CN102675471B - Porcine Foot-and-Mouth Disease Virus Type O Broad Spectrum Multi-epitope Recombinant Antigen and Its Application - Google Patents

Abstract

The invention discloses a porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope recombinant antigen and application thereof, belonging to the field of biological vaccines. The present invention adopts the "antigenized antibody" strategy, rationally concatenates the main epitopes of multiple strains of porcine foot-and-mouth disease virus type O, and then couples them with the constant region of the heavy chain of pig IgG to construct a broad-spectrum porcine foot-and-mouth disease virus type O The multi-epitope recombinant antigen, quantified by the Bio-Rad protein quantification kit, is compatible with the recombinant foot-and-mouth disease virus 3D protein to prepare a vaccine. The results of animal immunity tests show that the vaccine can stimulate the body to produce High-titer protective antibody, the antibody level is higher than the national standard, and has a good application prospect.

Description

Porcine Foot-and-Mouth Disease Virus Type O Broad Spectrum Multi-epitope Recombinant Antigen and Its Application

technical field

The invention relates to a porcine foot-and-mouth disease virus recombinant antigen and its application, in particular to a porcine foot-and-mouth disease virus O-type broad-spectrum multi-epitope recombinant antigen and its application, and also relates to a vaccine prepared from the antigen, which belongs to the field of biological vaccines.

Background technique

Foot-and-mouth disease, as a major animal disease that infects pigs, cattle and sheep as the main economic livestock, not only seriously threatens the healthy development of animal husbandry, but also involves the safety of animal-derived food and its foreign trade export. Once an epidemic occurs, the losses will be heavy and the impact will be severe. Inactivated vaccines, as the main prevention and control materials, play a very important role in the control of FMD epidemics. However, the production of such vaccines requires the construction of high-level biosafety production workshops to prevent pathogens from escaping, and it is necessary to distinguish between vaccine immunity and naturally infected animals. What's more serious is that incomplete virus inactivation has the risk of causing the epidemic of vaccine strains. For this reason, EU member states banned the use of inactivated vaccines for epidemic prevention and control after 1991, and the United States also banned the production and use of inactivated vaccines in its own country. The economic loss and impact of the 2001 FMD pandemic in the United Kingdom has sparked new discussions around the world about FMD control strategies. The development of safe and efficient new FMD vaccines has become a hot issue, but low immune efficacy and short duration of immunity are still technical bottlenecks restricting the development of new vaccines. Therefore, how to improve the immune efficacy of new vaccines is also the main problem to be solved at present.

The rapid development of modern immunology, genomics, molecular biology and bioinformatics technology, the screening of antigenic epitopes and the confirmation of immunogenicity have laid the foundation for the development of new molecular vaccines, and the use of reverse vaccine research technology to develop new vaccines It has become the trend of future vaccine development. The uncertainty of the biosafety of traditional inactivated vaccines has accelerated the process of researching new vaccines against foot-and-mouth disease. However, the vaccine assembly system based on small molecule target antigens and the ability to maximize the ability to simulate natural infection immunity are still technical bottlenecks restricting the research of small molecule epitope vaccines. In the early 1980s, researchers discovered that the VP1 structural protein of foot-and-mouth disease virus can induce the body to generate an immune response and resist homologous virus attack. In 1982, Bittle and others confirmed for the first time that the 140-160 amino acid synthetic peptide of the VP1 protein of FMD virus could produce FMD virus neutralizing antibodies after immunizing animals, which opened the prelude to the research of small molecule vaccines of FMD virus. Since then, researchers have used chemically synthesized peptides or genetic engineering techniques to develop molecular vaccines. The most widely studied is the use of the main epitopes (140-160 and 200-213 amino acid sequences) of the VP1 structural protein of the foot-and-mouth disease virus to construct genetically engineered subunit vaccines, DNA vaccines and synthetic peptides. Although all other molecular vaccines can induce (mice, guinea pigs) and some of these animals produce protective antibodies to protect animals against virus attack. However, the large-scale animal experiments all ended in failure (the protection rate of the virus attack did not reach the national standard), mainly because the small molecule antigens developed could not stimulate the body to produce a protective level of neutralizing antibodies, and some even could produce a certain level of protection. Antibodies, but the duration is short, which cannot meet the needs of epidemic prevention and control. In addition, the developed viral particle-like capsid protein can induce the body to produce high-titer protective antibodies, and immunized animals can resist the challenge of homologous viruses, but the low yield and high cost of the antigen limit the application of the vaccine. Recently, people have increased the immunogenicity of vaccines by increasing T cell epitopes and foreign carrier proteins to enhance antigen presentation capabilities, increase molecular weight, and promote B cell maturation and differentiation, enhance immune response, and increase the level of peripheral blood circulating antibodies and duration, although the immunogenicity of the antigen has been improved to a certain extent, the anti-foreign carrier protein antibody produced after multiple immunizations has affected the immune effect of the target antigen; in addition, because the T cell epitope used by the researchers It is not a universal epitope, that is, it cannot be recognized by the major histocompatibility complex (MHC-I and II) of all host animals, so it cannot induce immune responses in all animals. In recent years, epitope vaccines constructed by conjugating major immune factors such as IFN-γ, IL-2, etc. with epitope small molecules have become a new breakthrough in the research of epitope vaccines, but the immune effect on this animal is not ideal.

