CN114437236B

CN114437236B - Recombinant African swine fever virus multi-epitope fusion protein, preparation and application thereof

Info

Publication number: CN114437236B
Application number: CN202210024635.XA
Authority: CN
Inventors: 邵军军; 张光磊; 刘伟; 杨思成; 常惠芸
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2023-06-27
Anticipated expiration: 2042-01-11
Also published as: CN114437236A

Abstract

The invention relates to a recombinant African swine fever virus multi-epitope fusion protein, a preparation method and an application thereof, and belongs to the field of biotechnology pharmacy. The recombinant African swine fever virus multi-antigen epitope fusion protein has a general formula: n-segment amino acid sequence of p72 epitope 1- (Linker) N-p72 epitope 2- (Linker) N-p72 epitope 3- (Linker) N-p72 epitope 4- (Linker) N-pE 248R. The recombinant protein has high expression and can be identified by African swine fever virus positive serum. The vaccine prepared by the recombinant protein can excite organisms to generate high-level specific antibodies and cellular immune responses, and can be used for preparing African swine fever subunit vaccine.

Description

Recombinant African swine fever virus multi-epitope fusion protein, preparation and application thereof

Technical Field

The invention belongs to the field of biotechnology pharmacy, and particularly relates to a recombinant African swine fever virus multi-epitope fusion protein, a preparation method and an application thereof.

Background

African swine fever (African swine fever, ASF) is an acute infectious disease of pigs caused by African swine fever virus (African swine fever virus, ASFV), the death rate can reach 100%, and no commercial vaccine exists at present. The world animal health organization (World Organization for Animal Health, OIE) lists it as an animal epidemic that must be reported, and our country lists it as a class of animal infectious diseases that is of major defense.

Because of the huge ASFV genome, numerous encoding proteins and complex virus structure, no safe and effective vaccine is used for epidemic prevention and control to date. Previous experience in ASF vaccine studies has shown that: inactivated vaccines induce higher levels of humoral immune responses but fail to provide immune protection; (2) Although it is possible to provide immune protection against homologous or heterologous strains after ASF attenuated live vaccination, vaccinated animals are prone to adverse reactions and risk of virulence reversion. (3) Some ASFV antigen subunits can induce to generate neutralizing antibodies, and provide partial immune protection, and the vaccine has the outstanding advantages of high safety and easy differential diagnosis, thus providing possibility for developing safe and effective ASF vaccine. With the continuous development of adaptive immune responses to ASFV, protective antigens and research of new adjuvants, the design of effective ASF subunit vaccines that induce protective antibodies and specific cellular immunity is one of the hot areas of research.

p72 is an important structural protein of ASFV, and is a major component constituting a viral particle capsid, encoded by gene B646L, and has a molecular weight of 73.2 kDa. p72 is capable of inducing the production of neutralizing antibodies that inhibit infection of host cells by ASFV. Studies have shown that these neutralizing antibodies are induced by some important epitopes on p 72. Another structural protein of interest is pE248R, which has been shown by studies to be a major endocapsule protein, a potential antigen for inducing a protective immune response.

Disclosure of Invention

In order to develop a safe and effective ASF vaccine, 4 epitopes of an ASFV structural protein p72 and N-segment amino acid sequences of pE248R are connected in series through a connecting peptide (Linker) to obtain a recombinant multi-epitope fusion protein, which is named as recombinant protein PPE, the vaccine containing the recombinant protein PPE can induce to generate high-level anti-p 72 and anti-pE 248R IgG antibodies and specific cell immunity after being inoculated into mice, and the induced antibodies obviously inhibit ASFV from infecting host cells in vitro, so that the recombinant protein PPE can be used for preparing ASF subunit vaccines.

The invention adopts the following specific scheme:

the invention provides a recombinant multi-epitope fusion protein, which is formed by fusing 4 epitopes of ASFV structural protein p72 and an N-segment amino acid sequence of pE248R through a connecting peptide (Linker), and has the following general formula: p72 epitope 1- (Linker) _n -p72 epitope 2- (Linker) _n -p72 epitope 3- (Linker) _n -p72 epitope 4- (Linker) _n -N-stretch of amino acid sequence of pE 248R;

wherein, the amino acid sequence of the p72 epitope 1 is shown in SEQ ID NO:3, the amino acid sequence of the p72 epitope 2 is shown as SEQ ID NO:4, the amino acid sequence of the p72 epitope 3 is shown as SEQ ID NO:5, the amino acid sequence of the p72 epitope 4 is shown as SEQ ID NO:6 is shown in the figure; the connecting peptide sequence is GGGGS, and n is 1, 2, 3 or 4.

