阅读说明：本技术 一种用于制备***疫苗的分子内佐剂及其应用和***疫苗 (Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine ) 是由 常惠芸 雷垚 邵军军 李扬帆 常艳燕 张永光 于 2019-09-20 设计创作，主要内容包括：本发明提供了一种用于制备口蹄疫疫苗的分子内佐剂及其应用和口蹄疫疫苗,属于基因工程疫苗技术领域,所述分子内佐剂的氨基酸序列如SEQ ID NO：1所示。本发明提供的分子内佐剂能够明显增强FMD的免疫应答,显著提高了多表位免疫原的抗原广谱性。(The invention provides an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine, application thereof and the foot-and-mouth disease vaccine, belonging to the technical field of genetic engineering vaccines, wherein the amino acid sequence of the intramolecular adjuvant is shown as SEQ ID NO: 1 is shown. The intramolecular adjuvant provided by the invention can obviously enhance the immune response of FMD, and obviously improve the broad-spectrum antigen of multi-epitope immunogen.)

1. An intramolecular adjuvant for preparing a foot-and-mouth disease vaccine is characterized in that the amino acid sequence of the intramolecular adjuvant is shown as SEQ ID NO: 1 is shown.

2. A gene encoding the amino acid sequence of the intramolecular adjuvant of claim 1, the nucleotide sequence of the gene being as set forth in SEQ ID NO: 3, respectively.

3. Use of an intramolecular adjuvant according to claim 1 in the preparation of a vaccine.

4. A foot and mouth disease vaccine comprising the intramolecular adjuvant according to claim 1 and a foot and mouth disease virus multi-epitope antigen.

5. The aftosa vaccine according to claim 4, wherein the aftosa vaccine is a recombinant protein obtained by fusion expression of an intramolecular adjuvant and a multi-epitope antigen of aftosa virus.

6. The foot-and-mouth disease vaccine of claim 4, wherein the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen is as shown in SEQ ID NO: 2, respectively.

7. A gene encoding the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen of claim 6, the nucleotide sequence of the gene is shown as SEQ ID NO: 4, respectively.

8. The gene according to claim 6, wherein the foot-and-mouth disease virus is a foot-and-mouth disease type A virus and/or a foot-and-mouth disease type O virus.

Technical Field

The invention relates to the technical field of genetic engineering vaccines, in particular to an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine, application thereof and the foot-and-mouth disease vaccine.

Background

Foot-and-mouth disease (FMD) is an acute, febrile and highly infectious animal epidemic disease caused by infecting cloven-hoofed animals such as pigs, cattle and sheep with FMDV. In recent years, a series of foot and mouth disease epidemics of type a and type O have occurred in many countries including china, causing serious economic losses. At present, the inactivated vaccine of the foot-and-mouth disease is still the main means for preventing and controlling the foot-and-mouth disease, but in view of the potential safety hazard of the inactivated vaccine in production, the development of a novel safe and effective subunit vaccine of the foot-and-mouth disease is not slow enough. With the continuous development of genetic engineering technology, development of novel foot-and-mouth disease vaccines has also achieved favorable achievements, and the vaccines mainly comprise synthetic peptide vaccines, multi-epitope vaccines, virus-like particle vaccines and the like. The epitope vaccine is one of vaccines with development prospect, has great advantages compared with the traditional vaccine, is mainly formed by serially combining a plurality of antigen epitopes of the virus, can adjust the epitope sequence according to the difference of epidemic strains at any time, and can add the antigen epitopes of different subtype strains to enhance the broad spectrum of the epitope vaccine.

The antigen polypeptide is used as a small molecule immunogen, and the immunogenicity generated by the antigen polypeptide is weaker, so that a strong immune response to a pathogen cannot be generated. A reasonably designed epitope vaccine mainly comprises three components: an epitope peptide, a presentation system, and an adjuvant. For multi-epitope vaccines, the immunopotentiation effect of conventional adjuvants is not ideal.

Disclosure of Invention

In view of the above, the present invention aims to provide an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine, an application thereof, and a foot-and-mouth disease vaccine. The intramolecular adjuvant provided by the invention can obviously enhance the immune response of FMD, and obviously improve the broad-spectrum antigen of multi-epitope immunogen.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine, wherein the amino acid sequence of the intramolecular adjuvant is shown as SEQ ID NO: 1 is shown.

The invention also provides a gene for coding the amino acid sequence of the intramolecular adjuvant in the scheme, and the nucleotide sequence of the gene is shown as SEQ ID NO: 3, respectively.