At present, although the porcine foot-and-mouth disease virus O-type recombinant vaccine developed by Professor Zheng Zhaoxin of Fudan University in Shanghai has applied for a patent and obtained the "Class I New Veterinary Drug Certificate" issued by the Ministry of Agriculture, the product has not been commercialized due to various reasons. In my country, inactivated vaccines are still used for O-type foot-and-mouth disease prevention in pigs. UBI synthetic peptide vaccines have unsatisfactory immune effects, and the market prospect is bleak. The inventor has previously applied for patents for sheep and cattle Asia 1 type multi-epitope vaccines, which are currently under review. Based on this, the present invention adopts molecular biology technology and advanced design concepts to design and synthesize the main epitope sequence covering the O-type strain of porcine foot-and-mouth disease virus isolated in my country, that is, a broad-spectrum multi-epitope recombinant vaccine, from Fundamentally solve the problems of low immune efficacy and narrow antigenic spectrum of current epitope vaccines and synthetic peptide vaccines, and avoid the occurrence of immune failure and immune escape caused by a single vaccine antigenic spectrum, which is of great importance for the control and purification of swine type O foot-and-mouth disease meaning.

Contents of the invention

One of the purposes of the present invention is to provide a broad-spectrum multi-epitope recombinant antigen of porcine foot-and-mouth disease virus type O, which covers the main epitope sequence of porcine foot-and-mouth disease virus type O strain isolated in my country;

The second object of the present invention is to provide the application of the recombinant antigen in the preparation of porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope vaccine;

The third object of the present invention is to provide a porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope vaccine, which contains the recombinant antigen of the present invention.

In order to achieve the stated purpose, the present invention adopts the following technical solutions:

The present invention uses DNAStar biological software to carry out sequence analysis to three strains of porcine foot-and-mouth disease virus O type representative strains (O/China/99, O/Miandian/98, O/ZK/93) that my country isolates, and determined that the dominant antigenic epitope is The 135-160 amino acid segment of O/China/99, O/ZK/93 and O/Miandian/98 virus strains, and the 200-213 amino acid segment of O/China/99 was identified as another B antigen epitope. In order to prevent the emergence of new epitopes when constructing genes, a spacer sequence GGSSGG that can ensure the correct display of each epitope structure is introduced between two adjacent epitopes, thereby constructing a tandem sequence of multi-epitope genes, multi-epitope genes The series sequence is O/China/99(135-160)-O/Miandian/98(135-160)-O/ZK/93(135-160)-O/China/99(200-213), in order to achieve For better immune effect, the tandem sequence is further connected in series with the porcine IgG heavy chain constant region gene (PIgG), and the complete nuclear sequence after the two tandem repeats of the multi-epitope gene and the pig IgG connection gene sequence is obtained. nucleotide sequence.

