CN103897065A - Bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine and preparation method and application thereof - Google Patents
Bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine and a preparation method and an application thereof and belongs to the field of veterinary biological vaccines. The bovini Asia 1/O type foot and mouth disease virus compound multi-epitope recombinant antigen is obtained by coupling dominant epitope of epidemic bovini O and Asia 1 type foot-and-mouth disease virus representative strains connected in series in China with a bovini IgG heavy chain constant region. Immune efficacy experiments verify that the bovini Asia 1/O type foot-and-mouth disease bivalent multi-epitope vaccine prepared by the invention has good immunogenicity, and high level protective antibodies can be generated by the body induced by immune guinea pig or immune bovine. The inoculated vaccine is safe and harmless to immune animals. The vaccine is a novel vaccine which is abroad in prospect, supports matter storage and technical support to prevention and control of bovini Asia 1/O type foot-and-mouth disease in China, and is of great importance.
Description
Technical field
The present invention relates to a kind of hostis pecoris recombinant antigen and its preparation method and application, particularly a kind of ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines and its preparation method and application, the invention belongs to biovaccine for animals 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 food safety with and foreign export.To suffer heavy losses once there is epidemic situation, political fallout is severe.Inactivated vaccine is being brought into play very important effect in prevention and control FMD epidemic situation, but need to build owing to producing this type of vaccine the high-level Biosafety production plant that prevents that pathogenic agent from escaping, and distinguishes vaccine immunity and natural infected animal (DIVA).More seriously inactivation of virus not exclusively has the danger that causes that vaccine strain is popular.In addition, foot and mouth disease virus not only serotype is numerous, and has the heritable variation ability of height, has quasispecies attribute.It is worth mentioning that between each serotype and protect without cross immunity, this attribute specification of cause of disease will be developed efficient vaccine corresponding thereto for each serotype, and the difficulty that this has increased foot and mouth disease prevention and control, has also increased funds expenditure.Therefore, develop efficient, safe multivalence new generation vaccine very urgent for the prevention and control of foot and mouth disease.
Adopt molecular biology, correlation technique development safety, the efficient FMD multivalence new generation vaccines such as protein science and reverse vaccinology have become possibility.Although there has been the multinomial patent about foot and mouth disease new generation vaccine granted, comprise the pig O type foot and mouth disease virus recombiant vaccine of Fudan University in Shanghai professor Zheng Zhaoxin development, the ox Asia1 type foot and mouth disease recombinant multi-epitope vaccine (patent No.: ZL200910259363.6) of this laboratory development, sheep Asia1 type foot and mouth disease recombinant multi-epitope vaccine (patent No.: ZL200910224025.9), swine foot-and-mouth disease virus O type wide spectrum multi-epitope recombinant antigen and application (patent No.: ZL201210130997.3) thereof.These are all the vaccines for single serotype, and cross immunity protection can not be provided.The present invention is in order to realize the object of " pin is anti-how sick " on the one hand, in line with both reducing immune cost, the effect of 2 kinds of cause of diseases of prevention and control simultaneously again, the present invention is taking the foot and mouth disease polyepitope vaccines developed as basis, adopt Protocols in Molecular Biology and advanced design concept, for the Main Antigenic sequence of the hoof-and-mouth disease poison strain of China's cattle O type and 2 main serotypes of Asia1 type, develop ox Asia1/O foot and mouth disease bivalent wide spectrum polyepitope vaccines, to fundamentally solving a current aftosa vaccine limitation for a serotype, on the other hand, the present invention is carrying out further optimization by further further investigation to antigen epitope sequences, improve on the original basis the immunogenicity of epitope, and the antigen between bivalent vaccine does not interfere with each other, further improve the immune protection effectiveness of the bivalent polyepitope vaccines being prepared by it, proposition of the present invention has great importance to the foot and mouth disease of controlling and purify China's ox Asia1 type and 2 serotypes of O type, also there is reference for developing the more foot and mouth disease new generation vaccine of multiple-effect valency.
Summary of the invention
One of object of the present invention is to provide a kind of ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen, and this antigen is compounded with the dominant antigen epi-position of the popular cattle O type of China and the representative strain of Asia1 type foot and mouth disease virus;
Two of object of the present invention is to provide described multi-epitope recombinant antigen at preparation ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines;
Three of object of the present invention is to provide a kind of ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines, contains ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen of the present invention.
