CN114480463A

CN114480463A - Novel coronavirus vaccine antigen presentation system for attenuated salmonella secreting and expressing RBD structural domain protein and application thereof

Info

Publication number: CN114480463A
Application number: CN202011168321.4A
Authority: CN
Inventors: 华子春; 吴乐阳; 李霖
Original assignee: Targetpharma Laboratories Jiangsu Co ltd; Nanjing Jiruikang Biotechnology Research Institute Co ltd
Current assignee: Targetpharma Laboratories Jiangsu Co ltd; Nanjing Jiruikang Biotechnology Research Institute Co ltd
Priority date: 2020-10-26
Filing date: 2020-10-27
Publication date: 2022-05-13
Anticipated expiration: 2040-10-27
Also published as: CN114480463B; WO2022087856A1

Abstract

The invention discloses a novel coronavirus vaccine antigen presentation system for attenuated salmonella secreting and expressing RBD structural domain protein and application thereof, which are used for preventing novel coronavirus (SARS-CoV-2). The invention constructs controllable and stable expression plasmids for secreting and expressing RBD structural domain proteins and engineering modification of attenuated salmonella, so that antigenic proteins can be efficiently delivered to antigen presenting cells through an oral route and after administration by virtue of a special secretion system. The delivered antigenic protein can be effectively processed and presented by antigen presenting cells, and finally, the activation/regulation of the immune system, the generation of antibodies and the function of vaccine are realized.

Description

Novel coronavirus vaccine antigen presentation system for attenuated salmonella secreting and expressing RBD structural domain protein and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel coronavirus vaccine antigen presentation system for attenuated salmonella to secrete and express RBD structural domain protein and application thereof.

Background

The 2019 pandemic of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is the most serious challenge for humans for a century, accumulating to infect more than 2700 million people and causing death of more than 80 million people by 1/9 of 2020. This novel coronavirus is approximately 82% similar to the genome of the SARS coronavirus (SARS-CoV) which developed in 2003, and both coronaviruses also share the same cellular receptor, angiotensin converting enzyme 2(ACE2) (Lan J et al, 2020, Nature 581: 215-220.). Despite these similarities, the novel coronaviruses spread more widely, more rapidly, and more lethally than SARS virus (Zhu N et al 2020, N Engl J Med 382: 727-. Therefore, in order to effectively prevent and treat the spread of the novel coronavirus, the development of a safe, efficient and cheap SARS-CoV-2 vaccine is urgently needed. During the early development of vaccines against SARS-CoV, researchers found that antibodies against the viral spike protein (S protein) were highly effective in neutralizing the virus and preventing infection (Yang ZY et al, 2005, Proc Natl Acad Sci U S A102: 797-801.), and thus the S protein against SARS-CoV-2, and in particular the RBD region in this protein, was the primary target for current antiviral and vaccine development (Walls AC et al, 2020, Cell 181:281-292.e 6.).

At present, various research institutes and pharmaceutical companies are in rapid development of novel coronavirus vaccines, which relate to various types such as 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-. Although live attenuated vaccines show higher protection, they are associated with high risks; inactivated vaccines, recombinant virus vaccines and nucleic acid vaccines face the problems of high cost, inconvenience in inoculation and the like; however, the direct inoculation of the conventional recombinant protein vaccine cannot provide a good protective effect because the conventional recombinant protein vaccine cannot stimulate the cellular immune response well, and often needs the addition of an additional adjuvant (Guy B et al, 2007, Nat Rev Microbiol 5: 505-517.). So far, all developed vaccines at home and abroad are administrated by injection, and the side effects of the new coronary vaccine are reported in a plurality of countries internationally at present, and even death cases are reported. Injection is the most direct route of administration, but is the most risky and costly route of administration of all routes of administration. To date, there is a lack of other more convenient and safe modes or routes of administration in the development of new corona vaccines.