A kind of porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope recombinant antigen of the present invention is characterized in that described recombinant antigen is after the main antigenic epitope of multiple strains of porcine foot-and-mouth disease virus O type is connected in series, and pig IgG heavy Chain constant region coupling construction, the recombinant antigen contains the amino acid sequence shown in (a) or (b):

(a) the amino acid sequence shown in SEQ ID NO: 8; or

(b) A protein derivative still having an antigenic epitope function obtained by replacing, deleting or inserting the amino acid sequence shown in SEQ ID NO: 8 by one or more amino acid residues.

In a specific embodiment of the present invention, the amino acid sequence of the recombinant antigen is shown in SEQ ID NO:8.

The present invention also provides the nucleotide sequence encoding the recombinant antigen, which is characterized by having the nucleotide sequence shown in (a), (b) or (c) below:

(a) has the nucleotide sequence shown in SEQ ID NO: 7; or

(b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8; or

(c) A nucleotide sequence encoding a protein derivative having an antigenic epitope function, which is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 8 .

In a specific embodiment of the present invention, the nucleotide sequence is shown in SEQ ID NO:7.

Furthermore, the present invention also provides a recombinant expression vector, which is characterized by containing the above-mentioned nucleotide sequence.

Furthermore, the present invention also provides host cells containing the recombinant expression vector.

Still further, the present invention provides the application of the recombinant antigen in the preparation of a vaccine for preventing porcine foot-and-mouth disease. as well as

The application of the nucleotide sequence in preparing a vaccine for preventing porcine foot-and-mouth disease.

The porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope vaccine of the present invention is characterized in that it contains the multi-epitope recombinant antigen of the present invention.

More preferably, it is characterized in that also containing the full-length amino acid sequence of foot-and-mouth disease virus 3D protein or its fragment, the full-length amino acid sequence of described foot-and-mouth disease virus 3D protein is as shown in SEQ ID NO: 10, and foot-and-mouth disease virus 3D protein fragment is selected from such as At least one of the amino acid sequences shown in SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16.

The present invention adopts the "antigenized antibody" strategy, that is, the main antigenic epitopes of multiple strains of porcine foot-and-mouth disease virus O type that are currently popular in China are rationally connected in series, and then coupled with porcine IgG immunostimulatory fragments (IgG heavy chain constant regions) Combined, cloned into a prokaryotic expression plasmid such as pET-30a(+) to construct a recombinant expression plasmid, transformed BL21(DE3) to express recombinant antigen and purified by Ni-NAT chromatography column, quantified by Bio-Rad protein quantification kit and combined with recombinant The 3D protein of foot-and-mouth disease virus is compatible to prepare a vaccine. The results of animal immunity tests show that the vaccine compatibility can stimulate the body to produce high-titer protective antibodies, and the antibody level is higher than the national standard, and has a good application prospect.

Description of drawings

Figure 1 is the determination of serum antibody titer of mice immunized with different fragments of 3D protein and recombinant antigen;

Fig. 2 is the result of efficacy test of recombinant epitope antigen alone or combined with 3D full-length protein to immunize pigs.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

The preparation of embodiment 1 recombinant antigen (EoIgG)

1. Bioinformatics analysis of O-type foot-and-mouth disease virus VP1 gene:

The structural protein VP1 of foot-and-mouth disease virus is the dominant antigen of the virus. Whether it is the isolated and purified natural VP1 protein or the recombinant expression product, it can induce the body to produce protective neutralizing antibodies, which is type-specific. The full length of the foot-and-mouth disease virus VP1 gene consists of 639 nucleotides, encoding a protein with 213 amino acids, and its main epitopes are concentrated in the 140-160 amino acid and the 200-213 amino acid segment. The present invention uses DNAStar biological software to carry out sequence analysis to three strains of porcine foot-and-mouth disease virus O type representative strains (O/China/99, O/Miandian/98, O/ZK/93) that my country isolates, and determined that the dominant antigenic epitope is The 135th-160th amino acid segment, and the 200th-213th amino acid segment of O/China/99 was determined as another B antigen epitope.

