Ferritin oral tumor vaccine delivered by engineering bacteria as well as preparation method and application of ferritin oral tumor vaccine

Abstract

The invention provides a ferritin oral tumor vaccine delivered by engineering bacteria and a preparation method and application of the ferritin oral tumor vaccine, and belongs to the technical field of oral tumor vaccines. A positive strain is obtained after the target plasmid 1 and the target plasmid 2 are introduced; the target plasmid 1 is prepared by inserting an OVA-RGD-Fn-TRP2 coding sequence into the no-load plasmid, and the target plasmid 2 is prepared by inserting a PhiX174 coding sequence into the no-load plasmid; the nucleotide sequence of the coding sequence of the OVA-RGD-Fn-TRP2 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the coding sequence of the PhiX174 is as shown in SEQ ID NO. 2. The oral tumor vaccine can overcome the technical problems of biological barrier, limited antigen immunogenicity and the like of the oral tumor vaccine, so that the prevention or treatment effect of the oral tumor vaccine on tumors is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral tumor vaccines, and particularly relates to an iron protein oral tumor vaccine delivered by engineered bacteria, and a preparation method and application thereof. Background Art

[0002] Melanoma is a highly malignant skin cancer, and the survival rate of patients with advanced melanoma is as low as 6%. Traditional cancer treatment methods such as surgical resection, radiotherapy and chemotherapy have the disadvantages of limited treatment effects and obvious side effects. Therefore, it is urgent to develop new treatment methods to achieve safe and efficient treatment of melanoma. Tumor vaccines activate the adaptive immunity of the body through tumor antigens, thereby specifically recognizing and killing tumor cells, and at the same time having an immune memory effect, and having better safety and patient tolerance compared with conventional therapies.

[0003] Compared with the limited local lymph node drainage of injectable tumor vaccines, oral vaccines can activate abundant immune cells in the intestine to produce a strong immune response. As one of the largest immune organs in the human body, the intestine is enriched with about 70% of the body's immune cells and is an advantageous site for vaccines to activate the body's immune system. The development of oral tumor vaccines faces two major challenges. One is the complex gastrointestinal environment and intestinal epithelial barrier faced by oral delivery; the other is the limited immunogenicity of tumor antigens. How to overcome the above two problems is the key to developing an efficient oral tumor vaccine. Summary of the Invention

[0004] In order to solve the problem of insufficient efficacy of existing oral tumor vaccines, the present invention provides an iron protein oral tumor vaccine delivered by engineered bacteria, which can overcome technical problems such as biological barriers and limited antigen immunogenicity faced by oral tumor vaccines, thereby effectively improving the preventive or therapeutic effect of oral tumor vaccines on tumors.

[0005] The present invention also provides a preparation method and application of an iron protein oral tumor vaccine delivered by engineered bacteria.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides an iron protein oral tumor vaccine delivered by engineered bacteria, and the oral tumor vaccine includes genetically engineered bacteria, and the genetically engineered bacteria include a positive strain obtained by using an intestinal bacterium as a starting strain and introducing a target plasmid 1 and a target plasmid 2;

[0008] The target plasmid 1 is prepared by inserting an OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid, and the target plasmid 2 is prepared by inserting a PhiX174 coding sequence into an empty vector plasmid;

[0009] The nucleotide sequence of the OVA-RGD-Fn-TRP2 coding sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the PhiX174 coding sequence is shown in SEQ ID NO.2.

[0010] Furthermore, the genetically engineered bacterium is a strain that has been treated with IPTG after introducing the target plasmid 1 and the target plasmid 2.

[0011] Furthermore, the starting strain includes Escherichia coli;

[0012] The target plasmid 1 is prepared by inserting the OVA-RGD-Fn-TRP2 coding sequence into the pET22b empty vector plasmid through the Nde I and BamHI restriction enzyme sites, and the target plasmid 2 is prepared by inserting the PhiX174 coding sequence into the pBAD33 empty vector plasmid through the Xba I and Sal I restriction enzyme sites.

[0013] Furthermore, the plasmid map of the target plasmid 1 is as Figure 18 shown, and the plasmid map of the target plasmid 2 is as Figure 19 shown.

[0014] Based on the same inventive concept, the present invention provides a preparation method of an engineered bacterium-delivered ferritin oral tumor vaccine, and the preparation method includes:

[0015] Insert the OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid to construct the target plasmid 1, and insert the phage lysis protein PhiX174 coding sequence into an empty vector plasmid to construct the target plasmid 2;

[0016] Co-introduce the target plasmid 1 and the target plasmid 2 into intestinal bacteria, and perform double-antibody screening to obtain positive strains;

[0017] Cultivate the positive strains in a medium containing IPTG for induction culture to induce the expression of functional ferritin and obtain genetically engineered bacteria;

[0018] Among them, the nucleotide sequence of the OVA-RGD-Fn-TRP2 coding sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the PhiX174 coding sequence is shown in SEQ ID NO.2.

[0019] Furthermore, the step of inserting the OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid to construct the target plasmid 1 and inserting the phage lysis protein PhiX174 coding sequence into an empty vector plasmid to construct the target plasmid 2 specifically includes:

[0020] The OVA-RGD-Fn-TRP2 coding sequence was inserted into the pET22b empty vector plasmid through the Nde I and BamHI restriction enzyme sites to construct the target plasmid 1. The phage lysis protein PhiX174 coding sequence was inserted into the pBAD33 empty vector plasmid through the Xba I and Sal I restriction enzyme sites to construct the target plasmid 2.

[0021] Furthermore, the co-introduction of the target plasmid 1 and the target plasmid 2 into intestinal bacteria and the screening with double antibiotics to obtain positive strains specifically include:

[0022] The target plasmid 1 and the target plasmid 2 were co-introduced into Escherichia coli BL21 competent cells, and positive strains were obtained through double-antibiotic screening with ampicillin and chloramphenicol.

[0023] Furthermore, the induction culture of the positive strains in a medium containing IPTG to induce the expression of functional ferritin to obtain genetically engineered bacteria specifically includes:

[0024] The positive strains were inoculated into a medium containing ampicillin and chloramphenicol for culture, and then IPTG (isopropyl β-D-thiogalactoside) with a final concentration of 1 mM was added, and induction culture was carried out at 30 ± 1 °C to induce the expression of functional ferritin to obtain genetically engineered bacteria.