As a further optimization of the fusion protein, the connecting peptide sequence is GGGGS, and n is 3.

As a further optimization of the above fusion protein, the amino acid sequence of the N-segment of the ASFV structural protein pE248R is SEQ ID NO: shown at 7.

As a further optimization of the scheme, the amino acid sequence of the recombinant multi-antigen epitope fusion protein is shown as SEQ ID NO: 2. Preferably, the nucleotide sequence encoding the recombinant protein is SEQ ID NO:1.

the second object of the present invention is to provide an expression vector formed by ligating a nucleotide sequence encoding the above fusion protein with a backbone plasmid; preferably, the backbone plasmid is pET-30a (+).

The third object of the present invention is to provide a preparation method of the recombinant protein, which comprises the following steps:

(1) And (3) constructing a carrier: constructing the expression vector;

(2) Screening of transformed and positive clones: transforming the expression vector in the step (1) into host bacteria, and obtaining a strain capable of expressing target proteins through induction and SDS-PAGE identification;

(3) Induction of expression: transferring the positive strain in the step (2) to grow to a certain concentration, and adding IPTG to perform induction expression;

(4) Protein purification: collecting thalli in the step (3), and obtaining target protein through ultrasonic crushing, inclusion body washing and dissolving, ni chelate affinity chromatography purification and dialysis renaturation;

(5) Identification of recombinant proteins: the recombinant protein obtained in (4) was identified by SDS-PAGE and Western blotting.

The fourth object of the invention is to provide the application of the recombinant protein in the preparation of ASF vaccine.

The fifth object of the present invention is to provide an ASF subunit vaccine comprising the fusion protein described above. Preferably, the ASF subunit vaccine obtained by combining the fusion protein with an ISA206 adjuvant has better effect.

Due to the adoption of the technical scheme, the invention has the following advantages:

1. the PPE expression plasmid can induce expression in a prokaryotic expression system (escherichia coli), and has the advantages of high expression quantity and easy purification;

2. when selecting pET vector series, expressing the recombinant protein PPE in the form of fusion protein; the C end of the recombinant protein contains a 6 XHis tag with the molecular weight of only 0.84 kDa, has no influence on the function of the fusion protein, and ensures that the purification step is simple, and the purity of the recombinant protein PPE obtained by one-step purification is more than 90 percent;

3. the recombinant protein PPE is capable of inducing high levels of specific antibodies and cellular immunity in vaccinated animals.

4. The recombinant protein PPE prepared by the invention can be immunized by subcutaneous (intramuscular) injection to excite the organism to generate high-titer IgG antibodies. Neutralization experiments prove that the antibody induced by the vaccine prepared by the recombinant protein can inhibit ASFV from infecting host cells in vitro, and has value as an ASF subunit vaccine.

Drawings

FIG. 1 shows the results of double digestion identification of the recombinant expression vector pET-30a (+) -PPE in the present invention. Lane 1: pET-30a (+) -PPENdeI-XhoIDouble enzyme digestion, lane 2: pET-30a (+) -PPE; lane M: DNA molecular weight standard (Marker);

FIG. 2 shows the SDS-PAGE results of recombinant protein PPE induced expression and purification in the examples of the present invention. Lane 1: before the induction of the positive strain; lane 2: protein molecular weight standard (Marker); lane 3: after the induction of the positive strain; lane 4: the thalli are crushed and then deposited; lane 5: dissolving inclusion bodies; lane 6: purified PPE;

FIG. 3 shows Western blotting results of recombinant protein PPE identified in the examples of the present invention using 6 XHis monoclonal antibody (A) and ASFV positive serum (B). Lane 1: bovine serum albumin; lane 2: purified PPE; lane M: protein molecular weight standard (Marker);

FIG. 4 dynamic attenuation of p 72-specific IgG in serum at various time points after initial immunization of mice in an embodiment of the invention;

