A novel coronavirus vaccine based on attenuated salmonella controllable secretion expression and a preparation method and application thereof

Abstract

The application discloses a novel coronavirus vaccine based on controllable secretion expression of attenuated salmonella, a preparation method and application thereof, and is used for preventing the novel coronavirus. The application constructs controllable, stable and secretory expression plasmids of different antigen domain proteins of the novel coronavirus and attenuated salmonella expression strains of the different antigen domain proteins; a plurality of attenuated salmonella antigen presentation strains which can controllably and secretively express in antigen presenting cells are mixed, and through an oral route, the plurality of different antigen proteins can be efficiently secretively expressed in the antigen presenting cells by means of the unique secretion system of the attenuated salmonella after administration. The secretively expressed antigenic proteins can be effectively processed and presented by the antigen presenting cells, and finally the immune system is activated / regulated, higher titer antibodies are generated, and the vaccine plays a role.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a novel coronavirus vaccine based on attenuated Salmonella controlled secretion expression and a preparation method and application thereof. BACKGROUND

[0002] Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) has about 82% similarity with the genome of SARS coronavirus, and the two coronaviruses also share the same cell receptor, angiotensin-converting enzyme 2 (ACE2) (Lan J et al., 2020, Nature 581: 215-220.). Despite these similarities, the novel coronavirus spreads more widely, more quickly, and is more deadly than the SARS virus (Zhu N et al., 2020, N Engl J Med, 382:727-733.). Therefore, in order to effectively prevent and treat the spread of the novel coronavirus, it is urgent to develop a safe, efficient and inexpensive SARS-CoV-2 vaccine. In the early development of SARS-CoV vaccine, researchers found that antibodies against the viral spike protein (S protein) can efficiently neutralize the virus and prevent infection (Yang ZY et al., 2005, Proc Natl Acad Sci U S A 102: 797-801.), so the S protein of SARS-CoV-2 is the primary target for the development of antiviral drugs and vaccines (Walls AC et al., 2020, Cell 181: 281-292.e6.).

[0003] At present, many research institutes and pharmaceutical companies are rapidly developing novel coronavirus vaccines, involving attenuated live vaccines, recombinant viral vector vaccines, inactivated virus vaccines, protein subunit vaccines, virus-like particle (VLP) vaccines, and nucleic acid vaccines (Jeyanathan M et al., 2020, Nat Rev Immunol 20: 615-632.). Although attenuated live vaccines show high protection, they are also associated with high risk; inactivated vaccines, recombinant virus vaccines and nucleic acid vaccines face problems such as high cost and inconvenience of vaccination; and direct vaccination with traditional recombinant protein vaccines cannot stimulate a good cellular immune response and therefore cannot provide good protection, often requiring the addition of additional adjuvants (Guy B et al., 2007, Nat Rev Microbiol 5: 505-517.). So far, all vaccines developed at home and abroad are administered by injection, which is the most direct administration route, but also the most risky and costly administration method among all administration routes. So far, there is still a lack of other more convenient and safe administration methods or routes in the development of new coronavirus vaccines.

[0004] The working principle of the vaccine is that when the vaccine antigen is inoculated into the animal body, the animal body's immune system is stimulated, the antigen presenting cells of the animal body process, process and present the vaccine to specific lymphocytes (T and B lymphocytes), and then the lymphocytes recognize, activate, proliferate and differentiate the vaccine, finally produce immune effector molecules (antibodies and cytokines) and immune effector cells, and eventually eliminate the antigen from the animal body, which is called immune response. As can be seen, the current international general new coronavirus vaccine development in which the antigen is injected, the injection of the antigen in the whole body tissue may produce some side effects, and the process of the vaccine in triggering immunity is actually produced by antigen presenting cells. Therefore, if the antigen is limited to the production of antigen presenting cells, it is possible to limit the side effects of the antigen in the whole body tissue, and to achieve a safer goal.

[0005] Oral vaccines based on attenuated strains are widely used in the development of vaccines for various infectious diseases due to their simple inoculation and low price. Attenuated Salmonella is a widely used bacterial oral vaccine carrier, and also a natural mucosal immune adjuvant, antigen expression and delivery tool. The recombinant vaccine strain modified by genetic engineering can enter the body through M cells in the intestinal tract after oral administration (Jensen VB et al., 1998, Infect Immun 66:3758-3766.), and the strain in the body environment can be rapidly phagocytosed by antigen presenting cells (APC). At this time, if the secretory system of the bacteria is used, the antigen protein can be effectively secreted into the APC cell, further effectively decomposed into polypeptide segments and displayed to T-helper cells (T-help cell) through the major histocompatibility complex MHC-I or MHC-II pathway, so as to stimulate the body to produce cellular, humoral and mucosal immune responses against the antigen molecule (Mei Y et al., 2017, Cancer Immunol Res 5:503-514.).

