CN114426981A

CN114426981A - African swine fever virus antigen protein recombinant expression vector, recombinant plant lactic acid bacteria, and preparation method and application thereof

Info

Publication number: CN114426981A
Application number: CN202210157630.4A
Authority: CN
Inventors: 王春凤; 杨桂连; 邹博识; 牛天明; 杨文涛; 石春卫
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2022-02-21
Filing date: 2022-02-21
Publication date: 2022-05-03
Anticipated expiration: 2042-02-21
Also published as: CN114426981B; CN116396974B; CN116396974A

Abstract

The invention relates to the technical field of microorganisms, in particular to an African swine fever virus antigen protein recombinant expression vector, a recombinant plant lactic acid bacterium, a preparation method and an application thereof, wherein the African swine fever virus antigen protein recombinant expression vector comprises P54, Fc, P30 and EGP gene segments; wherein, P54 and P30 are the gene sequences of the cell membrane structure proteins which are encoded in the gene type II SY-18 strain of the African swine fever virus and are involved in the absorption and the internalization of a host; the Fc is a swine IgG3Fc gene sequence, and the EGP is an intestinal cell HSPGs receptor targeting ligand polypeptide sequence. The recombinant lactobacillus plantarum obtained by the invention can efficiently express P54/P30 antigen protein and has good immunogenicity. Compared with the original strain L.plantarum NC8, the strain can induce mucosal immunity, cellular immunity and humoral immunity response, and has good practical application value.

Description

African swine fever virus antigen protein recombinant expression vector, recombinant plant lactic acid bacteria, and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to an African swine fever virus antigen protein recombinant expression vector, a recombinant plant lactic acid bacterium, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

African Swine Fever (ASF) is an acute, highly contagious, virulent infectious disease caused by African Swine Fever Virus (ASFV), with high mortality. Because no effective commercial vaccine or specific medicine is available for preventing and treating the epidemic disease at present, the African swine fever virus has variation in the process of propagation among hosts, so that a new strain with weakened virulence appears recently, the disease condition is light, the incubation period is long, once the infection is discovered in a short time, the situation of preventing and controlling the non-epidemic situation is still complex.

There are many vaccine forms currently studied for prevention of african swine fever, but the ASFV differs from other nucleoplasmic macroviruses in that it has a multilayer structure and an overall icosahedral morphology, the immunobiology has many unknown features, and knowledge about the composition and structure of infectious virions and viral proteins responsible for recognition and induction of protective immune responses is very different, which hinders the development of effective vaccines. Research finds that structural proteins such as ASFV P54, P30, CD2v, P12, P17 and P72 participate in adsorption and internalization of a host, the P54 protein is positioned on an outer lipid membrane of an ASFV virion, the transport function in a host cell is controlled by combining with an dynein light chain, the structural proteins play an important role in a virus infection process, the structural proteins have better immunogenicity and conservation, can induce the generation of neutralizing antibodies and inhibit the internalization of viruses, and the P54 and P30 proteins are used as main candidate antigens of ASFV vaccines. Although it has been reported that immunization with baculovirus or vaccinia virus vectors displaying antigens P54 and P30 on their surfaces delays the onset of clinical symptoms of ASF, and also reduces viral genome load in blood and lymphoid tissues, while immunized piglets survive challenge with the E75 strain, but are not resistant to challenge with highly pathogenic genotype strains.

The molecular epidemiological research shows that the African swine fever virus introduced into China belongs to the II type of gene, and the research of the vaccine which can be used for preventing the II type ASFV strain is particularly important. The African swine fever virus can code hundreds of proteins, wherein the structural protein p14.5 coded by the E120R gene is identified as a key virulence factor and a late-stage expression protein of the virus, and can cause the body to generate humoral immunity. Traditional vaccine methods, such as inactivating viruses, have proven ineffective. More successful has been through natural attenuation of isolates or modification of live viruses. However, protection is generally only against homologous strains of the same genotype, and is not effective against infection by heterologous viruses. In addition, live attenuated strains often have associated side effects such as skin lesions and joint swelling that do not allow for safe vaccine use. Live virus vaccines can also lead to chronic or persistent infections and return a strong risk of toxicity. Another problem with live attenuated vaccines is the lack of stable producer cell lines, since ASFV replicates preferentially in primary monocytes or macrophages.

The ASFV mainly infects mucous membrane parts of piglets through the digestive tract and respiratory tract of piglets, wherein the digestive tract mucous membrane is regarded as the main infection part of the ASFV. The inventors found that although african swine fever virus spreads as aerosol or in direct contact and infects host mucosa, the specific mechanism of invasion of the mucosal barrier by ASFV is unclear and whether protection of piglets by the mucosal system for the immune pathway of antigen delivery is effective in blocking african swine fever virus infection deserves intensive studies.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide an African swine fever virus antigen protein recombinant expression vector, a recombinant plant lactic acid bacteria and a preparation method thereof, on one hand, the invention uses food-grade lactobacillus plantarum L.Plantarum NC8 as a starting strain, uses an ASFV gene type II SY-18 virulent strain sequence as a basis, and carries out optimization according to the codon preference of the lactobacillus plantarum by connecting a related protective antigen site fragment with a porcine immunoglobulin IgG Fc fragment and a heparan sulfate polysaccharide ligand polypeptide in series according to the research of protective antigen sites, thereby successfully constructing and obtaining an NC 8-pSIP-pgsA' -P54-Fc-P30-EGP (NC 8-pSIP-PPFPP) strain. Proved by experimental research, the recombinant lactobacillus plantarum obtained by the invention can efficiently express P54/P30 antigen protein and has good immunogenicity. Compared with the original strain L.plantarum NC8, the strain can induce mucosal immunity, cellular immunity and humoral immunity response, and has good practical application value.

On the other hand, the E120R gene of African swine fever virus coding p14.5 protein, murine IL-33, an enzymatic activity CTA1 subunit based on Cholera Toxin (CT) and an adjuvant CTA1-DD artificially formed by a D domain dimer of staphylococcus aureus protein A are selected to construct three new functional recombinant lactobacillus plantarum strains, NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D, and research results prove that the recombinant lactobacillus plantarum disclosed by the invention has the improvement effects on humoral immunity, cellular immunity and mucosal immunity after being fed to mice in an oral mode.

In order to achieve the above object, the technical solution of the present invention is as follows:

in a first aspect of the invention, a recombinant expression vector for an antigen protein of African swine fever virus is provided, which comprises P54, Fc, P30 and EGP gene segments;

wherein, P54 and P30 are the gene sequences of the cell membrane structure proteins which are encoded in the gene type II SY-18 strain of the African swine fever virus and are involved in the absorption and the internalization of a host; the Fc is a swine IgG3Fc gene sequence, and the EGP is an intestinal cell HSPGs receptor targeting ligand polypeptide sequence.

In one or more embodiments, the recombinant expression vector is P54-Fc-P30-egp (pfpp);

specifically, the P54, Fc, P30 and EGP gene fragments are expressed in tandem with pgsA' gene, and the nucleotide sequence of P54-Fc-P30-EGP is shown as SEQ ID NO. 1;

furthermore, the amino acid sequence of the P54-Fc-P30-EGP coding protein is shown as SEQ ID NO. 2.

In a second aspect of the invention, a recombinant lactobacillus is provided, wherein the recombinant lactobacillus is transformed with the African swine fever virus antigen protein recombinant expression vector.

In order to improve the effect of the exogenous antigen presented by the protein coded by the exogenous gene on the surface of the recombinant lactobacillus plantarum vector, the recombinant lactobacillus utilizes a pgsA 'gene fragment and pSIP409 which is anchored by bacillus subtilis membrane protein pgsA' and has surface display antigen as an expression vector.

Thus, in one of its specific embodiments, the recombinant lactobacillus comprises pgsA' anchored P54, Fc, P30 and EGP gene fragments.

In a specific embodiment, the recombinant lactobacillus is NC8-pSIP409-pgsA' -PFPP.

In a third aspect of the present invention, there is provided a method for constructing the above recombinant lactobacillus, the method comprising:

s1, inserting the P54, Fc, P30 and EGP gene segments into an anchoring expression vector pSIP409-pgsA' to construct a recombinant expression vector P54-Fc-P30-EGP;

s2, transferring the recombinant expression vector obtained in the step S1 into lactobacillus plantarum.

In a specific embodiment, in step S1, the nucleotide sequence of P54-Fc-P30-EGP is shown as SEQ ID NO. 1; the amino acid sequence of the P54-Fc-P30-EGP coding protein is shown as SEQ ID NO. 2;

in a specific embodiment, in step S2, the lactobacillus plantarum is specifically lactobacillus plantarum NC 8.

In the fourth aspect of the present invention, there is provided an african swine fever virus antigen protein recombinant expression vector, wherein the african swine fever virus antigen protein recombinant expression vector is: pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p 14.5-D-D;

wherein p14.5 is the E120R gene sequence of the African swine fever virus coding p14.5 protein; IL-33 is a murine IL-33 gene sequence; CTA1-DD is: an adjuvant CTA1-DD gene sequence artificially composed of an enzymatically active CTA1 subunit based on Cholera Toxin (CT) and a D-domain dimer of Staphylococcus aureus protein A;

preferably, the nucleotide sequence of pLP-S-p14.5 is shown in SEQ ID NO. 3;

the nucleotide sequence of pLP-S-p14.5-IL-33 is shown in SEQ ID NO. 4;

the nucleotide sequence of pLP-S-CTA1-p14.5-D-D is shown in SEQ ID NO. 5;

further preferably, the amino acid sequence of the protein coded by pLP-S-p14.5 is shown in SEQ ID NO. 6;

the amino acid sequence of the pLP-S-p14.5-IL-33 coding protein is shown in SEQ ID NO. 7;

the amino acid sequence of the protein coded by pLP-S-CTA1-p14.5-D-D is shown in SEQ ID NO. 8;

in a specific embodiment, the construction method of the African swine fever virus antigen protein recombinant expression vector comprises the following steps:

selecting a gene (E120R) sequence encoding P14.5 protein, optimizing two adjuvant sequences IL-33 and CTA1-DD, respectively placing the optimized adjuvant sequences at 5'3' end (CTA1-DD) and 3' end (IL-33) of E120R gene, P14.5, CTA1-P14.5-D-D and P14.5-IL-33-Mus sequence, synthesizing, and inserting into a single anchoring expression vector pLP-S with erm as a screening marker to obtain three African swine fever virus antigen protein recombinant expression vectors pLP-S-P14.5, pLP-S-P14.5-IL-33 and pLP-S-1-P14.5-D-D.

Protein P14.5 is synthesized late in infection, is located in the virus factory, is a protein necessary for the transfer of virus particles from the virus factory to the plasma membrane, and is combined with protein P72 to form the capsid of the virus, and can cause the body to generate humoral immunity. It is considered that the protein p14.5 may play a role in the embedding process of ASFV DNA.

Vaccine adjuvants are substances that can nonspecifically alter or enhance the body's specific immune response to an antigen, exerting an adjuvant effect. The adjuvant can induce the organism to generate long-term and efficient specific immune response, improve the protective capacity of the organism, reduce the dosage of immune substances and reduce the production cost of the vaccine; CTA1-DD is an adjuvant artificially composed of active CTA1 subunit based on cholera toxin and D-domain dimer of Staphylococcus aureus protein A, and the molecule is nontoxic and safe. It can stimulate strong and balanced CD4⁺T cell responses, greatly enhancing the production of specific antibodies; after systemic and mucosal immunization, FDCs (follicular dendritic cells) in peripheral lymph nodes can be efficiently matured, increasing germinal center B cell and Tfh responses.

IL-33 is a multifunctional gene, secreted when cells are damaged, plays a role in regulating immunity, can directly react with DC, and stimulates the differentiation of naive T cells to Th2 or helper T cells; studies have shown that IL-33 can enhance Th1 and CD8 by promoting expansion of NK and NKT cells during viral infection⁺T cell responses to help clear the virus.

The S-layer protein is a monomolecular crystal structure protein on the outermost surface of a cell, is considered as the outermost structure of a cell membrane in many archaea, and is used for displaying foreign proteins on the surface; the S-layer protein derived from lactobacillus has the capability of anchoring and expressing foreign protein, also has adhesion property, and can be used as an adjuvant for stimulating the body to generate immune response.

In a fifth aspect of the present invention, there is provided a recombinant lactobacillus plantarum transformed with the african swine fever virus antigen protein recombinant expression vector of the first aspect, comprising: NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p 14.5-D-D.

In a sixth aspect of the present invention, there is provided a method for constructing the recombinant lactobacillus plantarum described in the second aspect, comprising:

s1, inserting the p14.5 gene, IL-33 and adjuvant CTA1-DD gene fragment into a single anchored expression vector pLP-S with erm as a screening marker to construct recombinant expression vectors pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p 14.5-D-D;

s2, obtaining the competence of lactobacillus plantarum NC 8;

s3, and electrotransformation of the recombinant expression vector obtained in the step S1 into the competence of lactobacillus plantarum NC8 obtained in the step S2.

In a seventh aspect of the present invention, there is provided a microbial agent comprising the above recombinant lactobacillus plantarum or a fermentation product thereof or a metabolite thereof;

the microbial inoculum is liquid or solid, preferably solid, and further preferably freeze-dried powder.

The metabolite of the invention comprises a thallus intracellular metabolite and/or an extracellular metabolite.

The term "fermentate" is used to refer to a fermentation product. The corresponding fermentation product may be a liquid obtained from the process of fermentative culture of recombinant lactobacillus plantarum and, therefore, may also be referred to as fermentation broth; the liquid may contain bacteria (bacteria cells), but does not necessarily need to contain bacteria. The liquid preferably contains metabolites produced by the recombinant lactobacillus plantarum bacteria of the invention.

And, in an embodiment of the present invention, the bacterial cells grown in the fermentation broth or culture broth are separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art, and the liquid remaining when the bacterial cells are removed is a "supernatant" (in an embodiment of the present invention, the supernatant is labeled as CFS), and in the present invention, the supernatant contains the extracellular metabolites of the recombinant lactobacillus plantarum of the present invention. In the embodiment of the present invention, the microbial agent may also contain the supernatant.

And, in the embodiment of the present invention, the fermentation liquid or culture liquid containing the bacterial cells is centrifuged, filtered, settled or separated from the liquid by other means known in the art to obtain the bacterial cells, the bacterial cells can be disrupted by ultrasound (such as ultrasonic cell disruption in ice bath) or other means known in the art, or further, the bacterial cell disruption is centrifuged to collect the supernatant, the supernatant is designated as cell-free extract, and the disrupted or cell-free extract contains the intracellular metabolite of the recombinant lactobacillus plantarum of the present invention. In the embodiment of the present invention, the microbial agent may contain a disrupted product or a cell-free extract of the microbial agent.

In an eighth aspect of the invention, an application of the recombinant lactobacillus plantarum and/or the microbial inoculum in preparation of an antiviral product is provided.

The recombinant lactobacillus plantarum and/or the microbial inoculum can improve the absorption effect of intestinal tracts on ASFV antigen protein delivered by the recombinant lactobacillus, thereby inducing high-efficiency mucosal immune response reaction.

In the ninth aspect of the invention, the invention provides an antiviral product, which is characterized in that the antiviral product comprises the recombinant lactobacillus plantarum and/or the microbial inoculum;

preferably, the product is an animal vaccine, feed additive or feed;

further preferably, the animal vaccine is a swine vaccine, and the vaccine dosage form can be oral freeze-dried powder;

the virus includes African Swine Fever Virus (ASFV).

P54-Fc-P30-EGP nucleotide sequence (SEQ ID NO.1)

ATGGATAGTGAATTTTTTCAACCGGTTTATCCGCGTCATTATGGTGAATGTCTGAGTCCGGTTACGACGCCGAACTTTTTTAGTACGCACATGTATACCATTCTGATTGCGATTGTTGTTCTGGTTATTATCATTATCGTTCTGATTTATCTGTTTAGTAGTCGTAAAAAGAAAGCGGCGGCCATTGAAGAGGAAGATATTCAATTTATTAATCCGTATCAAGATCAACAATGGGTTGAAGTTACGCCGCAACCGGGTACGAGTAAACCGGCGGGTGCGACGACGGCGAGTGTTGGTAAGCCGGTTACGGGTCGTCCGGCCACGAATCGCCCGGCGACGAATAAACCGGTTACGGATAATCCGGTTACGGACCGTCTGGTGATGGCCACGGGTGGCCCGGCGGCCGCGCCGGCGGCCGCGAGTGCGCCGGCCCATCCGGCGGAACCGTATACGACGGTGACGACGCAGAATACGGCGAGTCAAACGATGAGTGCGATTGAAAATCTGCGTCAACGTAATACGTATACGCATAAAGATCTGGAAAATAGTCTGGGTAGTGGTGGTGGCGGTAGTGGTGGCGGTGGTAGCGGTAGTGACATTGAACCGCCGACGCCGATTTGTCCGGAAATTTGTAGTTGTCCGGCGGCGGAAGTTCTGGGTGCGCCGAGTGTTTTTCTGTTTCCGCCGAAACCGAAAGATATTCTGATGATTAGTCGTACGCCGAAAGTTACGTGTGTTGTGGTTGATGTTAGTCAAGAAGAAGCGGAAGTTCAATTTAGTTGGTATGTTGATGGTGTTCAACTGTATACGGCGCAAACGCGTCCGATGGAAGAACAATTTAATAGTACGTATCGTGTTGTTAGTGTTCTGCCGATTCAACATCAAGATTGGCTGAAAGGTAAAGCGTTTGCGTGTGCGGTTAATAATAAAGATCTGCTGAGTCCGATTACGCGTACGATTAGTAAAGCGACGGGTCCGAGTCGTGTTCCGCAAGTTTATACGCTGCCGCCGGCGTGGGAAGAACTGAGTAAAAGTAAAGTTAGTATTACGTGTCTGGTTACGGGTTTTTATCCGCCGGATATTGATGTTGAATGGCAAAGTAATGGTCAACAAGAACCGGAAGGTAATTATCGTACGACGCCGCCGCAACAAGATGTTGATGGTACGTATTTTCTGTATAGTAAACTGGCGGTTGATAAAGTTCGTTGGCAACGTGGTGATCTGTTTCAATGTGCGGTTATGCATGAAGCGCTGCATAATCACTATACGCAAAAAAGTATTAGTAAAACGCAAGGTAAAGAAGCGGCCGCGAAAGAAGCCGCGGCGAAAGAAGCGGCGGCCAAGGAAGCCGCGGCGAAGATGGATTTTATTCTGAATATTAGTATGAAAATGGAAGTTATTTTTAAAACGGATCTGCGTAGTAGCAGTCAAGTTGTTTTTCATGCGGGTAGTCTGTACAATTGGTTTAGTGTTGAAATTATTAATAGTGGTCGTATTGTTACGACGGCGATTAAAACGCTGCTGAGTACGGTTAAATATGATATTGTTAAAAGTGCGCGTATTTATGCGGGTCAAGGTTATACGGAACATCAAGCGCAAGAAGAATGGAATATGATTCTGCATGTTCTGTTTGAAGAGGAAACGGAAAGTAGTGCGAGTAGTGAAAATATTCATGAGAAAAATGATAATGAAACGAATGAATGTACGAGTAGTTTTGAAACGCTGTTTGAACAAGAACCGAGTAGTGAAGTTCCGAAAGATAGTAAACTGTATATGCTGGCGCAAAAAACGGTTCAACATATTGAACAATATGGTAAAGCGCCGGATTTTAATAAAGTTATTCGTGCGCATAATTTTATTCAAACGATTTATGGTACGCCGCTGAAAGAAGAGGAAAAAGAAGTTGTTCGTCTGATGGTTATTAAACTGCTGAAGAAAATTAACTTTTTTCTGACGTATATTGGTAGTGGTGGTGGCGGTAGTGGTAGTAAACGTAAAAAGAAAGGTAAAGGTCTGGGTAAAAAACGTGATCCGTGTCTGCGTAAATATAAA

