CN102657864B - A kind of immune adjuvant of foot-and-mouth disease vaccine and its application - Google Patents
A kind of immune adjuvant of foot-and-mouth disease vaccine and its application Download PDFInfo
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Abstract
本发明公开了一种口蹄疫疫苗的免疫佐剂及其应用，属于生物疫苗领域。本发明的一种免疫佐剂为口蹄疫病毒3D蛋白片段。实验证明，表达的3D蛋白片段与抗原配伍后免疫动物，能够加强多表位抗原的免疫原性，产生高水平的保护性中和抗体。体外刺激实验显示，口蹄疫病毒3D蛋白片段单独或与多表位抗原配伍后能够发生淋巴细胞增殖反应，说明本发明的免疫佐剂不仅能够增强免疫反应，并且可用于区分感染和免疫动物，是一种很好的免疫刺激剂和疫苗分子标记。The invention discloses an immune adjuvant for a foot-and-mouth disease vaccine and an application thereof, belonging to the field of biological vaccines. One immune adjuvant of the present invention is the 3D protein fragment of foot-and-mouth disease virus. Experiments have shown that immunizing animals with the expressed 3D protein fragments in combination with antigens can enhance the immunogenicity of multi-epitope antigens and produce high levels of protective neutralizing antibodies. In vitro stimulation experiments show that the 3D protein fragment of foot-and-mouth disease virus can produce lymphocyte proliferation reaction alone or after being compatible with multi-epitope antigens, indicating that the immune adjuvant of the present invention can not only enhance the immune response, but also can be used to distinguish infection and immune animals. A good immunostimulant and vaccine molecular marker.
Description
Technical field
The present invention relates to a kind of vaccine adjuvant and application thereof, particularly a kind of immunological adjuvant for the preparation of foot-and-mouth disease vaccine and application thereof.Belong to the biovaccine field.
Background technology
Foot and mouth disease, as the sustainable health development of great animal epidemic serious threat animal husbandry, also affects animal foodstuff safety and foreign export simultaneously.Once the morbidity loss is great, makes a very bad impression.Inactivated vaccine is brought into play very important effect in controlling the FMD epidemic situation as main prevention and control material, but need to build owing to producing this type of vaccine the high-level bio-safety workshop that prevents that pathogen from escaping, and distinguishes vaccine immunity and natural infected animal.More seriously inactivation of virus not exclusively has the danger that causes that vaccine strain is popular.For this reason, after 1991, EU member country bans use of inactivated vaccine to carry out epidemic prevention and control, and the U.S. also forbids producing and use inactivated vaccine in its this country.Calendar year 2001 the be very popular economic loss that causes and impact of Britain's foot and mouth disease caused the new discussion of the whole world to foot and mouth disease prevention and control strategy.Research and development novel FMD vaccine safely and efficiently become hot issue, and immune efficacy lowly reaches the short technical bottleneck that remains the development of restriction new generation vaccine of immune duration.Therefore, the immune efficacy that how to promote new generation vaccine is also the current subject matter that will solve.Comprise that at present the multiple adjuvant of water-soluble adjuvant (aluminium hydroxide) and oily adjuvant (ISA206) is used to improve the immune effect of vaccine, but be not very good for the effect of multi-epitope micromolecule vaccine.
Adopt and increase epitope for micromolecule vaccines such as epiposition vaccines at present, the many kinds of measures such as t cell epitope and introducing protein carrier improve the immunogenicity of vaccines, strengthen level and the persistent period of immunne response ability, lifting Peripheral Circulation antibody, although obviously improved to a certain extent the immunogenicity of antigen, but do not reached the effect of expectation.Result of study shows recently, and the FMD virus nonstructural protein 3D conservative at FMDV evolution camber can not only infect with the FMDV of all serotypes serum generation immunoreation, and enriched can be by the t cell epitope of different pig cattle MHC molecular alleles identifications.Also find that restructuring 3D albumen can the inducing cell immunne response; the reaction of part protective immune response is provided; be that immune swine symptom of immune animal after strong virus attack obviously alleviates, disease time postpones, but can't detect the FMDV neutrality antibody in immune serum.
Result of study demonstration of the present invention, 3D protein fragments and recombination epitope albumen combined immunization animal not only can be improved FMDV specific antibody titre, and can bring out the cellullar immunologic response reaction.It is worth mentioning that, the 3D n-end of albumen protein fragments of expressing through the present invention has not only been brought into play immunological enhancement, and with FMDV, do not infect serum generation immunoreation, it is a kind of very good immunological adjuvant, not only can strengthen immunoreation after adding vaccine, and be conducive to distinguish the infection and immunity animal, be a kind of good immunostimulant and vaccine molecular marker.