FIG. 5 dynamic attenuation of pE 248R-specific IgG in serum at various time points after the first immunization of mice in an embodiment of the invention;

FIG. 6 shows the levels of cytokines secreted by spleen lymphocytes 28 days after initial immunization of mice in the examples of the invention;

FIG. 7 shows lymphocyte proliferation levels of spleen lymphocytes after antigen stimulation 28 days after first immunization of mice in an embodiment of the invention;

FIG. 8 shows the genomic copy number (A) and the viral neutralization rate (B) of ASFV cultured after incubation of ASFV with serum 28 days after primary immunization of mice in the present invention.

Detailed Description

The invention connects the coding genes of 4 antigen epitopes in ASFV structural protein p72 and the coding genes of N-segment amino acid sequences of pE248R in series to form a multi-antigen epitope fusion DNA sequence, and the recombinant multi-antigen epitope fusion protein (named as recombinant protein PPE) is obtained through prokaryotic expression and purification, and the vaccine containing the recombinant protein PPE can induce to generate high-level anti-p 72 and anti-pE 248R IgG antibodies and specific cellular immunity after receiving mice, and the induced antibodies obviously inhibit ASFV from infecting host cells in vitro, thereby prompting that the recombinant protein PPE can be used for preparing ASF subunit vaccine.

For a better understanding of the present invention, the following description will further explain the present invention in conjunction with specific embodiments, but the present invention is not limited to the following examples.

Construction and identification of recombinant plasmids

The verified 4 ASFV p72 epitopes (epitopes ID 141844, 141941, 141989, 142069) in the The Immune Epitope Database (IEDB) database were passed (GGGGS) ₃ The flexible Linker is sequentially connected in series, and the p72 epitope after the connection is passed through (GGGGS) ₃ The flexible Linker is connected with N-segment amino acid sequence (Met 1-Lys 198) of ASFV-SY18 structural protein pE248R (protein ID: AYW 34102.1) in series to obtain amino acid sequence of ASFV multi-epitope antigen fusion protein PPE, the sequence is converted into nucleotide sequence according to codon preference of colibacillus, and specific enzyme cutting sites are respectively introduced at 5 'end and 3' endNdeIAndXhoIthe recombinant plasmid pET-30a (+) -PPE is obtained by entrusting the synthesis of Nanjing Jinsri biotechnology Co.Ltd and cloning the recombinant plasmid into pET-30a (+) expression vector. The recombinant plasmid is transformed into DH5 alpha, positive clones are selected and inoculated into a kana-resistant LB culture medium, and shake culture is carried out at 37 ℃ for overnight. According to the steps of the specification, plasmids of positive clones were extracted using a rapid plasmid miniprep kit. By passing throughNdeIAndXhoIenzyme cutting is carried out in a 37 ℃ water bath for 1.5 hours. The system is as follows: recombinant plasmid 5 mu L,NdeI1 mu L of enzyme,XhoIEnzyme 1 [ mu ] L, 10X Cutsmart buffer 1 [ mu ] L, ddH ₂ O2 [ mu ] L. And respectively adding 2.5 mu L of 5 xLoding buffer into the enzyme digestion reaction system, and observing enzyme digestion results by a UV scanner after electrophoresis for 25 minutes by using 1.0% agarose gel 130V.

The experimental results are shown in fig. 1: the size of the recombinant plasmid pET-30a (+) -PPE before enzyme digestion is more than 5000 bp, which accords with the expectations. Warp yarnNdeIAndXhoIafter double digestion, the recombinant plasmid pET-30a (+) -PPE is cut into 2 fragments, the large fragment about 5200 bp is the pET-30a (+) part of the expression vector, the small fragment about 1400 bp, and the fragment encoding the PPE is inserted, which indicates pET-30a (+) -for insertionThe construction of the PPE recombinant plasmid was successful, and the sequencing result of the recombinant plasmid further confirmed the conclusion.

