Listeria monocytogenes multi-epitope fusion antigen, multi-epitope based nano-particles and application of multi-epitope based nano-particles

Abstract

The invention discloses a listeria monocytogenes multi-epitope fusion antigen, a listeria monocytogenes multi-epitope fusion antigen-based nano-particle and application of the listeria monocytogenes multi-epitope fusion antigen-based nano-particle. Amino acid sequences of important antigen protein gene encoding proteins of LM antigens are analyzed, dominant antigen epitopes are screened out and connected in series according to a certain permutation and combination, a multi-epitope fusion antigen gene LM-MeAg is constructed and synthesized, and the listeria monocytogenes multi-epitope fusion antigen-based nano-particle is obtained. And further expressing the LM multi-epitope fusion antigen LM-MeAg gene in escherichia coli, and constructing a pT-LM-MeAg5 recombinant plasmid through a molecular cloning technology. According to the invention, dominant antigen epitopes of important antigens LLO, ActA, InlB and MPL of LM are assembled together in series according to a certain permutation and combination through nucleotides for coding flexible peptide amino acids, an LM multi-epitope fusion antigen gene is constructed, and an LM-MeAg5-PLGA nano-particle molecule with strong immunogenicity of the LM multi-epitope fusion antigen protein is further prepared. The method can be used for researching, developing and preparing efficient and safe anti-LM vaccines or drugs.

Description

Technical Field

[0001] The present invention relates to a multi-epitope fusion antigen of Listeria monocytogenes, a nano-particle thereof and applications, belonging to the field of biotechnology. Background Art

[0002] Listeria monocytogenes ( Listeria monocytogenes , hereinafter referred to as LM) is an important Gram-positive zoonotic pathogen, widely existing in natural environments such as soil and sewage, and having strong environmental tolerance. LM can infect humans and more than 40 kinds of animals, and can cross the blood-brain barrier, blood-fetal barrier and blood-testis barrier, causing serious diseases such as septicemia, meningitis and abortion. In addition, as an important foodborne bacterium, when humans ingest food contaminated with LM and get infected, it can cause food poisoning, posing a great threat especially to people with low immunity.

[0003] Therefore, this pathogen is listed as a foodborne pathogen that must be detected in the industry. In livestock, LM mainly infects various herbivores such as sheep, goats and cattle through contaminated silage and drinking water, etc., causing serious harm to the livestock breeding industry.

[0004] LM mainly enters the body's digestive tract through contaminated feed or food and infects various tissues and organs of the whole body. LM uses its surface proteins InlA and InlB to achieve the internalization of LM through the interaction with their respective receptors E-cadherin (E-Cad) and Met, and can cross epithelial cells, the blood-brain barrier and the placental barrier, and then spread to important organs such as the spleen and liver, causing systemic infection. During the process of LM infection, it mainly involves stages such as cell adhesion, invasion, internalization, vacuole lysis, intracellular proliferation and intercellular spread.

[0005] Existing studies have shown that the virulence factors expressed by LM are mainly concentrated within a 9 Kb pathogenicity island (Listeria pathogenicity island 1, LIPI-1) in its genome, encoding virulence factors such as the positive regulatory factor (prfA), phospholipase Cα (plcA), listeriolysin O (LLO), zinc-dependent metalloprotease (mlp), actin-polymerizing protein (actA), and phosphatidylinositol-specific phospholipase C (plcB). LM can utilize the above-mentioned virulence factors it expresses to complete the process of infecting host cells and reproduce and survive in cells for a long time. First, after the host ingests food contaminated with LM, LM adheres to the gastrointestinal epithelium under the action of internalin and then completes internalization. When LM completes internalization, it can escape from the phagosomal vacuole through the cytolysins of listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (plcA). Since the host cell provides sufficient nutrients, LM then multiplies abundantly in the cytoplasm. During this process, LM can further express the actin-polymerizing protein (ActA) to move within the cytoplasm and spread to adjacent cells, and then restart its infection cycle.