[0025] Based on the same inventive concept, the present invention provides an application of an iron protein oral tumor vaccine delivered by genetically engineered bacteria in the preparation of drugs for preventing and treating melanoma lung metastasis and / or drugs for preventing and treating melanoma subcutaneous tumors.

[0026] Based on the same inventive concept, the present invention provides a drug for preventing or treating melanoma, and the active ingredient of the drug includes the above-mentioned iron protein oral tumor vaccine delivered by genetically engineered bacteria.

[0027] Preferably, the drug for preventing or treating melanoma includes drugs for preventing and treating melanoma lung metastasis and / or drugs for preventing and treating melanoma subcutaneous tumors;

[0028] The active ingredient of the drug further includes arabinose.

[0029] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0030] 1. An iron protein oral tumor vaccine delivered by genetically engineered bacteria according to the present invention includes genetically engineered bacteria, and the genetically engineered bacteria are positive strains obtained by using intestinal bacteria as the starting strain and introducing the target plasmid 1 and the target plasmid 2. The target plasmid 1 integrates OVA 257-264 、OVA 323-339 、TRP2 180-188The coding fragments of peptides, ferritin Fn, and RGD. The destination plasmid 2 is loaded with the coding sequence of the phage lysis protein PhiX174. The two destination plasmids are introduced into intestinal bacteria. After IPTG induction, the destination plasmid 1 expresses functional ferritin in intestinal bacteria. The functional ferritin is a cage-shaped protein formed by the self-assembly of 24 subunits. The model antigen OVA is fused and expressed at the N-terminus of the ferritin subunit. 257-264 With OVA 323-339 Peptide segments and RGD fragments, and TRP2 is fused and expressed at the C-terminus. 180-188 Peptide segments, where the RGD fragment endows the functional ferritin with the ability to target intestinal microfold cells (M cells) and cross the intestinal epithelial barrier. The functional ferritin platform enhances the immunogenicity of the antigen. The multimeric OVA antigen clusters and TRP2 antigen clusters achieve the efficient presentation of tumor antigens and the activation of adaptive immunity, ultimately enhancing the therapeutic effects on melanoma lung metastasis and subcutaneous tumors.

[0031] 2. An engineered bacteria-delivered ferritin oral tumor vaccine of the present invention. The oral tumor vaccine uses Escherichia coli in intestinal bacteria as a drug delivery vector, which can efficiently overcome the complex gastrointestinal environment. After entering the intestinal environment, the phage lysis protein PhiX174 expressed by the destination plasmid 2 can achieve the controlled release of vaccine molecules in engineered bacteria in the intestine. The released functional ferritin targets intestinal microfold cells and crosses the intestinal epithelial barrier through the RGD fragment, thus overcoming the biological barrier problem faced by oral tumor vaccines. At the same time, the functional ferritin platform enhances the immunogenicity of the antigen, thereby synergistically improving the preventive or therapeutic effect of the oral tumor vaccine on tumors.

[0032] 3. An engineered bacteria-delivered ferritin oral tumor vaccine of the present invention. The execution program of this oral vaccine system includes the in vitro induction expression of functional ferritin in genetically engineered bacteria and the in vivo controlled release in the intestine. It can efficiently overcome the gastrointestinal environment and the intestinal epithelial barrier, and achieve the efficient presentation of multivalent tumor antigens and the activation of the body's adaptive immunity with the help of functional ferritin. It can significantly inhibit the growth of metastatic tumors and subcutaneous tumors in animals and exhibit good biosafety. This oral vaccine system relies on the biosynthetic system and does not involve other modification means, with the advantages of easy synthesis, good stability, and low cost.

[0033] 4. A drug for preventing or treating melanoma according to the present invention, wherein the ferritin oral tumor vaccine can overcome the harsh gastrointestinal environment including gastric acid, gastrointestinal proteases, etc., and by orally administering arabinose (Ara), induce the expression of the phage lysis protein PhiX174, thereby initiating the lysis of genetically engineered bacteria and the controlled release of functional ferritin vaccine. The 24-mer of ferritin can achieve the presentation of multiple tumor antigens and increase its immunogenicity, which helps to trigger strong cellular and humoral immunity in the body. The drug of the present invention shows excellent anti-tumor effects in melanoma lung metastasis models and subcutaneous tumor models, and has a long-term immune memory effect, which can provide protection for the body and prevent the occurrence of melanoma, and has great prospects in the development and application of future tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 Related diagrams for the construction and functional verification of genetically engineered bacteria: A shows different groups; B shows a schematic diagram of WB primary antibody incubation. After the protein is separated by electrophoresis and transferred to a PVDF membrane, the PVDF membrane is first co-incubated with an anti-HA tag primary antibody. After color development, the primary antibody is eluted, and then co-incubated with a cMyc primary antibody and then developed; C shows the WB verification results of genetically engineered bacteria in different groups; D shows the SEC analysis chart of the purified functional ferritin expressed by genetically engineered bacteria in different groups.

[0036] Figure 2 TEM characterization chart of the purified functional ferritin expressed by genetically engineered bacteria in different groups.

[0037] Figure 3 Hydration particle size analysis chart of the purified functional ferritin expressed by genetically engineered bacteria in different groups.

[0038] Figure 4 Zeta potential distribution chart of the purified functional ferritin expressed by genetically engineered bacteria in different groups.

[0039] Figure 5 WB verification results of the successful expression of PhiX174 lysis protein after co-transfection with different ferritins and induction by arabinose.

[0040] Figure 6Diagram related to the lysis of engineered bacteria induced by Ara: A shows the OD of engineered bacteria co-transfected with OVA-R-Fn-TRP2 and phage iX174 target plasmids at different concentrations of Ara and different time points. 600 Statistical line graph; B shows the colony plating diagrams of engineered bacteria co-transfected with OVA-R-Fn-TRP2 and phage iX174 target plasmids with / without Ara induction at different times; C shows the statistical graph of the colony numbers in B.

[0041] Figure 7 Diagram related to the function of engineered bacteria in the simulated gastrointestinal environment: A shows the OD of engineered bacteria co-transfected with OVA-R-Fn-TRP2 and phage iX174 target plasmids at different time points in different environments. 600 Statistical line graph; B shows the TEM characterization diagrams of engineered bacteria co-transfected with OVA-R-Fn-TRP2 and phage iX174 target plasmids with / without Ara induction for 6 h in LB medium, simulated intestinal fluid, and simulated gastric fluid.

