCAACTTTTTACCTCAGCCACACCTTTAGAAGAGTTTCCATCATGTTTGACTCTTCCGTGTCTTGGCCTGGCAATGACAGGCTGCTTACCCCCAATGAGTTTGAGATTAAAAGGTATGTAGACGGTGAAGGTTACAATGTGGCCCAGTCCAACATGACAAAAGACTGGTTCATGGTTCAAATGCTAGCCCACTACAACATCGGCTACCAAGGCTACCACCTGCCAGAAAGCTACAAGGACAGAATGTACTCCTTCCTAAGAAACTTTGAGCCCATGTGCAGACAGTTGGTGGACGTGGCCAACTATGCTGCCTACCAGCCGGTTACCGTGGGCCACCAGCATAACAATTCTGGTTATGCTAGCGCCCTTTCGGCCTTTAACCCGCGTGAGGGGCACCCATACCCAGCAAACTGGCCTTACCCACTCATTGGAGCCAATGCAGTACCCACTGTCACCCAGAAAAAGTTCCTCTGCGACAGGTCCCTGTGGCGCATCCCATTCTCCTCCAACTTTATGTCTATGGGAACCCTCACTGACCTGGGCCAAAACCTGCTGTACTCTAACTCCGCCCACGCCCTTGACATGACTTTTGAGGTTGATGCCATGAATGAGCCCACTCTGTTGTACGTTTTGTTTGAAGTGTTCGACGTGGCACGCGTCCATCAGCCCCACCGGGGGGTGATTGAGGTAGTGTACCTCAGAACTCCCTTCTCCGCCGGCAACGCCACCACCTAA
Hexonmer gene order in recombiant vaccine CAV2/HVR2-GAP:
ATGGCAACCCCGTCGATGCTGCCACAATGGTCTTACATGCACATTGCTGGCCAGGACGCCGCCGAATACTTGTCTCCCGCCCTGGTTCAGTTTGCCCAAGCAACCAGTTCTTACTTTAAGTTGGACAACAAGTTCAGAAACCCCACTGTGGCCCCCACCCACGATGTGACCACTGAGAGGTCGCAGCGCTTGCAGCTGCGCTTTGTGCCAGTCATGCAAGAGGATGGCCAGTACACTTACAAAACCCGCTTCCAGCTTGCGGTGGGAGACAACAGGGTGCTGGACATGGCCAGTACTTACTTCGATATCAGGGGTACCCTAGACAGAGGCCCCTCCTTCAAGCCTTACAGCGGCACCGCCTACAATGCCCTCGCCCCCAAGGCCGGGGCTAACAACTGTCTTTTTAATGGACAGGGTGCCAATATTAACACTTTAGCCCAGGTGCCCTCTGCAGGTACTGTG
GCTGTCACAAACAATACCTACCAGCCAGAGCCCCAGCTGGGCCCTGAAAGCTGGGTCGATGGCAGCCTAGCAGAGCTGGGGGATGCGTCTGGCCGTGCCCTTAAGGCTTCAACCCCGCGCATGCCTTGCTATGGTTCCTATGCTCCCCCCACCAACGAAAATGGAGGTACTGGTCCAGTGGAATCCAGATTTTATAAGGTGACCACCAACAATAACAATGAAGCAGATGCCATGCTATACACTGAAGATGTAAACCTGCAGGCCCCAGACACCCACCTGGTGCACCAAGTGCCAGAGGGTCAGGTTACAGGGGTGCAAGGGCTGGGCCAGCAGGCTGCGCCCAACAGGCCGAACTACATAGGCTTCAGGGACAACTTCATAGGCCTCATGTACTACAATAGTAATGGAAACCTAGGGGTGCTGGCGGGTCAGTCATCTCAGCTCAATGCCGTGGTGGACTTGCAAGACAGAAACACAGAGCTCTCTTACCAGCTGCTGCTGGATGCCCTCACAGACAGGTCCCGCTACTTTTCCATGTGGAACCAGGCTGTAGATAGCTATGACCAGGATGTTAGGATTATTGACAACCATGGCGTGGAAGATGATATGCCCAACTATTGCTACCCACTGAGCGGCATGGGGCCCCTAACAAACATGACCACCATGAAGGTTTTTCAGGCAGAAAATACCAATGTGGGGCCCATTCAAAAGATTGGTTTTGGAAATGTTGAGGCCATGGAAATCAACCTCAATGCCAACCTCTTCAAAAGCTTCCTTTACTCCAATGTGGCCTTATACTTGCCTGATGCCTTTAAATACACACCTGAAAACATTGTGGCCCCTGCCAATGTGAATACCTATGCTTACATGAATGTTAGATTACCCGCCGCCAACCTTATAGATACCTTTGTAAATATTGGCGCCAGATGGTCACCAGATGTAATGGACACTGTTAATCCTTTCAACCACCACAGAAATGCAGGACTCCGCTACCGTTCACAACTGCTTGGCAATGGCCGCTATTGCTCGTTCCATATTCAGGTCCCTCAAAAATTTTTTGCAATCAAAAATCTCCTCCTACTGCCTGGAACGTACACGTACGAGTGGTCTTTCAGAAAGGATGTAAACATGATCCTTCAAAGCAGCTTGGGCAATGACCTCCGAGTGGATGGGGCCACCATCAACATTCAGAGCATCAACCTATATGCAAGCTTTTTCCCAATGGCACACAACACTGCCTCCACTCTGGAAGCCATGCTGCGCAACGATGTAAATGACCAGTCCTTTGCAGACTACCTGTCTTCTGCCAACATGCTTTATCCCATCCCTGCCAACACTACTAACCTGCCAATCTCCATTCCCGCCAGAAACTGGGCGGGATTTAGAGGGTGGAGCTTTACCAGAATTAAGCAACGAGAAACTCCTGCCCTGGGCTCGCCTTATGATCCCTACTTCACTTATTCGGGCAGTATTCCATATCTGGATGCAACTTTTTACCTCAGCCACACCTTTAGAAGAGTTTCCATCATGTTTGACTCTTCCGTGTCTTGGCCTGGCAATGACAGGCTGCTTACCCCCAATGAGTTTGAGATTAAAAGGTATGTAGACGGTGAAGGTTACAATGTGGCCCAGTCCAACATGACAAAAGACTGGTTCATGGTTCAAATGCTAGCCCACTACAACATCGGCTACCAAGGCTACCACCTGCCAGAAAGCTACAAGGACAGAATGTACTCCTTCCTAAGAAACTTTGAGCCCATGTGCAGACAGTTGGTGGACGTGGCCAACTATGCTGCCTACCAGCCGGTTACCGTGGGCCACCAGCATAACAATTCTGGTTATGCTAGCGCCCTTTCGGCCTTTAACCCGCGTGAGGGGCACCCATACCCAGCAAACTGGCCTTACCCACTCATTGGAGCCAATGCAGTACCCACTGTCACCCAGAAAAAGTTCCTCTGCGACAGGTCCCTGTGGCGCATCCCATTCTCCTCCAACTTTATGTCTATGGGAACCCTCACTGACCTGGGCCAAAACCTGCTGTACTCTAACTCCGCCCACGCCCTTGACATGACTTTTGAGGTTGATGCCATGAATGAGCCCACTCTGTTGTACGTTTTGTTTGAAGTGTTCGACGTGGCACGCGTCCATCAGCCCCACCGGGGGGTGATTGAGGTAGTGTACCTCAGAACTCCCTTCTCCGCCGGCAACGCCACCACCTAA
Hexonmer gene order in recombiant vaccine CAV2/HVR3-GAP:
ATGGCAACCCCGTCGATGCTGCCACAATGGTCTTACATGCACATTGCTGGCCAGGACGCCGCCGAATACTTGTCTCCCGCCCTGGTTCAGTTTGCCCAAGCAACCAGTTCTTACTTTAAGTTGGACAACAAGTTCAGAAACCCCACTGTGGCCCCCACCCACGATGTGACCACTGAGAGGTCGCAGCGCTTGCAGCTGCGCTTTGTGCCAGTCATGCAAGAGGATGGCCAGTACACTTACAAAACCCGCTTCCAGCTTGCGGTGGGAGACAACAGGGTGCTGGACATGGCCAGTACTTACTTCGATATCAGGGGTACCCTAGACAGAGGCCCCTCCTTCAAGCCTTACAGCGGCACCGCCTACAATGCCCTCGCCCCCAAGGCCGGGGCTAACAACTGTCTTTTTAATGGACAGGGTGCCAATATTAACACTTTAGCCCAGGTGCCCTCTGCAGGTACTGTGGCTGTCACAAACAATACCTACCAGCCAGAGCCCCAGCTGGGCCCTGAAAGCTGGGTCGATGGCAGCCTAGCAGAGCTGGGGGATGCGTCTGGCCGTGCCCTTAAGGCTTCAACCCCGCGCATGCCTTGCTATGGTTCCTATGCTCCCCCCACCAACGAAAATGGAGGT
ACTGGTCCAGTGGAATCCAGATTTTATAAGGTGACCACCAACAATAACAATGAAGCAGATGCCATGCTATACACTGAAGATGTAAACCTGCAGGCCCCAGACACCCACCTGGTGCACCAAGTGCCAGAGGGTCAGGTTACAGGGGTGCAAGGGCTGGGCCAGCAGGCTGCGCCCAACAGGCCGAACTACATAGGCTTCAGGGACAACTTCATAGGCCTCATGTACTACAATAGTAATGGAAACCTAGGGGTGCTGGCGGGTCAGTCATCTCAGCTCAATGCCGTGGTGGACTTGCAAGACAGAAACACAGAGCTCTCTTACCAGCTGCTGCTGGATGCCCTCACAGACAGGTCCCGCTACTTTTCCATGTGGAACCAGGCTGTAGATAGCTATGACCAGGATGTTAGGATTATTGACAACCATGGCGTGGAAGATGATATGCCCAACTATTGCTACCCACTGAGCGGCATGGGGCCCCTAACAAACATGACCACCATGAAGGTTTTTCAGGCAGAAAATACCAATGTGGGGCCCATTCAAAAGATTGGTTTTGGAAATGTTGAGGCCATGGAAATCAACCTCAATGCCAACCTCTTCAAAAGCTTCCTTTACTCCAATGTGGCCTTATACTTGCCTGATGCCTTTAAATACACACCTGAAAACATTGTGGCCCCTGCCAATGTGAATACCTATGCTTACATGAATGTTAGATTACCCGCCGCCAACCTTATAGATACCTTTGTAAATATTGGCGCCAGATGGTCACCAGATGTAATGGACACTGTTAATCCTTTCAACCACCACAGAAATGCAGGACTCCGCTACCGTTCACAACTGCTTGGCAATGGCCGCTATTGCTCGTTCCATATTCAGGTCCCTCAAAAATTTTTTGCAATCAAAAATCTCCTCCTACTGCCTGGAACGTACACGTACGAGTGGTCTTTCAGAAAGGATGTAAACATGATCCTTCAAAGCAGCTTGGGCAATGACCTCCGAGTGGATGGGGCCACCATCAACATTCAGAGCATCAACCTATATGCAAGCTTTTTCCCAATGGCACACAACACTGCCTCCACTCTGGAAGCCATGCTGCGCAACGATGTAAATGACCAGTCCTTTGCAGACTACCTGTCTTCTGCCAACATGCTTTATCCCATCCCTGCCAACACTACTAACCTGCCAATCTCCATTCCCGCCAGAAACTGGGCGGGATTTAGAGGGTGGAGCTTTACCAGAATTAAGCAACGAGAAACTCCTGCCCTGGGCTCGCCTTATGATCCCTACTTCACTTATTCGGGCAGTATTCCATATCTGGATGCAACTTTTTACCTCAGCCACACCTTTAGAAGAGTTTCCATCATGTTTGACTCTTCCGTGTCTTGGCCTGGCAATGACAGGCTGCTTACCCCCAATGAGTTTGAGATTAAAAGGTATGTAGACGGTGAAGGTTACAATGTGGCCCAGTCCAACATGACAAAAGACTGGTTCATGGTTCAAATGCTAGCCCACTACAACATCGGCTACCAAGGCTACCACCTGCCAGAAAGCTACAAGGACAGAATGTACTCCTTCCTAAGAAACTTTGAGCCCATGTGCAGACAGTTGGTGGACGTGGCCAACTATGCTGCCTACCAGCCGGTTACCGTGGGCCACCAGCATAACAATTCTGGTTATGCTAGCGCCCTTTCGGCCTTTAACCCGCGTGAGGGGCACCCATACCCAGCAAACTGGCCTTACCCACTCATTGGAGCCAATGCAGTACCCACTGTCACCCAGAAAAAGTTCCTCTGCGACAGGTCCCTGTGGCGCATCCCATTCTCCTCCAACTTTATGTCTATGGGAACCCTCACTGACCTGGGCCAAAACCTGCTGTACTCTAACTCCGCCCACGCCCTTGACATGACTTTTGAGGTTGATGCCATGAATGAGCCCACTCTGTTGTACGTTTTGTTTGAAGTGTTCGACGTGGCACGCGTCCATCAGCCCCACCGGGGGGTGATTGAGGTAGTGTACCTCAGAACTCCCTTCTCCGCCGGCAACGCCACCACCTAA
Hexonmer gene order in recombiant vaccine CAV2/HVR4-GAP:
ATGGCAACCCCGTCGATGCTGCCACAATGGTCTTACATGCACATTGCTGGCCAGGACGCCGCCGAATACTTGTCTCCCGCCCTGGTTCAGTTTGCCCAAGCAACCAGTTCTTACTTTAAGTTGGACAACAAGTTCAGAAACCCCACTGTGGCCCCCACCCACGATGTGACCACTGAGAGGTCGCAGCGCTTGCAGCTGCGCTTTGTGCCAGTCATGCAAGAGGATGGCCAGTACACTTACAAAACCCGCTTCCAGCTTGCGGTGGGAGACAACAGGGTGCTGGACATGGCCAGTACTTACTTCGATATCAGGGGTACCCTAGACAGAGGCCCCTCCTTCAAGCCTTACAGCGGCACCGCCTACAATGCCCTCGCCCCCAAGGCCGGGGCTAACAACTGTCTTTTTAATGGACAGGGTGCCAATATTAACACTTTAGCCCAGGTGCCCTCTGCAGGTACTGTGGCTGTCACAAACAATACCTACCAGCCAGAGCCCCAGCTGGGCCCTGAAAGCTGGGTCGATGGCAGCCTAGCAGAGCTGGGGGATGCGTCTGGCCGTGCCCTTAAGGCTTCAACCCCGCGCATGCCTTGCTATGGTTCCTATGCTCCCCCCACCAACGAAAATGGAGGTACTGGTCCAGTGGAATCCAGATTTTATAAGGTGACCACCAACAATAACAATGAAGCAGATGCCATGCTATACACTGAAGATGTAAACCTGCAGGCCCCAGACACCCACCTGGTGCACCAAGTGCCAGAGGGTCAGGTTACAGGGGTGCAAGGGCTGGGCCAGCAGGCTGCGCCCAACAGGCCGAACTACATAGGCTTCAGGGACAACTTCATAGGCCTCATGTACTACAATAGTAATGGAAACCTAGGGGTGCTGGCGGGTCAGTCATCTCAGCTCAATGCCGTGGTGGACTTGCAAGACAGAAACACAGAGCTCTCTTACCAGCTGCTGCTGGATGCCCTCACAGACAGGTCCCGCTACTTTTCCATGTGGAACCAGGCTGTAGATAGCTATGACCAGGATGTTAGGATTATTGACAACCATGGCGTGGAAGATGATATGCCCAACTATTGCTACCCACTGAGCGGCATGGGGCCCCTAACAAACATGACCACCATGAAGGTT