[0006] However, developing an efficient and practical oral attenuated Salmonella vaccine for preventing the novel coronavirus still faces the following technical difficulties: a) maintaining the phenotypic stability of recombinant bacteria, using attenuated bacteria as an expression tool for foreign antigens requires the establishment of a suitable expression strategy to optimize antigen expression and plasmid compatibility, otherwise problems such as excessive toxicity of the strain, severe loss of engineering plasmid and loss of immune effect may occur; b) the effectiveness of antigen proteins secreted into cells by bacteria, as most bacteria are wrapped in a membrane-enclosed vesicular structure (SCV) after entering antigen-presenting cells, which greatly limits the effective presentation of antigen molecules (Zhang XL et al., 2008, Cell Mol Immunol 5:91-97.), if true effective presentation cannot be achieved, the preventive effect of the vaccine will be greatly reduced; c) what kind of promoter should be selected to make the antigen molecules only expressed in antigen-presenting cells, but not in blood and normal tissues, to provide the safety of antigen molecules. d) the optimality of virus antigen epitope screening and region selection, as the SARS-CoV-2 S protein has a large molecular weight and a complex structure (Hsieh CL et al., 2020, Science 369: 1501-1505.), it is necessary to screen different epitopes in different domains of the protein to avoid the ineffective secretion of overly complex protein structures, and to induce better immune responses by presenting as many effective antigen determinants as possible.

[0007] Therefore, to obtain an efficient SARS-CoV-2 coronavirus vaccine based on an attenuated Salmonella secretion expression system, a safe, efficient and stable Salmonella secretion expression vector must be constructed to express antigen molecules in Salmonella and effectively secret them into antigen-presenting cells to induce efficient immune responses in the body, which is the problem to be solved by the present application.

[0008] Different proteins have different primary sequences, resulting in large differences in the hydrophobic properties and charge distribution of different proteins, and different primary sequences of different proteins result in different spatial structures or higher structures of different proteins, leading to large differences in the spatial configuration of proteins and the physical and chemical properties of the protein surface. Therefore, it is extremely difficult to achieve efficient, stable and secretory expression of different antigen proteins in Salmonella, which cannot be predicted or inferred and requires individual research and exploration for different proteins, requiring creative labor.

[0009] More importantly, for a multi-domain protein like the S protein of the new coronavirus, the number of amino acids in the mature protein is more than 1368 amino acid residues, and different antigen epitope clusters are located in different domain segments and different spatial positions. When the S protein is used as a new coronavirus vaccine, the common method for all vaccines currently developed internationally is to use the whole protein as a vaccine combined with an adjuvant, regardless of the specific form of the vaccine, whether it is a protein vaccine, a virus vaccine, or a nucleic acid vaccine, etc. However, if each subunit of the S protein is enhanced with an adjuvant and then different subunit vaccines are used in combination, can the immune effect of each subunit vaccine be fully and better exhibited? The immune effect of the combined use of each subunit vaccine is theoretically better than that of the whole protein as an immune unit.

[0010] However, this technical path is difficult to implement because, in theory, it has higher research and development and preparation costs and more complex technology. One of the inventive points of the present application is to use a simple, low-cost and safe technical path to achieve a new approach to the development of a new coronavirus vaccine that has not been attempted by anyone internationally so far. SUMMARY

[0011] The main purpose of the present application is to construct efficient attenuated Salmonella vectors for intracellular controllable secretion expression of antigen-presenting cells, and to screen out the best immune response induced by the coronavirus antigen epitope domain, to establish a stable, safe and efficient coronavirus single antigen domain vaccine using attenuated Salmonella as a transport vector; on this basis, the attenuated Salmonella vaccine strains expressing different coronavirus single antigen epitope domains are mixed for application, to develop a stable, safe and efficient novel coronavirus oral vaccine.

[0012] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a novel coronavirus vaccine based on attenuated Salmonella controllable secretion expression, containing an expression vector of a novel coronavirus vaccine antigen and an antigen presentation system.

[0013] Further, the antigen presentation system is an attenuated Salmonella antigen presentation cell intracellular inducible secretion expression oral vaccine presentation system adapted to different domains of the S protein; the antigen presentation system is a Salmonella type III secretion antigen expression system induced by the intracellular environment of antigen presentation cells, including a type III secretion system promoter and a signal peptide sequence.

[0014] The present application is based on the analysis of the intracellular microenvironment of antigen presenting cells such as macrophages, and uses biological information methods and molecular cloning techniques to clone bacterial promoters and secretion signals; for each new coronavirus single antigen domain, the intracellular inducible promoter is used to regulate the bacterial secretion signal to secrete and express the domain antigen, and the plasmid loss prevention element is added to improve the stability of the plasmid in the bacteria, thereby constructing an efficient and stable intracellular regulated attenuated Salmonella secretion expression single antigen domain oral antigen presentation system.

