CN102675471A - Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof - Google Patents
Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof Download PDFInfo
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Abstract
The invention discloses a pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof and belongs to the field of biological vaccines. The pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen adopts a strategy of an antigenized antibody, after main antigen epitopes of a plurality of strains of pig foot-and-mouth disease virus O-type are connected in series reasonably, the plurality of strains of pig foot-and-mouth disease virus O-type are coupled with a pig intravenous gamma globulin (IgG) heavy chain constant region to construct the pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen, and after ration through a Bio-Rad protein ration kit, the pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and recombination foot-and-mouth disease virus 3D protein are matched to prepare the vaccines. Animal immunity testing results show that the vaccines can stimulate an organism to generate high-titer protective antibodies when the vaccines are used independently or matched with the recombination foot-and-mouth disease virus 3D protein to be used, an antibody level is higher than a national standard, and good application prospects are achieved.
Description
Technical field
The present invention relates to a kind of Schweineseuche virus recombinant antigen and application thereof, particularly a kind of Schweineseuche virus O type wide spectrum multi-epitope recombinant antigen and application thereof also relate to the vaccine that is obtained by this antigen prepd, belong to the biovaccine field.
Background technology
Foot and mouth disease is as the not only sound development of serious threat livestock industry of great animal epidemic of infecting main economic poultry boar, ox and sheep, and relate to animal derived food safety with and foreign export.Suffer heavy losses in case epidemic situation takes place, make a very bad impression.Inactivated vaccine is being brought into play crucial effect as main prevention and control material in control FMD epidemic situation; But need build the high-level Biosafety production plant that prevents that pathogenic agent from escaping owing to produce this type of vaccine, and need to distinguish vaccine immunity and natural infected animal.Even more serious is that inactivation of virus not exclusively has the vaccine strain of causing popular dangerous.For this reason, EU member country bans use of inactivated vaccine to carry out the epidemic situation prevention and control after 1991, and the U.S. also forbids producing and use inactivated vaccine in its this country.Calendar year 2001, the be very popular financial loss that causes of Britain's foot and mouth disease caused the new discussion of the whole world to foot and mouth disease prevention and control strategy with influence.Researching and developing novel safely and efficiently FMD vaccine becomes hot issue, and immune efficacy lowly reaches the technical bottleneck that the immune duration weak point remains the development of restriction new generation vaccine.Therefore, the immune efficacy that how to promote new generation vaccine also is the current subject matter that will solve.
The fast development of modern immunology, genomics, molecular biology and bioinformatics technique; The sieve of epitope obtains and the immunogenic development novel molecular vaccine that turns out to be is laid a good foundation, and utilizes reverse vaccine research technology development new generation vaccine to become following vaccine Development Trend.The uncertainty of tradition inactivated vaccine Biosafety has been quickened the process of research foot and mouth disease new generation vaccine.But the ability of vaccine assembly system that is the basis with small molecules target antigen and maximization simulating nature infection immunity still is the technical bottleneck of restriction small molecules epiposition vaccine research.At the beginning of the eighties in last century, the investigator finds that foot and mouth disease virus VP1 structural protein can induce body to produce immunne response, and can resist the homology virus attack.Nineteen eighty-two, Bittle etc. have confirmed can produce the foot and mouth disease virus neutralizing antibody behind the synthetic peptide immune animal of the proteic 140-160 amino acid of foot and mouth disease virus VP1 first, have opened the prelude of foot and mouth disease virus small molecules vaccine research.After this, the investigator adopts chemical synthesising peptide or genetic engineering technique research molecular vaccine one after another.Research is that the major antigen epi-position (140-160 and 200-213 aminoacid sequence) with foot and mouth disease virus VP1 structural protein makes up genetic engineering subunit vaccine and dna vaccination and synthetic peptide the most widely; Though every other molecular vaccine can induce small animal model (mouse, cavy) and this animal of part to produce protection antibody, the opposing virus attack watches for animals.But this animal experiment all end in failure (attack malicious protection ratio and do not reach national standard) on a large scale; Mainly be because the small molecules antigen of development can not stimulate body to produce the neutralizing antibody of protection level; Some promptly enables to produce the protection antibody of certain level; But the time length is short, can not satisfy the needs of eqpidemic disease prevention and control.In addition, the Virosome-like capsid protein of development can induce body to produce high protection antibody of tiring, and immune animal can be resisted the attack of homology virus, but this antigenic low yield and the expensive application that limits this vaccine.Recently; People are through increasing t cell epitope and foreign vector albumen to strengthen the immunogenicity that means such as antigen presentation ability, increase molecular weight, the differentiation of promotion B cell maturation improve vaccine; The level and the time length of enhancing immunity responsibility, lifting peripheral blood circulating antibody; Though improved immunogenicity of antigens to a certain extent, repeatedly the immunity back produces anti-foreign vector protein antibodies, has influenced the antigenic immune effect of target on the contrary; In addition, because the t cell epitope that the investigator adopts is not the versatility epi-position, promptly can not be by the main histocompatibility complex of all host animals (MHC-I and II) identification, so can not induce all animals to produce immunne response.In recent years, become the new breakthrough point that epiposition vaccine is studied with the epiposition vaccine that the coupling of epitope small molecules makes up with main immune factor such as IFN-γ, IL-2 etc., but unsatisfactory to the immune effect of this animal.