TTTCAGGCAGAAAATACCAATGTGGGGCCCATTCAAAAGATTGGTTTTGGAAATGTTGAGGCCATGGAAATCAACCTCAATGCCAACCTCTTCAAAAGCTTCCTTTACTCCAATGTGGCCTTATACTTGCCTGATGCCTTTAAATACACACCTGAAAACATTGTGGCCCCTGCCAATGTGAATACCTATGCTTACATGAATGTTAGATTACCCGCCGCCAACCTTATAGATACCTTTGTAAATATTGGCGCCAGATGGTCACCAGATGTAATGGACACTGTTAATCCTTTCAACCACCACAGAAATGCAGGACTCCGCTACCGTTCACAACTGCTTGGCAATGGCCGCTATTGCTCGTTCCATATTCAGGTCCCTCAAAAATTTTTTGCAATCAAAAATCTCCTCCTACTGCCTGGAACGTACACGTACGAGTGGTCTTTCAGAAAGGATGTAAACATGATCCTTCAAAGCAGCTTGGGCAATGACCTCCGAGTGGATGGGGCCACCATCAACATTCAGAGCATCAACCTATATGCAAGCTTTTTCCCAATGGCACACAACACTGCCTCCACTCTGGAAGCCATGCTGCGCAACGATGTAAATGACCAGTCCTTTGCAGACTACCTGTCTTCTGCCAACATGCTTTATCCCATCCCTGCCAACACTACTAACCTGCCAATCTCCATTCCCGCCAGAAACTGGGCGGGATTTAGAGGGTGGAGCTTTACCAGAATTAAGCAACGAGAAACTCCTGCCCTGGGCTCGCCTTATGATCCCTACTTCACTTATTCGGGCAGTATTCCATATCTGGATGCAACTTTTTACCTCAGCCACACCTTTAGAAGAGTTTCCATCATGTTTGACTCTTCCGTGTCTTGGCCTGGCAATGACAGGCTGCTTACCCCCAATGAGTTTGAGATTAAAAGGTATGTAGACGGTGAAGGTTACAATGTGGCCCAGTCCAACATGACAAAAGACTGGTTCATGGTTCAAATGCTAGCCCACTACAACATCGGCTACCAAGGCTACCACCTGCCAGAAAGCTACAAGGACAGAATGTACTCCTTCCTAAGAAACTTTGAGCCCATGTGCAGACAGTTGGTGGACGTGGCCAACTATGCTGCCTACCAGCCGGTTACCGTGGGCCACCAGCATAACAATTCTGGTTATGCTAGCGCCCTTTCGGCCTTTAACCCGCGTGAGGGGCACCCATACCCAGCAAACTGGCCTTACCCACTCATTGGAGCCAATGCAGTACCCACTGTCACCCAGAAAAAGTTCCTCTGCGACAGGTCCCTGTGGCGCATCCCATTCTCCTCCAACTTTATGTCTATGGGAACCCTCACTGACCTGGGCCAAAACCTGCTGTACTCTAACTCCGCCCACGCCCTTGACATGACTTTTGAGGTTGATGCCATGAATGAGCCCACTCTGTTGTACGTTTTGTTTGAAGTGTTCGACGTGGCACGCGTCCATCAGCCCCACCGGGGGGTGATTGAGGTAGTGTACCTCAGAACTCCCTTCTCCGCCGGCAACGCCACCACCTAA
2. fiber protein gene sequence in recombiant vaccine CAV2/Fiber-GAP
3. IX protein sequence in recombiant vaccine CAV2/IX-G '
<110 > MILITARY VETERINARY INST ACADE
<120 > the dog II type adenovirus live vector recombinant vaccine of displaying protective antigen of rabies virus
<160> 5
<170> PatentIn Version 2.1
<210> 1
<211> 2718
<212> DNA
<213 > dog II type adenovirus (canine adenovirus)
<220>
<221> CDS
<222> (1)...(2718)
<223>
<220>
<221> CDS
<222> (1)...(2718)
<400> 1
ATGGCAACCC CGTCGATGCT GCCACAATGG TCTTACATGC ACATTGCTGG CCAGGACGCC 60
GCCGAATACT TGTCTCCCGC CCTGGTTCAG TTTGCCCAAG CAACCAGTTC TTACTTTAAG 120
TTGGACAACA AGTTCAGAAA CCCCACTGTG GCCCCCACCC ACGATGTGAC CACTGAGAGG 180
TCGCAGCGCT TGCAGCTGCG CTTTGTGCCA GTCATGCAAG AGGATGGCCA GTACACTTAC 240
AAAACCCGCT TCCAGCTTGC GGTGGGAGAC AACAGGGTGC TGGACATGGC CAGTACTTAC 300
TTCGATATCA GGGGTACCCT AGACAGAGGC CCCTCCTTCA AGCCTTACAG CGGCACCGCC 360
TACAATGCCC TCGCCCCCAA GGCCGGGGCT AACAACTGTC TTTTTAATGG ACAGGGTGCC 420
AATATTAACA CTTTAGCCCA GGTGCCCTCT GCAGGTCTCG GATCCTGGAG CCCTATTGAC 480
ATACTAGGTT CAACTGTGGC TGTCACAAAC AATACCTACC AGCCAGAGCC CCAGCTGGGC 520
CCTGAAAGCT GGGTCGATGG CAGCCTAGCA GAGCTGGGGG ATGCGTCTGG CCGTGCCCTT 600
AAGGCTTCAA CCCCGCGCAT GCCTTGCTAT GGTTCCTATG CTCCCCCCAC CAACGAAAAT 660
GGAGGTACTG GTCCAGTGGA ATCCAGATTT TATAAGGTGA CCACCAACAA TAACAATGAA 720
GCAGATGCCA TGCTATACAC TGAAGATGTA AACCTGCAGG CCCCAGACAC CCACCTGGTG 780
CACCAAGTGC CAGAGGGTCA GGTTACAGGG GTGCAAGGGC TGGGCCAGCA GGCTGCGCCC 840
AACAGGCCGA ACTACATAGG CTTCAGGGAC AACTTCATAG GCCTCATGTA CTACAATAGT 900
AATGGAAACC TAGGGGTGCT GGCGGGTCAG TCATCTCAGC TCAATGCCGT GGTGGACTTG 960
CAAGACAGAA ACACAGAGCT CTCTTACCAG CTGCTGCTGG ATGCCCTCAC AGACAGGTCC 1020
CGCTACTTTT CCATGTGGAA CCAGGCTGTA GATAGCTATG ACCAGGATGT TAGGATTATT 1080
GACAACCATG GCGTGGAAGA TGATATGCCC AACTATTGCT ACCCACTGAG CGGCATGGGG 1140
CCCCTAACAA ACATGACCAC CATGAAGGTT TTTCAGGCAG AAAATACCAA TGTGGGGCCC 1200