The invention also provides application of the intramolecular adjuvant in the technical scheme in preparation of vaccines.

The invention also provides a foot-and-mouth disease vaccine which comprises the intramolecular adjuvant and the foot-and-mouth disease virus multi-epitope antigen.

Preferably, the foot-and-mouth disease vaccine is a recombinant protein obtained after fusion expression of an intramolecular adjuvant and a multi-epitope antigen of the foot-and-mouth disease virus.

Preferably, the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen is shown as SEQ ID NO: 2, respectively.

The invention also provides a gene for coding the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen in the technical scheme, and the nucleotide sequence of the gene is shown as SEQ ID NO: 4, respectively.

Preferably, the foot-and-mouth disease virus is a foot-and-mouth disease type A virus and a foot-and-mouth disease type O virus.

The invention has the beneficial effects that: the invention provides an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine. The intramolecular adjuvant provided by the invention is heparin-binding hemagglutinin (HBHA) of mycobacterium tuberculosis, and after the HBHA fragment is subjected to fusion expression with multi-epitope antigen of foot-and-mouth disease virus, the vaccine immune animal is prepared, so that the immunogenicity of the multi-epitope antigen can be obviously enhanced, and high-level protective neutralizing antibody and cellular immune response are generated; it can promote the maturation and differentiation of dendritic cell, the proliferation of specific T lymphocyte and the expression of cell factor obviously. The intramolecular adjuvant provided by the invention can obviously enhance the humoral and cellular immune response of the multi-epitope immunogen, and is a good immunostimulant.

Drawings

FIG. 1 is a graph showing the dynamic change of type A FMDV-specific IgG in each immune group serum sample;

FIG. 2 is a graph showing the dynamic change of O-type FMDV-specific IgG in each immune group serum sample;

FIG. 3 is a graph of FMDV neutralizing antibody levels for each immune group serum sample;

FIG. 4 is a graph showing the proliferation level of lymphocytes from each immune group after stimulation with inactivated FMDV antigen;

FIG. 5 is a graph showing the levels of cytokines in serum of each immune group.

Detailed Description

The invention provides an intramolecular adjuvant for preparing a foot-and-mouth disease vaccine, wherein the amino acid sequence of the intramolecular adjuvant is shown as SEQ ID NO: 1, specifically: MAENPNIDDLPAPLLAALGAADLALATVNDLIANLRERAEETRAETRTRVEERRARLTKFQEDLPEQFIELRDKFTTEELRKAAEGYLEAATNRYNELVERGEAALQRLRSQTAFEDASARAEGYVDQAVELTQEALGTVASQTRAVGERAAKLVGIELPGKAEAAGKKAQKAIAKAPAKKASAKKAPAKKAPAKKAAAKKVTQK are provided.

The invention also provides a gene for coding the amino acid sequence of the intramolecular adjuvant in the scheme, and the nucleotide sequence of the gene is shown as SEQ ID NO: 3, specifically: ATGGCGGAAAACCCGAACATCGACGACTTGCCAGCCCCGCTGCTCGCGGCCCTGGGCGCGGCCGACCTGGCCCTGGCCACGGTCAACGACCTGATCGCCAACCTGCGCGAGCGGGCCGAGGAGACCCGCGCCGAGACCCGCACCCGGGTCGAGGAGCGCCGCGCCCGGCTGACCAAGTTCCAGGAGGACCTGCCCGAGCAGTTCATCGAGCTGCGCGACAAGTTCACCACCGAGGAGCTGCGCAAGGCGGCCGAGGGCTACCTGGAGGCGGCGACCAACCGGTACAACGAGCTGGTCGAGCGCGGCGAGGCGGCCCTGCAGCGGCTGCGCAGCCAGACCGCCTTCGAGGACGCCTCCGCGCGCGCCGAGGGCTACGTGGACCAGGCCGTCGAGCTGACCCAGGAGGCGCTGGGCACCGTCGCGTCGCAGACCCGCGCGGTCGGTGAGCGCGCCGCCAAGCTGGTGGGCATCGAGCTGCCGGGCAAGGCCGAGGCCGCCGGCAAGAAGGCCCAGAAGGCCATCGCCAAGGCCCCCGCCAAGAAGGCGTCGGCCAAGAAGGCCCCCGCCAAGAAGGCGCCGGCCAAGAAGGCCGCGGCCAAGAAGGTCACCCAGAAG are provided.