At present, although patent has been obtained in the Schweineseuche virus O type recombiant vaccine Shen of Fudan University in Shanghai professor Zheng Zhaoxin development, and obtain " one type of new veterinary drug certificate " of Ministry of Agriculture's promulgation, owing to this product of multiple reason is not slowly realized commercialization.Inactivated vaccine is still used in the epidemic prevention of China pig O type foot and mouth disease, and the UBI synthetic peptide vaccine is because immune effect is undesirable, and market outlook are dull, and the patent of having applied for sheep and ox Asia 1 type polyepitope vaccines before the inventor is examined the stage at present.Based on this; The present invention adopts Protocols in Molecular Biology and advanced design concept; Designed and synthesized the major antigen epitope sequences of the Schweineseuche virus O type strain of containing isolated in China; Be the wide spectrum multi-epitope recombinant vaccine, fundamentally solve present epiposition vaccine and the synthetic peptide vaccine immune efficacy is low, the problem that spectrotype is narrow; Maximum possible avoids because the single generation that causes immuning failure and immunologic escape phenomenon of vaccine antigen spectrum has great importance for control and purification China pig O type foot and mouth disease.
Summary of the invention
One of the object of the invention is to provide a kind of Schweineseuche virus O type wide spectrum multi-epitope recombinant antigen, and this antigen has been contained the major antigen epitope sequences of the Schweineseuche virus O type strain of isolated in China;
Two of the object of the invention is to provide the application of said recombinant antigen in preparation Schweineseuche virus O type wide spectrum polyepitope vaccines;
Three of the object of the invention is to provide a kind of Schweineseuche virus O type wide spectrum polyepitope vaccines, and it contains recombinant antigen of the present invention.
In order to reach said purpose, the present invention has adopted following technical scheme:
The present invention represents strain (O/China/99 with the DNAStar biosoftware to three strain Schweineseuche virus O types of isolated in China; O/Miandian/98; O/ZK/93) carry out sequential analysis; Confirmed that the dominant antigen epi-position is O/China/99, the amino acid section of the 135-160 position of O/ZK/93 and O/Miandian/98 virus strain, and confirm that the 200-213 amino acids section with O/China/99 is an another one B epitope.In order to prevent new epi-position when making up gene, to occur; Can guarantee the correct spacer sequence GGSSGG that shows of each epi-position structure through between adjacent two epi-positions, introducing; Thereby make up the tandem sequence that obtains multi-epitope gene; The series sequence of multi-epitope gene is O/China/99 (135-160)-O/Miandian/98 (135-160)-O/ZK/93 (135-160)-O/China/99 (200-213); In order to reach better immune effect, further this tandem sequence is repeated the series connection back and be connected with pig IgG weight chain constant area gene (PIgG), obtain twice series connection repetition multi-epitope gene and be connected the complete nucleotide sequence after gene order connects with pig IgG.