ATTCAAAAGA TTGGTTTTGG AAATGTTGAG GCCATGGAAA TCAACCTCAA TGCCAACCTC 1260
TTCAAAAGCT TCCTTTACTC CAATGTGGCC TTATACTTGC CTGATGCCTT TAAATACACA 1320
CCTGAAAACA TTGTGGCCCC TGCCAATGTG AATACCTATG CTTACATGAA TGTTAGATTA 1380
CCCGCCGCCA ACCTTATAGA TACCTTTGTA AATATTGGCG CCAGATGGTC ACCAGATGTA 1440
ATGGACACTG TTAATCCTTT CAACCACCAC AGAAATGCAG GACTCCGCTA CCGTTCACAA 1500
CTGCTTGGCA ATGGCCGCTA TTGCTCGTTC CATATTCAGG TCCCTCAAAA ATTTTTTGCA 1560
ATCAAAAATC TCCTCCTACT GCCTGGAACG TACACGTACG AGTGGTCTTT CAGAAAGGAT 1620
GTAAACATGA TCCTTCAAAG CAGCTTGGGC AATGACCTCC GAGTGGATGG GGCCACCATC 1680
AACATTCAGA GCATCAACCT ATATGCAAGC TTTTTCCCAA TGGCACACAA CACTGCCTCC 1740
ACTCTGGAAG CCATGCTGCG CAACGATGTA AATGACCAGT CCTTTGCAGA CTACCTGTCT 1800
TCTGCCAACA TGCTTTATCC CATCCCTGCC AACACTACTA ACCTGCCAAT CTCCATTCCC 1860
GCCAGAAACT GGGCGGGATT TAGAGGGTGG AGCTTTACCA GAATTAAGCA ACGAGAAACT 1920
CCTGCCCTGG GCTCGCCTTA TGATCCCTAC TTCACTTATT CGGGCAGTAT TCCATATCTG 1980
GATGCAACTT TTTACCTCAG CCACACCTTT AGAAGAGTTT CCATCATGTT TGACTCTTCC 2040
GTGTCTTGGC CTGGCAATGA CAGGCTGCTT ACCCCCAATG AGTTTGAGAT TAAAAGGTAT 2100
GTAGACGGTG AAGGTTACAA TGTGGCCCAG TCCAACATGA CAAAAGACTG GTTCATGGTT 2160
CAAATGCTAG CCCACTACAA CATCGGCTAC CAAGGCTACC ACCTGCCAGA AAGCTACAAG 2220
GACAGAATGT ACTCCTTCCT AAGAAACTTT GAGCCCATGT GCAGACAGTT GGTGGACGTG 2280
GCCAACTATG CTGCCTACCA GCCGGTTACC GTGGGCCACC AGCATAACAA TTCTGGTTAT 2340
GCTAGCGCCC TTTCGGCCTT TAACCCGCGT GAGGGGCACC CATACCCAGC AAACTGGCCT 2400
TACCCACTCA TTGGAGCCAA TGCAGTACCC ACTGTCACCC AGAAAAAGTT CCTCTGCGAC 2460
AGGTCCCTGT GGCGCATCCC ATTCTCCTCC AACTTTATGT CTATGGGAAC CCTCACTGAC 2520
CTGGGCCAAA ACCTGCTGTA CTCTAACTCC GCCCACGCCC TTGACATGAC TTTTGAGGTT 2580
GATGCCATGA ATGAGCCCAC TCTGTTGTAC GTTTTGTTTG AAGTGTTCGA CGTGGCACGC 2640
GTCCATCAGC CCCACCGGGG GGTGATTGAG GTAGTGTACC TCAGAACTCC CTTCTCCGCC 2700
GGCAACGCCA CCACCTAA 2718
<210> 2
<211> 2718
<212> DNA
<213 > dog II type adenovirus (canine adenovirus)
<220>
<221> CDS
<222> (1)...(2718)
<223>
<220>
<221> CDS
<222> (1)...(2718)
<400> 2
ATGGCAACCC CGTCGATGCT GCCACAATGG TCTTACATGC ACATTGCTGG CCAGGACGCC 60
GCCGAATACT TGTCTCCCGC CCTGGTTCAG TTTGCCCAAG CAACCAGTTC TTACTTTAAG 120
TTGGACAACA AGTTCAGAAA CCCCACTGTG GCCCCCACCC ACGATGTGAC CACTGAGAGG 180
TCGCAGCGCT TGCAGCTGCG CTTTGTGCCA GTCATGCAAG AGGATGGCCA GTACACTTAC 240
AAAACCCGCT TCCAGCTTGC GGTGGGAGAC AACAGGGTGC TGGACATGGC CAGTACTTAC 300
TTCGATATCA GGGGTACCCT AGACAGAGGC CCCTCCTTCA AGCCTTACAG CGGCACCGCC 360
TACAATGCCC TCGCCCCCAA GGCCGGGGCT AACAACTGTC TTTTTAATGG ACAGGGTGCC 420
AATATTAACA CTTTAGCCCA GGTGCCCTCT GCAGGTACTG TGCTCGGATC CTGGAGCCCT 480
ATTGACATAC TAGGTTCAGC TGTCACAAAC AATACCTACC AGCCAGAGCC CCAGCTGGGC 540
CCTGAAAGCT GGGTCGATGG CAGCCTAGCA GAGCTGGGGG ATGCGTCTGG CCGTGCCCTT 600
AAGGCTTCAA CCCCGCGCAT GCCTTGCTAT GGTTCCTATG CTCCCCCCAC CAACGAAAAT 660
GGAGGTACTG GTCCAGTGGA ATCCAGATTT TATAAGGTGA CCACCAACAA TAACAATGAA 720
GCAGATGCCA TGCTATACAC TGAAGATGTA AACCTGCAGG CCCCAGACAC CCACCTGGTG 780
CACCAAGTGC CAGAGGGTCA GGTTACAGGG GTGCAAGGGC TGGGCCAGCA GGCTGCGCCC 840
AACAGGCCGA ACTACATAGG CTTCAGGGAC AACTTCATAG GCCTCATGTA CTACAATAGT 900
AATGGAAACC TAGGGGTGCT GGCGGGTCAG TCATCTCAGC TCAATGCCGT GGTGGACTTG 960
CAAGACAGAA ACACAGAGCT CTCTTACCAG CTGCTGCTGG ATGCCCTCAC AGACAGGTCC 1020
CGCTACTTTT CCATGTGGAA CCAGGCTGTA GATAGCTATG ACCAGGATGT TAGGATTATT 1080
GACAACCATG GCGTGGAAGA TGATATGCCC AACTATTGCT ACCCACTGAG CGGCATGGGG 1140
CCCCTAACAA ACATGACCAC CATGAAGGTT TTTCAGGCAG AAAATACCAA TGTGGGGCCC 1200
ATTCAAAAGA TTGGTTTTGG AAATGTTGAG GCCATGGAAA TCAACCTCAA TGCCAACCTC 1260
TTCAAAAGCT TCCTTTACTC CAATGTGGCC TTATACTTGC CTGATGCCTT TAAATACACA 1320
CCTGAAAACA TTGTGGCCCC TGCCAATGTG AATACCTATG CTTACATGAA TGTTAGATTA 1380