2. Gene cloning and protein expression, purification

Design and synthesis of multi-epitope genes:

In order to prevent the emergence of new epitopes when constructing genes, a spacer sequence GGSSGG that can ensure the correct display of each epitope structure is introduced between two adjacent epitopes. The tandem sequence of multi-epitope genes is O/China/99(135- 160)-O/Miandian/98(135-160)-O/ZK/93(135-160)-O/China/99(200-213), and at the 5'-end and 3'-end of the gene respectively Introduce specific enzyme cutting sites such as EcoRI and HindIII, the nucleotide sequence of the multi-epitope gene is shown in SEQ ID No.1, and the amino acid sequence encoded by it is shown in SEQ ID No.2, the multi-epitope gene The nucleotide sequence was synthesized by Dalian Bao Biological Company. At the same time, a specific primer was designed for multi-epitope gene amplification and synthesis for repeated tandem. The primers were: 3PU-BamHI5-cccggatccAAGTATGACGAGAGCCCCGT-3, 3PL-EcoRI5-cccgaattcggtggctctagcggcggtCAAAAGCTGTTTCACAGGCG-3. The synthesized multi-epitope gene and prokaryotic expression vector were respectively digested with EcoRI and HindIII, purified and recovered, inserted into the linearized pET-30a(+) vector (Novagen) with the corresponding enzyme, and the recombinant expression plasmid p3FOEN was constructed, and transformed into JM109 competent Positive screening, positive recombinants were determined by sequence determination. Use the above-mentioned specific primers to amplify the multi-epitope gene by PCR using the recombinant expression plasmid p3FOEN as a template. The amplified target gene is digested with BamHI/EcoRI double enzymes, purified and recovered, and then inserted into the p3FOEN recombinant plasmid linearized with the corresponding enzyme to construct all The polyepitope repeat expression plasmid p3FOEN2 with epitope repeated twice, wherein the nucleotide sequence of the polyepitope gene repeated twice in tandem is shown in SEQ ID No.3, and the encoded amino acid sequence is shown in SEQ ID No.4 ,.

PCR amplification of porcine IgG heavy chain constant region gene (PIgG):

ggtggctctagcggcggtGGGCCCTCGGTCTTCATC-3，pIgG xhoI5-cctcaTTTACCCGGAGTCTTGG A-3对目的基因进行扩增、纯化、回收，其中猪IgG重链恒定区核苷酸序列如SEQ ID No.5所示，其所编码的氨基酸序列如SEQ ID No.6所示，用HindIII/xhoI双酶切后，插入相应酶切线性化的重组表达质粒p3FOEN2构建重组表达质粒p3FOEN2-PIgG，该质粒经酶切、PCR及序列测定为阳性后-20℃保存备用，两次串联重复多表位基因与猪IgG连接基因序列连接后的完整核苷酸序列如SEQ ID No.7所示。Use the primer5.0 primer software to design specific primers for amplifying the full length of the porcine IgG heavy chain constant region gene, the positive strand primer: 5-AAGACGGCCCCATCGGT-3, the negative strand primer: 5-TTTACCCGGAGTCTTGGA-3, and obtain the target with a full length of 987kb Gene, and then use the specific primer pIgG HindIII5-gct with restriction site

ggtggctctagcggcggtGGGCCCTCGGTCTTCATC-3, pIgG xhoI5-cc tcaTTTACCCGGAGTCTTGG A-3 amplifies, purifies, and recovers the target gene, wherein the nucleotide sequence of the heavy chain constant region of porcine IgG is shown in SEQ ID No.5, and the encoded amino acid sequence is shown in SEQ ID No.6. After double digestion with HindIII/xhoI, insert the corresponding linearized recombinant expression plasmid p3FOEN2 to construct the recombinant expression plasmid p3FOEN2-PIgG. The plasmid is positive after digestion, PCR and sequence determination, and stored at -20°C for future use. Twice in series The complete nucleotide sequence after the repeated multi-epitope gene is linked with the pig IgG linking gene sequence is shown in SEQ ID No.7.

