CN114456240B

CN114456240B - African swine fever virus genetic engineering subunit oral vaccine

Info

Publication number: CN114456240B
Application number: CN202210208640.6A
Authority: CN
Inventors: 蔡秀磊; 赵赛赛; 张洪亮; 单虎; 秦志华; 张皓杰
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2023-08-15
Anticipated expiration: 2042-03-04
Also published as: CN114456240A

Abstract

The invention provides an African swine fever virus genetic engineering subunit oral vaccine, which comprises genetic engineering lactococcus lactis for recombinant expression of African swine fever virus antigen protein, wherein the antigen protein is ASFV p30 protein with an amino acid sequence of SEQ ID NO. 1, ASFV p54 protein with an amino acid sequence of SEQ ID NO. 3 or ASFV p72 protein with an amino acid sequence of SEQ ID NO. 5. The antigen protein is connected with a heat-labile enterotoxin B subunit LTB gene protein of escherichia coli. The African swine fever virus genetic engineering subunit oral vaccine can effectively stimulate intestinal tract local immune cells to produce secretory IgA, stimulate organisms to produce mucous membrane reaction, and further stimulate the organisms to produce cellular immunity and humoral immunity through blood circulation.

Description

African swine fever virus genetic engineering subunit oral vaccine

Technical Field

The invention belongs to the technical field of vaccine preparation, and particularly relates to an African swine fever virus genetic engineering subunit oral vaccine.

Background

African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious, fatal animal infectious disease caused by African swine fever virus (African swine fever virus, ASFV). ASFV is the only member of the African swine fever virus family (Asfarviridae) African swine fever virus genus (Asfivirus), the only arbovirus currently known to have a DNA genome. The onset pigs mainly show body temperature rise, skin mucosa extensive bleeding, pregnant sow abortion and the like. African swine fever mortality rate is up to 100%, and serious harm is brought to pig industry.

ASF, unlike other animal epidemic diseases, has been shown to be over 100 years since the first outbreak in 1921. At present, the prevention measures for African swine fever mainly comprise strengthening supervision force, strengthening disinfection and source extinguishment and making biological safety protection measures, and once outbreaks are only completely eradicated for the whole group, the loss is extremely large for the pig industry. The existing market still lacks effective vaccines, wherein inactivated vaccines cannot resist attack of ASF virulent strains and cannot provide effective immunity protection, so that the inactivated vaccines cannot be used as an important direction for future research; however, attenuated live vaccines still have hidden danger in terms of biological safety, and the safety, effectiveness and the like of the live vaccines after vaccination are to be further evaluated.

The genetic engineering vaccine has low cost, obvious advantages in the aspects of safety, differential diagnosis and the like, more ASFV protective antigens or immune adjuvants are researched, and the effect of stimulating the organism to generate immune response is the key for developing ASFV subunit vaccines at present, so that the ASFV subunit vaccines have great research potential. At present, research on live carrier vaccines of African swine fever is still under exploration, and selection and setting of carriers and protective antigens and optimization of adjuvants are key directions of research;

oral mucosal immunity not only generates immune response on mucosal tissues, but also can cause systemic humoral immune response, is simple and safe to use, and is an important direction for developing vaccines at present. The complex microenvironment in the alimentary canal is a key factor influencing the immunity of the oral mucosa, and the oral vaccine developed by utilizing the lactococcus lactis expression system has the characteristics of easy absorption through the mucosa and no side effect, and can generate good immunity effect through different immunity paths by antigen presentation through the gastrointestinal mucosa.

Disclosure of Invention

The invention mainly aims to provide an African swine fever virus genetic engineering subunit oral vaccine, namely the African swine fever virus genetic engineering subunit oral vaccine which is safe and nontoxic and can stimulate organisms to generate mucosal immune response.