Induction expression and purification of recombinant protein PPE

The positive recombinant plasmid pET-30a (+) -PPE is transformed into competent cells of escherichia coli BL21 (DE 3), and positive clones are selected and inoculated into 5 mL-Caragana-resistant LB culture medium for culture at 37 ℃ and 220 rpm. OD of the liquid to be sterilized ₆₀₀ When reaching 0.6-0.8, 1mL bacterial liquid is remained and transferred into a sterilizing EP tube to be used as a strain for expansion culture, the rest 4 mL bacterial liquid is averagely divided into 2 parts, one part is added with 1 mM IPTG for induction, the other part is used as a control, the culture is carried out for 4 hours at the temperature of 37 ℃ and 220 rpm, and the bacterial bodies are respectively collected for SDS-PAGE detection (as shown in figure 2), and the result shows that the bacterial bodies induced by the IPTG have obvious bands at the 48 kDa and are consistent with the expected size of the recombinant protein PPE. The 1mL bacterial liquid is inoculated into LB culture medium with 1L Carna resistance according to the ratio of 1:100 after the propagation, and cultured at 37 ℃ and 220 rpm. OD of the liquid to be sterilized ₆₀₀ When reaching 0.6-0.8, 1 mM IPTG was added for induction, and the culture was continued at 220 rpm at 37℃for 4 hours. 8000 The cells were collected by centrifugation at rpm for 7 minutes, and 50 mL binding buffer (300 mM NaCl, 20 mM NaH) was added ₂

PO

₄ 5. 5 mM imidazole, pH 8.0), subjecting the cells to ultrasonic lysis for 40 min, centrifuging at 10000 rpm for 25 min, collecting inclusion bodies and supernatant, and performing SDS-PAGE electrophoresis detection to show that the recombinant protein PPE is expressed as inclusion bodies. The inclusion bodies were dissolved in a binding buffer containing 8M urea, and the supernatant collected by centrifugation at 10000 rpm for 25 min was bound to Ni-excel affinity medium at 4℃for 2 hours, and washed with 20 mL wash buffer (8M urea, 300 mM NaCl, 20 mM NaH) ₂

PO

₄ 20. 20 mM imidazole, pH 8.0) and finally eluting with 20 mL elution buffer (8M urea, 300 mM NaCl, 20 mM NaH) ₂

PO

₄ 500. 500 mM imidazole, pH 8.0) to elute the bound recombinant protein. The eluted recombinant protein was sequentially washed in 8 M,6 M,4 M,2 M,0M-containing dialysate (20 mM NaH ₂ PO ₄ Renaturation in 300 mM NaCl, 2 mM beta-mercaptoethanol, 0.4% arginine, 10% glycerol, pH 7.5), collecting renatured recombinant protein, taking part of the product for SDS-PAGE detection, and the result shows that: renatured recombinant protein moleculesThe amount was 48 kDa, consistent with the size of the protein induced by the positive strain transformed with the recombinant plasmid, as shown in FIG. 2.

Concentration determination and Western blotting identification of recombinant protein PPE

The concentration of recombinant protein PPE was measured by Bradford method, 200 ng recombinant protein PPE was mixed with 5 x loading buffer and boiled for 10 minutes, SDS-PAGE (80V 30 minutes, 120V 1 hour 10 minutes) was performed, then electrotransferred onto nitrocellulose membrane (PVDF), and blocked with 5% nonfat milk powder at room temperature for 2 hours. Incubation resistance: the blocked PVDF membrane was incubated overnight at 4℃with murine anti-6 XHis monoclonal antibody (1:5000 dilution) and ASFV positive porcine serum (1:300 dilution), respectively, and washed 6 times with 1 XPBST. Secondary antibody incubation: peroxidase-labeled goat anti-mouse IgG (1:5000-fold dilution) and goat anti-pig IgG (1:5000-fold dilution) were added to the corresponding PVDF membranes, respectively, incubated for 1 hour at room temperature, and washed 6 times with 1 XPBST. Color development: ECL luminous liquid is prepared according to the instruction, uniformly paved on a PVDF film, after color development, the reaction is stopped by using deionized water, and the image is acquired by a multifunctional imager.

The Western blotting result is shown in figure 3, and the recombinant protein PPE can be recognized by a mouse anti-6 XHis monoclonal antibody and has good reactivity with ASFV positive serum, so that the recombinant multi-antigen epitope fusion protein has potential application value.