CCCGCCGCCA ACCTTATAGA TACCTTTGTA AATATTGGCG CCAGATGGTC ACCAGATGTA 1440
ATGGACACTG TTAATCCTTT CAACCACCAC AGAAATGCAG GACTCCGCTA CCGTTCACAA 1500
CTGCTTGGCA ATGGCCGCTA TTGCTCGTTC CATATTCAGG TCCCTCAAAA ATTTTTTGCA 1560
ATCAAAAATC TCCTCCTACT GCCTGGAACG TACACGTACG AGTGGTCTTT CAGAAAGGAT 1620
GTAAACATGA TCCTTCAAAG CAGCTTGGGC AATGACCTCC GAGTGGATGG GGCCACCATC 1680
AACATTCAGA GCATCAACCT ATATGCAAGC TTTTTCCCAA TGGCACACAA CACTGCCTCC 1740
ACTCTGGAAG CCATGCTGCG CAACGATGTA AATGACCAGT CCTTTGCAGA CTACCTGTCT 1800
TCTGCCAACA TGCTTTATCC CATCCCTGCC AACACTACTA ACCTGCCAAT CTCCATTCCC 1860
GCCAGAAACT GGGCGGGATT TAGAGGGTGG AGCTTTACCA GAATTAAGCA ACGAGAAACT 1920
CCTGCCCTGG GCTCGCCTTA TGATCCCTAC TTCACTTATT CGGGCAGTAT TCCATATCTG 1980
GATGCAACTT TTTACCTCAG CCACACCTTT AGAAGAGTTT CCATCATGTT TGACTCTTCC 2040
GTGTCTTGGC CTGGCAATGA CAGGCTGCTT ACCCCCAATG AGTTTGAGAT TAAAAGGTAT 2100
GTAGACGGTG AAGGTTACAA TGTGGCCCAG TCCAACATGA CAAAAGACTG GTTCATGGTT 2160
CAAATGCTAG CCCACTACAA CATCGGCTAC CAAGGCTACC ACCTGCCAGA AAGCTACAAG 2220
GACAGAATGT ACTCCTTCCT AAGAAACTTT GAGCCCATGT GCAGACAGTT GGTGGACGTG 2280
GCCAACTATG CTGCCTACCA GCCGGTTACC GTGGGCCACC AGCATAACAA TTCTGGTTAT 2340
GCTAGCGCCC TTTCGGCCTT TAACCCGCGT GAGGGGCACC CATACCCAGC AAACTGGCCT 2400
TACCCACTCA TTGGAGCCAA TGCAGTACCC ACTGTCACCC AGAAAAAGTT CCTCTGCGAC 2460
AGGTCCCTGT GGCGCATCCC ATTCTCCTCC AACTTTATGT CTATGGGAAC CCTCACTGAC 2520
CTGGGCCAAA ACCTGCTGTA CTCTAACTCC GCCCACGCCC TTGACATGAC TTTTGAGGTT 2580
GATGCCATGA ATGAGCCCAC TCTGTTGTAC GTTTTGTTTG AAGTGTTCGA CGTGGCACGC 2640
GTCCATCAGC CCCACCGGGG GGTGATTGAG GTAGTGTACC TCAGAACTCC CTTCTCCGCC 2700
GGCAACGCCA CCACCTAA 2718
<210> 3
<211> 2718
<212> DNA
<213 > dog II type adenovirus (canine adenovirus)
<220>
<221> CDS
<222> (1)...(2718)
<223>
<220>
<221> CDS
<222> (1)...(2718)
<400> 3
ATGGCAACCC CGTCGATGCT GCCACAATGG TCTTACATGC ACATTGCTGG CCAGGACGCC 60
GCCGAATACT TGTCTCCCGC CCTGGTTCAG TTTGCCCAAG CAACCAGTTC TTACTTTAAG 120
TTGGACAACA AGTTCAGAAA CCCCACTGTG GCCCCCACCC ACGATGTGAC CACTGAGAGG 180
TCGCAGCGCT TGCAGCTGCG CTTTGTGCCA GTCATGCAAG AGGATGGCCA GTACACTTAC 240
AAAACCCGCT TCCAGCTTGC GGTGGGAGAC AACAGGGTGC TGGACATGGC CAGTACTTAC 300
TTCGATATCA GGGGTACCCT AGACAGAGGC CCCTCCTTCA AGCCTTACAG CGGCACCGCC 360
TACAATGCCC TCGCCCCCAA GGCCGGGGCT AACAACTGTC TTTTTAATGG ACAGGGTGCC 420
AATATTAACA CTTTAGCCCA GGTGCCCTCT GCAGGTACTG TGCTCGGATC CTGGAGCCCT 480
ATTGACATAC TAGGTTCAAC TGGTCCAGTG GAATCCAGAT TTTATAAGGT GACCACCAAC 540
AATAACAATG AAGCAGATGC CATGCTATAC ACTGAAGATG TAAACCTGCA GGCCCCAGAC 600
TATGCTCCCC CCACCAACGA AAATGGAGGT CTCGGATCCT GGAGCCCTAT TGACATACTA 660
GGTTCAACTG GTCCAGTGGA ATCCAGATTT TATAAGGTGA CCACCAACAA TAACAATGAA 720
GCAGATGCCA TGCTATACAC TGAAGATGTA AACCTGCAGG CCCCAGACAC CCACCTGGTG 780
CACCAAGTGC CAGAGGGTCA GGTTACAGGG GTGCAAGGGC TGGGCCAGCA GGCTGCGCCC 840
AACAGGCCGA ACTACATAGG CTTCAGGGAC AACTTCATAG GCCTCATGTA CTACAATAGT 900
AATGGAAACC TAGGGGTGCT GGCGGGTCAG TCATCTCAGC TCAATGCCGT GGTGGACTTG 960
CAAGACAGAA ACACAGAGCT CTCTTACCAG CTGCTGCTGG ATGCCCTCAC AGACAGGTCC 1020
CGCTACTTTT CCATGTGGAA CCAGGCTGTA GATAGCTATG ACCAGGATGT TAGGATTATT 1080
GACAACCATG GCGTGGAAGA TGATATGCCC AACTATTGCT ACCCACTGAG CGGCATGGGG 1140
CCCCTAACAA ACATGACCAC CATGAAGGTT TTTCAGGCAG AAAATACCAA TGTGGGGCCC 1200
ATTCAAAAGA TTGGTTTTGG AAATGTTGAG GCCATGGAAA TCAACCTCAA TGCCAACCTC 1260
TTCAAAAGCT TCCTTTACTC CAATGTGGCC TTATACTTGC CTGATGCCTT TAAATACACA 1320
CCTGAAAACA TTGTGGCCCC TGCCAATGTG AATACCTATG CTTACATGAA TGTTAGATTA 1380
CCCGCCGCCA ACCTTATAGA TACCTTTGTA AATATTGGCG CCAGATGGTC ACCAGATGTA 1440
ATGGACACTG TTAATCCTTT CAACCACCAC AGAAATGCAG GACTCCGCTA CCGTTCACAA 1500
CTGCTTGGCA ATGGCCGCTA TTGCTCGTTC CATATTCAGG TCCCTCAAAA ATTTTTTGCA 1560
ATCAAAAATC TCCTCCTACT GCCTGGAACG TACACGTACG AGTGGTCTTT CAGAAAGGAT 1620