[0042] Figure 8 Function verification of engineered bacteria in mice: A shows the WB verification results of the successful expression of luciferase protein with arabinose induction; B shows the IVIS imaging results under arabinose induction; C shows the ex vivo fluorescence imaging of the gastrointestinal tract of mice at different time points after oral administration of engineered bacteria and subsequent gavage of Ara or PBS.

[0043] Figure 9 Statistical graph of the proportion of mature DCs analyzed by flow cytometry after co-incubating functional ferritin or free polypeptide fragments of different components with DC2.4 cells for 24 h.

[0044] Figure 10 Antitumor research diagram of the ferritin oral tumor vaccine delivered by engineered bacteria in a mouse melanoma lung metastasis model: A shows the treatment flow chart; B shows the pictures of lung tissues of mice after receiving different component treatments; C shows the statistical graph of lung metastasis foci of mice in different groups after treatment (n = 5).

[0045] Figure 11 Antitumor mechanism analysis diagram of the ferritin oral tumor vaccine delivered by engineered bacteria in a mouse melanoma lung metastasis model: A shows the treatment process and treatment groups; B shows the analysis of the proportions of CD8 + T cells and B cells in the single-cell suspensions of spleens of mice in different groups after treatment by flow cytometry; C shows the analysis of IFNγ secretion using an ELISA Spot kit after stimulating the spleen cells of mice in different groups with antigen peptides after treatment; D shows the analysis of CD8 + T cell infiltration in the metastatic foci after collecting lung tissues of mice in different groups, fixing them with 4% paraformaldehyde, and preparing paraffin sections after treatment.

[0046] Figure 12 Therapeutic safety assessment of ferritin oral tumor vaccine delivered by engineered bacteria: A shows the administration process and mouse grouping; B shows the graph of the body weight change of each group of mice over time during the treatment; C shows the blood biochemical analysis graph of the mice in different components after the treatment, with AST and ALT evaluating liver function and CREA evaluating kidney function; D shows the H&E staining analysis graph of the sections of the main organs including the heart, liver, spleen, lung, and kidney of each group of mice after the treatment.

[0047] Figure 13 Antitumor study of ferritin oral vaccine delivered by engineered bacteria in a murine subcutaneous melanoma model: A shows the treatment process and treatment grouping; B shows the subcutaneous tumor pictures of each group of mice after the treatment; C shows the tumor weights of each group of mice after the treatment; D shows the tumor volume growth curve graph of the mice receiving different treatments during the treatment, V = 1 / 2 length × width 2 。

[0048] Figure 14 Analysis of the antitumor mechanism of ferritin oral vaccine delivered by engineered bacteria in treating murine subcutaneous melanoma: A shows the statistical graph of the proportion of intratumoral infiltrating CD8 + T cells; B shows the proportion of intratumoral infiltrating mature DCs; C shows the proportion of intratumoral infiltrating B cells.

[0049] Figure 15 Safety assessment of oral vaccine in treating subcutaneous melanoma: A shows the treatment process and mouse grouping graph; B shows the colon pictures of mice in different groups after the treatment; C shows the statistical analysis graph of the ratio of colon weight to length of mice in different groups after the treatment; D shows the H&E staining analysis graph of colon sections of mice in different groups after the treatment.

[0050] Figure 16 Bio - safety analysis of oral tumor vaccine: A shows the change in the OUT abundance of feces of two groups of mice; B shows the non - metric multidimensional scaling (NMDS) difference analysis of fecal microorganisms of two groups of mice at the OTU level.

[0051] Figure 17 Immune effect of ferritin oral vaccine system delivered by engineered bacteria: A shows the oral vaccine inoculation and detection flow chart and mouse grouping graph; B and C show the concentrations of specific TRP2 IgG antibody and OVA IgG antibody in the blood of mice detected at different time points after three inoculations of different components of the oral vaccine; D and E show the proportions of central memory T cells and effector memory T cells in the spleen of mice on the 50th day after three inoculations of the oral vaccine; F shows the lung metastasis foci of mice after re - inoculation with tumor cells on the 50th day after three inoculations of the oral vaccine.

[0052] Figure 18 It is the map of plasmid 1 for the purpose.

[0053] Figure 19 It is the map of plasmid 2 for the purpose. Specific implementation manners

[0054] The present invention will be specifically described below in combination with specific implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation manners and examples are used to illustrate the present invention rather than to limit the present invention.

[0055] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0056] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0057] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0058] The technical principle of the present invention is as follows:

[0059] At present, the development of oral tumor vaccines faces two major challenges. One is the complex gastrointestinal environment and intestinal epithelial barrier faced by oral delivery; the other is the limited immunogenicity of tumor antigens. How to overcome the above two problems is the key to developing an efficient oral tumor vaccine.

[0060] Regarding the challenge of oral delivery, in recent years, intestinal bacteria have received extensive attention in the field of drug delivery due to various advantages. Escherichia coli has been used in a variety of oral drug delivery systems due to its easy gene editing and good biosafety. However, the controllability of the in vivo behavior of engineered bacteria is the key to ensuring safety and efficiency. In the present invention, we propose to use the phage Phix174 lysis protein E to control the intestinal controllable release of vaccine molecules in engineered bacteria. Regarding the dilemma of low immunogenicity of tumor antigens, we achieve the efficient presentation of tumor multivalent antigens by means of the ferritin platform. Ferritin is a cage-shaped protein self-assembled from 24 subunits, with advantages such as good stability, uniform size, and easy editing. By fusing and expressing the model antigen OVA 257-264 (SI INFEKL) and OVA 323-339 (ISQAVHAAHAEINEAGR) peptide segments and RGD fragments at the N-terminus of the ferritin subunit, and fusing and expressing TRP2 at the C-terminus 180-188(SVYDFFVWL) peptide, where the RGD fragment confers the ability of functional ferritin to target intestinal microfold cells (M cells) and cross the intestinal epithelial barrier, the ferritin platform enhances the immunogenicity of antigens, and the multimeric OVA antigen clusters and TRP2 antigen clusters achieve efficient presentation of tumor antigens and activation of adaptive immunity, ultimately enhancing the therapeutic effects on melanoma lung metastasis and subcutaneous tumors.

