CN114606250B - Recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof - Google Patents

Recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof Download PDF

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CN114606250B
CN114606250B CN202111277332.0A CN202111277332A CN114606250B CN 114606250 B CN114606250 B CN 114606250B CN 202111277332 A CN202111277332 A CN 202111277332A CN 114606250 B CN114606250 B CN 114606250B
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CN114606250A (en
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王春凤
杨桂连
牛荟
杨文涛
石春卫
牛天明
王红
谷巍
单宝龙
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Jilin Agricultural University
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Abstract

The application provides recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof. The recombinant lactic acid bacteria provided by the invention have a fusion gene which comprises at least one group of sequences in the following 1) to 3) connected with a DCpep gene; 1) the gene sequence intercepted from 1414 th and 3351 th sections of the G1211R gene; 2) comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4; 3) the nucleotide sequence with at least 90 percent of sequence homology with the sequence in 1) or 2), the recombinant lactic acid bacteria can express fusion protein with immunogenicity, and the recombinant lactic acid bacteria anchor and express African swine fever virus DNA polymerase, p72, DNA polymerase and p72 fusion antigen on the surface of lactobacillus plantarum respectively. The recombinant lactic acid bacteria can immunize animals in an oral mode, can induce the activation of the Peyer's patch DC, induce the generation of specific T cell reaction and induce B cell reaction, and show good immune effect.

Description

Recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof
Technical Field
The application relates to the technical field of genetic engineering, in particular to recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof.
Background
The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known as prior art to a person skilled in the art.
African Swine Fever Virus (ASFV) is a double-stranded DNA virus, the only member of the African swine fever family, varies in number of base from 170 to 190+ kb for different strains, and encodes over 150 proteins, of which up to 68 structural proteins have been identified. The African swine fever is used as an acute, hot and high-contact disease of pigs, because ASFV immune protection mechanism is unclear, structure is complex, most gene function is unknown, mechanism of interaction between virus and host cell is not clear, and other factors exist, and cellular immunity and humoral immunity play different roles in the process of specific immune response, the research and development of the vaccine are difficult, and a safe and ideal vaccine is lacked, thereby causing huge economic loss in the world. Attempts are being made around the world to control and eradicate ASFV, which is further classified as a disease that must be addressed by the world animal health Organization (OIE). Scientists have identified many proteins such as p72, CD2v, p11.5, p54, p22, p12, p30, p17, p10, p14.5, p150, p37, p34 and p14, and have been devoted to research on african swine fever vaccines, from early inactivated vaccines, attenuated live vaccines to current novel vaccines, including gene-deletion attenuated live vaccines, subunit vaccines, viral live vector vaccines, DNA vaccines, monocycle virus vaccines, etc., but the inactivated vaccines are low in protection, the subunit vaccines, nucleic acid vaccines and viral live vector vaccines can only provide partial immune protection, and natural or attenuated vaccines have the risk of virulence reversion and sustained virus spread, and great safety hazard, thus developing safe and effective genetic engineering attenuated live vaccines are at the forefront. For centuries, the fermentation of Food products with lactic acid bacteria has been the traditional one, and these Lactic Acid Bacteria (LAB) are Food-grade safe microorganisms recognized by the United States Food and Drug Administration (USFDA), have the effects of increasing the beneficial intestinal flora, improving the gastrointestinal function of the human body, regulating immunity, etc., and have been used in many fields. In the age of general banning of resistance, lactic acid bacteria are more and more concerned by people as a probiotic. However, the novel vaccine developed by using the genetic engineering technology generally has the defects of weak immunogenicity, incapability of inducing an organism to generate effective immune response and the like.
Disclosure of Invention
The important roles of Dendritic Cells (DCs) are in the uptake, processing and presentation of antigens, stimulating the body to mount an immune response. DCs are a class of cells that, when mature, have many dendritic processes and are therefore named. When the cells are mature, the cells can recognize, take and process exogenous antigens and present antigen peptides to initial T cells so as to induce the T cells to activate and proliferate the antigen presenting cells with the strongest functions. DCs are the initiators of adaptive immune responses in the body and are also the "bridge" connecting innate and adaptive immune responses. Based on the three groups of fusion genes, the invention starts with the idea of cutting off the formation and the replication of viruses and constructs three groups of fusion genes, the fusion gene of the invention comprises a DCpep gene sequence, and simultaneously comprises a partial sequence in a G1211R gene and/or a partial epitope gene of p72, and the three groups of fusion genes are connected in a specific combination. The DCpep consists of 12 amino acids, can well target dendritic cells so as to cause immune response of an organism, has the advantage of short sequence, can solve the problem of label expression, and simultaneously provides convenience for antigen preparation.
In the design concept of the present invention, the inventors considered that the G1211R gene encodes a DNA polymerase (DNApol) of a virus, which is closely involved in viral replication. The 1211 amino acid of DNA polymerase contains 3633 bases, is a non-structural protein, but is highly conserved, and can induce strong cell-mediated immunity, such as natural killer cell and T cell response. And, the p72 protein (with 646 amino acids, 1941 bases) encoded by the B646L gene is the major capsid protein, involved in viral entry into the host, and important in viral capsid formation later in viral infection. Through specific selection, the inventor combines a partial sequence of the gene with a DCpep sequence of a targeted dendritic cell so as to realize a good immune effect.
Based on the conception, the invention provides a fusion gene with a specific structure and a fusion protein coded by the gene, wherein the fusion protein can well stimulate the immunity of the organism, including but not limited to inducing the activation of lymph node DC, inducing the specific T cell response and inducing the activation of B cells; meanwhile, the invention also provides an expression vector and a recombinant engineering bacterium carrying the gene, the bacterium has good growth performance, can mature and express the fusion gene, can adopt the bacterium to carry out organism immunity in an oral form, anchors an expressed antigen on the surface of a bacterium body, and enhances the immune effect.
Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.
In a first aspect of the present invention, there is provided a fusion gene comprising at least one set of sequences described in 1) to 3) below linked to a DCpep gene;
1) the gene sequence intercepted from 1414 th and 3351 th sections of the G1211R gene;
2) comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4;
3) a nucleotide sequence having at least 90% sequence homology with the sequence described in 1) or 2).
In some embodiments of the invention, the DCpep gene comprises a nucleotide sequence identical to SEQ ID NO: 5 has at least 90% sequence homology. Wherein, the connection mode with the DCpep gene is tandem connection.
In some embodiments of the invention, the fusion gene comprises a DNApol gene fragment, wherein the DNApol gene fragment comprises a gene sequence truncated from the 1414 th and 3351 th segments of G1211R and DCpep sequences, wherein the gene sequence truncated from the 1414 th and 3351 th segments of G1211R is connected with three sets of repeated DCpep sequences.
In some embodiments of the invention, the fusion gene comprises a p72 gene fragment, and the p72 gene fragment comprises the nucleotide sequence as shown in SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 and a DCpep sequence, wherein the nucleotide sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 are respectively connected in series after three groups of repetition (sequentially connected in series), and then are connected with three groups of repeated DCpep sequences.
In some embodiments of the present invention, DCpep is composed of 12 amino acids, such a composition makes spatial conformation thereof easily changed, and the conventional advantages of DCpep are difficult to realize, and through research, the inventors found that when 3 dcpeps are connected in series, the spatial conformation can be prevented from being changed, the dcpeps can be fully exposed, the dcpeps have better effects on DC cells, and the dcpeps can better assist in stimulating stronger immune response of the body on the premise of exerting the conventional advantages.
And compared with the mode that the target fragment needs to be connected with an expression vector, then is converted into a bacterium for amplification expression, and then is immunized to a white rabbit after protein purification to obtain antibody serum in the traditional method, in the embodiment of the invention, 3 DCpeps are taken as the target fragments, polyclonal antibody serum can be obtained by immunizing DCpep polypeptide to an animal such as the white rabbit, and then the expression of the recombinant bacterium can be more conveniently detected, so that the complex steps of the traditional method are omitted on the basis of achieving the verification purpose.
And, in some embodiments of the present invention, the inventors found that selecting a single epitope without repeating the epitope presents the problem that the expressed antigenic molecule is small and difficult to be exposed sufficiently, the obtained recombinant bacterium is difficult to produce beneficial effects, and, on the basis of this, it is attempted to repeat the single epitope so that the sequence thereof becomes as long as possible and the expressed antigenic molecule becomes as large as possible, but the improvement of the immune effect is very limited. In other embodiments of the present invention, the inventors have selected 4 p72 epitopes, each of which has 10, 16, 15, and 14 amino acids, and performed 3 repeats, and as a result, they have found that such a treatment can enlarge the antigenic molecules and achieve better exposure, so that the immune cells in the body have more opportunities to recognize the antigens, and thus generate stronger immune response, and better improve the immune effect.
In some embodiments of the invention, the fusion gene comprises a DNApol-p72 gene fragment, and the DNApol-p72 gene fragment comprises a DNApol gene fragment and a p72 gene fragment, wherein the DNApol gene fragment and the p72 gene fragment are connected with an SD sequence (SEQ ID NO: 6).
In some embodiments of the invention, the fusion gene comprises a sequence set forth in any one of 1) to 3) below:
1) DNApol gene fragment sequence: which comprises the amino acid sequence as shown in SEQ ID NO: 7, or a nucleotide sequence that hybridizes under high stringency conditions with SEQ ID NO: 7, or a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 7 has at least 90 percent of sequence homology and has nucleotide sequences with the same functions;
2) p72 gene fragment sequence: which comprises the amino acid sequence as shown in SEQ ID NO: 8, or a nucleotide sequence that hybridizes under high stringency conditions with SEQ ID NO: 8, or a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 8 has at least 90 percent of sequence homology and nucleotide sequences with the same functions;
3) DNApol-p72 gene fragment sequence: which comprises the amino acid sequence as shown in SEQ ID NO: 9, or a nucleotide sequence that hybridizes under high stringency conditions with SEQ ID NO: 9, or a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 9 has at least 90 percent of sequence homology and has nucleotide sequences with the same functions.
In an embodiment of the present invention, the fusion gene may further include an initiator, a terminator, and a restriction enzyme site gene sequence; preferably, the sequence of the restriction site gene is Xba I and Hind III restriction site gene sequences, and the Xba I restriction site gene sequence: TCTAGA; the HindIII enzyme cutting site gene sequence: AAGCTT.
For example, in some embodiments of the invention, the fusion gene is a DNApol gene, a p72 gene, or a DNApol-p72 gene, the structure of which is shown in fig. 2.
In a second aspect of the present invention, the present invention provides a fusion protein for stimulating the body against African swine fever infection, which is encoded by the fusion gene of the first aspect, and the amino acid sequence of the fusion protein comprises the sequence shown in any one of 1) to 3) below:
1) and SEQ ID NO: 10 has at least 90% sequence homology and has the same function;
2) and SEQ ID NO: 11, and sequences having at least 90% sequence homology and identical functions;
3) and SEQ ID NO: 12 has at least 90% sequence homology and has the same function.
In a third aspect of the present invention, there is provided an expression vector comprising the fusion gene described in the first aspect above.
In some embodiments of the present invention, the vector is pWCF, and the nucleotide sequence thereof is shown in SEQ ID NO: 13, which is obtained by connecting a pWCF vector with the fusion gene in the first aspect; preferably, the pWCF vector is linked to the fusion gene by T4 DNA ligase.
In a fourth aspect of the invention, there is provided a transgenic cell line comprising a fusion gene as described in the first aspect above.
In a fifth aspect of the present invention, there is provided an engineered bacterium comprising the fusion gene of the first aspect. In some embodiments of the invention, the starting strain of the engineered bacterium is lactobacillus plantarum, preferably lactobacillus NC8, more preferably lactobacillus plantarum NC8 Δ alr deficient in the alanine racemase gene (alr). The engineering bacteria are non-antibiotic expression recombinant bacteria.
Specifically, in the embodiment of the invention, an inducible escherichia coli-lactic acid bacteria shuttle expression vector is adopted, wherein an aspartate-beta semialdehyde dehydrogenase (asd) gene and an alanine racemase gene (alr) are used as nonreactive screening markers, escherichia coli (E.coli χ 6212) with asd gene deficiency is used as a plasmid to construct an intermediate host bacterium, and an alr gene deletion strain NC8/Δ alr is used as an expression bacterium.
In some embodiments of the invention, the recombinant lactic acid bacterium expresses a protein that elicits an anti-african swine fever infection in the body as described in the second aspect above. For example, the recombinant bacteria are recombinant lactobacillus NC8 delta-pWCF-DNAPol, NC8 delta-pWCF-p 72 or NC8 delta-pWCF-DNAPol-p 72 which can respectively express and generate fusion antigens DNAPol (SEQ ID NO: 10), p72(SEQ ID NO: 11) and DNAPol-p72(SEQ ID NO: 12) capable of anchoring on the surface of lactobacillus plantarum.
In a sixth aspect of the invention, the invention provides a pharmaceutical composition or a pharmaceutical preparation or a feed, which comprises the fusion protein for stimulating the body against African swine fever infection as described in the second aspect or the engineered bacterium as described in the fifth aspect.
