CN110606875A

CN110606875A - Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine

Info

Publication number: CN110606875A
Application number: CN201910891539.3A
Authority: CN
Inventors: 常惠芸; 雷垚; 邵军军; 李扬帆; 常艳燕; 张永光
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2019-12-24

Abstract

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.

Description

Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine

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.

Example 1

Preparation of foot-and-mouth disease virus multi-epitope antigen (HAO)

1. Design and gene synthesis of recombinant bivalent multi-epitope antigen of A-type and O-type foot-and-mouth disease

According to the existing gene sequences of A-type FMDV (accession number: KF450794.1) and O-type FMDV (accession number: AJ318831.1) in GenBank, two B cell tables, namely VP1134-161 and 200-213 amino acids, on A-type FMDVVP1 and O-type FMDVVP1 are respectively selected, each epitope is subjected to two repeated tandem connection, and the epitopes are connected through a linker sequence GGSSGG to form a new epitope box HAO, the nucleotide sequence of which is shown as SEQ ID NO: 4, and sending the gene sequence of the tandem epitope box to Nanjing Kingsrey Biotechnology GmbH for synthesis, directly synthesizing the gene sequence to a pET-28a expression vector, wherein the enzyme cutting sites at two ends are BamH I and Hind III, obtaining a plasmid pET-28a-HAO, and the name is pHAO.

2. Expression and purification of recombinant antigen HAO

Positive plasmid, pHAO, was transformed into BL21 competent cells. Selecting a monoclonal antibody to be placed in 5mL of Carna resistance-containing LB culture medium, carrying out shake culture at 37 ℃ and 220rmp overnight, transferring 5mL of bacterial liquid into 500mL of Carna LB culture medium according to the transfer ratio of 1%, continuing to carry out shake culture at 37 ℃ and 220rmp, adding IPTG (isopropyl-beta-thiogalactoside) for induction when OD600 of the bacterial liquid reaches 0.4-0.6 to enable the final concentration to be 0.5mM, then carrying out shake culture at 37 ℃ and 220rmp for 8h, carrying out 5000rmp centrifugation to collect thalli, washing the collected thalli with PBS, then using binning buffer (0.5M NaCl,50mM Tris,5mM imidazole and pH 8.0) to carry out thalli resuspension, carrying out ultrasonic lysis, carrying out 12000rmp centrifugation, dividing the thalli into an inclusion body and a supernatant, and carrying out SDS-PAGE (sodium dodecyl sulfate electrophoresis) electrophoretic analysis on the two parts respectively, wherein the HAO expression form is in the inclusion body form. The recombinant protein HAO is purified from the inclusion body protein according to the specification of a Ni-NTA histidine purification column (GE), and the purified protein is renatured with the inclusion body protein by a urea gradient dialysis method. Filling the purified protein into a dialysis bag, sealing two ends of the dialysis bag, putting the dialysis bag into a renaturation solution (0.5M NaCl,50mM Tris, 0.5M EDTA, 6M Urea, 10% glycerol, 2mM GSH,0.2mM GSSG and pH 7.3), changing the dialysis solution every 8h, reducing the concentration of Urea (6M, 4M, 2M and 0M) in sequence, and finally dialyzing the dialysis solution for 8h by PBS to obtain the renatured recombinant protein. The purified protein is analyzed by SDS-PAGE electrophoresis and Westernblotting, the size of the recombinant protein HAO is consistent with the expectation, and the recombinant protein HAO can generate immunoreaction with FMDV (type A and type O) infected serum, so that the expressed recombinant protein has biological activity, and the amino acid sequence of the expressed recombinant protein is shown as SEQ ID No. 2.

Example 2

Preparation of recombinant protein HBHA

1. Synthesis of HBHA Gene

According to the gene sequence (accession number KC920678.1) of HBHA on GenBank, the gene sequence is sent to Nanjing Kingsry biological science and technology limited company for synthesis, the nucleotide sequence is shown as a sequence table SEQ ID No.3, the gene sequence is synthesized into a cloning vector pUC57, enzyme cutting sites at two ends are Hind III and Xho I, and the plasmid is named as pUC 57-HBHA. A pair of primers containing enzyme cutting sites BamH1(GGATCC) and XhoI (CTCGAG) is designed by taking the synthesized HBHA gene as a template, PCR amplification is carried out on the HBHA gene, and the primers are (SEQ ID NO: 5: 5'-CGGGATCCATGGCCGAAAACCCG-3', SEQ ID NO: 6: 5'-ccgctcgagtttctgggtcactttttt-3'), so that an HBHA nucleic acid product containing a BamH1 enzyme cutting site at the 5 'end and an XhoI enzyme cutting site at the 3' end is obtained. After the amplification product was purified by the PCR product purification kit, the plasmid pET-28a-HBHA named pHBHA was constructed by double-digesting with BamH I and Xho I, and simultaneously double-digesting the plasmid pET-28a with both of these endonucleases, and ligating them with T4 ligase.