[0061] The following will combine examples and experimental data to elaborate in detail on an engineered bacteria-delivered ferritin oral tumor vaccine of the present invention, its preparation method, and its application.

[0062] Example 1

[0063] In this example, engineered bacteria expressing functional ferritin and PhiX174 lysis protein were constructed, including the following operations:

[0064] 1. Plasmid design and construction

[0065] The amino acid sequence of Pyrococcus furiosus ferritin (Fn) is from the NCBI database. To construct the target ferritin plasmid, the RGD sequence (RGDRGD) and OVA 257-264 (SIINFEKL) and OVA 323-339 (ISQAVHAAHAEINEAGR) were linked to the N-terminus of Fn through a flexible amino acid sequence GGGGS, and influenza hemagglutinin (HA) was used as a tag protein; TRP2 180-188 (SVYDFFVWL) was fusion-expressed at the C-terminus of Fn through a flexible amino acid sequence GGGSGGGSGGGS, and cMyc was used as a tag protein. The above target gene was synthesized by Sangon Biotech and cloned into the pET22b plasmid through NdeI and BamHI restriction enzyme cleavage sites. The control ferritin vector for fusion-expressing a single antigen was also constructed by a similar method.

[0066] The amino acid sequence of phage lysis protein E is from the NCBI database, and Flag tag protein was fusion-expressed. The sequence was synthesized by Sangon Biotech and cloned into the pBAD33 plasmid through XbaI and SalI restriction enzyme cleavage sites.

[0067] The amino acid sequence of luciferase reporter gene (Luciferin) is from the NCBI database, and Flag tag protein was fusion-expressed. The sequence was synthesized by Sangon Biotech and cloned into the pBAD33 plasmid through XbaI and SalI restriction enzyme cleavage sites.

[0068] The amino acid sequences of each target protein used in the present invention are shown in Table 1. The plasmid names in Table 1 refer to the plasmids containing the coding sequences of the target proteins.

[0069] Table 1 Summary of amino acid sequences of each target protein

[0070]

[0071]

[0072] 2. Construction of genetically engineered bacteria

[0073] The PhiX174 plasmid was co-transformed with R-Fn, OVA-R-Fn, TRP2-R-Fn, and OVA-R-Fn-TRP2 plasmids (the nucleotide sequences of the 5 plasmids are shown in Table 2) into Escherichia coli BL21(DE3) competent cells. The specific method is as follows: 100 μL of BL21(DE3) competent cells were thawed on ice, and 1 μL of the PhiX174 plasmid and 1 μL of different ferritin plasmids were added respectively. After standing on ice for 30 min, the competent cells were heat-shocked in a 42 °C water bath for 70 s, and then 900 μL of LB medium without antibiotics was added. The cells were shaken at 37 °C and 180 rpm for 1 h. After centrifugation of each group of bacteria, 800 μL of the supernatant was discarded, and the remaining bacteria were resuspended and an appropriate amount was spread on a solid medium containing the corresponding antibiotics, and cultured overnight at 37 °C in an inverted position.

[0074] Table 2 Nucleotide sequences of target proteins of different plasmids

[0075]

[0076]

[0077]

[0078] Example 2

[0079] This example explores the induced expression and identification of different groups of functional ferritins.

[0080] The genetically engineered bacteria of different groups were cultured in LB medium containing chloramphenicol (50 μg / mL) and ampicillin (60 μg / mL) at 37 °C and 180 rpm with shaking. The grouping of the engineered bacteria is shown in Figure 1 A. When the OD of the bacterial solution 600When = 0.6 - 0.8, 1 mM IPTG was added and induced at 30 °C for 8 h. The induced bacterial solution was centrifuged at 4000 g for 15 min, and the bacterial cell precipitate was collected and resuspended in 20 mM Tris (pH 8.0) solution. It was broken using an ultrasonic crusher with the working conditions of a frequency of 20 kHz, a power of 150 W, ultrasonicated for 15 min under ice bath, lasting for 60 s and with an interval of 60 s. The sample was centrifuged at 12000 rpm at 4 °C for 15 min, and the supernatant was collected. The protein concentration was detected using a BCA kit, and 5× loading buffer was added and boiled for 10 min, then cooled for standby. Prepare a 10% separating gel SDS-PAGE gel and electrophoresis solution, and add the ferritin samples of different groups into different wells with a sample amount of 20 μg per well. The stacking gel was electrophoresed at a constant voltage of 80 V, and the separating gel was electrophoresed at a constant voltage of 120 V. After electrophoresis, the protein samples were transferred onto a PVDF membrane. After blocking with 3% BSA, the corresponding primary antibody was incubated overnight at 4 °C, then washed three times with TBST solution, incubated with the secondary antibody, and then developed. Regarding the membrane regeneration step, after the developed bands were washed in the membrane regeneration buffer for 30 min, the washing solution was discarded, washed three times with TBST, 5 min each time, incubated with a new primary antibody and secondary antibody again, and then developed and imaged again. The incubation schematic diagram is shown in Figure 1 B, The WB result diagram is shown in Figure 1 C, The results showed that the ferritin modified with double antigens could develop double tags, indicating that both single-antigen and double-antigen ferritins were successfully induced and expressed.

[0081] Example 3

[0082] This example explores the purification, identification, and characterization of the functional ferritin in Example 2

[0083] (1) Purification of functional ferritin

[0084] When the engineered bacteria grew to the logarithmic phase, IPTG was used to induce for 8 - 10 h. The expressed bacterial cell precipitate was resuspended in 20 mM Tris (pH 8.0) solution and homogenized and broken in a high-pressure homogenizer. The homogenized sample was centrifuged at 12000 g for 30 min, and the supernatant was collected. The supernatant was further heat-treated in a water bath at 80 °C for 20 min to denature and remove most of the impurity proteins. The sample was centrifuged again at 12000 g for 30 min, and the supernatant was collected for separation and purification using an anion exchange column (Q-Sepharose Fast Flow). <^

[0085] (2) High-performance liquid chromatography size exclusion (SEC) characterization of the purified ferritin

[0086] Ferritin samples with the same concentration were further purified by size exclusion chromatography (SuperdexTM 200 10 / 300 GL) to obtain the target ferritin, and the protein absorption at 280 nm was detected.