The working principle of the vaccine is as follows: after the vaccine antigen is inoculated to the animal body, the immune system of the animal body is stimulated, the antigen presenting cell of the animal body processes, processes and presents the vaccine to specific lymphocyte (T and B lymphocyte), then the lymphocyte recognizes, activates, proliferates and differentiates the vaccine to finally generate immune effector molecule (antibody and cell factor) and immune effector cell, and finally the antigen is eliminated from the animal body, and the process is called immune response. Therefore, the administration mode of the injected antigen in the development of the novel coronavirus vaccine which is currently widespread internationally is likely to cause some side effects of the injected antigen in the tissues of the whole body, and the process of developing the vaccine to trigger the immunity is actually generated by antigen presenting cells. Therefore, if the antigen is restricted to be produced in the antigen-presenting cells, it is possible to limit the side effects that the antigen may produce in the tissues of the whole body, and achieve a safer target.

The oral vaccine based on the attenuated strain is widely applied to the development of vaccines for various infectious diseases due to the advantages of simple inoculation, low price and the like. Attenuated salmonella is used as a carrier of oral vaccine of bacteria with wide application, and is also a natural mucosal immune adjuvant, antigen expression and delivery tool. Recombinant vaccine strains which are modified through genetic engineering enter the body through M cells in intestinal tracts after being orally taken (Jensen VB et al, 1998, infection Immun 66: 3758-.

However, the following technical problems are still faced in developing a highly effective and highly applicable oral attenuated salmonella vaccine for preventing new coronavirus: a) the phenotypic stability of recombinant bacteria is maintained, attenuated bacteria are used as an expression tool of exogenous antigens, and a proper expression strategy needs to be established to optimize the compatibility of antigen expression quantity and plasmids, otherwise, the problems of overhigh toxicity of strains, serious loss of engineering plasmids, loss of immune effect and the like can occur; b) the effectiveness of antigen protein secreted into cells by thallus, because most bacteria are wrapped by a membrane-wrapped vesicle Structure (SCV) after entering antigen presenting cells, the structure limits the effective presentation of antigen molecules to a great extent (Zhang XL et al, 2008, Cell Mol Immunol 5:91-7.), if the real effective presentation can not be realized, the prevention effect of the vaccine is greatly reduced; c) the promoter is selected so that the antigenic molecule is expressed only in the antigen presenting cells and not in blood and normal tissues, providing safety to the antigenic molecule. d) The optimality of epitope screening and region selection for the virus is that, because the S protein of the new coronavirus is large in molecular weight and complex in structure (Hsieh CL et al 2020, Science 369:1501-1505.), it is necessary to screen different epitopes in multiple domains thereof, so as to avoid that the complex protein structure cannot be effectively secreted, and to induce generation of better immune response by presenting as many effective epitopes as possible.

Therefore, to obtain a highly effective (SARS-CoV-2) new coronavirus vaccine based on attenuated Salmonella secretory expression system, it is necessary to construct a safe, highly effective and stable Salmonella secretory expression vector to achieve the expression of antigen molecules in Salmonella and to allow efficient secretion into antigen presenting cells for highly effective induction of immune response in the body, which is the problem to be solved by the present invention.

The hydrophobic properties and charge distributions of different proteins vary widely due to the different proteins having different primary sequences, which in turn cause different proteins to have different spatial or higher structures, resulting in spatial configurations of the proteins and also physical and chemical properties of the protein surface varying widely. Therefore, it is extremely difficult to achieve efficient, stable, and secretory expression of different antigenic proteins in salmonella, and it is impossible to predict or infer them, and it is necessary to study and search them one by one according to different proteins, and creative efforts are required.

Disclosure of Invention

The invention mainly aims to construct an efficient attenuated salmonella expression secretion vector and screen out a new coronavirus epitope combination capable of inducing the optimal immune response, thereby laying a foundation for establishing a novel coronavirus oral vaccine which is stable in expression and high in protective efficiency and takes attenuated salmonella as a transport vector.

