CN114957480B - A type foot-and-mouth disease vaccine using human replication defective recombinant adenovirus as carrier - Google Patents

Abstract

The invention provides a novel A-type foot-and-mouth disease vaccine taking human 5-type replication defective adenovirus as a vector. The vaccine takes replication defective human type 5 adenovirus with E1 and E3 combined deletion as Sup>A vector, HEK293 cells integrating adenovirus E1 genes as Sup>A packaging cell line, inserted antigen genes are A-type foot-and-mouth disease structure and non-structural genes (FMDV-A) which are optimally designed, and finally recombinant A-type foot-and-mouth disease virus (Ad 5-FMDV-A) with human replication defective adenovirus as Sup>A vector is formed. The vaccine prepared by the virus shows good immunogenicity on a mouse model, and can induce the mouse to generate obvious cellular immunity 7 days after immunization. The neutralizing antibodies are generated in the low, medium and high dose groups 7 days after immunization, the titer of the neutralizing antibodies is continuously increased after 21 days of immunization, and the difference between the high, medium and low dose groups is obvious, so that obvious dose dependency is presented.

Description

A type foot-and-mouth disease vaccine using human replication defective recombinant adenovirus as carrier

Technical Field

The invention relates to the field of bioengineering, in particular to an A-type foot-and-mouth disease vaccine taking human replication defective recombinant adenovirus as a vector.

Background

foot-and-M-outer disease (FM D) is an acute, virulent, contagious infectious disease of even animals caused by foot-and-M outh disease virus, fmd V. Foot and Mouth Disease Virus (FMDV) has O, A, C, SAT, SAT2, SAT3 (i.e., african foot and mouth disease virus types 1, 2, 3) and Asia1 (asian type 1) 7 serotypes. There was little cross-immune protection between the types. The type O and the type A are popular in China. At present, in the countries with underdeveloped economy, the traditional inactivated vaccine immunization is a main means for controlling and eradicating foot-and-mouth disease, but the traditional vaccine has a plurality of problems including short immunization duration, high vaccine cost, incomplete inactivation, easy toxicity dispersion to the surrounding environment and the like, so people are always seeking a safer and more effective FMD vaccine. The FM D V genome comprises one open reading frame (openreading fram e, ORF), which is divided into L, P, P2, P3 4 regions, P1 region encoding V P4 (1A), V P2 (1B), V P (1D) and V P3 (1C) 4 structural proteins, respectively; the P2 region encodes 3 proteins, namely 2A, 2B and 2C in sequence; the P3 region encodes 47 proteins, in turn 3A, 3B (VP g 1), 3B (VPg 2), 3B (VP g 3), 3C and 3D. At present, the type A foot-and-mouth disease vaccine used by economic animals in China mainly takes an inactivated vaccine prepared by the type A foot-and-mouth disease virus as a main part, and some novel vaccines, such as polypeptide vaccines, also have certain use amount.

Disclosure of Invention

In order to better prevent foot-and-mouth disease in animals, the first aspect of the present invention provides any one of the following proteins:

(a1) The protein comprises the following amino acid sequences from the N end to the C end: the amino acid sequence of structural protein P1 of the A-type foot-and-mouth disease virus, the amino acid sequence of non-structural protein 2A of the A-type foot-and-mouth disease virus, the amino acid sequence of non-structural protein 2B of the A-type foot-and-mouth disease virus, the amino acid sequence of partial protein 3B of the A-type foot-and-mouth disease virus and the amino acid sequence of non-structural protein 3C of the A-type foot-and-mouth disease virus;

(a2) The protein comprises the following amino acid sequences from the N end to the C end: methionine, amino acid sequence of structural protein P1 of type A foot-and-mouth disease virus, amino acid sequence of non-structural protein 2A of type A foot-and-mouth disease virus, amino acid sequence of non-structural protein 2B of type A foot-and-mouth disease virus, amino acid sequence of non-structural protein 3B part protein of type A foot-and-mouth disease virus, amino acid sequence of non-structural protein 3C protein of type A foot-and-mouth disease virus, and amino acid sequence of tag protein.

Optionally, the structural protein P1 of the A-type foot-and-mouth disease virus is structural protein P1 of an A/Wuhan strain; the non-structural protein 2A of the A-type foot-and-mouth disease virus is the non-structural protein 2A of an A/Wuhan strain; the non-structural protein 2B of the A-type foot-and-mouth disease virus is the non-structural protein 2B of an A/Wuhan strain;

the non-structural protein 3B part of protein of the foot-and-mouth disease virus A is 3B part of protein of an A/A24 strain or an A/A12 strain;

the A-type foot-and-mouth disease virus nonstructural protein 3C protein is 3C protein of an A/A24 strain or an A/A12 strain;

alternatively, the amino acid sequence of the structural protein P1 of the A/Wuhan strain is shown in SEQ ID NO:2 nd to 735 th bits; the nucleotide sequence of the coding DNA molecule is shown as SEQ ID NO:1, positions 4-2205;

alternatively, the amino acid sequence of the nonstructural protein 2A of the A/Wuhan strain is shown in SEQ ID NO: bits 736 to 753 of 2; the nucleotide sequence of the coding DNA molecule is shown as SEQ ID NO:1, positions 2206-2259;

alternatively, the amino acid sequence of the non-structural protein 2B of the A/Wuhan strain is shown in SEQ ID NO:2 bits 754 to 917; the nucleotide sequence of the coding DNA molecule is shown as SEQ ID NO: positions 2260 to 2751 of 1;

optionally, the amino acid sequence of the partial protein 3B of the nonstructural protein of the A/A24 strain is shown as SEQ ID NO:2 from 918 th bit to 1009 th bit from the N end; the nucleotide sequence of the coding DNA molecule is shown as SEQ ID NO: positions 2752-3027 of 1;

optionally, the amino acid sequence of the non-structural protein 3C protein of the A/A24 strain is shown in SEQ ID NO:2 from 1010 th to 1196 th bits from the N end; the nucleotide sequence of the coding DNA molecule is shown as SEQ ID NO:1 from 3028 to 3588.