P54-Fc-P30-EGP amino acid sequence (SEQ ID NO.2)

MDSEFFQPVYPRHYGECLSPVTTPNFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLGSGGGGSGGGGSGSDIEPPTPICPEICSCPAAEVLGAPSVFLFPPKPKDILMISRTPKVTCVVVDVSQEEAEVQFSWYVDGVQLYTAQTRPMEEQFNSTYRVVSVLPIQHQDWLKGKAFACAVNNKDLLSPITRTISKATGPSRVPQVYTLPPAWEELSKSKVSITCLVTGFYPPDIDVEWQSNGQQEPEGNYRTTPPQQDVDGTYFLYSKLAVDKVRWQRGDLFQCAVMHEALHNHYTQKSISKTQGKEAAAKEAAAKEAAAKEAAAKMDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKINFFLTYIGSGGGGSGSKRKKKGKGLGKKRDPCLRKYK

3 p14.5 nucleotide sequence (SEQ ID NO.3)

ATGGCTGATTTTAATTCACCCATACAATATCTCAAAGAAGACTCTCGCGATCGTACCTCGATCGGCAGCTTGGAATATGATGAAAATGCAGATACGATGATTCCGAGCTTTGCGGCGGGTCTGGAAGAGTTCGAGCCGATTCCAGACTACGACCCGACCACGTCCACCTCCCTTTACAGCCAGCTGACCCATAACATGGAAAAGATCGCCGAGGAGGAGGACTCTAACTTCCTGCATGATACCCGTGAATTCACTTCATTGGTTCCGGACGAGGCGGACAACAAGCCGGAAGATGATGAGGAAAGCGGTGCTAAACCGAAAAAGAAGAAGCACCTGTTTCCGAAGCTGAGCAGCCATAAAAGCAAA

4 p14.5-IL-33-Mus nucleotide sequence (SEQ ID NO.4)

ATGGCTGATTTTAATTCACCCATACAATATTTGAAGGAGGACAGCAGAGATCGTACCTCCATCGGTAGCCTGGAGTATGATGAGAACGCCGACACGATGATTCCGAGCTTTGCGGCTGGTCTGGAAGAGTTCGAGCCGATTCCGGATTACGACCCAACGACCTCCACCTCCCTGTATAGCCAACTCACCCATAATATGGAAAAGATCGCCGAAGAGGAGGACTCTAACTTCCTGCACGATACGCGTGAATTCACCTCTTTAGTTCCGGATGAGGCGGATAATAAGCCGGAAGACGACGAAGAAAGCGGTGCTAAACCGAAGAAGAAGAAGCACCTGTTTCCGAAACTGAGCTCCCATAAAAGCAAAGGTTCTGGCGGTGGCGGCTCTGGCGGCGGCGGGTCAGGTTCCATGCGTCCGCGTATGAAATACTCCAATAGCAAGATTAGCCCGGCGAAATTCAGCAGCACTGCCGGTGAACGTAGCGTCCCACCGTGCAAAATCCGCCGTAGTCAGCAGAAAACCAAAGAGTTCTGCCATGTTTACTGCATGCGCCTGCGCTCGGGCCTGACCATTCGTAAAGAAACCTCTTACTTCCGCAAAGAGCCGACTAAGCGCTACTCCCTTAAAAGCGGTACCAAACATGAAGAGAACTTTAGCGCATATCCGCGTGATAGCCGTAAGCGCAGCTTGCTGGGTTCTATCCAAGCGTTTGCGGCGAGTGTGGATACCCTGTCGATCCAGGGTACATCGTTGCTGACCCAGAGCCCGGCGAGCTTATCTACCTATAACGACCAGAGCGTCAGCTTCGTTTTGGAGAACGGTTGTTATGTGATCAACGTGGACGATTCCGGCAAGGACCAAGAACAAGATCAAGTGTTGCTGCGTTACTATGAAAGCCCGTGCCCGGCTTCGCAAAGCGGTGATGGTGTGGACGGCAAAAAGCTGATGGTTAATATGAGCAGCATCAAGGACACCGACATCTGGCTGCATGCAAACGATAAAGACTACTCTGTGGAACTGCAGCGTGGTGATGTTAGCCCGCCTGAACAGGCGTTTTTTGTACTTCACAAAAAGAGCTCAGATTTTGTGTCCTTCGAGTGCAAGAACTTGCCGGGTACGTACATTGGTGTTAAAGACAACCAGCTGGCTCTGGTTGAAGAGAAGGACGAGAGCTGTAATAATATTATGTTCAAACTGTCGAAAATT

5 CTA1-p14.5-D-D nucleotide sequence (SEQ ID NO.5)

ATGAATGATGACAAACTATATAGGGCTGATAGCCGTCCGCCAGATGAGATCAAACAATCTGGTGGTCTGATGCCGCGCGGTCAGTCCGAATACTTCGATAGAGGAACCCAGATGAACATTAACCTGTACGACCACGCGACCCAGACCGGTTTTGTGCGTCATGATGATGGCTACGTCAGCACTTCTATTAGCTTACGTAGCGCGCATTTGGTTGGTCAGGAGGTTAGCGCGCTGGGTGGTATCCCGTATAGCCAGATCTATGGTTCGTACCGCGTGCATTTTGGTGTTCTGGACGAACAGCTGCATCGTAACCGTGGTTATCGTTATTACTCTAACCTCGACATCCCGCCGGCGGCCGACGGCTACGGCCTAGCGGGTTTCCCGCCGGAGCACCGCGCATGGCGCCAGGAGCCGTGGATTCACCATGCTCCGCCAGGTTGCGGTAATGCCCCACGTAGCAGCGGCTCTGGCGGCGGTGGCTCAGGCGGTGGCGGTAGCGGTTCTATGGCTGACTTCAACAGCCCGATCCAGTATCTGAAAGAAGACTCTCGCGATCGTACCTCGATCGGTTCATTGGAATATGATGAGAATGCCGATACTATGATCCCGAGCTTTGCAGCGGGCCTGGAAGAATTTGAACCGATTCCGGATTATGATCCGACCACCTCGACGTCACTTTACAGTCAACTGACCCATAATATGGAAAAAATCGCTGAAGAGGAGGATTCCAACTTTCTGCACGATACGCGTGAGTTCACCAGCCTTGTTCCGGACGAGGCTGACAACAAACCGGAGGACGACGAGGAAAGCGGCGCAAAACCGAAAAAGAAGAAGCATCTGTTTCCGAAACTGAGCAGCCACAAAAGCAAGGGTTCCGGTGGCGGTGGTAGCGGCGGCGGTGGCTCTGGGAGCGCCGACGCACAGCAGAATAATTTCAACAAGGACCAACAAAGCGCGTTTTACGAAATTTTGAATATGCCGAATTTGAATGAGGCGCAACGCAACGGCTTCATCCAGTCCTTGAAAGACGATCCGTCCCAGAGCACCAACGTGTTGGGTGAGGCGAAAAAGCTGAATGAGTCCCAAGCGCCCAAGGGCAGCGGTGGCGGCGGTTCCGGTGGAGGCGGCAGTGGGAGCGCTGATGCGCAGCAAAACAACTTCAACAAAGACCAACAGTCTGCGTTCTACGAAATTCTGAACATGCCGAACCTGAACGAAGCACAGCGTAATGGTTTCATTCAAAGCCTGAAGGACGACCCGAGCCAATCTACGAACGTGCTGGGCGAAGCCAAAAAGCTGAACGAGAGCCAAGCGCCTAAG

6 p14.5 amino acid sequence (SEQ ID NO.6)

MADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFL HDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSK

7P 14.5-IL-33-Mus amino acid sequence (SEQ ID NO.7)

MADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSKGSGGGGSGGGGSGSMRPRMKYSNSKISPAKFSSTAGERSVPPCKIRRSQQKTKEFCHVYCMRLRSGLTIRKETSYFRKEPTKRYSLKSGTKHEENFSAYPRDSRKRSLLGSIQAFAASVDTLSIQGTSLLTQSPASLSTYNDQSVSFVLENGCYVINVDDSGKDQEQDQVLLRYYESPCPASQSGDGVDGKKLMVNMSSIKDTDIWLHANDKDYSVELQRGDVSPPEQAFFVLHKKSSDFVSFECKNLPGTYIGVKDNQLALVEEKDESCNNIMFKLSKI

8 CTA1-P14.5-D-D amino acid sequence (SEQ ID NO.8)

MNDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHATQTGFVRHDDGYVSTSISLRSAHLVGQEVSALGGIPYSQIYGSYRVHFGVLDEQLHRNRGYRYYSNLDIPPAADGYGLAGFPPEHRAWRQEPWIHHAPPGCGNAPRSSGSGGGGSGGGGSGSMADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSKGSGGGGSGGGGSGSADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKGSGGGGSGGGGSGSADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK

The specific embodiment of the invention has the following beneficial effects:

on one hand, two recombinant plant lactic acid bacteria NC8-pSIP409-pgsA '-PFPP and NC8-pSIP409-pgsA' -PmFPP are constructed, oral gavage immunity experiments are carried out on mice, PBS and NC8 groups are compared, after one week of immunity, the influence of the recombinant plant lactic acid bacteria on the cell immunity of the mice after immunity is detected, and the result shows that the two recombinant plant lactic acid bacteria can induce the spleen of the mice to generate CD4⁺IL-4⁺And CD8⁺IFN-γ⁺The cell shows that the fusion protein expressed by the recombinant plant lactobacillus is used as an antigen for inducing the cellular immunity of an organism, the reaction of T lymphocytes is successfully induced, and possibly two T cell subsets play a role in protection;

the IgA content in mouse Peyer's patches B cells after the recombinant plant lactobacillus immunization is detected by using flow cytometry, and the result shows that B220 in the recombinant plant lactobacillus⁺IgA⁺The number of the recombinant lactobacillus is obviously higher than that of PBS and NC8 groups, which shows that the secretion of the antibody is obviously increased under the stimulation of the recombinant lactobacillus plantarum, and the reaction of B lymphocytes is successfully activated;

the two recombinant plant lactic acid bacteria can stimulate CD80 in Peyer's patches after oral gavage⁺And CD86⁺The activation of the surface marker indicates that the two recombinant plant lactic acid bacteria can activate dendritic cells and further exert the antigen uptake and presentation functions of the dendritic cells;

the mice are immunized in a gastric perfusion mode, the two recombinant plant lactic acid bacteria can better stimulate the activation of DC cells, induce the mice to generate cell factors, promote the proliferation of T cells, improve the activation level of B cells, provide conditions for the formation of a hair center and promote the expression of IgA.

On the other hand, the invention successfully constructs three recombinant lactobacillus plantarum NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D, and verifies the successful expression of the three in vitro; since lactobacillus can colonize in mouse intestinal tract and stimulate lactobacillus to express p54 protein and p54-IL-33, CTA1-p14.5-D-D fusion protein under intestinal tract environment, organism T cell can recognize p54 protein and p54-IL-21 fusion protein and generate specific antibody; the result of a preliminary animal test by using a mouse shows that the feeding of the recombinant lactobacillus plantarum has a certain effect of improving the immune effect of the mouse;

flow cytometry experiments show that the secretion amount of B220 and IgA in PP node lymphocytes of mice fed with the recombinant lactobacillus plantarum group and 11C⁺CD80 and 11C⁺The content of CD86 is greater than that of the control group; CD4 in spleen⁺IFN-γ⁺CD4⁺IL-4 and CD8⁺IFN-γ⁺CD8⁺The expression level of IL-4 is also obviously enhanced.

The experimental results of the invention show that the immunity of mice is obviously improved after the recombinant lactobacillus plantarum is fed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a diagram: the vector was obtained by double digestion in example 1; 1: double enzyme digestion;

FIG. 2 is a diagram of: the pUC-GW-Kan-PFPP double digestion and the pUC-GW-Kan-PmFPP double digestion in the embodiment 1 are carried out; 1: double enzyme digestion of pUC-GW-Kan-PFPP; 2: double enzyme digestion of pUC-GW-Kan-PmFPP;

FIG. 3 is a diagram of: double restriction enzyme digestion verification of the recombinant plasmids pSIP409-pgsA '-PFPP and pSIP409-pgsA' -PmFPP in example 1; 1: the recombinant plasmid pSIP409-pgsA' -PFPP is subjected to double enzyme digestion; 2: the recombinant plasmid pSIP409-pgsA' -PmFPP is subjected to double enzyme digestion;

FIG. 4 is a diagram of: the pSIP409-pgsA' -PFPP/PmFPP plasmid map in example 1;

FIG. 5 is a diagram: the colony morphology of NC8-pSIP409-pgsA' -PFPP in example 1;

FIG. 6 is a diagram of: the growth profiles of the strains of NC8(pSIP 409-pgsA'), NC8(pSIP409-PFPP), NC8(pSIP409-PmFPP) in example 1;

FIG. 7 is a diagram of: westen blot results in example 1;

FIG. 8 is a diagram of: the animal immunization procedure in example 2;

FIG. 9 is a diagram of: CD4+ IL-4+ and CD8+ IFN-. gamma. + expression in the spleen in example 2;

FIG. 10 is a diagram: expression of B220+ IgA + in Peyer's patches in example 2;

FIG. 11 is a diagram of: activation of dendritic cells in Peyer's patches in example 2.

FIG. 12 is a diagram: plasmid double restriction verification of pLP-S-p14.5 in example 3 (M: DL15000 marker; 1-4: cloning cleavage results No. 1-4);

FIG. 13 is a graph of: the plasmid pLP-S-CTA1-p14.5-D-D in example 3 was verified by double digestion (M: DL15000 marker; 1-2: cloning digestion results No. 1-2);

FIG. 14 is a graph of: the plasmid pLP-S-p14.5-IL-33-Mus in example 3 was verified by double digestion (M: DL10000 marker; 1: No.1 cloning digestion result);

FIG. 15 is a schematic diagram of: the successfully constructed NC8-pLP-S-p14.5 plasmid map in example 3;

FIG. 16 is a graph showing: the successfully constructed NC8-pLP-S-p14.5-IL-33-Mus plasmid map in example 3;

FIG. 17 is a graph of: the successfully constructed NC8-pLP-S-CTA1-p14.5-D-D plasmid map in example 3;

FIG. 18 is a graph of: electrophorogram of p14.5 PCR product in example 3 (M: DL2000 marker; 1: p14.5 PCR product);

FIG. 19 is a graph of: the double digestion verification of BL21-pET-28a-p14.5 plasmid in example 3 (M: DL10000 marker; 1: No.1 cloning digestion result);

FIG. 20 is a graph showing the growth of NC8-pLP-S, NC8-pLP-S-P14.5, NC8-pLP-S-P14.5-IL-33-Mus, NC8-pLP-S-CTA1-P14.5-D-D strains in example 3;

FIG. 21 is a schematic view of: secretion patterns and histograms in spleen lymphocytes of the PBS group, NC8-pLP-S group, NC8-pLP-S-P14.5 group, NC8-pLP-S-CTA1-P14.5-D-D group, NC8-pLP-S-P14.5-IL-33-Mus group in example 4; wherein (a) is the IFN-gamma secretion of the PBS group; (b) the secretion amount of IFN-gamma in NC8-pLP-S group, (c) the secretion amount of IFN-gamma in NC8-pLP-S-P14.5 group, (D) the secretion amount of IFN-gamma in NC8-pLP-S-CTA1-P14.5-D-D group, (e) the secretion amount of IFN-gamma in NC8-pLP-S-P14.5-IL-33-Mus group;

FIG. 22 is a schematic diagram of: in example 4, CD3 among splenic lymphocytes of PBS group, NC8-pLP-S group, NC8-pLP-S-P14.5 group, NC8-pLP-S-CTA1-P14.5-D-D group, NC8-pLP-S-P14.5-IL-33-Mus group⁺CD4⁺A graph and histogram of IL-4 secretion in T cells, wherein (a) is the IL-4 secretion in PBS group; (b) the secretion amount of IL-4 in NC8-pLP-S group, (c) the secretion amount of IL-4 in NC8-pLP-S-P14.5 group, (D) the secretion amount of IL-4 in NC8-pLP-S-CTA1-P14.5-D-D group, (e) the secretion amount of IL-4 in NC8-pLP-S-P14.5-IL-33-Mus group;

FIG. 23 is a schematic representation of: example 4 CD3 in spleen lymphocytes⁺CD8⁺The amount of IL-4 secreted in T cells;

FIG. 24 shows: example 4 CD3 in spleen lymphocytes⁺CD8⁺IFN-gamma secretion in T cells;

FIG. 25 is a graph showing: example 4 11C in Pepper's lymph node Collection (PP) cells⁺80⁺Content (c);

FIG. 26 is a diagram: example 4 11C in Pepper's lymph node Collection (PP) cells⁺86⁺Content (c);

FIG. 27 is a graph of: example 4B 220 in Pepper's lymph node Collection (PP) cells⁺IGA⁺And (4) content.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

Construction and verification of recombinant plant lactic acid bacteria for expressing African swine fever virus fusion antigen

1.1 materials and methods

The strains and plasmids used in this experiment are shown in Table 1.