[0006] To effectively prevent and control LM infection, the development of inactivated vaccines and attenuated vaccines against LM has been successively carried out at home and abroad. In the research on inactivated vaccines, Jiao Xin'an et al. (2022) inactivated the Listeria monocytogenes strain of serotype 4h by mixing it with a β-lactone inactivator, and then mixed and emulsified it with an oil-in-water nanoemulsion adjuvant to obtain the LM inactivated vaccine; it was confirmed by subcutaneous injection that this inactivated vaccine could stimulate cellular and humoral immune responses and provide a certain degree of immune protection. In the research on attenuated vaccines, since LM has strong virulence, its virulence genes need to be molecularly modified to attenuate its virulence before developing live vaccines. Jiao Xin'an et al. (2018) constructed an actA, plcB, and orfX triple-gene deletion attenuated mutant strain of LM, and the virulence of this mutant strain was significantly reduced (794 times lower than that of the wild type), and at the same time, it had a good immune protection effect. Zeng Haijuan et al. (2018) from the University of Shanghai for Science and Technology constructed an attenuated LM that can be used to deliver and express foreign antigens with four genes deleted, namely actA, inlB, dal, and dat. Song Houhui et al. (2020) constructed an attenuated LM strain Lemo-C07 with a mutation in the hly gene (the asparagine at position 478 and valine at position 479 were mutated to alanine) using the wild-type LM strain EGD-e.

[0007] Currently, although certain progress has been made in the research on LM inactivated vaccines and attenuated vaccines, the LM inactivated vaccine has the disadvantages of weak immunogenicity and inability to effectively induce cellular immunity in the body; although the LM attenuated vaccine has good immunogenicity, there are problems such as virulence reversion and safety. Therefore, developing a new type of highly efficient and safe LM vaccine is of great significance for the prevention and control of this disease. s

[0008] Poly(lactic-co-glycolic acid) (PLGA), also known as poly(lactide-co-glycolide), is a biodegradable and biocompatible polymer synthesized by chemically reacting lactic acid (LA) and glycolic acid (GA) monomers in the presence of a catalyst. PLGA has received extensive attention in the field of vaccine adjuvants due to its good safety, biocompatibility, drug encapsulation efficiency, and drug sustained-release properties. PLGA nanoparticles can encapsulate drugs or antigens within the nanoparticles, protecting them from enzymatic degradation and enabling long-term, slow release, thereby achieving long-term drug action or long-term antigen-mediated immune protection. Additionally, PLGA has characteristics such as low molecular weight, high hydrophilicity, and low crystallinity. PLGA degrades in the body through the cleavage of ester bonds and is ultimately hydrolyzed into the original monomers, lactic acid and glycolic acid, and thus has no significant toxicity. At the same time, PLGA nanoparticles can increase uptake and cross-presentation by mimicking the size and shape of invading pathogens.

[0009] Currently, many studies have encapsulated protein antigens in PLGA nanoparticles, which can effectively induce cellular and humoral immune responses in the body.

[0010] In the study of porcine reproductive and respiratory syndrome (PRRS) vaccines, the inactivated PRRS virus (PRRSV) vaccine encapsulated in PLGA nanoparticles (NP-KAg), after nasal immunization, showed higher IFN-γ levels in porcine lung homogenates and sera, stimulating innate and adaptive immune responses and effectively protecting against PRRSV infection.

[0011] In the study of porcine epidemic diarrhea (PED) vaccines, PLGA nanoparticles (PLGA-KAg) encapsulating inactivated porcine epidemic diarrhea virus (PEDV) antigen (KAg), after immunization of pregnant sows, significantly increased the titers of PEDV-specific IgG and IgA antibodies and provided protection to piglets.

[0012] In the study of swine influenza vaccines, PLGA nanoparticles (PLGA-KAg) encapsulating swine influenza virus (SwIV) H1N2 antigen (KAg), after nasal immunization, increased antigen-specific lymphocyte proliferation, enhanced the frequencies of T helper / memory and CTL in peripheral blood mononuclear cells (PBMC), and reduced the heterologous virus load challenged in the porcine airway.

