CN114456240A - African swine fever virus genetic engineering subunit oral vaccine - Google Patents

African swine fever virus genetic engineering subunit oral vaccine Download PDF

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CN114456240A
CN114456240A CN202210208640.6A CN202210208640A CN114456240A CN 114456240 A CN114456240 A CN 114456240A CN 202210208640 A CN202210208640 A CN 202210208640A CN 114456240 A CN114456240 A CN 114456240A
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蔡秀磊
赵赛赛
张洪亮
单虎
秦志华
张皓杰
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Qingdao Agricultural University
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Abstract

The invention provides an African swine fever virus genetic engineering subunit oral vaccine, which comprises genetic engineering lactococcus lactis for recombining and expressing 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 LTB gene protein of B subunit of heat-labile enterotoxin of Escherichia coli. The African swine fever virus genetic engineering subunit oral vaccine can effectively stimulate local immune cells of intestinal tracts to generate secretory IgA, stimulate organisms to generate mucosal reaction, and further stimulate the organisms to generate 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 (ASF) is an acute, febrile, highly contagious, lethal infectious disease of animals caused by African Swine Fever Virus (ASFV). ASFV is the only member of African swine fever virus family (Asfarviridae) African swine fever virus genus (Asfivirus), and is the only arbovirus with DNA genome known at present. The affected pigs mainly show the symptoms of body temperature rise, skin mucosa extensive bleeding, pregnant sow abortion and the like. The fatality rate of African swine fever is as high as 100%, which brings serious harm to the pig industry.
Unlike other animal epidemic diseases, ASF has been developed for over 100 years since 1921. At present, the prevention measures for African swine fever mainly comprise the steps of strengthening supervision, strengthening disinfection and source elimination and well taking biological safety protection measures, once outbreak happens, the outbreak can be eradicated completely by killing the whole group, and the loss is great for the pig raising industry. At present, an effective vaccine is still lacking in the market, wherein the inactivated vaccine cannot resist the attack of ASF virulent strains and provide effective immune protection, so that the inactivated vaccine cannot be used as a key direction for future research; the attenuated live vaccine still has hidden troubles in the aspect of biological safety, and the safety, the effectiveness and the like after vaccination need to be further evaluated.
The genetic engineering vaccine has low cost and obvious advantages in the aspects of safety, differential diagnosis and the like, and the research on more ASFV protective antigens or immunologic adjuvants and the stimulation of the immune response effect of an organism are the key for developing the ASFV subunit vaccine at present, so the ASFV subunit vaccine has larger research potential. Currently, the research on live vector vaccines of African swine fever is still under investigation, and the selection and setting of vectors and protective antigens and the optimization of adjuvants are the key directions of the research;
oral mucosal immunization not only generates immune response in mucosal tissues, but also can cause systemic humoral immune response, is simple and safe to use, and is an important direction for the research and development of vaccines at present. The complex microenvironment in the digestive tract is a key factor influencing the immunity of the oral mucosa, 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 the antigen presentation is carried out through the gastrointestinal mucosa, so that a good immune effect can be generated through different immune pathways.
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 an organism to generate mucosal immune response.
The invention firstly provides an antigen protein which can be used for preparing an 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 coding amino acid sequence of SEQ ID NO. 1 has the sequence of SEQ ID NO. 2;
the gene of ASFV p54 protein with the coding amino acid sequence of SEQ ID NO. 3 and the sequence of the gene is SEQ ID NO. 4;
the gene of ASFV p72 protein with the coding 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 LTB gene protein of Escherichia coli heat-labile enterotoxin B subunit;
the amino acid sequence of the ASFV p30 protein connected with LTB is SEQ ID NO. 7; the sequence of the coding gene is SEQ ID NO. 8;
the amino acid sequence of the ASFV p54 protein connected with LTB is SEQ ID NO. 9; the sequence of the coding gene is SEQ ID NO. 10;
the amino acid sequence of the ASFV p72 protein connected with LTB is SEQ ID NO. 11; the sequence of the coding gene is SEQ ID NO 12;
the invention also provides a genetic engineering lactococcus lactis, wherein the genetic engineering lactococcus lactis carries a recombinant expression vector for encoding the antigen protein;
as a specific description of an example, the expression vector is 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 generate secretory IgA, stimulates an organism to generate mucosal reaction, and further stimulates the organism to generate cellular immunity and humoral immunity through blood circulation. To overcome the potential for the immunogen to be degraded or inactivated before reaching the mucosa of the small intestine, a live carrier system must be used to deliver the intact antigenic component. The purpose can be achieved by taking bacterial viruses as a live vector system, lactococcus lactis is used as a live vector system of various pathogens to carry out presentation of foreign proteins and obtain a good effect, but the foreign proteins are not reported in the development of vaccines of African swine fever.