[0015] The present application provides a novel coronavirus oral vaccine presentation system induced by attenuated Salmonella secretion expression, which is characterized by using the Gram-negative bacteria type III secretion system to secrete the single antigen domain of the novel coronavirus, and fusing the type III secretion signal with the domain antigen molecule to realize the secretion of the novel coronavirus antigen. The type III secretion signal is Salmonella virulence island 2 (SPI-2) effector protein SseJ, and the expression of SseJ is regulated by SseJ promoter and SifB promoter; the domain antigen molecule is the amino acid sequence of SARS-CoV-2 spike protein (S protein) NTD, RBD, S2 domain part or all. The system has been proved to be suitable for the secretion expression of the above three different sizes, structures and functions by a large number of comparative test research.

[0016] The present application provides an attenuated Salmonella antigen presentation cell intracellular inducible secretion expression oral vaccine presentation system suitable for different domains of S protein, which is characterized by being a Salmonella type III secretion antigen expression system induced by the intracellular environment of antigen presentation cells, including type III secretion system promoter and signal peptide sequence.

[0017] A variety of attenuated Salmonella antigen presentation strains that can controllably secrete and express different antigen domain proteins of the novel coronavirus in the intracellular environment of antigen presentation cells are mixed to prepare a high-efficiency, safe, antigen presentation cell intracellular expression, low-cost and convenient novel coronavirus vaccine.

[0018] Further, attenuated Salmonella antigen presentation strains that can controllably, stably and efficiently secrete and express different antigen domain proteins of the novel coronavirus in the intracellular environment of antigen presentation cells are constructed.

[0019] Further, the antigen is secreted by using the antigen presentation cell intracellular inducible promoter to regulate the bacterial secretion signal, and the antigen is secreted by using the Salmonella secretion expression system, and the plasmid loss prevention element is added to improve the plasmid stability in the expression vector in the Salmonella, so as to obtain an antigen that is suitable for different antigen domains to realize efficient, stable and intracellular regulated secretion expression in attenuated Salmonella.

[0020] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0021] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0022] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0023] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0024] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0025] Further, the combination of the SARS-CoV-2 antigen epitope domains that can induce the best immune response is the SARS-CoV-2 spike protein (S protein) RBD domain, located at amino acids 319-541 of the entire amino acid sequence of the S protein; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6.

[0026] Further, a mixed strain vaccine of multiple recombinant attenuated Salmonella is prepared, and the mixed strain must contain two kinds of recombinant attenuated Salmonella respectively loaded with the Ah-BJ-RBD and Ah-BJ-NTD expression plasmids, and the number of strains in the mixed strain must be not less than the above two kinds of strains.

[0027] The application of the preparation method of the novel coronavirus vaccine based on the attenuated Salmonella controllable secretion expression provided by the application can induce the body to produce higher titer antibodies in a mouse model by orally administering the mixed strain of attenuated Salmonella containing the expression vector of the novel coronavirus vaccine antigen.

[0028] The preparation method of the novel coronavirus vaccine based on the attenuated Salmonella controllable secretion expression provided by the application is applied to the preparation of the novel coronavirus vaccine and the prevention of the novel coronavirus drug.

[0029] The application provides a novel method for preparing a novel coronavirus vaccine, specifically, a mixed bacteria vaccine is prepared by mixing recombinant attenuated Salmonella expression strains expressing single antigen domains (such as NTD, RBD, S2 and the like) of SARS-CoV-2 spike protein (the RBD domain is located at amino acids 319-541 in the overall amino acid sequence of the S protein; the S2 domain is located at amino acids 886-1077 in the overall amino acid sequence of the S protein; and the NTD domain is located at amino acids 13-303 in the overall amino acid sequence of the S protein) in a certain proportion, so that the body can be induced to produce the best immune response after vaccination, thereby providing high protection.

[0030] Beneficial effects: The application provides a novel, efficient, safe, antigen-presenting cell intracellular expression, low-cost, convenient, novel coronavirus vaccine preparation method and application for humans and animals. The application constructs controllable, stable and secretory expression expression plasmids of different antigen domain proteins of the novel coronavirus and attenuated Salmonella expression strains thereof; multiple attenuated Salmonella antigen-presenting strains that can be controllably secreted in antigen-presenting cells are mixed, and multiple different antigen proteins can be efficiently secreted in antigen-presenting cells by means of the unique secretion system thereof through the oral route after administration. The secreted antigenic proteins can be effectively processed and presented by the antigen-presenting cells, and finally the immune system can be activated / regulated to produce higher titer antibodies, thereby playing the role of the vaccine.

[0031] Compared with the existing and the novel coronavirus vaccines in the development process, the application has the following characteristics and innovations:

[0032] (1) The application discloses a novel coronavirus vaccine antigen expression vector using a Salmonella type III secretion system signal, which realizes controllable secretion expression of antigen molecules of different domains of the novel coronavirus and induces high-titer antibodies in a mouse model.

[0033] (2) The application discloses a novel method for preparing a novel coronavirus vaccine by using mixed bacteria, wherein the vaccine is prepared by using novel attenuated Salmonella as a carrier.