In some embodiments of the present invention, the feed of the present invention may further comprise edible substances required for the growth of other animals, especially pigs, such as food substances including soybean, soybean meal, corn, grains, etc., oil, meat and bone meal, feed additives, amino acids, vitamins, trace elements, fish meal whey powder, miscellaneous meal, etc., in addition to at least one of the fusion proteins of the present invention or at least one of the recombinant lactobacillus plantarum of the present invention. And the pharmaceutical composition or the pharmaceutical preparation comprises at least one protein for stimulating the body to resist the African swine fever infection in the second aspect or at least one engineering bacterium for stimulating the body to resist the African swine fever infection in the fifth aspect, and on the basis, the pharmaceutical composition or the pharmaceutical preparation can also comprise at least one pharmaceutical carrier or pharmaceutically acceptable auxiliary materials or other therapeutically effective agents. Suitable pharmaceutical Excipients may be of a kind known in the art, such as solvents, buffers, diluents and the like, and may be, for example, those described in the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients) by the authors Paul J Sheskey et al.
In a seventh aspect of the present invention, the present invention provides a method for expressing the fusion protein for stimulating the body against African swine fever infection as described in the second aspect, which comprises: constructing an expression vector containing the fusion gene described in the first aspect, introducing the constructed expression vector into a host cell to obtain a recombinant strain, culturing the recombinant strain, and finally separating the fusion protein from the culture.
In an eighth aspect of the present invention, the present invention provides the use of the fusion gene of the first aspect, or the fusion protein of the second aspect for stimulating the body to resist african swine fever infection, or the engineered bacterium of the fifth aspect for preparing a vaccine, a drug or a feed for preventing and/or treating african swine fever.
Through one or more technical means, the following beneficial effects can be achieved:
the invention successfully constructs three recombinant lactic acid bacteria of NC8 delta-pWCF-DNAPol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNAPol-p 72, wherein the recombinant lactic acid bacteria can mature and express fusion protein DNAPol protein, p72 protein and DNAPol-p72 protein with good immunogenicity, and can be anchored and expressed on the surface of lactobacillus plantarum. The recombinant lactic acid bacteria can immunize animals in an oral mode, can induce the activation of the Peyer's patch DC, induce the generation of specific T cell reaction and induce the activation of B cells, and show good immune effect.
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The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein: in the following pictures, p < 0.05; p < 0.01; p < 0.001; p < 0.0001.
FIG. 1: DNApol was analyzed for surface accessibility (yellow), antigenic properties (pink), hydrophilicity (blue) using DNAstar protein software to predict the truncation site of the protein.
FIG. 2: and (3) synthesizing the target fragment.
FIG. 3: and (3) immunization and blood collection time of the white rabbits.
FIG. 4 is a schematic view of: double enzyme digestion verification of the recombinant plasmids pWCF-DNAPol (A), pWCF-p72(B) and pWCF-DNAPol-p72 (C); wherein, in the A, B, C three pictures, M is DL10,000 DNA Marker; in A, 1: recombinant plasmid pWCF-DNApol; 2: double enzyme digestion verification of the recombinant plasmid; in B, 1: a pWCF-p72 recombinant plasmid; 2: double enzyme digestion verification of the recombinant plasmid; in C, 1: recombinant plasmid pWCF-DNApol-p 72; 2: and (5) double enzyme digestion verification of the recombinant plasmid.
FIG. 5: PCR verification of the recombinant lactic acid bacterium plasmid NC 8. delta. -pWCF-DNApol (A); m: DL10,000 DNA Marker; 1: PCR validation of recombinant lactic acid bacteria plasmid NC 8. delta. -pWCF-DNApol. PCR verification of the recombinant lactic acid bacteria plasmid NC8 delta-pWCF-p 72 (B); m: DL2,000 DNA Marker; 1: PCR validation of recombinant Lactobacillus plasmid NC8 Δ -pWCF-p 72. PCR verification (C) of the recombinant lactic acid bacterium plasmid NC 8. delta. -pWCF-DNApol-p 72; m: DL5,000 DNA Marker; 1: PCR verification of recombinant lactic acid bacteria plasmid NC8 delta-pWCF-DNApol-p 72.
FIG. 6: western Blot detection expressed in NP DCpep recombinant lactobacillus treated by an ultrasonic disruption method; m: protein marker; 1: expression product of NP DCpep.
FIG. 7: strain growth profile.
FIG. 8: and (5) detecting the result of the recombinant lactobacillus by flow cytometry.
FIG. 9: recombinant lactobacillus immunofluorescence; NC8 delta-pWCF white light and fluorescence; NC8 delta-pWCF-DNAPol white light and fluorescence; NC8 delta-pWCF-p 72 white light and fluorescence; g, H, NC8 delta-pWCF-DNAPol-p 72 white light and fluorescence.
FIG. 10: animal immunization procedure.
FIG. 11: the activation results of DC in PPs, the left image flow cytometry detection results, and the right image analysis statistics results.
FIG. 12: CD4 in MLN + IFN-γ + Expression of (A), CD8 in MLN + IFN-γ + Expression of (A) (B).
FIG. 13: CD4 in spleen + IFN-γ + Expression of (A), CD8 in spleen + IFN-γ + Expression of (A) (B).
FIG. 14: CD4 in MLN + T cell proliferation results (A), CD8 in MLN + T cell proliferation results (B).
FIG. 15: CD4 in spleen + T cell proliferation results (A), CD8 in spleen + T cell proliferation results (B).
FIG. 16: b220 in PP + IgA + The number of cells varied.
FIG. 17: CD80 expression in bone marrow-derived DCs (a) and CD86 expression in bone marrow-derived DCs (B).
Detailed Description
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in the conventional manner in the art or in the product specification. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are exemplary only.
The DCpep consists of 12 amino acids, can be specifically targeted and combined with Dendritic Cells (DCs), is helpful for the DCs to recognize antigens and induce immune response, has a short sequence and plays an important role in immune homeostasis, however, in some embodiments of the present invention, the inventors found that the DCpep consisting of 12 amino acids has a composition that allows the spatial conformation thereof to be easily changed, and the conventional advantages of DCpep are difficult to realize, so that the inventors have conducted many studies on the above problems, and found that the problems can be improved when DCpep is used in series in the research process, and particularly, the inventors found that the spatial conformation can be prevented from being changed and can be fully exposed when 3 DCpep are used in series, and the DCpep has a better effect on DC cells, and can better assist the body to stimulate a stronger immune response on the premise of exerting the conventional advantages thereof.
Specifically, in the research process, the inventors constructed 5 groups of recombinant lactic acid bacteria (original starting strain is NC 8/delta alr) with 1-5 DCpep tandem as target fragments and pWCF as a vector. In vitro DC stimulation experiments were then performed. The specific test method comprises the following steps: SPF grade female C57BL/6 mice were sacrificed by decapitation; washing bone marrow cells from femur of mouse with sterile RPMI 1640 culture medium in super clean bench; washing bone marrow cells 2 times with complete RPMI 1640 medium; after washing, adding complete RPMI 1640 culture medium into the bone marrow cells, adding 25ng/mL recombinant GM-CSF, and culturing in a 37 ℃ incubator; on day 8, purity of the DCs was identified by flow cytometry with CD11c marker; in 96-well plates, DC (2X 10) 5 Individual cell) and each group of recombinant lactic acid bacteria (2X 10) 6 CFU) as per 1: 10 for 12h, and then detecting the molecules of CD80 and CD86 on the surface of the DC by flow cytometry.
As a result: the mouse bone marrow-derived DC is co-cultured with recombinant lactic acid bacteria NC8 delta-pWCF-1 × DCpep, NC8 delta-pWCF-2 × DCpep, NC8 delta-pWCF-3 × DCpep, NC8 delta-pWCF-4 × DCpep and NC8 delta-pWCF-5 × DCpep for 12h respectively. In the detection of CD80 molecules, the CD80 of the surface of the 3 × DCpep group is very significantly higher than that of the 1 × DCpep group (P < 0.001); the molecules of CD80 were significantly higher in the 4 × DCpep and 5 × DCpep groups than in the 1 × DCpep group (P < 0.01); the 3 × DCpep, 4 × DCpep and 5 × DCpep groups CD80 molecules were all significantly up-regulated compared to the 2 × DCpep group (P < 0.05), with the results shown in fig. 17 (a). In the detection of CD86 molecules, both the 3 × DCpep and 5 × DCpep groups CD86 molecules were significantly up-regulated (P < 0.05) compared to the 1 × DCpep group, and the results are shown in fig. 17 (B). In addition, the inventors found that the sequence is too long to facilitate the tag expression and the antigen preparation, therefore, the combined results show that the 3 DCpep tandem can achieve better effect on DC, and the activation of DC surface molecules is not significantly different with the increase of DCpep number, so that the 3 DCpep tandem effect is optimal. Thus, in a specific embodiment of the invention, 3 dcpeps are selected in tandem.
Example 1Construction and verification of recombinant lactic acid bacteria for expressing African swine fever virus fusion antigen
The inventor finds that DNA polymerase is important in the replication link of the virus in the research of African swine fever virus protein, and p72 protein is responsible for the formation of virus capsid. The present example provides the construction and verification of three strains of NC8 delta-pWCF-DNApol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNApol-p 72 non-antibiotic recombinant lactic acid bacteria from the idea of cutting off the formation and replication of viruses. Then, rabbit DCpep polyclonal antibody serum is prepared, and the antibody is verified by a western blot technology. Finally, the immunogenicity of the target protein is verified by flow cytometry, and the anchoring expression condition of the target protein is detected by using an immunofluorescence technique, and the embodiment proves that the induced recombinant lactic acid bacteria have the immunogenicity and can be anchored and expressed on the surface of NC 8/delta alr lactobacillus plantarum.
1.1 materials and methods
1.1.1 materials
The strains and plasmids used in this example are shown in Table 1-1.
TABLE 1-1 strains and plasmids
Figure GDA0003700011840000061
Preparation of a culture medium:
(1) LB liquid culture Medium
The components: NaCl 10.0g, Tryptone 10.0g, Yeast extract 5.0g, distilled water (ddH) 2 O)1L。
Sterilizing with high pressure steam at 121 deg.C for 20min, and storing at room temperature.
(2) MRS liquid medium
The components: beef extract (Lab-Lemco Powder)10.0g, Tryptone (Tryptone)10.0g, glucose (C) 6 H 12 O 6 )20.0g, sodium acetate (CH) 3 COONa 5.0g, Yeast extract (Yeast extract)0.05g, Tween 80(Tween-80)1.0g, and dipotassium hydrogen phosphate (KH) 2 PO 4 )2.0g of diammonium hydrogen citrate (C) 6 H 14 N 2 O 7 )2.0g magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 O)0.2g, manganese sulfate monohydrate (MnSO) 4 ·H 2 O)0.05g, distilled water (ddH) 2 O)1L。
Sterilizing with high pressure steam at 115 deg.C for 15min, and storing at room temperature.
Preparing an electrotransformation solution:
(1) and (3) configuring a shock buffer solution:
consists of the following components: 34.21g of sucrose, MgCl 2 0.029g、ddH 2 O 100mL。
Adjusting pH to 7.4, sterilizing with high pressure steam, and storing at-20 deg.C.
(2) Washing buffer solution preparation:
consists of the following components: na (Na) 3 PO 4 0.19g、MgCl 2 0.009g、ddH 2 O 100mL。
Adjusting pH to 7.4, sterilizing with high pressure steam, and storing at-20 deg.C.
1.1.2 test methods
1.1.2.1 construction and identification of recombinant lactic acid bacteria
1.1.2.1.1 Synthesis of target Gene
Nucleotide sequence information of three groups of target genes:
first set of genes of interest (DNApol gene fragments): the nucleotide sequence is shown as SEQ ID NO: shown in fig. 7.
Second set of genes of interest (p72 gene fragment): the nucleotide sequence is shown as SEQ ID NO: shown in fig. 8.
Third group of genes of interest (DNApol-p72 gene fragment): the nucleotide sequence is shown as SEQ ID NO: shown in fig. 9.
Synthesis of three groups of genes of interest:
the sequence of ASFV-SY18 gene (MH766894.1) is inquired in GenBank to construct three groups of target genes, and the target genes are connected with a pUC-GW-Kan vector to obtain three groups of target fragments pUC-DNApol, pUC-p72 and pUC-DNApol-p72 respectively.
According to the analysis of DNAstar Protean software, the DNA polymerase protein coded by the G1211R gene has generally good hydrophilicity, the antigen index dense region is more concentrated in the latter half section, and a plurality of transmembrane regions are analyzed through surface accessibility, which is shown in figure 1. The G1211R 1414 th and 3351 th gene sequences (472 th and 1117 th amino acids) are intercepted and connected with the three groups of repeated DCpep sequences to be used as a first group of target genes.