In order to achieve the purpose, the invention provides the following technical scheme: the invention relates to a novel coronavirus vaccine antigen presentation system for secretion expression of RBD structural domain protein by attenuated salmonella, which comprises a III type secretion system promoter and a signal peptide sequence; the secretory expression of the novel coronavirus antigen in an antigen presenting cell can be realized by attenuated salmonella, and an organism is induced to generate high titer antibodies in a mouse model.

Furthermore, an antigen for secretory expression of a bacterial secretion signal is regulated and controlled by an intracellular inducible promoter of the antigen presenting cell, the antigen can be secreted by a salmonella secretory expression system, and a plasmid loss prevention element is added to improve the plasmid stability of an expression vector in salmonella, so that the efficient and stable antigen presenting cell intracellular regulated and controlled antigen presenting system for secretory expression of attenuated salmonella is obtained.

Furthermore, the novel coronavirus vaccine antigen is a screened novel coronavirus antigen epitope combination capable of inducing the optimal immune response, specifically, the novel coronavirus antigen epitope combination capable of inducing the optimal immune response is a SARS-CoV-2 spike protein (S protein) RBD structure domain, the gene sequence of the RBD structure domain is a nucleotide sequence shown as SEQ ID No.6, and the RBD structure domain is positioned at 319-541 bit amino acids of the whole amino acid sequence of the S protein.

Furthermore, the antigen presenting cell intracellular inducible promoter is a salmonella sifB promoter; the gene sequence of the salmonella sifB promoter is a nucleotide sequence shown in SEQ ID No. 4.

Furthermore, the intracellular inducible promoter of the antigen presenting cell is an escherichia coli NirB promoter, or a salmonella SseA promoter, or a salmonella SseJ promoter; the gene sequence of the escherichia coli sifB promoter is a nucleotide sequence shown in SEQ ID No.1, the gene sequence of the salmonella SseA promoter is a nucleotide sequence shown in SEQ ID No.2, and the gene sequence of the salmonella SseJ promoter is a nucleotide sequence shown in SEQ ID No. 3.

Further, the bacterial secretion signal secretion expression system is a salmonella III type secretion expression system, the III type secretion signal is a salmonella virulence island 2(SPI-2) effector protein SseJ signal peptide, and the gene sequence of the salmonella virulence island 2(SPI-2) effector protein SseJ signal peptide is a nucleotide sequence shown in SEQ ID No. 5.

Furthermore, the plasmid anti-loss element is an AT element sequence, and the gene sequence of the AT element sequence is a nucleotide sequence shown in SEQ ID No. 8.

Further, expression of the vaccine antigen is achieved by attenuated salmonella, which is VNP20009, htrA gene-deficient attenuated salmonella VNP20009 (Ah-1).

The application of the novel coronavirus vaccine antigen presentation system for the attenuated salmonella secretly expressing the RBD structural domain protein induces the body to generate high-titer antibodies in a mouse model by orally taking the attenuated salmonella containing the novel coronavirus vaccine antigen presentation system for the attenuated salmonella secretly expressing the RBD structural domain protein.

The invention discloses an application of a novel coronavirus vaccine antigen presentation system for secretion expression of RBD structural domain protein by attenuated salmonella in preparation of a novel coronavirus vaccine and a medicament for preventing a novel coronavirus.

Has the advantages that: the invention relates to a novel coronavirus vaccine secretion expression vector which is brand-new, efficient, safe, low-cost and convenient, can express antigen presenting cells in cells and can be used for human and animals, and application thereof. The invention can efficiently deliver antigenic protein to antigen presenting cells through oral route and by virtue of a special secretion system after administration by constructing a controllable and stable expression plasmid for secreting and expressing RBD structural domain protein and engineering modification of attenuated salmonella. The delivered antigenic protein can be effectively processed and presented by antigen presenting cells, and finally, the activation/regulation of the immune system, the generation of antibodies and the function of vaccine are realized.

Compared with the existing and developing novel coronavirus vaccines, the invention has the characteristics and innovation points that:

(1) the invention discloses a novel coronavirus vaccine antigen expression vector guided by salmonella III type secretion system signals, realizes the secretory expression of a novel coronavirus antigen, and induces an organism to generate a high-titer antibody in a mouse model.