Expression of recombinant protein and identification of its biological activity:

Transform the positive recombinant expression plasmid into BL21(DE3) (Novagen), select a single clone to inoculate in LB culture medium (Kan+), induce expression with IPTG and identify the expression form, express the recombinant protein on a large scale, that is, pick a single colony from the LAB plate and inoculate it 5ml of LB culture solution containing kanamycin, cultivate overnight in a 37°C incubator at 220rpm, add 1% of the overnight culture into the freshly prepared sterile LB culture solution (kan+), and cultivate to OD600 in a 37°C incubator at 220rpm When ≈0.4 to 0.6, add 0.4mM IPTG to induce expression for 4 to 6 hours under aseptic conditions in an ultra-clean bench, centrifuge at 2000rpm for 30 minutes to harvest the culture, add protein lysate according to 20% of the original culture volume, and place in an ice bath Perform ultrasonication (30 min), centrifuge at 20,000 g for 20 min to collect the precipitate (4°C), and discard the supernatant. Purify the protein according to the instructions of Ni-NTA histidine purification column (Novagen). The purified protein was analyzed by SDS-PAGE electrophoresis and Western blotting. The size of the recombinant protein 3FOEN2-PIgG was in line with expectations, and it could be compatible with FMDV (type O) infected serum and horseradish The immune reaction of the peroxidase-labeled rabbit anti-pig IgG indicates that the expressed recombinant protein has biological activity, and the amino acid sequence of the expressed recombinant protein is shown in SEQ ID No.8.

The preparation of embodiment 2 foot-and-mouth disease virus 3D protein different fragments

1. Bioinformatics analysis of 3D protein gene of foot-and-mouth disease virus:

The non-structural protein 3D of foot-and-mouth disease virus is a viral RNA polymerase, which is highly conserved in seven serotypes of foot-and-mouth disease virus. The full length of the 3D gene is 1410 nucleotides, encoding a protein with a length of 470 amino acids. Using DNAStar biological software to analyze the sequences of 46 representative strains of seven serotypes obtained and compare them with the GenBank database, the results show that the nucleotides are homologous The sex is higher than 90%, the amino acid homology is higher than 97%, and it is highly conserved. In this study, the 3D sequence of the Asia1 type FMD virus JS/China/05 strain was used as a reference sequence to design primers to amplify the target gene fragment and express the protein.

