CN114606250A

CN114606250A - Recombinant lactic acid bacteria for expressing African swine fever fusion antigen and application thereof

Info

Publication number: CN114606250A
Application number: CN202111277332.0A
Authority: CN
Inventors: 王春凤; 杨桂连; 牛荟; 杨文涛; 石春卫; 牛天明; 王红; 谷巍; 单宝龙
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2022-06-10
Anticipated expiration: 2041-10-29
Also published as: CN114606250B

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 of 1414-3351 segment of the G1211R gene is intercepted; 2) comprises the amino acid sequence shown as SEQ ID NO: 1. the amino acid sequence of 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 virus family, varies in number of bases from 170 to 190+ kb for different strains, and encodes more than 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 engaged in 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 the development of safe and effective genetic engineering attenuated live vaccines is 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 have many dendritic processes when mature 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 constructs three groups of fusion genes by starting from the idea of cutting off the formation and replication of viruses, 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 acids of DNA polymerase, containing 3633 bases, is a non-structural protein, but highly conserved, and is capable of inducing strong cell-mediated immunity, such as natural killer cell and T cell responses. And, the p72 protein (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 the gene sequence truncated from the 1414 th and 3351 th segments of G1211R and a DCpep sequence, and wherein the gene sequence truncated from the 1414 th and 3351 th segments of G1211R is linked to 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 amino acid sequence shown as SEQ ID NO: 1. 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. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: the nucleotide sequences shown in 4 are respectively connected in series after three groups of repetitions are carried out (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.

Compared with the traditional method that the target fragment needs to be connected with an expression vector, then is transformed into a bacterium for amplification expression, and then is immunized into a white rabbit after protein purification to obtain antibody serum, in the embodiment of the invention, 3 DCpeps are taken as the target fragments, the animal such as the white rabbit is immunized with DCpep polypeptide to obtain polyclonal antibody serum, 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 segment, and the DNApol-p72 gene segment comprises a DNApol gene segment and a p72 gene segment, wherein the DNApol gene segment and the p72 gene segment are connected by 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 has 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 the 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 an organism to resist African swine fever infection, which is encoded by the fusion gene of the first aspect, and comprises a sequence shown in any one of the following 1) to 3) in the amino acid sequence:

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 of the first aspect.

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, the invention provides a transgenic cell line comprising the fusion gene 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 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, asd gene-deficient escherichia coli (e.coli χ 6212) is used as a plasmid to construct an intermediate host bacterium, and an alr gene-deficient 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 grain 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 against african swine fever infection or the engineered bacterium of the fifth aspect for preparing a vaccine or a medicament 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, and 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.

Drawings

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 Protean software to predict the truncation site of the protein.

FIG. 2: and (3) synthesizing the target fragment.

FIG. 3: and (3) immunizing white rabbits and collecting blood.

FIG. 4: 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 DL10,000DNA 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: the plasmid map of pWCF-DNApol (A), the plasmid map of pWCF-p72l (B), and the plasmid map of pWCF-DNApol-p72 (C).

FIG. 6: PCR verification of the recombinant lactic acid bacterium plasmid NC 8. delta. -pWCF-DNApol (A); m: DL10,000DNA Marker; 1: PCR verification of the recombinant lactic acid bacteria plasmid NC8 delta-pWCF-DNApol. PCR verification of the recombinant lactic acid bacterium plasmid NC 8. delta. -pWCF-p72 (B); m: DL2,000DNA 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: DL5,000DNA Marker; 1: PCR verification of recombinant lactic acid bacteria plasmid NC8 delta-pWCF-DNApol-p 72.

FIG. 7: 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. 8: strain growth profile.

FIG. 9: and (5) detecting the result of the recombinant lactobacillus by flow cytometry.

FIG. 10: performing immunofluorescence on the recombinant lactobacillus; 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. 11: animal immunization procedure.

FIG. 12: the activation results of DC in PPs, the left image flow cytometry detection results, and the right image analysis statistics results.

FIG. 13: CD4 in MLN⁺IFN-γ⁺Expression of (A), CD8 in MLN⁺IFN-γ⁺Expression of (A) (B).

FIG. 14: CD4 in spleen⁺IFN-γ⁺Expression of (A), CD8 in spleen⁺IFN-γ⁺Expression of (A) (B).

FIG. 15: CD4 in MLN⁺T cell proliferation results (A), CD8 in MLN⁺T cell proliferation results (B).

FIG. 16: CD4 in spleen⁺T cell proliferation results (A), CD8 in spleen⁺T cell proliferation results (B).