TABLE 1 strains and plasmids

1.1.1.2 enzymes and Primary test reagents

Restriction enzymes Xba I and Hind III, PrimeSTAR Max Premix (2X), DNA Marker (DL5000, DL10000, DL15000), 10XLoading Buffer, nucleic acid electrophoresis gel agarose were purchased from Takara bioengineering (Dalian) Inc. (Takara); a Rapid DNA Ligation Kit (Rapid DNA Ligation Kit), a DNA end smoothing Kit (DNA Blunting Kit) and a BCA Protein concentration determination Kit (Enhanced type) (Enhanced BCA Protein Assay Kit) are purchased from Biyuntian biology Ltd; SppIP is preserved by the research center of animal microecological preparation engineering in Jilin province; beef powder, available from OXOID, UK; DAP, D-alanine were purchased from Sigma; DNA gel recovery and purification kit

Gel Extraction Kit is purchased from Omega company, Fast Pure Plasmid Mini Kit, 180kDa Prestained Protein Marker is purchased from Vazyme company, SDS-PAGE sample buffer is purchased from Beijing kang century Biotechnology Co., Ltd; kanamycin sulfate powder, erythromycin powder, isopropyl-beta-D-thiogalactoside (IPTG) powder were purchased from Beijing Solebao technologies, Inc.; super GelBlue fluorescent nucleic acid gel staining reagent (EVERBRIGHT, USA); seamless Cloning Kit (Thailand Biotechnology Co., Ltd.); PBS phosphate buffer was purchased from Solarbio; other reagents required by the test are domestic analytical pure products or imported.

1.1.1.4.1 preparation of culture Medium

(1) LB liquid medium: sodium chloride 10.0g/L, tryptone 10.0g/L, yeast extract 5.0g/L, ultra pure water dissolved, 121 degrees C high pressure sterilization for 20 min.

(2) LB solid medium: 10.0g/L of sodium chloride, 10.0g/L of tryptone, 5.0g/L of yeast extract and 15g/L of bacteriological agar powder; dissolving in ultrapure water, and autoclaving at 121 deg.C for 20 min.

(3) MRS liquid medium: 10.0g/L beef extract, 10.0g/L tryptone, 20.0g/L D-anhydrous glucose, 5.0g/L sodium acetate trihydrate, 0.05g/L yeast extract, tween-801.0 g/L, 2.0g/L potassium hydrogen phosphate, 2.0g/L triammonium citrate, 2.0g/L dipotassium hydrogen phosphate trihydrate, 0.2g/L magnesium sulfate heptahydrate and 0.05g/L manganese sulfate monohydrate; dissolving in ultrapure water, and autoclaving at 115 deg.C for 15 min.

(4) MRS solid medium: 10.0g/L of beef extract, 10.0g/L of tryptone, 20.0g/L of D-anhydrous glucose, 5.0g/L of sodium acetate trihydrate, 0.05g/L of yeast extract, 801.0 g/L of tween-80, 2.0g/L of potassium hydrogen phosphate, 2.0g/L of triammonium citrate, 2.0g/L of dipotassium hydrogen phosphate trihydrate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate and 15g/L of bacteriological agar powder; dissolving in ultrapure water, and autoclaving at 115 deg.C for 15 min.

1.1.1.4.2 nucleic acid electrophoresis solution configuration

(1)50 × TAE electrophoresis buffer stock: 57.1mL of glacial acetic acid, 242g of Tris, 100mL of 0.5mol/L EDTA (8.0) and 1L of ultrapure water;

(2) 1% agarose gel: 1 × TAE 100mL, agarose 1 g;

heating to dissolve, cooling to 65 deg.C, adding 10 μ l Super GelBlue, mixing, and cooling for 30 min.

1.1.1.4.3 Heat shock conversion solution:

0.1mol/L CaCl₂sterilizing the solution at 121 deg.C for 20 min; 0.1mol/L CaCl₂(containing 15% glycerol), sterilizing at 121 deg.C for 20 min.

1.1.1.4.4 electric shock conversion solution preparation

(1) And (3) configuring a shock buffer solution: 34.21g of sucrose, MgCl₂0.029g, 100mL of ultrapure water, adjusting the pH value to 7.4, sterilizing with high-pressure steam at 121 ℃ for 20min, and storing at-20 ℃.

(2) Washing buffer solution preparation: na (Na)₃PO₄ 0.19g，MgCl₂0.009g, 100mL of ultrapure water, adjusting pH to 7.4, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at-20 deg.C.

(3)0.5mol/L sucrose preparation: sucrose 17.1g, ultrapure water 100mL, 121 ℃ high pressure steam sterilization for 20min, 4 ℃ storage.

(4) 2% glycine configuration: glycine 2g, ultrapure water 100mL, in sterile environment operation, dissolved using 0.22um sterilization filter sterilization.

1.1.1.4.5Western blotting electrophoresis solution preparation

(1) Preparation of 5 XSDS-PAGE electrophoresis buffer: 15.1g of Tris alkali, 600mL of ultrapure water, 94.0g of glycine, 50mL of 10% SDS and 350mL of ultrapure water;

(2)10 times of the formula of the film transfer liquid: 15.1g of Tris, 72.0g of glycine, 1.85g of SDS and 400mL of ultrapure water, stirring until the Tris is completely dissolved, and then fixing the volume to 500 mL.

(3)10 × TBS solution: 236g of NaCl, 72.6g of Tris and 2L of ultrapure water; stirring until completely dissolved, diluting to 3L, and adjusting pH to 7.6 with concentrated hydrochloric acid.

(4) TBST solution: 10 × TBS 100mL, Tween 201 mL and ultrapure water 1L; stirring to dissolve completely, and storing at 4 deg.C.

(5) Blocking buffer: 5g of skimmed milk powder and 100mL of TBST, stirring until the skimmed milk powder is completely dissolved, and storing at 4 ℃.

1.1.1.4.6 preparation of antibiotics

(1) Preparation of 10mg/mL kanamycin: glycerol 1g and ultrapure water 100mL were subjected to aseptic processing, dissolved, sterilized with a 0.22um sterilizing filter, and stored at-20 ℃ in a 1.5mL sterile EP tube.

(2) Preparation of 20mg/mL erythromycin: 2g of glycerol and 100mL of absolute ethanol, and the mixture is sterilized by a 0.22um sterilizing filter after being dissolved in a sterile environment and stored at-20 ℃ in a 1.5mL sterile EP tube.

1.1.1.4.7 preparation of the rest solution

(1) Preparation of 80% glycerol: 80mL of glycerol and 20mL of ultrapure water, wherein the sterilization temperature is 121 ℃, and the sterilization time is 20 min.

(2) Preparation of PBS phosphate buffer: 1 bag of PBS phosphate buffer solution powder, 2L of ultrapure water, and the sterilization temperature of 121 ℃ and the sterilization time of 20 min.

(3) Preparation of 200mg/mL IPTG: IPTG 2g and ultrapure water 8mL, after complete dissolution, the volume was adjusted to 10mL with ultrapure water, sterilized with a 0.22um sterilizing filter, and then dispensed into 1.5EP tubes at 1 mL/tube, and stored at-20 ℃.

1.1.2 test methods

1.1.2.1 construction and identification of recombinant plant lactic acid bacteria

1.1.2.1.1 Synthesis of target Gene

The target gene is as follows: P54-Fc-P30-EGP nucleotide sequence (SEQ ID NO.1)

1.1.2.1.2 primer design

In order to identify the correctness of the recombinant plasmid, universal primers aiming at the pSIP409-pgsA '-Erm vector are designed, and a gene fragment between Xba I and Hind III after a pgsA' sequence can be identified for PCR identification and sequencing. The primer sequences are as follows:

F 5’-AGATATTGTTGGTGCTGG-3’

R 5’-TCAATCAAAGCAACACG-3’

1.1.2.1.3 vector and acquisition of target fragment

Production of Coli DH 5. alpha. competence 1.1.2.1.3.1E

a. Adding 0.1mol/L CaCl₂Solution, 0.1mol/L CaCl containing 15% glycerol₂Placing the solution in a refrigerator for precooling at 4 ℃, and precooling a 1.5mL centrifuge tube in the refrigerator at-80 ℃;

b. activating and recovering escherichia coli DH5 alpha, and carrying out shake aerobic culture at 200rpm and 37 ℃ for 12 h;

c. streaking, inoculating and culturing for 16h on an LB solid culture medium by using an inoculating loop;

d. a single colony of DH5 alpha is selected and inoculated in 5mL liquid LB culture medium to be cultured for 12h at 37 ℃ and 200 rpm;

e. inoculating 1mL of the bacterial solution into 100mL of liquid LB medium, culturing at 37 ℃ and 180rpm to OD₆₀₀To 0.4-0.5;

f. transferring the bacterial liquid into a 50mL sterilized centrifugal tube, and carrying out ice bath for 15 min;

g.4 ℃, centrifuging for 10min at 4000rpm, and discarding the supernatant; using 15mL of 0.1mol/L CaCl₂The solution is gently floated and mixed with thalli, and ice bath is carried out for 10 min;

centrifuging at h.4 deg.C and 4000rpm for 10min, and removing supernatant; using 15mL of 0.1mol/L CaCl₂The solution is gently suspended and mixed with thalli evenly, and ice bath is carried out for 30 min;

centrifuging at i.4 deg.C and 4000rpm for 10min, and removing supernatant; 2mL of 0.1mol/L CaCl containing 15% of glycerol₂Suspending the solution, mixing thallus uniformly, and performing ice bath for 30 min;

j.100 μ L/tube, subpackaging in sterilized 1.5mL centrifuge tube precooled at-80 deg.C, quick freezing with liquid nitrogen, and storing at-80 deg.C

1.1.2.1.3.2 plasmid of pUC-GW-Kan vector containing target fragment was transferred to E.coli DH 5. alpha. for amplification and storage

a. pUC-GW-Kan-PFPP and pUC-GW-Kan-PmFPP plasmid freeze-dried powder (2 mu g) synthesized by the company (GENEWIZ) are centrifuged at 12000rpm for 2min and then diluted by 10 mu L of ultrapure water;

b. taking out DH5 alpha from a refrigerator at-80 ℃, placing on ice, adding 1 mu L plasmid when the competent bacteria is in an ice-water mixture state, gently mixing uniformly, and carrying out ice bath for 20 min;

c. performing heat shock in 42 deg.C water bath for 1min 30s, immediately placing back on ice for ice bath for 5 min;

d. adding 600 μ L LB liquid culture medium preheated at 37 deg.C, mixing, and recovering for 1h in shaking culture at 200rpm on a shaker at 37 deg.C;

e.4000rpm for 5min, discarding 600. mu.L of liquid, mixing the thalli precipitates, and plating on LB solid culture medium added with 10. mu.g/mL kanamycin;

f, after the incubator at 37 ℃ is placed upright and absorbed for 1 hour, the incubator is inversely cultured for more than 12 hours;

g. selecting a single colony to be cultured in 5mL LB liquid culture medium added with 10 mug/mL kanamycin for 12 hours;

h. sucking 800 μ L of bacterial liquid and 200 μ L of 80% glycerol, and storing in a refrigerator at-80 deg.C;

i. extracting Plasmid from residual bacteria liquid (detailed method is shown in Vazyme FastSt Plasmid Mini Kit instruction), and storing in a refrigerator at 20 ℃.

1.1.2.1.4 order fragment and vector connection

The laboratory-stored clonal bacteria containing the pSIP409-pgsA' plasmid were cultured in LB liquid medium supplemented with 200. mu.g/mL of erythromycin for 12 h.

Plasmids were extracted (detailed methods see the use instruction of the Vazyme FastPreplasmid Mini Kit).

HindIII and Xho I were used to double-cleave three plasmids, pSIP409-pgsA', pUC-GW-Kan-PFPP and pUC-GW-Kan-PmFPP. The enzyme digestion system is as follows: plasmid 20. mu.L, Xba I0.5. mu.L, Hind III 0.5. mu.L, 10xBufferM 3. mu.L, BSA 3. mu.L, Ultrapure water 3. mu.L, in total 30. mu.L;

after 6h of digestion at 37 ℃, 3. mu.L of 10XLoading Buffer was added and the mixture was electrophoresed on 1% agarose gel 100V nucleic acid for 35 min. Performing gel recovery to obtain linear DNA (gel recovery method is described in Omega

Gel Extraction Kit instructions).

The recovered products of PFPP and PmFPP were ligated to the pSIP 409-pgs' linearized vector by Rapid T4DNA ligase, respectively. According to the carrier: amount ratio of fragment material 1:3 ligation was performed, the vector amount was set to 75ng based on Rapid T4DNA ligase ligation ability, and the dsDNA average molecular weight was calculated as 650 (Dal/bp). According to the 6160bp vector and 2085bp fragment, the fragment mass is about 76 ng.

The pSIP409-pgsA' -PFPP ligation reaction system is as follows: 2 XT 4DNA Ligase Buffer 10. mu.L, Vector (pSIP 409-pgsA') 0.65. mu.L, insert (PFPP) 0.3. mu.L, Rapid T4DNA Ligase 1. mu.L, Ultrapure water 8.05. mu.L, total 20. mu.L;

the pSIP409-pgsA' -PmFPP ligation reaction system is as follows: 2 XT 4DNA ligand Buffer 10. mu.L, Vector (pSIP 409-pgsA') 0.65. mu.L, insert (PmFPP) 0.2. mu.L, Rapid T4DNA ligand 1. mu.L, Ultrapure water 8.15. mu.L, total 20. mu.L; incubating at 25 deg.C for 30min, and storing at-20 deg.C.

1.1.2.1.5pSIP 409-pgsA' empty vector construction

The pSIP409-pgsA' linear DNA vector was obtained by double digestion at 1.1.2.1.4, and Xba I and HindIII restriction endonuclease cleaved sticky ends were converted to blunt ends by DNA end smoothing kit.

The reaction system is as follows:

pSIP409-pgsA' cohesive end smoothing reaction system: t4DNA Polymerase 1. mu.L, Reaction Buffer (5X), 4. mu.L, dNTP mix 0.8. mu.L, pSIP409-pgsA' (linear DNA) 7. mu.L, Ultrapure water 7.2. mu.L, total 20. mu.L; the reaction was stopped by incubation at 25 ℃ for 10min and at 70 ℃ for 15 min.

Linear DNA was self-circularized using a rapid DNA ligation kit. The reaction system is as follows: pSIP409-pgsA' blunt end cyclization reaction system: 2 XT 4DNA Ligase buffer 5. mu.L, Rapid T4DNA Ligase 1. mu.L, pSIP409-pgsA' (linear DNA) 2. mu.L, Ultrapure water 2. mu.L, in total 10. mu.L; incubating at 25 deg.C for 30min, and storing at-20 deg.C.

1.1.2.1.6 Heat shock method ligation products were transformed into E.coli DH5a competent and preserved

a. Taking out DH5 alpha from a refrigerator at minus 80 ℃, placing on ice, adding 10 mu L of the ligation product when the allelochemicals are in an ice-water mixture state, gently mixing uniformly, and carrying out ice bath for 20 min;

b. heating in 42 deg.C water bath for 1min 30s, immediately returning to ice, and ice-cooling for 5 min;

c. adding 600 μ L LB liquid culture medium preheated at 37 deg.C, mixing, and recovering for 1h in shaking culture at 200rpm on a shaker at 37 deg.C;

e.4000rpm for 5min, discarding 600. mu.L of liquid, mixing the thalli precipitates, and plating on LB solid culture medium added with 200. mu.g/mL of erythromycin;

g. selecting a single colony to be cultured in 5mL LB liquid culture medium added with 200 mug/mL erythromycin for 12 h;

Identification of recombinant plasmid in 1.1.2.1.7 E.coli DH5a

1.1.2.1.7.1 enzyme digestion identification

The restriction enzyme identification system of pSIP409-pgsA '-PFPP and pSIP409-pgsA' -PmFPP is as follows:

enzyme digestion system: 1. mu.L of Plasmid, 0.2. mu.L of XbaI, 0.2. mu.L of Hind III, 1. mu.L of 10xBufferM, 1. mu.L of BSA, 6.6. mu.L of Ultrapure water, and 10. mu.L in total;

the pSIP409-pgsA' self-ligating plasmid restriction identification system is as follows: 2 μ L of Plasmid, 1 μ L of BamHI, 1 μ L of 10xBufferK, 6 μ L of Ultrapure water, 10 μ L in total;

after 4h of metal bath at 37 ℃, 1 μ L of 10XLoading Buffer was added and the correct band size was identified by electrophoresis on a 1% agarose gel at 120V for 25 min.

1.1.2.1.7.2 PCR identification

After the plasmid is diluted to 10 ng/. mu.L, PCR is carried out, and the PCR identification system is as follows: forward primer 1.25. mu.L, Reverse primer 1.25. mu.L, Plasmid 2. mu.L, Prime

Max DNA Polymerase 25. mu.L, Ultrapure water 20.5. mu.L, total 50. mu.L;

the PCR amplification conditions were as follows:

the PCR product was added to 5. mu.L of 10Xloading Buffer, and subjected to 120V nucleic acid electrophoresis on 1% agarose gel for 25min to determine whether the band size was correct.

1.1.2.1.7.3 plasmid sequencing

And (3) after the enzyme digestion and the PCR identification are correct, sending the plasmid to Jilin province, Shumei biotechnology limited for sequencing.

1.1.2.1.8 transformation of recombinant plasmid into NC8

1.1.2.1.8.1 NC8 competent cells

a. Pre-cooling an electric shock buffer solution and a cleaning buffer solution in a refrigerator at 4 ℃, and pre-cooling a 1.5m LEP tube in a refrigerator at-80 ℃;

b. placing NC8 frozen bacteria in 5mL MRS liquid culture medium, performing anaerobic activation recovery culture at 37 ℃ for 12 h;

c. using a bacterium inoculation ring to dip bacterium liquid, scribing on an MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 16-18 h;

d. selecting NC8 single colony, inoculating in 5mL MRS liquid culture medium, adding 100 μ L2% Gly 37 deg.C, and anaerobically culturing for 7-8 h;

e. inoculating 2mL of bacterial liquid into 100mL of MRS liquid culture medium, adding 2mL of 2% Gly at 37 ℃, and performing anaerobic subculture until OD is reached₆₀₀To 0.2-0.3;

f. transferring the bacterial liquid into two 50mL centrifuge tubes, carrying out ice bath for 10min, centrifuging at 4 ℃ and 5000rpm for 10min, and removing the supernatant;

g. resuspending the precipitate in 10mL precooled washing buffer solution, centrifuging at 4 ℃ and 5000rpm for 10min, and discarding the supernatant;

h. repeating the steps, then re-suspending the precipitate by using a pre-cooled cleaning buffer solution, centrifuging at 4 ℃ and 5000rpm for 10min, and removing a supernatant;

i. the thalli are suspended in 1mL of electric shock buffer solution in two tubes respectively, ice bath is carried out for 10min, 100 mu L/tube is subpackaged into 1.5mL of EP tube, and the thalli are stored in a refrigerator at minus 80 ℃ after being frozen by liquid nitrogen.