[0034] (3) The application discloses a novel coronavirus vaccine prepared by using mixed bacteria, wherein the vaccine uses attenuated Salmonella as a carrier to express various antigen epitopes or domains of the novel coronavirus, thereby enhancing the immune response ability of the body to the vaccine and making up for the deficiency that a single antigen epitope / domain or a whole protein containing multiple domains cannot induce a strong immune response due to the difficulty in fully displaying the multiple domain epitopes.

[0035] (4) The application discloses a novel coronavirus vaccine preparation method by using mixed bacteria, wherein the vaccine is prepared by using novel attenuated Salmonella as a carrier.

[0036] (5) The application discloses a novel coronavirus vaccine expressed in antigen presenting cells, which is only expressed in antigen presenting cells, can effectively produce the immune effect of the vaccine, can effectively avoid the existence of antigens in normal tissues and possible side effects, and can realize higher safety.

[0037] (6) The application discloses a safe and controllable oral vaccine system, which uses novel attenuated Salmonella as a carrier and can be effectively and quickly eliminated by taking conventional Salmonella sensitive antibiotics.

[0038] (7) Although the RBD, NTD and S2 domain recombinant engineering bacteria express and secrete the three domain proteins at the same level, and the use amount of each recombinant engineering bacteria is the same in all experimental groups, the immune effects produced by them and combinations are quite different, which cannot be found and obtained by developing a single antigen domain or based on theoretical prediction, thereby highlighting the necessity and innovation of the design of the application. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1The schematic diagram of the different expression systems of the novel coronavirus RBD domain of the application is constructed.

[0040] 1. Ah-JP-RBD plasmid, using J23100 promoter, pelB signal peptide, expressing RBD protein; 2. Ah-NS-RBD plasmid, using NirB promoter, the gene sequence of the NirB promoter is the nucleotide sequence shown in SEQ ID No. 1; SseJ signal peptide, expressing RBD protein; 3. Ah-SS-RBD plasmid, using SseA promoter, the gene sequence of the sseA promoter is the nucleotide sequence shown in SEQ ID No. 2; SseJ signal peptide, expressing RBD protein; 4. Ah-JJ-RBD plasmid, using SseJ promoter, the gene sequence of the sseJ promoter is the nucleotide sequence shown in SEQ ID No. 3; SseJ signal peptide, expressing RBD protein; 5. Ah-BJ-RBD plasmid, using SifB promoter, SseJ signal peptide, expressing RBD protein. The three recombinant attenuated Salmonella of Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD can effectively induce the body to produce corresponding antibodies, and the antibody induction production effect of the Ah-BJ-RBD recombinant attenuated Salmonella is significantly better than that of the other three recombinant bacteria. The ELISA method for determining RBD protein titer of the Ah-BJ-RBD recombinant attenuated Salmonella is 6.03, 3.55 and 2.19 times higher than that of the Ah-NC, Ah-JP-RBD and Ah-JJ-RBD recombinant attenuated Salmonella, respectively.

[0041] Figure 2 The schematic diagram of the controllable secretion expression system of the novel coronavirus RBD, NTD and S2 domain of the application is constructed.

[0042] 1. Ah-BJ-RBD plasmid, using SifB promoter, SseJ signal peptide, expressing RBD domain protein. 2. Ah-BJ-NTD plasmid, using SifB promoter, SseJ signal peptide, expressing NTD domain protein. 3. Ah-BJ-S2 plasmid, using SifB promoter, SseJ signal peptide, expressing S2 domain protein. The Western blot result shows that the recombinant attenuated Salmonella engineering bacteria containing the three expression plasmids express and secrete RBD, NTD and S2 domain proteins in the intracellular macrophages; and do not express in the LB culture medium.

[0043] Figure 3The recombinant attenuated Salmonella engineering bacteria of the immunofluorescence detection of the present application express and secrete RBD, NTD and S2 domain proteins in macrophages. 1. The recombinant attenuated Salmonella is loaded with an expression plasmid without protein sequence and HA tag but with the same other elements; 2. Ah-BJ-RBD recombinant attenuated Salmonella; 3. Ah-BJ-NTD recombinant attenuated Salmonella; 4. Ah-BJ-S2 recombinant attenuated Salmonella. The nucleus is stained with DAPI, and the Ah-1 is stained with a Salmonella fluorescent antibody (arrow), and the RBD-HA, NTD-HA and S2-HA are stained with a HA tag fluorescent antibody (arrow). The three kinds of recombinant attenuated Salmonella engineering bacteria express and secrete RBD, NTD and S2 domain proteins in macrophages. The same HA antibody is used to compare the expression and secretion levels of the three domain proteins expressed and secreted by the RBD, NTD and S2 domain recombinant engineering bacteria, and the results show that the levels of the three domain proteins expressed and secreted by the RBD, NTD and S2 domain recombinant engineering bacteria are the same.

[0044] Figure 4 is an evaluation of the antibody production induced by oral administration of the new coronavirus vaccine prepared by mixing the recombinant attenuated Salmonella of the present application.