Second, the present invention also provides a biomaterial as described in any one of the following:

1) A DNA molecule encoding the above protein;

2) An expression cassette, recombinant vector or recombinant microorganism comprising 1) said DNA molecule;

optionally, the DNA molecule is any one of the following:

1) The nucleotide sequence is SEQ ID NO:1 from position 4 to position 3588;

2) The nucleotide sequence is SEQ ID NO:1, and a DNA molecule shown in 1.

Optionally, the recombinant microorganism is a recombinant virus; optionally, the recombinant virus is a recombinant adenovirus; alternatively, the recombinant adenovirus is a human replication-defective recombinant adenovirus; alternatively, the human replication defective recombinant adenovirus is a human replication defective adenovirus type 5 (Ad 5).

Third, the invention provides a foot-and-mouth disease virus vaccine, the active ingredient of which is the recombinant adenovirus.

Optionally, the foot-and-mouth disease virus is a type a foot-and-mouth disease virus.

The protein or the biological material is applied to the preparation of vaccines for preventing foot-and-mouth disease viruses.

The foot-and-mouth disease virus is A-type foot-and-mouth disease virus;

or, the vaccine is in the form of injection, nose drops or spray;

optionally, the vaccine is a monovalent vaccine, a bivalent vaccine or a trivalent vaccine; the monovalent vaccine is a monovalent vaccine for foot-and-mouth disease; the bivalent vaccine is an O-type and A-type foot-and-mouth disease bivalent vaccine; trivalent vaccines are trivalent vaccines for foot and mouth disease type O, type a and asian type 1.

Optionally, the active ingredient of the A-type foot-and-mouth disease vaccine in the monovalent vaccine, the bivalent vaccine or the trivalent vaccine is the recombinant adenovirus.

Fourth, the present invention provides a method for preparing a recombinant adenovirus, comprising the steps of:

(1) Constructing a recombinant shuttle plasmid vector comprising a DNA molecule encoding the protein of claim 1 or 2;

(2) Transfecting the recombinant shuttle plasmid vector of the step (1) together with an adenovirus backbone plasmid into a host cell;

(3) Culturing the host cell of step (2);

(4) Harvesting the recombinant adenovirus released from the cells of step (3);

(5) Performing amplification culture on the recombinant adenovirus in the step (4);

(6) Purifying the culture product in step (5).

Optionally, the shuttle plasmid vector of step (1) is pADV-mcv-MCS-3 xFLAG; the recombinant shuttle plasmid vector is obtained by inserting a DNA molecule between EcoRI and XbaI of pADV-mcV-MCS-3 xFLAG; the nucleotide sequence (5 '-3') of the DNA molecule is shown in SEQ ID NO:1 is shown in the specification;

alternatively, the backbone plasmid of step (2) is pBHGloxdelE13cre.

The recombinant adenovirus is a human type 5 replication-defective virus; in particular Ad5-FMDV-A.

The technical scheme of the invention has the following advantages:

the invention obtains a recombinant A-type foot-and-mouth disease vector virus strain which can correctly express A-type foot-and-mouth disease virus (A/Wuhan) structural protein P1 and partial non-structural protein (2A-2B-3B (partial protein) -3C protein) and takes human replication defective adenovirus as a vector. Experiments of vaccines prepared by the strain in vitro and in animals prove that the structural gene P1 carried by the strain can be effectively cut, and form A-type foot-and-mouth disease (A/Wuhan) virus-like particles (VLPs). The experimental result of mice (C57 BL/6) shows that the vaccine has good immunogenicity, and can induce animals to generate specific humoral and cellular immunity in a short period of time.

The A-type foot-and-mouth disease vaccine using the human replication defective recombinant adenovirus as a vector provided by the invention has the following characteristics:

(1) Has good immunogenicity, and can induce animals to generate humoral and cellular immunity after intramuscular injection.

(2) The vaccine takes human type 5 replication defective adenovirus as a vector, does not replicate in vivo after immunization of animals, and has good safety.

(3) The vaccine does not need to add any adjuvant component.

(4) The neutralizing antibodies can be induced in the high, medium and low dose groups 7 days after immunization. After 21 days of immunization, the antibody titers of the dose groups increased continuously and exhibited significant dose dependence; the difference between the high, medium and low dose groups was significant (P < 0.05). The results show that: after one immunization, strong neutralizing antibody levels were induced and the antibody titers exhibited a clear dose-dependence.

(5) The vaccine can be produced in large scale under the biosafety level 2 condition, and the biosafety risk possibly caused by using the foot-and-mouth disease virulent strain for production is avoided.