In order to reach described object, the present invention has adopted following technical scheme:
The present invention adopts brand-new reverse vaccine operation to learn a skill and strategy, the dominant antigen epi-position of the representative strain of cattle O type popular China and Asia1 type foot and mouth disease virus is optimized and reasonably after series connection, and with ox IgG immunostimulation segment (IgG CH) coupling being optimized, be cloned into prokaryotic expression plasmid as pET-30a(+) the middle recombinant expression plasmid that builds, transform BL21(DE3) express recombinant antigen and adopt after Ni-NAT chromatography column purifying, after Bio-Rad protein quantification test kit is quantitative, find that the recombinant antigen amount obtaining obviously increases after above optimization, by it separately or prepare vaccine with recombined foot-and-mouth disease virus 3D albumen compatibility, animal immune experiment result shows, this vaccine compatibility can stimulate body to produce the protection antibody for the high-titer of 2 serotypes simultaneously, also confirmed that the present invention adopts the antigen between the bivalent vaccine of brand-new design research not interfere with each other, can reach the object of " pin is anti-how sick ", have a good application prospect.
A kind of ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen of the present invention, it is characterized in that described recombinant antigen is after the dominant antigen epi-position of cattle O type popular China and the representative strain of Asia1 type foot and mouth disease virus is connected and the coupling of ox IgG CH obtains, wherein, the aminoacid sequence of the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus is as shown in SEQ ID NO.1, the aminoacid sequence of the dominant antigen epi-position of the representative strain of ox Asia1 type foot and mouth disease virus is as shown in SEQ ID NO.3, the aminoacid sequence of ox IgG CH is as shown in SEQ ID NO.5.
For determining of the aminoacid sequence of the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus, the present invention is by representing strain O/China/99(GenBank accession number:AF506822 with DNAStar biosoftware to the cattle O type foot and mouth disease virus of isolated in China) carry out sequential analysis, determined that dominant antigen epi-position is 140 – 160 amino acid, 200 – 213 amino acid are another one B epitope.Select 21 – 40 amino acid of VP1 protein region, 20 – 34 amino acids of VP4 is t cell epitope simultaneously, and its aminoacid sequence is optimized, the aminoacid sequence of the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus obtaining after optimization is as shown in SEQ ID NO.1.In the present invention, preferred, the aminoacid sequence of described recombinant antigen is as shown in SEQ ID NO.7.
Further, the invention allows for the nucleotide sequence of the described ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen of coding.
In the present invention, preferred, described nucleotide sequence is as shown in SEQ ID NO.8.
The host cell that contains described nucleotide sequence recombinant expression vector and contain this recombinant expression vector is also within protection scope of the present invention.
Further, the present invention proposes the application of described ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen in preparation prevention ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines.And
Described nucleotides sequence is listed in the application in preparation prevention ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines.
A kind of ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines of the present invention, is characterized in that containing the multi-epitope recombinant antigen described in above any one.
Preferably, in described ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines, also contain full length amino acid sequence or its fragment of Protein 3 D of Foot-and-mouth, the full length amino acid sequence of described Protein 3 D of Foot-and-mouth is as shown in SEQ ID NO:10, and Protein 3 D of Foot-and-mouth fragment is selected from least one in the aminoacid sequence as shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
Immune efficacy experiment confirms; the ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines that the present invention prepares has good immunogenicity; no matter be after immune guinea pig or immune cattle, all to induce body to produce high-caliber protectiveness neutralizing antibody; and safe and harmless to immune animal after inoculation; it is a kind of new generation vaccine with bright prospects; to provide reserve supply and technical support to China ox Asia1 and the prevention and control of O type foot and mouth disease, have great importance.
Compared to prior art, the invention has the advantages that:
1, adopt Protocols in Molecular Biology and advanced design concept, for the Main Antigenic sequence of the hoof-and-mouth disease poison strain of China's cattle O type and 2 main serotypes of Asia1 type, develop ox Asia1/O foot and mouth disease bivalent wide spectrum polyepitope vaccines, the object that has realized " pin is anti-how sick ", fundamentally solves a current aftosa vaccine limitation for a serotype; Also greatly reduce immune cost, realized the prevention and control to 2 different serotypes cause of diseases, improved Immune efficiency simultaneously;
2, epitope has been carried out to further optimization, made after rationally connecting, can keep to greatest extent immunogenicity each other from the epitope of 2 different serotypes viruses, thereby provide higher, more fully protection to immune animal.