2. Gene cloning and protein expression, purification

Use 3D protein-specific primers to amplify the full-length 3D protein gene and its three fragments. The full-length nucleotide sequence of the foot-and-mouth disease virus 3D protein gene is shown in SEQ ID No.9, and its encoded amino acid sequence is shown in SEQ ID No. As shown in 10, the full-length primer for amplifying the 3D protein gene, the positive strand primer is 5-GGATTGATAGTTGACACCAGAGA-3, and the negative strand primer is 5-TGCGTCACCGCACACGGCGTTC-3. The nucleotide sequence encoding the N-terminal fragment (represented by 3D1) is shown in SEQ ID No.11, and the encoded amino acid sequence is as shown in SEQ ID No.12. The primers used to amplify the N-terminal fragment: positive strand Primer 3D-1U/BamHI5'-CCCGGATCCGGATTGATAGTTGACACCAG-3', negative strand primer 3D-1L/HindIII5'-CCCAAGCTTTCATTTCTCCATGAGCTCTAAGGC-3'; the nucleotide sequence of the coding middle fragment (expressed as 3D2) is shown in SEQ ID No.13, The encoded amino acid sequence is shown in SEQ ID No.14, and the primers used to amplify the middle fragment: 3D-2U/EcoRI5'-CCCGAATTCGCCTTAGAGCTCATGGAGAAA-3', 3D-2L/HindIII5'-CCCAAGCTTTCAGATGCTTGTTGCGGAACAACCA-3'; encoding C The nucleotide sequence of the terminal fragment (represented by 3D3) is as shown in SEQ ID No.15, and the amino acid sequence encoded by it is as shown in SEQ ID No.16. The primers for amplifying the C-terminal fragment: 3D-3U/ BamHI 5'-CCCGGATCCGGTTGTTCCGCAACAAGCATC-3', 3D-3L/1HindIII 5'-CCCAAGCTTTCATGCGTCACCGCACACGGCGTT-3'. The obtained target genes were respectively inserted into the expression plasmid pET-30a(+) (Novagen) linearized with the corresponding enzymes HindIII and BamHI, and the corresponding recombinant expression plasmids p3DN (containing the nucleotide sequence encoding the N-terminal fragment), respectively, were constructed. p3DM (containing the nucleotide sequence encoding the middle fragment) and p3DC (containing the nucleotide sequence encoding the C-terminal fragment). The recombinant expression plasmid was transformed into BL21(DE3) (Novagen) after sequencing and identification, and a single clone was selected and inoculated in LB culture medium (kan+). After IPTG-induced expression and expression form identification, the recombinant protein was expressed on a large scale, that is, singled from the LAB plate Inoculate 5ml of LB culture solution containing kanamycin with the colony, culture overnight at 220rpm in a 37°C incubator, add 1% of the overnight culture into the newly prepared sterile LB culture solution (kan+), and culture at 220rpm in a 37°C incubator When QD600≈0.4～0.6, add 0.4mM IPTG to induce expression for 4～6 hours under the aseptic condition of ultra-clean bench, centrifuge at 2000rpm for 30min to harvest the culture, add protein lysate according to 20% of the original culture volume, and place on ice Ultrasonic crushing (30min) was performed under the conditions, the precipitate was collected by centrifugation at 20000g for 20min (4°C), and the supernatant was discarded. The protein was purified according to the instructions of the Ni-NTA histidine purification column (Novagen). The purified protein was analyzed by SDS-PAGE electrophoresis and Western blotting. The results showed that the expressed N-terminal fragment (3D1), middle fragment (3D2), and C-terminal fragment ( 3D3) The sizes are all as expected. Except for the N-terminal protein fragment of 3D protein, the rest of the proteins can react with FMDV infectious serum; all proteins can react with anti-histidine monoclonal antibody, indicating that the expressed recombinant protein has biological activity. Wherein, the amino acid sequence of the expressed N-terminal fragment (3D1) is shown in SEQ ID No.12, the amino acid sequence of the middle fragment (3D2) is shown in SEQ ID No.14, and the amino acid sequence of the C-terminal fragment (3D3) is shown in Shown in SEQ ID No.16.

Embodiment 3, vaccine preparation and immune efficacy experiment:

1. Vaccine preparation

The purified recombinant antigen prepared in Example 1 and the purified 3D protein full-length or its different fragments (3D1\3D2\3D3) prepared in Example 2 were quantified by the Bio-Rad quantitative kit and then diluted into appropriate Concentration, after the purified 3D protein fragments and recombinant antigens were configured in a ratio of 1:2 (V/V), an equal volume of oil adjuvant ISA206 (France) was added to emulsify into vaccine preparations, and the recombinant antigens were contained in 1ml/tube. 200 μg, 100 μg of the full-length 3D protein or its fragments.