[0013] Listeria monocytogenes (LM) is a Gram-positive intracellular parasite. Therefore, the developed vaccine must be able to induce a high level of cellular and humoral immune responses in the body. Due to the characteristics of PLGA such as drug controlled release and sustained release, low cytotoxicity, tissue and cell biocompatibility, and targeted delivery, antigens carried can be presented to dendritic cells (DCs) through nanoparticles, thus inducing a strong cellular immunity in the body. Therefore, it is a good nanoadjuvant for vaccines.

[0014] By analyzing the amino acid sequences of the important antigens LLO, ActA, InlB, and MPL proteins of Listeria monocytogenes (LM) and their dominant linear antigenic epitopes, the sequences of the common dominant linear antigenic epitopes were selected. The nucleotide sequences corresponding to the predicted different epitopes (amino acid sequences) were ligated in a certain order with the flexible peptide coding sequence to synthesize the fusion antigen LM-MeAg gene containing multiple LM epitopes. The multi-epitope fusion antigen LM-MeAg gene was inserted into the Escherichia coli expression vector pET28a(+), and electrotransformed into the Escherichia coli BL21(DE3) expression strain for the expression of the multi-epitope fusion antigen LM-MeAg. SDS-PAGE and Western blot analyses confirmed that the recombinant multi-epitope fusion protein LM-MeAg had strong reactogenicity and specificity. The expressed recombinant antigen protein LM-MeAg was purified according to the Ni-NTA column and mixed with the poly(lactic-co-glycolic acid) (PLGA) emulsion to prepare LM-MeAg-PLGA nanoparticles. After immunizing animals with LM-MeAg-PLGA, through mouse and sheep tests, it was confirmed that the LM-MeAg5-PLGA nanoparticles could induce strong humoral and cellular immune responses, providing a new candidate antigen molecule for the development of an efficient and safe new subunit vaccine against LM infection. Therefore, a multi-epitope fusion antigen of Listeria monocytogenes, its nanoparticles, and their applications came into being. Summary of the Invention

[0015] The object of the present invention is to prepare a multi-epitope fusion antigen of Listeria monocytogenes and its nanoparticles and use them in a new subunit vaccine or drug for efficient and safe anti-LM infection.

[0016] The present invention analyzes the amino acid sequences of the proteins encoded by the genes of the important antigens LLO, ActA, InlB, and MPL of LM, screens out the dominant antigenic epitopes, and concatenates the selected dominant antigenic epitopes in a certain permutation and combination to construct and synthesize the multi-epitope fusion antigen gene LM-MeAg.

[0017] After predicting and analyzing the rare codons of Escherichia coli in the fusion gene LM-MeAg of rare codons, and replacing the rare codons of Escherichia coli with the preferred codons of Escherichia coli encoding the same amino acids, the constructed and synthesized multi-epitope fusion gene does not contain rare codons of Escherichia coli and is used for the next step of expression in Escherichia coli.

[0018] Furthermore, through animal immune experiments and screening, the LM multi-epitope fusion antigen gene of the present invention is obtained as: LM-MeAg5, whose DNA is as shown in sequence <210>5 in the sequence listing, and the amino acids encoded by its DNA are as shown in sequence <210>10 in the sequence listing.

[0019] Furthermore, the expression of the above multi-epitope fusion antigen LM-MeAg5 gene in Escherichia coli: constructing a pT-LM-MeAg5 recombinant plasmid through molecular cloning technology.

[0020] Using Eco R I and Xho I to perform double digestion on the pT-LM-MeAg plasmid and the pET-28a(+) plasmid, recover the LM-MeAg5 target fragment and the vector fragment, and use T4 DNA ligase to construct the pET-LM-MeAg5 recombinant expression plasmid.

[0021] Purification: After the above recombinant bacteria are induced to express, add the bacterial liquid to a centrifuge tube, place it in a high-speed refrigerated centrifuge at 4°C (±0.5) and centrifuge at 8000 - 10000 r / min for 10 min to collect the bacteria; add 10 mL of Lysis Buffer lysate, pipette and mix well, and then freeze-thaw 3 times repeatedly under liquid nitrogen or corresponding low temperature and at 42°C (±1.0), then place it in an ultrasonic cell disruptor to break until the bacteria are clarified, and after centrifugation, add 5 mL of 8 M urea to completely dissolve the inclusion bodies; purify the expressed recombinant LM-MeAg according to the Ni-NTA purification kit instructions. After measuring the purified protein concentration, the concentration of the recombinant protein LM-MeAg purified by the Ni column is 30 mg / mL. Dry the purified recombinant protein LM-MeAg with a freeze dryer to obtain a solid powder of the recombinant protein LM-MeAg, and store it at -20°C (±1) for standby.