A kind of Schweineseuche virus O type wide spectrum multi-epitope recombinant antigen of the present invention; It is characterized in that described recombinant antigen is after the major antigen epi-position of a plurality of strains of Schweineseuche virus O type is connected; Make up with the coupling of pig IgG CH and to obtain, described recombinant antigen contain following (a) or (b) shown in aminoacid sequence:
(a) aminoacid sequence shown in the SEQ ID NO:8; Or
(b) protein derivatives that still has the epitope function that replacement, disappearance or the insertion of the aminoacid sequence shown in the SEQ ID NO:8 through one or more amino-acid residues is obtained.
In a specific embodiment of the present invention, the aminoacid sequence of described recombinant antigen is shown in SEQ ID NO:8.
The present invention also provides the nucleotide sequence of the said recombinant antigen of encoding, it is characterized in that having following (a) and (b) or (c) shown in nucleotide sequence:
(a) has the nucleotide sequence shown in the SEQ ID NO:7; Or
(b) nucleotide sequence of the aminoacid sequence shown in the coding SEQ ID NO:8; Or
(c) coding has the nucleotide sequence of the protein derivatives of epitope function, and this protein derivatives obtains through one or more amino-acid residue replacements, disappearance or the insertion with the aminoacid sequence shown in the SEQ ID NO:8.
In a specific embodiment of the present invention, described nucleotide sequence is shown in SEQ ID NO:7.
Further, the present invention also provides a kind of recombinant expression vector, it is characterized in that containing above-described nucleotide sequence.
Further, the present invention also provides the host cell that contains described recombinant expression vector.
Further again, the invention provides the application of described recombinant antigen in preparation prevention foot and mouth disease vaccine.And
Described nucleotides sequence is listed in the application in the preparation prevention foot and mouth disease vaccine.
A kind of Schweineseuche virus O type wide spectrum polyepitope vaccines of the present invention is characterized in that containing multi-epitope recombinant antigen of the present invention.
Preferred; It is characterized in that also containing the proteic full length amino acid sequence of foot and mouth disease virus 3D or its fragment; The proteic full length amino acid sequence of described foot and mouth disease virus 3D is shown in SEQ ID NO:10, and foot and mouth disease virus 3D protein fragments is selected from least a in the aminoacid sequence shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
The present invention adopts " antigenized antibody " strategy; After the major antigen epi-position that is about to a plurality of strains of present China popular Schweineseuche virus O type is reasonably connected; With pig IgG immunostimulation segment (IgG CH) coupling; Be cloned into prokaryotic expression plasmid such as pET-30a (+) and make up recombinant expression plasmid, after transforming BL21 (DE3) express recombinant antigen and adopting Ni-NAT chromatography column purifying, after Bio-Rad protein quantification test kit is quantitative, prepare vaccine with recombined foot-and-mouth disease virus 3D albumen compatibility; The animal immune test-results shows; This vaccine compatibility can stimulate body to produce high protection antibody of tiring, and antibody horizontal is higher than national standard, has a good application prospect.
Description of drawings
Fig. 1 is that 3D albumen different fragments and recombinant antigen combined immunization mice serum antibody titer are measured;
Fig. 2 for the reorganization epitope antigen separately or with 3D full-length proteins combined immunization pig body potency test result.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
The preparation of embodiment 1 recombinant antigen (EoIgG)
1, the bioinformatic analysis of O type foot and mouth disease virus VP1 gene:
Foot and mouth disease virus structural protein VP1 is the dominant antigen of virus, no matter is that the natural VP1 albumen or the recombination expression product of separation and purification can both induce body to produce the protectiveness neutralizing antibody, has type specificity.Foot and mouth disease virus VP1 full length gene is made up of 639 Nucleotide, encodes one to have 213 amino acid whose albumen, and its major antigen epi-position concentrates on the amino acid of 140-160 position and the amino acid section of 200-213 position.The present invention represents strain (O/China/99 with the DNAStar biosoftware to three strain Schweineseuche virus O types of isolated in China; O/Miandian/98; O/ZK/93) carry out sequential analysis; Confirmed that the dominant antigen epi-position is the amino acid section of 135-160 position, and confirmed that the amino acid section with the 200-213 position of O/China/99 is an another one B epitope.