Vaccine preparation and mouse immunization protocol

Diluting the renaturated recombinant protein PPE to 200 mug/mL, dividing the PPE into 2 parts averagely, mixing 1 part with an equal volume of ISA206, and fully emulsifying to prepare a water/oil/water dosage form vaccine; the other part is mixed with PBS with equal volume to prepare the vaccine without adjuvant. ISA206 adjuvant was also set up as a negative control mixed with an equal volume of PBS, PBS as a blank. 20 female BALB/c mice of SPF class 6-8 weeks old were randomly divided into 4 groups (5/each group), and the prepared vaccine was boosted 1 time 14 days after priming by subcutaneous multipoint immunization, according to the immunization group and immunization dose in the following table.

Group of	Immune component/Only	Injection volume uL/only
			Experiment group 1	20 µg PPE	200
Experiment group 2	20 µg PPE+ISA206	200
			Negative control	ISA 206	200
Blank control	PBS	200

Specific IgG detection

All immunized BALB/c mice were bled for

tail vein blood

7, 14, 21 and 28 days before and after the first immunization, the serum was isolated by centrifugation at 4000 rpm for 10 minutes, and the immune serum was detected by established p72 and pE248R indirect ELISA. The specific operation is as follows: purified recombinant p72 and pE248R (expressed and purified separately) were diluted to 1 μg/mL with coating buffer (carbonate buffer, pH 9.6) 100 μl per well overnight at 4 ℃; the coating liquid is discarded, PBST containing 5% skimmed milk powder is added for sealing (200 mu L/hole), and the mixture is incubated for 2 hours at 37 ℃; the blocking solution was discarded, the plate was washed 5 times with 1 XPBST, and 1:100 dilution of immune miniprepMouse serum, 100 μl/well, incubated for 1 hour at 37 ℃; the liquid is discarded, the plate is washed 5 times by 1 XPBST, HRP marked goat anti-mouse IgG diluted by 1:10000 is added, 100 MuL/hole is incubated for 1 hour at 37 ℃; removing liquid, washing the plate for 5 times by using 1 XPBST, adding TMB substrate color development liquid, 100 mu L/hole, and keeping out of light at 37 ℃ for 10-15 min; adding 2M H ₂ SO ₄ The reaction was stopped (100. Mu.L/well) and the OD at 450nm was determined.

Results and analysis: as shown in fig. 4 and 5, neither adjuvant immunized nor PBS groups detected specific antibodies to p72 and pE248R during immunization, no statistical differences between groups (p>0.05). The individual immunization group of the recombinant protein PPE and the ISA206 can detect specific IgG antibodies aiming at p72 and pE248R 7 days after the first immunization, compared with the first immunization, the antibody level after the boosting is obviously increased, and the specific IgG antibody level of the anti-p 72 and anti-pE 248R of the recombinant protein PPE and the ISA206 group is obviously higher than that of the individual immunization group of the recombinant protein PPEp <0.001). The result shows that the recombinant protein PPE has good immunogenicity and better immune effect after being compatible with ISA 206.

Lymphocyte secretion cytokine level assay

28 days after the first immunization, 3 mice were randomly selected from each group to be euthanized by dislocation, and soaked in 75% alcohol for about 5 minutes; taking out spleen aseptically in an ultra clean bench, soaking in RPMI-1640 culture medium, and lightly grinding with 5mL syringe piston to obtain spleen cell suspension; centrifuging at 1500rpm for 5min, discarding supernatant, adding 2mL of erythrocyte lysate to cell pellet, and performing room temperature lysis for 3 min; the reaction was then stopped by adding 10mL of PBS, the cells were collected by centrifugation at 1500rpm for 5 minutes and washed 2 times with PBS; the collected spleen cells were resuspended in RPMI-1640 medium containing 10% FBS and the cell density was adjusted to 1X 10 ⁶ /mL; then inoculating 12-well plates, 1mL each, adding purified recombinant protein p72 and pE248R at a final concentration of 5. Mu.g/mL, 5% CO at 37 ℃ ₂ Culturing for 72 hours, collecting supernatant, and detecting the content of IL-2, IFN-gamma and TNF-alpha in the supernatant by using a mouse cell seed detection kit.