[0022] Preparation method of LM-MeAg-PLGA nanoparticles: Preparation of PLGA nanoparticles: Accurately measure 50 mL of deionized water and place it in a 100 mL beaker as the aqueous phase; Weigh 20 mg of PLGA and 5 mg of LM-MeAg5 separately using an electronic balance, mix them, add 4.5 - 5.5 mL of a mixed solvent of absolute ethanol and acetone, and ultrasonically dissolve it to obtain the oil phase. Under magnetic stirring at a speed of 500 - 600 r / min, slowly and uniformly drip the oil phase into the aqueous phase. After the dripping is completed, place the beaker in a fume hood and continue stirring for 1 h (±0.15) to remove the organic solvent and fully solidify the nanoparticles; Centrifuge and wash 3 times with a 100 kD ultrafiltration tube (3500 - 4500 r / min, 10 min), and collect the concentrated nanoparticle suspension in the upper layer to obtain LM-MeAg-PLGA nanoparticles.

[0023] Furthermore, by applying the above multi-epitope fusion antigen gene or Listeria monocytogenes multi-epitope fusion antigen nanoparticles to vaccines or drugs against Listeria or listeriosis, highly efficient and safe anti-LM vaccines or drugs can be obtained.

[0024] The present invention has the following advantages: By using nucleotides encoding flexible peptide amino acids, the dominant antigenic epitopes of the important LM antigens LLO, ActA, InlB, and MPL are tandemly assembled in a certain arrangement and combination to construct a LM multi-epitope fusion antigen gene, and further prepare a LM multi-epitope fusion antigen protein LM-MeAg5-PLGA nanoparticle molecule with strong immunogenicity, which can be used for the research and development of highly efficient and safe anti-LM vaccines or drugs. Description of the Drawings

[0025] Figure 1 Table 1 shows the dominant linear epitope sequences of important LM antigen proteins.

[0026] Figure 2 Table 2 shows the construction strategy of the multi-epitope fusion antigen protein LM-MeAg.

[0027] Figure 3 Table 3 shows the detection results of cytokines in the sera of mice immunized with LM-MeAg5-PLGA nanoparticles.

[0028] Figure 4 Table 4 shows the detection results of specific IgG antibodies in the sera of sheep immunized with LM-MeAg5-PLGA nanoparticles.

[0029] Figure 5 Table 5 shows the detection results of cytokines in the sera of sheep immunized with LM-MeAg5-PLGA nanoparticles.

[0030] Figure 6 It is the control for the safety evaluation test of inoculating mice with LM-MeAg5-PLGA nanoparticles. Specific implementation manners

[0031] It mainly includes the following processes: 1. Gene cloning of important LM antigen proteins LLO, ActA, InlB, and MPL; 2. Prediction and analysis of dominant antigenic epitopes of important LM antigen proteins; 3. Construction method of LM-MeAg multi-epitope fusion antigen gene; 4. Expression of LM-MeAg multi-epitope fusion antigen gene in Escherichia coli; 5. Purification of LM-MeAg multi-epitope fusion antigen recombinant antigen protein; 6. Preparation method of LM-MeAg-PLGA nanoparticles; 7. Immune test of LM-MeAg-PLGA nanoparticles on mice; 8. Safety evaluation test of LM-MeAg-PLGA nanoparticles; 9. Immune test of LM-MeAg5-PLGA nanoparticles on sheep.

[0032] Gene cloning of important LM antigen proteins LLO, ActA, InlB, and MPL: According to the existing DNA sequences of LM genomic antigen genes LLO and MPL, specific primers for amplifying the above genes were designed respectively.