[0087] (3) Transmission electron microscopy imaging of purified ferritin

[0088] Take 10 μL of ferritin samples from different groups (0.5 mg / mL) and drop them onto a copper grid. After standing for 10 min for adsorption, use filter paper to absorb the excess sample. Then, add phosphotungstic acid for negative staining for 2 - 3 min, and use filter paper to absorb the excess staining solution. After air drying, use transmission electron microscopy to characterize the morphology.

[0089] (4) Dynamic light scattering (DLS) analysis of potential particle size

[0090] Take purified ferritin samples from different groups (0.2 mg / mL) and disperse them in PBS buffer. Use a Malvern particle size analyzer to analyze the hydrodynamic particle size and Zeta potential.

[0091] As Figure 1 shown in D, the SEC results proved that the elution positions of different purified ferritins (R-Fn, OVA-R-Fn, TRP2-R-Fn, OVA-R-Fn-TRP2) were similar, indicating that the structures of each component of ferritin were similar. The transmission electron microscopy results showed that the purified ferritins in each group exhibited a uniform nanocage structure, and the modification of single antigen or double antigen peptide segments did not affect the structure of ferritin ( Figure 2 ). The DLS particle size results showed that the ferritin nanocages of each component exhibited good monodispersity and particle size uniformity, and the single antigen or double antigen modification had little effect on the particle size of ferritin ( Figure 3 ). Figure 4 The Zeta potential results showed that the potential of each component of ferritin was distributed around -10 mV, and there was no significant difference among the groups.

[0092] Example 4

[0093] This example explores the induced expression and functional verification of engineered bacterial lysate proteins

[0094] (1) WB verification of the expression of engineered bacterial system PhiX174 lysate proteins

[0095] Genetically engineered bacteria from different groups were cultured with shaking at 37 °C and 180 rpm in LB medium containing chloramphenicol (50 μg / mL) and ampicillin (60 μg / mL). When the OD of the bacterial solution 600When = 0.6 - 0.8, 1 mM IPTG was added and induced at 30 °C for 8 h. Then, arabinose (2 mg / mL) was added to the bacterial solution and induced for 12 h. After that, it was centrifuged at 4000 g for 15 min to collect the bacterial cell precipitate. The cell precipitate was lysed with RIPA strong cell lysis buffer on ice for 30 min, and then centrifuged at 12000 rpm for 15 min. The protein supernatant was taken, the protein concentration was measured using BCA, boiled after adding loading buffer, subjected to SDS-PAGE gel electrophoresis, then transferred to a membrane and incubated with the anti-Flag tag primary antibody, and the secondary antibody was incubated. Finally, development was carried out. Figure 5 The results showed that after co-transfection of PhiX174 lysis protein with different ferritins, they could all be expressed under the induction of arabinose.

[0096] (2) Verification of the function of arabinose-induced lysis of engineered bacteria

[0097] The engineered bacteria co-transfected with OVA-R-Fn-TRP2 and PhiX174 target plasmids were induced at different concentrations of Ara, and the absorbance values at 600 nm were detected at different time points using a spectrophotometer to analyze the survival of bacteria. Compared with the group without arabinose induction, arabinose at 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL could significantly induce bacterial lysis ( Figure 6 A). When the arabinose concentration was 0 or 2 mg / mL, the bacterial solution after induction for different times was plated to analyze the colony-forming units (CFU) ( Figure 6 B) and statistical analysis was carried out ( Figure 6 C). After 8 h of arabinose induction, the number of CFU decreased by 3 orders of magnitude, indicating the feasibility of the function of arabinose-induced bacterial lysis.

[0098] Example 5

[0099] This example explored the verification of the lysis function of engineered bacteria in simulated body fluid.

[0100] The engineered bacteria co-transfected with OVA-R-Fn-TRP2 and PhiX174 target plasmids were cultured in LB medium, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) with or without 2 mg / mL arabinose, and samples were taken at different time points to detect the OD 600 absorbance value and statistical analysis was carried out. The engineered bacteria in different environments after 6 h of culture were observed for morphological changes using TEM.

[0101] Figure 7 It was shown that the OD of the engineered bacteria in simulated intestinal fluid containing arabinose 600The value gradually decreases with the extension of time, which is similar to its arabinose-induced lysis in LB medium, indicating that the intestinal fluid environment does not affect the lysis behavior of engineered bacteria. Under the simulated gastric fluid environment, the OD 600 value of the engineered bacteria remains almost unchanged, which may be due to the self-protection mechanism of the bacteria in the strong acid environment. The TEM results show that after 6 h of induction, the morphology of the engineered bacteria in LB medium and simulated intestinal fluid is severely damaged, and the bacterial lysis is obvious, further proving the feasibility of the lysis behavior of the engineered bacteria.

[0102] Example 6

[0103] This example explores the functional verification of engineered bacteria in mice

[0104] The luciferase reporter gene Lucifer in-HA was used to replace the PhiX174 sequence and inserted into the pBAD33 backbone plasmid, and co-transformed with the OVA-R-Fn-TRP2 target plasmid into Escherichia coli to obtain luciferase (LUC) engineered bacteria.

[0105] (1) In vitro verification of the successful construction of LUC engineered bacteria

[0106] The LUC engineered bacteria were cultured in LB medium containing chloramphenicol (50 μg / mL) and ampicillin (60 μg / mL) at 37 °C and 180 rpm. When the OD 600 value reached 0.6 - 0.8, IPTG was added for overnight induction of the expression of functional ferritin. The engineered bacteria were centrifuged at 4000 rpm for 15 min, and the bacterial cells were redispersed in fresh LB medium containing chloramphenicol (60 μg / mL), ampicillin (100 μg / mL), and arabinose (2 mg / mL). After 8 h of induction, the bacterial cells were centrifuged to collect the cells, washed with PBS, added with strong RIPA lysis buffer, lysed on ice for 30 min, centrifuged at 4 °C and 12,000 rpm for 30 min, and the supernatant protein was collected. The protein concentration was detected by BCA, and after adding loading buffer and boiling, the expression of the luciferase reporter gene was analyzed by WB. After induction with IPTG and arabinose, the luciferase substrate was added to the LUC engineered bacteria, and the fluorescence was detected by the IVIS imaging system.