[0045] Figure 4A Figure 4 is an evaluation of the antibody production induced by oral administration of the new coronavirus vaccine prepared by mixing the recombinant attenuated Salmonella of the present application.

[0046] Figure 4B Figure 4 is an evaluation of the antibody production induced by oral administration of the new coronavirus vaccine prepared by mixing the recombinant attenuated Salmonella of the present application. DETAILED DESCRIPTION

[0047] The application will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are used to illustrate the application, but are not used to limit the scope of the application. The S protein region of the novel coronavirus SARS-COV-2 (including RBD, NTD, S2) is used as an example of delivering antigens. The attenuated Salmonella used in the examples is the htrA-deficient VNP20009 attenuated strain (referred to as Ah-1). If the specific conditions are not specified in the examples, the routine conditions or the conditions recommended by the manufacturer are used. Example 1

[0048] Construction of antigen protein novel coronavirus protein RBD delivery plasmid

[0049] RBD is a partial structural region in the S protein of the novel coronavirus. Structural analysis shows that the RBD protein plays a key role in the process of S protein binding to ACE2 (angiotensinase 2). Considering the large structural region of S, a large number of, multi-angle bioinformatics analysis and mutual comparison and prediction found that RBD located at 319-541 of the overall amino acid sequence of S protein contains more antigenic determinant sites. After a large number of experimental studies and attempts of various reported common bacterial secretion systems, secretion signal peptides and matching promoters, the strong constitutive promoter J23100 and the pelB signal peptide were selected for use in the constitutive expression secretion system Ah-JP-RBD plasmid. For the inducible expression type III secretion expression plasmids Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD, the inventors respectively tried to use the anoxic promoter NirB, the type III secretion system related promoters SseA, SseJ, and SifB, and the type III secretion system related signal peptide SseJ in the four plasmids. The N-terminus of the above five plasmids is coupled to the NTD protein sequence of the antigen molecule by means of a linker to realize its secretion. In order to facilitate subsequent detection, the RBD is coupled with an HA tag Figure 1 ). The gene sequence of the Linker sequence is the nucleotide sequence shown in SEQ ID No. 9;

[0050] The NirB, SseA, SseJ, and SifB promoters, SseJ, PelB signal peptide, and RBD in the SARS-COV-2 S protein region were obtained by PCR method. The related primer sequences are:

[0051] PNirB P1; the gene sequence of the PNirB P1 primer is the nucleotide sequence shown in SEQ ID No. 11;

[0052] PNirB P2; the gene sequence of the PNirB P2 primer is the nucleotide sequence shown in SEQ ID No. 12;

[0053] PsseA P1; the gene sequence of the PsseA P1 primer is the nucleotide sequence shown in SEQ ID No. 13;

[0054] PsseA P2; the gene sequence of the PsseA P2 primer is the nucleotide sequence shown in SEQ ID No. 14;

[0055] PsifB P1: 5'-caaaatcccttataagaattctgccctaccgctaaacatc-3'; the gene sequence of the PsifB P1 primer is the nucleotide sequence shown in SEQ ID No. 15;

[0056] PsifB P2: 5'-tgtccaacactcaatggcatccacaagtgattatatgata-3'; the gene sequence of the PsifB P2 primer is the nucleotide sequence shown in SEQ ID No. 16;

[0057] SseJ P1: 5'-tatcatataatcacttgtggatgccattgagtgttggaca-3'; the gene sequence of the SseJ P1 primer is the nucleotide sequence shown in SEQ ID No. 17;

[0058] SseJ P2: 5'-gccttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the SseJ P2 primer is the nucleotide sequence shown in SEQ ID No. 18;

[0059] PsseJ-sseJ P1: 5'-caaaatcccttataagaatttcacataaaacactagcact-3'; the gene sequence of the PsseJ-sseJ P1 primer is the nucleotide sequence shown in SEQ ID No. 19;

[0060] PsseJ-sseJ P2: 5'-GCCttcagtggaataatgatgagctataaaactttctaac-3'; the gene sequence of the PsseJ-sseJ P2 primer is the nucleotide sequence shown in SEQ ID No. 20;

[0061] 50 ng of Salmonella genomic DNA as a template.

[0062] RBD P1: 5'-agcggaggtggaggcagcccgaacatcaccaacctg-3'; the gene sequence of the RBD P1: primer is the nucleotide sequence shown in SEQ ID No. 21;

[0063] RBD P2: 5'-tctggaacatcgtatgggtacggcgcgtgcagcagttc-3'; the gene sequence of the RBD P2: primer is the nucleotide sequence shown in SEQ ID No. 22;

[0064] Vec P1: 5'-tacccatacgatgttccagattacg-3'; the gene sequence of the RBD P2: primer is the nucleotide sequence shown in SEQ ID No. 23;

[0065] Vec P2: 5'-gctgcctccacctccgctgc-3'; the gene sequence of the RBD P2: primer is the nucleotide sequence shown in SEQ ID No. 24;

[0066] The plasmid pQE30 with AT element in the laboratory was used as a template. The Linker sequence between the signal peptide and the target protein was obtained by hot annealing self-ligation method. After obtaining each fragment by PCR, the corresponding fragments were assembled by homologous recombination method, and finally various protein expression and secretion vectors of type III secretion system were obtained, including Ah-JP-RBD, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD.