[0033] The LM EGD-e strain stored at -80 °C was streaked and inoculated on a BHI medium agar plate, and cultured in a constant temperature incubator at 37 °C with 5% CO2 for 20 h. Then, single colonies were picked and inoculated into 5 mL of BHI liquid medium, and cultured with shaking at 37 °C and 180 r / min for 24 hours. Then, the LM genomic DNA was extracted using a bacterial DNA extraction kit. Using the LM genomic DNA as a template, PCR amplification was performed separately with the designed primers. The amplified PCR products were cloned into the pMD19-T vector, and after sequencing, bioinformatics analysis was performed on the amino acid sequences of the encoded proteins of the above genes.

[0034] Prediction and analysis of dominant antigenic epitopes of important LM antigen proteins: The amino acid sequences of the encoded proteins of important LM antigen genes LLO, ActA, OppA, and EF-Tu were analyzed for dominant antigenic epitopes, and based on the results of different analysis methods, the sequences of common dominant linear antigenic epitopes were screened out.

[0035] As a result, dominant linear antigenic epitopes were screened out from important LM antigen proteins, as shown in Table 2.

[0036] Among them, 6 dominant linear antigenic epitopes were screened from the LLO protein: EP1: VAYGRQVYL; EP2: LTYPGALVKA; EP3: KIDYDDEMAY; EP4: EVISFKQIYYNV; EP5: SESQLIAKFGTA; EP6: NAISSLTYPGALV.

[0037] 3 dominant linear antigenic epitopes were screened from the ActA protein: EP7: SINMPSLPV; EP8: DSELESLTY; EP9: ETAPSLDSSFTSGDLASL.

[0038] 3 dominant linear antigenic epitopes were screened from the InlB protein: EP10: NTDYMSGNDFTLY; EP11: HLPEFTNEV; EP12: YMSGNDFTLY.

[0039] 2 dominant linear antigenic epitopes were screened from the MPL protein: EP13: KINDLFYLV; EP14: EVASWIVQV.

[0040] Construction of the LM multi-epitope fusion antigen LM-MeAg gene: The gene fragments encoding the dominant antigenic epitopes were concatenated in a certain arrangement through the nucleotide sequence (GGCCCGGGC) encoding flexible amino acids (GPG), and a series of multi-epitope fusion antigen genes LM-MeAg were constructed.

[0041] The constructed multi-epitope fusion antigen genes containing LM-MeAg are shown in Table 2 as LM-MeAg1 to LM-MeAg5.

[0042] Through prediction and analysis of the rare codons of Escherichia coli in the fusion gene LM-MeAg using rare codons, and replacing the rare codons of Escherichia coli with the preferred codons of Escherichia coli encoding the same amino acids, the constructed and synthesized multi-epitope fusion gene does not contain rare codons of Escherichia coli and is used for the next step of expression in Escherichia coli.

[0043] After optimization, the LM multi-epitope fusion antigen gene of the present invention was obtained: LM-MeAg5, whose DNA is shown as sequence <210>5 in the sequence listing, and the amino acids encoded by its DNA are shown as sequence <210>10 in the sequence listing.

[0044] Expression of the LM multi-epitope fusion antigen LM-MeAg gene in Escherichia coli: Construction of the recombinant expression vector: The recombinant plasmid pT-LM-MeAg5 was constructed by molecular cloning technology.

[0045] Using Eco R I and XhoThe pT-LM-MeAg plasmid and the pET-28a(+) plasmid were double digested, the LM-MeAg5 target fragment and the vector fragment were recovered, and the pET-LM-MeAg5 recombinant expression plasmid was constructed using T4 DNA ligase. The recombinant expression plasmid was transferred into competent cells E.coli DH5α, and spread on an LB plate containing ampicillin and cultured at 37 °C (±0.5) for 12 - 18 h.

[0046] Single colonies were picked for verification by colony PCR, the positive colonies were sent to BGI for sequencing, and at the same time, the plasmids of the positive colonies were extracted for double digestion verification.