(2) The invention discloses a novel coronavirus vaccine prepared from novel oral bacteria, which uses novel attenuated salmonella as a vector, and the novel attenuated salmonella can be used as a high-efficiency adjuvant due to weak pathogenicity, so that additional adjuvant addition is omitted.

(3) The invention discloses a novel oral bacteria coronavirus vaccine with low cost, which uses novel attenuated salmonella as a vector, and has the characteristics of low production cost, greatly shortened vaccine preparation time and simplified operation process due to the characteristic that attenuated strains can be automatically propagated, so that the finally obtained product has stronger price advantage.

(4) The novel coronavirus vaccine expressed in the antigen presenting cells is only expressed in the antigen presenting cells, so that the immune effect of the vaccine can be effectively generated, and the existence of the antigen in normal tissues and possible side effects can be effectively avoided, so that higher safety can be realized.

(5) The present invention is a safe and controlled oral vaccine system using novel attenuated salmonella as a carrier that can achieve effective and rapid regression by administering conventional salmonella-sensitive antibiotics.

Drawings

FIG. 1 is a schematic representation of the novel coronavirus RBD domain of the present invention and its receptor binding;

FIG. 1A is a schematic diagram showing the structure of the new coronavirus RBD protein domain of the present invention at the position in the S protein;

FIG. 1B is a schematic diagram showing the binding of the novel coronavirus RBD protein domain of the present invention and its receptor ACE2 protein, 1.ACE2 protein; an RBD protein domain; an enlarged schematic diagram of the binding region of ACE2 and RBD protein domain.

FIG. 2 is a schematic diagram showing the construction of different novel coronavirus RBD protein domain expression systems of the present invention;

Ah-JP-RBD plasmid, using J23100 promoter, pelB signal peptide, expression of RBD protein; 2. the Ah-NS-RBD plasmid uses a salmonella NirB promoter, and the gene sequence of the salmonella NirB promoter is a nucleotide sequence shown as SEQ ID No. 1; an SseJ signal peptide, expressing an RBD protein; 3. an Ah-SS-RBD plasmid, wherein an SseA promoter is used, and the gene sequence of the SseA promoter is a nucleotide sequence shown in SEQ ID No. 2; an SseJ signal peptide, expressing an RBD protein; an Ah-JJ-RBD plasmid, wherein an SseJ promoter is used, and the gene sequence of the SseJ promoter is a nucleotide sequence shown as SEQ ID No. 3; an SseJ signal peptide, expressing an RBD protein; 5, Ah-BJ-RBD plasmid, using a SifB promoter, wherein the gene sequence of the SifB promoter is a nucleotide sequence shown in SEQ ID No. 4; SseJ signal peptide, using SifB promoter, RBD protein was expressed.

FIG. 3 is a diagram showing the expression and secretion of RBD protein of a novel coronavirus of recombinant attenuated Salmonella of WB assay in different expression systems according to the present invention;

FIG. 4 is a diagram showing the intracellular expression and secretion of the novel coronavirus RBD protein of the immunofluorescence assay recombinant attenuated Salmonella engineered bacteria of the present invention;

1. recombinant attenuated salmonella is loaded with a plasmid that does not contain the RBD-HA sequence, but the remaining elements are identical; 2. Ah-JJ-RBD recombinant attenuated salmonella; Ah-BJ-RBD recombinant attenuated Salmonella. DAPI stains the nucleus, Ah-1 by Salmonella fluorescent antibody (arrow), and RBD-HA by corresponding fluorescent antibody (arrow).