Summary of the invention
One of purpose of the present invention is to provide a kind of foot-and-mouth disease vaccine that can be used for preparing, and strengthens immunoreactive immunological adjuvant;
Two of purpose of the present invention is to provide the application of described immunological adjuvant in preparing foot-and-mouth disease vaccine;
Three of purpose of the present invention is to provide a kind of vaccine that contains immunological adjuvant of the present invention.
The objective of the invention is to reach by the following technical programs:
A kind of foot-and-mouth disease vaccine immunological adjuvant of the present invention, it is characterized in that described immunological adjuvant have following (a) or (b) shown in aminoacid sequence:
(a) aminoacid sequence shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16; Or
(b) protein derivatives that still there is foot and mouth disease virus nonstructural protein 3D function that replacement, disappearance or the insertion by one or more amino acid residues obtains by the aminoacid sequence shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
In a specific embodiment of the present invention, the aminoacid sequence of described immunological adjuvant is as shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
The encode nucleotide sequence of described immunological adjuvant, it is characterized in that thering is following (a), (b) or (c) shown in nucleotide sequence:
(a) nucleotide sequence shown in SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15; Or
(b) nucleotide sequence of the aminoacid sequence shown in coding SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16; Or
(c) coding has the nucleotide sequence of the protein derivatives of foot and mouth disease virus nonstructural protein 3D function, and this protein derivatives is replaced, lacked or insert by one or more amino acid residues of the aminoacid sequence by shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 and obtains.
In a specific embodiment of the present invention, described nucleotide sequence is as shown in SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15.
Further, the present invention also provides a kind of recombinant expression carrier, it is characterized in that containing above-described nucleotide sequence.
Further, the present invention also provides a kind of Host Strains, it is characterized in that containing above-described recombinant expression carrier.
Further, the present invention also provides the application of described immunological adjuvant in preparing foot-and-mouth disease vaccine.And
Described nucleotides sequence is listed in the application prepared in foot-and-mouth disease vaccine.
Finally, the invention provides a kind of foot-and-mouth disease vaccine, it is characterized in that containing immunological adjuvant of the present invention.
The present invention adopts the 3D sequence of Asia 1 type FMD virus JS/China/05 strain as reference primers amplifying target genes fragment, and expressing protein.Experiment showed, immune animal after the 3D protein fragments of expression and antigen compatibility, can strengthen the immunogenicity of multi-epitope antigen, produce high-caliber protectiveness neutralizing antibody.The stimulated in vitro experiment shows, the Protein 3 D of Foot-and-mouth fragment separately or with the multi-epitope antigen compatibility after lymphproliferation response can occur, illustrate that immunological adjuvant of the present invention can strengthen immunoreation, and can be used for distinguishing the infection and immunity animal, is a kind of good immunostimulant and vaccine molecular marker.
The accompanying drawing explanation
Fig. 1 is that 3D albumen different fragments is measured separately or with antigen combined immune serum antibody titer;
Fig. 2 is the lymphocyte proliferation assay result;
Fig. 3 is In vitro culture lymphocyte supernatant IL-4 horizontal detection result after the FMDV inactivation antigen stimulates;
Fig. 4 IFN-γ level that is the In vitro culture lymphocyte after the FMDV inactivation antigen stimulates in culture supernatant;
Fig. 5 is epiposition vaccine immune swine body potency test result.
Specific embodiments
Further describe the present invention below in conjunction with specific embodiment, 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 modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 Protein 3 D of Foot-and-mouth different fragments
1, the bioinformatic analysis of Protein 3 D of Foot-and-mouth gene:
The foot and mouth disease virus nonstructural protein 3D is viral rna polymerase, conservative at seven serotype camber of foot and mouth disease virus, albumen can with seven serotype viral infection serum generation immunoreation.The 3D full length gene is 1410 nucleotide, encode one long be 470 amino acid whose albumen, with the DNAStar biosoftware, 46 of seven serotypes that obtain are represented to strain sequence analysis and GenBank data base comparison, result shows that nucleotide homology is higher than 90%, amino acid identity is higher than 97%, high conservative.This research adopts the 3D sequence of Asia 1 type FMD virus JS/China/05 strain as reference primers amplifying target genes fragment, and expressing protein.