[0047] SDS-PAGE electrophoresis analysis: The correctly identified recombinant expression plasmids were respectively transformed into the expression engineering bacteria E.coli BL21, cultured at 37 °C (±0.5) for 12 - 18 h, colonies were picked and screened by PCR, the screened positive bacteria were transferred to a liquid LB medium containing kanamycin and cultured at 37 °C (±0.5) until the OD 600nm reached 0.8, IPTG with a final concentration of 1 mmol / L was added for induction, the bacterial solution was collected after 8 - 12 h of induction, and at the same time, an empty vector control and a recombinant bacterium without IPTG induction were set as controls for SDS-PAGE electrophoresis analysis.

[0048] The results of SDS-PAGE electrophoresis analysis showed that in Escherichia coli, the LM-MeAg5 target protein was successfully expressed ( Figure 5 ), and the molecular weight was about 17.2 kDa, which was consistent with the expected size.

[0049] Western blot analysis: The gel after SDS-PAGE electrophoresis was transferred to an NC membrane through a semi-dry transfer electrophoresis tank, rinsed 3 times with TBST Buffer, blocked overnight with a blocking buffer, and Western blot analysis was performed using a polyclonal antibody against LM bacterial protein in mice as the primary antibody and goat anti-mouse HPRT-IgG as the secondary antibody.

[0050] The results of Western blot analysis showed that the expressed LM-MeAg1 - LM-MeAg5 all had specific immunological reactions with the anti-LM positive serum.

[0051] Purification of the recombinant protein of LM-MeAg multi-epitope fusion antigen: After the recombinant bacteria were induced and expressed according to the above method, the bacterial solution was added to a 50 mL centrifuge tube and centrifuged at 8000 - 10000 r / min for 10 min in a low-temperature high-speed refrigerated centrifuge at 4℃ (±0.5) to collect the bacteria. 10 mL of LysisBuffer lysate was added, and after pipetting and mixing evenly, it was repeatedly frozen and thawed 3 times under liquid nitrogen and 42℃ conditions, and then placed in an ultrasonic crusher to break the bacteria until the bacterial solution became clear. After centrifugation, 5 mL of 8 M urea was added to completely dissolve the inclusion bodies. The recombinant LM-MeAg was purified according to the Ni-NTA purification kit instructions. After measuring the protein concentration of the purified product, the concentration of the recombinant protein LM-MeAg purified by the Ni column was 30 mg / mL. The purified recombinant protein LM-MeAg was dried with a freeze dryer to obtain a solid powder of the recombinant protein LM-MeAg, which was stored at -20℃ for later use.

[0052] 6. Preparation of LM-MeAg-PLGA nanoparticles: Preparation of PLGA nanoparticles: Accurately measure 50 mL of deionized water and place it in a 100 mL beaker as the aqueous phase. Weigh 20 mg of PLGA and 5 mg of LM-MeAg accurately using an electronic balance, mix them, and then add 4.8 mL of a mixed solvent of anhydrous ethanol and acetone (the volume ratio of the two is 4:6), and ultrasonically dissolve it to serve as the oil phase. Under magnetic stirring at a rotation speed of 550 r / min, the oil phase was uniformly and slowly added dropwise to the aqueous phase. After the addition was completed, the beaker was placed in a fume hood and stirred for another 1 h to remove the organic solvents and fully solidify the nanoparticles. Centrifugally wash 3 times with a 100 kD ultrafiltration tube at a rotation speed of 4000 r / min for about 10 min, and collect the concentrated nanoparticle suspension in the upper layer to obtain LM-MeAg-PLGA nanoparticles.

[0053] Characterization of LM-MeAg-PLGA nanoparticles: Incubate a few drops of the PLGA nanoparticle suspension on a special copper mesh for transmission electron microscopy for 3 min, and observe the particle morphology using a transmission electron microscope; take an appropriate amount of PLGA nanoparticles, sputter them with gold, and observe their morphological characteristics through a scanning electron microscope. Dilute an appropriate amount of PLGA nanoparticles with water and measure the emulsion parameters using a ZCEC nanoparticle size and zeta potential analyzer. The observation results of the scanning electron microscope and the transmission electron microscope showed that the prepared nanoparticles were spherical, with a smooth surface and uniform size, and no large-area aggregation occurred. The average value of the polydispersity index (PDI) obtained by DLS was 0.259, which was lower than the threshold of 0.300, indicating that the prepared LM-MeAg-PLGA nanoparticles had a uniform particle size distribution.