FIG. 17: b220 in PP⁺IgA⁺The number of cells varied.

FIG. 18: 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 below 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 intended to be 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)⁵Individual cell) and each group of recombinant lactic acid bacteria (2X 10)⁶CFU) as per 1: 10 for 12h, and then detecting CD on the surface of the DC by a flow cytometer80 and CD86 molecules.

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 (P < 0.05) compared to the 2 × DCpep group, with the results shown in fig. 18 (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. 18 (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. Starting from the idea of cutting off the formation and replication of viruses, the embodiment 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. 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

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)₂O)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)₆H₁₂O₆)20.0g, sodium acetate (CH)₃COONa 5.0g, Yeast extract 0.05g, Tween 80(Tween-80)1.0g, and dipotassium hydrogen phosphate (KH)₂PO₄)2.0g of diammonium hydrogen citrate (C)₆H₁₄N₂O₇)2.0g magnesium sulfate heptahydrate (MgSO)₄·7H₂O)0.2g, manganese sulfate monohydrate (MnSO)₄·H₂O)0.05g, distilled water (ddH)₂O)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 and MgCl₂ 0.029g、ddH₂O 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)₃PO₄ 0.19g、MgCl₂ 0.009g、ddH₂O 100mL。

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 target genes:

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 to 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 stronger immune responses were selected by reference to Mallory E et al (Linear epitopes in influenza virus p72 purified antigenic peptides-expressed epitopes) and shown in Table 1-1. In this example, four groups of antigens are duplicated in three groups respectively, and then are connected in series with the duplicated Dcpep genes in three groups to serve as a second group of target genes.

TABLE 1-1 p72 protein epitopes

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

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 mesh fragment and vector ligation

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

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 into E.coli χ 6212 competent

Preparation of cold bite x 6212 competence 1.1.2.1.6.1E

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 centrifugal tube in a super clean bench; centrifuging at 2500rpm and 4 deg.C for 10 min; discarding the supernatant, adding the pre-cooled ddH₂Supplementing 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 with 100 mu L, quickly freezing with 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 enzyme digestion and PCR identification are carried out.

1.1.2.1.7.1 enzyme digestion identification

The enzyme digestion identification system is as follows:

TABLE 1-4 enzyme digestion System

After being subjected to metal bath at 37 ℃ for 8 hours, 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 to be added into 5mL of MRS liquid culture medium added with D-alanine (the 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 transferred 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 electrotransformation instrument for electric shock, and having the following conditions: 2000V, 400. omega., 25. mu.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 flat plate, and carrying out inverted culture at a constant temperature of 37 ℃ in an anaerobic workstation 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 NC 8/delta alr recombinant plasmid verification

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

The PCR amplification conditions were as follows:

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 bacteria⁹CFU (optimal number of mice fed with Lactobacillus is 0.5X 10)⁹～1×10⁹CFU) how much volume of bacteria solution 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: adding DCpep rabbit polyclonal antibody serum diluted at a ratio of 1:300 into a centrifugal tube, and shaking overnight at 4 ℃; washing with 1mL of 0.2% Tween-20-added PBS for three times, and collecting the precipitate; 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 (0.2% Tween-20) for 3 times, removing supernatant, and collecting precipitate; and (4) performing flow detection, mixing the solution with 400 mu L of PBS, and loading the solution on a 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, as shown in FIG. 2.

DNApol gene structure (2062 base pairs total): xba I-DNA pol 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 structure of the p72 gene fragment is as follows: 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 verified by double digestion, and a clear band was visible at 2062bp (FIG. 4, A).

The recombinant plasmid pWCF-p72 was double-digested and verified to have a clear band at 619bp (FIG. 4, B).

The recombinant plasmid pWCF-DNApol-p72 was double-digested and verified, and a clear band was visible at 2683bp (FIG. 4, C).

1.2.1.3.2 plasmid sequencing

The plasmid sequencing results were correct and the plasmid map was generated using SnapGene software, FIG. 5.

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. Clear bands were visible at 2062, 619, 2683bp (FIG. 6).

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 can be seen at the position of 55kDa (figure 7), 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

The results (24h) of the growth curve measurements (OD 600, 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 after 3 cultivations and measurements were plotted using GraphPad Prism software, see FIG. 8. 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 three-time bacterial colony plate counting assay, and the feed time points were 1 × 10 in 0.2mL of NC8/Δ alr, NC8 Δ -pWCF, 0.22mL of NC8 Δ -pWCF-DNApol, 1.3mL of NC8 Δ -pWCF-p72, and 1.66mL of NC8-pWCF-DNApol-p72 bacterial solution⁹CFU。

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 9, on the abscissa axis, the more the image is displayed to the right, the stronger the fluorescence intensity carried by the detection 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. 10B; 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. 10D, F, H.