[0067] Example 2. Electroporation transformation of recombinant attenuated Salmonella:

[0068] Preparation of Salmonella electroporation competent: Inoculate fresh attenuated Salmonella into 200 mL LB medium, 37°C incubator to OD value between 0.4-0.6, centrifugal 5000 rpm, 5 min to collect bacteria, washed once with sterile double distilled water, 5000 rpm, 5 min, and washed bacteria with sterilized 10% glycerol 3-5 times, centrifugal 5000 rpm, 5 min, resuspended with 500 μL 10% glycerol, 50 μL / tube for electroporation. Recombinant vaccine DNA vector was transformed into attenuated Salmonella by electroporation method: 0.5-5 μg of constructed recombinant vector was added to the electroporation competent under sterile conditions, mixed, and then transferred to a 2 mm electroporation cup for electroporation at 1.8 kV, 25 μF, and 500 Ω. After electroporation, the colonies were screened on kanamycin plates, and the obtained colonies were selected for sequencing verification. Example 3

[0069] Detection of effective secretion of antigenic protein

[0070] The obtained recombinant attenuated Salmonella was cultured in kanamycin-resistant liquid LB medium to OD600 0.8-1.0, the bacteria were collected and adjusted to OD600 value of about 1.0 with PBS. Store at 4°C for standby. Use 100 ng / mL LPS to induce macrophage cell line RAW264.7 to obtain M1 type macrophages, and the induction time is 24 hours (hereinafter referred to as RAW264.7 (M1)). The obtained RAW264.7 (M1) was co-cultured with the above-mentioned four kinds of recombinant attenuated Salmonella Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD at a ratio of 1:10 for 90 minutes, the supernatant was discarded and washed with PBS for 2-3 times, and cultured in cell culture medium (10% serum, no double antibody) added with 100 ng / mL gentamicin for 6 hours. Collect the cells and collect the total protein by heat lysis method, i.e. resuspend the cells with 100 μL PBS, add 25 μL 5X Loading Buffer, and lyse at 100°C for 10-15 minutes. After centrifugation at 9,000 rpm for 5 minutes to collect the four kinds of recombinant bacteria in LB, the total protein in the bacteria was collected by the same method.

[0071] For the Ah-JP-RBD recombinant attenuated Salmonella, the inoculated bacteria were expanded in 50 mL kanamycin-resistant liquid LB to an OD600 of about 1.0. The total protein in the supernatant was collected using the TCA (trichloroacetic acid)-acetone precipitation method. Briefly, the supernatant was transferred to a 50 ml centrifuge tube, and an ultracentrifuge was used at 15,000 g, 4 degrees, for 10 min. The supernatant was transferred to a new 50 ml centrifuge tube, 10% TCA was added, and the mixture was vortexed to mix well and then placed on ice for 30 min. The mixture was centrifuged at 7,000 g, 4 degrees, for 20 min, resuspended with 300 μl PBS, and transferred to a 1.5 ml sterile EP tube. 1.2 ml pre-cooled acetone was added (pre-cooled at -20), and the mixture was centrifuged at 17,000 g, 4 degrees, for 20 min. The supernatant was removed, 300 μl PBS was added again, and the above operation was repeated. The supernatant was removed, 40 μl PBS was added for resuspension, and the total protein secreted by the bacteria in the supernatant was obtained. The centrifugally collected bacteria were added to the loading buffer, boiled at 100 degrees for 10 min, and the total protein of the bacteria was obtained. The collected total protein was detected for the presence or absence of the target protein using the Western blot (WB) method. Rabbit monoclonal HA tag antibody was used as the primary antibody, and HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody.

[0072] The detection results showed that the Ah-JP-RBD recombinant attenuated Salmonella could effectively express and secrete RBD protein. For the Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, and Ah-BJ-RBD recombinant attenuated Salmonella, when the bacteria were present in liquid LB, none of the four bacteria expressed and secreted RBD protein. When the bacteria were inside macrophages, they were induced and stimulated by the intracellular environment, and the Ah-JJ-RBD and Ah-BJ-RBD recombinant attenuated Salmonella effectively expressed RBD protein with a large band with a signal peptide, and the protein was effectively secreted into the cell and the signal peptide was cut off, thus showing a smaller RBD protein band. However, the Ah-NS-RBD and Ah-SS-RBD engineering bacteria did not effectively express and secrete RBD protein. Example 4

[0073] Immunization procedure and method

[0074] 6-8 week old female C57BL / 6 mice were grouped according to 4-5 mice per group, and each mouse was orally administered 1×10 9 CFU bacterial dose of empty bacteria or Ah-JP-RBD, Ah-JJ-RBD, and Ah-BJ-RBD recombinant attenuated Salmonella was administered once a week, and one week after the third time, the mice were subjected to eye blood collection to detect the concentration of specific antibodies in the serum. Example 5

[0075] Antibody detection of novel coronavirus S protein RBD domain protein

[0076] Preparation of immune serum: After obtaining the blood of the immunized mice by the above method, stand at room temperature for 2-4 hours, centrifuge at 3,000 rpm at 4 degrees for 15 minutes, collect the serum and store at -80 degrees for standby.