GTAAACATGA TCCTTCAAAG CAGCTTGGGC AATGACCTCC GAGTGGATGG GGCCACCATC 1680
AACATTCAGA GCATCAACCT ATATGCAAGC TTTTTCCCAA TGGCACACAA CACTGCCTCC 1740
ACTCTGGAAG CCATGCTGCG CAACGATGTA AATGACCAGT CCTTTGCAGA CTACCTGTCT 1800
TCTGCCAACA TGCTTTATCC CATCCCTGCC AACACTACTA ACCTGCCAAT CTCCATTCCC 1860
GCCAGAAACT GGGCGGGATT TAGAGGGTGG AGCTTTACCA GAATTAAGCA ACGAGAAACT 1920
CCTGCCCTGG GCTCGCCTTA TGATCCCTAC TTCACTTATT CGGGCAGTAT TCCATATCTG 1980
GATGCAACTT TTTACCTCAG CCACACCTTT AGAAGAGTTT CCATCATGTT TGACTCTTCC 2040
GTGTCTTGGC CTGGCAATGA CAGGCTGCTT ACCCCCAATG AGTTTGAGAT TAAAAGGTAT 2100
GTAGACGGTG AAGGTTACAA TGTGGCCCAG TCCAACATGA CAAAAGACTG GTTCATGGTT 2160
CAAATGCTAG CCCACTACAA CATCGGCTAC CAAGGCTACC ACCTGCCAGA AAGCTACAAG 2220
GACAGAATGT ACTCCTTCCT AAGAAACTTT GAGCCCATGT GCAGACAGTT GGTGGACGTG 2280
GCCAACTATG CTGCCTACCA GCCGGTTACC GTGGGCCACC AGCATAACAA TTCTGGTTAT 2340
GCTAGCGCCC TTTCGGCCTT TAACCCGCGT GAGGGGCACC CATACCCAGC AAACTGGCCT 2400
TACCCACTCA TTGGAGCCAA TGCAGTACCC ACTGTCACCC AGAAAAAGTT CCTCTGCGAC 2460
AGGTCCCTGT GGCGCATCCC ATTCTCCTCC AACTTTATGT CTATGGGAAC CCTCACTGAC 2520
CTGGGCCAAA ACCTGCTGTA CTCTAACTCC GCCCACGCCC TTGACATGAC TTTTGAGGTT 2580
GATGCCATGA ATGAGCCCAC TCTGTTGTAC GTTTTGTTTG AAGTGTTCGA CGTGGCACGC 2640
GTCCATCAGC CCCACCGGGG GGTGATTGAG GTAGTGTACC TCAGAACTCC CTTCTCCGCC 2700
GGCAACGCCA CCACCTAA 2718
<210> 4
<211> 2718
<212> DNA
<213 > dog II type adenovirus (canine adenovirus)
<220>
<221> CDS
<222> (1)...(2718)
<223>
<220>
<221> CDS
<222> (1)...(2718)
<400> 4
ATGGCAACCC CGTCGATGCT GCCACAATGG TCTTACATGC ACATTGCTGG CCAGGACGCC 60
GCCGAATACT TGTCTCCCGC CCTGGTTCAG TTTGCCCAAG CAACCAGTTC TTACTTTAAG 120
TTGGACAACA AGTTCAGAAA CCCCACTGTG GCCCCCACCC ACGATGTGAC CACTGAGAGG 180
TCGCAGCGCT TGCAGCTGCG CTTTGTGCCA GTCATGCAAG AGGATGGCCA GTACACTTAC 240
AAAACCCGCT TCCAGCTTGC GGTGGGAGAC AACAGGGTGC TGGACATGGC CAGTACTTAC 300
TTCGATATCA GGGGTACCCT AGACAGAGGC CCCTCCTTCA AGCCTTACAG CGGCACCGCC 360
TACAATGCCC TCGCCCCCAA GGCCGGGGCT AACAACTGTC TTTTTAATGG ACAGGGTGCC 420
AATATTAACA CTTTAGCCCA GGTGCCCTCT GCAGGTACTG TGCTCGGATC CTGGAGCCCT 480
ATTGACATAC TAGGTTCAAC TGGTCCAGTG GAATCCAGAT TTTATAAGGT GACCACCAAC 540
AATAACAATG AAGCAGATGC CATGCTATAC ACTGAAGATG TAAACCTGCA GGCCCCAGAC 600
TATGCTCCCC CCACCAACGA AAATGGAGGT ACTGGTCCAG TGGAATCCAG ATTTTATAAG 660
GTGACCACCA ACAATAACAA TGAAGCAGAT GCCATGCTAT ACACTGAAGA TGTAAACCTG 720
CAGGCCCCAG ACACCCACCT GGTGCACCAA GTGCCAGAGG GTCAGGTTAC AGGGGTGCAA 780
GGGCTGGGCC AGCAGGCTGC GCCCAACAGG CCGAACTACA TAGGCTTCAG GGACAACTTC 840
ATAGGCCTCA TGTACTACAA TAGTAATGGA AACCTAGGGG TGCTGGCGGG TCAGTCATCT 900
CAGCTCAATG CCGTGGTGGA CTTGCAAGAC AGAAACACAG AGCTCTCTTA CCAGCTGCTG 960
CTGGATGCCC TCACAGACAG GTCCCGCTAC TTTTCCATGT GGAACCAGGC TGTAGATAGC 1020
TATGACCAGG ATGTTAGGAT TATTGACAAC CATGGCGTGG AAGATGATAT GCCCAACTAT 1080
TGCTACCCAC TGAGCGGCAT GGGGCCCCTA ACAAACATGA CCACCATGAA GGTTCTCGGA 1140
TCCTGGAGCC CTATTGACAT ACTAGGTTCA TTTCAGGCAG AAAATACCAA TGTGGGGCCC 1200
ATTCAAAAGA TTGGTTTTGG AAATGTTGAG GCCATGGAAA TCAACCTCAA TGCCAACCTC 1260
TTCAAAAGCT TCCTTTACTC CAATGTGGCC TTATACTTGC CTGATGCCTT TAAATACACA 1320
CCTGAAAACA TTGTGGCCCC TGCCAATGTG AATACCTATG CTTACATGAA TGTTAGATTA 1380
CCCGCCGCCA ACCTTATAGA TACCTTTGTA AATATTGGCG CCAGATGGTC ACCAGATGTA 1440
ATGGACACTG TTAATCCTTT CAACCACCAC AGAAATGCAG GACTCCGCTA CCGTTCACAA 1500
CTGCTTGGCA ATGGCCGCTA TTGCTCGTTC CATATTCAGG TCCCTCAAAA ATTTTTTGCA 1560
ATCAAAAATC TCCTCCTACT GCCTGGAACG TACACGTACG AGTGGTCTTT CAGAAAGGAT 1620
GTAAACATGA TCCTTCAAAG CAGCTTGGGC AATGACCTCC GAGTGGATGG GGCCACCATC 1680
AACATTCAGA GCATCAACCT ATATGCAAGC TTTTTCCCAA TGGCACACAA CACTGCCTCC 1740
ACTCTGGAAG CCATGCTGCG CAACGATGTA AATGACCAGT CCTTTGCAGA CTACCTGTCT 1800