2, gene clone and protein expression thereof, purification
By 3D protein-specific primer amplification 3D protein gene total length and 3 fragments thereof, Protein 3 D of Foot-and-mouth full length gene nucleotide sequence is as shown in SEQ ID No.9, its coded aminoacid sequence is as shown in SEQ ID No.10, amplification 3D protein gene total length primer, positive strand primer is 5-GGATTGATAGTTGACACCAGAGA-3, and the minus strand primer is 5-TGCGTCACCGCACACGGCGTTC-3.The nucleotide sequence of coding N end fragment (meaning with 3D1) is as shown in SEQ ID No.11, its coded aminoacid sequence is as shown in SEQ ID No.12, primer for the N end fragment that increases: positive strand primer 3D-1U/BamHI 5 '-CCCGGATCC GGATTGATAGTTGACACCAG-3 ', minus strand primer 3D-1L/HindIII 5 '-CCCAAGCTTTCA TTTCTCCATGAGCTCTAAGGC-3 '; The nucleotide sequence of coding intermediate segment (meaning with 3D2) is as shown in SEQ ID No.13, its coded aminoacid sequence is as shown in SEQ ID No.14, primer for the intermediate segment that increases: 3D-2U/EcoRI 5 '-CCCGAATTCGCCTTAGAGCTCATGGAGAAA-3 ', 3D-2L/HindIII 5 '-CCCAAGCTTTCAGATGCTTGTTGCGGAACAACCA-3 '; The nucleotide sequence of coding C end fragment (meaning with 3D3) is as shown in SEQ ID No.15, its coded aminoacid sequence is as shown in SEQ ID No.16, primer for the C end fragment that increases: 3D-3U/BamHI 5 '-CCCGGATCCGGTTGTTCCGCAACAAGCATC-3 ', 3D-3L/1HindIII 5 '-CCCAAGCTTTCATGCGTCACCGCAC ACGGCGTT-3 '.By the genes of interest of acquisition insert respectively with corresponding enzyme HindIII and the linearizing expression plasmid pET-30a of BamHI (+) (Novagen) in, build respectively corresponding recombinant expression plasmid p3DN (nucleotide sequence that contains coding N end fragment), p3DM (nucleotide sequence that contains the intermediate segment of encode) and p3DC (nucleotide sequence that contains the C end fragment of encoding).Recombinant expression plasmid transforms BL21 (DE3) (Novagen) after order-checking is identified, select monoclonal inoculation LB culture fluid (kan+) after IPTG abduction delivering and expression-form evaluation, large-scale is expressed recombiant protein, choose the LB culture fluid of single colony inoculation 5ml containing kanamycin from the LAB flat board, in 37 ℃ of incubator 220rpm incubated overnight, overnight culture is added in freshly prepd aseptic LB culture fluid (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 0.4mM under the super-clean bench aseptic condition, the centrifugal 30min results of 2000rpm culture, add protein lysate by 20% of stock culture volume, carry out ultrasonication processing (30min) under condition of ice bath, the centrifugal 20min collecting precipitation of 20000g (4 ℃), abandon supernatant.According to Ni-NTA histidine purification column (Novagen) description purifying protein, purifying protein is analyzed through SDS-PAGE electrophoresis and Western blotting, and the N end fragment (3D1) that Explicit Expression obtains as a result, intermediate segment (3D2), C end fragment (3D3) size all conform to expection.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, illustrate that the recombiant protein of expressing has biological activity.Wherein, the aminoacid sequence of the N end fragment (3D1) that expression obtains is as shown in SEQ ID No.12, the aminoacid sequence of intermediate segment (3D2) is as shown in SEQ ID No.14, and the aminoacid sequence of C end fragment (3D3) is as shown in SEQ ID No.16.
The preparation of embodiment 2 recombinant antigens (EoIgG)
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 the protectiveness neutralizing antibody, has type specificity.The VP 1 Gene of Foot-and-Mouth Disease virus total length is comprised of 639 nucleotide, encodes one and has 213 amino acid whose albumen, and its Main Antigenic concentrates on the aminoacid of 140-160 position and the aminoacid section of 200-213 position.The present invention represents strain (O/China/99 with the DNAStar biosoftware to three strain swine foot-and-mouth disease virus O types of isolated in China, O/Miandian/98, O/ZK/93) carry out sequence analysis, determined that the dominant antigen epi-position is O/China/99, the aminoacid section of the 135-160 position of O/Miandian/98 and O/ZK/93, and determine that take the aminoacid section of 200-213 position of O/China/99 is another one B epitope.