Based on the characteristics, the invention can provide a safe and effective means for preventing, controlling and purifying foot-and-mouth disease in China, and provides a new choice for vast farmers to prevent and treat type A foot-and-mouth disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of Western-Blot detection of FMDV-A protein expression in example 1;

FIG. 2 shows the results of the detection of neutralizing antibody titers in example 2;

FIG. 3 shows CD3+, CD107a+ T cell ratio, CD3 in example 3 ⁺ ,CD4 ⁺ ,CD107a ⁺ T cell ratio, CD3 ⁺ ,CD8 ⁺ ,CD107a ⁺ T cell ratio results;

FIG. 4 is CD3 in example 3 ⁺ ，IL-2 ⁺ T cell ratio, CD3 ⁺ ，CD4 ⁺ ，IL-2 ⁺ T cell ratio, CD3 ⁺ ，CD8 ⁺ ，IL-2 ⁺ T cell ratio results;

FIG. 5 is CD3 in example 3 ⁺ ，TNF-α ⁺ T cell ratio, CD3 ⁺ ，CD4 ⁺ ，TNF-α ⁺ T cell ratio, CD3 ⁺ ，CD8 ⁺ ，TNF-α ⁺ T cell ratio results;

FIG. 6 is CD3 ⁺ ，IFN-γ ⁺ T cell ratio, CD3 ⁺ ，CD4 ⁺ ，IFN-γ ⁺ T cell ratio, CD3 ⁺ ，CD8 ⁺ ，IFN-γ ⁺ T cell ratio results;

FIG. 7 is Sup>A schematic representation of the recombinant shuttle vector pADV-mcv-FMDV-A-3 xFLAG.

Detailed Description

The preparation method of the dialysis buffer solution comprises the following steps: 50g sucrose, 10mL 1M Tris-HCl solution at pH 8.0, 2mL 1M MgCl ₂ The volume of the solution is fixed to 1000mL;

example 1 preparation of A-type foot-and-mouth disease vaccine Using human replication defective recombinant adenovirus (Ad 5-FMDV-A) as vector

Acquisition of Gene (one)

The P1 gene, the 2A gene, the 2B gene, the A/A24 strain 3B gene and the A/A24 strain 3C gene of the foot-and-mouth disease virus A/Wuhan strain are subjected to codon optimization respectively, so that the foot-and-mouth disease virus A/Wuhan strain is more suitable for expression in mammalian cells, and the optimized gene sequences are shown in the following table.

TABLE 1

The optimized gene sequences are added with an initiation codon and a termination codon, and the nucleotide sequence of a gene (5 '-3') formed according to the sequence of the initiation codon-P1-2A-2B-3B-3C-termination codon is shown as SEQ ID NO:1, the gene is named as FMDV-A, and the amino acid sequence of FMDV-A protein coded by the gene is shown as SEQ ID NO: 2. pMD18-T-FMDV-A was synthesized directly by Bio-Inc.

Construction of recombinant shuttle vector

The above pMD18-T-FMDV-A vector was digested with EcoRI and XbaI, and the digested product was recovered and ligated with the shuttle plasmid pADV-mCMV-MCS-3xFLAG (and Metro, cat. H225) of the AdMax adenovirus system digested with the same enzymes (EcoRI and XbaI). Coli DH5 alpha is competent by the ligation product transformation, LB plates (Amp resistance) are coated, bacterial liquid PCR identification is carried out by selecting monoclonal, and clones identified as positive are sent to a sequencing company for sequencing, and PCR identification and sequencing primers are shown in the following table. The recombinant shuttle vector with correct sequencing is named pADV-mcv-FMDV-A-3 xFLAG, and the map is shown in FIG. 7, and the vector is obtained by inserting FMDV-A gene between EcoRI and XbaI of the shuttle plasmid pADV-mcv-MCS-3 xFLAG.

TABLE 2

(III) packaging and identification of recombinant adenovirus Ad5-FMDV-A

1) The day before transfection, HEK293 cells are inoculated into a 6-well plate, and the density of the cells during transfection is controlled to be 70% -80%;

2) Taking out the cell culture plate 1 hour before transfection, removing the original cell culture medium, adding 1.5mL of Opti-MEM culture medium, and placing the cells back into the incubator;

3) Transfection:

preparation of plasmid dilutions: 4 mug of the viral vector plasmid to be transfected is dissolved in an Opti-MEM culture medium, the total volume is 250 mug, and the mixture is uniformly mixed; the virus vector plasmid to be transfected consists of a recombinant shuttle plasmid and a skeleton plasmid pBHGloxdelE13cre (belonging to AdMax adenovirus system); the backbone plasmid of the AdMax adenovirus system pBHGloxdelE13cre: the mass ratio of the recombinant shuttle plasmid is=1:1;

preparation of transfection reagent dilutions: mu.L of transfection reagent 2000 ^TM (life science) is dissolved in Opti-MEM culture medium, the total volume is 250 mu L, and the mixture is uniformly mixed to obtain the compound feed;

dripping the transfection reagent dilution liquid into the plasmid dilution liquid, uniformly mixing, and carrying out room temperature for 20min to obtain a DNA-transfection reagent complex;

taking out the cell culture plate, adding the DNA-transfection reagent complex prepared above into the cell culture plate, marking, and placing back into the incubator;

after 6h, the medium was aspirated, washed once with PBS, and 2mL of fresh complete medium was added for cultivation;

the liquid is changed once every three days, virus plaques appear in 7-15 days, the supernatant is collected after complete lesions, and the virus in the supernatant is the positive recombinant virus which is named as Ad5-FMDV-A.

Ad5-FMDV-A virus identification

Extracting Ad5-FMDV-A genome. The following primer pairs are used: f1:5'-AACGGAACCTCCAAATACAG-3' (SEQ ID NO: 5); and R1:5'-TTGCTCCGTAGTTGAAAGAG-3' (SEQ ID NO: 6) as a primer for PCR amplification. The PCR reaction system is as follows: 1. Mu.L of template, 0.5. Mu.L of primer upstream and downstream respectively (primer concentration: 5. Mu.M), 1. Mu.L of dNTP,DNA polymerase(NEB)0.5uL，5X Q 5reaction Buffer 10uL，ddH ₂ O 32.5uL。

PCR reaction conditions: 94 ℃ for 5min;94℃for 30s,56℃for 30s,72℃for 50s,30 cycles; and at 72℃for 5min. And (3) sequencing the recombinant viruses amplified by the PCR to be positive, and carrying out subsequent experiments, wherein the sequencing result is correct.