1.3 summary

The experiments in this section 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-p72 groups. Treating each group of recombinant lactobacillus bacterial liquid, wherein the number of live bacteria fed to each mouse is 1.0 multiplied by 10⁹CFU。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

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 was examined by flow cytometry at day 29. As shown in fig. 11.

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 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. Spleen, mesenteric lymph node and Peyer's lymph node were harvested, sterilized copper mesh was placed in a 35mm sterile plate and soaked in 1mL1640 complete medium, tissue was placed in the copper mesh, and 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, 500. mu.L of PBS is added to the spleen cells after 3min on ice, 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. The cells of each group were diluted and counted, and the number of cells of different tissues was different, so that the spleen was dividedDirty 100 fold dilution and required 1.0x10⁷One cell, MLN 50-fold diluted and required 1.0X10⁷One cell, PPs 20-fold diluted and required 1.0 × 10⁶And (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.l of CD16/CD32 Pure per tube at 4 ℃ for 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 in 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)⁶Individual 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 (antigen peptide nos. 1,2,3,4), group B (antigen peptide nos. 1,2,3,4), group C (antigen peptide nos. 1,2), group D (antigen peptide nos. 1,3,4), and group E (antigen 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. The incubation was prepared by adding 10. mu.L each of CD3-percpcy5.5 (40-fold dilution), CD4-FITC (60-fold dilution), and CD8-APCCY7 (60-fold dilution) to the mixture at 4 ℃ in the dark for 20 min. 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 detecting T cell proliferation of 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 cultured in a cell culture chamber 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 peyer's patch, the mice were fed with the recombinant lactic acid bacteria orally and immunologically, and the condition of DCs in the mouse peyer's patch was examined one week after the booster immunization. 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 show that the recombinant lactic acid bacteria have activating effect on mouse P's lymph node DC, and the results are shown in FIG. 12.

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. 13(A), and compared with PBS control group, the CD4 of NC8 delta-pWCF-DNApol-p 72 recombinant lactobacillus group is orally taken⁺IFN-γ⁺The proportion of cells is greatly 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 differed significantly (p < 0.01) compared with the PBS group; 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. 13(B), and compared with the PBS control group, CD8 of the oral NC8 delta-pWCF-DNAPol-p 72 recombinant lactic acid bacteria and the oral NC8 delta-pWCF-DNAPol 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 P.mice⁺IFN-γ⁺The results of the cytokine assay are shown in FIG. 14(A), and compared with the PBS control group, the CD4 of the oral NC8 delta-pWCF-DNAPol-p 72 recombinant lactobacillus group and the 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⁺The highest proportion of cells; the DNApol-p72 group differed significantly (p < 0.001) compared to the unloaded group; the DNApol group has very significant difference (p is less than 0.01) compared with the unloaded group; the DNApol group and DNApol-p72 group differed significantly (p < 0.01) compared to the p72 group. While detecting CD8⁺IFN-γ⁺In the cells, the results are shown in FIG. 14(B), and CD8 of NC8 delta-pWCF-DNAPol-p 72 recombinant lactic acid bacteria 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), and 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 lactic acid bacteria 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. 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 proliferation of T cells is very significantly increased (p < 0.001), and the CD4 of DNApol group and p72 group⁺The proliferation of T cells was significantly increased (p < 0.05) and among the groups, DNApol-p72 group CD4⁺T cells proliferate maximally; compared with the unloaded group, the DNApol-p72 group has a very significant difference in value-added conditions (p < 0.001), and the DNApol group has a significant difference in value-added conditions (p < 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. 15(B), and CD8 of the recombinant lactic acid bacteria group was orally administered NC 8. delta. -pWCF-DNApol-p72, as compared with the PBS control group⁺The proliferation of T cells is very significantly increased (p < 0.001), and the proliferation of CD8 of p72 group⁺The proliferation of T cells was significantly increased (p < 0.05) and among the groups, DNApol-p72 group CD8⁺T cells proliferate maximally; compared with the unloaded group, the value-added situation of the DNApol-p72 group is remarkably different (p is less than 0.001), and the value-added situation of the p72 group is remarkably different (p is less than 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. 16(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. 16(B), and CD8 of the recombinant lactic acid bacteria group was orally administered with NC 8. delta. -pWCF-DNAPol-p72, compared to the PBS control group⁺Proliferation of T cellsThe situation is remarkably 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 anchoring expression African swine fever virus fusion antigen recombinant lactobacillus can induce the activation of the mouse B cells, and detects the condition of the B cells in the mouse PPs after the boosting immunity. The results are shown in FIG. 17, comparing the p72 group with the PBS control group, the empty vector group, and the DNApol group, B220⁺IgA⁺The percentage of 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