[0107] (2) In vivo evaluation of the distribution of LUC engineered bacteria

[0108] C57BL / 6 mice were divided into two groups: the experimental group and the control group, with 12 mice in each group. The mice were orally administered LUC engineered bacteria (10 8(CFU / mouse), mice in the experimental group were orally administered arabinose solution (2 mg / mL) simultaneously, and mice in the control group were orally administered an equal volume of normal saline. Mice were sacrificed at 1 h, 2 h, 4 h, and 6 h after gavage, 3 mice were sacrificed at each time point, the gastrointestinal tissues of the mice were dissected and collected, and after soaking in the luciferase substrate solution, imaging was performed using the IVIS system.

[0109] As Figure 8 shown, the WB results showed that the luciferase protein was successfully expressed when induced by arabinose, and the luciferase protein was not expressed without arabinose induction (8A). Similarly, under arabinose induction, IVIS imaging showed obvious fluorescence (8B). The in vivo imaging results showed that in mice orally administered arabinose and LUC engineered bacteria, the fluorescence signal appeared in the anterior half of the intestine at 1-2 h after gavage, the fluorescence signal was mainly concentrated in the colon at 4 h after gavage, and there was no obvious fluorescence signal in the intestine at 6 h. In contrast, no fluorescence signal was observed in the intestine of mice orally administered only LUC engineered bacteria without arabinose at each time point. These results indicate that the in vivo expression of the target protein of the engineered bacteria can be controlled by orally administered arabinose and the background signal is low (8C).

[0110] Example 7

[0111] This example explores the function of activating dendritic cells by the purified functional ferritin in Example 3

[0112] DC2.4 cells were added to a 6-well plate at 10 5 cells / well. After overnight culture and attachment, the medium was replaced with fresh RPMI-1640 medium containing double antibodies. PBS, TRP2, OVA, R-Fn, TRP2-R-Fn, OVA-R-Fn, and OVA-R-Fn-TRP2 (the unified Fn concentration was 200 μg / mL) were added and co-incubated for 24 h. Then, the cells were digested with trypsin and collected. After washing twice with PBS, the cells in each group were resuspended in 200 μL of PBS, and APC-CD11c flow antibody, FITC-CD80 flow antibody, and PerCP-CD86 flow antibody were added. The cells were co-incubated at 4 °C in the dark for 30 min, and mixed every 10 min in the middle. After the incubation, the cells were washed once with cold PBS, resuspended in 200 μL of PBS, and the proportion of mature DCs (CD11c + CD80 + CD86 + ) was analyzed using a flow cytometer.

[0113] As Figure 9As shown, compared with the free antigen peptides TRP2 and OVA, the single antigen groups TRP2-R-Fn and OVA-R-Fn displayed by ferritin increased the DC maturation ratio to 32.3% and 30.17% respectively. In addition, the dual antigen-displaying ferritin OVA-R-Fn-TRP2 had the highest stimulation of DC maturation ratio (44.39%), further indicating that the functional ferritin released by the lysis of engineered bacteria could effectively activate the maturation of DC cells.

[0114] Example 8

[0115] This example explores the anti-lung metastasis treatment effect and biosafety of the oral vaccine system. The specific operations are as follows:

[0116] (1) Animals

[0117] Six-week-old female C57BL / 6 mice were purchased from Beijing Speywood (SPF) Biotechnology Co., Ltd. (Beijing, China). The animals were housed in an SPF-level environment. The temperature of the animal room was set at a constant 24 ± 2 °C. All cages, bedding, feed, and drinking water were sterilized. The breeding environment of the mice was clean and hygienic, and there was sufficient drinking water and food. All animal experiments were conducted in accordance with the regulations of the Henan Provincial Laboratory Animal Management Regulations and the guiding principles of the Animal Ethics Committee of Zhengzhou University.

[0118] (2) Establishment of a mouse lung metastasis model

[0119] The mouse melanoma cells B16F10-OVA cultured to the logarithmic growth phase were digested with trypsin. The collected cells were washed once with sterile PBS and resuspended in PBS. The cell suspension was injected into C57BL / 6 mice via the tail vein at a dose of 2×10 5 cells / mouse to establish a lung metastasis model.

[0120] (3) Evaluation of the anti-lung metastasis tumor effect of the oral vaccine

[0121] The C57BL / 6 mice injected with melanoma cells B16F10-OVA via the tail vein were randomly divided into 5 groups (n = 5) ( Figure 11A), respectively: PBS, R-Fn, TRP2-R-Fn, OVA-R-Fn, and OVA-R-Fn-TRP2. At 3, 7, and 14 days, mice were gavaged with PBS or orally inoculated with vaccine engineering bacteria of different components. Mice were euthanized on day 21, and lung tissues, blood, and major organs (heart, liver, spleen, lung, kidney) were collected. After the lung tissues were fixed with 4% paraformaldehyde, paraffin sections were made, and immunohistochemistry was used to analyze the infiltration of CD8+ T cells in tumor tissues; mouse blood was used for blood biochemical analysis; part of the spleen was ground into a single-cell suspension, and flow cytometry was used to analyze the infiltration of immune cells. Part of the spleen cells were stimulated with OVA and TRP2 antigen peptides, and an ELISA SPOT detection kit was used to analyze the IFNγ secretion; paraffin sections and H&E staining were performed on the major organs for pathological analysis.

[0122] Figure 10 The results showed that compared with the PBS, R-Fn, TRP2-R-Fn, and OVA-R-Fn control groups, the treatment effect of the OVA-R-Fn-TRP2 oral vaccine group was the best ( Figure 10 B), the statistically counted number of lung metastases was significantly lower than that of other control groups ( Figure 10 C). The above results indicated that this oral vaccine could inhibit the growth of lung metastases of murine melanoma.

[0123] Figure 11 The results showed that the proportion of CD8+ T cells in the spleen of the OVA-R-Fn-TRP2 oral vaccine group was as high as about 12%, which was more than doubled compared with PBS (about 5%), and was also significantly higher than that of the single-antigen ferritin oral vaccine group. In addition, the proportion of B cells (CD45+CD19+) was significantly higher than that of other control groups, indicating that this oral vaccine system activated cellular immunity and humoral immunity to jointly eliminate tumor cells ( Figure 11 B). After the spleen cells of different groups were stimulated with antigens, the spleen cells of the OVA-R-Fn-TRP2 oral vaccine group secreted the most IFNγ, which was consistent with the highest infiltration ratio of cytotoxic T cells ( Figure 11 C). Further, the lung tissue sections of mice after treatment also showed that the OVA-R-Fn-TRP2 oral vaccine group had the most intratumoral infiltrating CD8+ T cells ( Figure 11 D). The above results revealed that the oral vaccine exerted anti-tumor effects by activating the adaptive immunity of the body, including cellular immunity and humoral immunity.