The invention firstly provides an antigen protein which can be used for preparing African swine fever virus genetic engineering subunit vaccine, wherein the antigen protein is ASFV p30 protein with an amino acid sequence of SEQ ID NO. 1, ASFV p54 protein with an amino acid sequence of SEQ ID NO. 3 or ASFV p72 protein with an amino acid sequence of SEQ ID NO. 5;

wherein the gene of ASFV p30 protein with the amino acid sequence of SEQ ID NO. 1, the sequence of which is SEQ ID NO. 2;

a gene of ASFV p54 protein with the amino acid sequence of SEQ ID NO. 3, and the sequence of the gene is SEQ ID NO. 4;

a gene of ASFV p72 protein with the amino acid sequence of SEQ ID NO. 5, and the sequence of the gene is SEQ ID NO. 6;

furthermore, the antigen protein is connected with a thermolabile enterotoxin B subunit LTB gene protein of escherichia coli;

wherein, the ASFV p30 protein connected with LTB has the amino acid sequence of SEQ ID NO. 7; the sequence of the coding gene is SEQ ID NO. 8;

wherein, the ASFV p54 protein connected with LTB has the amino acid sequence of SEQ ID NO 9; the sequence of the coding gene is SEQ ID NO. 10;

wherein, the ASFV p72 protein connected with LTB has the amino acid sequence of SEQ ID NO. 11; the sequence of the coding gene is SEQ ID NO. 12;

the invention also provides a genetically engineered lactococcus lactis, wherein the genetically engineered lactococcus lactis carries a recombinant expression vector for encoding the antigen protein;

as one specific example, the expression vector is a pMG36e expression vector.

The invention also provides an African swine fever virus genetic engineering subunit oral vaccine, wherein the antigen comprises the genetic engineering lactococcus lactis.

The African swine fever virus genetic engineering subunit oral vaccine can be orally taken, can effectively stimulate local immune cells of intestinal tracts to produce secretory IgA, stimulate organisms to produce mucous membrane reaction, and further stimulate the organisms to produce cellular immunity and humoral immunity through blood circulation. To overcome the potential for degradation or inactivation of the immunogen prior to reaching the mucosa of the small intestine, a live carrier system must be used to deliver intact antigen components. The aim can be achieved by taking bacterial viruses as a live carrier system, and lactococcus lactis is used as a live carrier system of various pathogens to present exogenous proteins and obtain good effects, but has not been reported in the development of African swine fever vaccine.

Drawings

Fig. 1: PCR identification results: lane 2MG1363/pMG36e-p30-His; lane 3MG1363/pMG36e-p54-His; lane 4MG1363/pMG36e-p72-His; lane 5MG1363/pMG36e-p54-LTB-His; lane 6MG1363/pMG36e-p72-LTB-His; lane 7MG1363/pMG36e-p30-LTB-His.

Fig. 2: SDS-PAGE and Western blot identification result diagram: lane 2MG1363/pMG36e-p30-His; lane 3MG1363/pMG36e-p54-His

Fig. 3: SDS-PAGE and Western blot identification result diagram: lane 2MG1363/pMG36e-p30-LTB-His.

Fig. 4: SDS-PAGE and Western blot identification result diagram: lane 2MG1363/pMG36e-p54-LTB-His

Fig. 5: SDS-PAGE and Western blot identification result diagram: lane 2MG1363/pMG36e-p72-His; lane 3MG1363/pMG36e-p72-LTB-His.

Detailed Description

The p30 protein or p32 protein used in the present invention is encoded by ASFV ORF CP204L gene and has a size of 30 KD. The protein function of the p30 protein is related to viral invasion. The p30 protein has excellent antigenicity and can induce body to produce strong humoral immune response, so as to stimulate animal body to produce neutralizing antibody. Due to its early and large expression in cells, it can be used as an antigen for detecting ASFV antibodies.

p54 protein (protein encoded by ASFV E183L gene, size of about 25 KD). The p54 protein is involved in viral invasion, transmembrane domain, and is essential for viral membrane protein formation. Transcription of its gene plays an important role in the late stages of viral infection and during the infection process. The p54 protein has good antigenicity, and the virus enters the body and stimulates the generation of antibodies against the protein, so that the virus has a certain protection effect.