[0054] 7. Immunization test of LM-MeAg-PLGA nanoparticles in mice: SPF-grade Balb / c mice aged 6-8 weeks were divided into 6 groups and injected with LM-MeAg1-PLGA to LM-MeAg5-PLGA nanoparticles and sterilized PBS buffer respectively. The first five groups were experimental groups and the last group was the control group.

[0055] The prepared LM-MeAg-PLGA nanoparticles were used to immunize the experimental group mice by subcutaneous injection. After 7 days, the mice were immunized for the second time. The control group mice were injected with LM-MeAg-PLGA nanoparticles or sterilized PBS buffer respectively.

[0056] Fifteen days after the second immunization, blood was collected from the eyeballs to separate serum, and different cytokine levels in the sera of the experimental group and the control group were detected using mouse IFN-γ, IL-6, TNF-α and IL-2 indirect ELISA kits respectively. At the same time, mouse peripheral blood lymphocytes were separated using mouse lymphocyte separation solution, and mouse peripheral blood CD4 / CD8 levels were analyzed by flow cytometry using mouse CD4 monoclonal antibody (FITC-labeled) and CD8 monoclonal antibody (PE-labeled).

[0057] The results of indirect ELISA analysis showed that the levels of IFN-γ, IL-6, TNF-α and IL-2 in the mice inoculated with LM-MeAg5-PLGA in the experimental group were significantly higher than those in the control group and also higher than those in the LM-MeAg1-PLGA, LM-MeAg2-PLGA, LM-MeAg3-PLGA and LM-MeAg5-PLGA groups, as shown in Table 3.

[0058] The results of flow cytometry analysis showed that the level of mouse peripheral blood CD4 / CD8 in the LM-MeAg5-PLGA experimental group was higher than that in the LM-MeAg1-PLGA, LM-MeAg2-PLGA, LM-MeAg3-PLGA, LM-MeAg4-PLGA groups, and there was a highly significant difference compared with the control group mice, P < 0.05.

[0059] The above experiments confirmed that LM-MeAg5-PLGA nanoparticles could induce strong humoral and cellular immune responses in mice.

[0060] 8. Safety evaluation test of LM-MeAg-PLGA nanoparticles: Twenty 6- to 8-week-old SPF-grade Balb / c mice were divided into two groups of 10 mice each. LM-MeAg5-PLGA nanoparticles and a sterilized PBS control group were inoculated into the mice via subcutaneous injection, and then the mice were continuously observed for 21 days after the first immunization and re-inoculation to observe whether there was redness, swelling, and inflammatory reactions at the inoculation site on the skin. On the 15th day after the second immunization, the hearts, livers, spleens, lungs, kidneys, and brains of the mice were taken, fixed with 10% formaldehyde tissue, sectioned, and then subjected to HE staining and pathological observation. The safety of LM-MeAg5-PLGA nanoparticles for mice was tested through the above experiments. The results showed that during the 21-day observation period after the first immunization and re-inoculation, there was no redness, swelling, and inflammatory reactions at the inoculation site on the skin of the mice. Histological section observations of the hearts, livers, spleens, lungs, kidneys, and brains of the inoculated mice showed that no pathological tissue damage occurred in the above organs, confirming that LM-MeAg5-PLGA nanoparticles had good safety, as Figure 6 shown.

[0061] 9. Immunization test of LM-MeAg5-PLGA nanoparticles in sheep: Twelve healthy 4-month-old lambs were randomly divided into 3 groups of 4 each (n = 4), namely the experimental group inoculated with LM-MeAg5-PLGA, the sterilized PBS control group, and the PLGA control group. The lambs were first subcutaneously inoculated with 1 ml (0.5 mg / ml) of LM-MeAg5-PLGA; 7 days after the injection, LM-MeAg5-PLGA was re-inoculated at the same dose and inoculation method. Peripheral blood of the lambs was collected 15 days after the second immunization, serum was separated and harvested, and the IgG antibody titer against Lm in the serum of the immunized lambs was detected by the indirect ELISA method using whole Lm-coated plates. At the same time, the levels of IFN-γ, IL-6, TNF-α, and IL-2 were detected according to the instructions of the ELISA kits for sheep IFN-γ, IL-6, TNF-α, and IL-2.