1.1.2.1.8.2 transformation and preservation of recombinant plasmid

a. Soaking a 2mm electric shock cup in 75% alcohol overnight;

b. completely drying under an ultraviolet lamp, and precooling on ice;

c. taking out NC8 competent bacteria from a refrigerator at-80 deg.C, placing on ice, adding 1 μ L plasmid when the competent bacteria is in ice-water mixture state, mixing, transferring into an electric shock cup, and ice-cooling for 10 min;

d. the conditions of the electroporation system are set as that the output voltage is 2000V, the capacitance is 25 muF, the sample resistance is 400 omega, and the electrode spacing of the electric shock cup is 2 mm; standing at room temperature for 3min after electric shock is finished;

e. adding 600 mu L of 30 ℃ preheated MRS liquid culture medium and 150 mu L of 0.5mol/L sucrose, uniformly mixing, transferring to a 1.5mL centrifuge tube, and recovering for 3h by 30 ℃ water bath culture;

centrifuging at 4000rpm for 5min, discarding 600 μ L of liquid, mixing thallus precipitates, plating on MRS solid culture medium containing erythromycin at 10 μ g/mL, performing anaerobic culture at 37 deg.C, absorbing at normal position for 1 hr, and performing inverted culture for 17 hr;

g. selecting a single colony, and anaerobically culturing the single colony in 5mL of mrs liquid culture medium added with 10 mu g/mL of erythromycin at 37 ℃ for 12 h;

i. extracting Plasmid from the residual bacterial liquid, adding 180 muL of 50 mug/muL lysozyme after the suspended bacteria are precipitated, carrying out water bath at 37 ℃ for 1h, operating the rest steps according to the use instruction of Vazyme FastPure Plasmid Mini Kit, and storing the Plasmid in a refrigerator at-20 DEG C

1.1.2.1.9NC8 recombinant plasmid validation

And carrying out PCR and enzyme digestion identification on the extracted recombinant plant lactobacillus plasmid. And amplifying the target fragment of the recombinant plasmid in NC8 by using a PCR (polymerase chain reaction) technology, thereby identifying whether the recombinant plasmid is successfully transformed into NC 8. The identification system and amplification procedure are 1.1.2.1.7.

1.1.2.2 preparation of polyclonal antibodies P30 and P54

1.1.2.2.1 construction of P30 and P54 prokaryotic expression vector

1.1.2.2.1.1 primer design

In order to obtain P54 and P30 single-antigen linear DNA P30a and P54a with pET28a homologous arms, an upstream primer and a downstream primer are designed for PCR amplification, and the sequences of the primers are as follows:

P54a F：

5-AAGAAGGAGATATACCATGGATGGATAGTGAATTTTTTCAACCG-3

P54a R：

5-TGGTGGTGGTGGTGCTCGAGCAGACTATTTTCCAGATCTTTATGCG-3

P30a F：

5-AAGAAGGAGATATACCATGGATGGATTTTATTCTGAATATTAGTATGAA-3

P30a R：

5-TGGTGGTGGTGGTGCTCGAGAATATACGTCAGAAAAAAGTTAATTTTC-3

1.1.2.2.1.2 obtaining P30a and P54a linear DNA

After pUC-GW-kan-PFPP plasmid is diluted to 10 ng/. mu.L respectively, PCR is carried out, and the PCR amplification system is as follows: forward primer 1.25. mu.L, Reverse primer 1.25. mu.L, Plasmid 2. mu.L, Prime

Max DNA Polymerase 25. mu.L, Ultrapure water 20.5. mu.L, total 50. mu.L;

the PCR amplification conditions were as follows:

adding 5. mu.L 10XLoading Buffer into the PCR product, performing gel recovery on the PCR product for 35min in 1% agarose gel 100V nucleic acid electrophoresis (see Omega for gel recovery method for details) to obtain P30a and P54a linear DNAs

Gel Extraction Kit instructions).

1.1.2.2.1.3 construction of prokaryotic expression plasmids pET28a-P30a and pET28a-P54a

The pET28a vector was linearized using Xho I and Nco I as follows: plasmid 10. mu.L, Xho I1.5. mu.L, Nco I1.5. mu.L, 10xBufferK 2. mu.L, Ultrapure water 5. mu.L, total 20. mu.L;

after 6h of digestion at 37 ℃, 2. mu.L of 10Xloading Buffer was added and the mixture was electrophoresed on a 1% agarose gel at 100V for 35 min. Performing gel recovery to obtain linear DNA (gel recovery method is described in Omega

Gel Extraction Kit instructions).

According to the carrier: amount ratio of fragment material 1:3 ligation was performed, the vector amount was set to 60ng based on Seamless Master Mix ligation capacity, and the dsDNA average molecular weight was calculated as 650 (Dal/bp). According to the vector pET28a 5369bp, the fragment P30a is 643bp, and the fragment P54a is 592bp, the mass of the fragment P30a is about 18ng, and the mass of the fragment P54a is about 16.6 ng. Using a seamless cloning kit for ligation, the pET28a-P30a ligation system was as follows: vector (pET28a) 3.75. mu.L, Insert (P30a) 1. mu.L, 2XSeamless Master Mix 5. mu.L, Ultrapure water 0.25. mu.L, 10. mu.L in total;

the pET28a-P54a connection system is as follows: vector (pET28a) 3.75. mu.L, Insert (P30a) 0.7. mu.L, 2XSeamless Master Mix 5. mu.L, Ultrapure water 0.55. mu.L, 10. mu.L in total; ligation was performed at 50 ℃ for 20 min.

1.1.2.2.1.4 prokaryotic expression plasmids pET28a-P30a and pET28a-P54a are transferred into DH5 alpha for amplification and preservation

The same step is 1.1.2.1.3.2

Identification of pET28a-P30a and pET28a-P54a recombinant plasmids in 1.1.2.2.1.5E.coli DH5a

The enzyme digestion identification system is as follows: plasmid 1. mu.L, Xho I0.5. mu.L, Nco I0.5. mu.L, 10xBufferK 1. mu.L, Ultrapure water 7. mu.L, total 10. mu.L;

1.1.2.2.1.6 prokaryotic expression plasmids pET28a-P30a and pET28a-P54a are transferred into BL21 competent plasmid

The same step is 1.1.2.1.3.2

1.1.2.2.2. Gel-cutting purified protein

a. Inoculating BL21-pET28a-P30a and BL21-pET28a-P54a to a liquid LB culture medium containing 10 ug/mL kanamycin, and culturing at 37 ℃ and 200rpm for 8 h;

b. transferring 10mL of the bacterial solution to 1L of liquid LB medium containing 10. mu.g/mL of kanamycin, culturing at 37 ℃ and 200rpm to OD₆₀₀To 0.6-0.8;

c. adding 250 mu L of 200mg/mL IPTG, and inducing at 37 ℃ and 200rpm for 4 h;

d. transferring the bacterial liquid into a plurality of 50mL centrifuge tubes for several times, centrifuging for 10min at 4 ℃ and 1000rpm, suspending and uniformly mixing thalli precipitates by PBS, and collecting the thalli together;

e. repeatedly freezing and thawing for 3 times by using liquid nitrogen;

carrying out ultrasonic crushing at 200W, wherein the ultrasonic program is 5s of ultrasonic treatment in every 10s, stopping for 5s and lasting for 10 min;

g.4 ℃, centrifuging at 10000rpm for 20min and discarding the supernatant;

h. suspending, uniformly mixing and precipitating with PBS containing 2M urea, centrifuging at 4 ℃ and 10000rpm for 5min, and discarding the supernatant;

i. suspending, mixing uniformly and precipitating with PBS containing 4M urea, centrifuging at 4 ℃ and 10000rpm for 5min, and removing supernatant;

j. suspending, mixing uniformly and precipitating with PBS containing 8M urea, and soaking on a shaking table at 4 ℃ for 7 h;

k. filtering with 0.22um sterilizing filter, centrifuging at 4 deg.C and 10000rpm for 20min, adding 800 μ L into 200 μ L5 xSDS PAGE Buffer, clamping with explosion-proof clamp, and decocting in boiling water for 10 min;

carrying out protein electrophoresis on a sample of 80V until bromophenol blue moves to the lower end of an electrophoresis tank;

m, determining the position of a protein strip by using 0.25M KCl treatment, cutting glue, mashing, and soaking for 8 hours by using PBS;

centrifuging at n.4 deg.C at 10000rpm for 20min, sucking supernatant, preparing 96-well plate, determining protein concentration with BCA protein concentration determination kit (enhanced type), supplementing volume with PBS, and performing other specific operation steps according to product instruction;

after incubation at o.37 ℃ for 30min, absorbance measurements were performed using an Epoch 2 microplate spectrophotometer and protein concentrations were calculated from standard curves using an ELISACalc regression/fitting procedure.

1.1.2.2.3 preparation of Rabbit-derived antibody

a. One rabbit was purchased at 6 weeks of age and observed for 3 days to adapt to environmental changes.

b. The purified antigen was combined with adjuvant and the first immunization dose was 200 μ g antigen, i.e., 1mL purified antigen plus 1mL complete Freund's adjuvant. A second immunization was performed 10 days apart at an immunization dose of 1mL of purified antigen plus 1mL of incomplete Freund's adjuvant. A third immunization was performed 10 days later at an immunization dose of 1mL of purified antigen plus 1mL of incomplete Freund's adjuvant.

c. Ten days after the third immunization, the rabbits were subjected to respiratory anesthesia, blood was collected from the heart, and centrifuged to obtain serum.

1.1.2.3 recombinant plant lactobacillus protein expression identification

1.1.2.3.1 treatment of vegetable lactic acid bacteria protein-like

There are two methods for the treatment of vegetable lactic acid bacteria protein-like, as follows:

ultrasonic crushing method

a. Activating recombinant plant lactic acid bacteria stored in a refrigerator at the temperature of-80 ℃, and inoculating the activated recombinant plant lactic acid bacteria to 5mL of MRS liquid culture medium and culturing overnight at 37 ℃ in an anaerobic workstation;

b. the next day, 1: 40 transfer to a 40mL centrifuge tube, anaerobic culture at 30 ℃ for 3h (OD)₆₀₀At about 0.3), adding SppIP induction peptide (12.5 mu L of each 5mL of MRS liquid culture medium), and carrying out induction culture until logarithmic growth phase;

c, centrifuging at 5000rpm for 10min, reserving 200 mu L of the solution, and adding PBS for suspension;

d. ultrasonically crushing for 10min, centrifuging again, and removing supernatant;

e. adding 1mL of PBS into the precipitate for suspension, carrying out 12000rpm, centrifuging for 1min, and extracting the supernatant for later use;

f. add 300. mu.L of ddH to the pellet₂O, then adding 100 mu L of 5xSDS PAGE Buffer and mixing evenly;

g. boiling the tube on an explosion-proof clamp in a boiling water bath at 100 ℃ for 10min, then centrifuging at 12000rpm for 1min, sampling with the supernatant, and storing the sample at-20 ℃.

(II) repeated freezing and thawing method

b. the next day, 1: 40 transferred into a 40mL centrifuge tube, and anaerobically cultured for 3h (OD) at 30 DEG C₆₀₀At about 0.3), adding SppIP induction state (12.5 mu L of each 5mL of MRS liquid culture medium), and performing induction culture until logarithmic growth phase;

c. centrifuging 2mL of bacterial liquid at 5000rpm for 5min, and removing a supernatant;

d. adding 1mL TES solution into the precipitate, suspending, and carrying out water bath at 37 ℃ for 30 min;

e. centrifuging after water bath, centrifuging for 10min at 2500g, and suspending the precipitate with 1mL of PBS;

f. then centrifuged at 2500g for 5min and treated with 500. mu.L ddH₂Resuspending, and repeatedly freezing and thawing in a refrigerator at-80 deg.C for 5 times;

centrifuging at 18000g at 4 deg.C for more than 2 times and 40 min;

h. adding 150. mu.L PBS and 50. mu.L 5xSDS PAGE Buffer to the precipitate and mixing;

i. boiling the tube on an explosion-proof clamp in a boiling water bath at 100 ℃ for 10min, then centrifuging at 12000rpm for 1min, sampling with the supernatant, and storing the sample at-20 ℃.

1.1.2.3.2 Western blot technique

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 plant lactobacillus sample is incubated to verify whether the antibody is successfully prepared. The method comprises the following steps: a. firstly, the glass plate for making glue is put in, and ddH is used₂Checking leakage for 20min, preferably no water surface drop; b. preparing separation glue (lower layer glue), mixing, adding into a space between glass plates, sealing with water to make the liquid level horizontal, standing for 20min to solidify the glue; c. preparing concentrated glue (upper layer glue), pouring water between glass plates, sucking with filter paper, slightly adding concentrated glue, inserting into a comb to avoid bubbles, standing for 20min to solidify the glue; d. after the upper layer glue is completely polymerized, carefully pulling out the comb, fixing the glass plate in an electrophoresis tank, and adding a1 xSDS solution to submerge the glue plate; e. sample adding: adding 10 mu L of the processed sample and a Marker into the glue hole; f. electrophoresis: connecting a power supply, wherein the voltage of the upper layer gel is 80V, the voltage of the lower layer gel is 120V, and the electrophoresis is stopped when the bromophenol blue moves to the lower end of the electrophoresis tank; g. slightly prying the glue-making glass plate, taking out the glue plate, removing the concentrated glue, trimming the glue by using a cutting knife, and soaking in a film transfer liquid; h. film transfer: the following materials were placed in order: black surface → sponge → filter paper → glue → NC membrane → filter paper → sponge, drive out the bubble, put into rotating the membrane trough, the black surface is to the black surface, add and rotate the membrane liquid, rotate the membrane on the ice; i. installing an electrode, and rotating for 1.5 hours at a constant current of 200 mA; j. and (3) sealing: adding 5% skimmed milk powder and 100ml TBST, sealing for 1 hr; k. incubating the primary antibody: the blocking solution was recovered, primary antibody (diluted with 5% BSA and TBST) was added and placed on a shaker at 4 ℃ overnight; recovering primary antibody, adding TBST, and washing the membrane on a shaking table for 3 times, each time for 10 min; m. incubation secondary antibody: adding a second antibodyDiluting with 5% skimmed milk powder and TBST), and shaking for 1h at normal temperature; n, recovering primary antibody, adding TBST, and washing the membrane on a shaking table for 3 times, 10min each time; and o, placing the film in an imaging system, and dripping the luminous liquid for exposure.

1.1.2.4 optimization of culture condition of lactobacillus plantarum

1.1.2.4.1 determination of growth curve of recombinant plant lactobacillus

a. NC8-pSIP409-pgsA ', NC8-pSIP409-pgsA ' -PFPP and NC8-pSIP409-pgsA ' -PmFPP were streaked onto plates supplemented with 200. mu.g/mL erythromycin, respectively, and cultured anaerobically at 37 ℃ for 16 h; selecting single colonies with medium size to inoculate in 5mL of MRS liquid culture medium containing 200 mug/mL of erythromycin for activation culture for 12 hours;

b. 1mL of activated NC8 bacterial liquid is inoculated into 50mL of MRS culture medium added with 200 mug/mL of erythromycin; adding plant lactobacillus inducer SppIP 125 μ L after 2 h;

c. 2mL of OD value was measured from each group of the solutions at intervals of 1 hour, and the growth curve was plotted after 12 hours of monitoring.

1.1.2.4.2 colony plate count

The bacterial liquid of 7h and 8h in 1.1.2.4.1 is diluted in gradient to 10⁶，10⁷，10⁸Then, 100. mu.L of the suspension was subjected to plate-drop counting on a solid medium containing 200. mu.g/mL of erythromycin.

1.2 results

1.2.1 construction and identification of recombinant plant lactic acid bacteria

1.2.1.1 Synthesis of the Gene of interest

The PFPP and PmFPP genes are 2085bp

1.2.1.2 vector and acquisition of target fragment

The vector and the target fragment are subjected to double enzyme digestion respectively, and a vector band can be seen at 6160bp (figure 1), and a target fragment band can be seen at 2085bp (figure 2).

Identification of recombinant plasmids in 1.2.1.3E.coli DH5 alpha

1.2.1.3.1 enzyme digestion identification

The recombinant plasmids pSIP409-pgsA '-PFPP and pSIP409-pgsA' -PmFPP were verified by double digestion, and a clear band was visible at 2085bp (FIG. 3).

1.2.1.3.2 plasmid sequencing

Plasmid sequencing was determined by Changchun Kumei corporation, and the sequencing results proved to be 100% correct without any mutation. The plasmid map is as follows (FIG. 4).

1.2.1.4 identification of recombinant plasmids in NC8

The recombinant lactobacillus obtained by the construction method is named as CFWDD-0369, and the characteristics of the recombinant lactobacillus plantarum CFWDD-0369 are as follows: the convex, medium-sized, slightly white edges were neat, conforming to the morphology of lactobacillus plantarum (fig. 5).

1.2.1.4.1 enzyme digestion identification

The recombinant lactobacillus plantarum NC8-pSIP409-pgsA' -PFPP double-enzyme digestion identification is carried out to obtain a vector with the length of 6161bp and a target band with the length of 2085bp, wherein the vector comprises a protective fusion antigen P54-Fc-P30-EGP gene fragment.

1.2.1.4.2 PCR identification

1.2.2 identification of P30 and P54 polyclonal antibodies

1.2.3 optimization of culture conditions for Lactobacillus plantarum

1.2.3.1 growth Curve determination

And (3) carrying out colony growth curve and colony counting determination on the recombinant plant lactobacillus NC8-pSIP409-pgsA' -PFPP, adding an inducer at the concentration of 500ng/mL, and adding the inducer when the recombinant plant lactobacillus is cultured in an MRS culture medium for 2 hours at a 30 ℃ anaerobic workstation, wherein the optimal induction time is 7-8 hours. Growth curve measurements (12h) were made using GraphPad Prism software, see FIG. 6.