2, gene clone and protein expression thereof, purifying
The design of multi-epitope gene and synthetic:
In order to prevent new epi-position when making up gene, to occur; Between adjacent two epi-positions, introduce and to guarantee the correct spacer sequence GGSSGG that shows of each epi-position structure; The series sequence of multi-epitope gene is O/China/99 (135-160)-O/Miandian/98 (135-160)-O/ZK/93 (135-160)-O/China/99 (200-213); And 5 of gene '-end with 3 '-hold and introduce specificity restriction enzyme site such as EcoRI and HindIII respectively; The nucleotide sequence of multi-epitope gene is shown in SEQ ID No.1, and its coded aminoacid sequence is shown in SEQ ID No.2, and the nucleotide sequence of multi-epitope gene entrusts the precious biotech firm in Dalian synthetic.It is synthetic to repeat series connection to have designed a primer amplified to multi-epitope gene simultaneously; Primer is: 3PU-BamHI5-cccggatccAAGTATGACGAGAGCCCCGT-3,3PL-EcoRI5-cccgaattcggtggctctagcggcggtCAAAAGCTGTTTCACAGG CG-3.Synthetic multi-epitope gene and prokaryotic expression carrier are cut with EcoRI and HindIII enzyme respectively; Insert after the purifying and recovering with corresponding enzyme linearizing pET-30a (+) carrier (Novagen); Make up recombinant expression plasmid p3FOEN; Transform the JM109 competence and carry out positive-selecting, confirm positive recombinant through sequencing.Use above-mentioned Auele Specific Primer with recombinant expression plasmid p3FOEN as template through the pcr amplification multi-epitope gene; The goal gene of amplification is through the BamHI/EcoRI double digestion; Insert with the linearizing p3FOEN recombinant plasmid of corresponding enzyme after the purifying and recovering, make up 2 multiple multi-epitopes of all epi-positions and repeat expression plasmid p3FOEN2, wherein the nucleotide sequence of twice series connection repetition multi-epitope gene is shown in SEQID No.3; Its coded aminoacid sequence shown in SEQ ID No.4.
The pcr amplification of pig IgG weight chain constant area gene (PIgG):
Auele Specific Primer with primer5.0 primer software design amplification pig IgG weight chain constant area gene total length; Positive strand primer: 5-AAGACGGCCCCATCGGT-3; Minus strand primer: 5-TTTACCCGGAGTCTTGGA-3; Obtaining total length is the goal gene of 987kb; Then with Auele Specific Primer pIgG HindIII5-gct
ggtggctctagcggcggtGGGCCCTCGGTCTTCATC-3 that has restriction enzyme site; PIgG xhoI5-cc
tcaTTTACCCGGAGTCTTGG A-3 to goal gene increase, purifying, recovery; Wherein pig IgG CH nucleotide sequence is shown in SEQ ID No.5; Its coded aminoacid sequence is shown in SEQ ID No.6; Behind the HindIII/xhoI double digestion; The recombinant expression plasmid p3FOEN2 that inserts corresponding linearization for enzyme restriction makes up recombinant expression plasmid p3FOEN2-PIgG; This plasmid through enzyme cut, PCR and sequencing positive after-20 ℃ of preservations subsequent use, twice series connection repetition multi-epitope gene is connected complete nucleotide sequence after the gene order connection shown in SEQ ID No.7 with pig IgG.
Recombinant Protein Expression and biological activity thereof are identified:
Positive recombinant expression plasmid is transformed BL21 (DE3) (Novagen), select mono-clonal inoculation LB nutrient solution (Kan+) after IPTG abduction delivering and expression-form evaluation, change express recombinant protein on a large scale; Promptly choose the LB nutrient solution that single colony inoculation 5ml contains kantlex from the LAB flat board; In 37 ℃ of incubator 220rpm incubated overnight, overnight culture is added in the freshly prepd aseptic LB nutrient solution (kan+) by 1%, be cultured to OD600 ≈ 0.4～0.6 o'clock in 37 ℃ of incubator 220rpm; The IPTG abduction delivering 4～6 hours that adds down 0.4mM in the super clean bench aseptic condition; The centrifugal 30min results of 2000rpm culture is pressed 20% of stock culture volume and is added protein lysate, carries out ultrasonication under the condition of ice bath and handles (30min); The centrifugal 20min collecting precipitation of 20000g (4 ℃) is abandoned supernatant.According to Ni-NTA Histidine purification column (Novagen) specification sheets purifying protein; Purifying protein is analyzed through SDS-PAGE electrophoresis and Western blotting; Recombinant protein 3FOEN2-PIgG size conforms to expection; Can infect the anti-pig IgG generation of the rabbit immunoreation of serum and horseradish peroxidase-labeled with FMDV (O type), the recombinant protein biologically active of expressing is described, the aminoacid sequence of the recombinant protein of expression is shown in SEQ ID No.8.