2, gene clone and protein expression thereof, purification
The design of multi-epitope gene and synthetic:
In order to prevent new epi-position occurring when building gene, introduce between adjacent two epi-positions and can guarantee the correct spacer sequence GGSSGG showed 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 and 3 '-hold and to introduce respectively specificity enzyme action site as EcoRI and HindIII, the nucleotide sequence of multi-epitope gene is as shown in SEQ ID No.1, its coded aminoacid sequence is as shown in SEQ ID No.2, the nucleotide sequence of multi-epitope gene entrusts the precious biotech firm in Dalian synthetic.Designed a primer amplified for multi-epitope gene synthetic to be repeated series connection simultaneously, primer is: 3PU-BamHI5-cccggatccAAGTATGACGAGAGCCCCGT-3,3PL-EcoRI5-cccgaattcggtggctctagcggcggtCAAAAGCTGTTTCACAGG CG-3.Synthetic multi-epitope gene and prokaryotic expression carrier are used respectively to EcoRI and HindIII enzyme action, after reclaiming, inserts with corresponding enzyme linearisation pET-30a (+) carrier (Novagen) purification, build recombinant expression plasmid p3FOEN, transform the JM109 competence and carry out positive-selecting, by sequencing, determine positive recombinant.With above-mentioned Auele Specific Primer take recombinant expression plasmid p3FOEN as template through the pcr amplification multi-epitope gene, the genes of interest of amplification is through the BamHI/EcoRI double digestion, after reclaiming, inserts with the linearizing p3FOEN recombiant plasmid of corresponding enzyme purification, build the multi-epitope repetition expression plasmid p3FOEN2 that all epi-positions repeat for 2 times, wherein twice series connection repeats the nucleotide sequence of multi-epitope gene as shown in SEQ ID No.3, its coded aminoacid sequence as shown in SEQ ID No.4.
The pcr amplification of pig IgG weight chain constant area gene (PIgG):
Auele Specific Primer by 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, the genes of interest that the acquisition total length is 987kb, then use the Auele Specific Primer pIgGHindIII5-gct with restriction enzyme site
ggtggctctagcggcggtGGGCCCTCGGTCTTCATC-3, pIgGxhoI5-cc
tcaTTTACCCGGAGTCTTGG A-3 is increased to genes of interest, purification, reclaim, wherein pig IgG CH nucleotide sequence is as shown in SEQ ID No.5, its coded aminoacid sequence is as shown in SEQ ID No.6, with after the HindIII/xhoI double digestion, the recombinant expression plasmid p3FOEN2 that inserts corresponding linearization for enzyme restriction builds recombinant expression plasmid p3FOEN2-PIgG, this plasmid is through enzyme action, after PCR and sequencing are positive ,-20 ℃ save backup, twice series connection repeats multi-epitope gene and is connected complete nucleotide sequence after the gene order connection with pig IgG as shown in SEQ ID No.7.
The expression of recombiant protein and Biological Activity Identification thereof:
Positive recombinant expression plasmid is transformed to BL21 (DE3) (Novagen), select monoclonal inoculation LB culture fluid (Kan+) after IPTG abduction delivering and expression-form evaluation, large-scale is expressed recombiant protein, choose the LB culture fluid of single colony inoculation 5ml containing kanamycin from the LAB flat board, in 37 ℃ of incubator 220rpm incubated overnight, overnight culture is added in freshly prepd aseptic LB culture fluid (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 0.4mM under the super-clean bench aseptic condition, the centrifugal 30min results of 2000rpm culture, add protein lysate by 20% of stock culture volume, carry out ultrasonication processing (30min) under condition of ice bath, the centrifugal 20min collecting precipitation of 20000g (4 ℃), abandon supernatant.According to Ni-NTA histidine purification column (Novagen) description purifying protein, purifying protein is analyzed through SDS-PAGE electrophoresis and Western blotting, recombiant protein 3FOEN2-PIgG size conforms to expection, can infect with FMDV (O type) the anti-pig IgG generation of the rabbit immunoreation of serum and horseradish peroxidase-labeled, illustrate that the recombiant protein of expressing has biological activity, the aminoacid sequence of the recombiant protein of expression is as shown in SEQ ID No.8.