Brief description of the drawings
Fig. 1 be recombinant multi-epitope antigens Asia1/O/BCIgG separately or with 3D albumen combined immunization cavy after the NAT result for O type foot and mouth disease virus that produces;
Result shows, recombinant antigen all can induce body to produce the protectiveness neutralizing antibody for the higher level of O type foot and mouth disease virus separately or with 3D albumen combined immunization cavy, after booster immunization, serum NAT further raises, and demonstrates after this recombinant antigen immune guinea pig and can induce body to produce the high-caliber specificity neutralizing antibody for O type foot and mouth disease virus;
Fig. 2 be recombinant multi-epitope antigens Asia1/O/BCIgG separately or with 3D albumen combined immunization cavy after the NAT result for Asia1 type foot and mouth disease virus that produces;
Result demonstration, recombinant antigen all can induce body to produce the high-caliber protectiveness neutralizing antibody for Asia1 type foot and mouth disease virus separately or with 3D albumen combined immunization cavy;
Fig. 3 is that recombinant multi-epitope antigens Asia1/O/BCIgG produces the NAT result for O type foot and mouth disease virus separately or with 3D albumen combined immunization Niu Tihou;
Result shows, recombinant antigen separately or with 3D albumen combined immunization ox after all can induce body to produce the high-caliber protectiveness neutralizing antibody for O type foot and mouth disease virus;
Fig. 4 be recombinant multi-epitope antigens Asia1/O/BCIgG separately or the NAT result for Asia1 type foot and mouth disease virus producing with 3D albumen combined immunization Niu Tihou;
Result shows, recombinant antigen separately or with 3D albumen combined immunization ox after all can induce body to produce the high-caliber protectiveness neutralizing antibody for Asia1 type foot and mouth disease virus;
Fig. 5 is two kinds of O type foot and mouth disease polyepitope vaccines immune guinea pig serum NAT comparative results;
Fig. 6 is two kinds of O type foot and mouth disease polyepitope vaccines immune cattle serum NAT comparative results;
Fig. 7 is O type foot and mouth disease univalent vaccine, the Asia1/O type foot and mouth disease bivalent vaccine immune guinea pig serum specificity neutralizing antibody serum titer for O type foot and mouth disease virus;
Fig. 8 is Asia1 type foot and mouth disease univalent vaccine, the Asia1/O type foot and mouth disease bivalent vaccine immune guinea pig serum specificity neutralizing antibody serum titer for Asia1 type foot and mouth disease virus;
Fig. 9 is O type foot and mouth disease univalent vaccine, after Asia1/O type foot and mouth disease bivalent vaccine immune cattle for the NAT of O type foot and mouth disease virus;
Figure 10 is Asia1 type foot and mouth disease univalent vaccine, after Asia1/O type foot and mouth disease bivalent vaccine immune cattle for the NAT of Asia1 type foot and mouth disease virus.
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of 1 N of Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen of embodiment (Asia1/O/BCIgG)
1, the bioinformatic analysis of O type VP 1 Gene of Foot-and-Mouth Disease virus:
Foot-and-mouth disease VP1 is viral dominant antigen, is no matter that natural VP1 albumen or the recombination expression product of separation and purification can induce body to produce protectiveness neutralizing antibody, has type specificity.This research represents strain O/China/99(GenBank accession number:AF506822 with DNAStar biosoftware to the cattle O type foot and mouth disease virus of isolated in China) carry out sequential analysis, determined that dominant antigen epi-position is 140 – 160 amino acid, 200 – 213 amino acid are another one B epitope.Select 21 – 40 amino acid of VP1 protein region, 20 – 34 amino acids of VP4 to be t cell epitope, and its aminoacid sequence is optimized simultaneously.
2, the bioinformatic analysis of Asia1 type foot-and-mouth disease virus gene:
Select Asia1 type foot and mouth disease virus JS/05 strain whole genome sequence (GenBank accession number:EF149009.1) to carry out the design of multi-epitope, choose two immunogenicity B cell epitopes (136-160aa, 198-211aa) on major structural protein gene VP1 as dominant antigen epi-position.