2. Immunity efficacy test:

Use the prepared vaccine to vaccinate pigs (200 μg antigen + 100 μg full-length 3D protein or its fragments) through the muscle route according to 1 ml per head. The results of immunization experiments show that whether the recombinant antigen alone or in combination with 3D protein can stimulate the body to produce High-titer FMDV protective antibody, and the addition of the full-length 3D protein or any of its three fragments can significantly enhance the antibody titer. 14 days after the booster immunization, the serum antibody titer increased significantly, and 85% of the serum antibodies of the immunized animals The titer exceeds 1:720 (LBP-ELISA), and 70% of the immunized animals have a serum antibody titer higher than 1:1024, suggesting a good immune effect. The results are shown in Figure 1 and Figure 2. In addition, it can be seen from the results of the recombinant epitope antigen alone or combined with 3D full-length protein (3DC) or its fragments (3D1\3D2\3D3) to immunize pigs with efficacy test results, it can be seen that 3D full-length protein was added 28 days after the first immunization Vaccines or their fragments (3D1\3D2\3D3) are more effective than epitope antigen alone immunization, but epitope antigen alone immunization effect is better after booster immunization, the results are shown in Figure 2.

3. Antivirus test

According to my country's FMD vaccine immune efficacy test standard, the immunized pigs were challenged with 1000ID50/2ml homologous virus (such as O-type strain O/China/99) to attack the animals 14 days after the booster immunization, and continued to observe After 10 days, the results showed that all the immunized animals did not observe any clinical symptoms and obtained 100% protection, except that the PBS control animals had typical clinical symptoms of foot-and-mouth disease (fever, blisters on the lips, heels and nose mirrors). It shows that the broad-spectrum vaccine developed by the present invention has good immune efficacy, and the results are shown in Table 1.

Wherein the O-type strain O/China/99 is a disclosed strain in the prior art, and this strain has been recorded in the 01st issue of "Acta Virology" in 2009 entitled foot-and-mouth disease virus pan-subtype O/CHINA/99 strain complete In the article "Long cDNA Molecular Cloning Construction and Infection Identification", the author is Lv Jianliang et al. It is now preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

Table 1 The results of the challenge protection test of pigs immunized with pig O-type broad-spectrum multi-epitope vaccine

Whether the recombinant antigen alone or in combination with 3D protein full-length or 3D protein fragments, there was no redness, swelling, fever, etc. at the injection site of the immunized animals, and no adverse reactions to inoculation, normal appetite, and good mental state, suggesting that the porcine foot-and-mouth disease prepared by the present invention Virus O-type multi-epitope vaccine has good immunogenicity, can produce high level of protective antibody after immunizing pigs, and is safe and harmless to immunized animals after vaccination. It is of great significance to provide material reserves and technical support for the prevention and control of O-type foot-and-mouth disease.

Claims (9)

1. a kind of porcine foot-and-mouth disease virus O type broad-spectrum multi-epitope recombinant antigen is characterized in that described recombinant antigen is after the main epitope of multiple strains of porcine foot-and-mouth disease virus O type is connected in series, and pig IgG heavy chain The constant region coupling construction is obtained, and the amino acid sequence of the recombinant antigen is shown in SEQ ID NO:8. 2. A nucleotide sequence encoding the recombinant antigen of claim 1. 3. The nucleotide sequence as claimed in claim 2, characterized in that said nucleotide sequence is as shown in SEQ ID NO:7. 4. A recombinant expression vector, characterized in that it contains the nucleotide sequence according to claim 2 or 3. 5. A host cell, characterized in that it contains the recombinant expression vector according to claim 4. 6. the application of recombinant antigen described in claim 1 in the preparation prevention porcine foot-and-mouth disease vaccine. 7. The use of the nucleotide sequence according to claim 2 or 3 in the preparation of a vaccine against porcine foot-and-mouth disease. 8. A porcine foot-and-mouth disease virus type O broad-spectrum multi-epitope vaccine, characterized in that it contains the multi-epitope recombinant antigen described in claim 1. 9. porcine foot-and-mouth disease virus O type broad-spectrum multi-epitope vaccine as claimed in claim 8 is characterized in that also containing the full-length amino acid sequence of foot-and-mouth disease virus 3D protein or its fragment, the full-length amino acid of described foot-and-mouth disease virus 3D protein The sequence is shown in SEQ ID NO: 10, and the foot-and-mouth disease virus 3D protein fragment is selected from at least one of the amino acid sequences shown in SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16.

Images