[0124] Figure 12 The results were used to illustrate the in vivo application safety of this oral vaccine system. Such as Figure 12As shown in Figure B, the body weight of the mice did not change significantly during the entire treatment period, indicating that the oral vaccine had no obvious systemic toxicity. The results of routine blood analysis in each group showed that after the mice were treated with the oral vaccine, the ALT, AST, and CREA indexes were not significantly abnormal and were all within the normal range( Figure 12 C), indicating that the inoculation of the oral vaccine had no obvious toxicity to the livers and kidneys of the mice. No obvious pathological damage was observed in the H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of the mice, further verifying its biosafety( Figure 12 D).

[0125] Example 9

[0126] This example explored the anti-tumor effect and biosafety evaluation of the oral vaccine system in a murine subcutaneous melanoma model. The specific operations are as follows:

[0127] After the murine melanoma cells B16F10-OVA were digested with trypsin, the cells were collected, washed once with sterile PBS, and resuspended in PBS. The cell suspension was subcutaneously injected into C57BL / 6 mice at a dose of 2×10 6 cells / mouse to construct a subcutaneous tumor model. The length and width of the tumor were measured using vernier calipers, and the tumor size was calculated. V = length × width × width × 1 / 2. Ten days after the subcutaneous tumor injection, the oral vaccine was inoculated and recorded as day 0. The mice were evenly divided into 6 groups( Figure 13 A), with 5 mice in each group. PBS or engineering bacteria vaccines of different groups (10 8 CFU / mouse) were orally administered on days 0, 3, and 7 respectively. In the 6th group, Poly(I:C) and TRP2 peptide and OVA peptide were subcutaneously inoculated to simulate clinical subcutaneous vaccination. Starting from day 0, the tumor volume and body weight changes of the mice were monitored every two days. The mice were euthanized on day 16, and the tumor tissues, feces (PBS group and OVA-R-Fn-TRP2 oral vaccine group), colon tissues, and main organs were collected. After the tumor tissues were weighed, they were ground into single-cell suspensions, and the proportions of infiltrating immune cells (cytotoxic T cells, B cells, and mature DC cells) in the tumors were analyzed using a flow cytometer. The colon tissues were sectioned for H&E analysis.

[0128] As Figure 13 shown in Figure B, the therapeutic effect of engineering bacteria orally loaded with single antigens of TRP2 or OVA was better than that of the PBS group, indicating that it could inhibit the growth of subcutaneous tumors to a certain extent. In contrast, the tumor volume of the engineering bacteria oral vaccine group receiving dual antigens was significantly lower than that of other control groups, which was similar to that of the group receiving Poly(I:C)+TRP2+OVA peptide treatment. The above results prove that the oral vaccine system can inhibit the growth of murine subcutaneous melanoma. The tumor weight analysis chart after the treatment further verified the therapeutic effect of the oral vaccine( Figure 13C). The tumor growth curve showed that compared with the PBS group and the engineered bacteria oral vaccine loaded with ferritin, the engineered bacteria vaccine loaded with single antigen ferritin moderately inhibited the growth of subcutaneous tumors, and the oral vaccine loaded with dual antigen ferritin greatly inhibited the growth of subcutaneous tumors ( Figure 13 D).

[0129] Figure 15 The anti-tumor mechanism of the oral vaccine in the treatment of subcutaneous tumors was further analyzed. Compared with the control group, the intratumoral infiltrating cytotoxic T cells in the final vaccine group ( Figure 14 A), mature DC cells ( Figure 14 B), and B cells ( Figure 14 C) were all significantly higher than those in other groups, indicating that the oral vaccine increased the infiltration of anti-tumor immune cells in the tumor to inhibit tumor cells. Figure 15 The safety of the oral vaccine during the treatment of subcutaneous tumors was analyzed. Pictures of the colon of mice in each group after treatment showed that the colon length did not significantly shorten ( Figure 15 B), and there were also no significant differences in the statistical chart of the ratio of colon length to weight among groups, further demonstrating the safety of the oral vaccine system for the intestine ( Figure 15 C). Analysis of the H&E staining results of colon sections of mice in each group showed no obvious histopathological damage ( Figure 15 D).

[0130] To further analyze the effect of the oral vaccine system on the abundance of intestinal microorganisms in mice, the mice were divided into two groups, with 5 mice in each group. The control group was orally administered PBS, and the experimental group was orally administered the ferritin tumor vaccine system delivered by engineered bacteria. After the treatment was completed, the feces of mice in the PBS group and the OVA-R-Fn-TRP2 oral vaccine group were collected for microbiome analysis. Figure 16 A showed that there was no statistical difference in the microorganisms in the intestinal feces of the two groups of mice at the operational taxonomic unit (OTU) level, and the principal coordinate (pCoA) analysis showed that the community structures were similar. Further non-metric multidimensional scaling (NMDS) analysis showed that the bacterial community structures of the oral vaccine group and the PBS group were similar ( Figure 16 B).

[0131] Example 10

[0132] This example explored the humoral immune effect and immune memory effect of the engineered bacteria vaccine. The specific operations are as follows:

[0133] The C57BL / 6 mice were divided into 5 groups ( Figure 17 A), with 10 mice in each group. The mice received different engineered bacteria oral vaccines on days 0, 3, and 7 (10 8(CFU / mouse) or PBS, and blood samples were collected from the fundus at 14 days, 21 days, and 28 days respectively for detecting the concentrations of anti-TRP2 IgG antibody and anti-OVA IgG antibody in serum at different time points after three oral vaccinations. On the 50th day, each group of mice was randomly divided into two parts, with 5 mice in each part. Five mice in one part of each group were injected with B16-OVA tumor cells (2×10 5 cells / mouse) via the tail vein to simulate lung metastasis behavior, and the mice were euthanized on the 65th day to collect lung tissues for analyzing metastatic foci. Five mice in one part of each group were euthanized on the 50th day to collect spleen tissues, which were ground into single-cell suspensions, and flow cytometry was used to analyze immune memory cells in the spleen.