The p72 protein, alternatively referred to as VP73 protein (a protein encoded by the ASFV B646L gene and having a size of approximately 73.09 KD). The p72 protein is an important component of the viral capsid, and also an important component of the viral icosahedron, and is generated in the later stage of viral infection, and is mainly involved in viral invasion. The protein sequence is highly conserved and has good antigenicity, and is often used for serodiagnosis of ASFV.

In the invention, in order to improve the recombinant expression quantity of p30, p54 and p72 proteins in lactococcus lactis, ASFV p30, p54 and p72 are modified, and the amino acid sequences of the modified SFV p30, p54 and p72 are SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively.

Meanwhile, in order to further provide immune effect, LTB genes with good antigenicity are connected to ASFV p30, p54 and p72 proteins. For simple identification of protein expression, a nucleotide sequence of 6 XHis Tag was ligated to a target gene, and the target protein was expressed by indirect His Tag expression.

On the basis of the obtained antigen protein, the African swine fever virus genetic engineering subunit oral vaccine is prepared through the following steps.

1. Expression vector constructs

The p30-His, p54-His, p72-His and p30-LTB-His, p54-LTB-His and p72-LTB-His gene fragments carrying SalI and HindIII enzyme cutting sites are synthesized, after sequencing identification is correct, quikCut SalI and QuikCutHindIII double enzyme cutting gene fragments and pMG36e expression vectors are used, after glue recovery and purification, the mixture is subjected to overnight connection at 16 ℃ and transformed into JM109 competent cells, positive monoclonal is selected, and correct standby is identified through bacterial liquid PCR and sequencing.

A pair of universal identification primers for pMG36e expression vector was designed for subsequent identification.

The general primer sequences were as follows:

pMG36e F：5’-gcctcctcatcctcttcatc-3’

pMG36e R：5’-aatatcgtagcgccggggta-3’

2. preparation and transformation of lactococcus lactis MG1363 competence;

picking a bacterial solution of the lactococcus lactis MG1363 full of the ring by using an inoculating ring, streaking on an MRS agar medium, picking bacteria from single bacterial colony, inoculating to 5ml of GM17 solution, and standing and culturing for 24 hours at 30 ℃; the next day was transferred to 25ml SGM17G medium at 2% inoculum size and cultured at 30℃for 0D ₆₀₀ =0.8. Centrifugally collecting thalli; suspending the thalli with 10ml of washing liquid, washing twice, and suspending with 400 mu l of washing liquid to obtain competent cells; the centrifuge tube was removed during transformation, placed in an ice bath, 1. Mu.l of plasmid was added to 50. Mu.l of competent cells, and then transferred to an electric beaker for 10 minutes in an ice bath, and subjected to an Ecol II mode electric shock, after 2 hours of stationary culture at 30℃for rapid transfer recovery, 100. Mu.l of the culture solution was applied to an erythromycin resistance plate, and then cultured overnight at 30℃for the next day, and transformants were examined.

3. Inducible expression of proteins

Selecting recombinant bacteria single colony, inoculating the recombinant bacteria single colony into a GM17 culture medium containing erythromycin, taking an empty plasmid strain as a negative control, standing and culturing at 30 ℃ for 48 hours, transferring the recombinant bacteria single colony into a fresh GM17 culture medium containing erythromycin according to 1% of inoculum size in the next day, standing and culturing at 30 ℃ for overnight, centrifuging, washing 2 times by adding 10ml of PBS, re-suspending thalli by using 2ml of PBS for cell disruption, adding 2X loading buffer solution, boiling for 10 minutes, and performing SDS-PAGE protein detection test: the results showed that the expressed target protein was approximately MG1363/pMG36e-p30-His with a specific band at 22.8KD (FIG. 2), MG1363/pMG36e-p54-His with a specific band at 20.4KD (FIG. 2), MG1363/pMG36e-p72-His with a specific band at 14.8KD (FIG. 5) and MG1363/pMG36e-p30-LTB-His with a specific band at 40.2KD (FIG. 3), MG1363/pMG36e-p54-LTB-His with a specific band at 38.8KD (FIG. 4), MG1363/pMG36e-p72-LTB-His with a specific band at 33.1KD (FIG. 5); after SDS-PAGE electrophoresis is finished, transferring the protein on the undyed polyacrylamide gel to a PVDF membrane, sealing by using 5% skimmed milk powder, reacting goat Anti-mouse IgG/horseradish enzyme labeled secondary antibody with the primary antibody by using the Anti-His Tag Monoclonal Antibody as the primary antibody, and finally developing by using HRP-DAB; western blot identification recombinant proteins have the reaction with p30-His, p54-His, p72-His, p30-LTB-His, p54-LTB-His and p72-LTB-His, and the good reactivity of the exogenous proteins expressed by recombinant bacteria is proved.