FIG. 5 is a schematic diagram of an experiment for immunizing a mouse with the recombinant bacterium of the present invention;

FIG. 6 is a graph showing the evaluation of Ah-JP-RBD, Ah-JJ-RBD, Ah-BJ-RBD3 recombinant attenuated Salmonella of the present invention inducing the production of antibodies against the RBD protein of the novel coronavirus;

a blank control group Ah-NC recombinant attenuated salmonella; Ah-JP-RBD recombinant attenuated Salmonella; 3, Ah-JJ-RBD recombinant attenuated salmonella; Ah-BJ-RBD recombinant attenuated Salmonella. The 3 recombinant attenuated salmonella can effectively induce a mouse to generate a corresponding antibody of a new coronavirus RBD protein, wherein the effect of inducing the Ah-BJ-RBD recombinant attenuated salmonella to generate the antibody is obviously better than that of other 3 recombinant bacteria, and compared with Ah-NC, Ah-JP-RBD and Ah-JJ-RBD recombinant attenuated salmonella ELISA RBD protein titer detection values, the titer of the Ah-NC, Ah-JP-RBD and Ah-JJ-RBD recombinant attenuated salmonella ELISA RBD protein is respectively improved by 6.03,3.55 and 2.19 times, which indicates that the system is more efficient.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The RBD protein region of the novel coronavirus SARS-COV-2S protein was used as an example for delivery antigen. The attenuated salmonella used in the examples was an htrA-deficient VNP20009 attenuated strain (designated Ah-1). The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Example 1

Construction of novel coronavirus protein RBD (radial basis function) delivery plasmid for antigen protein

RBD is a partial structural region in the S protein of the novel coronavirus (figure 1), and structural analysis shows that the RBD protein plays a key role in the binding process of the S protein and ACE2 (angiotensin enzyme 2). Considering the larger structural region of S, the RBD at position 319-541 of the overall amino acid sequence of the S protein is predicted to contain more epitope sites through a large amount of multi-angle bioinformatics analysis and mutual comparison verification. A great deal of experimental research and trial is carried out on various reported common bacterial secretion systems, secretion signal peptides and matched promoters. In Ah-JP-RBD plasmid of a constitutive expression secretion system, through screening, the invention selects and uses a strong constitutive promoter J23100 and pelB signal peptide. In inducible expression III type secretion expression plasmids Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD and Ah-BJ-RBD, the invention respectively tries to use hypoxic promoters NirB, III type secretion system related promoters SseA, SseJ and SifB, and 4 plasmids all use III type secretion system related signal peptides SseJ. The N ends of the 5 plasmids are coupled with an antigen molecule NTD protein sequence by virtue of a Linker to realize the secretion of the antigen molecule NTD protein sequence, and the gene sequence of the Linker sequence is a nucleotide sequence shown as SEQ ID No. 7. To facilitate subsequent detection, RBDs were conjugated to HA tags (fig. 2).

NirB, sseA, sseJ and sifB promoter, sseJ, PelB signal peptide, RBD in SARS-COV-2S protein region, etc. are obtained by PCR method.

The sequences of the related primers are as follows: the gene sequence of the PNirB P1 primer is the nucleotide sequence shown as SEQ ID No. 9.

PNirB P2; the gene sequence of the PNirB P2 primer is a nucleotide sequence shown as SEQ ID No. 10.

PsseA P1; the gene sequence of the PsseA P1 primer is the nucleotide sequence shown in SEQ ID No. 11.

PsseA P2; the gene sequence of the PsseA P2 primer is the nucleotide sequence shown in SEQ ID No. 12.

PsifB P1: 5'-caaaatcccttataagaattctgccctaccgctaaacatc-3'; the gene sequence of the PsifB P1 primer is the nucleotide sequence shown in SEQ ID No. 13.

PsifB P2: 5'-tgtccaacactcaatggcatccacaagtgattatatgata-3', respectively; the gene sequence of the PsifB P2 primer is the nucleotide sequence shown in SEQ ID No. 14.

SseJ P1: 5'-tatcatataatcacttgtggatgccattgagtgttggaca-3'; the gene sequence of the SseJ P1 primer is the nucleotide sequence shown in SEQ ID No. 15.

SseJ P2: 5'-gccttcagtggaataatgatgagctataaaactttctaac-3', respectively; the gene sequence of the SseJ P2 primer is the nucleotide sequence shown in SEQ ID No. 16.