1.2.3.2 colony plate count results

1.2.4 expression identification of recombinant plant lactic acid bacteria

1.2.4.2 flow cytometry identification of fusion protein expression

Through Western-blot test detection, the induction conditions can successfully induce the recombinant plant lactobacillus NC8-pSIP409-pgsA '-PFPP surface to contain P54-Fc-P30-EGP antigen, successfully express surface anchoring fusion protein pgsA' -P54-Fc-P30-EGP with the size of about 98KDa, and the results of Westen blot are shown in FIG. 7.

1.2.4.2 flow cytometry identification of fusion protein expression

1.2.4.3 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.

Example 1 test successfully constructed NC8-pSIP409-pgsA '-PFPP and NC8-pSIP409-pgsA' -PmFPP recombinant plant lactic acid bacteria. Furthermore, rabbit derived P30 and P54 polyclonal antibody sera were successfully prepared. The immunogenicity of the fusion protein expressed by the 2 groups of non-antibiotic recombinant lactobacillus plantarum is verified, and the recombinant lactobacillus plantarum fuses the African swine fever virus EGP polypeptide and the P30 protein and connects the P54-Fc fusion protein through a helical structure protein linker and is anchored and expressed on the surface of the lactobacillus plantarum.

Example 2

Immune effect of the recombinant plant lactic acid bacteria constructed in example 1:

the plant lactobacillus vaccine has the advantages of low cost and convenient large-scale production, can be planted in intestinal tracts of organisms for a long time and continuously plays a role, and the plant lactobacillus is probiotics and can improve the immunity of the organisms and promote the mucosal immunity. The oral immunization mode also makes it more convenient. Therefore, the recombinant plant lactobacillus vaccine has wide development prospect. The recombinant plant lactobacillus constructed in the experiment is non-antibiotic plant lactobacillus, is more friendly to the environment and avoids antibiotic tolerance for animals.

Example 2 experiments set up primary and booster immunization programs to induce expression of 2 groups of recombinant plant lactic acid bacteria, and set up control groups 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 plant lactobacillus is evaluated.

2.1 materials and methods

2.1.1 materials

2.1.1.1 test strains

NC8-pSIP409-pgsA ' -PFPP recombinant plant lactic acid bacteria, NC8-pSIP409-pgsA ' -PmFPP recombinant plant lactic acid bacteria and NC8-pSIP409-pgsA ' empty vector plant lactic acid bacteria are constructed by the inventors.

2.1.1.2 test animal

40 SPF grade 6 week old mice were purchased from Beijing Huafukang, Inc.

2.1.1.3 test reagents

Nylon screens were purchased from Solarbio corporation; protease inhibitors (PMSF) and erythrocyte lysates were purchased from Biyuntian biotechnology, Inc.; 200 mesh copper net; 4% paraformaldehyde universal tissue fixative available from biosharp; cell fixation/cell disruption kits were purchased from BD; reagents related to flow cytometry, such as antibodies related to flow cytometry, are stored in laboratories or configured by the inventors.

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 PBS groups, NC8-pSIP409-pgsA ', NC8-pSIP409-pgsA ' -PFPP and NC8-pSIP409-pgsA ' -PmFPP groups for a total of 4 groups of 10 mice each. Animal immunization protocol and groups are shown in table (table 2).

Table 2 animal immunization protocol and groupings

2.1.2.1.2 immunization procedure

Four groups are immunized once on

days

1, 2 and 3, twice on

days

11, 12 and 13, and three times on

days

21, 22 and 23, and feces are collected every three days. Blood was collected on

days

10, 20, and 30 and flow cytometry analysis was performed, and the immunization protocol was as shown in fig. 8.

2.1.2.2 flow experiment

The experimental steps are as follows: the recombinant lactobacillus plantarum is counted firstly, and when the bacterial quantity reaches 1x10⁹And (3) carrying out oral immunization on the mice at the CFU/mL, and ensuring that no overflow exists during each gastric lavage. On

day

10, 3 mice were taken from each group and flow-tested. The method comprises the following steps:

(1) blood sampling and sacrifice are respectively carried out on mice by eyeballs.

(2) In a clean bench, spleen, PP nodule, and mesenteric lymph node MLN of the mice were placed on ice.

(3) The spleens were ground, 1mL of complete medium was added to the copper mesh, ground and pipetted into a 1.5EP tube, centrifuged at 2000rpm in a horizontal centrifuge at 4 ℃ for 5min, and the supernatant was discarded. Wash once with 1mL PBS and discard the supernatant and add 1mL complete medium.

(4) MLN was added to 1mL of complete medium in a copper mesh, ground, pipetted into a 1.5EP tube, centrifuged for 5min at 2000rpm at 4 ℃ in a horizontal centrifuge, washed once with 1mL of PBS and the supernatant discarded and added to 1mL of complete medium.

(5) When the pp knots were ground, 1mL of complete medium was added to the copper mesh, ground and pipetted into a 1.5EP tube, centrifuged at 2000rpm for 5min at 4 ℃ in a horizontal centrifuge, washed once with 1mL of PBS and 1mL of complete medium was added to the supernatant.

(6) Cell counting: on ice, spleen cell stock solution was diluted 100-fold (10. mu.L plus 900. mu.L PBS), MLN cells 20-fold (50. mu.L plus 950. mu.L PBS), PP nodule cells 20-fold (50. mu.L plus 950. mu.L PBS), and counted by a hemocytometer to calculate 1X10⁶How much μ L of stock solution is required for the cells.

(7) Dilution of the antibody: separately to the flow antibody B220⁺IgA、CD11⁺、CD80⁺CD86⁺CD3 CD4 CD8/IFN-y IL-4 were diluted, protected from light, and placed on ice.

(8) PP surface (generation center) ISO single B220⁺IgA，B220⁺(APC, 110x), IgA (FITC, 40x), pp intracellular: adding 10 mu L of B220APC into 100 mu L of sample, washing the sample once with PBS (1% BSA) at 4 ℃ in a dark place for 20min, centrifuging and discarding the supernatant, adding 250 mu L of formaldehyde fixing solution, adding 800 mu L of membrane penetrating solution and mixing the solution (diluted by Waahaha 1: 9), immediately centrifuging the solution, discarding the supernatant, adding 1mL of membrane penetrating solution for the second time, standing the solution at room temperature in a dark place for 5min, centrifuging and discarding the supernatant (not washing with PBS), directly adding IgA + FITC (100 mu L), diluting the solution by 40 times, incubating the solution for 20min at 4 ℃ in a dark place, centrifuging and discarding the supernatant after incubation, washing the solution once with 1mL of PBS, adding 250 mu L of LPBS and mixing the solution. And (5) passing through a film and processing.

(9)CD11c⁺，(APC，40x)、CD80⁺，(FITC，60x)、CD86⁺(PEcy7, 40X), 10. mu.L of each sample + 30. mu.L (three diluted antibodies may be mixed in advance)Together). After washing once with 1mL PBS at 4 ℃ in the dark for 20min, the supernatant was discarded, and 250. mu.L LPBS was added and mixed. And (5) passing through a film and processing.

(10) MLN, Spleen (24-well plate) ISO single-label CD3 CD4 CD8/IFN-y and IL-4, 2x10 are respectively taken⁶Spreading the cells/well in 48-well plate, adding ionomycin (1: 10), PMA (1: 100) and blocker (1: 40), stimulating for 6h, washing the cells in 1.5mL centrifuge tube, adding antibody CD3 CD4 CD8, washing with PBS 1% BSA once at 4 deg.C in dark for 20min, centrifuging, discarding supernatant, adding 250 μ L formaldehyde fixing solution, washing at 4 deg.C in dark for 20min, adding 800 μ L membrane-penetrating solution, mixing (diluting with Onhaha 1: 9), centrifuging immediately, discarding supernatant, adding 1mL membrane-penetrating solution again, standing at room temperature in dark for 5min, centrifuging, discarding supernatant (not washing with PBS), and leaving 100 μ L IgA directly and adding IgA, adding 1 μ L membrane-penetrating solution⁺FITC, (40 fold dilution), protected from light and incubated at 4 ℃ for 20 min. After incubation, the supernatant was centrifuged and washed with 1mL PBS, and the supernatant was discarded and mixed with 250. mu.L LPBS. And (5) passing through a film and processing.

2.1.2.3 ELISA test

2.1.2.3.1 detection of secreted SIgA in feces

Treating collected excrement, adding PMSF protein inhibitor three times the weight of the excrement into the excrement, treating the excrement, centrifuging at 4 ℃ overnight at 3000rpm for 10min, and sucking supernatant for later use. The method for detecting the secretory SIgA in the excrement is as follows:

(1) coating antigen, diluting the purified protein antigen to 3 mug/mL by using a coating buffer solution, sealing in a 96-well plate at 100 mug/mL, and standing at 4 ℃ overnight;

(2) washing the plate, adding 300 μ L of PBST to each well of the 96-well plate, and washing the plate 5 times for 3-5min each time. Lightly shaking and patting dry;

(3) sealing, adding 300 μ L of sealing liquid into each hole, sealing membrane, and sealing at 37 deg.C for 1 hr;

(4) washing the plate, adding 300 mu L of PBST into each hole of a 96-hole plate, washing the plate for 5 times, 3-5min each time, slightly shaking and patting to dry;

(5) adding primary antibody, wherein the primary antibody is the excrement supernatant prepared above, and performing gradient dilution, membrane sealing, 37 ℃ and 1h on the excrement supernatant respectively;

(6) washing the plate, adding 300 μ L of PBST to each well of the 96-well plate, and washing the plate 5 times for 3-5min each time. Lightly shaking and patting dry;

(7) adding enzyme-labeled secondary antibody, using anti-mouse SIgA antibody (1: 100000 dilution), sealing membrane, 37 deg.C, 1 h;

(8) washing the plate, adding 300 mu L of PBST into each hole of a 96-hole plate, washing the plate for 5 times, 3-5min each time, slightly shaking and patting to dry;

(9) developing substrate, adding 100 μ L substrate developing solution into each well, and maintaining at 37 deg.C for 10 min;

(10) stopping the reaction, and adding 50 mu L of planting solution (the stop solution is 2M concentrated sulfuric acid) into each hole;

(11) detecting the result by an enzyme-labeling instrument, detecting the wavelength of 450nm, vibrating the plate for 30s and reading in the enzyme-labeling instrument;

(12) and (4) misjudging the result: if the OD of the hole to be detected is more than 0.1 and is more than 2.1 times of the negative control hole (P/N is more than 2.1) P, the OD value measured at a certain dilution factor of the sample to be detected, N: the sample was negative, and the OD value measured at the corresponding dilution was determined to be positive. And taking the highest dilution factor of the positive samples as the antibody titer.

2.1.2.3.2 detection of IgG in serum

Blood collected from the eyeball is incubated in an incubator at 37 ℃ for 4h, centrifuged at 5000rpm for 10min, and the separated serum is sucked for later use. The measurement of IgG in serum was performed as follows:

(1) coating antigen, diluting the purified protein to 3 mug/mL by using a coating buffer solution, sealing in a 96-well plate at 4 ℃ overnight, wherein the volume of the wells is 100 mug; (2) washing the plate, adding 300 mu L of PBST into each hole of a 98-hole plate, washing the plate for 5 times, 3-5min each time, slightly shaking and patting to dry; (3) sealing, adding 300 μ L of sealing solution into each hole, sealing at 37 deg.C for 1 h; (4) washing the plate, adding 300 mu L of PBST into each hole of the 86-hole plate, washing the plate for 5 times, 3-5min each time, slightly shaking and patting to dry; (5) adding primary antibody, wherein the primary antibody is the serum prepared above, diluting the serum, sealing the membrane, and keeping the temperature at 37 ℃ for 1 h; (6) washing the plate, adding 300 mu L of PBST into each hole of the 86-hole plate, washing the plate for 5 times, 3-5min each time, slightly shaking and patting to dry; (7) adding enzyme-labeled secondary antibody, diluting with anti-mouse IgG antibody (1: 100000), sealing membrane, and keeping at 37 deg.C for 1 hr; (8) washing the plate, adding 300 mu L of PBST into a 86-pore plate, washing the plate for 5 times, each time for 3-5min, slightly shaking, and patting to dry; (9) developing the substrate, adding 100 mu L of substrate developing solution into each hole, and carrying out reaction at 37 ℃ for 1 h; (10) stopping the reaction, and adding 50 mu L of stop solution (the stop solution is 2M concentrated sulfuric acid) into each hole; (11) detecting the result by an enzyme-labeling instrument, detecting the wavelength of 450nm, vibrating the plate for 30s and reading in the enzyme-labeling instrument; (12) and (4) judging the result wrongly: if the OD of the to-be-detected hole is more than 0.1 and is more than 2.1 times (P/N is more than 2.1) of the negative control hole P, the OD value measured at a certain dilution factor, N: the negative sample was judged to be positive by the OD value measured at the corresponding dilution. And taking the highest dilution factor of the positive samples as the antibody titer.

2.1.2.3.3 CCK-8 colorimetric method for detecting T lymphocyte proliferation 96-well plate

(1) Spleen lymph node cells were labeled 5x10⁵Uniformly spreading the mixture in a 96-well plate; (2) a blank control group (ccK 8 was added later) containing only the complete culture medium of PRMI1640 and an experimental group containing lymphocyte suspension were prepared. (three wells with cells only and no stimulus) three replicate wells per set (PBS added to the outermost of 96 well plates to maintain ambient humidity, 87 wells total, media added to the remaining wells); (3) adding 10 μ L ConA or protein with concentration of 10 μ g/mL into each well, mixing, and standing at 37 deg.C with 5% CO₂Culturing for 44h in an incubator; (4) the medium supernatant was carefully removed, 90. mu.L of fresh PRMI1640 complete medium was added, 10. mu.L of cck-8 solution was added and the mixture was continued at 37 ℃ with 5% CO₂Culturing for 4h under the condition; before reading the plate, it can be gently mixed on a shaker. Then measuring the absorbance at 450nm by using a microplate reader; (5) calculating stimulation index SI [ (experiment group A value-blank control group A value)/(PBS control group A value-blank control group A value)]。

2.2 results

2.2.1 flow cytometry results

2.2.1.1 Effect of recombinant plant lactic acid bacteria on cytokines in spleen of immunized mice

To detect the immune response, CD4 in the mouse spleen was flow cytometry for one week after immunization⁺IL-4⁺And CD8⁺IFN-γ⁺The cells were examined. The results show that the PBS group compared with the PmFPP group in CD4⁺IL-4⁺Differential poleIs significant (P)<0.001); CD4 in PBS versus PFPP groups⁺IL-4⁺The difference is extremely remarkable (P)<0.01); NC8 group compared to PmFPP group CD4⁺IL-4⁺The difference is extremely remarkable (P)<0.01); NC8 group CD4 compared to PFPP⁺IL-4⁺Significant difference (P)<0.05); CD8 in PBS versus PmFPP groups⁺IFN-γ⁺The difference is extremely remarkable (P)<0.001); CD8 in PBS versus PFPP groups⁺IFN-γ⁺Significant difference (P)<0.05); NC8 group compared to PmFPP group CD8⁺IFN-γ⁺The difference is extremely remarkable (P)<0.01). The results showed that spleen of mouse could effectively induce cell immune response under the action of recombinant plant lactobacillus, as shown in fig. 9.

2.2.1.2 Effect of recombinant plant lactic acid bacteria on B cells in Peyer's Patchoiei lymph node of immunized mice

To examine the effect of recombinant plant lactic acid bacteria on B cells in PP (propene Polymer) nodules of mice, B220 was examined⁺IgA⁺. The results show B220 in the PBS group and PmFPP group⁺IgA⁺The difference is extremely remarkable (P)<0.01); b220 in PBS group and PFPP group⁺IgA⁺The difference is extremely remarkable (P)<0.01); NC8 group and B220 in PmFPP group⁺IgA⁺Significant difference (P)<0.05); NC8 group and B220 in PFPP group⁺IgA⁺Significant difference (P)<0.05). The results show that recombinant plant lactic acid bacteria efficiently induce B cell activation in the PP nodules of mice, see fig. 10.

2.2.1.3 Effect of recombinant plant lactic acid bacteria on dendritic cell activation of Peyer's patches of mice after immunization

In order to evaluate the influence of the recombinant plant lactic acid bacteria, the recombinant plant lactic acid bacteria are fed to the mice in an oral intragastric administration immunization mode, and after one week of immunization, the expression conditions of the dendritic cell surface activation markers CD80 and CD86 in the Peyer's patches are detected. The results showed that CD80 differed very significantly in the PBS group compared to the PmFPP group (P < 0.0001); CD80 differed significantly between the PBS and PFPP groups (P < 0.01); CD80 was significantly different in NC8 versus PmFPP (P < 0.001); CD80 differed significantly in NC8 group from PFPP group (P < 0.01); CD80 differed significantly in the PmFPP group and PFPP group (P < 0.05); CD86 differed significantly in the PBS group and the PmFPP group (P < 0.05); the CD86 difference between the PBS group and the PFPP group is significant (P <0.05), and the results show that the recombinant lactobacillus plant expressing the African swine fever fusion protein has the activation effect on dendritic cells of the mouse P-type lymph nodes, and the result is shown in figure 11.

2.3 discussion

Mice were subjected to oral gavage immunization experiments using two recombinant plant lactic acid bacteria NC8-pSIP409-pgsA '-PFPP and NC8-pSIP409-pgsA' -PmFPP, and PBS and NC8 groups were compared to verify the immunization effect of the recombinant plant lactic acid bacteria.

2.3.1 flow assay of various immunological indicators after immunization

After one week of immunization, various indexes are detected by using flow cytometry, and the influence of the recombinant plant lactobacillus on the cellular immunity of mice is detected. The results show that both recombinant plant lactic acid bacteria can induce mouse spleen to produce CD4⁺IL-4⁺And CD8⁺IFN-γ⁺The cell shows that the fusion protein expressed by the recombinant plant lactobacillus is used as an antigen for inducing the cellular immunity of an organism, the reaction of T lymphocytes is successfully induced, and possibly, two T cell subsets play a role in protection.

The IgA content in mouse Peyer's patches B cells after the recombinant plant lactobacillus immunization is detected by using flow cytometry, and the result shows that B220 in the recombinant plant lactobacillus⁺IgA⁺The number of the recombinant lactobacillus is remarkably higher than that of the PBS and NC8 groups, which shows that the secretion amount of the antibody is remarkably increased under the stimulation of the recombinant lactobacillus plantarum, and the B lymphocyte reaction is successfully activated.