[0134] Figure 17 B shows that after three oral vaccinations with engineered bacterial vaccines loaded with TRP2, compared with the PBS control group, the concentration of anti-TRP2 IgG antibody in the serum of mice increased significantly, while the concentration of anti-OVA IgG antibody did not change significantly; similarly, after three oral vaccinations with engineered bacterial vaccines loaded with OVA, compared with the PBS control group, the concentration of anti-OVA IgG antibody in the serum of mice increased significantly, while the concentration of anti-TRP2 IgG antibody did not change significantly ( Figure 17 C). It is worth mentioning that after three oral vaccinations with engineered bacterial vaccines loaded with OVA-R-Fn-TRP2, compared with other control groups, both anti-OVA IgG antibody and anti-TRP2 IgG antibody in the blood of mice remained at the highest level within two weeks. To explore the immune memory effect of oral vaccines, on the 50th day of mice, some mice were sacrificed and flow cytometry was used to analyze immune memory cells. The results showed that compared with other control groups, the proportion of effector memory T cells ( Figure 17 D) and central memory T cells ( Figure 17 E) in mice vaccinated orally with engineered bacterial vaccines loaded with OVA-R-Fn-TRP2 was the highest, indicating that oral vaccines could have a good immune memory effect. To explore the preventive and protective effects of oral vaccines, some mice were inoculated with tumor cells via the tail vein again on the 50th day and sacrificed on the 65th day to collect lung tissues. Figure 17 F shows that compared with other control groups, the number of lung metastatic foci in mice vaccinated orally with engineered bacterial vaccines loaded with OVA-R-Fn-TRP2 was the least, further demonstrating that the oral vaccine system has a long-term immune surveillance effect and can prevent the occurrence of tumors.

[0135] Finally, it should also be noted that the term "including", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0136] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0137] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. An engineered bacteria-delivered ferritin oral tumor vaccine, characterized in that, The oral tumor vaccine includes genetically engineered bacteria, and the genetically engineered bacteria include positive strains obtained by using intestinal bacteria as the starting strain and introducing target plasmid 1 and target plasmid 2; The target plasmid 1 is prepared by inserting the OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid, and the target plasmid 2 is prepared by inserting the PhiX174 coding sequence into an empty vector plasmid; The nucleotide sequence of the OVA-RGD-Fn-TRP2 coding sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the PhiX174 coding sequence is as shown in SEQ ID NO.

2.

2. The ferritin oral tumor vaccine delivered by engineered bacteria according to claim 1, wherein The genetically engineered bacteria are strains treated with IPTG after introducing the target plasmid 1 and the target plasmid 2.

3. An engineered bacteria-delivered ferritin oral tumor vaccine according to claim 1 or 2, wherein, The starting strain includes Escherichia coli; The target plasmid 1 is prepared by inserting the OVA-RGD-Fn-TRP2 coding sequence into the pET22b empty vector plasmid through the Ndel and BamHI restriction enzyme sites, and the target plasmid 2 is prepared by inserting the PhiX174 coding sequence into the pBAD33 empty vector plasmid through the Xbal and Sal l restriction enzyme sites.

4. An engineered bacteria-delivered ferritin oral tumor vaccine according to claim 1 or 2, characterized in that, The plasmid map of the target plasmid 1 is shown in Figure 18, and the plasmid map of the target plasmid 2 is shown in Figure 19.

5. The preparation method of an engineered bacteria-delivered ferritin oral tumor vaccine according to any one of claims 1-4, characterized in that, The preparation method includes: Inserting the OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid to construct the target plasmid 1, and inserting the phage lysis protein PhiX174 coding sequence into an empty vector plasmid to construct the target plasmid 2; Co-introducing the target plasmid 1 and the target plasmid 2 into intestinal bacteria, and screening with double antibiotics to obtain positive strains; Inducing and culturing the positive strains in a medium containing IPTG to induce the expression of functional ferritin to obtain genetically engineered bacteria; Among them, the nucleotide sequence of the OVA-RGD-Fn-TRP2 coding sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the PhiX174 coding sequence is as shown in SEQ ID NO.

2.

6. The preparation method of an engineered bacteria-delivered ferritin oral tumor vaccine according to claim 5, wherein, The step of inserting the OVA-RGD-Fn-TRP2 coding sequence into an empty vector plasmid to construct the target plasmid 1 and inserting the phage lysis protein PhiX174 coding sequence into an empty vector plasmid to construct the target plasmid 2 specifically includes: Inserting the OVA-RGD-Fn-TRP2 coding sequence into the pET22b empty vector plasmid through the Ndel and BamHI restriction enzyme sites to construct the target plasmid 1, and inserting the phage lysis protein PhiX174 coding sequence into the pBAD33 empty vector plasmid through the Xbal and Sal l restriction enzyme sites to construct the target plasmid 2.

7. The preparation method of an engineered bacteria-delivered ferritin oral tumor vaccine according to claim 5, characterized in that, The step of co-introducing the target plasmid 1 and the target plasmid 2 into intestinal bacteria and screening with double antibiotics to obtain positive strains specifically includes: Co-introducing the target plasmid 1 and the target plasmid 2 into Escherichia coli BL21 competent cells, and screening with ampicillin and chloramphenicol double antibiotics to obtain positive strains.

8. The preparation method of an engineered bacteria-delivered ferritin oral tumor vaccine according to claim 5, characterized in that, The positive strain is induced and cultured in a medium containing IPTG to induce the expression of functional ferritin, thereby obtaining genetically engineered bacteria, which specifically includes: The positive strain is inoculated into a medium containing ampicillin and chloramphenicol for culture, and then IPTG with a final concentration of 1 mM is added, followed by induction culture at 30 ± 1 °C to induce the expression of functional ferritin, thereby obtaining genetically engineered bacteria.

9. Use of a ferritin oral tumor vaccine delivered by an engineered bacterium according to any one of claims 1 - 4 in the preparation of a drug for preventing and treating melanoma lung metastasis and / or a drug for preventing and treating melanoma subcutaneous tumor.

10. A drug for preventing or treating melanoma, characterized in that, The active ingredient of the drug includes a ferritin oral tumor vaccine delivered by an engineered bacterium according to any one of claims 1 - 4.

11. A drug for preventing or treating melanoma according to claim 10, characterized in that, The drug for preventing or treating melanoma includes a drug for preventing and treating melanoma lung metastasis and / or a drug for preventing and treating melanoma subcutaneous tumor; The active ingredient of the drug further includes arabinose.

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