PsseJ-sseJ P1: 5'-caaaatcccttataagaatttcacataaaacactagcact-3'; the gene sequence of the PsseJ-sseJ P1 primer is the nucleotide sequence shown in SEQ ID No. 17.

PsseJ-sseJ P2: 5'-GCCttcagtggaataatgatgagctataaaactttctaac-3', respectively; the gene sequence of the PsseJ-sseJ P2 primer is the nucleotide sequence shown in SEQ ID No. 18.

50ng of Salmonella genomic DNA was used as a template.

RBD P1: 5'-agcggaggtggaggcagcccgaacatcaccaacctg-3'; the gene sequence of the RBD P1 primer is a nucleotide sequence shown in SEQ ID No. 19.

RBD P2: 5'-tctggaacatcgtatgggtacggcgcgtgcagcagttc-3', respectively; the gene sequence of the RBD P2 primer is a nucleotide sequence shown in SEQ ID No. 20.

Commercial plasmid MC _0101082 was used as template;

vec P1: 5'-tacccatacgatgttccagattacg-3'; the gene sequence of the Vec P1 primer is a nucleotide sequence shown as SEQ ID No. 21.

Vec P2: 5'-gctgcctccacctccgctgc-3'; the gene sequence of the Vec P2 primer is a nucleotide sequence shown in SEQ ID No. 22.

Plasmid pQE30 with AT element was used as template in this laboratory. The Linker sequence between the signal peptide and the target protein is obtained by a thermal annealing self-linking method. After each fragment is obtained by PCR, the corresponding fragment is assembled by a homologous recombination method, and various protein expression secretion vectors of a III type secretion system are finally obtained, wherein the protein expression secretion vectors comprise Ah-JP-RBD, Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD and Ah-BJ-RBD.

Example 2

Electroporation transformation of recombinant attenuated salmonella:

preparing electrical transformation competence of salmonella: inoculating fresh attenuated salmonella into 200mL LB culture medium, shaking culturing at 37 ℃ until OD value is between 0.4-0.6, centrifuging at 5000rpm for 5min, collecting thallus, washing once with sterile double distilled water, centrifuging at 5000rpm for 5min, washing thallus with sterilized 10% glycerol for 3-5 times, centrifuging at 5000rpm for 5min, re-suspending with 500. mu.L 10% glycerol, and subpackaging at 50. mu.L/tube for electric conversion. Transforming the recombinant vaccine DNA vector into attenuated salmonella using electroporation: under the aseptic condition, 0.5-5 mu g of the constructed recombinant vector is added into an electrotransformation competence, is uniformly mixed and then is transferred into an electrotransfer cup with the diameter of 2mm for electric shock, and the electrotransfer condition is 1.8kV,25 mu F and 500 omega. After electric conversion, the bacterial colony is coated on a kanamycin plate for screening, the grown bacterial colony is the constructed recombinant bacterium, and a monoclonal bacterial strain is selected for sequencing verification.

Example 3

Efficient secretion assay for antigenic proteins

The obtained recombinant attenuated salmonella was cultured in a kanamycin-resistant liquid LB medium to OD 6000.8-1.0, and the cells were collected and adjusted to an OD600 value of about 1.0 with PBS. And placing for 4 degrees for later use. The macrophage cell line RAW264.7 was induced using 100ng/mL LPS for 24 hours to obtain M1-type macrophages (hereinafter referred to as RAW264.7 (M1)). The obtained RAW264.7(M1) and the Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD, Ah-BJ-RBD obtained above were combined to obtain 4 recombinant attenuated Salmonella bacteria in a ratio of 1: 10 Co-culture for 90 minutes, the supernatant was aspirated and washed with PBS 2-3 times, and cultured in cell culture medium (10% serum, no double antibody) supplemented with 100ng/mL gentamicin for 6 hours. The cells were harvested and total cellular protein was harvested by thermal lysis, i.e., 100. mu.L of PBS resuspended cells, and after addition of 25. mu.L of 5 Xloading Buffer, lysed at 100 ℃ for 10-15 minutes. After 4 recombinant bacteria in LB were collected by centrifugation at 9,000rpm for 5 minutes, total protein in the cells was collected by the same method.