Dendritic cells, when stimulated by antigen, differentiate to produce CD80⁺And CD86⁺Molecule, therefore this experiment detected CD80 in Peyer's patches dendritic cells⁺And CD86⁺The molecular and the two recombinant plant lactic acid bacteria can stimulate CD80 in Peyer's patches after oral gavage⁺And CD86⁺The activation of the surface marker indicates that both recombinant plant lactic acid bacteria can activate dendritic cells to further exert the antigen uptake and presentation functions of the dendritic cells. Wherein, the immunization effect of the porcine IgG3Fc fragment mutation group NC8-pSIP409-pgsA' -PmFPP is better than that of the non-mutation group NC8-pSIP409-pgsA' -PmFPP, probably because porcine-derived Fc γ cannot bind to murine Fc γ R.

2.4 summary

Example 3

Construction of recombinant lactobacillus plantarum and protein expression verification and purification

1.1 materials

1.1.1 plasmids

An African swine fever virus strain BA71V is taken as a reference sequence, a sequence of a coding P14.5 protein gene (E120R) in the strain is selected, and optimized and synthesized by Nanjing King Muscoy Biotech limited according to a lactobacillus plantarum expression codon. Simultaneously, after two adjuvant sequences are optimized, the two adjuvant sequences are respectively placed at the 5'3' end (CTA1-DD) and the 3' end (IL-33) of the E120R gene, P14.5, CTA1-P14.5-D-D, P14.5-IL-33-Mus sequences are synthesized and then inserted into a single anchored expression vector pLP-S taking erm as a screening marker, and three plasmids pLP-S-P14.5, pLP-S-P14.5-IL-33 and pLP-S-CTA1-P14.5-D-D are obtained.

1.1.2 strains and plasmid vectors

NC8 lactic acid bacteria were stored in the laboratory, and the surface display system of lactic acid bacteria (pLP-S) was stored in the laboratory.

1.1.3 Primary reagents

Restriction enzymes (SalI and XhoI), SppIP were purchased from Bao bioengineering Dalian Co., Ltd; 10 XH buffer, DNA Marker (DL-15000, DL-10000) purchased from Takara Bio Inc.; erythromycin was purchased from Beijing Sorley technologies, Inc.; the plasmid mini-extraction kit box is purchased from Beijing Quanzijin biology company; the electric stun cup was purchased from Bio-Rad; DNA gel recovery kit was purchased from Omega Bio-Tek, USA; the inducer SppIP is stored in a laboratory; the BCA protein concentration assay kit was purchased from Biyuntian Biotech.

1.1.4 preparation of Primary reagents

1) The LB medium formula: 10g of peptone, 5g of yeast and 10g of NaCl;

dissolving in 800mL distilled water, adding to 1L after completely dissolving, subpackaging in test tube or conical flask, and autoclaving at 121 deg.C for 20min for use (liquid). Adding 1.5% agar powder into prepared LB liquid culture medium, and autoclaving at 121 deg.C for 20min for use (solid).

2) MRS culture medium formula: 10g of peptone, 5g of yeast, 10g of glucose, 10g of beef protein powder, 5g of sodium acetate, 2g of diammonium citrate, 2g of dipotassium phosphate, 801 mL of tween-801, 0.2g of magnesium sulfate heptahydrate and 0.05g of manganese sulfate monohydrate;

dissolving the mixture in 800mL of distilled water, supplementing the solution to 1L after complete dissolution, sterilizing the solution for 15min at 115 ℃ by using an autoclave, and adjusting the pH value to 6.2-6.4 (liquid). Adding 1.5% agar powder into the prepared MRS liquid culture medium, and autoclaving at 121 deg.C for 20min for use (solid).

3)50 XTAE electrophoresis buffer (pH8.5) formulation: tris 242g, Na₂EDTA·2H₂O 37.2g；

Note that: dissolving in 1000mL distilled water, and storing at room temperature.

4) 5x SDS electrophoresis buffer formulation: tris 15.1g, Glycine 94g, SDS 5 g; note that: dissolved in 1000ml of distilled water and stored at 4 ℃.

5) TBST formula: 8.8g of NaCl, 20mL of 1M/L Tris-HCL (PH8.0), 800mL of deionized water and 200.5 mL of Tween; note that: dissolved in 1000mL of distilled water and stored at 4 ℃.

6) The formula of the film transfer liquid is as follows: 2.9g of Glycine, 5.8g of Tris, 0.37g of SDS, 200mL of methanol and 600mL of deionized water;

note that: dissolved in 1000ml of distilled water and stored at 4 ℃.

7) PBS buffer: PBS buffer 0.02M, 1800mL of distilled water; note that: sterilizing at 121 deg.C under high pressure, and storing at room temperature.

8) Electrotransformation solution

And (3) electric shock buffer solution: MgCl₂0.029g, sucrose 34.21g, dH₂O80 mL, adjusting the pH of the solution to 7.4 by using dilute hydrochloric acid, adding distilled water to a constant volume of 100mL, sterilizing at 121 ℃ for 20min, and storing at 4 ℃ for later use.

Washing buffer solution: na (Na)₃PO₄ 0.19g、MgCl₂ 0.009g；dH₂Adjusting pH to 7.4 with diluted hydrochloric acid (O80 mL), adding distilled water to 100mL, sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.

9) Protein electrophoresis solution (5 × Running Buffer): 7.575g Tris powder, 35.1g glycine powder, 2.5g SDS powder, ddH₂O is dissolved uniformly, and finally the volume is increased to 500mL and the solution is used at a ratio of 1:5 (v/v).

10) 30% acrylamide solution: acrylamide 87.6g, methylene bisacrylamide 2.4g, add dd H₂Dissolving and uniformly mixing O, metering to 300mL, filling in a brown bottle, and storing in dark.

11)1.5M Tris-Cl, pH 8.8: tris powder 56.013g was added to dd H₂Mixing in O, slowly adding 1M HCl dropwise to adjust the pH of the solution to 8.8, and finally adding dd H₂And O is metered to 300 mL.

12)0.5M Tris-Cl, pH 6.8: tris powder 36.342g was added to dd H₂Mixing in O, slowly adding 1M HCl dropwise to adjust the pH of the solution to 6.8, and finally adding ddH₂And O is added to the volume of 300 mL.

13) 10% APS solution: 0.3g of APS (ammonium persulfate) powder, dd H was added₂Dissolving O, mixing uniformly, metering to 5mL, subpackaging (500 mu L/part) and freezing at-20 ℃ for later use.

14) Alkaline Phosphatase (AP) buffer: NaCl 0.58g, MgCl₂·6H₂O0.11 g, Tris 1.21g, add dd H₂And O, stirring and mixing uniformly, metering to 100mL, and storing at 4 ℃ for later use.

1.2 methods

1.2.1 obtaining sequences of target genes p14.5, IL-33-Mus, CTA1-DD

The nucleotide sequence and amino acid sequence of p14.5 and IL-33-Mus were obtained by NCBI, and the nucleotide sequence and amino acid sequence of CTA1-DD were obtained by literature query.

1.2.2 construction of plasmids

A P14.5, CTA1-P14.5-D-D and P14.5-IL-33-Mus sequence is optimized by Nanjing Kingsler Biotech Co., Ltd according to a lactobacillus plantarum expression codon, and is inserted into a single-anchored expression vector pLP-S taking erm as a screening marker after synthesis to obtain three plasmids pLP-S-P14.5, pLP-S-P14.5-IL-33 and pLP-S-CTA 1-P14.5-D-D.

1.2.3 plasmid double digestion and sequencing identification

(1) The plasmid samples were sent to Jilin province, Kuumei Biotech for sequencing and identification.

(2) The 15. mu.L enzyme digestion identification system is as follows: plasmid 10.5. mu.L, enzyme SalI 1.5. mu.L, enzyme XhoI 1.5. mu.L, Buffer 1.5. mu.L.

1.2.4 gel electrophoresis test of digestion products

Taking 1-10 mu L of enzyme digestion product, mixing with 10 multiplied by Loading Buffer 2 mu L, adding into an agarose gel hole, carrying out electrophoresis test under the condition of 120V/20min, taking off the gel after electrophoresis, putting into a rubber-shooting table, and determining whether the size of the gel is consistent with the expectation through a table type ultraviolet imaging analyzer.

1.2.5 acquisition of Lactobacillus plantarum NC8 competence

(1) Inoculating lactobacillus plantarum NC8 cryopreserved at-80 ℃ into 5mL of MRS liquid culture solution containing Erm (5 mu g/mL), and culturing in an anaerobic workstation; (2) uniformly smearing 0.1mL of cultured bacterial liquid on an MRS solid culture medium containing Erm (5 mu g/mL), and placing the cultured bacterial liquid in an anaerobic workstation for culturing until a single colony grows out (16-18 h); (3) inoculating 5mL of an MRS liquid culture medium (containing 2% Gly) of Erm (5 mu g/mL) to a single colony on a solid plate, and culturing in an anaerobic workstation until the OD600 value of the bacterial liquid is 0.6-0.8; (4) inoculating 50mL of MRS liquid culture medium (containing 2% Gly and 5 μ g/mL Erm) containing the bacteria liquid to culture 1mL of the bacteria liquid, and continuously culturing under anaerobic condition until the OD600 value of the bacteria liquid is 0.3; (5) carrying out ice bath on the collected bacterial liquid for 10min, centrifuging at 8000r/min at 4 ℃ for 5min, and collecting thalli precipitate; (6) resuspending the thallus precipitate with ice-cold 2mL cleaning buffer solution, centrifuging at 8000r/min at 4 deg.C for 10min, washing twice, and collecting thallus precipitate; (7) the thalli sediment is resuspended in ice-cold 200 mu L of electric shock buffer solution, subpackaged with 100 mu L of each tube after ice bath for 10min, and frozen by liquid nitrogen and then placed in a refrigerator at minus 80 ℃ for later use.

1.2.6 electrotransformation of plasmids into Lactobacillus plantarum NC8

(1) Respectively adding 5 mu L of recombinant plasmids pLP-S-P14.5, pLP-S-P14.5-IL-33-Mus and pLP-S-CTA1-P14.5-D-D into 100 mu L of lactobacillus NC8 competence of a three-tube ice bath, gently and uniformly mixing, transferring into an electric shock cup, standing in the ice bath for 5min, and then placing into an electric converter (2.5Kv, 6ms) for electric shock;

(2) after the electric shock is finished, continuing ice-bath for 3-5min, adding 800 mu LMRS culture solution into an electric shock cup, uniformly mixing, sucking all liquid in the electric shock cup into a centrifugal tube of 1.5mL, sealing by a sealing film, and culturing for 2h in an anaerobic workstation;

(3) the ep tube is taken out, 4000rpm is carried out, centrifugation is carried out for 3min, supernatant liquid is discarded, 100 mu L of the supernatant liquid is left to be evenly coated on MRS solid culture medium containing Erm (5 mu g/mL) by using a sterile coating rod, and anaerobic culture is carried out until single colony with proper size grows out, and the time is about 16-20 h.

1.2.7 extraction and identification of recombinant Lactobacillus plantarum plasmids

Inoculating single lactobacillus colony in solid culture medium into 5 mM MRS culture solution containing Erm (5 μ g/mL), culturing under anaerobic condition for 16-20h, and performing plasmid extraction with plasmid miniprep kit (using lysozyme for wall breaking). The extracted plasmid is subjected to double enzyme digestion by SalI/XhoI, and gel electrophoresis is used for identifying whether the plasmid is successfully transferred into lactobacillus NC 8.

1.2.8 determination of growth curve of recombinant Lactobacillus plantarum

a. Respectively streaking NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D on a plate added with EM resistance, culturing for 16h in an anaerobic box at 37 ℃, selecting single colonies with proper size, and selecting the single colonies to be cultured in 5mL MRS test tubes added with EM resistance overnight for activation; b. inoculating activated NC8 bacterial liquid 1:30 into 50mL of a centrifuge tube added with EM resistance; adding lactobacillus inducer SppIP (12.5 muL/5 mL) when the OD values of the three groups of bacteria liquid are all between 0.3 and 0.4; c. 2mL of OD600 values were measured from each group of the culture solutions at intervals of 1h, and the culture was monitored for 12 hours and a growth curve was prepared.

1.2.9 plate count of colonies dropped

Through multiple tests, the OD values of NC8-pLP-S, NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D bacterial solutions when cultured for 12 hours are respectively 2.09, 1.64, 1.42 and 1.59.

Respective gradient dilution

10⁶，10⁷，10⁸Then, 100. mu.L of the resulting suspension was counted and found to be 5.5X 10 in 0.1mL of NC8-pLP-S-p14.5⁹The number of individual colonies; 0.5mL of NC8-pLP-S-p14.5 had a 1X10⁹The number of individual colonies; 0.8mL of NC8-pLP-S-p14.5-IL-33-Mus had a 1X10⁹The number of individual colonies; 1.1mL of NC8-pLP-S-CTA1-p14.5-D had a 1X10⁹The number of colonies (the optimal number of the mice fed with Lactobacillus is 0.5X 10)⁹-1×10⁹)

1.2.10 Westen blot assay for recombinant Lactobacillus plantarum

(1) Protein sample processing

a. Taking the preserved bacterial liquid NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D out of a refrigerator at the temperature of-80 ℃, inoculating the liquid in 5mL of MRS liquid containing Erm (5 mu g/mL) and culturing the liquid overnight; b. transferring to 50mL of MRS liquid added with Erm (5 mu g/mL) on the next day, culturing for 1h at 37 ℃ in an anaerobic workstation, respectively adding SppIP 125 mu L (50mg/mL), inducing and culturing for 6h, centrifuging, washing the precipitate for 3 times with PBS, and respectively re-suspending with 5mL of PBS; c. the samples were sonicated for 20min at 60W and the bacterial supernatants and pellets were collected separately in Ep tubes.

(2)Western blot

a. Firstly, preparing gel; NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D, respectively taking 10 mu L of bacterial liquid, adding 5 mu L of 5 Xloading buffer solution, and boiling in 100 ℃ boiling water for 10 min; c. adding the sample into a rubber groove, running the upper layer rubber for 45min at 80V, and running the lower layer rubber for 30min at 150V; d. soaking membrane-transferring filter paper in a membrane-transferring solution in advance, taking out gel, cutting off an NC membrane with proper size according to the required size, paying attention to the neat edge, soaking in methanol for 30s, activating, and soaking in a transfer buffer solution; e. placing sponges, filter paper, NC membranes, gel, filter paper and sponges in a transfer printing clamp in sequence from top to bottom in a film transfer solution, paying attention to the fact that the white side is on the top and the black side is on the bottom, removing bubbles generated between layers by using a small roller, connecting one side of the NC membrane with a positive electrode and one side of the gel with a negative electrode, keeping the current constant at 300mA/1h, and performing the whole film transfer process on ice; f. and (3) sealing: the NC membrane was removed and washed once quickly with 1 XPBS in order to wash off excess electrotransfer solution. Adding PBST solution, and sealing at room temperature for 1 h; g. antibody incubation: the blocking solution was discarded, 1 XPBST was washed twice, 1 XPPBS was washed once, 5 min/time, and the excess blocking solution was washed away. Adding into appropriate amount of antibody diluent, and incubating overnight at 4 deg.C on a shaking bed; ect color development: uniformly mixing the solution A and the solution B of the ECT color development kit according to a ratio of 1:1, uniformly coating the mixture on an NC membrane, putting the NC membrane into an AI 600 instrument for color development, and obtaining an image result.

1.2.11 construction of recombinant Escherichia coli BL21-Pet-28a-p14.5

(1) Primer design

In order to obtain a p14.5 gene fragment with enzyme cutting sites Nco I and Xho I, an upstream and downstream primer is designed for PCR amplification, and the Nco I enzyme cutting site is added at the upstream 5' end of the ASFV p514.5 sequence.

F：5'CATGCCATGGGTCGACATGGCTGATTTTAATTCACCC 3'

R：5'CTCGAGGTGGTGATGGTGGTGGTG 3'

(2) PCR amplification

The PCR amplification system is as follows: plasmid (P14.5-pLP-S) 1. mu.L, PrimeSTAR Max 25. mu.L, P14.5F 2. mu.L, P14.5R 2. mu.L, ddH₂O20. mu.L, 50. mu.L in total;

the PCR reaction procedure is shown in Table 3

TABLE 3

Step 2-4 repeat 35 cycles;

(3) glue recovery

The amplified fragment was 402bp in size and recovered from gel.

(4) Ligation of Peasy-Blunt-zero, transformation into T1 competence

a. Mixing the gel recovery product with 4:1 carrier, namely 4 microliter PCR product and 1 microliter carrier, and connecting by using T4 ligase at 16 ℃; b. carefully adding the ligation product into T1 competence (dissolved on ice), flicking, mixing, and standing on ice for 30 min; c, thermally shocking the mixture for 90 seconds in a water bath kettle at 42 ℃, and standing the mixture on ice for 5 min; d. adding 300 μ L of liquid LB balanced to room temperature into T1 competence, and shake culturing at 37 deg.C for 1 h; e. taking out the cultured bacterial liquid, centrifuging at 4000rpm for 5min, removing the supernatant, leaving 100 μ L suspended thallus precipitate, uniformly coating on an LB plate added with Kana resistance, and inversely placing the plate in a 37 ℃ incubator for culture;

(5) selecting bacteria to identify plasmid, enzyme cutting to recover target fragment

a. Selecting a single colony from the plate, adding the single colony into a Kana resistant liquid LB test tube for overnight culture, and extracting plasmids for double enzyme digestion identification and sequencing identification;

b. storing the target fragments recovered after double enzyme digestion in a refrigerator at the temperature of-20 ℃;

(6) the target fragment was ligated to pET28a expression vector and transformed into BL21(DE3) competent

mu.L of the target fragment (gel recovery product) was ligated with 0.5. mu.L of pET-28a expression vector in a 16 ℃ water bath overnight, the ligation product was transferred into BL21(DE3) competence and plated on solid LB plate with Kana resistance (50 mg/mL); selecting single bacterial colony with proper size on the plate, selecting bacteria to culture in liquid LB, extracting plasmid, double enzyme digestion determination, and sequencing plasmid.

1.2.12 purification of p14.5 protein

a. Inoculating BL21-pET-28a-p14.5, taking out the preserved bacteria from-80 ℃ and culturing in 5mL of Kana resistant LB culture medium for 8 h; b. transferring, adding 10mL of a bacterial liquid in a into 800mLkana resistant LB liquid culture medium, and culturing until od600 is 0.6-0.8; c. inducing, namely adding 250 mu L of IPTG (200mg/mL) inducer into the bacterial liquid to induce and express for 4 h; d. centrifuging, centrifuging the induced bacterium liquid at 1000rpm for 10min, and resuspending the thallus precipitate by 5mL of PBS; e. centrifuging at 10000rpm at 4 deg.C for 20min after ultrasonication, and collecting precipitate (collecting supernatant for detection); f. washing once with 2M urea PBS, once with 4M urea PBS, once with 8M urea PBS, filtering with 0.22 μ M sterilizing filter;

g. centrifuging to obtain supernatant, and decocting in boiling water for 10 min;

h. running a western machine, treating the glue with 0.25M Kcl, cutting the glue into pieces, and soaking the pieces in PBS for 7-8 h; centrifuging and sucking the supernatant.