4. Preparation of the vaccine.

The cell concentration was about 1.0X10 ⁸ CFU/mL bacterial liquid is prepared into freeze-dried powder by freeze-drying technology for preservation. Taking the freeze-dried powder, adding 1ml of physiological saline, uniformly mixing, and directly taking orally.

The present invention will be described in detail with reference to the following examples and the accompanying drawings.

Example 1: preparation of African swine fever virus genetic engineering subunit oral vaccine

1. Optimizing the gene fragment:

(1) according to the amino acid sequences of ASFV p30 (GenBank accession number: MN 270980.1), p54 (GenBank accession number: MN 393476.1) and p72 (GenBank accession number: MN 886930.1), antigen proteins of ASFV p30, p54 and p72, the amino acid sequences of which are SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, were obtained by modification, and were used as target proteins expressed by host bacteria.

(2) To enhance its immune effect, LTB genes with good antigenicity were linked to ASFV p30, p54, p72 with the function of eliminating toxicity while retaining adjuvant activity.

(3) For simple identification of protein expression, a nucleotide sequence of 6 XHis Tag was ligated to a target gene, and the target protein was expressed by indirect His Tag expression.

(2) Expression vector construction

The gene fragments of p30-His, p54-His, p72-His and p30-LTB-His, p54-LTB-His and p72-LTB-His carrying SalI and HindIII enzyme cutting sites are constructed by a company, after identification is correct, the QuikCut SalI and QuikCutHindIII enzyme cutting pMG36e expression vector is used, after glue recovery and purification, the vector is subjected to overnight connection at 16 ℃ to be transformed into JM109 competent cells, positive monoclonal is selected, and bacterial liquid PCR identification and sequencing identification are correct.

The general primer sequences were as follows:

pMG36e F：5’-gcctcctcatcctcttcatc-3’

pMG36e R：5’-aatatcgtagcgccggggta-3’

pMG36e and the designed gene sequence were cut with QuikCut SalI and QuikCut HindIII double-cutting enzymes, subjected to ligation transformation, and identified as correct by sequencing.

3. Preparation and transformation of lactococcus lactis MG1363 competence

Picking a bacterial solution of the lactococcus lactis MG1363 full of the ring by using an inoculating ring, streaking on an MRS agar medium, picking bacteria from single bacterial colony, inoculating to 5ml of GM17 solution, and standing and culturing for 24 hours at 30 ℃; the next day was transferred to 25ml SGM17G medium at 2% inoculum size, and the OD was cultured at 30℃with standing ₆₀₀ =0.8. Centrifugally collecting thalli; suspending the thalli with 10ml of washing liquid, washing twice, and suspending with 400 mu l of washing liquid to obtain competent cells; the centrifuge tube was removed during transformation, placed in an ice bath, 1. Mu.l of plasmid was added to 50. Mu.l of competent cells, and then transferred to an electric beaker for 10 minutes in an ice bath, and subjected to an Ecol II mode electric shock, after 2 hours of stationary culture at 30℃for rapid transfer recovery, 100. Mu.l of the culture solution was applied to an erythromycin resistance plate, and then cultured overnight at 30℃for the next day, and transformants were examined.