For Ah-JP-RBD recombinant attenuated Salmonella, the inoculated cells were expanded and cultured in 50mL of Carna-resistant liquid LB to an OD600 of about 1.0. Total protein was collected in the supernatant using TCA (trichloroacetic acid) -acetone precipitation, briefly transferred to a 50ml centrifuge tube and centrifuged for 10min using an ultracentrifuge at 15,000g, 4 degrees. The supernatant was transferred to a new 50ml centrifuge tube, 10% TCA was added, vortexed to mix well, and allowed to stand on ice for 30 min. After centrifugation at 4 degrees for 20min at 7,000g, the pellet was resuspended in 300. mu.l PBS and transferred to a 1.5ml sterile EP tube. 1.2ml of pre-cooled acetone (pre-20 ℃ C.), 17,000g, 4 ℃ C. was added and centrifuged for 20 min. The supernatant was removed and 300. mu.l PBS was added again and the procedure was repeated. And removing the supernatant, and adding 40 mu l of PBS for resuspension to obtain the total protein secreted by the thalli in the supernatant. And adding the centrifugally collected thalli into a Loading buffer, and boiling for 10 minutes at 100 ℃ to obtain the total protein of the thalli. The collected total protein was examined for the presence or absence of production and secretion of the target protein by Western Blotting (WB). A rabbit monoclonal HA-tagged antibody was used as the primary antibody and a HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody.

The detection result shows that the Ah-JP-RBD recombinant attenuated salmonella can effectively express and secrete to produce the RBD protein (figure 3). For 4 recombinant attenuated Salmonella of Ah-NS-RBD, Ah-SS-RBD, Ah-JJ-RBD and Ah-BJ-RBD, when the thallus exists in the liquid LB, the expression and secretion of RBD protein do not occur in 4 bacteria, and when the thallus is positioned in the macrophage, the thallus is induced and stimulated by the intracellular environment, the recombinant attenuated Salmonella of Ah-JJ-RBD and Ah-BJ-RBD effectively expresses RBD protein (larger strip) with signal peptide, and the signal peptide (smaller strip) is cut off after the protein is effectively secreted into the cell. However, the expression and secretion of RBD protein could not be effectively realized in the Ah-NS-RBD and Ah-SS-RBD engineering bacteria, so the two engineering bacteria were discarded in the subsequent experiments (FIG. 3).

The expression and secretion of antigenic proteins by Ah-JJ-RBD, Ah-BJ-RBD recombinant bacteria in cells were analyzed by immunofluorescence, and after RAW264.7(M1) cell slide phagocytosing Ah-BJ-NC, Ah-JJ-RBD or Ah-BJ-RBD recombinant bacteria was obtained as described above, washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed 3 times with PBS. The wells were punched with 0.5% TritonX-100 in PBS for 30 minutes at room temperature. PBS was washed 3 times, after blocking with 3% BSA for 30min at room temperature, PBS was washed 3 times, using rabbit monoclonal HA-tagged antibody as the primary antibody for 4-degree overnight incubation slide. After PBST was washed 3 times, the antibody was incubated with a monkey anti-rabbit fluorescent secondary antibody and a Salmonella fluorescent antibody for 1 hour at room temperature in the dark. After PBST washing 3 times, the nuclear dye DAPI was added and photographed using fluorescence microscope observation. Fluorescence imaging results show that the Ah-JJ-RBD or Ah-BJ-RBD recombinant attenuated salmonella existing in macrophages can effectively express and secrete RBD-HA protein, and HA-related fluorescence signals are not detected by recombinant attenuated salmonella carrying no-load plasmids (figure 4).