2. Expression and purification of recombinant protein HBHA

The recombinant plasmid pHBHA is expressed according to the protein expression program in the example 1, and the recombinant protein HBHA is soluble expression after SDS-PAGE verification, the soluble protein is purified by the recombinant protein HBHA according to the specification of a Ni-NTA histidine purification column (GE), and the purified protein is analyzed by SDS-PAGE electrophoresis and Western blotting, so that the result shows that the protein HBHA can not have immunoreaction with FMDV infectious serum; and can have immunoreaction with the anti-histidine monoclonal antibody, which shows that the expressed recombinant protein has biological activity, and the amino acid sequence of the expressed recombinant protein is shown as SEQ ID No. 1.

Example 3

Preparation of recombinant fusion protein HAO-HBHA

1. Construction of recombinant fusion protein HAO-HBHA expression plasmid

The two plasmids pET-28a-HAO and pUC57-HBHA were double-digested with Hind III and Xho I, respectively, and ligated by T4 ligase, transformed, and the like. Thereby constructing a recombinant plasmid pET-28a-HAO-HBHA which is named as pHAO-HBHA.

2. Expression and purification of recombinant fusion protein HAO-HBHA

The recombinant plasmid pHAO-HBHA is expressed according to the protein expression program in the example 1, and the SDS-PAGE verifies that the recombinant protein HAO-HBHA is soluble expression, the recombinant protein HBHA purifies the soluble protein according to the specification of a Ni-NTA histidine purification column (GE), and the SDS-PAGE electrophoresis and Western blotting analysis of the purified protein show that the protein HAO-HBHA can perform immunoreaction with FMDV (type A and type O) infectious serum; and the recombinant protein can have immunoreaction with the anti-histidine monoclonal antibody, which indicates that the expressed recombinant protein has biological activity.

Example 4

Vaccine preparation and immunopotency assay:

1. preparation of vaccines

The purified recombinant antigen and HBHA protein obtained in example 3 and example 1 were quantified with a Bio-Rad quantification kit and diluted to appropriate concentrations. The purified HAO protein fragment and the recombinant protein HBHA are respectively mixed according to the ratio of 1: 2(V/V) configuration or equivalent configuration with oil adjuvant ISA206(Seppic), and emulsifying into vaccine preparation. Meanwhile, HAO-HBHA is not added with any immunologic adjuvant, and the recombinant protein is used as a vaccine preparation.

2. Immunopotentiality test

56 female SPF-grade BALB/c mice 6-8 weeks old were randomly divided into 7 groups (8 mice/group), and the prepared immunogens were vaccinated 3 times at weeks 0, 2 and 4, respectively, according to the immunization groups and immunization doses in the following table. The main groups are as follows: HAO immunization group, 50 μ g/mouse; group HAO + ISA206, 50 μ g/mouse; HAO + HBHA group, 50 μ g +50 μ g/mouse; 50 μ g/HAO-HBHA group; HBHA group, 50 μ g/mouse; PBS + ISA206 group, 100 μ L/mouse; and in the inactivated vaccine group, the immune result shows that: except FMDV specific antibodies which cannot be detected by the HBHA group and the PBS group, A-type and O-type FMDV specific antibodies can be detected by other immunization groups, wherein the antibody level of the HAO-HBHA immunization group is not obviously different from that of the inactivated vaccine group, and is the highest relative to other recombinant antigen immunization groups, and particularly, the antibody response level of immunization when HBHA and HAO epitope antigen are fused to form fusion protein for immunization is obviously higher than that of immunization after the HBHA and HAO epitope antigen are mixed for immunization or after the HAO alone is emulsified by 206 adjuvant. The results are shown in FIGS. 1 and 2.

The neutralizing antibody titers of FMDV types A and O in the serum of immunized mice are determined by an in vitro neutralization test, and the results show that 5 groups can induce the reaction of generating neutralizing antibodies of FMDV types A/O in addition to the HBHA and PBS immunized groups, wherein the neutralizing antibody titer of the combined immune group of HAO + HBHA and the immune group of HAO +206 adjuvant is far lower than that of the immune group of HAO-HBHA and the bivalent inactivated vaccine of FMDV types A/O. In addition, the neutralizing antibody titer induced by HAO-HBHA was not significantly different from that of the inactivated vaccine group. The results are shown in FIG. 3.

Splenic lymphocytes of mice were isolated 42d after the first immunization, stimulated in vitro with inactivated FMDV, and lymphocyte proliferation responses were detected using MTT lymphocyte proliferation assay. The proliferation capacity of the lymphocytes of each experimental group and the commercial vaccine group is obviously higher than that of the HBHA group and the PBS group. The proliferation capacity of the lymphocytes in the HBHA-HAO group after being stimulated by the A type or O type FMDV antigen is higher than that of the HAO +206 adjuvant group and the HAO immune group, while the HBHA-HAO group has no significant difference compared with the commercial inactivated vaccine group, and the results are shown in FIG. 4. The levels of cytokines associated with Th1 type, Th2 type and monocyte differentiation were measured in 42d serum samples after priming, and the results are shown in fig. 5.

Moreover, the injection part of the immune animal does not have the phenomena of red swelling, fever and the like, and does not have adverse reaction of inoculation, the appetite is normal, the mental state is good, and the HBHA protein prepared by the invention not only can play the effect of enhancing the immunity of FMDVA type and O type broad-spectrum multivalent epitope vaccine, but also is safe and harmless to the immune animal.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

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.