Four groups of p72 protein epitopes that produce a strong immune response were selected by reference to Mallory E et al (Linear epitopes in African swine farm animal viruses p72 purified epitopes-expressed antigens), see tables 1-1. In this example, four groups of antigens were duplicated in three groups, and then concatenated together with the duplicated Dcpep genes in three groups as a second group of target genes.
TABLE 1-1 p72 protein epitopes
Figure GDA0003700011840000071
The first group of target genes and the third group of target genes are connected by an SD sequence of 12 bases to form a third group of target genes.
The three groups of target genes are respectively added with an initiator and a terminator, and Xba I and Hind III enzyme cutting site gene sequences are respectively arranged at two ends. Wherein the enzyme cutting site gene sequence, the DCpep gene sequence and the SD sequence information are as follows:
xba I cleavage site gene sequence: TCTAGA;
hind iii restriction site gene sequence: AAGCTT;
the nucleotide sequence of the DCpep gene is shown as SEQ ID NO: 5 is shown in the figure;
the SD sequence is shown as SEQ ID NO: shown in fig. 6.
In addition, the gene sequence is optimized aiming at the protein expression of the lactobacillus plantarum.
1.1.2.1.2 primer design
In order to identify the correctness of the recombinant plasmid, universal primers aiming at the pSIP 409-pgsA' -alr vector are designed and used for PCR identification and sequencing. The primer sequences are as follows:
F 5’-AGATATTGTTGGTGCTGG-3’(SEQ ID NO：14)
R 5’-TCAATCAAAGCAACACG-3’(SEQ ID NO：15)
1.1.2.1.3 vector and acquisition of target fragment
The pWCF vector was obtained from the laboratory-stored plasmid 409ata by double digestion for future use. Similarly, the desired fragment was excised from the pUC vector by double digestion for use. The enzyme digestion system is as follows:
TABLE 1-2 enzyme digestion System
Figure GDA0003700011840000081
And (3) carrying out gel recovery after enzyme digestion at 37 ℃ overnight, wherein the gel recovery method is operated according to the kit instruction.
1.1.2.1.4 order fragment and vector connection
The 3 groups of recovered products were linked to the vector by T4 DNA ligase, respectively. The linking system is as follows:
TABLE 1-3 ligation reaction System
Figure GDA0003700011840000082
Ligation conditions were 16 ℃ overnight.
Construction of 1.1.2.1.5 pWCF empty vector
The vector was obtained by double digestion in 1.1.2.1.3, after gel recovery, both ends of the cleavage site were smoothed with a DNA end smoothing kit, and then both ends of the nick were ligated with T4 DNA ligase to obtain pWCF empty vector (SEQ ID NO: 13) for use.
1.1.2.1.6 ligation products were transformed to E.coli χ 6212 competent
Preparation of competence 1.1.2.1.6.1 E.coli χ 6212
Coli χ 6212 was streaked on a plate of LB medium supplemented with DAP (final concentration of 50. mu.g/ml), and cultured overnight at 37 ℃; picking single colony every other day, culturing in 100ml LB liquid culture medium added with DAP (final concentration is 50 mug/ml) at 37 ℃, measuring OD600 between 0.8-1.0, and taking out bacterial liquid; transferring the bacterial liquid to a 50ml precooled centrifuge tube in a super clean bench; centrifuging at 2500rpm and 4 deg.C for 10 min; discard the supernatant and add the pre-cooled ddH 2 Supplementing O to 40ml, gently mixing on ice, and washing once; washing with 10% glycerol for 3 times; suspended with 1ml 10% glycerol on ice; precooling in a 1.5ml centrifuge tube, subpackaging each tube by 100 mu L, quickly freezing by liquid nitrogen, and transferring to a refrigerator at-80 ℃ for storage.
1.1.2.1.6.2 conversion of ligation products
The ligation product was transferred to e.coli χ 6212 competence by electrotransformation. Melting competence on ice, sucking 2-5 mu L of the ligation product into the competence, transferring the competence into a clean 0.2 cm-distance electric shock cup, and placing the electric shock cup on ice for 5 min; using an electrotransformation instrument for electric shock under the conditions of 2500V, 200 omega and 25 muF; sucking 600 mu LLB liquid culture medium in an electric shock cup, transferring the mixed solution into a centrifuge tube, and performing shaking culture at the constant temperature of 37 ℃ for 1 h; uniformly coating the bacterial liquid on an LB culture medium plate, and culturing in an inverted incubator at 37 ℃ for more than 14 h; picking single colony in a 5ml LB liquid culture medium test tube, and preserving the bacteria after culturing at 37 ℃.
Identification of recombinant plasmid in 1.1.2.1.7 E.coli chi 6212
Taking bacterial liquid to extract a small amount of plasmids, and the method is described in the specification. Then carrying out enzyme digestion and PCR identification.
1.1.2.1.7.1 enzyme digestion identification
The enzyme digestion identification system is as follows:
TABLE 1-4 enzyme digestion System
Figure GDA0003700011840000091
After a metal bath at 37 ℃ for 8h, the band size was identified by agarose gel electrophoresis.
1.1.2.1.7.2 plasmid sequencing
After the enzyme digestion identification is correct, the plasmid is sequenced by Jilin province Shumei Biotech limited.
1.1.2.1.8 transformation of recombinant plasmid into NC 8/. DELTA.alr
1.1.2.1.8.1 NC8/Δ alr competence preparation
Taking 50 mu L of NC 8/delta alr cryopreserved bacteria, adding into 5mL of MRS liquid culture medium added with D-alanine (final concentration is 0.2mg/mL), and activating NC 8/delta alr; dipping NC 8/delta alr bacterial liquid by using an inoculating loop, streaking the liquid in an MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 16-18 h; selecting a single colony on an MRS solid culture medium plate, adding D-alanine (with the final concentration of 0.2mg/mL) and 100 mu L Gly (2%) in 5mL of MRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 7-8 h until the OD600 is between 0.6 and 0.8; subculturing until OD600 is between 0.2 and 0.3, and taking out; carrying out ice-bath on the bacterial liquid for 20min, centrifuging at 4 ℃ and 5000rpm for 10min, removing supernatant, and collecting thalli; washing with 1mL of pre-cooled washing buffer for 2 times (1 mL each time), and discarding the supernatant; resuspend the pellet with 400. mu.L shock buffer, then dispense 100. mu.L per tube into pre-cooled 1.5mL centrifuge tubes, snap-freeze with liquid nitrogen, and transfer to-80 deg.C refrigerator for storage.
1.1.2.1.8.2 transformation of recombinant plasmid
The constructed plasmid was transformed into NC 8/. DELTA.alr by the electrotransformation method. Pre-cooling before the motor cup is lifted on ice; melting NC 8/delta alr competent ice, adding the recombinant plasmid, and flicking fingers to mix evenly; transferring the mixed solution of the NC 8/delta alr receptor bacterial cells and the recombinant plasmids into a motor cup, and placing for 5min on ice; using an electrotransfer instrument for electric shock, and adopting the following conditions: 2000V, 400 Ω, 25 μ F; sucking 800 μ L of MRS liquid culture solution which is placed in a 30 ℃ water bath kettle in advance and preheated into an electric shock cup, sucking out the mixed solution in the electric shock cup into a 2.0mL centrifuge tube, adding 150 μ L of sucrose solution (0.5mol/L), and standing in the 30 ℃ water bath kettle for 3 h; absorbing bacterial liquid, uniformly coating the bacterial liquid on an MRS solid culture medium plate, and carrying out constant-temperature inverted culture in an anaerobic workstation at 37 ℃ for more than 18 h; and (4) picking a single colony in a 5ml MRS liquid culture medium test tube, and preserving the bacteria after culturing at 37 ℃.
1.1.2.1.9 verification of recombinant plasmid in NC 8/delta alr
And (3) extracting recombinant lactic acid bacteria plasmid according to the operation of the small gram-positive bacteria plasmid kit instruction, and performing PCR identification.
The PCR system was as follows:
TABLE 1-5 PCR identification System
Figure GDA0003700011840000101
The PCR amplification conditions were as follows:
Figure GDA0003700011840000102
1 μ L of the PCR product was subjected to agarose gel electrophoresis to identify the band size.
1.1.2.2 preparation of DCpep polyclonal antibody
1.1.2.2.1 preparation of polyclonal antibodies
A DCpep12 amino acid short peptide was synthesized and observed for 7 days at 6 weeks of age to adapt to the environment. Dissolving and diluting the short peptide to 1mg/mL, adding 1mL of complete Freund's adjuvant into the short peptide with the first immunization dose of 1mL, and uniformly mixing by using a homogenizer. The second immunization was performed at 14-day intervals, and the immunization dose was 0.5mL of short peptide plus 0.5mL of incomplete Freund's adjuvant. Blood was collected from the marginal vein at 7 day intervals and centrifuged to obtain serum, see FIG. 3.
1.1.2.2.2 identification of polyclonal antibodies
After the lactobacillus protein sample is treated by an ultrasonic disruption method, by a Western blot technology, serum is used as a primary antibody, goat anti-rabbit HRP is used as a secondary antibody, and a DCpep positive lactobacillus sample is incubated to verify whether the antibody is successfully prepared.
1.1.2.3 expression identification of recombinant lactic acid bacteria
1.1.2.3.1 optimization of culture condition of lactic acid bacteria
Inoculating 50 μ L of the frozen stock solution to 5mM MRS culture solution, and performing anaerobic culture at 37 ℃ overnight. On the next day, 4mL of overnight fresh bacterial liquid was subcultured in a 50mL centrifuge tube containing 40mL of LMRS culture solution, and anaerobic culture was performed at 30 ℃.2mL of the culture solution was taken every hour to measure OD600, and when the OD600 value was about 0.3, induced SPPIP (12.5. mu.L/5 mL) was added. After the induction of the bacterial liquid, taking the bacterial liquid in the logarithmic growth phase for gradient dilution, counting the number of the dropping plates, and calculating 1 multiplied by 10 groups of 3 groups of recombinant lactic acid bacteria 9 CFU (optimal number of mice fed with Lactobacillus is 0.5X 10) 9 ～1×10 9 CFU) how much volume of inoculum each needs.
1.1.2.3.2 flow cytometry identification
Activating the frozen recombinant lactobacillus, streaking on an MRS solid culture medium plate, and selecting a single bacterium to culture in a 5mL MRS liquid culture medium test tube overnight; 1:40 transferring to a new MRS liquid culture medium test tube, carrying out anaerobic culture at 30 ℃ for 3 hours, and adding 12.5 mu L of SppIP for induction culture to logarithmic phase; taking 0.5mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging for 1min at 12000r, collecting the precipitate, and resuspending with 1mL of PBS; adding 1 mL/1% BSA PBS, placing in a shaking table at 4 ℃ to block for 1h, and washing with 1mL PBS for three times after finishing; incubating the primary antibody: 1:300 diluted DCpep rabbit polyclonal antibody serum is added into a centrifuge tube and shaken overnight at 4 ℃; washing for three times by using 1mL of PBS (phosphate buffer solution) added with 0.2% Tween-20, and collecting precipitates; incubation of secondary antibody: adding FITC labeled anti-rabbit secondary antibody diluted by 1:800 under the condition of keeping out of the sun, and uniformly mixing the thalli in a 1.5mL centrifuge tube; shaking table at room temperature for 1.5h in dark place; centrifuging to remove supernatant, washing with PBS (phosphate buffer solution) added with 0.2% Tween-20 for 3 times, removing supernatant, and collecting precipitate; flow detection, mixing with 400 μ L PBS, and loading onto machine after membrane filtration.
1.1.2.3.3 immunofluorescent microscopy
And uniformly mixing 200 mu L of PBS, applying the thalli of the primary antibody and the secondary antibody, dripping 10 mu L of bacterial liquid on a glass slide, naturally drying, dripping a fluorescent anti-attenuation agent to cover the bacterial liquid, covering a cover glass, keeping away from light with tinfoil, and observing under a microscope.
1.2 results
1.2.1 construction and identification of recombinant lactic acid bacteria
1.2.1.1 Synthesis of the Gene of interest
The sizes of the three finally constructed target genes are 2062, 619 and 2683nt respectively, and are shown in figure 2.
DNApol gene structure (2062 base pairs total): xba I-DNApol Gene fragment-3 group Dcpep tandem fragment-Hind III.
p72 gene structure (619 base pairs total): xba I-p72 gene fragment-3 group Dcpep tandem fragment-Hind III, wherein the p72 gene fragment has the following structure: 3 groups of epitopes 1 are connected in series-three groups of epitopes 2 are connected in series-three groups of epitopes 3 are connected in series-three groups of epitopes 4 are connected in series.
DNApol-p72 gene structure (2683 base pairs total): xba I-DNApol-p72 gene fragment-3 group Dcpep tandem fragment-Hind III, wherein the DNApol-p72 gene fragment has the following structure: DNA pol gene fragment-group 3 Dcpep tandem fragment-SD sequence-p 72 gene fragment-group 3 Dcpep tandem fragment.