In the present invention, the SEQ ID NO: the 3 sequence was synthesized by Nanjing Kingsry Biotech, Inc. based on the gene sequence of HBHA on GenBank (accession No. KC 920678.1).

The invention also provides application of the intramolecular adjuvant in the technical scheme in preparation of vaccines. In the invention, when the intramolecular adjuvant is used for preparing the vaccine, the intramolecular adjuvant is preferably prepared by mixing and emulsifying the intramolecular adjuvant and a multi-epitope antigen of foot-and-mouth disease virus; the volume ratio of the intramolecular adjuvant to the multi-epitope antigen of the foot-and-mouth disease virus is 2: 1. In the present invention, when the intramolecular adjuvant is used for preparing a vaccine, the intramolecular adjuvant is more preferably fused and expressed with a multi-epitope antigen of foot-and-mouth disease virus, and the obtained recombinant protein itself is used as a vaccine preparation. The intramolecular adjuvant provided by the invention can obviously enhance the immunogenicity of a multi-epitope antigen.

The invention also provides a foot-and-mouth disease vaccine which comprises the intramolecular adjuvant and the foot-and-mouth disease virus multi-epitope antigen. In the invention, the foot-and-mouth disease vaccine is a recombinant protein obtained after fusion expression of an intramolecular adjuvant and a multi-epitope antigen of the foot-and-mouth disease virus. In the invention, the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen is shown as SEQ ID NO: 2, specifically: MAENPNIDDLPAPLLAALGAADLALATVNDLIANLRERAEETRAETRTRVEERRARLTKFQEDLPEQFIELRDKFTTEELRKAAEGYLEAATNRYNELVERGEAALQRLRSQTAFEDASARAEGYVDQAVELTQEALGTVASQTRAVGERAAKLVGIELPGKAEAAGKKAQKAIAKAPAKKASAKKAPAKKAPAKKAAAKKVTQK are provided.

The invention also provides a gene for coding the amino acid sequence of the foot-and-mouth disease virus multi-epitope antigen in the technical scheme, and the nucleotide sequence of the gene is shown as SEQ ID NO: 4, specifically: GCGGCGATCGAGTTCTTTGAGGGTATGGTGCACGACAGCATTAAGGGTGGCAGCAGCGGTGGCAAATACAGCGCGCCGCAGAACCGTCGTGGTGATAGCGGTCCGCTGGCGGCGCGTCTGGCGGCGCAACTGCCGGCGAGCTTCGGTGGCAGCAGCGGTGGCAAGTATAGCGCGCCGGCGACCCGTCGTGGTGACCTGGGCACCTGGCGGCGCGCTTAGCTGCTCAATTACCGGCGAGCTTTGGTGGCAGCAGCGGTGGCAAATATAGCACCGGTAACGCGGGTCGTCGTGGTGATTTAGGCAGCCTGGCTGCGCGTGTTGCGGCGCAGTTACCTGCGAGCTTCGGTGGCAGCAGCGGTGGCCGTCACAAGCAGAAAATCATTGCGCCGGCGAAGCAACTGCTGGGTGGCAGCAGCGGTGGCAAATACGGCGAGAGCCCGGTGACCAACGTTCGTGGCGACCTGCAGGTTCTGGCGCAAAAAGCGGCGCGTACCCTGCCGGGTGGCAGCAGCGGTGGCAAATATGCGGGTGGCAGCCTGCCGAACGTGCGTGGTGATCTGCAGGTGCTGGCGCAGAAAGCGGCGCGTCCGTTACCGGGTGGCAGCAGCGGTGGCAGGCACAAGCAGAAAATCGTGGCGCCGGTTAAACAGTTACTGGGTGGCAGCAGCGGTGGCGCGGCGATTGAATTCTTTGAGGGCATGGTTCACGACAGCATTAAG are provided.

In the invention, the foot-and-mouth disease virus is a foot-and-mouth disease A type virus and/or a foot-and-mouth disease O type virus. In the invention, the foot-and-mouth disease virus multi-epitope antigen is formed by respectively selecting two B cell tables, namely VP1134-161 amino acid and 200-213 amino acid, on A-type FMDV VP1 and O-type FMDV VP1 according to the existing gene sequences of the A-type FMDV (accession number: KF450794.1) and the O-type FMDV (accession number: AJ318831.1) in GenBank, performing two repeated tandem connection on each epitope, and connecting the epitopes through a linker sequence GGSSGG.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.