(IV) recombinant adenovirus Ad5-FMDV-A mass amplification and purification

HEK293 cells were plated in 40 plates of 10cm diameter until the cells grew to 70% -80% and each plate was added to the supernatant of the previous step (containing FMDV-A expressing recombinant virus at Sup>A titer of 10) ⁷ -10 ⁸ IFU/mL) 10. Mu.L, infected cells, after all lesions, 500. Mu.L of 10% (v/v) Nonidet P40 (Biyun) were added to each plate to lyse the cells. Cell lysates were collected and centrifuged at 12000rpm for 10min, and supernatants were collected. 50mL of a virus pellet (20% (v/v) PEG8000,2.5M NaCl) was added to each 100mL of the supernatant, the mixture was centrifuged at 12000rpm for 20min on ice to pellet the virus, the supernatant was discarded, the pellet was suspended in 10mL of CsCl solution (density: 1.10 g/mL), and the virus suspension was collected by centrifugation at 7000rpm at 4℃for 5min.

Addition to Beckman ultracentrifuge tube2.0mL of CsCl solution (1.40 g/mL). A further 3.0mL of CsCl solution (1.30 g/mL) was added. Finally, 5mL of virus suspension was added. 22800rpm,4℃for 2.5h. The virus bands with a density between 1.30-1.40g/mL were collected into 1mL dialysis bags (10 mM EDTA Na before use of the dialysis bag) ₂ Boiling for 10 min). The dialysis buffer was dialyzed overnight at 4℃with an intermediate exchange of the dialysis solution. The virus was collected and stored at-80 ℃.

(V) Western-Blot detection of FMDV-A protein expression

The purified recombinant adenovirus Ad5 FMDV A and positive control (foot-and-mouth disease inactivated virus) are subjected to 12% SDS-PAGE electrophoresis separation, rabbit Anti-foot-and-mouth disease VP1 enzyme-labeled antibody (Bioss: rabbit Anti-FMDV VP1 polyclonal Antibody bs-4521R) is used, sup>A color development solution (Beijing Soy Bao technology Co., ltd.) is added according to the specification for exposing and developing, and the expression of FMDV-A protein is detected. Actin served as an internal reference, actin antibody: solarbio K200058M.

The experimental results are shown in fig. 1: the sample has obvious color development and clear bands.

Example 2 detection of human replication defective recombinant adenovirus (Ad 5-FMDV-A) as vector A foot and mouth disease vaccine-induced humoral immune response in the organism

The immunization dose of the recombinant viral vector FMDV vaccine was studied on a mouse model and the humoral immunity induced by the vaccine was evaluated.

The experimental steps are as follows:

1. 60 female C57BL/6 mice of 5-8 weeks old, 5 female C57BL/6 mice/group, and 12 female C57BL/6 mice/group were selected and designated as a blank group, a negative control group (day 0, day 21), a high dose group, a medium dose group, and a low dose group (day 7, day14, day 21), respectively. The injection dosages and injection modes are shown in the following table.

TABLE 3 Table 3

The negative control group is that the recombinant virus in the high-dose group is replaced by virus diluent (0.01 MPBS), and the dose and the mode are unchanged; the blank group was not immunized. The negative control group, the high dose group and the medium dose group collect peripheral blood 7 days after immunization (i.e., day 7), 14 days (i.e., day 14), 21 days (i.e., day 21), and the negative control group collect peripheral blood 7 days after immunization (i.e., day 7), 21 days (i.e., day 21).

2. All collected peripheral blood is kept stand at 37 ℃ for 50min, centrifuged at 8000rpm for 15min, serum is collected, and the serum is stored at-80 ℃ after sub-packaging.

3. Neutralizing antibody titers were determined using a liquid phase blocking ELISA method, kit (purchased from the veterinary institute of lanzhou, 20201202134).

Test serum was split and diluted in a gradient with PBS buffer on a dilution plate. The initial dilution multiple is 4X, the subsequent multiple dilution is carried out, the volume after dilution is 50 mu L/hole per dilution gradient, 50 mu L/hole of antigen working solution is added, negative and positive control serum and virus antigen holes are added according to instructions, sealing plates are used, shaking is carried out, and incubation is carried out for 30 minutes at 37 ℃. PBST plate was washed 3 times, added with foot-and-mouth disease A type enzyme-labeled antibody working solution, 50. Mu.L/well, sealed and incubated at 37℃for 30 minutes. PBST plates were washed 3 times with solution A and B, 50. Mu.L/well, and developed for 15 minutes at 37 ℃. Adding stop solution 50 mu L/hole, and reading OD by enzyme label instrument ₄₅₀ Values. Positive controls and negative controls were operated according to instructions.

The experimental results are shown in fig. 2, and the results show that the high, medium and low dose groups can induce the generation of neutralizing antibodies 7 days after immunization. After 21 days of immunization, the antibody titers of the dose groups increased continuously and exhibited significant dose dependence; the difference between the high, medium and low dose groups was significant (P < 0.05). The results show that: after one immunization, strong neutralizing antibody levels were induced and the antibody titers exhibited a clear dose-dependence.