1.2.1.2 vector and acquisition of fragments of interest
The vector and the target fragment are respectively subjected to double enzyme digestion, and vector bands, 2062bp, 619bp and 6683bp, can be seen at 8121bp
Identification of recombinant plasmid in 1.2.1.3 E.coli chi 6212
1.2.1.3.1 enzyme digestion identification
The recombinant plasmid pWCF-DNApol was double-digested and verified, and a clear band was visible at 2062bp (FIG. 4, A).
The recombinant plasmid pWCF-p72 was verified by double digestion, and a clear band was visible at 619bp (FIG. 4, B).
The recombinant plasmid pWCF-DNApol-p72 was verified by double digestion, and a clear band was visible at 2683bp (FIG. 4, C).
1.2.1.3.2 plasmid sequencing
The plasmid sequencing result is correct.
1.2.1.4 identification of recombinant plasmids in NC8/Δ alr
And amplifying the target fragment of the recombinant plasmid in NC 8/delta alr by using a PCR (polymerase chain reaction) technology so as to identify whether the recombinant plasmid is successfully transformed into NC 8/delta alr. A clear band was visible at 2062, 619, 2683bp (FIG. 5).
1.2.2 identification of the DCpep polyclonal antibody
The DCpep rabbit polyclonal antibody serum is identified by the Western blot technology, a clear protein band is visible at 55kDa (figure 6), and the result shows that the preparation of the polyclonal antibody serum is successful.
1.2.3 expression identification of recombinant lactic acid bacteria
1.2.3.1 optimization of culture conditions for lactic acid bacteria
After 3 times of cultivation and determination of OD600, the results (24h) of the growth curve determination of NC 8/. DELTA.alr, NC 8. DELTA. -pWCF, NC 8. DELTA. -pWCF-DNApol, NC 8. DELTA. -pWCF-p72 and NC 8. DELTA. -pWCF-DNApol-p72 were plotted using GraphPad Prism software, see FIG. 7. The optimal induction time is 3 hours after the bacterial liquid is transferred. The feeding time point at 9h (6 h after induction) was selected according to the results of the triple colony plate count assay, and the bacterial solutions of 0.2mL NC 8/delta alr, NC8 delta-pWCF, 0.22mL NC8 delta-pWCF-DNApol, 1.3mL NC8 delta-pWCF-p 72, and 1.66mL NC8-pWCF-DNApol-p72 had 1X 10 bacterial solutions 9 CFU。
1.2.3.2 flow cytometry identification of fusion protein expression
NC8 delta-pWCF, NC8 delta-pWCF-DNAPol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNAPol-p 72 are subjected to protein induction expression and then are detected by a flow cytometer, as shown in figure 8, on the abscissa axis, the more the displayed image is to the right, the stronger the fluorescence intensity carried by the checking substance is, and the more the detected substance is combined, the result shows that the constructed recombinant plasmid is expressed in the lactobacillus, and the recombinant lactobacillus has good antigenicity.
1.2.3.3 immunofluorescence identification of fusion protein expression
FITC labeled secondary antibody can be combined with rabbit-derived primary antibody, so that the fusion protein anchored and expressed on the surface of lactobacillus plantarum has green fluorescence. As a result, NC 8. delta. -pWCF did not fluoresce, as in FIG. 9B; fluorescence was seen for each of NC 8. delta. -pWCF-DNAPol, NC 8. delta. -pWCF-p72, and NC 8. delta. -pWCF-DNAPol-p72, as shown in FIGS. 9D, F, H.
1.3 summary
The test in this chapter successfully constructed NC8 delta-pWCF-DNApol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNApol-p 72 recombinant lactic acid bacteria. Furthermore, rabbit-derived DCpep polyclonal antibody sera were successfully prepared. The immunogenicity of the fusion protein expressed by 3 groups of non-antibiotic recombinant lactic acid bacteria is verified, and the recombinant lactic acid bacteria anchor and express African swine fever virus DNA polymerase, p72, DNA polymerase and p72 fusion antigen on the surface of lactobacillus plantarum respectively.
Example 2Research on immune effect of recombinant lactic acid bacteria
The lactobacillus vaccine has the advantages of low cost and convenient large-scale production, can be permanently planted in intestinal tracts of organisms to continuously play a role, and can improve the immunity of the organisms and promote the mucosal immunity because the lactobacillus is probiotics. The oral immunization mode also makes it more convenient. Therefore, the recombinant lactobacillus vaccine has wide development prospect. The recombinant lactic acid bacteria constructed in the embodiment 1 of the invention are non-antibiotic lactic acid bacteria, are more environment-friendly and avoid antibiotic tolerance for animals.
In this example, primary immunization and boosting immunization programs were set to induce the expression of 3 groups of recombinant lactic acid bacteria, and control groups were set to perform oral gavage immunization on each group of mice. And after the immunization is finished, the immunization indexes of the mice of each group are detected, and then the immunization effect of the recombinant lactic acid bacteria is evaluated.
2.1 materials and methods
2.1.1 materials
2.1.1.1 test strains
NC8 delta-pWCF-DNAPol recombinant lactobacillus, NC8 delta-pWCF-p 72 recombinant lactobacillus, NC8 delta-pWCF-DNAPol-p 72 recombinant lactobacillus and NC8 delta-pWCF empty vector lactobacillus.
2.1.1.2 test animal
50 SPF grade 6 week old C57BL/6J mice were purchased from Henan Scout Biotechnology Ltd.
2.1.2 methods
2.1.2.1 grouping and immunization scheme for experimental animals
2.1.2.1.1 immunization group
Mice were randomly divided into 5 groups of 10 each of PBS, NC8 Δ -pWCF, NC8 Δ -pWCF-DNApol, NC8 Δ -pWCF-p72 and NC8 Δ -pWCF-DNApol-p 72. Treating each group of recombinant lactobacillus bacterial liquid, wherein the number of live bacteria fed to each mouse is 1.0 multiplied by 10 9 And (4) CFU. The culture method and required volume of lactobacillus are 1.2.3.1. The induced bacteria solution of the corresponding volume was taken, washed three times with sterile PBS, resuspended with 200. mu.L PBS, and the mice were gavaged. Animal immunization protocol and groups are shown in Table (Table 2-1).
TABLE 2-1 animal immunization protocol and groupings
Figure GDA0003700011840000131
2.1.2.1.2 immunization procedure
Primary immunization on days 1,3 and 5, and booster immunization on days 15, 17 and 19. The status of the mice was observed after the booster immunization and flow cytometry was performed on day 29. As shown in fig. 10.
2.1.2.2 polypeptide Synthesis
Synthesizing 4 polypeptides as cell stimulation antigen peptides required by detecting specific T cell related indexes by flow cytometry, wherein the amino acid sequences of the cell stimulation antigen peptides are respectively as follows: 1. DCpep (SEQ ID NO: 16), 2, DNA polymerase partial amino acids (SEQ ID NO: 17), 3, p72 epitope 2(SEQ ID NO: 19), 4, p72 epitope 3(SEQ ID NO: 20).
2.1.2.3 flow cytometry
Each group of 3 mice were sacrificed by cervical dislocation.
2.1.2.3.1 preparation of cell suspension
Spleen, Mesenteric Lymph Nodes (MLN), and lymph nodes Peyer-Patches (PPs) were removed from the clean bench, taking care to strictly control the aseptic process. The spleen, mesenteric lymph nodes and Peyer's lymph nodes were harvested, sterilized copper mesh was placed in a 35mm sterile plate and soaked in 1mL1640 complete medium, the tissue was placed in the copper mesh, and the tissue was ground with the tail of a 1mL syringe. After the tissue is ground, the liquid is absorbed and transferred into a centrifuge tube, the centrifuge tube is centrifuged for 5min at the temperature of 4 ℃ and the rpm of 2000, and the supernatant is discarded. Spleen cells need to be lysed, 500. mu.L of erythrocyte lysate is added to the spleen cells, the spleen cells are placed on ice for 3min and then 500. mu.L of PBS is added to the spleen cells, the mixture is placed on ice for 2min, and the supernatant is discarded after centrifugation. Spleen, MLN and PPs cell fluid were washed 1 time with 1mL PBS, centrifuged, and the supernatant was discarded, followed by addition of 1mL1640 complete medium. When the cells of each group are diluted and counted, the number of the cells of different tissues is different, so that the spleen is 100 times diluted and needs 1.0x10 7 One cell, MLN 50-fold diluted and required 1.0X10 7 One cell, PPs 20-fold diluted and required 1.0 × 10 6 And (4) cells. After counting, the corresponding volume of the cell stock solution in the tube was washed once and then filled to 1mL with PBS to prepare a cell suspension. The PPs cell dilution was centrifuged at 2000rpm for 5min at 4 ℃ and 900. mu.L of the supernatant was discarded, leaving 100. mu.L and mixing well. Two tubes were prepared for use.
2.1.2.3.2 detection of P's lymph node cell DC surface CD80 + And CD86 +
To 100. mu.L of the prepared cell suspension, 5. mu.g of CD16/CD32 Pure per tube, 4 ℃ and 5min were mixed. 10. mu.L each of CD11c-APC (40-fold dilution), CD80-FITC (60-fold dilution), CD86-PE-Cy7 (20-fold dilution), and MHC-II-PerCy-Cy5.5) antibody was added, and incubated at 4 ℃ for 20min in the absence of light. Washing with PBS once, reserving 100 μ L, mixing with 200 μ L PBS, sieving with nylon sieve, and loading on machine.
2.1.2.3.3 detection of B cell activation level of Peyer's patches
To each set of prepared 100. mu.L of P.pai-shi lymph node cell suspension was mixed diluted CD16/CD32 Pure 5. mu.l/tube at 4 ℃ for 5 min. Add 10. mu.L of B220-APC (110-fold dilution) antibody, protect from light at 4 ℃ for 20min, and wash once with PBS. Adding 250 μ L formaldehyde fixing solution, and fixing at 4 deg.C in dark for 20 min. After twice membrane-piercing with the membrane-piercing liquid, IgA-FITC (diluted 40 times) was added, and the mixture was protected from light at 4 ℃ for 20min, and the supernatant was centrifuged. After washing away the residual antibody with 1mL PBS, 200. mu.L PBS was added and mixed, and the mixture was screened through a nylon screen and then loaded onto a machine.
2.1.2.3.4 detection of cytokines in spleen and mesenteric lymph node cells
Cell plating in 24-well plates: each group of prepared spleen and mesenteric lymph node cell suspensions was aspirated at 150. mu.L (1.5X 10) 6 Individual cells) were added with 350. mu.L of complete medium, 40ng/mL of PMA as a stimulator, and 4. mu.g/mL of each of the corresponding antigen peptides, with the respective numbers of the antigen peptides in each group being: group a (antigenic peptide nos. 1,2,3,4), group B (antigenic peptide nos. 1,2,3,4), group C (antigenic peptide nos. 1,2), group D (antigenic peptide nos. 1,3,4), and group E (antigenic peptide nos. 1,2,3, 4).
The cell-plated plates were incubated at 37 ℃ for 8 hours. Add 1 μ L of blocker (protein transport inhibitor) at a dose to complete medium ratio of 1: incubate at 40, 37 ℃ for an additional 4 hours. After the incubation, the mixture was aspirated into a 1.5mL centrifuge tube, centrifuged, and the supernatant was removed, washed once with 1mL PBS, and 100. mu.L of the remaining solution was obtained. Then, 10-fold diluted CD16/CD32 Pure was mixed at 5. mu.s/tube, 4 ℃ for 5 min. Incubation was performed under conditions of addition of light-shielding at 4 ℃ for 20min, and 10. mu.L each of CD3-percpcy5.5 (40-fold dilution), CD4-FITC (60-fold dilution), and CD8-APCCY7 (60-fold dilution) was added thereto. Washing once and keeping precipitate, adding 250 μ L formaldehyde fixing solution, and keeping away from light at 4 deg.C for 20 min. After the incubation is finished, the cell is directly subjected to membrane penetration by using the membrane penetration solution twice, 800 mu L of the membrane penetration solution is used for the first time, 1mL of the membrane penetration solution is added for the second time, the cell is incubated for 5min in a dark place and then centrifuged, and the precipitate and 100 mu L of supernatant are reserved. IFN-gamma-APC (30-fold dilution) was added at 10. mu.L per tube and incubated for 20min at 4 ℃ in the absence of light. Washing once and keeping the precipitate, uniformly mixing 200 mu LPBS, passing through a nylon screen, and detecting on a machine.
2.1.2.3.5 test the proliferation of T cells in spleen and mesenteric lymph node
300 μ L of the prepared cell suspension was stained with CFSE (final concentration 1 μ L/mL), incubated at 37 ℃ for 10min, and 300 μ L of serum FBS was added to stop the reaction, washed 1 time with PBS and 1 time with 1640 complete medium. After leaving 200. mu.L, 1mL of 1640 complete culture medium was added thereto, and 1.2. mu.L of PMA was diluted 1000-fold, and 2.4. mu.L of each of the antigen peptides (1mg/mL) was suspended and mixed well. The wells were plated in 96-well plates at 200. mu.L per well and each sample was repeated 6 times. The plated plates were incubated in a cell incubator at 37 ℃ for 3 days. The cells were transferred to a 1.5mL centrifuge tube, supplemented with PBS to 1mL, centrifuged, washed 1 time with 1mL PBS, and 100. mu.L of the remaining cell pellet was mixed. Mix in CD16/CD32 Pure (10 fold dilution) 5 μ,4 ℃ per tube for 5 min. Add antibody CD4-PERCP (60-fold diluted) CD8-APC (60-fold diluted) 10. mu.L each sample, incubate at 4 ℃ for 20min in the dark. The sample was washed once with PBS, and 200. mu.L of PBS was mixed well and precipitated through a nylon mesh and placed on a machine.