3, the pcr amplification of ox IgG weight chain constant area gene (CIgG):
Comprise the Auele Specific Primer of the whole weight chain constant area gene total length of ox IgG with the amplification of primer5.0 primer software design, positive strand primer: 5 '-AAGACGGCCCCATCGGT-3 ', minus strand primer:
5 '-TTTACCCGGAGTCTTGGA-3 ', obtains the DNA fragmentation that comprises IgG CH goal gene, then uses the Auele Specific Primer Cattle IgG HC Hind III with restriction enzyme site,
5′-gctaagcttgcggtggtggctctGGTGCCCTGAAGAGCG?GCGT-3′；Cattle?IgG?HC?xho1
5 '-gctctcgagtcaTTTACCCGCAGACTTAGAGGTGGAC-3 ' to goal gene increase, purifying, recovery, save backup with 20 DEG C of Hind III/xhol double digestion purifying Hou ， –.The nucleotide sequence of the coding ox IgG CH arriving of amplification is as shown in SEQ ID NO.6, and its corresponding aminoacid sequence is as shown in SEQ ID NO.5.
4, gene clone and protein expression thereof, purifying
The design of 4.1 multi-epitope genes and synthetic:
In order to prevent occurring new epi-position in the time building gene, between adjacent two epi-positions, introduce and can ensure correct spacer sequence GGGS or the GGSSGG showing of each epi-position structure, the series sequence of O type foot-and-mouth disease virus multi-epitope gene is 21 – 40-GGGS-20 – 34-GGSSGG-140 – 160-GGSSGG-200 – 213-GGSSGG-140 – 160-GGS SGG-200 – 213, and introduce respectively specificity restriction enzyme site as EcoR I and Hind III at the 5'-of gene end and 3'-end, the aminoacid sequence of the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus obtaining after optimization is as shown in SEQ ID NO.1, its corresponding nucleotide sequence is as shown in SEQ ID NO.2, the series sequence of Asia1 type foot-and-mouth disease virus multi-epitope gene is: 136-160-GGSSGG-198-211-GGSSGG-136-160-GGSSGG-198-211, introduce respectively specificity restriction enzyme site BamH I and EcoR I at the 5'-of gene end and 3'-end, obtain the aminoacid sequence of dominant antigen epi-position of the representative strain of ox Asia1 type foot and mouth disease virus as shown in SEQ ID NO.3, its corresponding nucleotide sequence is as shown in SEQ ID NO.4.The precious biotechnology (Dalian) of all tandem genes company limited is synthetic.By the synthetic multi-epitope gene directed prokaryotic expression carrier pET-30a(+ that inserts respectively), build recombinant expression plasmid pAsia1/O.The ox IgG weight chain constant area gene that purifying reclaims corresponding enzyme linearizing recombinant expression plasmid pAsia1/O for insertion after Hind III/Xho1 enzyme is cut, build recombinant expression plasmid pAsia1/O/BCIgG, transform JM109 competence and carry out positive-selecting, determine positive recombinant by sequencing.
The expression of 4.2 recombinant proteins and Biological Activity Identification thereof:
Positive recombinant expression plasmid pAsia1/O/BCIgG is transformed to BL21(DE3) (Novagen), select mono-clonal inoculation LB nutrient solution (Kan+) after IPTG abduction delivering and expression-form qualification, the extensive recombinant protein of expressing, choose the LB nutrient solution of single colony inoculation 5ml containing kantlex from LAB flat board, in 37 DEG C of incubator 220rpm incubated overnight, overnight culture is added in freshly prepd aseptic LB nutrient solution (kan+) by 1%, be cultured to OD600 ≈ 0.4～0.6 o'clock in 37 DEG C of incubator 220rpm, under super clean bench aseptic condition, add the IPTG abduction delivering 4～6 hours of 0.4mM, the centrifugal 30min results of 2000rpm culture, add protein lysate by 20% of stock culture volume, under condition of ice bath through ultrasonication processing (30min), the centrifugal 20min collecting precipitation of 20000g (4 DEG C), abandon 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 A sia1/O/BCIgG size conforms to expection, respectively can with the antibody generation immune response of the anti-ox IgG of rabbit of Asia1 type and O type mouth disease virus infection serum and horseradish peroxidase-labeled, illustrate that the recombinant protein of expression has biological activity.
The aminoacid sequence of recombinant protein (antigen) Asia1/O/BCIgG after the purifying obtaining is as shown in SEQ ID NO.7, and the nucleotide sequence of this recombinant protein of encoding is as shown in SEQ ID NO.8.