Example 3 detection of cellular immune response induced by A foot and mouth disease vaccine Using human replication defective recombinant adenovirus (Ad 5-FMDV-A) as vector

The immunization dose of Sup>A vaccine using human replication-defective recombinant adenovirus (Ad 5-FMDV-A) as Sup>A vector was studied in Sup>A mouse model, and the level of cellular immunity induced by the vaccine was evaluated.

The experimental steps are as follows:

1. 15, 5 and three groups of C57BL/6 5-8 week old mice were taken, and the groups were negative control group, experimental group (7 day) and experimental group (14 day). The specific injection doses and injection modes are shown in the following table.

TABLE 4 Table 4

Negative control group: the negative control group is obtained by replacing recombinant virus in experimental group with virus diluent (0.01 MPBS), and the injection dosage and injection mode are unchanged. The experimental group is: 2.5X10 ⁸ IFU/mL Ad5-FMDV-A was injected at Sup>A dose of 100. Mu.L by bilateral injection of the lateral thigh muscle of the mouse, 50. Mu.L on each side.

2. Mice were sacrificed 7 days post-immunization in experimental group (7 day), 14 days post-immunization in experimental group (14 day), and 14 days post-immunization in negative control group; taking spleens of mice in an experimental group and a negative control group, separating spleen lymphocytes, incubating lymphocyte surface staining antibodies, stimulating by using a cell stimulating solution, incubating, lysing erythrocytes after incubation, blocking Fc receptors, staining by surface antibodies, fixing cells, breaking membranes and staining by intracellular factor antibodies. Detection of CD3 ⁺ ，CD107a ⁺ T cells; CD3 ⁺ ,CD4 ⁺ ,CD107a ⁺ T cells; CD3 ⁺ ,CD8 ⁺ ,CD107a ⁺ T cells; CD3 ⁺ ，IL-2 ⁺ T cells, CD3 ⁺ ，CD4 ⁺ ，IL-2 ⁺ T cells; CD3 ⁺ ，CD8 ⁺ ，IL-2 ⁺ T cells; CD3 ⁺ ，IFN-γ ⁺ T cells, CD3 ⁺ ，CD4 ⁺ ，IFN-γ ⁺ T cells; CD3 ⁺ ，CD8 ⁺ ，IFN-γ ⁺ T cells; CD3 ⁺ ，TNF-α ⁺ T cells, CD3 ⁺ ，CD4 ⁺ ，TNF-α ⁺ T cells; CD3 ⁺ ，CD8 ⁺ ，TNF-α ⁺ T cells; all experimental procedures were performed as per BD flow cytometry antibody instructions.

The method comprises the following specific steps:

(a) Obtaining lymphocyte suspensions

The spleens of mice were stripped and placed in 1mL 1640 cell culture medium. The spleen was ground until the spleen was dissociated, and cells were washed with Stain Buffer into a centrifuge tube, filtered through a nylon mesh (200 mesh), and centrifuged at 200g for 5 minutes. Cells were resuspended with Stain Buffer and counted.

(b) Preparation of a solution for cell stimulation

Adding 2 mu L of activator into 1 x 10-6 cells, calculating the volume of the activator, and adding into 1640 cell culture medium containing 10% FBS.

(c) Lymphocyte surface staining antibody incubation

Every 1×10 ⁶ Adding 2 μl of CD107a IgG and CD107a antibody into cells, mixing, adding 5% CO at 37deg.C ₂ Is incubated for 4 hours in the incubator.

(d) Lymphocyte activation and incubation

Every 3×10 ⁶ Adding 2mL of stimulating solution into individual cells, mixing, and adding 5% CO at 37deg.C ₂ Is incubated for 6 hours in the incubator.

(e) Lysing erythrocytes

After incubation, the incubation was completed at 1X 10 ⁶ Each cell was lysed by adding 1mL of erythrocyte lysate (BD, cat# 555899) at room temperature for 15 minutes in the absence of light. Centrifuge at 200g for 5min, discard supernatant. 1mL of Stain Buffer was added and washed 1 time. Cells were resuspended with Stain Buffer at 100. Mu.L of 1X 10≡6 cells.

(f) Blocking Fc receptors

According to 1X 10 ⁶ mu.L of Fc Block was added to each cell and incubated at 4℃for 15 minutes. Cells were washed 1 time with Stain Buffer.

(g) Surface antibody staining

According to 1X 10 ⁶ mu.L of CD3 antibody, 1. Mu.L of CD4 antibody and 3. Mu.L of CD8 antibody were added to each cell, and after mixing, the cells were incubated at 4℃for 30 minutes. Cells were washed 2 times with Stain Buffer.

(h) Cell fixation and rupture of membranes

According to 1X 10 ⁶ The cells were resuspended by adding 100. Mu. L Staining Buffer to each cell, and 250. Mu.L of the fixed membrane-disrupting solution was added at 4 ℃Incubate for 20 minutes. Cells were washed 2 times with wash solution (BD, cat# 554714).

(i) Intracellular factor antibody staining

According to 1X 10 ⁶ mu.L IFN-gamma (BD, 554412) and isotype antibody (BD, cat# 554685) were added to each cell; according to 1X 10 ⁶ mu.L of IL-2 (BD, 554428) and isotype antibody (BD, cat# 556925) were added to each cell, and after mixing, incubated at 4℃for 30 minutes. Cells were washed 2 times with Stain Buffer.

(g) And (5) detecting on the machine.

The results showed that after 7 days of immunization (D7 vaccine group in FIG. 3), the experimental group had CD3+ and CD107a in the spleen cells ⁺ T cell ratio was significantly increased, CD3 ⁺ ,CD4 ⁺ ,CD107a ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,CD107a ⁺ T cell rate up-regulation was evident (P<0.05 A) is provided; after 14 days of immunization (D14 vaccine group in fig. 3), CD3 ⁺ ,CD8 ⁺ ,CD107a ⁺ The T cell ratio remained high, with a clear difference from the negative control group, see figure 3.