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FIG. 1: and (3) PCR identification result: lane 2MG1363/pMG36e-p 30-His; lane 3MG1363/pMG36e-p 54-His; lane 4MG1363/pMG36e-p 72-His; lane 5MG1363/pMG36e-p 54-LTB-His; lane 6MG1363/pMG36e-p 72-LTB-His; lane 7MG1363/pMG36e-p 30-LTB-His.
FIG. 2: SDS-PAGE and Western blot identification result chart: lane 2MG1363/pMG36e-p 30-His; lane 3MG1363/pMG36e-p54-His
FIG. 3: SDS-PAGE and Western blot identification result chart: lane 2MG1363/pMG36e-p 30-LTB-His.
FIG. 4: SDS-PAGE and Western blot identification result chart: lane 2MG1363/pMG36e-p54-LTB-His
FIG. 5: SDS-PAGE and Western blot identification result chart: lane 2MG1363/pMG36e-p 72-His; lane 3MG1363/pMG36e-p 72-LTB-His.
Detailed Description
The p30 protein or p32 protein used in the invention is a protein with the size of 30KD and is coded by ASFV ORF CP204L gene. The protein function of the p30 protein is involved in viral entry. 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. Because of the early and large-scale expression in cells, it can be used as an antigen for detecting ASFV antibodies.
p54 protein (protein with a size of about 25KD encoded by ASFV E183L gene). The p54 protein is involved in viral entry, the transmembrane domain, and is essential for viral membrane protein formation. The transcription of its genes 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 production of antibodies against the protein, thereby having a certain protective effect.
The p72 protein or VP73 protein (protein encoded by ASFV B646L gene, size is about 73.09 KD). The p72 protein is an important component of the capsid of the virus and also of the icosahedron of the virus, which is produced in the later stages of viral infection and is mainly involved in virus entry. The protein sequence is highly conserved and has good antigenicity, and is often used for serological diagnosis 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 an immune effect, LTB genes with good antigenicity are connected to ASFV p30, p54 and p72 proteins. In order to identify protein expression simply, the nucleotide sequence of 6 XHis Tag is linked to the target gene, and the target protein is expressed by indirect expression His Tag method.
On the basis of obtaining the antigen protein, the African swine fever virus genetic engineering subunit oral vaccine is prepared by the following steps.
1. Expression vector constructs
Synthesizing gene segments of p30-His, p54-His, p72-His, p30-LTB-His, p54-LTB-His and p72-LTB-His carrying SalI and HindIII enzyme cutting sites, after sequencing and identifying the gene segments to be correct, using Quikcut SalI and Quikcut HindIII double enzyme cutting gene segments and a pMG36e expression vector, recovering and purifying by using glue, carrying out overnight connection at 16 ℃, transforming to JM109 competent cells, selecting positive single clone, and identifying the correct gene segments to be used by bacterial liquid PCR and sequencing.
A pair of universal identifying primers of pMG36e expression vectors is designed for subsequent identification.