4. Inducible expression of proteins

Selecting recombinant bacteria single colony, inoculating the recombinant bacteria single colony into a GM17 culture medium containing erythromycin, taking an empty plasmid strain as a negative control, standing and culturing at 30 ℃ for 48 hours, transferring the recombinant bacteria single colony into a fresh GM17 culture medium containing erythromycin according to 1% of inoculum size in the next day, standing and culturing at 30 ℃ for overnight, centrifuging, washing 2 times by adding 10ml of PBS, re-suspending thalli by using 2ml of PBS for cell disruption, adding 2X loading buffer solution, boiling for 10 minutes, and performing SDS-PAGE protein detection test: the results showed that the expressed target protein was approximately MG1363/pMG36e-p30-His with a specific band at 22.8KD, MG1363/pMG36e-p54-His with a specific band at 20.4KD, MG1363/pMG36e-p72-His with a specific band at 14.8KD, MG1363/pMG36e-p30-LTB-His with a specific band at 40.2KD, pMG36e-p54-LTB-His with a specific band at 38.8KD, MG1363/pMG36e-p72-LTB-His with a specific band at 33.1 KD. The method comprises the steps of carrying out a first treatment on the surface of the After SDS-PAGE electrophoresis is finished, transferring the protein on the undyed polyacrylamide gel to a PVDF membrane, sealing by using 5% skimmed milk powder, reacting goat Anti-mouse IgG/horseradish enzyme labeled secondary antibody with the primary antibody by using the Anti-His Tag Monoclonal Antibody as the primary antibody, and finally developing by using HRP-DAB; western blot identification proves that the exogenous protein expressed by the recombinant bacterium has good reactivities.

5. Preparation of vaccine

Example 2: combined oral vaccine immune efficacy test against non-target animals

1 materials and methods

1.1 materials

1.1.1 test animals 40 animals weighing about 2kg, female New Zealand white rabbits, were purchased from Qingdao corporation.

1.1.2 testing of 2 batches of recombinant vaccine products prepared in the pharmaceutical laboratory, the number of viable bacteria detected after dilution was 1.0X10 ⁸ /ml。

1.2 test methods

1.2.1 immunization test

40 rabbits weighing about 2kg, female New Zealand white rabbits, were divided into 4 groups of 10, vaccination group without LTB: namely, the combined immunization of MG1363/pMG36e-p30-His, MG1363/pMG36e-p54-His, MG1363/pMG36e-p72-His group; vaccination group with LTB: combination immunization of MG1363/pMG36e-p30-LTB-His, MG1363/pMG36e-p54-LTB-His, MG1363/pMG36e-p72-LTB-His group; empty carrier control group and PBS control group, and 3ml of recombinant bacteria are orally combined for each big rabbit in the recombinant bacteria group, which is about 1.0X10 ⁸ 3ml of oral air bacterium of air bacterium control group, about 1.0X10 ⁸ /ml. The control group was orally administered 3ml of sterilized PBS.

Immunization procedure: immunization was performed 2 times, with booster immunizations every 2 weeks, 3 days in succession, 1 time per day.

1.2.2 sample collection and handling

Serum samples: on the 14 th day after primary immunization and the 14 th day after booster immunization, the venous blood of the white rabbits were collected in groups, and the collected blood samples were placed obliquely at 4℃for standing for 1h and centrifuged at 3000rpm for 5min. The upper clear serum was aspirated and placed in a quiet centrifuge tube and stored at-80 ℃ for testing.

Mucous membrane sample: on the 14 th day after primary immunization and the 14 th day after booster immunization of the white rabbits, small intestines with the length of 5cm are fully dissolved in PBS buffer solution with the length of 0.01mol/L, the mixture is placed for 1.5 hours at the temperature of 4 ℃, and the supernatant is collected by centrifugation and stored at the temperature of minus 80 ℃ for detection.

1.2.3 measurement of intestinal mucosa sIgA of immunized white Rabbit

And (3) detecting by using an indirect ELISA detection kit.

1.2.4 detection of IL-4 level in immunized white rabbits

Detection was performed using an ELISA detection kit.

1.2.5 detection of IFN-gamma level in immunized white rabbits

Detection was performed using an ELISA detection kit.

2 results

From the experimental data shown in table 1, it can be analyzed that the combined oral vaccine produced significant sIgA antibodies after 2 consecutive immunizations of white rabbits. Antibody levels were significantly higher than the empty vector control and PBS control (P < 0.05), vaccination group with LTB reached maximum siga antibody levels at booster 14 d.