2.1.2.4 data processing and analysis
Flow cytograms were analyzed using Flowjo 6.2.1 software. The data were plotted and counted using GraphPad Prism software, and differences between the groups of data were counted using one-way ANOVA (p, 0.05;. p, 0.01;. p, 0.001;. p < 0.0001).
2.2 results
2.2.1 Effect of recombinant lactic acid bacteria on post-immunization mouse Peyer's Patch DC activation
The important roles of Dendritic Cells (DCs) are in the uptake, processing and presentation of antigens, stimulating the body to mount an immune response. To evaluate the effect of the recombinant lactic acid bacteria NC8 delta-pWCF-DNAPol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNAPol-p 72 on the mouse P-lymph node, the mice were fed with the recombinant lactic acid bacteria orally and immunologically, and after one week of booster immunization, the condition of DCs in the mouse P-lymph node was examined. The test is used for detecting the expression of activation markers CD80, CD86 and MHC-II on the surface of the dendritic cells. The results show MFI in PPs (CD11 c) + CD80 + The MFI) DNApol-p72 group differed significantly (p < 0.05) compared to the PBS group, and the DNApol-p72 group differed significantly (p < 0.05) compared to the unloaded group; MFI in PPs (CD11 c) + CD86 + ) The DNApol-p72 group is significantly different (p < 0.05) compared with the DNA-pol group; MFI in PPs (CD11 c) + MHC-Ⅱ + ) The DNApol-p72 group differed significantly (p < 0.0001) compared with the PBS group, the DNApol-p72 group differed significantly (p < 0.01) compared with the unloaded group, the DNApol-p72 group differed significantly (p < 0.05) compared with the DNApol group, and the DNApol and p72 groups differed significantly (p < 0.01) compared with the PBS group. The above results demonstrate that recombinant lactic acidThe bacteria had activating effect on mouse P.pekinensis DC, and the result is shown in FIG. 11.
2.2.2 Effect of recombinant lactic acid bacteria on mouse specific cytokines after immunization
Following booster immunization, flow cytometry was used to detect CD4 in mouse mesenteric lymph nodes and spleen + IFN-γ + And CD8 + IFN-γ + The cell is detected, and the result shows that the cell immune reaction of the MLN and the spleen of the mouse can be activated by the recombinant lactic acid bacteria anchoring and expressing the African swine fever virus fusion antigen.
CD4 in mouse MLN + IFN-γ + The results of cytokine detection are shown in FIG. 12(A), and compared with the PBS control group, the CD4 of the recombinant lactobacillus group was orally administered with NC 8. delta. -pWCF-DNApol-p72 + IFN-γ + The proportion of cells was very significantly increased (p < 0.001) and among the groups, DNApol-p72 group CD4 + IFN-γ + The highest proportion of cells; the DNApol group is very different from the PBS group (p is less than 0.001); the p72 group has very significant difference compared with the PBS group (p < 0.01); the DNApol-p72 group differed significantly (p < 0.01) compared to the unloaded group. While detecting CD8 + IFN-γ + In cells, the results are shown in FIG. 12(B), and compared with the PBS control group, CD8 of the NC8 delta-pWCF-DNApol-p 72 oral administration recombinant lactic acid bacteria and NC8 delta-pWCF-DNApol oral administration recombinant lactic acid bacteria group + IFN-γ + The cell proportion is very remarkably increased (p is less than 0.001), and the CD8 of the p72 group + IFN-γ + The cell ratio was very significantly increased (p < 0.01), but the group DNApol-p72 had CD8 + IFN-γ + The highest proportion of cells; DNApol-p72 and DNApol group CD8 compared to the unloaded group + IFN-γ + The cell proportion is remarkably increased (p is less than 0.01); DNApol-p72 differed significantly (p < 0.05) compared to the p72 group.
CD4 in spleen of para mouse + IFN-γ + The results of cytokine detection are shown in FIG. 13(A), and compared with PBS control group, CD4 of oral NC8 delta-pWCF-DNAPol-p 72 recombinant lactobacillus group and oral NC8 delta-pWCF-DNAPol recombinant lactobacillus group + IFN-γ + The proportion of cells was very significantly increased (p < 0.001) and among the groups, DNApol-p72 group CD4 + IFN-γ + Cell ratioThe case is the highest; the DNApol-p72 group differed significantly (p < 0.001) compared to the unloaded group; the DNApol group is very different from the unloaded group (p is less than 0.01); the DNApol group and DNApol-p72 group differed significantly (p < 0.01) compared to the p72 group. While detecting CD8 + IFN-γ + In cells, the results are shown in FIG. 13(B), and NC 8. delta. -pWCF-DNApol-p72 recombinant lactic acid bacteria CD8 was orally administered compared to PBS control group + IFN-γ + Very significant increase in cell fraction (p < 0.001), CD8 in DNApol group + IFN-γ + The cell ratio was significantly increased (p < 0.05), and DNApol-p72 group CD8 + IFN-γ + The highest proportion of cells; DNApol-p72 group CD8 compared to the empty group + IFN-γ + The cell ratio is remarkably increased (p is less than 0.001), the DNApol group is remarkably increased (p is less than 0.05); DNApol-p72 differed significantly (p < 0.05) compared to the p72 group.
2.2.3 Effect of recombinant lactic acid bacteria on proliferation of MLN and splenic T cells in immunized mice
After the boosting immunization, the T cell proliferation in the mesenteric lymph node and the spleen of the mouse is detected by flow cytometry, and the result shows that the MLN and the T cell proliferation of the spleen of the mouse can be activated by the recombinant lactobacillus anchoring and expressing the African swine fever virus fusion antigen.
CD4 in mouse MLN + The results of the detection of T cell proliferation are shown in FIG. 14(A), and compared with the PBS control group, the CD4 of the recombinant lactic acid bacteria group is orally administered with NC8 delta-pWCF-DNApol-p 72 + The proliferation of T cells was very significantly increased (p < 0.001), and CD4 in DNApol and p72 groups + The proliferation of T cells was significantly increased (p < 0.05) and among the groups, DNApol-p72 group CD4 + T cell proliferation is highest; compared with the unloaded group, the value-added situation of the DNApol-p72 group is very obvious in difference (p is less than 0.001), and the value-added situation of the DNApol group is obvious in difference (p is less than 0.05); the DNApol-p72 group differed significantly (p < 0.05) compared to the p72 group. While detecting CD8 + The results of T cell proliferation are shown in FIG. 14(B), and CD8 of the recombinant lactic acid bacteria group was orally administered NC 8. delta. -pWCF-DNApol-p72, compared with the PBS control group + The proliferation of T cells is very significantly increased (p < 0.001), and the proliferation of CD8 in the p72 group + The proliferation of T cells was significantly increased (p < 0.05) and in each group, DNApol-p7Group 2 CD8 + T cells proliferate maximally; compared with the unloaded group, the DNApol-p72 group has a very significant difference in increment (p < 0.001), and the p72 group has a significant difference in increment (p < 0.05); the DNApol-p72 group differed significantly (p < 0.05) compared to the DNApol group.
CD4 in spleen of P.mice + The results of the detection of T cell proliferation are shown in FIG. 15(A), and compared with the PBS control group, the CD4 of the recombinant lactobacillus group was orally administered with NC 8. delta. -pWCF-DNApol-p72 + The T cell proliferation is very significantly increased (p < 0.01), CD4 in DNApol group + T cell proliferation was significantly increased (p < 0.05), and among the groups, DNApol-p72 group CD4 + T cells proliferate the highest. While detecting CD8 + The results of T cell proliferation are shown in FIG. 15(B), and CD8 of the recombinant lactic acid bacteria group was orally administered NC 8. delta. -pWCF-DNApol-p72, compared with the PBS control group + The proliferation of T cells is obviously increased (p is less than 0.05); the value added situation of the DNApol-p72 group compared with the empty vector group is remarkably different (p < 0.05).
2.2.4 Effect of recombinant lactic acid bacteria on B cells of immunized mice
2.2.4.1 Effect of recombinant lactic acid bacteria on B cells of P-type lymph node of immunized mice
The test researches whether the recombinant lactobacillus of the fusion antigen of the African swine fever virus which is expressed by anchoring can induce the activation of the B cells of the mice or not, and detects the condition of the B cells in PPs of the mice after the immunity is strengthened. The results are shown in FIG. 16, comparing the p72 group with the PBS control group, the empty vector group, and the DNApol group, B220 + IgA + The percentage of the cells is remarkably increased (p is less than 0.05); DNApol-p72 group B220 + IgA + The percentage of cells tended to increase, but there was no significant difference.
2.3 subsection
This example demonstrates that the recombinant lactic acid bacteria NC8 delta-pWCF-DNAPol, NC8 delta-pWCF-p 72 and NC8 delta-pWCF-DNAPol-p 72 can induce the activation of mouse peyer's patch DC and induce the mouse to generate specific T cell reaction, and overall, NC8 delta-pWCF-DNAPol-p 72 has better effect. All 3 groups of recombinant lactic acid bacteria had an effect on the B cell response of mice.
Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Jilin university of agriculture
<120> recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof
<130> 202126140
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
acgcttgtag atccctttgg aagacctatt 30
<210> 2
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<212> DNA
<213> Artificial sequence
<400> 2
cagcgaacgt gcagccatac caacccgaaa ttcctttcac aacatttt 48
<210> 3
<211> 45
<212> DNA
<213> Artificial sequence
<400> 3
tttcccgaga actctcacaa tatccaaaca gcaggtaaac aagat 45
<210> 4
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<212> DNA
<213> Artificial sequence
<400> 4
gcaggtaaac aagatattac tcctattacg gacgcaacgt at 42
<210> 5
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<212> DNA
<213> Artificial sequence
<400> 5
ttttatccat catatcattc aactccacaa cgtcca 36
<210> 6
<211> 12
<212> DNA
<213> Artificial sequence
<400> 6
aggaaacaga cc 12
<210> 7
<211> 1984
<212> DNA
<213> Artificial sequence
<400> 7
gccatacatg atgagtatgg tagaattgct tgtagtacta ttgctagagg taaaagagaa 60
catggtaaat atcctggtgc ttttgttatt gatcctgtta aaggtttaga acaagataaa 120
ccaactactg gtttagattt tgctagctta tatccatccc ttatcatggc ttataatttt 180
agtcctgaaa aatttgttgc tagtagagat gaagctaata gtttaatggc caaaggtgaa 240
agtcttcatt atgttagttt tcattttaat aatagattag ttgaaggttg gtttgttaga 300
cataataatg ttcctgataa aatggggctg taccctaaag ttttgattga tttattaaac 360
aagagaactg ctttaaaaca agaactaaaa aagttaggag aaaaaaaaga atgtattcat 420
gaaagtcatc ctggttttaa agaattacaa tttagacatg ctatggtaga tgcaaagcag 480
aaagccttaa aaattttcat gaatactttt tatggtgaag ctggtaataa tttaagtcca 540
ttttttttat taccattagc tggtggggtt actagtagtg gtcagtataa tcttaagtta 600
gtttacaact ttgttattaa caaaggttat ggaattaaat atggtgatac tgatagtctt 660
tacatcactt gtcctgattc cttatatact gaggtgacag atgcttactt aaacagtcag 720
aagactatta aacactatga acaactatgt catgaaaaag tcctgttaag tatgaaagct 780
atgagtactt tatgtgctga agttaatgaa tatttaagac aagataatgg tactagttat 840
ttaagaatgg catatgaaga ggttttattt cctgtttgtt tcactggcaa gaagaagtac 900
tatggtatag ctcatgttaa caccccaaat ttcaatacta aagagttatt tatcagaggt 960
attgacataa taaagcaagg tcaaactaaa ttaactaaaa ctattggtac tagaattatg 1020
gaagaaagta tgaaattaag aagacctgaa gatcatagac caccattaat agaaattgtt 1080
aaaactgtct taaaagatgc tgttgttaat atgaaacagt ggaattttga agattttatt 1140
caaactgatg cttggagacc tgataaagat aataaagctg ttcaaatttt tatgagtaga 1200
atgcatgcta gaagagaaca attaaaaaaa catggtgctg ctgctagtca atttgctgaa 1260
cctgaacctg gtgaaagatt tagttatgtt attgttgaaa aacaagtcca atttgatatt 1320
caaggtcata gaactgatag tagtagaaaa ggtgacaaaa tggaatatgt tagtgaggct 1380
aaggctaaaa atttgccaat tgacattcta ttctacataa ataactatgt tttaggtcta 1440
tgtgctagat ttattaatga aaatgaagaa tttcaaccac ctgataatgt tagtaataaa 1500
gatgaatatg ctcaaagaag agctaagagt tacctacaaa aatttgttca atccattcat 1560
ccaaaagaca agagtgttat aaaacaaggt aatgtacaca gacaatgcta caagtacata 1620
catcaagaaa tcaaaaagaa aattggcatc tttgctgact tatacaagga gtttttcaat 1680
aatactacaa atccaattga aagttttatc caatctactc aatttatgat tcaatacttt 1740
gatggtgaac aaaaagtgaa ccatagtatg aaaaaaatgg ttgaacaaca tgctactgct 1800
agtaatagag ctggtaaacc tgctggtaac cctgctggta atgctttaat gagagctatt 1860
tttacccaat tgataactga agaaaagaag attgttcaag ctctatataa taaaggggat 1920
gctattcatg atttactgtt ttacccatcc taccacagta ccccacaaag acctttctat 1980
ccaa 1984
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<212> DNA
<213> Artificial sequence
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accttggttg acccatttgg tagaccaatt actctagttg acccctttgg tagaccaatt 60
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tttttatcac aacatttcca aagaacttgc agtcacacca acccaaagtt tctaagccaa 180
cactttcaaa gaacttgctc acatactaac ccaaagttcc tgagtcaaca ctttttccct 240
gagaacagtc ataatattca gactgctggt aagcaagatt tccctgaaaa tagtcataac 300
atacaaactg ctggtaagca agattttcct gaaaattccc ataatattca gactgctggt 360
aagcaagatg ctggtaaaca agatattacc cctataactg atgctaccta tgctgggaaa 420
caagatataa ccccaatcac agatgcaaca tatgctggta aacaagacat tactcctatt 480
actgatgcca cttattttta cccaagttat cacagtactc cacaaagacc attttaccca 540
agttaccatt caactccaca gagaccattc tacccaagtt accactcaac tccacaaaga 600
cca 603
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<212> DNA
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gcgattcatg atgaatatgg tcggattgct tgtagtacga ttgctcgggg taaacgggaa 60
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ccaacgacgg gtttagattt cgcttcgcta tatccgtctc ttattatggc ttataatttt 180
agtccggaaa aatttgttgc tagtcgggat gaagctaata gtttaatggc taaaggggaa 240
agtttacatt atgtcagttt tcattttaat aatcggttag ttgaaggttg gtttgtcaga 300
cacaataacg ttccggataa gatgggttta tacccaaaag tcctcataga tttattgaac 360
aaacggacgg ctttaaagca agaattaaaa aagttaggtg aaaaaaaaga atgtattcat 420
gaaagtcatc cgggttttaa agaattacaa tttcggcacg caatggtgga tgccaagcag 480
aaggcactaa aaatattcat gaatacgttt tatggggaag ctggtaacaa tttaagtcca 540
ttttttttat taccattagc tggaggtgtc acctcaagtg gccaatataa tctgaaatta 600
gtttacaatt ttgttattaa taaagggtac ggcattaagt acggtgatac ggattccctc 660
tatattacgt gtccggatag tctatatacg gaagtgacag atgcttatct caatagtcag 720
aagacgatta agcattacga acaactatgc cacgagaaag ttttattaag tatgaaagct 780
atgagtacgt tatgtgctga agttaatgaa tatttacggc aagataatgg tacgagttat 840
ttacggatgg cttatgaaga agtgttattt ccggtttgct ttacgggtaa gaaaaaatat 900
tatggtatag ctcacgttaa caccccaaat tttaatacga aagagttatt tatcagaggt 960
attgatatca ttaagcaagg tcagacgaaa ttaacgaaaa cgattggtac gcggattatg 1020
gaagaaagta tgaaattacg gcggccggaa gatcatcggc caccattaat cgaaattgtt 1080
aagactgttt tgaaagacgc tgttgttaat atgaagcagt ggaattttga agattttatt 1140
caaacggatg cttggcggcc ggataaagat aataaagctg ttcagatttt tatgagtcgt 1200
atgcatgctc ggcgggaaca attaaaaaaa catggtgctg ctgctagtca atttgctgaa 1260
ccggaaccgg gtgaacggtt tagttatgtt attgttgaaa aacaagtgca gtttgatatt 1320
caaggtcatc ggacggatag tagtcggaag ggagataaaa tggaatatgt tagtgaagcc 1380
aaggcgaaaa acttgccaat tgatatactg ttttatatta ataattacgt tcttggtctg 1440
tgtgcccggt ttattaatga aaatgaagaa tttcaaccac cggataatgt tagtaataaa 1500
gatgaatatg ctcaacgccg ggcgaaaagt tatttacaga aatttgttca aagtattcat 1560
cccaaggaca aaagtgttat aaaacaaggt aacgtgcacc ggcaatgcta caaatatatc 1620
caccaagaaa tcaagaagaa gattggcatt tttgcggacc tgtacaagga atttttcaat 1680
aatacgacga atccaattga gagctttatt cagtcgacgc aattcatgat tcaatatttt 1740
gatggcgagc aaaaagttaa ccacagtatg aaaaaaatgg ttgaacaaca tgctacggct 1800
agtaatcggg ctggtaaacc ggctggtaat cctgcgggta atgcattaat gcgggctatt 1860
tttacgcaac tcattactga agagaaaaaa attgttcaag cattatataa taaaggtgat 1920
gcaatccacg atttattatt ctatccaagt tatcattcta cgccacaacg gcctttctac 1980
ccctcatatc atagtacgcc gcagcggcca ttctacccct cctaccatag tactccgcag 2040
aggccttaaa ggaaacagac catgaccttg gttgacccat ttggtcgccc aattacgcta 2100
gttgacccct ttggtcggcc aattactctc gtcgatccat tcggtcgacc aattcaaagg 2160
acgtgtagtc acacgaatcc aaaattttta tcacaacatt tccaacggac gtgcagtcac 2220
accaacccaa agtttctaag ccaacacttt caacgtactt gctcacatac taacccaaag 2280
ttcctgagtc aacacttttt ccctgagaac agtcataata ttcagacggc tggtaagcaa 2340
gatttccccg aaaattcgca taacatacaa acggctggta agcaagattt tccggaaaat 2400
tcccataata ttcagacggc tggtaagcaa gacgctggta aacaagatat tacccctata 2460
acggacgcta cctacgctgg gaaacaagat ataaccccaa tcacagatgc aacatatgct 2520
ggtaaacaag acattacgcc tattaccgat gccacgtatt tttacccaag ttatcacagt 2580
acgccacaac ggccatttta cccatcgtac cattcaactc cacagcggcc attctaccca 2640
agttaccact caactccaca aagacca 2667
<210> 10
<211> 874
<212> PRT
<213> Artificial sequence
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Met Gly Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr
1 5 10 15
Lys Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro
20 25 30
Ile Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu
35 40 45
Thr Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe
50 55 60
Val Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln
65 70 75 80
Lys Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala
85 90 95
Ser Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys
100 105 110
Asn Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu
115 120 125
Ser Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala
130 135 140
Asn Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu
145 150 155 160
Gly Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser
165 170 175
Leu Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Ser Arg Met
180 185 190
Ala Ile His Asp Glu Tyr Gly Arg Ile Ala Cys Ser Thr Ile Ala Arg
195 200 205
Gly Lys Arg Glu His Gly Lys Tyr Pro Gly Ala Phe Val Ile Asp Pro
210 215 220
Val Lys Gly Leu Glu Gln Asp Lys Pro Thr Thr Gly Leu Asp Phe Ala
225 230 235 240
Ser Leu Tyr Pro Ser Leu Ile Met Ala Tyr Asn Phe Ser Pro Glu Lys
245 250 255
Phe Val Ala Ser Arg Asp Glu Ala Asn Ser Leu Met Ala Lys Gly Glu
260 265 270
Ser Leu His Tyr Val Ser Phe His Phe Asn Asn Arg Leu Val Glu Gly
275 280 285
Trp Phe Val Arg His Asn Asn Val Pro Asp Lys Met Gly Leu Tyr Pro
290 295 300
Lys Val Leu Ile Asp Leu Leu Asn Lys Arg Thr Ala Leu Lys Gln Glu
305 310 315 320
Leu Lys Lys Leu Gly Glu Lys Lys Glu Cys Ile His Glu Ser His Pro
325 330 335
Gly Phe Lys Glu Leu Gln Phe Arg His Ala Met Val Asp Ala Lys Gln
340 345 350
Lys Ala Leu Lys Ile Phe Met Asn Thr Phe Tyr Gly Glu Ala Gly Asn
355 360 365
Asn Leu Ser Pro Phe Phe Leu Leu Pro Leu Ala Gly Gly Val Thr Ser
370 375 380
Ser Gly Gln Tyr Asn Leu Lys Leu Val Tyr Asn Phe Val Ile Asn Lys
385 390 395 400
Gly Tyr Gly Ile Lys Tyr Gly Asp Thr Asp Ser Leu Tyr Ile Thr Cys
405 410 415
Pro Asp Ser Leu Tyr Thr Glu Val Thr Asp Ala Tyr Leu Asn Ser Gln
420 425 430
Lys Thr Ile Lys His Tyr Glu Gln Leu Cys His Glu Lys Val Leu Leu
435 440 445
Ser Met Lys Ala Met Ser Thr Leu Cys Ala Glu Val Asn Glu Tyr Leu
450 455 460
Arg Gln Asp Asn Gly Thr Ser Tyr Leu Arg Met Ala Tyr Glu Glu Val
465 470 475 480
Leu Phe Pro Val Cys Phe Thr Gly Lys Lys Lys Tyr Tyr Gly Ile Ala
485 490 495
His Val Asn Thr Pro Asn Phe Asn Thr Lys Glu Leu Phe Ile Arg Gly
500 505 510
Ile Asp Ile Ile Lys Gln Gly Gln Thr Lys Leu Thr Lys Thr Ile Gly
515 520 525
Thr Arg Ile Met Glu Glu Ser Met Lys Leu Arg Arg Pro Glu Asp His
530 535 540
Arg Pro Pro Leu Ile Glu Ile Val Lys Thr Val Leu Lys Asp Ala Val
545 550 555 560
Val Asn Met Lys Gln Trp Asn Phe Glu Asp Phe Ile Gln Thr Asp Ala
565 570 575
Trp Arg Pro Asp Lys Asp Asn Lys Ala Val Gln Ile Phe Met Ser Arg
580 585 590
Met His Ala Arg Arg Glu Gln Leu Lys Lys His Gly Ala Ala Ala Ser
595 600 605
Gln Phe Ala Glu Pro Glu Pro Gly Glu Arg Phe Ser Tyr Val Ile Val
610 615 620
Glu Lys Gln Val Gln Phe Asp Ile Gln Gly His Arg Thr Asp Ser Ser
625 630 635 640
Arg Lys Gly Asp Lys Met Glu Tyr Val Ser Glu Ala Lys Ala Lys Asn
645 650 655
Leu Pro Ile Asp Ile Leu Phe Tyr Ile Asn Asn Tyr Val Leu Gly Leu