The sequences of the universal primers are as follows:
pMG36e F：5’-gcctcctcatcctcttcatc-3’
pMG36e R：5’-aatatcgtagcgccggggta-3’
2. preparing and transforming lactococcus lactis MG1363 competence;
selecting a bacterial liquid of lactococcus lactis MG1363 with an inoculating loop, streaking the bacterial liquid on an MRS agar culture medium, selecting bacteria from a single colony to be inoculated on 5ml of GM17 solution, and standing and culturing for 24 hours at 30 ℃; inoculating into 25ml SGM17G medium with 2% inoculum size the next day, and standing at 30 deg.C for 0D6000.8. Centrifugally collecting thalli; suspending the thalli by 10ml of washing liquid, washing twice, and finally suspending by 400 mul of washing liquid to obtain competent cells; taking out the centrifuge tube during transformation, placing the centrifuge tube on ice for ice bath, then adding 1 mu l of plasmid into 50 mu l of competence, then transferring the centrifuge tube into an electric rotating cup for ice bath for 10 minutes, carrying out Ecol II mode electric shock, taking 100 mu l of culture solution to coat an erythromycin resistance plate after carrying out static culture for 2 hours at 30 ℃ for rapid transfer recovery culture, then carrying out culture overnight at 30 ℃, and checking transformants the next day.
3. Inducible expression of proteins
Selecting a recombinant bacterium single colony to be inoculated into a GM17 culture medium containing erythromycin, taking a strain carrying empty plasmids as a negative control, carrying out static culture at 30 ℃ for 48 hours, transferring the recombinant bacterium single colony into a fresh GM17 culture medium containing erythromycin according to the inoculation amount of 1% the next day, carrying out static culture at 30 ℃ overnight, centrifuging, washing for 2 times by adding 10ml PBS, carrying out cell disruption by using 2ml PBS to resuspend the bacterium, then adding 2 times of loading buffer solution, boiling for 10 minutes, and carrying out SDS-PAGE protein detection test: the results showed that the expressed proteins of interest showed a specific band at 22.8kD for MG1363/pMG36e-p30-His (FIG. 2), at 20.4kD for MG1363/pMG36e-p54-His (FIG. 2), at 14.8kD for MG1363/pMG36e-p72-His (FIG. 5) and at 40.2kD for MG1363/pMG36e-p30-LTB-His (FIG. 3), at 38.8kD for MG1363/pMG36e-p54-LTB-His (FIG. 4), at 33.1kD for MG1363/pMG36e-p72-LTB-His (FIG. 5); after SDS-PAGE electrophoresis is finished, transferring proteins on the undyed polyacrylamide gel onto a PVDF membrane, sealing the PVDF membrane by using 5% skimmed milk powder, taking Anti-His Tag Monoclonal Antibody as a primary Antibody, reacting a goat Anti-mouse IgG/horseradish enzyme-labeled secondary Antibody with the primary Antibody, and finally developing the color by using HRP-DAB; western blot identifies that the recombinant protein has the reaction with anti-p 30-His, p54-His, p72-His, p30-LTB-His, p54-LTB-His and p72-LTB-His, and proves that the foreign protein expressed by the recombinant bacteria has good reactogenicity.
4. And (3) preparing the vaccine.
The cell concentration was about 1.0X 108And (5) preparing the CFU/mL bacterial solution into lyophilized powder by using a freeze-drying technology for storage. Adding 1ml of normal saline into the freeze-dried powder, mixing uniformly, and directly taking orally.
The present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1: preparation of African swine fever virus genetic engineering subunit oral vaccine
1. Optimizing the gene segment:
the antigen proteins of ASFV p30, p54 and p72 with the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 are obtained by modifying according to the amino acid sequences of ASFV p30(GenBank accession number: MN270980.1), p54(GenBank accession number: MN393476.1) and p72(GenBank accession number: MN886930.1), and the antigen proteins are used as target proteins expressed by host bacteria.
② in order to enhance the immune effect, the LTB gene which has the function of eliminating toxicity and retaining adjuvant activity and has good antigenicity is connected with ASFV p30, p54 and p 72.