TCTGCCAACA TGCTTTATCC CATCCCTGCC AACACTACTA ACCTGCCAAT CTCCATTCCC 1860
GCCAGAAACT GGGCGGGATT TAGAGGGTGG AGCTTTACCA GAATTAAGCA ACGAGAAACT 1920
CCTGCCCTGG GCTCGCCTTA TGATCCCTAC TTCACTTATT CGGGCAGTAT TCCATATCTG 1980
GATGCAACTT TTTACCTCAG CCACACCTTT AGAAGAGTTT CCATCATGTT TGACTCTTCC 2040
GTGTCTTGGC CTGGCAATGA CAGGCTGCTT ACCCCCAATG AGTTTGAGAT TAAAAGGTAT 2100
GTAGACGGTG AAGGTTACAA TGTGGCCCAG TCCAACATGA CAAAAGACTG GTTCATGGTT 2160
CAAATGCTAG CCCACTACAA CATCGGCTAC CAAGGCTACC ACCTGCCAGA AAGCTACAAG 2220
GACAGAATGT ACTCCTTCCT AAGAAACTTT GAGCCCATGT GCAGACAGTT GGTGGACGTG 2280
GCCAACTATG CTGCCTACCA GCCGGTTACC GTGGGCCACC AGCATAACAA TTCTGGTTAT 2340
GCTAGCGCCC TTTCGGCCTT TAACCCGCGT GAGGGGCACC CATACCCAGC AAACTGGCCT 2400
TACCCACTCA TTGGAGCCAA TGCAGTACCC ACTGTCACCC AGAAAAAGTT CCTCTGCGAC 2460
AGGTCCCTGT GGCGCATCCC ATTCTCCTCC AACTTTATGT CTATGGGAAC CCTCACTGAC 2520
CTGGGCCAAA ACCTGCTGTA CTCTAACTCC GCCCACGCCC TTGACATGAC TTTTGAGGTT 2580
GATGCCATGA ATGAGCCCAC TCTGTTGTAC GTTTTGTTTG AAGTGTTCGA CGTGGCACGC 2640
GTCCATCAGC CCCACCGGGG GGTGATTGAG GTAGTGTACC TCAGAACTCC CTTCTCCGCC 2700
GGCAACGCCA CCACCTAA 2718
<210> 5
<211> 1550
<212> DNA
<213 > dog II type adenovirus (canine adenovirus)
<220>
<221> CDS
<222> (1)...(1550)
<223>
<220>
<221> CDS
<222> (1)...(1550)
<400> 5
GTCGACGGTG CCCCCAGCAG AAGTATCGAC TGCATGCTAA TTATTAACAA ACCAAAACCC 60
GGGTGGAGCC CGATCGATAT AGGCGCCTCT CTAAAAGGCG CATGCAAACT CAAGTTATGT 120
GGAGTTCTTG GACTTAGACT TATGGACGGA ACATGGGGGC CCAGTAATGA GACCAAATGG 180
TGTCCTCCCG ATCAGTTGGT TAATCTGCAC GACTTTCGCT CAGACGAAAT TGAGCATCTT 240
GTTGTAGAGG AGGCTAGTGG CGTTGCCACT TACACCCTTA CCTTTAGGTT TTTAAACTTT 300
AACAGACTAA GCGGAGGTAC CCTGTTTAAA ACTGATGTCT TAACCTTTAC CTATGTAGGC 360
GAAAATCAAT AAAACCAGAA AAAAATAAGT TTAAAAGCTT TATTTTTCAT ACACGCGAGC 420
GGTAAGGCTG CCGCCTTCAG GAAAAGTTAC TCTGTAAACA GTTCTTTCAC AACAGCACAA 480
AACATAGGTA TTAGTTAACA GTTCATTTGG GCTATAATAA TATACATTTT CTTGGGTGGC 540
AAAGCAAGGG TCGGTAATCT CAACAAAACC ATCAACTGGA ATGCAAGAAT AGTCCAGCAC 600
GGTGGGTTCA ATCTAAAAAT GAAGAAACGC GTTGAGGTTC ACTAAGCACA GGTTTTGAAT 660
CTGTCGGCAG CGTCCATGCA TCATAGCTTG TCTCAAAGCA GATTGTCTTC TTTCCTCTGC 720
CTTGGAAGTG GTTTGGTGAA GCACTACAGG TGTCTTTTCA ACCTCTTTCA GCACCCGCAC 780
TATTACAGAT CTCACCCACA CAGCACAGTT TTTAAGAGAA CAATAGTTTT GAAGGCTACA 840
AGATTTACAC TTAAGCACCA GCCAGTAATT ATAAGTGCTT TTAAGAACTA CCCCTAGCTC 900
AGGGTTAATG CACCTTTTAA TGGCCTCCAT GCAGGCTTTA TGGACAGTTC TAAAAAAAGA 960
CAGTCTAAAA TAAATGTAGT GAGTGTTTCT AAATATAATA CTCCCCACAT AGTTAATTTC 1020
ATCAGGCCTG CTAGAATTTA CAAACTCTCG GTACCACATA TACTTTTTAT TCATAGCCCC 1080
ACCCTTAATA AAGTCCTCAA TCACTTTCTG AACCACATGC TTGCTAGCCA TGCATTGTAA 1140
AGACAAGCTG TTAGAGCAGT GACAGTGTAC TCGCCACGTT TGAGCCTCTG CCAGGCAGCA 1200
GTGCTTAGTT ACTATCAACT CAATACCCGC ATTGCATGTA AACCCCCCAA AGAGCAGTTT 1260
TTCATGCCTG TGTAGCACAT CATCCCACAA AATAGGAATT TCATAGCATA AAGCAAAGCA 1320
ATTACAATAT TTAGGAACTC TCACCACAGC AGTCACGTGA CATGTTGTCT CAGCAGTGCA 1380
GTTGCCTTCC ATCCTACAAT TATGAACAAA AACTAAACAC TTCTAACAAA GATACAGTGA 1440
CAATCTCCCT TCCTCTAAAA GCATTGTTTA CATTAGGGTG ATTATTAACA ACGTCAGAAA 1500
TTTCTTTAAT TAAAGTGCCT TTAAAATGTG CAAGAGCATC ATCATACTCA 1550
Claims (3)
1. a dog II type adenovirus live vector recombinant vaccine that utilizes IX albumen displaying protective antigen of rabies virus; it is characterized in that: the dog II type adenovirus of take is carrier; gene and the SV40 virus poly A transcription stop signals of rabies virus glucoprotein film outskirt inserted in restriction enzyme A se I site in its IX protein gene, makes IX albumen and glycoprotein film outskirt utilize the same reading frame of transcribing at adenovirus particles outer surface amalgamation and expression.