660 665 670
Cys Ala Arg Phe Ile Asn Glu Asn Glu Glu Phe Gln Pro Pro Asp Asn
675 680 685
Val Ser Asn Lys Asp Glu Tyr Ala Gln Arg Arg Ala Lys Ser Tyr Leu
690 695 700
Gln Lys Phe Val Gln Ser Ile His Pro Lys Asp Lys Ser Val Ile Lys
705 710 715 720
Gln Gly Asn Val His Arg Gln Cys Tyr Lys Tyr Ile His Gln Glu Ile
725 730 735
Lys Lys Lys Ile Gly Ile Phe Ala Asp Leu Tyr Lys Glu Phe Phe Asn
740 745 750
Asn Thr Thr Asn Pro Ile Glu Ser Phe Ile Gln Ser Thr Gln Phe Met
755 760 765
Ile Gln Tyr Phe Asp Gly Glu Gln Lys Val Asn His Ser Met Lys Lys
770 775 780
Met Val Glu Gln His Ala Thr Ala Ser Asn Arg Ala Gly Lys Pro Ala
785 790 795 800
Gly Asn Pro Ala Gly Asn Ala Leu Met Arg Ala Ile Phe Thr Gln Leu
805 810 815
Ile Thr Glu Glu Lys Lys Ile Val Gln Ala Leu Tyr Asn Lys Gly Asp
820 825 830
Ala Ile His Asp Leu Leu Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln
835 840 845
Arg Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro Phe Tyr
850 855 860
Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
865 870
<210> 11
<211> 393
<212> PRT
<213> Artificial sequence
<400> 11
Met Gly Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr
1 5 10 15
Lys Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro
20 25 30
Ile Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu
35 40 45
Thr Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe
50 55 60
Val Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln
65 70 75 80
Lys Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala
85 90 95
Ser Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys
100 105 110
Asn Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu
115 120 125
Ser Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala
130 135 140
Asn Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu
145 150 155 160
Gly Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser
165 170 175
Leu Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Ser Arg Met
180 185 190
Thr Leu Val Asp Pro Phe Gly Arg Pro Ile Thr Leu Val Asp Pro Phe
195 200 205
Gly Arg Pro Ile Thr Leu Val Asp Pro Phe Gly Arg Pro Ile Gln Arg
210 215 220
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Gln Arg
225 230 235 240
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Gln Arg
245 250 255
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Phe Pro
260 265 270
Glu Asn Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Phe Pro Glu
275 280 285
Asn Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Phe Pro Glu Asn
290 295 300
Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Ala Gly Lys Gln Asp
305 310 315 320
Ile Thr Pro Ile Thr Asp Ala Thr Tyr Ala Gly Lys Gln Asp Ile Thr
325 330 335
Pro Ile Thr Asp Ala Thr Tyr Ala Gly Lys Gln Asp Ile Thr Pro Ile
340 345 350
Thr Asp Ala Thr Tyr Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg
355 360 365
Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro Phe Tyr Pro
370 375 380
Ser Tyr His Ser Thr Pro Gln Arg Pro
385 390
<210> 12
<211> 1081
<212> PRT
<213> Artificial sequence
<220>
<221> misc_feature
<222> (875)..(875)
<223> Xaa can be any naturally occurring amino acid
<400> 12
Met Gly Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr
1 5 10 15
Lys Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro
20 25 30
Ile Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu
35 40 45
Thr Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe
50 55 60
Val Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln
65 70 75 80
Lys Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala
85 90 95
Ser Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys
100 105 110
Asn Tyr Lys Gln Ala Asp Lys Glu Ile His Leu Gln Thr Asn Lys Glu
115 120 125
Ser Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala
130 135 140
Asn Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu
145 150 155 160
Gly Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser
165 170 175
Leu Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Ser Arg Met
180 185 190
Ala Ile His Asp Glu Tyr Gly Arg Ile Ala Cys Ser Thr Ile Ala Arg
195 200 205
Gly Lys Arg Glu His Gly Lys Tyr Pro Gly Ala Phe Val Ile Asp Pro
210 215 220
Val Lys Gly Leu Glu Gln Asp Lys Pro Thr Thr Gly Leu Asp Phe Ala
225 230 235 240
Ser Leu Tyr Pro Ser Leu Ile Met Ala Tyr Asn Phe Ser Pro Glu Lys
245 250 255
Phe Val Ala Ser Arg Asp Glu Ala Asn Ser Leu Met Ala Lys Gly Glu
260 265 270
Ser Leu His Tyr Val Ser Phe His Phe Asn Asn Arg Leu Val Glu Gly
275 280 285
Trp Phe Val Arg His Asn Asn Val Pro Asp Lys Met Gly Leu Tyr Pro
290 295 300
Lys Val Leu Ile Asp Leu Leu Asn Lys Arg Thr Ala Leu Lys Gln Glu
305 310 315 320
Leu Lys Lys Leu Gly Glu Lys Lys Glu Cys Ile His Glu Ser His Pro
325 330 335
Gly Phe Lys Glu Leu Gln Phe Arg His Ala Met Val Asp Ala Lys Gln
340 345 350
Lys Ala Leu Lys Ile Phe Met Asn Thr Phe Tyr Gly Glu Ala Gly Asn
355 360 365
Asn Leu Ser Pro Phe Phe Leu Leu Pro Leu Ala Gly Gly Val Thr Ser
370 375 380
Ser Gly Gln Tyr Asn Leu Lys Leu Val Tyr Asn Phe Val Ile Asn Lys
385 390 395 400
Gly Tyr Gly Ile Lys Tyr Gly Asp Thr Asp Ser Leu Tyr Ile Thr Cys
405 410 415
Pro Asp Ser Leu Tyr Thr Glu Val Thr Asp Ala Tyr Leu Asn Ser Gln
420 425 430
Lys Thr Ile Lys His Tyr Glu Gln Leu Cys His Glu Lys Val Leu Leu
435 440 445
Ser Met Lys Ala Met Ser Thr Leu Cys Ala Glu Val Asn Glu Tyr Leu
450 455 460
Arg Gln Asp Asn Gly Thr Ser Tyr Leu Arg Met Ala Tyr Glu Glu Val
465 470 475 480
Leu Phe Pro Val Cys Phe Thr Gly Lys Lys Lys Tyr Tyr Gly Ile Ala
485 490 495
His Val Asn Thr Pro Asn Phe Asn Thr Lys Glu Leu Phe Ile Arg Gly
500 505 510
Ile Asp Ile Ile Lys Gln Gly Gln Thr Lys Leu Thr Lys Thr Ile Gly
515 520 525
Thr Arg Ile Met Glu Glu Ser Met Lys Leu Arg Arg Pro Glu Asp His
530 535 540
Arg Pro Pro Leu Ile Glu Ile Val Lys Thr Val Leu Lys Asp Ala Val
545 550 555 560
Val Asn Met Lys Gln Trp Asn Phe Glu Asp Phe Ile Gln Thr Asp Ala
565 570 575
Trp Arg Pro Asp Lys Asp Asn Lys Ala Val Gln Ile Phe Met Ser Arg
580 585 590
Met His Ala Arg Arg Glu Gln Leu Lys Lys His Gly Ala Ala Ala Ser
595 600 605
Gln Phe Ala Glu Pro Glu Pro Gly Glu Arg Phe Ser Tyr Val Ile Val
610 615 620
Glu Lys Gln Val Gln Phe Asp Ile Gln Gly His Arg Thr Asp Ser Ser
625 630 635 640
Arg Lys Gly Asp Lys Met Glu Tyr Val Ser Glu Ala Lys Ala Lys Asn
645 650 655
Leu Pro Ile Asp Ile Leu Phe Tyr Ile Asn Asn Tyr Val Leu Gly Leu
660 665 670
Cys Ala Arg Phe Ile Asn Glu Asn Glu Glu Phe Gln Pro Pro Asp Asn
675 680 685
Val Ser Asn Lys Asp Glu Tyr Ala Gln Arg Arg Ala Lys Ser Tyr Leu
690 695 700
Gln Lys Phe Val Gln Ser Ile His Pro Lys Asp Lys Ser Val Ile Lys
705 710 715 720
Gln Gly Asn Val His Arg Gln Cys Tyr Lys Tyr Ile His Gln Glu Ile
725 730 735
Lys Lys Lys Ile Gly Ile Phe Ala Asp Leu Tyr Lys Glu Phe Phe Asn
740 745 750
Asn Thr Thr Asn Pro Ile Glu Ser Phe Ile Gln Ser Thr Gln Phe Met
755 760 765
Ile Gln Tyr Phe Asp Gly Glu Gln Lys Val Asn His Ser Met Lys Lys
770 775 780
Met Val Glu Gln His Ala Thr Ala Ser Asn Arg Ala Gly Lys Pro Ala
785 790 795 800
Gly Asn Pro Ala Gly Asn Ala Leu Met Arg Ala Ile Phe Thr Gln Leu
805 810 815
Ile Thr Glu Glu Lys Lys Ile Val Gln Ala Leu Tyr Asn Lys Gly Asp
820 825 830
Ala Ile His Asp Leu Leu Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln
835 840 845
Arg Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro Phe Tyr
850 855 860
Pro Ser Tyr His Ser Thr Pro Gln Arg Pro Xaa Arg Lys Gln Thr Met
865 870 875 880
Thr Leu Val Asp Pro Phe Gly Arg Pro Ile Thr Leu Val Asp Pro Phe
885 890 895
Gly Arg Pro Ile Thr Leu Val Asp Pro Phe Gly Arg Pro Ile Gln Arg
900 905 910
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Gln Arg
915 920 925
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Gln Arg
930 935 940
Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Phe Pro
945 950 955 960
Glu Asn Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Phe Pro Glu
965 970 975
Asn Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Phe Pro Glu Asn
980 985 990
Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp Ala Gly Lys Gln Asp
995 1000 1005
Ile Thr Pro Ile Thr Asp Ala Thr Tyr Ala Gly Lys Gln Asp Ile
1010 1015 1020
Thr Pro Ile Thr Asp Ala Thr Tyr Ala Gly Lys Gln Asp Ile Thr
1025 1030 1035
Pro Ile Thr Asp Ala Thr Tyr Phe Tyr Pro Ser Tyr His Ser Thr
1040 1045 1050
Pro Gln Arg Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg
1055 1060 1065
Pro Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
1070 1075 1080
<210> 13
<211> 8127
<212> DNA
<213> Artificial sequence
<400> 13
ccatgggcaa gaaagaatta agtttccacg agaagttatt aaaattgact aaacaacaaa 60
aaaagaagac taacaagcat gtgtttattg ctattccaat tgttttcgtt ttaatgtttg 120
cttttatgtg ggcaggtaaa gctgagactc caaaagttaa gacttatagt gatgacgttt 180
tgagtgcttc atttgtcggc gacattatga tgggtcgtta cgttgagaaa gtcacggaac 240
aaaagggtgc agatagtatt ttccaatatg ttgaaccgat tttccgtgct agtgattatg 300
ttgctggcaa ttttgaaaat cctgttactt atcagaaaaa ctacaaacaa gctgataaag 360
agattcattt acagactaat aaggaaagtg ttaaagtttt aaaggatatg aattttactg 420
tcttaaatag tgctaataat catgctatgg attatggtgt tcaaggtatg aaagatacgt 480
taggtgagtt tgctaaacag aatttagata ttgttggtgc tggttattca ttaagtgacg 540
ctaagaagaa aattagttac cagaaagtgt ctagaaagct tcaaattaca gcacgtgttg 600
ctttgattga tagccaaaaa gcagcagttg ataaagcaat tactgatatt gctgaaaaat 660
tgtaatttat aaataaaaat caccttttag aggtggtttt tttatttata aattattcgt 720
ttgatttcgc tttcgataga acaatcaaag cgagaataag gaagataaat cccataaggg 780
cgggagcaga atgtccgaga ctaattcatg accaaaatcc cttaacgtga gttttcgttc 840
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 900
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 960
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1020
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1080
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 1140
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 1200
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 1260
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 1320
ccggtaagcg gcagggtcgg aacaggaggc gcacgaggga gcttccaggg ggaaacgcct 1380
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 1440
gctcgtcagg ggggcggagc ctatcgaaaa acgccagcaa cgcggccttt ttacggttcc 1500
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 1560
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 1620
gcagcgagtc agtgagcgag aaggattatt cggctggttg agacgttaaa atgataaagg 1680
ttgtattaat cttatattac ggttataatg tactcaactt aataaatgaa cgcaaaaaaa 1740
agaaccctca acttagcaga gttaggattc acgacttatc agcacaacct gataagattt 1800
tcgatagcaa gtactaccaa tacaagctat ctaacttggt actattataa catgtaggct 1860
aagtttttca accattgata cttaaagtaa acggttgtta tcgggaatct taacagaaac 1920
ctgatagcaa ccgttttttt gttattcaat ggttagcaac catcaaagca actaaaggct 1980
ggaaacctgt tcttagctag taaaacctcc cgtgagtgtc gttcgtgacc ccgcttgcag 2040