1.3 results

1. The nucleotide sequences of plasmids pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p14.5-D-D are respectively shown in SEQ ID NO. 3-5;

2. the amino acid sequences of plasmids pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p14.5-D-D are respectively shown in SEQ ID NO. 6-8;

3. identification of plasmids pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p14.5-D-D

The synthesized plasmid was digested with SalI and XhoI to obtain the vector and the desired fragment, respectively, as shown in FIGS. 12, 13 and 14.

4. Plasmid map

The successfully constructed NC8-pLP-S-p14.5 is shown in FIG. 15, NC8-pLP-S-p14.5-IL-33-Mus is shown in FIG. 16, and NC8-pLP-S-CTA1-p14.5-D-D is shown in FIG. 17.

P14.5 amplification of PCR results

The PCR amplification product of the P14.5 gene is verified by nucleic acid electrophoresis, and the size of the amplified gene fragment is 402, as shown in FIG. 18;

6. successfully constructed enzyme digestion identification result of BL21-pET-28a-p14.5

Carrying out double enzyme digestion on the constructed BL21-pET-28a-p14.5 plasmid by using Xho I and Nco I, wherein the result is consistent with the expectation; as shown in fig. 19.

7 growth Curve determination and optimal dilution multiple colony count

Measurement results of growth curves of NC8-pLP-S, NC8-pLP-S-P14.5, NC8-pLP-S-P14.5-IL-33-Mus and NC8-pLP-S-CTA1-P14.5-D-D (12h) and multiple bacterial drop plate counting measurement results 12h is selected as the optimal feeding time point (i.e. 8h after adding an inducer SppIP). The strain growth curves are shown in FIG. 20.

Example 4

Animal experiments with recombinant Lactobacillus plantarum

In example 1, three strains of recombinant lactobacillus plantarum have been successfully constructed and anchoring expression in vitro is successful, in this example, mouse C57BL/6 is selected as an experimental animal to perform preliminary animal experiments, the mice are immunized by oral feeding, and changes in humoral immunity, cellular immunity, mucosal immunity and the like of the immunized mice are detected by flow cytometry, lymphocyte proliferation tests, ELISA and the like, so as to study the immunity enhancing effect of the novel functional lactobacillus on the immunized animals.

2.1 materials

2.1.1 strains

NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus, NC8-pLP-S-CTA1-p14.5-D-D were obtained from example 1;

2.1.2 Experimental animals

SPF rated female C57BL/6 mice 50 at 5-6 weeks of age were purchased only from Beijing Huafukang Biotech GmbH; the breeding is carried out in an SPF-level breeding room of an innovative building of Jilin agriculture university, and the test is carried out after the breeding room is adapted to the environment for 1 week.

2.1.3 Primary reagents

Mouse ear tag, scissors, tweezers, 1.5mL EP tube, gavage needle small-size, eyeball blood collection tube, PBS, 24-hole cell culture plate, 96-hole enzyme-linked immunosorbent assay (ELISA) detection plate, and cell 1640 culture solution are all purchased from Beijing Quanjin biology, Inc.; the flow antibodies CD3, CD4, CD8, CD11, CD80, CD86, B220, IgA, IFN-. gamma.and IL-4 were all purchased from BD.

2.1.4 Main Instrument

Low temperature ultracentrifuge (Eppendorf 5810R); SW-CJ-2FD type single-sided double-person superclean bench (Shanghai Boxun); HRLM-80 model full-automatic autoclave, BCD-649WDCE model refrigerator (Haier Corp.); G70D20CN1P-D2(S0)20L type microwave oven (Grignard, China); innova 40R type constant temperature shaking table (U.S. NBS).

2.2 methods

2.2.1 Experimental groups

50 mice were randomly divided into 5 groups of 10 mice each. Each group was fed with PBS, NC8-pLP-S, NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus, NC8-pLP-S-CTA1-p14.5-D-D, numbered on the right ear of each mouse, and each group was assigned a natural number of 10.

2.2.2 lactic acid bacteria feeding

The new functional recombinant lactobacillus plantarum strain was fed orally to mice by inoculating and culturing in the same manner as in example 3 under the known optimal feeding conditions of 1.3.7.

2.2.3 Ocular vein blood sampling

And (3) well finding the canthus position of the mouse by using a vein blood taking needle, slightly rotating the needle, draining into a 1.5mL EP tube after bleeding, and taking 0.2mL of blood.

2.2.4 feces Collection

Feces were collected every three days.

2.2.5 method for preparing Single cell suspension

Excess fat was removed in a super clean bench by peeling Mesenteric Lymph Node (MLN), Peyer's Patches (PP) and spleen with ophthalmic scissors and ophthalmic forceps (autoclaved). The stacked 200-mesh sterile filter screens were placed in a sterile small plate, and 1mL of RPMI-1640 culture medium was added. The spleen was placed in a sieve and gently ground with the end of a sterile 1mL syringe until sufficient grinding was achieved, and the liquid was pipetted into a 1.5mL EP tube and placed in a precooled centrifuge at 2000rpm at 4 ℃ for 5 min. Then, the supernatant was discarded, 0.5mL of erythrocyte lysate was added, and after 3min of lysis on ice, 0.5mL of PBS was added, mixed well and left to stand for 2 min. Centrifuging at 2000rpm and 4 deg.C for 5min, and discarding the supernatant. 1mL PBS wash 1 pass and discard the supernatant plus 1mL complete medium. After 100-fold dilution, the cells were counted on a cell counting plate. MLN, PP nodules were treated in the same way, but without lysis with erythrocyte lysate. After the treatment, the cells were diluted 20 times and counted.

2.2.6 antibody staining

The staining method is described in the specification of flow antibody staining.

2.2.7 flow type machine

2.2.8 ELISA assays

Stool was tested for secreted SIgA and serum IgG.

(1) Coating: coating purified P14.5 antigen (with a concentration of 1. mu.g/mL) in each sample well of a 96-well culture plate, standing overnight at 4 ℃ in 100. mu.L per well; (2) washing: PBS containing 0.05% Tween-20 is added into each sample well for washing at room temperature, 300 mu L/well, and washing is carried out once every 5min for three times; (3) and (3) sealing: PBS containing 1% BSA was added to each sample well, blocked overnight at 4 ℃ and 150. mu.L/well; (4) washing: repeating the step 2; (5) a first antibody: adding a sample to be detected (a sample after the feces and the serum are treated), and sealing a membrane; (6) washing: repeating the step 2; (7) secondary antibody: the second antibody is diluted anti-mouse SIgA and anti-mouse IgG, and is sealed; (8) washing: repeating the step 2; (9) developing a substrate, adding 100 mu L/hole of developing solution, and reacting for 1h in a dark place at 37 ℃; (10) and (3) terminating the reaction: adding 10% H of stop solution₂SO₄50 μ L/well; (11) the result and detection of the enzyme label instrumentThe wavelength is 450nm, and data are reserved; (12) and (6) analyzing the result.

2.2.9CCK-8 colorimetric method for detecting T lymphocyte proliferation 96-well plate

1. Preparing a cell suspension: counting the cells; 2. plating into 96-well plates: according to the appropriate number of plated cells (about 1-2X 10)⁴) Approximately 100ul of cell suspension per well, 4-6 replicates of the same sample can be made; 3. culturing in an incubator at 37 ℃: the cells need to be cultured for about 4 hours after being inoculated and if the cells do not need to be attached, the step can be omitted; 4. add 10 μ L CCK 8: because the amount of CCK8 added into each well is small, errors are caused by the fact that reagents are attached to the walls of the wells, and the gun head is recommended to be immersed into the culture solution for adding and the culture plate is slightly knocked after the reagents are added so as to help uniform mixing. Or directly preparing culture medium containing 10% CCK8 (prepared at present), and adding in liquid-changing manner; 5. culturing for 0.5-4 hours: the amount of formed Formazan varied from cell type to cell type. If the color development is not sufficient, the culture can be continued to confirm the optimum conditions (it is recommended to search for the time point in advance). In particular, the Formazan formed by blood cells is very little, and longer color development time (5-6 hours) is needed; 6. measurement of absorbance at 450 nm: it is recommended to use dual wavelengths for the measurement, the detection wavelength 450-.

2.3 results

2.3.1 flow cytometry assay results

2.3.1.1 CD3 in spleen lymphocytes⁺CD4⁺IL-4, IFN-gamma and CD3 in T cells⁺CD8⁺The content of IL-4 and IFN-gamma in T cells is obviously increased

Spleen is a major peripheral immune organ, where T cells and B cells colonize, and a major organ for producing antibodies in vivo, and plays an important role in body defense and immune response. CD3, CD4, CD8 are surface markers for lymphocytes and can label cytotoxic T cells and helper T cells. IL-IL-4 is mainly produced by activated T cells, and has immunoregulation action on B cells, T cells, mast cells, macrophages and hematopoietic cells, and IFN-gamma has antiviral, antitumor and immunoregulation actions, and can promote NK cell activity, antigen presentation and macrophage lysosome activity.

The recombinant Lactobacillus plantarum enhances the immune effect on the body, and the CD3 in spleen lymphocytes⁺CD4⁺IL-4, IFN-gamma and CD3 in T cells⁺CD8⁺The content of IL-4 and IFN-gamma in T cells is obviously increased; as shown in fig. 21-25.

2.3.1.2 Pegyi lymph node (PP) cell 11C⁺80⁺And 11C⁺86⁺Variation of the content

Peyer's patch (Peyer's patch), also known as Peyer's patch, PP node, intestinal tract's patch, Peyer's patch, etc., is an important component of the intestinal mucosal immune system, a group of lymphatic follicles in the small intestine mucosa.

Lymphoid follicles consist of B cells and T cells (predominantly CD 4) covered on their surface by a layer of microfold cells, also known as M cells. It can recognize many antigens present in gastrointestinal tract, mainly phagocytize virus and enteric pathogenic bacteria, and the presented swallowed intestinal cavity antigen is transferred to immune cell, and the immune cell can process, transport and present pathogenic antigen. In the process, activated immune cells return to the inherent layer of the intestinal mucosa through the process of circulating homing, become plasma cells mainly secreting IgA and effector T cells and participate in local immune reaction of the intestinal tract. The ileal region is mainly present.

CD80 is a co-stimulatory factor when CD86 activates T lymphocytes and plays an important role in autoimmune monitoring, humoral immune response and transplantation response. CD86 interacts with the inducer CD28 and the inhibitor CTLA4 and is a major co-factor inducing T lymphocyte proliferation and IL-2 production.

Recombinant lactobacillus remarkably enhances 11C in PP⁺80⁺And 11C⁺86⁺(ii) expression of (a); as shown in fig. 26 and 27.

2.3.1.3 Pegyi lymph node (PP) cells B220⁺And IgA⁺Variation of the content

PP is a secondary lymphoid organ of the small intestine of a host, and various cells such as B cells, M cells and the like are gathered, wherein the M cells play an important role in the process of taking up antigens. PP is present from the mucosa to the submucosa, B cells are located in the germinal center, and T cells are in its periphery. Among them, PP plays a role in monitoring mucosal immune response of intestinal lumen antigens, and B220 and IgA are B cell activation markers, so that the level of activated B cells in PP is an important index for evaluating body immunity.

Recombinant lactobacillus remarkably enhances B220 in PP⁺IgA⁺(ii) expression of (a); as shown in fig. 18.

2.4 example 1 NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p14.5-D-D were successfully constructed and their successful expression in vitro was verified. Since lactobacillus can colonize in mouse intestinal tract and stimulate lactobacillus to express p54 protein and p54-IL-33, CTA1-p14.5-D-D fusion protein under intestinal tract environment, body T cells recognize p54 protein and p54-IL-21 fusion protein and generate specific antibody. The result of a preliminary animal test by using a mouse shows that the feeding of the recombinant lactobacillus plantarum has a certain effect of improving the immune effect of the mouse.

The results of a series of experiments show that the immunity of the mice is obviously improved after the new functional recombinant lactobacillus plantarum is fed.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Jilin university of agriculture

<120> African swine fever virus antigen protein recombinant expression vector, recombinant plant lactic acid bacteria, and preparation method and application thereof

<130> 202126361

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 2052

<212> DNA

<213> P54-Fc-P30-EGP nucleotide sequence

<400> 1

atggatagtg aattttttca accggtttat ccgcgtcatt atggtgaatg tctgagtccg 60

gttacgacgc cgaacttttt tagtacgcac atgtatacca ttctgattgc gattgttgtt 120

ctggttatta tcattatcgt tctgatttat ctgtttagta gtcgtaaaaa gaaagcggcg 180

gccattgaag aggaagatat tcaatttatt aatccgtatc aagatcaaca atgggttgaa 240

gttacgccgc aaccgggtac gagtaaaccg gcgggtgcga cgacggcgag tgttggtaag 300

ccggttacgg gtcgtccggc cacgaatcgc ccggcgacga ataaaccggt tacggataat 360

ccggttacgg accgtctggt gatggccacg ggtggcccgg cggccgcgcc ggcggccgcg 420

agtgcgccgg cccatccggc ggaaccgtat acgacggtga cgacgcagaa tacggcgagt 480

caaacgatga gtgcgattga aaatctgcgt caacgtaata cgtatacgca taaagatctg 540

gaaaatagtc tgggtagtgg tggtggcggt agtggtggcg gtggtagcgg tagtgacatt 600

gaaccgccga cgccgatttg tccggaaatt tgtagttgtc cggcggcgga agttctgggt 660

gcgccgagtg tttttctgtt tccgccgaaa ccgaaagata ttctgatgat tagtcgtacg 720

ccgaaagtta cgtgtgttgt ggttgatgtt agtcaagaag aagcggaagt tcaatttagt 780

tggtatgttg atggtgttca actgtatacg gcgcaaacgc gtccgatgga agaacaattt 840

aatagtacgt atcgtgttgt tagtgttctg ccgattcaac atcaagattg gctgaaaggt 900

aaagcgtttg cgtgtgcggt taataataaa gatctgctga gtccgattac gcgtacgatt 960

agtaaagcga cgggtccgag tcgtgttccg caagtttata cgctgccgcc ggcgtgggaa 1020

gaactgagta aaagtaaagt tagtattacg tgtctggtta cgggttttta tccgccggat 1080

attgatgttg aatggcaaag taatggtcaa caagaaccgg aaggtaatta tcgtacgacg 1140

ccgccgcaac aagatgttga tggtacgtat tttctgtata gtaaactggc ggttgataaa 1200

gttcgttggc aacgtggtga tctgtttcaa tgtgcggtta tgcatgaagc gctgcataat 1260

cactatacgc aaaaaagtat tagtaaaacg caaggtaaag aagcggccgc gaaagaagcc 1320

gcggcgaaag aagcggcggc caaggaagcc gcggcgaaga tggattttat tctgaatatt 1380

agtatgaaaa tggaagttat ttttaaaacg gatctgcgta gtagcagtca agttgttttt 1440

catgcgggta gtctgtacaa ttggtttagt gttgaaatta ttaatagtgg tcgtattgtt 1500

acgacggcga ttaaaacgct gctgagtacg gttaaatatg atattgttaa aagtgcgcgt 1560

atttatgcgg gtcaaggtta tacggaacat caagcgcaag aagaatggaa tatgattctg 1620

catgttctgt ttgaagagga aacggaaagt agtgcgagta gtgaaaatat tcatgagaaa 1680

aatgataatg aaacgaatga atgtacgagt agttttgaaa cgctgtttga acaagaaccg 1740

agtagtgaag ttccgaaaga tagtaaactg tatatgctgg cgcaaaaaac ggttcaacat 1800

attgaacaat atggtaaagc gccggatttt aataaagtta ttcgtgcgca taattttatt 1860

caaacgattt atggtacgcc gctgaaagaa gaggaaaaag aagttgttcg tctgatggtt 1920

attaaactgc tgaagaaaat taactttttt ctgacgtata ttggtagtgg tggtggcggt 1980

agtggtagta aacgtaaaaa gaaaggtaaa ggtctgggta aaaaacgtga tccgtgtctg 2040

cgtaaatata aa 2052

<210> 2

<211> 684

<212> PRT

<213> P54-Fc-P30-EGP amino acid sequence

<400> 2

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

1 5 10 15

Cys Leu Ser Pro Val Thr Thr Pro Asn Phe Phe Ser Thr His Met Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Gly Gly Gly Ser Gly Ser Asp Ile Glu Pro Pro Thr Pro Ile Cys Pro