The preparation of 2 Ns of Asia1/O type foot and mouth disease bivalent polyepitope vaccines of embodiment and immune efficacy experiment
1, vaccine preparation:
The purifying protein Asia1/O/BCIgG that embodiment 1 obtains is diluted to 1mg/ml after Bio-Rad quantification kit is quantitative, press 1:2(concentration ratio with recombinant antigen separately or with the total length 3D albumen (shown in SEQ ID NO:10) of purifying) configure after, add isopyknic oily adjuvant ISA206(France) be emulsified into vaccine preparation.
2, cavy immuning effect test:
With the vaccine of preparation, inoculate cavy (50 μ g antigens or 50 μ g antigen+25 μ g3D albumen) 5 cavys of immunity respectively by every cavy 0.5ml through intramuscular routes, immunization experiment result shows, after first immunisation 28 days, be no matter Asia1/O/BCIgG recombinant antigen separately or with 3D albumen combined immunization cavy all can stimulate body produce higher level for Asia1 type foot and mouth disease virus specificity neutralizing antibody, and for the specific antibody level lower (Fig. 1) of O type foot and mouth disease virus, after booster immunization, neutralizing antibody level obviously raises.Compared with O type neutralizing antibody level, after first immunisation 28 days, specificity NAT for Asia1 type is higher, and after booster immunization, antibody titer further raises, reach the positive criteria (Fig. 2) that OIE specifies, point out the Asia1/O type foot and mouth disease bivalent polyepitope vaccines of this research development to there is good immune effect.
3, ox body immuning effect test
With the vaccine of preparation, by every part 2ml through intramuscular routes immunoprophylaxis ox (500 μ g antigens or 500 μ g antigen+250 μ g3D albumen) respectively 5 of immunity without foot and mouth disease specific antibody and Nonstructural Protein antibody health ox.Result shows, head exempts from latter 28 days, no matter be that recombinant antigen Asia1/O/BCIgG all can stimulate body to produce the specificity neutralizing antibody for Asia1 type and O type foot and mouth disease virus separately or with 3D albumen combined immunization ox, for the neutralizing antibody level higher (Fig. 4) of Asia1 type foot and mouth disease virus, and for the neutralizing antibody level relatively low (Fig. 3) of O type foot and mouth disease virus.But after booster immunization 14 days; no matter be for Asia1 type foot and mouth disease virus (Fig. 4); or the specificity NAT of O type foot and mouth disease virus obviously raise (Fig. 3); all reach OIE specified standards; prompting Asia1/O type foot and mouth disease bivalent polyepitope vaccines can induce ox to produce the protectiveness neutralizing antibody of high-titer, has good immune effect.There is not the phenomenon such as redness, heating in immune animal injection site; there is not inoculating untoward reaction yet; appetite is normal; the mental status is good; ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines prepared by prompting the present invention has good immunogenicity; no matter be that cavy or ox immunity all can induce body to produce high-caliber protection antibody afterwards; and safe and harmless to immune animal after inoculation; it is a kind of new generation vaccine with bright prospects; to provide reserve supply and technical support to China ox Asia1 and the prevention and control of O type foot and mouth disease, have great importance.
Comparative example 1
In order to further illustrate the present invention's technical superiority compared with prior art, the present invention has carried out following contrast experiment:
1, the contrast of the immune efficacy of two kinds of O type foot-and-mouth disease virus multi-epitope vaccines
Experimental group: adopt the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus obtaining after optimization of the present invention, its aminoacid sequence is as shown in SEQ ID NO.1, and its corresponding nucleotide sequence is as shown in SEQ ID NO.2.
Comparative group: adopt the epitope tandem sequence of disclosed O/China/99 in the patent that the patent No. is ZL201210130997.3, the order of connection is as follows:
135–160-GGSSGG-200–213-GGSSGG—135–160-GGSSGG-200–213
The expression of epitope antigen albumen, the preparation of epiposition vaccine and immune efficacy experiment etc. are all identical with embodiment 1 and 2.
Two kinds of O type foot and mouth disease polyepitope vaccines immune guinea pig serum NAT comparative results as shown in Figure 5.Result shows that the NAT (experimental group) of the O type foot-and-mouth disease virus multi-epitope vaccine after improving is better than (comparative group) before improvement.