The preparation of embodiment 2 foot and mouth disease virus 3D albumen different fragments
1, the bioinformatic analysis of foot and mouth disease virus 3D protein gene:
Foot and mouth disease virus Nonstructural Protein 3D is a viral rna polymerase, and is conservative at seven serotype camber of foot and mouth disease virus, albumen can with seven serotype virus infection serum generation immunoreations.3D full length gene 1410 Nucleotide; Encode one long be 470 amino acid whose albumen; With the DNAStar biosoftware 46 of seven serotypes that obtain are represented strain sequential analysis and the comparison of GenBank DB; The result shows that nucleotide homology is higher than 90%, and amino acid identity is higher than 97%, high conservative.The 3D sequence of this research employing Asia1 type FMD virus JS/China/05 strain is sequences Design primer amplification target gene fragment as a reference, and expressing protein.
2, gene clone and protein expression thereof, purifying
With 3D protein-specific primer amplification 3D protein gene total length and 3 fragments thereof; Foot and mouth disease virus 3D protein gene full length nucleotide sequence is shown in SEQ ID No.9; Its coded aminoacid sequence is shown in SEQ ID No.10; Amplification 3D protein gene total length primer, positive strand primer is 5-GGATTGATAGTTGACACCAGAGA-3, the minus strand primer is 5-TGCGTCACCG CACACGGCGTTC-3.The nucleotide sequence of coding N end fragment (representing with 3D1) is shown in SEQ ID No.11; Its coded aminoacid sequence is shown in SEQ ID No.12; The primer of N end fragment is used to increase: positive strand primer 3D-1U/BamHI5 '-CCCGGATCCGGATTGATAGTTGACACCAG-3 ', minus strand primer 3D-1L/HindIII5 '-CCCAAGCTTTCATTTCTCCATGAGCTCTAAGGC-3 '; The nucleotide sequence of coding intermediate segment (representing with 3D2) is shown in SEQ ID No.13; Its coded aminoacid sequence is shown in SEQ ID No.14; The primer of intermediate segment is used to increase: 3D-2U/EcoRI5 '-CCCG AATTCGCCTTAGAGCTCATGGAGAAA-3 ', 3D-2L/HindIII5 '-CCCAAGCTTTCA GATGCTTGTTGCGGAACAACCA-3 '; The nucleotide sequence of coding C end fragment (representing with 3D3) is shown in SEQ ID No.15; Its coded aminoacid sequence is shown in SEQ ID No.16; The primer of C end fragment is used to increase: 3D-3U/BamHI5 '-CCCGGATCCGGTTGTTCCGCAACAAGCATC-3 ', 3D-3L/1HindIII5 '-CCCAAGCTTTCATGCGTCACCGCAC ACGGCGTT-3 '.With the goal gene that obtains insert respectively with corresponding enzyme HindIII and the linearizing expression plasmid pET-30a of BamHI (+) (Novagen) in, make up corresponding recombinant expression plasmid p3DN (nucleotide sequence that contains coding N end fragment), p3DM (nucleotide sequence that contains the intermediate segment of encoding) and p3DC (nucleotide sequence that contains the C end fragment of encoding) respectively.Recombinant expression plasmid transforms BL21 (DE3) (Novagen) after order-checking is identified, select mono-clonal inoculation LB nutrient solution (kan+) after IPTG abduction delivering and expression-form evaluation, changes express recombinant protein on a large scale; Promptly choose the LB nutrient solution that single colony inoculation 5ml contains kantlex from the LAB flat board; In 37 ℃ of incubator 220rpm incubated overnight, overnight culture is added in the freshly prepd aseptic LB nutrient solution (kan+) by 1%, be cultured to QD600 ≈ 0.4～0.6 o'clock in 37 ℃ of incubator 220rpm; The IPTG abduction delivering 4～6 hours that adds down 0.4mM in the super clean bench aseptic condition; The centrifugal 30min results of 2000rpm culture is pressed 20% of stock culture volume and is added protein lysate, carries out ultrasonication under the condition of ice bath and handles (30min); The centrifugal 20min collecting precipitation of 20000g (4 ℃) is abandoned supernatant.According to Ni-NTA Histidine purification column (Novagen) specification sheets