The example proves 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 lymph node DC and induce the mouse to generate specific T cell reaction, and overall, the 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, or improvement 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

<211> 48

<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

<211> 42

<212> DNA

<213> Artificial sequence

<400> 4

gcaggtaaac aagatattac tcctattacg gacgcaacgt at 42

<210> 5

<211> 36

<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

<210> 8

<211> 603

<212> DNA

<213> Artificial sequence

<400> 8

accttggttg acccatttgg tagaccaatt actctagttg acccctttgg tagaccaatt 60

actttagttg atccatttgg tagaccaatt caaaggactt gtagtcacac taatccaaaa 120

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

<210> 9

<211> 2667

<212> DNA

<213> Artificial sequence

<400> 9

gcgattcatg atgaatatgg tcggattgct tgtagtacga ttgctcgggg taaacgggaa 60

catggtaaat atccgggtgc ttttgttatt gatccggtta aaggtttaga acaagataaa 120

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

<400> 10

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

1. A fusion gene comprising at least one set of sequences described in 1) to 3) below linked to a DCpep gene;

2) comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4;

2. The fusion gene of claim 1, wherein the DCpep gene comprises a nucleotide sequence identical to SEQ ID NO: 5 having at least 90% sequence homology;

preferably, the linkage to the DCpep gene is in tandem.

3. The fusion gene as claimed in claim 1 or 2, wherein the fusion gene comprises a DNApol gene fragment, the DNApol gene fragment comprises the gene sequence truncated from paragraphs 1414 and 3351 of G1211R and the DCpep sequence, wherein the gene sequence truncated from paragraphs 1414 and 3351 of G1211R is linked with three sets of repeated DCpep sequences;

preferably, the fusion gene comprises a p72 gene fragment, and the p72 gene fragment comprises the nucleotide sequence shown as SEQ ID NO: 1. 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, the nucleotide sequences shown in the sequence table are respectively connected in series after three groups of repetitions are carried out, and then are connected with three groups of repeated DCpep sequences;

preferably, the fusion gene comprises a DNApol-p72 gene segment, and the DNApol-p72 gene segment comprises a DNApol gene segment and a p72 gene segment, wherein the two segments are connected by an SD sequence.

4. The fused gene according to claim 3, wherein the fused gene comprises a sequence represented by any one of the following 1) to 3):

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 the nucleotide sequence set forth in 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;

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;

preferably, the fusion gene further comprises 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 sequence.

5. A fusion protein for stimulating the body to resist African swine fever infection, which is encoded by the fusion gene of any one of claims 1 to 4, and the amino acid sequence of the fusion protein comprises the sequence shown in any one of the following 1) to 3):

6. An expression vector containing the fusion gene according to any one of claims 1 to 4;

preferably, the vector is pWCF, and the nucleotide sequence of the vector is shown in SEQ ID NO: 13, obtained by connecting a pWCF vector with a fusion gene;

preferably, the pWCF vector is linked to the fusion gene by T4 DNA ligase.

7. A transgenic cell line comprising the fusion gene of any one of claims 1 to 4.

8. An engineered bacterium containing the fusion gene according to any one of claims 1 to 4;

preferably, the starting strain of the engineering bacteria is lactobacillus plantarum, preferably lactobacillus NC8, more preferably lactobacillus plantarum NC8 Δ alr deficient in alanine racemase gene (alr);

preferably, the engineering bacteria express and produce a sequence anchored on the surface of lactobacillus plantarum as shown in SEQ ID NO: 10, and the sequence of the fusion antigen DNApol is shown as SEQ ID NO: 11 or p72 as shown in SEQ ID NO: 12, DNApol-p 72.

9. 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 claim 5 or the engineered bacterium as described in claim 7.

10. A method of expressing the fusion protein of claim 5 for stimulating the body against African swine fever infection, comprising: constructing an expression vector containing the fusion gene according to any one of claims 1 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.

11. Use of the fusion gene of any one of claims 1 to 4 or the fusion protein for stimulating the body against African swine fever infection of claim 5 or the engineered bacterium of claim 8 in the preparation of vaccine or medicament or feed for preventing and/or treating African swine fever.