[0062] The results showed that 15 days after the second immunization, the sheep immunized with LM-MeAg5-PLGA nanoparticles induced a high level of specific IgG antibodies in the body, and the IgG antibody titer was significantly higher than that of the PLGA control group and the PBS control group, with a significant difference (P < 0.05) (Table 4). At the same time, compared with the PLGA control group and the PBS control group, the secretion levels of INF-γ, IL-6, TNF-α, and IL-2 in the serum of the sheep in the LM-MeAg5-PLGA inoculation group were all significantly increased (P < 0.05) (Table 5).

[0063] The above experiments confirmed that LM-MeAg5-PLGA nanoparticles could induce strong humoral and cellular immune responses in sheep.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention. The protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, which should also be regarded as within the protection scope of the present invention.

Claims

1. A Listeria monocytogenes multi-epitope fusion antigen, characterized in that, The multi-epitope fusion antigen gene described above is: LM-MeAg5, and its DNA is as shown in sequence <210>5 in the sequence listing, and the amino acids encoded by its DNA are as shown in sequence <210>10 in the sequence listing.

2. A Listeria monocytogenes multi-epitope fusion antigen nanoparticle, characterized in that It is prepared through the following process. Expression of the multi-epitope fusion antigen LM-MeAg5 gene described in claim 1 in Escherichia coli: Construct the pT-LM-MeAg5 recombinant plasmid through molecular cloning technology. Adopt Eco R I and Xho Double digest the pT-LM-MeAg plasmid and the pET-28a(+) plasmid with I, recover the LM-MeAg5 target fragment and the vector fragment, and construct the pET-LM-MeAg5 recombinant expression plasmid using T4 DNA ligase; Purification: After the above-mentioned recombinant bacteria are induced to express, add the bacterial liquid to a centrifuge tube, place it in a high-speed refrigerated centrifuge at 4°C (±0.5) for centrifugation at 8000 - 10000 r / min for 10 min to collect the bacterial cells. Add Lysis Buffer lysate, pipette and mix well, and then repeatedly freeze-thaw 3 times under liquid nitrogen or corresponding low temperature and 42°C (±1.0), then place it in an ultrasonic crusher to break until the bacterial cells are clarified. After centrifugation, add urea to completely dissolve the inclusion bodies; purify the expressed recombinant LM-MeAg according to the Ni-NTA purification kit instructions, dry the purified recombinant protein LM-MeAg with a freeze dryer to obtain a solid powder of recombinant protein LM-MeAg, and store it at -20°C (±1) for later use. Preparation method of LM-MeAg-PLGA nanoparticles: Preparation of PLGA nanoparticles: Take deionized water as the aqueous phase; weigh 20 mg of PLGA and 5 mg of LM-MeAg5 respectively, mix them, add 4.5 - 5.5 mL of a mixed solvent of anhydrous ethanol and acetone thereto, and ultrasonically dissolve it fully as the oil phase. Under magnetic stirring at a rotation speed of 500 - 600 r / min, slowly and uniformly drip the oil phase into the aqueous phase. After dripping, place the beaker in a fume hood and continue stirring for 1 h (±0.15) to remove the organic solvents and fully solidify the nanoparticles; centrifuge and wash 3 - 4 times with a 100 kD ultrafiltration tube, with a centrifugation speed of 3500 - 4500 r / min and a time of 10 - 15 min), and collect the concentrated nanoparticle suspension in the upper layer to obtain LM-MeAg-PLGA nanoparticles.

3. Application of a Listeria monocytogenes multi-epitope fusion antigen, characterized in that: Apply the multi-epitope fusion antigen gene described in claim 1 or the Listeria monocytogenes multi-epitope fusion antigen nanoparticles described in claim 2 to vaccines or drugs against Listeria or listeriosis.

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