Thirdly, in order to simply identify the protein expression, the nucleotide sequence of 6 × His Tag is connected to the target gene, and the target protein is expressed by an indirect expression His Tag method.
(2) Expression vector construction
The gene segments of p30-His, p54-His, p72-His, p30-LTB-His, p54-LTB-His and p72-LTB-His carrying SalI and HindIII enzyme cutting sites are constructed by a company, after the gene segments are identified correctly, a pMG36e expression vector is subjected to double enzyme cutting by QuikCut SalI and QuikCut HindIII, after the gene segments are recovered and purified by glue, the enzyme cutting is performed overnight at 16 ℃ and is transformed to JM109 competent cells, a positive monoclonal is selected, and the PCR identification and sequencing identification of bacterial liquid are correct.
A pair of universal identifying primers of pMG36e expression vectors is designed for subsequent identification.
The sequences of the universal primers are as follows:
pMG36e F：5’-gcctcctcatcctcttcatc-3’
pMG36e R：5’-aatatcgtagcgccggggta-3’
pMG36e and the designed gene sequence were cleaved by double digestion with QuikCut SalI and QuikCut HindIII, ligated and transformed, and the correct sequence was identified by sequencing.
3. Preparation and transformation of lactococcus lactis MG1363 competence
Selecting a bacterial liquid of lactococcus lactis MG1363 with an inoculating loop, streaking the bacterial liquid on an MRS agar culture medium, selecting bacteria from a single colony to be inoculated on 5ml of GM17 solution, and standing and culturing for 24 hours at 30 ℃; transferring into 25ml SGM17G medium with 2% inoculum size the next day, and performing static culture at 30 deg.C to obtain OD6000.8. Centrifugally collecting thalli; suspending the thalli by 10ml of washing liquid, washing twice, and finally suspending by 400 mul of washing liquid to obtain competent cells; the centrifuge tube was removed during transformation, placed on ice for ice bath, then 1. mu.l of plasmid was added to 50. mu.l of competence, and then transferred to an electric rotor for ice bath for 10 minutes, and electric shock was applied in Ecol II mode, after rapid transfer recovery culture was performed for 2 hours at 30 ℃ and static culture was performed, 100. mu.l of culture medium was smeared on an erythromycin-resistant plate, and further cultured overnight at 30 ℃ and the transformants were checked the next day.
4. Inducible expression of proteins
Selecting a recombinant bacterium single colony to be inoculated into a GM17 culture medium containing erythromycin, taking a strain carrying empty plasmids as a negative control, carrying out static culture at 30 ℃ for 48 hours, transferring the recombinant bacterium single colony into a fresh GM17 culture medium containing erythromycin according to the inoculation amount of 1% the next day, carrying out static culture at 30 ℃ overnight, centrifuging, washing for 2 times by adding 10ml PBS, carrying out cell disruption by using 2ml PBS to resuspend the bacterium, then adding 2 times of loading buffer solution, boiling for 10 minutes, and carrying out SDS-PAGE protein detection test: the results showed that the expressed proteins of interest showed a specific band at 22.8kD for MG1363/pMG36e-p30-His, a specific band at 20.4kD for MG1363/pMG36e-p54-His, a specific band at 14.8kD for MG1363/pMG36e-p72-His, a specific band at 40.2kD for MG1363/pMG36e-p30-LTB-His, a specific band at 38.8kD for pMG36e-p54-LTB-His, a specific band at 33.1kD for MG1363, MG1363/pMG36e-p 72-LTB-His. (ii) a After SDS-PAGE electrophoresis is finished, transferring proteins on the undyed polyacrylamide gel onto a PVDF membrane, sealing the PVDF membrane by using 5% skimmed milk powder, taking Anti-His Tag Monoclonal Antibody as a primary Antibody, reacting a goat Anti-mouse IgG/horseradish enzyme-labeled secondary Antibody with the primary Antibody, and finally developing the color by using HRP-DAB; western blot identification proves that the foreign protein expressed by the recombinant bacteria has good reactogenicity.