Table 1: experimental large rabbit intestinal mucosa sIgA antibody level detection table

From the experimental data shown in Table 2, it can be analyzed that the combination oral vaccine produced significant IL-4 antibodies after 2 consecutive immunizations of white rabbits. IL-4 levels were significantly higher than the empty vector control and PBS control (P < 0.05), vaccination group with LTB at boost 14d IL-4 antibody levels reached a maximum.

Table 2: experimental white rabbit IL-4 level detection table

From the experimental data shown in Table 3, it can be analyzed that the combined oral vaccine produced significant IFN-gamma antibodies after 2 consecutive immunizations of white rabbits. IFN-gamma levels were significantly higher than the empty vector control and PBS control (P < 0.05), vaccination group without LTB at 14d boost, IFN-gamma antibody levels reached a maximum.

Table 3: experimental white rabbit IFN-gamma level detection table

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Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn

290 295 300

Ser Ile Ala Ala Ile Ser Met Glu Lys Gly Leu Gln Gly Gly Gly Gly

305 310 315 320

Gly Val Thr Arg Val Val Thr His Glu Met Ala His Ala Leu Gly Thr

325 330 335

Pro Ala Ala Asn Ile Ala Ser Arg Tyr Asp Gln Ser Thr Ile Ile Thr

340 345 350

Ile Thr

<210> 8

<211> 1064

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gattttattt taagcttaaa tatatccatg aaaatggagg tcatcttcaa aacggattta 60

agatcatctt cacaagttgt gtttcatgcg ggtagcttgt ataattggtt ttctgttgag 120

attatcaata gcggtagaat tgttacgacc gctataaaaa cattgctcag tactgttaag 180

tatgatattg tgaaatctgc tcatatatat gcagggcaag ggtatactga acatcaggct 240

caagaagaat ggaatatgat tctgcatgtg ctgtttgaag aggagacaga atcctcagca 300

tcatcggaaa gcattcatga aaaaaatgat aatgaaacca atgaatgcac atcctccttt 360

gaaacattgt ttgagcaaga gccctcatca gaggaaccta aagactccaa gctgtatatg 420

cttgcacaaa agactgtgca acatattgaa caatatggaa aggcacctga ttttaacaag 480

gttattagag cacataactt tattcaaacc attcatggaa cccctctaaa ggaagaagaa 540

aaagaggtgg taagactcat ggtcattaaa cttttaaaaa aaaaaggtgg cggtggctct 600

ggcggaggtg ggagcggcgg tggtggcagc gctcctcagt ctattacaga actatgttcg 660

gaatatcaca acacacaaat atatacgata aatgacaaga tactatcata tacggaatcg 720

atggcaggca aaagagaaat ggttatcatt acatttaaga gcggcgcaac atttcaggtc 780

gaagtcccgg gcagtcaaca tatagactcc caaaaaaaag ccattgaaag gatgaaggac 840

acattaagaa tcacatatct gaccgagacc aaaattgata aattatgtgt atggaataat 900

aaaaccccca attcaattgc ggcaatcagt atggaaaaag gcctgcaggg tggtggtggt 960

ggtgtcacgc gtgtcgtcac gcacgagatg gcgcacgcgc tcggtacccc cgcggcgaac 1020

attgcgtcgc ggtacgacca gtcgaccatc atcaccatca ccat 1064

<210> 9

<211> 342

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Asp Ser Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly Glu Cys