As shown in FIG. 6, recombinant protein PPE+ISA206 immune group and recombinant protein PPE alone immune groupAfter in vitro stimulation, spleen lymphocytes evenly secrete IL-2, IFN-gamma and TNF-alpha, but the immune group of PPE+ISA206 is obviously higher than the independent immune group of recombinant protein PPEp <0.001 A) is provided; the levels of IL-2, IFN-gamma and TNF-alpha were low in both the adjuvant alone immunized group and the PBS control group compared to the experimental group after in vitro stimulation. Experimental results prove that: the vaccine prepared by compatibility trial of the recombinant protein PPE and the ISA206 can obviously activate immune cells after immunization, and can improve the secretion level of cytokines.

1.7 lymphocyte proliferation assay

Spleen cell isolation As described in 2.6, the isolated spleen cells were adjusted to 5X 10 ⁵ Per mL, 96-well plates (100 μl/well) were inoculated, and then p72 and pE248R recombinant proteins were added at a final concentration of 2.5 μg/mL, while RPMI-1640 medium control, normal cell control and ConA positive control were established. 37. DEG C5% CO ₂ After culturing for 72 hours, 10 mu L of CCK8 reaction solution and 5% CO at 37 ℃ are added into each hole ₂ After incubation for 3.5 hours, OD was detected _450nm Stimulation index si= (experimental group OD) ₄₅₀ Blank OD ₄₅₀ ) /(negative control OD) ₄₅₀ Blank OD ₄₅₀ )。

As shown in FIG. 7, the recombinant protein PPE+ISA206 immunized group spleen lymphocytes have the highest proliferation level (percentage) of lymphocytes after in vitro antigen stimulation, and the average value of lymphocyte stimulation indexes is more than 3 and obviously higher than that of other three groups of spleen lymphocytesp <0.001). The independent immune group of the recombinant protein PPE is obviously higher than the adjuvant immune groupp <0.01 And PBS groupp <0.05). Experimental results prove that: the level of the recombinant protein PPE which is matched with the ISA206 adjuvant to induce the cellular immune response is obviously superior to that of a group without the adjuvant, which indicates that the cellular immune response capability of the recombinant protein can be obviously improved by matching with the adjuvant.

Serum neutralization assay

Neutralization assays were performed with serum before and 28 days after the first immunization, and were performed as follows: serum was subjected to 1 respectively with sterilized PBS: 5 dilution and filter sterilized with a 0.22 μm needle filter, inactivated in a water bath at 56℃for 30 min. ASFV-CN/SC/2019 (MOI=0.01, department of agricultural China)BSL-3 laboratory store at the institute of veterinary, academy of langerhans) was mixed with inactivated mouse serum overnight at 37 ℃. The overnight virus mixture was inoculated into PAM monolayers, 200 μl per well (24 well plate), incubated for 1 hour at 37 ℃ and gently shaken 1 every 10 minutes. The virus serum mixture was discarded, washed 3 times with sterile PBS, followed by the addition of 500. Mu.L of RPMI-1640 containing 5% FBS, 5% CO at 37 ℃ ₂ Culturing for 48 hours. Collecting cultured cells, respectively extracting ASFV genome of each hole by using a virus genome extraction kit, amplifying ASFV genes by using a qPCR kit, calculating copy numbers of the ASFV genome in each sample according to Ct values and established standard curves, and determining virus neutralization rate: viral neutralization (%) = 100-100 x post-immune serum incubation ASFV copy number/post-immune serum incubation copy number of ASFV genome.

As shown in FIG. 8A, compared with preimmune serum, the recombinant protein PPE alone immune group and the recombinant protein PPE+ISA206 immune serum can obviously reduce the copy number of ASFV genomep <0.001 The average inhibition of ASFV in vitro was calculated to be 71.5% and 78.4% for both immune sera based on ASFV genome copy number (fig. 8B). No significant difference between genome copy number of ASFV and serum before immunizationp >0.05 I.e. no neutralizing antibodies in serum. The serum of the recombinant protein PPE independent immune group and the serum of the recombinant protein PPE+ISA206 group can obviously inhibit the ASFV from infecting PAM in vitro, but the virus inhibition rate of the recombinant protein PPE+ISA206 group is obviously higher than that of the recombinant protein PPE independent immune groupp <0.01). The recombinant protein PPE constructed by the invention induces the ASFV neutralizing antibody, and the ASFV neutralizing antibody level is further improved by matching with an ISA206 adjuvant, so that the recombinant protein PPE is an important immunoprotection recombinant protein form.