195 200 205

Glu Ile Cys Ser Cys Pro Ala Ala Glu Val Leu Gly Ala Pro Ser Val

210 215 220

Phe Leu Phe Pro Pro Lys Pro Lys Asp Ile Leu Met Ile Ser Arg Thr

225 230 235 240

Pro Lys Val Thr Cys Val Val Val Asp Val Ser Gln Glu Glu Ala Glu

245 250 255

Val Gln Phe Ser Trp Tyr Val Asp Gly Val Gln Leu Tyr Thr Ala Gln

260 265 270

Thr Arg Pro Met Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

275 280 285

Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys Gly Lys Ala Phe Ala

290 295 300

Cys Ala Val Asn Asn Lys Asp Leu Leu Ser Pro Ile Thr Arg Thr Ile

305 310 315 320

Ser Lys Ala Thr Gly Pro Ser Arg Val Pro Gln Val Tyr Thr Leu Pro

325 330 335

Pro Ala Trp Glu Glu Leu Ser Lys Ser Lys Val Ser Ile Thr Cys Leu

340 345 350

Val Thr Gly Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn

355 360 365

Gly Gln Gln Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln Gln

370 375 380

Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val Asp Lys

385 390 395 400

Val Arg Trp Gln Arg Gly Asp Leu Phe Gln Cys Ala Val Met His Glu

405 410 415

Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Lys Thr Gln Gly

420 425 430

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

435 440 445

Glu Ala Ala Ala Lys Met Asp Phe Ile Leu Asn Ile Ser Met Lys Met

450 455 460

Glu Val Ile Phe Lys Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe

465 470 475 480

His Ala Gly Ser Leu Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser

485 490 495

Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys

500 505 510

Tyr Asp Ile Val Lys Ser Ala Arg Ile Tyr Ala Gly Gln Gly Tyr Thr

515 520 525

Glu His Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His Val Leu Phe

530 535 540

Glu Glu Glu Thr Glu Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys

545 550 555 560

Asn Asp Asn Glu Thr Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe

565 570 575

Glu Gln Glu Pro Ser Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr Met

580 585 590

Leu Ala Gln Lys Thr Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro

595 600 605

Asp Phe Asn Lys Val Ile Arg Ala His Asn Phe Ile Gln Thr Ile Tyr

610 615 620

Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu Val Val Arg Leu Met Val

625 630 635 640

Ile Lys Leu Leu Lys Lys Ile Asn Phe Phe Leu Thr Tyr Ile Gly Ser

645 650 655

Gly Gly Gly Gly Ser Gly Ser Lys Arg Lys Lys Lys Gly Lys Gly Leu

660 665 670

Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr Lys

675 680

<210> 3

<211> 366

<212> DNA

<213> p14.5 nucleotide sequence

<400> 3

atggctgatt ttaattcacc catacaatat ctcaaagaag actctcgcga tcgtacctcg 60

atcggcagct tggaatatga tgaaaatgca gatacgatga ttccgagctt tgcggcgggt 120

ctggaagagt tcgagccgat tccagactac gacccgacca cgtccacctc cctttacagc 180

cagctgaccc ataacatgga aaagatcgcc gaggaggagg actctaactt cctgcatgat 240

acccgtgaat tcacttcatt ggttccggac gaggcggaca acaagccgga agatgatgag 300

gaaagcggtg ctaaaccgaa aaagaagaag cacctgtttc cgaagctgag cagccataaa 360

agcaaa 366

<210> 4

<211> 1206

<212> DNA

<213> p14.5-il-33-mus nucleotide sequence

<400> 4

atggctgatt ttaattcacc catacaatat ttgaaggagg acagcagaga tcgtacctcc 60

atcggtagcc tggagtatga tgagaacgcc gacacgatga ttccgagctt tgcggctggt 120

ctggaagagt tcgagccgat tccggattac gacccaacga cctccacctc cctgtatagc 180

caactcaccc ataatatgga aaagatcgcc gaagaggagg actctaactt cctgcacgat 240

acgcgtgaat tcacctcttt agttccggat gaggcggata ataagccgga agacgacgaa 300

gaaagcggtg ctaaaccgaa gaagaagaag cacctgtttc cgaaactgag ctcccataaa 360

agcaaaggtt ctggcggtgg cggctctggc ggcggcgggt caggttccat gcgtccgcgt 420

atgaaatact ccaatagcaa gattagcccg gcgaaattca gcagcactgc cggtgaacgt 480

agcgtcccac cgtgcaaaat ccgccgtagt cagcagaaaa ccaaagagtt ctgccatgtt 540

tactgcatgc gcctgcgctc gggcctgacc attcgtaaag aaacctctta cttccgcaaa 600

gagccgacta agcgctactc ccttaaaagc ggtaccaaac atgaagagaa ctttagcgca 660

tatccgcgtg atagccgtaa gcgcagcttg ctgggttcta tccaagcgtt tgcggcgagt 720

gtggataccc tgtcgatcca gggtacatcg ttgctgaccc agagcccggc gagcttatct 780

acctataacg accagagcgt cagcttcgtt ttggagaacg gttgttatgt gatcaacgtg 840

gacgattccg gcaaggacca agaacaagat caagtgttgc tgcgttacta tgaaagcccg 900

tgcccggctt cgcaaagcgg tgatggtgtg gacggcaaaa agctgatggt taatatgagc 960

agcatcaagg acaccgacat ctggctgcat gcaaacgata aagactactc tgtggaactg 1020

cagcgtggtg atgttagccc gcctgaacag gcgttttttg tacttcacaa aaagagctca 1080

gattttgtgt ccttcgagtg caagaacttg ccgggtacgt acattggtgt taaagacaac 1140

cagctggctc tggttgaaga gaaggacgag agctgtaata atattatgtt caaactgtcg 1200

aaaatt 1206

<210> 5

<211> 1320

<212> DNA

<213> CTA1-p14.5-D-D nucleotide sequence

<400> 5

atgaatgatg acaaactata tagggctgat agccgtccgc cagatgagat caaacaatct 60

ggtggtctga tgccgcgcgg tcagtccgaa tacttcgata gaggaaccca gatgaacatt 120

aacctgtacg accacgcgac ccagaccggt tttgtgcgtc atgatgatgg ctacgtcagc 180

acttctatta gcttacgtag cgcgcatttg gttggtcagg aggttagcgc gctgggtggt 240

atcccgtata gccagatcta tggttcgtac cgcgtgcatt ttggtgttct ggacgaacag 300

ctgcatcgta accgtggtta tcgttattac tctaacctcg acatcccgcc ggcggccgac 360

ggctacggcc tagcgggttt cccgccggag caccgcgcat ggcgccagga gccgtggatt 420

caccatgctc cgccaggttg cggtaatgcc ccacgtagca gcggctctgg cggcggtggc 480

tcaggcggtg gcggtagcgg ttctatggct gacttcaaca gcccgatcca gtatctgaaa 540

gaagactctc gcgatcgtac ctcgatcggt tcattggaat atgatgagaa tgccgatact 600

atgatcccga gctttgcagc gggcctggaa gaatttgaac cgattccgga ttatgatccg 660

accacctcga cgtcacttta cagtcaactg acccataata tggaaaaaat cgctgaagag 720

gaggattcca actttctgca cgatacgcgt gagttcacca gccttgttcc ggacgaggct 780

gacaacaaac cggaggacga cgaggaaagc ggcgcaaaac cgaaaaagaa gaagcatctg 840

tttccgaaac tgagcagcca caaaagcaag ggttccggtg gcggtggtag cggcggcggt 900

ggctctggga gcgccgacgc acagcagaat aatttcaaca aggaccaaca aagcgcgttt 960

tacgaaattt tgaatatgcc gaatttgaat gaggcgcaac gcaacggctt catccagtcc 1020

ttgaaagacg atccgtccca gagcaccaac gtgttgggtg aggcgaaaaa gctgaatgag 1080

tcccaagcgc ccaagggcag cggtggcggc ggttccggtg gaggcggcag tgggagcgct 1140

gatgcgcagc aaaacaactt caacaaagac caacagtctg cgttctacga aattctgaac 1200

atgccgaacc tgaacgaagc acagcgtaat ggtttcattc aaagcctgaa ggacgacccg 1260

agccaatcta cgaacgtgct gggcgaagcc aaaaagctga acgagagcca agcgcctaag 1320

<210> 6

<211> 122

<212> PRT

<213> p14.5 amino acid sequence

<400> 6

Met Ala Asp Phe Asn Ser Pro Ile Gln Tyr Leu Lys Glu Asp Ser Arg

1 5 10 15

Asp Arg Thr Ser Ile Gly Ser Leu Glu Tyr Asp Glu Asn Ala Asp Thr

20 25 30

Met Ile Pro Ser Phe Ala Ala Gly Leu Glu Glu Phe Glu Pro Ile Pro

35 40 45

Asp Tyr Asp Pro Thr Thr Ser Thr Ser Leu Tyr Ser Gln Leu Thr His

50 55 60

Asn Met Glu Lys Ile Ala Glu Glu Glu Asp Ser Asn Phe Leu His Asp

65 70 75 80

Thr Arg Glu Phe Thr Ser Leu Val Pro Asp Glu Ala Asp Asn Lys Pro

85 90 95

Glu Asp Asp Glu Glu Ser Gly Ala Lys Pro Lys Lys Lys Lys His Leu

100 105 110

Phe Pro Lys Leu Ser Ser His Lys Ser Lys

115 120

<210> 7

<211> 402

<212> PRT

<213> P14.5-IL-33-Mus amino acid sequence

<400> 7

Met Ala Asp Phe Asn Ser Pro Ile Gln Tyr Leu Lys Glu Asp Ser Arg

1 5 10 15

Asp Arg Thr Ser Ile Gly Ser Leu Glu Tyr Asp Glu Asn Ala Asp Thr

20 25 30

Met Ile Pro Ser Phe Ala Ala Gly Leu Glu Glu Phe Glu Pro Ile Pro

35 40 45

Asp Tyr Asp Pro Thr Thr Ser Thr Ser Leu Tyr Ser Gln Leu Thr His

50 55 60

Asn Met Glu Lys Ile Ala Glu Glu Glu Asp Ser Asn Phe Leu His Asp

65 70 75 80

Thr Arg Glu Phe Thr Ser Leu Val Pro Asp Glu Ala Asp Asn Lys Pro

85 90 95

Glu Asp Asp Glu Glu Ser Gly Ala Lys Pro Lys Lys Lys Lys His Leu

100 105 110

Phe Pro Lys Leu Ser Ser His Lys Ser Lys Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Ser Met Arg Pro Arg Met Lys Tyr Ser

130 135 140

Asn Ser Lys Ile Ser Pro Ala Lys Phe Ser Ser Thr Ala Gly Glu Arg

145 150 155 160

Ser Val Pro Pro Cys Lys Ile Arg Arg Ser Gln Gln Lys Thr Lys Glu

165 170 175

Phe Cys His Val Tyr Cys Met Arg Leu Arg Ser Gly Leu Thr Ile Arg

180 185 190

Lys Glu Thr Ser Tyr Phe Arg Lys Glu Pro Thr Lys Arg Tyr Ser Leu

195 200 205

Lys Ser Gly Thr Lys His Glu Glu Asn Phe Ser Ala Tyr Pro Arg Asp

210 215 220

Ser Arg Lys Arg Ser Leu Leu Gly Ser Ile Gln Ala Phe Ala Ala Ser

225 230 235 240

Val Asp Thr Leu Ser Ile Gln Gly Thr Ser Leu Leu Thr Gln Ser Pro

245 250 255

Ala Ser Leu Ser Thr Tyr Asn Asp Gln Ser Val Ser Phe Val Leu Glu

260 265 270

Asn Gly Cys Tyr Val Ile Asn Val Asp Asp Ser Gly Lys Asp Gln Glu

275 280 285

Gln Asp Gln Val Leu Leu Arg Tyr Tyr Glu Ser Pro Cys Pro Ala Ser

290 295 300

Gln Ser Gly Asp Gly Val Asp Gly Lys Lys Leu Met Val Asn Met Ser

305 310 315 320

Ser Ile Lys Asp Thr Asp Ile Trp Leu His Ala Asn Asp Lys Asp Tyr

325 330 335

Ser Val Glu Leu Gln Arg Gly Asp Val Ser Pro Pro Glu Gln Ala Phe

340 345 350

Phe Val Leu His Lys Lys Ser Ser Asp Phe Val Ser Phe Glu Cys Lys

355 360 365

Asn Leu Pro Gly Thr Tyr Ile Gly Val Lys Asp Asn Gln Leu Ala Leu

370 375 380

Val Glu Glu Lys Asp Glu Ser Cys Asn Asn Ile Met Phe Lys Leu Ser

385 390 395 400

Lys Ile

<210> 8

<211> 440

<212> PRT

<213> CTA1-P14.5-D-D amino acid sequence

<400> 8

Met Asn Asp Asp Lys Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp Glu

1 5 10 15

Ile Lys Gln Ser Gly Gly Leu Met Pro Arg Gly Gln Ser Glu Tyr Phe

20 25 30

Asp Arg Gly Thr Gln Met Asn Ile Asn Leu Tyr Asp His Ala Thr Gln

35 40 45

Thr Gly Phe Val Arg His Asp Asp Gly Tyr Val Ser Thr Ser Ile Ser

50 55 60

Leu Arg Ser Ala His Leu Val Gly Gln Glu Val Ser Ala Leu Gly Gly

65 70 75 80

Ile Pro Tyr Ser Gln Ile Tyr Gly Ser Tyr Arg Val His Phe Gly Val

85 90 95

Leu Asp Glu Gln Leu His Arg Asn Arg Gly Tyr Arg Tyr Tyr Ser Asn

100 105 110

Leu Asp Ile Pro Pro Ala Ala Asp Gly Tyr Gly Leu Ala Gly Phe Pro

115 120 125

Pro Glu His Arg Ala Trp Arg Gln Glu Pro Trp Ile His His Ala Pro

130 135 140

Pro Gly Cys Gly Asn Ala Pro Arg Ser Ser Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Gly Ser Met Ala Asp Phe Asn Ser Pro Ile

165 170 175

Gln Tyr Leu Lys Glu Asp Ser Arg Asp Arg Thr Ser Ile Gly Ser Leu

180 185 190

Glu Tyr Asp Glu Asn Ala Asp Thr Met Ile Pro Ser Phe Ala Ala Gly

195 200 205

Leu Glu Glu Phe Glu Pro Ile Pro Asp Tyr Asp Pro Thr Thr Ser Thr

210 215 220

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

225 230 235 240

Glu Asp Ser Asn Phe Leu His Asp Thr Arg Glu Phe Thr Ser Leu Val

245 250 255

Pro Asp Glu Ala Asp Asn Lys Pro Glu Asp Asp Glu Glu Ser Gly Ala

260 265 270

Lys Pro Lys Lys Lys Lys His Leu Phe Pro Lys Leu Ser Ser His Lys

275 280 285

Ser Lys Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser

290 295 300

Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe

305 310 315 320

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly

325 330 335

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

340 345 350

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gly Ser Gly

355 360 365

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Ala Asp Ala Gln Gln

370 375 380

Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn

385 390 395 400

Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu

405 410 415

Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys

420 425 430

Leu Asn Glu Ser Gln Ala Pro Lys

435 440

Claims

1. An African swine fever virus antigen protein recombinant expression vector is characterized in that the recombinant expression vector comprises P54, Fc, P30 and EGP gene segments;

wherein, P54 and P30 are the gene sequences of the cell membrane structure proteins which are encoded in the gene type II SY-18 strain of the African swine fever virus and are involved in the absorption and the internalization of a host; the Fc is a swine IgG3Fc gene sequence, and the EGP is an intestinal cell HSPGs receptor targeting ligand polypeptide sequence;

preferably, the recombinant expression vector is P54-Fc-P30-EGP, and P54, Fc, P30 and EGP gene fragments are expressed in tandem with pgsA' gene;

further preferably, the nucleotide sequence of P54-Fc-P30-EGP is shown in SEQ ID NO. 1.

2. A recombinant Lactobacillus plantarum transformed with the recombinant expression vector for African swine fever virus antigen protein according to claim 1;

preferably, the recombinant lactobacillus plantarum comprises pgsA' anchored P54, Fc, P30 and EGP gene fragment;

the P54, Fc, P30 and EGP gene fragments are expressed in tandem with the pgsA' gene;

further preferably, the nucleotide sequence of the recombinant lactobacillus plantarum is shown as SEQ ID No. 1;

furthermore, the amino acid sequence of the recombinant lactobacillus plantarum coding protein is shown as SEQ ID NO. 2.

3. A method for constructing a recombinant Lactobacillus plantarum strain according to claim 1 or 2, comprising:

s2, transferring the recombinant expression vector obtained in the step S1 into lactobacillus plantarum to obtain the lactobacillus plantarum;

preferably, in step S1, the nucleotide sequence of P54-Fc-P30-EGP is shown as SEQ ID NO. 1; the amino acid sequence of the P54-Fc-P30-EGP coding protein is shown as SEQ ID NO. 2;

alternatively, in step S2, the lactobacillus plantarum is specifically lactobacillus plantarum NC 8.

4. The African swine fever virus antigen protein recombinant expression vector is characterized in that the African swine fever virus antigen protein recombinant expression vector is as follows: pLP-S-p14.5, pLP-S-p14.5-IL-33 and pLP-S-CTA1-p 14.5-D-D;

wherein p14.5 is the E120R gene sequence of the African swine fever virus coding p14.5 protein; IL-33 is a murine IL-33 gene sequence; CTA1-DD is: the enzymatic activity of Cholera Toxin (CT) -based CTA1 subunit and the D-domain dimer of S.aureus protein A were artificially constructed as adjuvant CTA1-DD gene sequences.

5. The African swine fever virus antigen protein recombinant expression vector of claim 4, wherein the nucleotide sequence of pLP-S-p14.5 is shown in SEQ ID No. 1;

the nucleotide sequence of pLP-S-p14.5-IL-33 is shown in SEQ ID NO. 2;

the nucleotide sequence of pLP-S-CTA1-p14.5-D-D is shown in SEQ ID NO. 3;

preferably, the amino acid sequence of the pLP-S-p14.5 encoding protein is shown in SEQ ID NO. 4;

the amino acid sequence of the pLP-S-p14.5-IL-33 coding protein is shown in SEQ ID NO. 5;

the amino acid sequence of the protein coded by pLP-S-CTA1-p14.5-D-D is shown in SEQ ID NO. 6.

6. The African swine fever virus antigen protein recombinant expression vector of claim 4, wherein the construction method of the African swine fever virus antigen protein recombinant expression vector comprises the following steps:

selecting a gene (E120R) sequence encoding P14.5 protein, optimizing two adjuvant sequences IL-33 and CTA1-DD, respectively placing the optimized adjuvant sequences at 5'3' end (CTA1-DD) and 3' end (IL-33) of E120R gene, P14.5, CTA1-P14.5-D-D and P14.5-IL-33-Mus sequence, synthesizing, inserting into a single anchoring expression vector pLP-S with erm as a screening marker, and obtaining three plasmids pLP-S-P14.5, pLP-S-P14.5-IL-33 and pLP-S-CTA 1-P14.5-D-D.

7. A recombinant Lactobacillus plantarum transformed with the African swine fever virus antigen protein recombinant expression vector of claim 4, comprising: NC8-pLP-S-p14.5, NC8-pLP-S-p14.5-IL-33-Mus and NC8-pLP-S-CTA1-p 14.5-D-D.

8. A method for constructing a recombinant Lactobacillus plantarum strain according to claim 7, comprising:

s2, obtaining the competence of lactobacillus plantarum NC 8;

s3, and electrically transforming the recombinant expression vector of the step S1 into the competence of lactobacillus plantarum NC8 obtained from S2.

9. A microbial preparation comprising the recombinant Lactobacillus plantarum strain of claim 5, or a fermentation product or metabolite thereof;

10. An antiviral product comprising the recombinant lactobacillus plantarum of claim 2 and/or the recombinant lactobacillus plantarum of claim 7 and/or the microbial inoculum of claim 7;

preferably, the virus comprises african swine fever virus;

preferably, the product is an animal vaccine, feed additive or feed;

preferably, the animal vaccine is a swine vaccine, and the vaccine dosage form can be oral freeze-dried powder.