1 5 10 15

Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr His Val Tyr Thr

20 25 30

Ile Leu Ile Ala Ile Val Val Leu Val Ile Ile Ile Ile Val Leu Ile

35 40 45

Tyr Leu Phe Ser Ser Arg Lys Lys Lys Ala Ala Ala Ile Glu Glu Glu

50 55 60

Asp Ile Gln Phe Ile Asn Pro Tyr Gln Asp Gln Gln Trp Val Glu Val

65 70 75 80

Thr Pro Gln Pro Gly Thr Ser Lys Pro Ala Gly Ala Thr Thr Ala Ser

85 90 95

Val Gly Lys Pro Val Thr Gly Arg Pro Ala Thr Asn Arg Pro Ala Thr

100 105 110

Asn Lys Pro Val Thr Asp Asn Pro Val Thr Asp Arg Leu Val Met Ala

115 120 125

Thr Gly Gly Pro Ala Ala Ala Pro Ala Ala Ala Ser Ala Pro Ala His

130 135 140

Pro Ala Glu Pro Tyr Thr Thr Val Thr Thr Gln Asn Thr Ala Ser Gln

145 150 155 160

Thr Met Ser Ala Ile Glu Asn Leu Arg Gln Arg Asn Thr Tyr Thr His

165 170 175

Lys Asp Leu Glu Asn Ser Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Gly Gly Gly Gly Ser Ala Pro Gln Ser Ile Thr Glu Leu Cys Ser

195 200 205

Glu Tyr His Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser

210 215 220

Tyr Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe

225 230 235 240

Lys Ser Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile

245 250 255

Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile

260 265 270

Thr Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn

275 280 285

Lys Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Glu Lys Gly Leu Gln

290 295 300

Gly Gly Gly Gly Gly Val Thr Arg Val Val Thr His Glu Met Ala His

305 310 315 320

Ala Leu Gly Thr Pro Ala Ala Asn Ile Ala Ser Arg Tyr Asp Gln Ser

325 330 335

Thr Ile Ile Thr Ile Thr

340

<210> 10

<211> 1028

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gattctgaat tttttcaacc ggtgtatccg cggcattatg gtgagtgttt gtcaccagtc 60

actacaccaa gcttcttctc cacacatgtg tatactattc tcattgctat cgtggtctta 120

gtcatcatta tcatcgttct aatctatcta ttctcttcaa gaaagaaaaa agctgctgct 180

attgaggagg aagatataca gtttataaat ccttatcaag atcagcagtg ggtagaagtc 240

actccacaac caggtacctc taaaccagct ggagcgacta cagcaagtgt aggcaagcca 300

gtcacgggca gaccggcaac aaacagacca gcaacaaaca aaccagttac ggacaacccg 360

gttacggaca gactagtcat ggcaactggc gggccggcgg ccgcacctgc ggccgcgagt 420

gctcctgctc atccggctga gccttacacg acagtcacta ctcagaacac tgcttcacaa 480

acaatgtcgg ctattgaaaa tttacgacaa agaaacacct atacgcataa agacctagaa 540

aactccttgg gtggcggtgg ctctggcgga ggtgggagcg gcggtggtgg cagcgctcct 600

cagtctatta cagaactatg ttcggaatat cacaacacac aaatatatac gataaatgac 660

aagatactat catatacgga atcgatggca ggcaaaagag aaatggttat cattacattt 720

aagagcggcg caacatttca ggtcgaagtc ccgggcagtc aacatataga ctcccaaaaa 780

aaagccattg aaaggatgaa ggacacatta agaatcacat atctgaccga gaccaaaatt 840

gataaattat gtgtatggaa taataaaacc cccaattcaa ttgcggcaat cagtatggaa 900

aaaggcctgc agggtggtgg tggtggtgtc acgcgtgtcg tcacgcacga gatggcgcac 960

gcgctcggta cccccgcggc gaacattgcg tcgcggtacg accagtcgac catcatcacc 1020

atcaccat 1028

<210> 11

<211> 293

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Glu Glu Thr His Leu Val His Leu Ser Phe Asn Ala His Phe Lys Pro

1 5 10 15

Tyr Gly Pro Gly Pro Arg Asn Gly Tyr Asp Trp Asp Asn Gln Thr Pro

20 25 30

Leu Gly Pro Gly Leu Tyr Glu Asn Val Arg Phe Asp Val Asn Gly Asn

35 40 45

Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr Thr Leu Val Arg Lys Phe

50 55 60

Cys Ile Pro Gly Pro Gly Lys Pro His Gln Ser Lys Pro Ile Leu Thr

65 70 75 80

Asp Glu Asn Asp Thr Gln Pro Gly Pro Gly Ile Thr Pro Ile Thr Asp

85 90 95

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

100 105 110

Gly His Val Val Asn Ala Gly Pro Gly Thr Thr Ala Asp Leu Val Val

115 120 125

Ser Ala Ser Ala Ile Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Ala Pro Gln Ser Ile Thr Glu Leu Cys Ser Glu