5. Preparation of vaccines
The cell concentration was about 1.0X 108And (5) preparing the CFU/mL bacterial solution into lyophilized powder by using a freeze-drying technology for storage. Adding 1ml of normal saline into the freeze-dried powder, mixing uniformly, and directly taking orally.
Example 2: immunopotency testing of combination oral vaccines against non-target animals
1 materials and methods
1.1 materials
1.1.1 test animals 40 female New Zealand white rabbits weighing about 2kg were purchased from Qingdao, Inc.
1.1.2 batches of recombinant vaccine products prepared in experimental drug laboratories, the number of viable bacteria detected after dilution is 1.0 multiplied by 108/ml。
1.2 test methods
1.2.1 immunization test
40 female New Zealand white rabbits weighing about 2kg, divided into 4 groups of 10 rabbits each, Vaccination group with out LTB: namely, combined immunization of MG1363/pMG36e-p30-His, MG1363/pMG36e-p54-His, MG1363/pMG36e-p72-His group; vaccination group with LTB: combined immunization of the MG1363/pMG36e-p30-LTB-His, MG1363/pMG36e-p54-LTB-His, MG1363/pMG36e-p72-LTB-His group; empty vector control group and PBS control group, 3ml of recombinant bacteria are orally combined in each large white rabbit of the recombinant bacteria group, and the volume is about 1.0 multiplied by 108Perml, empty bacteria control group orally takes empty bacteria 3ml, about 1.0X 108And/ml. The control group was orally administered 3ml of sterile PBS.
Immunization procedure: immunizations were performed 2 times, booster every 2 weeks for 3 days, 1 time per day.
1.2.2 sample Collection and handling
Serum samples: on the 14 th day after the primary immunization and the 14 th day after the boosting immunization of the white rabbits, respectively collecting the auricular venous blood of the white rabbits according to groups, obliquely placing the collected blood samples at 4 ℃, standing for 1h, and centrifuging for 5min at 3000 rpm. Sucking the clear serum at the upper layer, placing the serum in a quiet centrifuge tube, and storing the serum at the temperature of minus 80 ℃ for detection.
Mucosal sample: on the 14 th day after the primary immunization and the 14 th day after the boosting immunization of the white rabbits, small intestines with the length of 5cm are fully dissolved in PBS buffer solution with the concentration of 0.01mol/L, placed for 1.5 hours at the temperature of 4 ℃, centrifuged to collect supernatant, and stored at the temperature of minus 80 ℃ for detection.
1.2.3 determination of intestinal mucosal sIgA in immunized white rabbits
And detecting by adopting an indirect ELISA detection kit.
1.2.4 detection of IL-4 level in immunized white rabbits
And (3) detecting by using an ELISA detection kit.
1.2.5 detection of IFN-. gamma.levels in immunized white rabbits
And (3) detecting by using an ELISA detection kit.
2 results
As can be analyzed from the experimental data shown in table 1, the combination 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), and the Vaccination group with LTB reached a maximum at 14d of boost.
Table 1: experimental rabbit intestinal mucosa sIgA antibody level detection table
Figure BDA0003532217590000111
As can be analyzed from the experimental data shown in Table 2, the combination oral vaccine produced significant IL-4 antibody after 2 consecutive immunizations of white rabbits. IL-4 levels were significantly higher than the empty vector control and PBS control (P < 0.05), while the Vaccination group with LTB reached a maximum IL-4 antibody level at 14d of the boost.
Table 2: experimental white rabbit IL-4 level detection table
Figure BDA0003532217590000121
As can be analyzed from the experimental data shown in Table 3, the combination 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), while the levels of IFN-gamma antibody reached a maximum at 14d of boost immunization with Vaccination group without LTB.