purifying protein; Purifying protein is analyzed through SDS-PAGE electrophoresis and Westem blotting, and N end fragment (3D1), intermediate segment (3D2), C end fragment (3D3) size reached of indicator gauge all conforms to expection as a result.Except 3D albumen n end protein fragments, all the other albumen all can with the infectious serum generation of FMDV immunoreation; All albumen all can with anti-Histidine monoclonal antibody generation immunoreation, the recombinant protein biologically active of expressing is described.Wherein, the aminoacid sequence of expressing the N end fragment (3D1) that obtains is shown in SEQ ID No.12, and the aminoacid sequence of intermediate segment (3D2) is shown in SEQ ID No.14, and the aminoacid sequence of C end fragment (3D3) is shown in SEQ ID No.16.
Embodiment 3, vaccine production and immune efficacy experiment:
1, the preparation of vaccine
3D albumen total length behind the purifying that recombinant antigen behind the purifying that embodiment 1 prepares and embodiment 2 prepare or its different fragments (3D2) after the Bio-Rad quantification kit is quantitative, be diluted to suitable concentration respectively; After the 3D protein fragments of purifying disposes by 1: 2 (V/V) with recombinant antigen respectively; Add isopyknic oily adjuvant ISA206 (France) and be emulsified into vaccine preparation; Press the packing of 1ml/ pipe, wherein contain recombinant antigen 200 μ g, 3D albumen total length or its fragment 100 μ g.
2, immuning effect test:
Vaccine with preparation; Inoculate pig (200 μ g antigens+100 μ g3D albumen total length or its fragments) by every part 1ml through intramuscular routes; The immunization experiment result shows, no matter is that recombinant antigen all can stimulate body to produce high FMDV protection antibody of tiring separately or with 3D albumen combined immunization pig, and adds 3D albumen total length or its 3 fragments and can significantly strengthen antibody titer after arbitrary; Behind the booster immunization 14 days; Serum antibody titer obviously raises, and 85% immune serum antibody titer surpasses 1: 720 (LBP-ELISA), and 70% immune serum antibody titer is higher than the 1:1024 prompting and has good immune effect result such as Fig. 1 and shown in Figure 2.In addition from recombination epitope antigen separately or with 3D full-length proteins (3DC) or its fragment (3D2) can find out the combined immunization pig body potency test result; Immunity for the first time added in back 28 days 3D full-length proteins or its fragment (3D2) vaccine than the independent good immune effect of epitope antigen; But the independent immune effect of epitope antigen is better behind the booster immunization, and the result is as shown in Figure 2.
3, challenge test
According to China's aftosa vaccine immuning effect test standard immune swine is attacked malicious protection test, promptly use 1000ID50/2ml homology virus (like O type strain O/China/99) that 14 days animal behind the booster immunization is attacked, continue to observe 10 days; The result shows; Except typical foot and mouth disease clinical symptom appears in the PBS control animal (bubble appears in heel and asoscope for fever, lip); All immune animals all do not observe any clinical symptom, obtain 100% protection.The wide spectrum vaccine that the present invention's development is described has good immune efficacy, and the result is as shown in table 1.
Wherein O type strain O/China/99 is a disclosed strain in the prior art; This strain has been documented in 2009 the 01st phases " viral journal " and has been entitled as foot and mouth disease virus Pan Asia type O/CHINA/99 strain full-length cDNA molecular cloning and makes up with infectious and identify that the author is Lv Jianliang etc. in the literary composition.Preserve by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences at present.