[0077] ELISA assay of total IgG antibody titer: 1. Antigen coating, dilute the recombinant S protein antigen of SARS-CoV-2 to 10 μg / mL with 50 mM, pH 9.6 carbonate buffer, add 100 μL per well, coat the enzyme-labeled plate at 4 degrees overnight, wash three times with PBST (5 minutes each time); 2. Blocking, add 300 μL of 3% BSA per well to the coated plate, block at 37 degrees for 3 hours, and wash three times with PBST (5 minutes each time); 3. Sample addition, add 100 μL of serum gradient dilution (50-400 times) to the well with PBS at 37°C for 1-2 hours, and wash three times with PBST (5 minutes each time); 4. Secondary antibody, add HRP (horseradish peroxidase) conjugated goat anti-mouse IgG secondary antibody (diluted 1:5000 with PBST), react at 37°C for 1 hour, and wash three times with PBST (5 minutes each time); 5. Color development, after washing the plate, add 100 μL of TMB substrate solution at room temperature for color development for 5-10 minutes, and when the color turns blue, add 100 μL of 2M sulfuric acid per well to stop the reaction, and measure the 450 nM absorbance.

[0078] The antibody titer detection results show that the three kinds of recombinant attenuated Salmonella, Ah-JP-RBD, Ah-JJ-RBD, and Ah-BJ-RBD, can effectively induce the production of corresponding antibodies to the RBD protein of the novel coronavirus in mice, and the Ah-BJ-RBD recombinant attenuated Salmonella has a significantly better effect on inducing the production of antibodies than the other three kinds of recombinant bacteria; the ELISA RBD protein titer detection value of the Ah-BJ-RBD recombinant attenuated Salmonella is 6.03, 3.55, and 2.19 times higher than that of the Ah-NC, Ah-JP-RBD, and Ah-JJ-RBD recombinant attenuated Salmonella, respectively, indicating that this system is more efficient. Example 6

[0079] Construction and evaluation of Ah-BJ-NTD and Ah-BJ-S2 recombinant bacteria based on the Ah-BJ-RBD system

[0080] The NTD and S2 domains in the S protein region of the novel coronavirus SARS-COV-2 are obtained by PCR method.

[0081] NTD P1: 5'-gcagcggaggtggaggcagcgtgaatctgaccacccgc-3';

[0082] NTD P2: 5'-tctggaacatcgtatgggtagctcttcagggtgcacttg-3';

[0083] S2 P1: 5'-agcggaggtggaggcagcggtgcgggtgcggcgctg-3'; the gene sequence of the S2 P1 primer is the nucleotide sequence shown in SEQ ID No. 25;

[0084] S2 P2: 5'-tctggaacatcgtatgggtacgccggcgcggtggtaaagt-3'; the gene sequence of the S2 P2 primer is the nucleotide sequence shown in SEQ ID No. 26;

[0085] The commercial plasmid MC_0101082 was used as a template;

[0086] Vec P1: 5'-tacccatacgatgttccagattacg-3'; the gene sequence of the Vec P1: primer is the nucleotide sequence shown in SEQ ID No. 27;

[0087] Vec P2: 5'-gctgcctccacctccgctgc-3'; the gene sequence of the Vec P2: primer is the nucleotide sequence shown in SEQ ID No. 28;

[0088] The Ah-BJ-RBD was used as a template. After obtaining each fragment by PCR, the corresponding fragments were assembled by homologous recombination to obtain the NTD and S2 protein expression and secretion vector of the type III secretion system, including Ah-BJ-NTD, Ah-BJ-S2 (Ah-BJ-NTD-S2) Figure 2 ). The obtained plasmid was subjected to electric transformation to obtain the Ah-BJ-NTD and Ah-BJ-S2 recombinant attenuated Salmonella according to the description in Example 2. The expression and secretion of the two recombinant attenuated Salmonella related proteins were detected by the Ah-BJ-RBD detection method described in Example 3. The WB detection results showed that the Ah-BJ-NTD and Ah-BJ-S2 recombinant attenuated Salmonella could effectively express and secrete NTD protein and S2 protein.