Table 3: experimental white rabbit IFN-gamma level detection table
Figure BDA0003532217590000122
Sequence listing
<110> Qingdao agricultural university
<120> African swine fever virus genetic engineering subunit oral vaccine
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 203
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Asp Phe Ile Leu Ser Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe
1 5 10 15
Lys Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser
20 25 30
Leu Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val
35 40 45
Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val
50 55 60
Lys Ser Ala His Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala
65 70 75 80
Gln Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr
85 90 95
Glu Ser Ser Ala Ser Ser Glu Ser Ile His Glu Lys Asn Asp Asn Glu
100 105 110
Thr Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro
115 120 125
Ser Ser Glu Glu Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys
130 135 140
Thr Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys
145 150 155 160
Val Ile Arg Ala His Asn Phe Ile Gln Thr Ile His Gly Thr Pro Leu
165 170 175
Lys Glu Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu
180 185 190
Lys Lys Lys Val Asp His His His His His His
195 200
<210> 2
<211> 609
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
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 aaaaagtcga ccatcatcac 600
catcaccat 609
<210> 3
<211> 191
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
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 Val Asp His His His His His His
180 185 190
<210> 4
<211> 573
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
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 tcgaccatca tcaccatcac cat 573
<210> 5
<211> 141
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
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 Lys Ser Thr Ile Ile Thr Ile Thr
130 135 140
<210> 6
<211> 425
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
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 agtcgaccat catcaccatc 420
accat 425
<210> 7
<211> 354
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Asp Phe Ile Leu Ser Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe
1 5 10 15
Lys Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser
20 25 30
Leu Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val
35 40 45
Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val
50 55 60
Lys Ser Ala His Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala
65 70 75 80
Gln Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr
85 90 95
Glu Ser Ser Ala Ser Ser Glu Ser Ile His Glu Lys Asn Asp Asn Glu
100 105 110
Thr Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro
115 120 125
Ser Ser Glu Glu Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys
130 135 140
Thr Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys
145 150 155 160
Val Ile Arg Ala His Asn Phe Ile Gln Thr Ile His Gly Thr Pro Leu
165 170 175
Lys Glu Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu
180 185 190
Lys Lys Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
195 200 205
Gly Ser Ala Pro Gln Ser Ile Thr Glu Leu Cys Ser Glu Tyr His Asn
210 215 220
Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr Thr Glu Ser
225 230 235 240
Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys Ser Gly Ala
245 250 255
Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys
260 265 270
Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr Tyr Leu Thr
275 280 285
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 (10)

1. The antigen protein for preparing the African swine fever virus genetic engineering subunit vaccine is characterized in that 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.
2. A gene encoding the antigenic protein of claim 1.
3. The gene as claimed in claim 2, which encodes ASFV p30 protein having the amino acid sequence of SEQ ID NO. 1, and the gene has the sequence of SEQ ID NO. 2; the sequence of the gene of ASFV p54 protein with the coding amino acid sequence of SEQ ID NO. 3 is SEQ ID NO. 4; the sequence of the gene of the ASFV p72 protein of which the coding amino acid sequence is SEQ ID NO. 5 is SEQ ID NO. 6.
4. The antigenic protein of claim 1 which is linked to the LTB gene protein of the B subunit of Escherichia coli heat-labile enterotoxin.
5. The antigenic protein of claim 4 which is ASFV p30 protein having the amino acid sequence of SEQ ID NO 7 linked to LTB; the sequence of the coding gene is SEQ ID NO. 8.
6. The antigenic protein of claim 4 which is ASFV p54 protein having the amino acid sequence of SEQ ID NO 9 linked to LTB; the sequence of the coding gene is SEQ ID NO 10.
7. The antigenic protein of claim 4 which is ASFV p72 protein having the amino acid sequence of SEQ ID NO 11 linked to LTB; the sequence of the coding gene is SEQ ID NO 12.
8. A genetically engineered lactococcus lactis bacterium carrying a recombinant expression vector encoding the antigenic protein of claim 1 or claim 4.
9. The genetically engineered lactococcus lactis bacterium of claim 8, wherein the expression vector is a pMG36e expression vector.
10. An African swine fever virus genetic engineering subunit oral vaccine, characterized in that, the antigen of the subunit oral vaccine comprises the genetic engineering lactococcus lactis of claim 8.
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Title
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