ttaacaacat aggtatgcta aaccttgtcg agatcaacgc gactaaagac gtggctggaa 2100
gactaggaaa tgatacggac aggctaacta ttaacgcaga ttattcgggt tgctgctaaa 2160
accaactcta ataatagtta gtgcaagggc tggttgagct taaattgtct gataaagagt 2220
tctctcttta tactgcaaaa gaagcgcagt tattcacgat taggataact gtttgagaga 2280
gcctaagggc ttgacccttg atggtttaag caccgctatg cgtgcgggat cctcttccct 2340
aaatttaaat ataaacaacg aattatctcc ttaacgtacg ttttcgttcc attggccctc 2400
aaacccctaa ttaggatcaa taaaacagcg acggaaatga ttcccttcct aacgcaaatt 2460
ccctgataat cgccactgga ctttctgctt gcgcggtaag gcaggataag tcgcattact 2520
gatggcttcg ctatcattga ttaatttcac ttgcgacttt ggctgctttt tgtatggtga 2580
aggatgcgcc acaggatact ggcgcgcata cacagcacat ctctttgcag gaaaaaaacg 2640
ctatgaaaaa tgttggtttt atcggctggc gcggaatggt cggctctgtt ctcatgcaac 2700
gcatggtaga ggagcgcgat ttcgacgcta ttcgccctgt tttcttttct acctcccagt 2760
ttggacaggc ggcgcccacc ttcggcgaca cctccggcac gctacaggac gcttttgacc 2820
tggatgcgct aaaagcgctc gatatcatcg tgacctgcca gggcggcgat tataccaacg 2880
aaatttatcc aaagctgcgc gaaagcggat ggcagggtta ctggattgat gcggcttcta 2940
cgctgcgcat gaaagatgat gccattatta ttctcgaccc ggtcaaccag gacgtgatta 3000
ccgacggcct gaacaatggc gtgaagacct ttgtgggcgg taactgtacc gttagcctga 3060
tgttgatgtc gctgggcggt ctctttgccc ataatctcgt tgactgggta tccgtcgcga 3120
cctatcaggc cgcctccggc ggcggcgcgc gccatatgcg cgagctgtta acccagatgg 3180
gtcagttgta tggccatgtc gccgatgaac tggcgacgcc gtcttccgca attcttgata 3240
ttgaacgcaa agttacggca ttgacccgca gcggcgagct gccggttgat aactttggcg 3300
taccgctggc gggaagcctg atcccctgga tcgacaaaca gctcgataac ggccagagcc 3360
gcgaagagtg gaaaggccag gcggaaacca acaagattct caatactgcc tctgtgattc 3420
cggttgatgg tttgtgtgtg cgcgtcggcg cgctgcgctg tcacagccag gcgttcacca 3480
tcaagctgaa aaaagaggta tccattccga cggtggaaga actgctggcg gcacataatc 3540
cgtgggcgaa agtggtgccg aacgatcgtg atatcactat gcgcgaatta accccggcgg 3600
cggtgaccgg cacgttgact acgccggttg gtcgtctgcg taagctgaac atggggccag 3660
agttcttgtc ggcgtttacc gtaggcgacc agttgttatg gggcgccgcc gagccgctgc 3720
gtcgaatgct gcgccagttg gcgtagtggc tattgcagcg cttatcgggc ctgcgtgtgg 3780
ttctgtaggc cggataaggc gcgtcagcgc cgccatccgg cggggaaatt tgtgttaaac 3840
caggggtgca tcgtcaccct ttttttgcgt aatacaggag taaacgcaga tgtttcattt 3900
ttatcaggag ttaagcagag cattggctat tctttaaggg tagcttaatc ccacgggtat 3960
taagcctaac ctgaaggtag gacgacgcag ataggatgca cagtgtgctg cgccgttcag 4020
gtcaaagaag tgtcactacc tgatgttgtg gacgaaaagc cctgacaacc ctcgttccta 4080
aaaaggaata agcgtttggt cagtaaataa tagaaataaa aaatcagacc taagactgat 4140
gacaaaaaga gcaaattttg ataaaatagt attagaatta aattaaaaag ggaggccaaa 4200
tataatgaaa aatatgaatg acaatgatgt tatggttgta attggggagc accgccacac 4260
acaagtcaca gtggacttgc aggcaattaa gacaaatatt agtaatgaaa tggcgcaaaa 4320
ggatgagttg accgagttat gggcagtcgt taaagcgaat ggttatggac atggaattat 4380
ccaagttgct caggccgcca aagaagccgg ggcgaccggc ttttgtgttg caatcctgga 4440
tgaggcctta gcgttgcggg ccgctggctt tgcggaaccc atcctagtac ttggaattac 4500
ggaaccggaa tacgccccac tggtagctga aaaggatatt tcactagctg ttggaacgca 4560
agattggctg actacggccg cagcaatttt agcggctaat caagtgacga caccacttca 4620
cgttcatctt gcattagata cgggtatggg acgaatcggg tttcagacgc ccgaagaatt 4680
ggcaacggcg gttacgactt tgcgtcaacc gcagtcacca tttgactttg aagggatttt 4740
tacgcatttt gcaacggctg accaggcaga tgatacgtat tttactcatc aattaaataa 4800
ttggaaacac ttgattgcag tggtggatga gctaccacgc tatgtccacg tgtccaattc 4860
ggccaccagt ctctggcatc aaacttgcaa tggcaacatg gtgcgctttg gggttgcact 4920
ctatggtcta aatccttctg gtcgcgaact cagcgcacca taccccttgc aacccgcgtt 4980
gtcgctaacg gcacgcttga cgtttgttaa acgcttggct cggggcaaat cggtcagcta 5040
tggtgccacg tatacggccg cacaggatga atggattggc acggtgccga ttgggtatgc 5100
ggacggctat gaacgccgat tacaaggctt ccatgtactt gttgatggtg agttttgcga 5160
aatcgtcgga cgggtctgca tggaccagct gatggttcgt ctgccacatg aagtaccggt 5220
tggagctaag gtaactttgg ttggcacgga cggtgctcgt accatttcgt tgcaagatat 5280
tgctgactat tgtgggacaa ttcattatga gattgcttgt gggttagcac cacgagtgcc 5340
gagagtttat atagattaat tctatgagtc gcttttttaa atttggaaag ttacacgtta 5400
ctaaagggaa tggagaccgg ggcttcaata gagttcttaa cgttaatccg aaaaaaacta 5460
acgttaatat taaaaaataa gatccgcttg tgaattatgt ataatttgat tagactaaag 5520
aataggagaa agtatgatga tatttaaaaa actttctcgt taagataggt tgttggtgag 5580
catgttatat acggatgtat cggtttcctt aatgcaaaat tttgttgcta tcttattaat 5640
ttttctatta tatagatata ttcaaagaaa gataacattt aaacggatca tattagatat 5700
tttaatagcg attatttttt caatattata tctgtttatt tcagatgcgt cattacttgt 5760
aatggtatta atgcgattag ggtggcattt tcatcaacaa aaagaaaata agataaaaac 5820
gactgataca gctaatttaa ttctaattat cgtgatccag ttattgttag ttgcggttgg 5880
gactattatt agtcagttta ccatatcgat tatcaaaagt gatttcagcc aaaatatatt 5940
gaacaatagt gcaacagata taactttatt aggtattttc tttgctgttt tatttgacgg 6000
cttgttcttt atattattga agaataagcg gactgaatta caacatttaa atcaagaaat 6060
cattgaattt tcgttagaaa aacaatattt tatatttata tttattttat ttatagtaat 6120
agaaattatt ttagcagttg ggaatcttca aggagtaaca gccacgatat tattaaccat 6180
tatcattatt ttttgtgtcc ttatcgggat gactttttgg caagtgatgc tttttttgaa 6240
ggcttattcg attcgccaag aagccaatga ccaattggtc cggaatcaac aacttcaaga 6300
ttatctagtc aatatcgaac agcagtacac cgaattacgg cgatttaagc atgattatca 6360
aaacatctta ttatcgttgg agagttttgc cgaaaagggc gatcagcaac agtttaaggc 6420
gtattaccaa gaattattag cacaacggcc aattcaaagt gaaatccaag gggcagtcat 6480
tgcacaactc gactacttga aaaatgatcc tattcgagga ttagtcattc aaaagttttt 6540
ggcagccaaa caggctggtg ttactttaaa attcgaaatg accgaaccaa tcgaattagc 6600
aaccgctaat ctattaacgg ttattcggat tatcggtatt ttattagaca atgcgattga 6660
acaagccgtt caagaaaccg atcaattggt gagttgtgct ttcttacaat ctgatggttt 6720
aatcgaaatt acgattgaaa atacggccag tcaagttaag aatctccaag cattttcaga 6780
gttaggctat tcaacgaaag gcgctggtcg ggggactggt ttagctaatg tgcaggattt 6840
gattgccaaa caaaccaatt tattcttaga aacacagatt gaaaatagaa agttacgaca 6900
gacattgatg attacggagg aaacttaatt tgtatcccgt ttatttatta gaggatgatt 6960
tacagcaaca agcgatttat cagcaaatta tcgcgaatac gattatgatt aacgaatttg 7020
caatgacttt aacatgcgct gccagtgata ctgagacatt gttggcggca attaaggatc 7080
agcaacgagg tttattcttt ttggatatgg aaattgagga taaccgccaa gccggtttag 7140
aagtggcaac taagattcgg cagatgatgc cgtttgcgca aattgtcttc attacaaccc 7200
acgaggaact gacattatta acgttagaac gaaaaatagc gcctttagat tacattctca 7260
aggaccaaac aatggctgaa atcaaaaggc aattgattga tgatctattg ttagctgaga 7320
agcaaaacga ggcggcagcg tatcaccgag aaaatttatt tagttataaa ataggtcctc 7380
gctttttctc attaccatta aaggaagttg tttatttata tactgaaaaa gaaaatccgg 7440
gtcatattaa tttgttagcc gttaccagaa aggttacttt tccaggaaat ttaaatgcgc 7500
tggaagccca atatccaatg ctctttcggt gtgataaaag ttacttagtt aacctatcta 7560
atattgccaa ttatgacagt aaaacacgga gtttaaaatt tgtagatggc agtgaggcaa 7620
aagtctcgtt ccggaaatca cgggaactag tggccaaatt aaaacaaatg atgtagcgcc 7680
tgcaggcacg ccaaatgatc ccagtaaaaa gccacccgca tggcgggtgg ctttttatta 7740
gccctagaag ggcttcccac acgcatttca gcgccttagt gccttagttt gtgaatcata 7800
ggtggtatag tcccgaaata cccgtctaag gaattgtcag ataggcctaa tgactggctt 7860
ttataatatg agataatgcc gactgtactt tttacagtcg gttttctaat gtcactaacc 7920
tgccccgtta gttgaagaag gtttttatat tacagctcca gatctaccgg tttaatttga 7980
aaattgatat tagcgtttaa cagttaaatt aatacgttaa taattttttt gtctttaaat 8040
agggatttga agcataatgg tgttatagcg tacttagctg gccagcatat atgtattcta 8100
taaaatacta ttacaaggag attttag 8127
<210> 14
<211> 18
<212> DNA
<213> Artificial sequence
<400> 14
agatattgtt ggtgctgg 18
<210> 15
<211> 17
<212> DNA
<213> Artificial sequence
<400> 15
tcaatcaaag caacacg 17
<210> 16
<211> 12
<212> PRT
<213> Artificial sequence
<400> 16
Phe Tyr Pro Ser Tyr His Ser Thr Pro Gln Arg Pro
1 5 10
<210> 17
<211> 20
<212> PRT
<213> Artificial sequence
<400> 17
Asn Thr Pro Asn Phe Asn Thr Lys Glu Leu Phe Ile Arg Gly Ile Asp
1 5 10 15
Ile Ile Lys Gln
20
<210> 18
<211> 10
<212> PRT
<213> Artificial sequence
<400> 18
Thr Leu Val Asp Pro Phe Gly Arg Pro Ile
1 5 10
<210> 19
<211> 16
<212> PRT
<213> Artificial sequence
<400> 19
Gln Arg Thr Cys Ser His Thr Asn Pro Lys Phe Leu Ser Gln His Phe
1 5 10 15
<210> 20
<211> 15
<212> PRT
<213> Artificial sequence
<400> 20
Phe Pro Glu Asn Ser His Asn Ile Gln Thr Ala Gly Lys Gln Asp
1 5 10 15
<210> 21
<211> 14
<212> PRT
<213> Artificial sequence
<400> 21
Ala Gly Lys Gln Asp Ile Thr Pro Ile Thr Asp Ala Thr Tyr
1 5 10

Claims (13)

1. A fusion protein for stimulating an organism to resist African swine fever infection has an amino acid sequence shown in any one of the following 1) to 3):
1) as shown in SEQ ID NO: 10;
2) as shown in SEQ ID NO: 11;
3) as shown in SEQ ID NO: 12.
2. An expression vector expressing the fusion protein of claim 1.
3. The expression vector of claim 2, wherein the vector is pWCF, and the nucleotide sequence of the vector is as shown in SEQ ID NO: 13, obtained by ligating the pWCF vector with the fusion gene.
4. The expression vector of claim 3, wherein the pWCF vector is linked to the fusion gene by T4 DNA ligase.
5. A transgenic cell line expressing the fusion protein of claim 1.
6. An engineered bacterium expressing the fusion protein of claim 1.
7. The engineering bacterium according to claim 6, wherein the starting strain of the engineering bacterium is Lactobacillus plantarum.
8. The engineering bacterium according to claim 6, wherein the starting strain of the engineering bacterium is Lactobacillus NC 8.
9. The engineering bacterium of claim 6, wherein the starting strain of the engineering bacterium is alanine racemase gene-deficient Lactobacillus plantarum NC8 Δ alr.
10. The engineered bacterium of claim 6, wherein the engineered bacterium is expressed to produce a lactobacillus plantarum surface anchored to a sequence shown in SEQ ID NO: 10, and the sequence of the fusion antigen DNApol is shown as SEQ ID NO: 11 or p72 or a sequence shown as SEQ ID NO: 12, DNApol-p 72.
11. A pharmaceutical composition or a pharmaceutical preparation or a feed comprising the fusion protein for stimulating the body against african swine fever infection as set forth in claim 1 or the engineered bacterium as set forth in any one of claims 6 to 10.
12. A method of expressing the fusion protein of claim 1 for stimulating the body against african swine fever infection, comprising: constructing an expression vector according to any one of claims 2 to 4, introducing the constructed expression vector into a host cell to obtain a recombinant strain, culturing the recombinant strain, and finally isolating the fusion protein from the culture.
13. Use of the fusion protein for stimulating the body to resist African swine fever infection of claim 1 or the engineering bacterium of any one of claims 6 to 10 in the preparation of vaccine or medicine or feed for preventing and/or treating African swine fever.
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