Embodiment 3, vaccine preparation and immune efficacy experiment:
1, the preparation of vaccine
The different fragments of the 3D albumen after the purification that the recombinant antigen after the purification that embodiment 2 prepares and embodiment 1 prepare (3D1 3D2 3D3) is diluted to suitable concentration through the Bio-Rad quantification kit after quantitatively respectively, after the 3D protein fragments of purification configures by 1: 2 (V/V) with recombinant antigen respectively, add isopyknic oily adjuvant ISA206 (France) to be emulsified into bacterin preparation, press the packing of lml/ pipe, wherein contain recombinant antigen 200 μ g, 3D protein fragments 100 μ g.
2, immuning effect test:
Vaccine with preparation, inoculate Cavia porcellus (the 3D protein fragments of 100 μ g antigens+50 μ g) and inoculate this animal pig (the 3D protein fragments of 200 μ g antigens+100 μ g) immunization experiment result by every part 1ml through intramuscular routes through intramuscular routes by every part 0.5ml and show, after the independent immune mouse of 3D albumen different fragments, can't detect special viral antibody, separately immunity or all can induce the FMDV specific antibody with 3D albumen different fragments combined immunization mice of recombinant antigen, and can significantly strengthen antibody titer after 3 fragments of interpolation 3D albumen, result is as shown in Figure 1.The stimulated in vitro experiment shows, except control sample, lymphproliferation response all can occur in all samples after the antigenic stimulus of FMDV inactivated whole virus, result as shown in Figure 2, the In vitro culture lymphocyte is after the FMDV inactivation antigen stimulates, the level of IL-4 and IFN-γ in the detection supernatant, result respectively as shown in Figure 3 and Figure 4.With independent use recombinant antigen, compare, after having added any one fragment in 3 fragments of 3D albumen (3D1 3D2 3D3), each antibody level of serum of organizing vaccine is apparently higher than the antibody horizontal that does not add the 3D protein fragments and only inject epitope antigen, and result as shown in Figure 5.
3, challenge test
According to China's foot-and-mouth disease vaccine immuning effect test standard, immune swine is carried out to protest test; use 1000ID50/2ml homology virus (as O type strain O/China/99) to be attacked the animal of 14 days after booster immunization; continue to observe 10 days; result shows; except typical foot and mouth disease clinical symptoms appears in the PBS control animal (blister appears in heel and asoscope for fever, lip); all immune animals all do not observe any clinical symptoms, obtain 100% protection.The broad-spectrum vaccine that the present invention's development is described has good immune efficacy, and result is as shown in table 1.
Wherein O type strain O/China/99 is published strain in prior art, this strain be documented in that the 01st phase in 2009 " viral journal " is entitled as that foot and mouth disease virus Pan Asia type O/CHINA/99 strain full-length cDNA clone builds and infection identification one literary composition in, the author is Lv Jianliang etc.Now by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences, preserved.
Protest test result after table 1 pig O type wide spectrum polyepitope vaccines immune swine body
Importantly, the phenomenons such as redness, heating do not appear in the immune animal injection site, the inoculation untoward reaction does not appear yet, appetite is normal, the mental status is good, three fragments of the 3D albumen of pointing out us to prepare can not only play the effect of immunostimulant, and are safe to immune animal, harmless.
Claims (8)
1. a foot-and-mouth disease vaccine immunological adjuvant, is characterized in that the aminoacid sequence of described immunological adjuvant is as shown in SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16.
2. the encode nucleotide sequence of immunological adjuvant claimed in claim 1.
3. nucleotide sequence as claimed in claim 2, is characterized in that described nucleotide sequence is as shown in SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15.
4. a recombinant expression carrier, is characterized in that containing the described nucleotide sequence of claim 2 or 3.
5. a Host Strains, is characterized in that containing recombinant expression carrier claimed in claim 4.
6. the application of immunological adjuvant claimed in claim 1 in preparing foot-and-mouth disease vaccine.
7. the described nucleotides sequence of claim 2 or 3 is listed in the application prepared in foot-and-mouth disease vaccine.
8. a foot-and-mouth disease vaccine, is characterized in that containing immunological adjuvant claimed in claim 1.
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| CN110624100B (en) * | 2019-09-06 | 2021-03-16 | 中国农业科学院兰州兽医研究所 | Application of foot-and-mouth disease virus 3D protein as activator of cellular TLR3 pathway |
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| 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 |
| CN101864434A (en) * | 2009-12-21 | 2010-10-20 | 中国农业科学院兰州兽医研究所 | Sheep foot-and-mouth disease virus Asia1 type multi-epitope recombinant vaccine and its preparation method |
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| 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 |
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