14 days after immunization (D14 vaccine group in FIG. 4), the experimental group had CD3 in the spleen cells ⁺ ，IL-2 ⁺ T cell ratio was significantly increased, CD3 ⁺ ,CD4 ⁺ ,IL-2 ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,IL-2 ⁺ T cell rate up-regulation was evident (P<0.05 A) is provided; after 7 days of immunization (D7 vaccine group in fig. 4), CD3 ⁺ ,CD4 ⁺ ,IL-2 ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,IL-2 ⁺ The T cell ratio was significantly different from the negative control group, see fig. 4.

14 days after immunization (D14 vaccine group in FIG. 6), the experimental group had CD3 in the spleen cells ⁺ ，IFN-γ ⁺ T cell ratio was significantly increased, CD3 ⁺ ,CD4 ⁺ ,IFN-γ ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,IFN-γ ⁺ T cell rate up-regulation was evident (P<0.05 A) is provided; after 7 days of immunization (D7 vaccine group in fig. 6), CD3 ⁺ ,CD4 ⁺ ,IFN-γ ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,IFN-γ ⁺ The T cell ratio was initially up-regulated compared to the negative control group (P>0.05). See fig. 6.

After 14 days of immunization (FIG. 5Medium D14 vaccine group), experimental group spleen intracellular CD3 ⁺ ，TNF-α ⁺ T cell ratio was significantly increased, CD3 ⁺ ,CD4 ⁺ ,TNF-α ⁺ T cells and CD3 ⁺ ,CD8 ⁺ ,TNF-α ⁺ T cell rate up-regulation was evident (P<0.05 A) is provided; after 7 days of immunization (D7 vaccine group in fig. 5), CD3 ⁺ ,CD4 ⁺ ,TNF-α ⁺ The T cell ratio was initially up-regulated (P < 0.05) compared to the negative control group, see FIG. 5.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

<110> Beijing micro-Bai biotechnology Co., ltd

<120> A type foot-and-mouth disease vaccine using human replication defective recombinant adenovirus as vector

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<170> SIPOSequenceListing 1.0

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tttggccaca tcgagaagct ggaactgccc accgaccata agggggtgta cggccagctg 540