[0089] The expression and secretion of antigenic proteins of the Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2 recombinant bacteria were analyzed by immunofluorescence analysis of intracellular Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2. After the RAW264.7 (M1) cells phagocytosed the Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2 recombinant bacteria according to the method described in Example 3, the cells were washed with PBS three times, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed with PBS three times. Triton X-100 was used to punch holes at room temperature for 30 minutes. After washing with PBS three times, 3% BSA was used to block the cells at room temperature for 30 minutes. After washing with PBS three times, a rabbit monoclonal HA tag antibody was used as a primary antibody to incubate the cells at 4°C overnight. After washing with PBST three times, a monkey anti-rabbit fluorescent secondary antibody and a salmonella fluorescent antibody were used to incubate the cells at room temperature for 1 hour. After washing with PBST three times, DAPI was added, and the cells were observed and photographed under a fluorescence microscope. The fluorescence imaging results showed that the Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2 recombinant attenuated salmonella effectively expressed and secreted RBD-HA, NTD-HA, and S2-HA proteins, while the recombinant attenuated salmonella carrying the empty plasmid did not detect HA-related fluorescent signals. Figure 3 . Example 7

[0090] Effect of a new coronavirus vaccine prepared by mixing Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2 recombinant attenuated salmonella on the production of S protein-specific antibodies.

[0091] On the basis of Example 4, the empty bacteria, Ah-BJ-RBD, Ah-BJ-NTD, and Ah-BJ-S2 recombinant attenuated salmonella were arranged and combined to prepare a new coronavirus vaccine. Figure 4A), and the administration method is the same as described in Example 4. Each group of induced mice to produce S protein-specific antibodies was detected according to the description in Example 5. The results showed that No. 5 was 12.02, 3.90, 2.90, 7.38, 2.67, and 2.28 times higher than No. 1, No. 2, No. 3, No. 4, No. 6, and No. 7, respectively. No. 8 was 12.63, 4.13, 3.06, 7.74, 2.81, and 2.40 times higher than No. 1, No. 2, No. 3, No. 4, No. 6, and No. 7, respectively. That is, the three strains of Ah-BJ-RBD + Ah-BJ-NTD + Ah-BJ-S2 and the three strains of Ah-BJ-RBD + Ah-BJ-NTD + Ah-BJ-NC can more efficiently induce the body to produce specific antibodies to the S protein of the new coronavirus. Therefore, the use of mixed recombinant strains to prepare a new coronavirus vaccine is a more effective method for preparing a vaccine to induce antibody production, and the vaccine prepared by mixing the two recombinant bacteria Ah-BJ-RBD and Ah-BJ-NTD has the best immune effect. Figure 4B This combination result is a never-before-seen discovery and innovative invention, which is of great significance for the development of a new coronavirus vaccine.

[0092] Although the RBD, NTD, and S2 domain recombinant engineering bacteria express and secrete the same levels of these three domain proteins, and the use amount of each recombinant engineering bacteria in all experimental groups is the same, their respective and combined immune effects are very different. This cannot be discovered and obtained by developing a single antigen domain or based on theoretical prediction, thereby highlighting the necessity and innovation of the design of the present application.

[0093] The above shows and describes the basic principles, main features, and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and the scope of protection of the present application is defined by the appended claims, the specification, and their equivalents.

Claims

1. A method for preparing a novel coronavirus vaccine based on attenuated Salmonella controllable secretion expression, characterized by: The mixed strain vaccine of multiple recombinant attenuated Salmonella is prepared, and the mixed strain must contain two recombinant attenuated Salmonella strains respectively loaded with the Ah-BJ-RBD and Ah-BJ-NTD expression plasmids, and the number of strains in the mixed strain must be not less than the above two strains; The attenuated Salmonella used is an htrA-deficient VNP20009 attenuated strain; The Ah-BJ-RBD expression plasmid uses a Salmonella SifB promoter, a Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide, and expresses a SARS-CoV-2 spike protein (S protein) RBD domain protein; The Ah-BJ-NTD expression plasmid uses a Salmonella SifB promoter, a Salmonella virulence island 2 (SPI-2) effector protein SseJ signal peptide, and expresses a SARS-CoV-2 spike protein (S protein) NTD domain protein; The gene sequence of the Salmonella SifB promoter is the nucleotide sequence shown in SEQ ID No. 4; the gene sequence of the SseJ signal peptide is the nucleotide sequence shown in SEQ ID No. 5; the gene sequence of the RBD domain is the nucleotide sequence shown in SEQ ID No. 6; and the gene sequence of the NTD domain is the nucleotide sequence shown in SEQ ID No. 7; The Ah-BJ-RBD expression plasmid and the Ah-BJ-NTD expression plasmid contain a plasmid loss-preventing element AT element, and the gene sequence of the AT element is the nucleotide sequence shown in SEQ ID No.

10.

2. Use of the preparation method of the novel coronavirus vaccine based on the controllable secretion expression of attenuated Salmonella in claim 1 in the preparation of a novel coronavirus vaccine.

3. Use of the preparation method of the novel coronavirus vaccine based on the controllable secretion expression of attenuated Salmonella in claim 1 in the preparation of a novel coronavirus vaccine.

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