145 150 155 160

Tyr His Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr

165 170 175

Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys

180 185 190

Ser Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

195 200 205

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr

210 215 220

Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys

225 230 235 240

Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Glu Lys Gly Leu Gln Gly

245 250 255

Gly Gly Gly Gly Val Thr Arg Val Val Thr His Glu Met Ala His Ala

260 265 270

Leu Gly Thr Pro Ala Ala Asn Ile Ala Ser Arg Tyr Asp Gln Ser Thr

275 280 285

Ile Ile Thr Ile Thr

290

<210> 12

<211> 881

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gaagaaacac atttggttca tttaagcttt aatgcgcatt ttaagcctta tggcccgggc 60

cctcgcaacg gatatgactg ggacaaccaa acacctttag gcccgggcct ttatgaaaac 120

gtaagattcg atgtaaatgg aaattccctg gacgaatata gttcggatgt cacaacgctt 180

gtgcgcaaat tttgcatccc aggcccgggc aagccgcacc aaagcaaacc tattcttacc 240

gatgaaaatg atacgcagcc aggcccgggc attactccta ttacggacgc aacgtatctg 300

gacataagac gtaatgttca tggcccgggc aagttcggac atgttgttaa cgccggcccg 360

ggcactacgg ctgatcttgt ggtatcggca tctgctatta acggtggcgg tggctctggc 420

ggaggtggga gcggcggtgg tggcagcgct cctcagtcta ttacagaact atgttcggaa 480

tatcacaaca cacaaatata tacgataaat gacaagatac tatcatatac ggaatcgatg 540

gcaggcaaaa gagaaatggt tatcattaca tttaagagcg gcgcaacatt tcaggtcgaa 600

gtcccgggca gtcaacatat agactcccaa aaaaaagcca ttgaaaggat gaaggacaca 660

ttaagaatca catatctgac cgagaccaaa attgataaat tatgtgtatg gaataataaa 720

acccccaatt caattgcggc aatcagtatg gaaaaaggcc tgcagggtgg tggtggtggt 780

gtcacgcgtg tcgtcacgca cgagatggcg cacgcgctcg gtacccccgc ggcgaacatt 840

gcgtcgcggt acgaccagtc gaccatcatc accatcacca t 881

Claims

1. The African swine fever virus genetic engineering subunit oral vaccine is characterized in that an antigen of the subunit oral vaccine comprises genetic engineering lactococcus lactis, and the genetic engineering lactococcus lactis carries a recombinant expression vector for expressing antigen proteins; the antigen protein is ASFV p30 protein with the amino acid sequence of SEQ ID NO. 1, ASFV p54 protein with the amino acid sequence of SEQ ID NO. 3 and ASFV p72 protein with the amino acid sequence of SEQ ID NO. 5; the p30 protein, the p54 protein and the p72 protein are respectively connected with the escherichia coli heat-labile enterotoxin B subunit LTB gene protein;

wherein the amino acid sequence of the p30 protein connected with the LTB is SEQ ID NO. 7, the amino acid sequence of the ASFV p54 protein connected with the LTB is SEQ ID NO. 9, and the amino acid sequence of the p72 protein connected with the LTB is SEQ ID NO. 11.

2. The genetically engineered subunit oral vaccine of claim 1, wherein the LTB-linked p30 protein has a coding gene sequence of SEQ ID No. 8.

3. The genetically engineered subunit oral vaccine of claim 1, wherein the LTB-linked ASFV p54 protein has a sequence of SEQ ID No. 10.

4. The genetically engineered subunit oral vaccine of claim 1, wherein the LTB-linked p72 protein has a coding gene sequence of SEQ ID No. 12.

5. The genetically engineered subunit oral vaccine of claim 1, wherein the recombinant expression vector is a pMG36e expression vector.