gtggactcct tcgcctacat gcgcaacggg tgggatgtgg aggtgagtgc cgtgggcaac 600

cagtttaatg gcggatgtct gctggtcgct atggtgcctg agttcaagga gtttaccacc 660

cgcgagaagt accagctgac cctgtttccc caccagttta tcagtcccag gaccaacatg 720

acagcccaca tcaccgtgcc atacctgggc gtgaaccggt acgatcagta taacaagcat 780

aagccatgga cactggtggt catggtggtc tcccccctga ccacctcctc catcggggcc 840

tcccagatca aggtgtacac aaatatcgcc cctacccacg tgcacgtcgc cggagagctg 900

ccttccaaag agggcatcgt gcccgtcgcc tgttccgacg gatatggcgg cctggtgact 960

actgacccaa agaccgctga cccggcctac ggcatggtgt ataatccccc tagaactaac 1020

tacccaggca ggttcaccaa cctcctggat gtggccgagg cctgtcctac ctttctgtgc 1080

tttgacgacg gaaaaccgta cgtggtgacc cggactgacg agcagcggct gttggctaaa 1140

ttcgatctgt cacttgccgc caagcatatg agcaatacct acctgagcgg catcgcccag 1200

tactacgccc agtatagcgg caccatcaac ctgcacttta tgttcactgg gagcaccgac 1260

agcaaggccc ggtacatggt ggcctacgtg cctcctggag tcacggcccc acctgataca 1320

cccgagagag cagcacactg catccatgcc gagtgggata caggcctgaa ttccaagttt 1380

acctttagca tcccctacgt gagcgccgcc gattacgcct acaccgctag cgatgtggcc 1440

gatactacta atgtgcaggg ctgggtgtgc atctaccaga tcacccatgg gaaagccgag 1500

caggacaccc tcgtcgtgtc cgtgagcgct gggaaggact ttgagctgag actgcctatc 1560

gaccctcggg cccagaccac agccaccggc gagtccgctg atccagtgac aaccacagtg 1620

gagaactacg gcggcgagac acaggtgcag cgacggcacc acaccgatgt gagcttcatc 1680

atggaccgct tcgtgcagat caagcccgtg tcaccgaccc acgtgatcga tctgatgcag 1740

acccaccagc acggcctggt gggagccatg ctgagagccg ccacctacta cttctccgac 1800

ctcgaaatcg tcgtgaacca tacaggccgc ctgacctggg tgcctaatgg agccccagaa 1860

gccgccctgg acaacaccag taacccaact gcttaccaca aggctccctt caccagactc 1920

gccctgccct acaccgcacc ccatagagtg ctggccaccg tctacaacgg aacctccaaa 1980

tacagcgccc ccgctacccg gaggggcgac ctcgggtctc tcgccgccag actggccgcc 2040

cagctgcctg cctctttcaa ctacggagca attcgggcca ccgagatcca ggagctgctg 2100

gtgagaatga aacgggctga actctactgc cccaggcctc tgctggccgt ggaggtgacc 2160

tcccaggacc ggcataagca gaagatcatt gctccagcca agcagctgct gaacctggac 2220

ctcctgaagc tcgccggcga cgtggagagt aaccccggcc ctttcttctt ctccgacgtg 2280

cgctcaaact tcaccaaact ggtggagacc attaatcaga tgcaggagga tatgtccacc 2340

aagcacggcc ctgacttcag ccggctggtg tccgccttcg aggagctcgc caccggagtg 2400

aaggccatcc ggaacggcct cgacgaggcc aaaccatggt acaagctgat caagctcctc 2460

tccagactga gctgtatggc cgcggtcgcc gccaggtcca aggatccggt gctggtggct 2520

atcatgctgg ccgacaccgg actggagatc ctggactcca cctttgtggt gaagaagatt 2580

tccgactccc tgagctctct cttccacgtc cccgcccccg tctttagctt cggcgccccc 2640

gtgctgctgg ctgggctggt gaaggtggct agcagtttct tcaggagcac ccccgaggac 2700

ctggagcgcg ccgagaaaca gctgaaagcc cgcgacatta acgatatctt cgccattctg 2760

gatgacgtga acagcgagcc cgcccaccca ggcgacgaac agccccaggc tgaaggccct 2820

tacgccggcc ctctggagcg gcagcgcccc ctgaaggtga gggcaaagct gccccagcag 2880

gaaggcccct acgccggccc catggagcgg cagaagcccc tcaaggtgaa agctaaagcc 2940

ccagtggtgc gggaaggccc ctatgaggga cccgtgaaga agcccgtcgc cctgaaggtg 3000

aaggccaaga atctgatcgt gaccgagagc ggggcgccac ctaccgatct gcagaagatg 3060

gtcatgggca acaccaagcc cgtcgagctg atcctcgacg gcaagaccgt ggccatttgc 3120

tgtgccaccg gggtgtttgg gaccgcctac ctggtgcccc ggcatctgtt tgccgaaaag 3180

tacgataaga tcatgctgga cgggcgggcc atgaccgact ccgactatcg cgtctttgag 3240

ttcgagatca aggtcaaggg gcaggacatg ctgtccgacg ccgccctgat ggtgctgcac 3300

agggggaaca gggtccgcga catcaccaag cacttccgcg acacagcccg gatgaaaaag 3360

gggacacccg tcgtgggggt ggtgaacaac gccgacgtgg gacgcctgat cttcagcggc 3420

gaggccctga cctataagga tatcgtggtg tgcatggacg gcgacaccat gcccggcctg 3480

ttcgcctata aggccgctac caaggccggc tactgcgggg gggccgtgct cgccaaggac 3540

ggcgccgaca ccttcatcgt gggcacccac agcgccggcg gcaacggcta a 3591

<210> 2

<211> 1196

<212> PRT

<213> artificial sequence

<400> 2

Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser

1 5 10 15

Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln

20 25 30

Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser

35 40 45

Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln

50 55 60

Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu

65 70 75 80

Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu

85 90 95

Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr

100 105 110

Gln Ser Ser Val Gly Val Thr Tyr Gly Tyr Ser Thr Gly Glu Asp His

115 120 125

Val Ser Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala

130 135 140

Glu Arg Phe Phe Lys Lys His Leu Phe Asp Trp Thr Thr Asp Lys Pro

145 150 155 160

Phe Gly His Ile Glu Lys Leu Glu Leu Pro Thr Asp His Lys Gly Val

165 170 175

Tyr Gly Gln Leu Val Asp Ser Phe Ala Tyr Met Arg Asn Gly Trp Asp

180 185 190

Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu

195 200 205

Val Ala Met Val Pro Glu Phe Lys Glu Phe Thr Thr Arg Glu Lys Tyr

210 215 220

Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met

225 230 235 240

Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln

245 250 255

Tyr Asn Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro

260 265 270

Leu Thr Thr Ser Ser Ile Gly Ala Ser Gln Ile Lys Val Tyr Thr Asn

275 280 285

Ile Ala Pro Thr His Val His Val Ala Gly Glu Leu Pro Ser Lys Glu

290 295 300

Gly Ile Val Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val Thr

305 310 315 320

Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Met Val Tyr Asn Pro

325 330 335

Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala

340 345 350

Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val

355 360 365

Val Thr Arg Thr Asp Glu Gln Arg Leu Leu Ala Lys Phe Asp Leu Ser

370 375 380

Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln

385 390 395 400

Tyr Tyr Ala Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr

405 410 415

Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Val Pro Pro

420 425 430

Gly Val Thr Ala Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile

435 440 445

His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile

450 455 460

Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Val Ala

465 470 475 480

Asp Thr Thr Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His

485 490 495

Gly Lys Ala Glu Gln Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys

500 505 510

Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Ala Gln Thr Thr Ala

515 520 525

Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly

530 535 540

Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser Phe Ile

545 550 555 560

Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His Val Ile

565 570 575

Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Met Leu Arg

580 585 590

Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Asn His Thr

595 600 605

Gly Arg Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ala Ala Leu Asp

610 615 620

Asn Thr Ser Asn Pro Thr Ala Tyr His Lys Ala Pro Phe Thr Arg Leu

625 630 635 640

Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn

645 650 655

Gly Thr Ser Lys Tyr Ser Ala Pro Ala Thr Arg Arg Gly Asp Leu Gly

660 665 670

Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn Tyr

675 680 685

Gly Ala Ile Arg Ala Thr Glu Ile Gln Glu Leu Leu Val Arg Met Lys

690 695 700

Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Glu Val Thr

705 710 715 720

Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln Leu

725 730 735

Leu Asn Leu Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro

740 745 750

Gly Pro Phe Phe Phe Ser Asp Val Arg Ser Asn Phe Thr Lys Leu Val

755 760 765

Glu Thr Ile Asn Gln Met Gln Glu Asp Met Ser Thr Lys His Gly Pro

770 775 780

Asp Phe Ser Arg Leu Val Ser Ala Phe Glu Glu Leu Ala Thr Gly Val

785 790 795 800

Lys Ala Ile Arg Asn Gly Leu Asp Glu Ala Lys Pro Trp Tyr Lys Leu

805 810 815

Ile Lys Leu Leu Ser Arg Leu Ser Cys Met Ala Ala Val Ala Ala Arg

820 825 830

Ser Lys Asp Pro Val Leu Val Ala Ile Met Leu Ala Asp Thr Gly Leu

835 840 845

Glu Ile Leu Asp Ser Thr Phe Val Val Lys Lys Ile Ser Asp Ser Leu

850 855 860

Ser Ser Leu Phe His Val Pro Ala Pro Val Phe Ser Phe Gly Ala Pro

865 870 875 880

Val Leu Leu Ala Gly Leu Val Lys Val Ala Ser Ser Phe Phe Arg Ser

885 890 895

Thr Pro Glu Asp Leu Glu Arg Ala Glu Lys Gln Leu Lys Ala Arg Asp

900 905 910

Ile Asn Asp Ile Phe Ala Ile Leu Asp Asp Val Asn Ser Glu Pro Ala

915 920 925

His Pro Gly Asp Glu Gln Pro Gln Ala Glu Gly Pro Tyr Ala Gly Pro

930 935 940

Leu Glu Arg Gln Arg Pro Leu Lys Val Arg Ala Lys Leu Pro Gln Gln

945 950 955 960

Glu Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln Lys Pro Leu Lys Val

965 970 975

Lys Ala Lys Ala Pro Val Val Arg Glu Gly Pro Tyr Glu Gly Pro Val

980 985 990

Lys Lys Pro Val Ala Leu Lys Val Lys Ala Lys Asn Leu Ile Val Thr

995 1000 1005

Glu Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly

1010 1015 1020

Asn Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala

1025 1030 1035

Ile Cys Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro

1040 1045 1050

Arg His Leu Phe Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly

1055 1060 1065

Arg Ala Met Thr Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile

1070 1075 1080

Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val

1085 1090 1095

Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg

1100 1105 1110

Asp Thr Ala Arg Met Lys Lys Gly Thr Pro Val Val Gly Val Val

1115 1120 1125

Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu

1130 1135 1140

Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro

1145 1150 1155

Gly Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala Gly Tyr Cys Gly

1160 1165 1170

Gly Ala Val Leu Ala Lys Asp Gly Ala Asp Thr Phe Ile Val Gly

1175 1180 1185

Thr His Ser Ala Gly Gly Asn Gly

1190 1195

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<400> 3

ggtataagag gcgcgaccag 20

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

gaaatttgtg atgctattgc 20

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

aacggaacct ccaaatacag 20

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<400>6

ttgctccgta gttgaaagag 20

Claims (11)

1. The proteins as follows:

the amino acid sequence comprises the following components from the N end to the C end in sequence: the amino acid sequence of structural protein P1 of the A-type foot-and-mouth disease virus, the amino acid sequence of non-structural protein 2A of the A-type foot-and-mouth disease virus, the amino acid sequence of non-structural protein 2B of the A-type foot-and-mouth disease virus, the amino acid sequence of partial protein 3B of the A-type foot-and-mouth disease virus and the amino acid sequence of non-structural protein 3C of the A-type foot-and-mouth disease virus;

the DNA molecule encoding the protein is any one of the following:

1) The nucleotide sequence is SEQ ID NO:1 from position 4 to position 3588;

2) The nucleotide sequence is SEQ ID NO:1, and a DNA molecule shown in 1.

2. A biological material as defined in any one of the following:

1) A DNA molecule encoding the protein of claim 1;

2) An expression cassette, a recombinant vector or a recombinant microorganism comprising the DNA molecule of 1).

3. The biomaterial of claim 2, wherein the recombinant microorganism is a recombinant virus.

4. A biomaterial according to claim 3 wherein the recombinant virus is a recombinant adenovirus.

5. The biomaterial of claim 4, wherein the recombinant adenovirus is a human replication-defective recombinant adenovirus.

6. The biomaterial of claim 5, wherein the human replication-defective recombinant adenovirus is a human replication-defective adenovirus of type 5.

7. A foot-and-mouth disease virus vaccine, the active ingredient of which is the recombinant adenovirus of claim 4.

8. Use of a protein according to claim 1, or a biomaterial according to any one of claims 2 to 6, for the preparation of a vaccine for the prevention of foot and mouth disease virus.

9. The use according to claim 8, wherein the foot-and-mouth disease virus is type a foot-and-mouth disease virus;

the vaccine is in the form of injection, nose drops or spray.

10. The use according to claim 9, wherein,

the vaccine is a monovalent vaccine, a bivalent vaccine or a trivalent vaccine; the monovalent vaccine is a monovalent vaccine for foot-and-mouth disease; the bivalent vaccine is an O-type and A-type foot-and-mouth disease bivalent vaccine; trivalent vaccines are trivalent vaccines for foot and mouth disease type O, type a and asian type 1.

11. A method of producing a recombinant adenovirus, said method comprising the steps of:

(1) Constructing a recombinant shuttle plasmid vector comprising a DNA molecule encoding the protein of claim 1;

(2) Transfecting the recombinant shuttle plasmid vector of the step (1) together with an adenovirus backbone plasmid into a host cell;

(3) Culturing the host cell of step (2);

(4) Harvesting the recombinant adenovirus released from the cells of step (3);

(5) Performing amplification culture on the recombinant adenovirus in the step (4);

(6) Purifying the culture product in step (5).