2. recombiant vaccine according to claim 1, it is characterized in that: utilize in pPOLYII-CAV2 two single restriction enzyme site EcoR I and Cla I to obtain the large fragment containing the IX protein gene, it is cloned into to pBluescriptII KS (+/-) carrier, obtain recombiant plasmid pBS-LIX, in recycling pBS-LIX, two single restriction enzyme site Mlu I of large fragment and EcoR I obtain containing the small fragment of IX protein gene, it is cloned into to the pEGFP-C1 carrier, obtain recombiant plasmid pEGFP-SIX, then, at IX albumen termination codon place, utilize the restriction enzyme site Ase I of himself to insert rabies virus glucoprotein film outskirt gene and SV40 poly A transcription terminator, realize IX albumen and glycoprotein film outskirt amalgamation and expression, finally, according to above-mentioned route, the back clone obtains the full genome of canine recombinant II type adenovirus.
3. recombiant vaccine according to claim 1, it is characterized in that: described rabies virus glucoprotein film outskirt gene order is:
GGCCCAAATTCCCTATTTACACGATACCAGACACACTTGGTCCCTGGAGACCGATCGATATACATCATCTCAGTTGCCCAAACAATTTGGTTGTGGAGGATGAAGGATGCACCAACCTGTCAGGGTTCTCCTACATGGAACTTAAAGTTGGACACATCTCAGCCATAAAGGTGAACGGGTTCACTTGCACAGGCGTTGTGACAGAGGCAGAAACGTACACTAACTTTGTTGGTTATGTCACCACCACGTTCAAAAGAAAGCATTTCCGCCCAACACCAGATGCATGTAGAGCCGCGTACAACTGGAAGATGGCCGGTGACCCCAGATATGAAGAGTCTCTACACAGTCCGTACCCTGACTACCATTGGCTTCGAACCGTAAAAACCACAAAGGAGTCTATCGTTATCATATCTCCAAGTGTGGCAAATTTGGACCCATATGATAACTCCCTTCACTTGAGGGTCTTCCCTAGCGGAAAGTGCTCAGGAATAACGGTGCCTTCTGTCTACTGCTCAACTAACCACGATTACACCGTTTGGATGCCTGAAATCCTGAGACTAGGGACATCTTGTGATATTTTTACTAATAGTAGAGGGAAGAGAGCATCCAAGGGGAGCAAGACCTGTGGCTTTGTAGATGAAAGGGGCCTATATAAGTCTCTAAAAGGCGCATGCAAACTCAAGTTATGTGGAGTTCCTGGACTTAGACTTATGGACGGAACATGGGTCTCGATGCAGACATCAAATGAGACCAAATGGTGTCCTCCCGGTCAGTTGGTTAATCTGCACGACCTTCACTCAGACGAAATTGAGCATCTTGTTGTAGAGGAGTTGGTCAAGAAAAGAGAGGAGTGTCTGGATGCACTAGAGTCCATCATAACCACCAAGTCAGTGAGTTTCAGACGTCTCAGTCATTTAAGAAAACTTGTCCCCGGGTTCGGAAAGGCATATACCATATTCAACAAGACCTTGATGGAGGCTGAAGCTCACTACAAGTCAGTCAGGACTTGGAATGAGATCATCCCCTCAAAAGGGTGTTTGAGAGTTGGAGGGCATGTTTTAATCCCAGAGATGCAATCATCCCTCCTCCAGCAACATATAGAGTTATTGGAATCCTCAGTTATTCCCCTGATGCACCCCCTTGCAGACCCGTTCACAGTTTTCAAGGACGGCGATGAGATTGAGGATTTTGTGGAAGTTCACCTTCCCGATGTGCACGAACAGGTCTCAGGGGTTGACCTGGGTCTCCCGAACTGGGGGAAG 。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102739118A CN102335424B (en) | 2007-08-22 | 2007-08-22 | II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using IX protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102739118A CN102335424B (en) | 2007-08-22 | 2007-08-22 | II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using IX protein |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100559862A Division CN101406698B (en) | 2007-08-22 | 2007-08-22 | Dog type II adenovirus live vector recombinant vaccine for displaying protective antigen of rabies virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102335424A CN102335424A (en) | 2012-02-01 |
| CN102335424B true CN102335424B (en) | 2013-12-04 |
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|---|---|---|---|
| CN2011102739118A Expired - Fee Related CN102335424B (en) | 2007-08-22 | 2007-08-22 | II type adenovirus live vector recombinant vaccine of dog for showing lyssa virus protective antigen by using IX protein |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1147837A (en) * | 1993-10-25 | 1997-04-16 | 坎吉公司 | Recombinant adenoviral vector and methods of use |
| CN1718242A (en) * | 2004-07-07 | 2006-01-11 | 中国人民解放军军事医学科学院军事兽医研究所 | The canine adenovirus type 2 recombiant vaccine of rabies virus sugar/structural protein such as nuclear |
| CN1827172A (en) * | 2005-04-27 | 2006-09-06 | 中国人民解放军军事医学科学院军事兽医研究所 | Pig viral infectious disease gene recombined live vaccine using canine II type adenovirus as carrier and preparation process thereof |
-
2007
- 2007-08-22 CN CN2011102739118A patent/CN102335424B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1147837A (en) * | 1993-10-25 | 1997-04-16 | 坎吉公司 | Recombinant adenoviral vector and methods of use |
| CN1718242A (en) * | 2004-07-07 | 2006-01-11 | 中国人民解放军军事医学科学院军事兽医研究所 | The canine adenovirus type 2 recombiant vaccine of rabies virus sugar/structural protein such as nuclear |
| CN1827172A (en) * | 2005-04-27 | 2006-09-06 | 中国人民解放军军事医学科学院军事兽医研究所 | Pig viral infectious disease gene recombined live vaccine using canine II type adenovirus as carrier and preparation process thereof |
Non-Patent Citations (8)
| Title |
|---|
| Characterization of capsid-modified adenovirus vectors containing heterologous peptides in the fiber knob, protein IX, or hexon;S Kurashi等;《Gene Therapy》;20060928;第14卷(第3期);266-274 * |
| Robert A. Meulenbroek等.Use of adenovirus protein IX (pIX) to display large polypeptides on the virion—generation of fluorescent virus through the incorporation of pIX-GFP.《Molecular Therapy》.2004,第9卷(第4期),617-624. |
| S Kurashi等.Characterization of capsid-modified adenovirus vectors containing heterologous peptides in the fiber knob, protein IX, or hexon.《Gene Therapy》.2006,第14卷(第3期),266-274. |
| Use of adenovirus protein IX (pIX) to display large polypeptides on the virion—generation of fluorescent virus through the incorporation of pIX-GFP;Robert A. Meulenbroek等;《Molecular Therapy》;20040430;第9卷(第4期);617-624 * |
| 王永志.表达狂犬病病毒糖蛋白膜外区重组犬2型腺病毒的构建及其免疫原性研究.《微生物学报》.2005,第45卷(第2期),213-217. |
| 表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定;赵忠鹏等;《微生物学报》;20070404;第47卷(第2期);335-339 * |
| 表达狂犬病病毒糖蛋白膜外区重组犬2型腺病毒的构建及其免疫原性研究;王永志;《微生物学报》;20050430;第45卷(第2期);213-217 * |
| 赵忠鹏等.表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定.《微生物学报》.2007,第47卷(第2期),335-339. |
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| CN102335424A (en) | 2012-02-01 |
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