Two kinds of O type foot and mouth disease polyepitope vaccines immune cattle serum NAT comparative results as shown in Figure 6.Result shows that the NAT (experimental group) that improves rear O type foot and mouth disease polyepitope vaccines is better than the vaccine (comparative group) before improvement, especially after booster immunization, the NAT of the vaccine after improvement will higher than improve before 1 titre of valence of vaccine antibody or more than, obviously strengthened immune efficacy.
2, Asia1/O type foot and mouth disease bivalent vaccine is compared to the immune efficacy evaluation of O type foot and mouth disease univalent vaccine and Asia1 type foot and mouth disease univalent vaccine
Asia1/O type foot and mouth disease bivalent vaccine: adopt the method preparation of the embodiment of the present invention 1 and embodiment 2.The vaccine that preparation contains total length 3D albumen (shown in SEQ ID NO:10) adjuvant simultaneously and the vaccine that does not contain total length 3D albumen (shown in SEQ ID NO:10) adjuvant.
O type foot and mouth disease univalent vaccine: adopt the method preparation of the embodiment of the present invention 1 and embodiment 2, distinguish the Asia1 type foot-and-mouth disease epitope that is not connect.The vaccine that preparation contains total length 3D albumen (shown in SEQ ID NO:10) adjuvant simultaneously and the vaccine that does not contain total length 3D albumen (shown in SEQ ID NO:10) adjuvant.
Asia1 type foot and mouth disease univalent vaccine: adopt the method preparation of the embodiment of the present invention 1 and embodiment 2, difference is not serial-connection O-shaped foot and mouth disease virus antigen epitope.The vaccine that preparation contains total length 3D albumen (shown in SEQ ID NO:10) adjuvant simultaneously and the vaccine that does not contain total length 3D albumen (shown in SEQ ID NO:10) adjuvant.
The expression of epitope recombinant protein, the preparation of epiposition vaccine and immune efficacy experiment etc. are all identical with embodiment 1 and 2.
O type foot and mouth disease univalent vaccine, Asia1/O type foot and mouth disease bivalent vaccine immune guinea pig serum are for the specificity neutralizing antibody serum titer result of O type foot and mouth disease virus as shown in Figure 7; Asia1 type foot and mouth disease univalent vaccine, Asia1/O type foot and mouth disease bivalent vaccine immune guinea pig serum are for the specificity neutralizing antibody serum titer result of Asia1 type foot and mouth disease virus as shown in Figure 8; O type foot and mouth disease univalent vaccine, after Asia1/O type foot and mouth disease bivalent vaccine immune cattle for the NAT result of O type foot and mouth disease virus as shown in Figure 9; Asia1 type foot and mouth disease univalent vaccine, after Asia1/O type foot and mouth disease bivalent vaccine immune cattle for the NAT result of Asia1 type foot and mouth disease virus as shown in figure 10.
Can find out that from the above results Asia1/O type foot and mouth disease bivalent vaccine of the present invention is compared to O type foot and mouth disease univalent vaccine or Asia1 type foot and mouth disease univalent vaccine; can stimulate body to produce the protection antibody for the high-titer of Asia1 type and 2 different serotypes of O type simultaneously; and antigen between bivalent vaccine does not interfere with each other; the object that can reach " pin is anti-how sick ", has a good application prospect.
Claims (10)
1. ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen, it is characterized in that described recombinant antigen is to connect and obtain with ox IgG CH after the dominant antigen epi-position of cattle O type popular China and the representative strain of Asia1 type foot and mouth disease virus is connected, wherein, the aminoacid sequence of the dominant antigen epi-position of the representative strain of cattle O type foot and mouth disease virus is as shown in SEQ ID NO.1, the aminoacid sequence of the dominant antigen epi-position of the representative strain of ox Asia1 type foot and mouth disease virus is as shown in SEQ ID NO.3, the aminoacid sequence of ox IgG CH is as shown in SEQ ID NO.5.
2. ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen as claimed in claim 1, is characterized in that the aminoacid sequence of described recombinant antigen is as shown in SEQ ID NO.7.
3. the nucleotide sequence of the ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen described in coding claim 1 or 2.
4. nucleotide sequence as claimed in claim 3, is characterized in that described nucleotide sequence is as shown in SEQ ID NO.8.