Attack malicious protection test result behind the table 1 pig O type wide spectrum polyepitope vaccines immune swine body
No matter be that recombinant antigen phenomenons such as redness, heating do not occur separately or with 3D albumen total length or 3D protein fragments combined immunization immune animal injection site; The inoculation untoward reaction also do not occur, appetite is normal, and the mental status is good; The Schweineseuche virus O type polyepitope vaccines of prompting the present invention preparation has good immunogenicity; Can produce high-caliber protection antibody behind the immune swine body, the inoculation back is safe and harmless to immune animal, is a kind of new generation vaccine with bright prospects; To also technical support of reserve supply be provided to the prevention and control of China pig O type foot and mouth disease, have great importance.
Claims (10)
1. Schweineseuche virus O type wide spectrum multi-epitope recombinant antigen; It is characterized in that described recombinant antigen is after the major antigen epi-position of a plurality of strains of Schweineseuche virus O type is connected; Make up with the coupling of pig IgG CH and to obtain, described recombinant antigen contain following (a) or (b) shown in aminoacid sequence:
(a) aminoacid sequence shown in the SEQ ID NO:8; Or
(b) protein derivatives that still has the epitope function that replacement, disappearance or the insertion of the aminoacid sequence shown in the SEQ ID NO:8 through one or more amino-acid residues is obtained.
2. recombinant antigen as claimed in claim 1, the aminoacid sequence that it is characterized in that described recombinant antigen is shown in SEQ ID NO:8.
3. the nucleotide sequence of coding claim 1 described recombinant antigen, it is characterized in that having following (a) and (b) or (c) shown in nucleotide sequence:
(a) has the nucleotide sequence shown in the SEQ ID NO:7; Or
(b) nucleotide sequence of the aminoacid sequence shown in the coding SEQ ID NO:8; Or
(c) coding has the nucleotide sequence of the protein derivatives of epitope function, and this protein derivatives obtains through one or more amino-acid residue replacements, disappearance or the insertion with the aminoacid sequence shown in the SEQ ID NO:8.
4. the nucleotide sequence shown in claim 4 is characterized in that described nucleotide sequence is shown in SEQ ID NO:7.
5. a recombinant expression vector is characterized in that containing claim 3 or 4 described nucleotide sequences.
6. a host cell is characterized in that containing the described recombinant expression vector of claim 5.
7. claim 1 or the 2 described recombinant antigens application in preparation prevention foot and mouth disease vaccine.
8. claim 3 or 4 described nucleotides sequences are listed in the application in the preparation prevention foot and mouth disease vaccine.
9. a Schweineseuche virus O type wide spectrum polyepitope vaccines is characterized in that containing the described multi-epitope recombinant antigen of claim 1 or claim 2.
10. Schweineseuche virus O type wide spectrum polyepitope vaccines as claimed in claim 8; It is characterized in that also containing the proteic full length amino acid sequence of foot and mouth disease virus 3D or its fragment; The proteic full length amino acid sequence of described foot and mouth disease virus 3D is shown in SEQ ID NO:10, and foot and mouth disease virus 3D protein fragments is selected from least a in the aminoacid sequence shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
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| CN104119442A (en) * | 2013-04-24 | 2014-10-29 | 中国农业科学院兰州兽医研究所 | Bovine A-type foot-and-mouth disease multi-epitope vaccine, and preparation method and application thereof |
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| CN108997493A (en) * | 2018-08-15 | 2018-12-14 | 中国农业科学院兰州兽医研究所 | A kind of preparation method of the O-shaped foot and mouth disease virus wide spectrum neutralizing antibody in full ox source |
| CN109851663A (en) * | 2019-03-14 | 2019-06-07 | 天津威特生物医药有限责任公司 | Method for purifying His-tag marked foot-and-mouth disease epitope protein antigen by nickel-combined nano magnetic beads |
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Inventor after: Shao Junjun Inventor after: Chang Huiyun Inventor after: Congguozheng Inventor after: Lin Tong Inventor after: Gao Shandian Inventor after: Independent military administration Inventor before: Chang Huiyun Inventor before: Shao Junjun Inventor before: Congguozheng Inventor before: Lin Tong Inventor before: Gao Shandian Inventor before: Independent military administration |