It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.

SEQUENCE LISTING

<110> the animal doctor institute of Lanzhou, china academy of agricultural sciences

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<400> 6

Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro Ile

1 5 10 15

Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asn Pro

20 25 30

<210> 7

<211> 198

<212> PRT

<213> ASFV

<400> 7

Met Gly Gly Ser Thr Ser Lys Asn Ser Phe Lys Asn Thr Thr Asn Ile

1 5 10 15

Ile Ser Asn Ser Ile Phe Asn Gln Met Gln Ser Cys Ile Ser Met Leu

20 25 30

Asp Gly Lys Asn Tyr Ile Gly Val Phe Gly Asp Gly Asn Ile Leu Asn

35 40 45

His Val Phe Gln Asp Leu Asn Leu Ser Leu Asn Thr Ser Cys Val Gln

50 55 60

Lys His Val Asn Glu Glu Asn Phe Ile Thr Asn Leu Ser Asn Gln Ile

65 70 75 80

Thr Gln Asn Leu Lys Asp Gln Glu Val Ala Leu Thr Gln Trp Met Asp

85 90 95

Ala Gly Thr His Asp Gln Lys Thr Asp Ile Glu Glu Asn Ile Lys Val

100 105 110

Asn Leu Thr Thr Thr Leu Ile Gln Asn Cys Val Ser Ser Leu Ser Gly

115 120 125

Met Asn Val Leu Val Val Lys Gly Asn Gly Asn Ile Val Glu Asn Ala

130 135 140

Thr Gln Lys Gln Ser Gln Gln Ile Ile Ser Asn Cys Leu Gln Gly Ser

145 150 155 160

Lys Gln Ala Ile Asp Thr Thr Thr Gly Ile Thr Asn Thr Val Asn Gln

165 170 175

Tyr Ser His Tyr Thr Ser Lys Asn Phe Phe Asp Phe Ile Ala Asp Ala

180 185 190

Ile Ser Ala Val Phe Lys

195

Claims

1. The recombinant multi-epitope fusion protein is formed by fusing 4 antigen epitopes of ASFV structural protein p72 and an N-segment amino acid sequence of pE248R through a connecting peptide (Linker), and has the following general formula: p72 epitope 1- (Linker) _n -p72 epitope 2- (Linker) _n -p72 epitope 3- (Linker) _n -p72 epitope 4- (Linker) _n -N-stretch of amino acid sequence of pE 248R;

wherein, the amino acid sequence of the p72 epitope 1 is shown in SEQ ID NO:3, the amino acid sequence of the p72 epitope 2 is shown as SEQ ID NO:4, the amino acid sequence of the p72 epitope 3 is shown as SEQ ID NO:5, the amino acid sequence of the p72 epitope 4 is shown as SEQ ID NO:6 is shown in the figure; the connecting peptide sequence is GGGGS, and n is 3; the amino acid sequence of the N section of the ASFV structural protein pE248R is SEQ ID NO: shown in figure 7;

the amino acid sequence of the recombinant multi-antigen epitope fusion protein is shown as SEQ ID NO: 2.

2. The fusion protein of claim 1, wherein: the nucleotide sequence encoding the fusion protein is SEQ ID NO:1.

3. an expression vector formed by a backbone plasmid modifiable ligation of a nucleotide sequence encoding the fusion protein of claim 1.

4. A preparation method of a recombinant multi-epitope fusion protein is characterized by comprising the following steps: the preparation method comprises the following steps:

(1) And (3) constructing a carrier: constructing the expression vector of claim 3;

(3) Induction of expression: transferring the positive strain in the step (2) to grow to a certain concentration, and adding IPTG to induce and express the recombinant protein;

(5) Identification of recombinant proteins: and (3) identifying the recombinant protein obtained in the step (4) by adopting SDS-PAGE and Western blotting.

5. Use of the fusion protein of claim 1 or 2 in the preparation of an ASF vaccine.

6. An ASF subunit vaccine comprising the fusion protein of claim 1 or 2.

7. The ASF subunit vaccine of claim 6, wherein the fusion protein is formulated with ISA206 adjuvant.