5. a recombinant expression vector, is characterized in that containing the nucleotide sequence described in claim 3 or 4.
6. a host cell, is characterized in that containing recombinant expression vector claimed in claim 5.
7. the application of the ox Asia1/O type foot and mouth disease virus composite multi-epitope recombinant antigen described in claim 1 or 2 in preparation ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines.
8. the nucleotides sequence described in claim 3 or 4 is listed in the application in preparation ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines.
9. an ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines, is characterized in that containing the multi-epitope recombinant antigen described in claim 1 or 2.
10. ox Asia1/O type foot and mouth disease bivalent polyepitope vaccines as claimed in claim 9, it is characterized in that also containing full length amino acid sequence or its fragment of Protein 3 D of Foot-and-mouth, the full length amino acid sequence of described Protein 3 D of Foot-and-mouth is as shown in SEQ ID NO:10, and Protein 3 D of Foot-and-mouth fragment is selected from least one in the aminoacid sequence as shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
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| CN108059685A (en) * | 2018-01-25 | 2018-05-22 | 中国农业科学院兰州兽医研究所 | Swine foot-and-mouth disease virus A type Fc polypeptide vaccines and its preparation method and application |
| CN108273054A (en) * | 2018-01-25 | 2018-07-13 | 中国农业科学院兰州兽医研究所 | Swine foot-and-mouth disease virus is O-shaped, A type Fc polypeptide bivalent vaccines and its preparation method and application |
| CN109232720A (en) * | 2018-09-13 | 2019-01-18 | 中国农业科学院兰州兽医研究所 | A kind of O-shaped virus sIgA antibody ELISA detection kit of aftosa and its application |
| CN110606875A (en) * | 2019-09-20 | 2019-12-24 | 中国农业科学院兰州兽医研究所 | Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine |
| CN117304277A (en) * | 2023-09-26 | 2023-12-29 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus VP4 protein T cell epitope polypeptide and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775399A (en) * | 2010-03-11 | 2010-07-14 | 中国农业科学院兰州兽医研究所 | Asia1 type multi-epitope recombinant vaccine of bovine foot-and-mouth disease viruses and preparation method thereof |
| US20110206718A1 (en) * | 2008-10-24 | 2011-08-25 | Ray Vaccine Limited | Anti-fmd vaccine composition and preparation and use thereof |
| CN102675471A (en) * | 2012-04-28 | 2012-09-19 | 中国农业科学院兰州兽医研究所 | Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof |
| CN103169962A (en) * | 2008-10-24 | 2013-06-26 | 法罗斯疫苗公司 | Anti-foot-and-mouth disease vaccine composition and preparation and application thereof |
| 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 |
-
2014
- 2014-03-13 CN CN201410093058.5A patent/CN103897065B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110206718A1 (en) * | 2008-10-24 | 2011-08-25 | Ray Vaccine Limited | Anti-fmd vaccine composition and preparation and use thereof |
| CN103169962A (en) * | 2008-10-24 | 2013-06-26 | 法罗斯疫苗公司 | Anti-foot-and-mouth disease vaccine composition and preparation and application thereof |
| CN101775399A (en) * | 2010-03-11 | 2010-07-14 | 中国农业科学院兰州兽医研究所 | Asia1 type multi-epitope recombinant vaccine of bovine foot-and-mouth disease viruses and preparation method thereof |
| CN102675471A (en) * | 2012-04-28 | 2012-09-19 | 中国农业科学院兰州兽医研究所 | Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof |
| 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 |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108059685A (en) * | 2018-01-25 | 2018-05-22 | 中国农业科学院兰州兽医研究所 | Swine foot-and-mouth disease virus A type Fc polypeptide vaccines and its preparation method and application |
| CN108273054A (en) * | 2018-01-25 | 2018-07-13 | 中国农业科学院兰州兽医研究所 | Swine foot-and-mouth disease virus is O-shaped, A type Fc polypeptide bivalent vaccines and its preparation method and application |
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| CN110606875A (en) * | 2019-09-20 | 2019-12-24 | 中国农业科学院兰州兽医研究所 | Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine |
| CN117304277A (en) * | 2023-09-26 | 2023-12-29 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus VP4 protein T cell epitope polypeptide and application thereof |
| CN117304277B (en) * | 2023-09-26 | 2024-03-08 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus VP4 protein T cell epitope polypeptide and application thereof |
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