Example 4

Immunization programs and methods

6-8 week-old female C57BL/6 mice were divided into groups of 4-5 mice each, each mouse was inoculated with either airborne bacteria or Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD3 recombinant attenuated Salmonella bacteria at an oral dose of 1X 109CFU, once a week apart, and one week after the third, and the serum concentration of specific antibodies was measured (FIG. 5).

Example 5

RBD protein antibody detection

Preparation of immune serum: after obtaining the blood of the immunized mouse according to the method, standing for 2-4 hours at room temperature, 4 ℃ at 3,000rpm, centrifuging for 15 minutes, collecting serum and storing the serum to-80 ℃ for later use.

ELISA assay total IgG antibody titer: 1. antigen coating, diluting the recombinant S protein antigen of SARS-CoV-2 with 50mM carbonate buffer solution with pH 9.6 at 10. mu.g/mL, dripping 100. mu.L of 4-degree coated ELISA plate into each hole, overnight, PBST washing three times (once for 5 min); 2. blocking, adding 300. mu.L of 3% BSA to each well of the coated plate, blocking at 37 ℃ for 3 hours, washing three times (once in 5 minutes) with PBST; 3. sample loading, serum gradient dilution (50-400 fold) into wells with 100 μ L PBS, reaction at 37 ℃ for 1-2 hours, PBST washing three times (once in 5 minutes); 4. adding secondary antibody, goat anti-mouse IgG secondary antibody (PBST 1:5000 dilution) coupled with HRP (horse radish peroxidase), reacting at 37 ℃ for 1 hour, and washing PBST three times (once in 5 minutes); 5. and (3) developing, adding 100 mu L of TMB substrate solution after washing the plate, developing for 5-10 minutes at room temperature, adding 100 mu L of 2M concentrated sulfuric acid into each hole when the color turns blue, stopping, and measuring the light absorption of 450 nM.

The result of antibody titer detection shows that 3 recombinant attenuated salmonella Ah-JP-RBD, Ah-JJ-RBD and Ah-BJ-RBD can effectively induce an organism to generate corresponding RBD protein antibodies, wherein the Ah-BJ-RBD recombinant strain has better induction effect (figure 6), and the system has stronger application value.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Sequence listing

<110> Jiangsu target biological medicine research institute limited of Nanjing Jiruikang Biotechnology research institute limited

<120> novel coronavirus vaccine antigen presentation system for attenuated salmonella secreting and expressing RBD structural domain protein and application thereof

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tgagtatcaa gccagctcat actcacgcca gcacactaaa atcaggagtg gcttcttttt 120

tagatctttg ccttagccag gcgcacactc aataatgata gcagtcagat aatatgtacc 180

aggcattaac ctcacgttgt tgatgatata tttacttcgt tgaaaaacaa taaacattgt 240

atgtatttta ttggcgacga aaaactgtta aagaagcgta attccatata caccatttac 300

ctgattactt ttcttgctaa tatttgctaa ttaattattt gctaaagcgt gtttaataaa 360

gtaaggagga cact 374

<210> 4

<211> 388

<212> DNA

<213> Artificial sequence (SifB promoter)

<400> 4

ctgccctacc gctaaacatc tcattgttgt tagcctaata atacttttag tttaacttct 60

tataagacaa tttctacacg gttgagcaac tatttacttt ctctaaaaat aatatagtgc 120

gtaattaatc attactcata gtacatgatg atgtgagaat taagaaaacc gttttacttt 180

cattcgtttt atctgacata tttcatggcc aggaggcgtg ggcatgacta aagctacggg 240

tcgatttgaa caattgaaca ataatgttga cggttcagga caaagcaaaa atcaggtgtt 300

tcaccgatag gcaaaccgat gggcaacatg ggataatatt tcgaatacca cctattccag 360

taatgaagta tcatataatc acttgtgg 388

<210> 5

<211> 1224

<212> DNA

<213> Artificial sequence (type III secretion signal is SseJ signal peptide of Salmonella virulence island 2 effector protein)

<400> 5

atgccattga gtgttggaca gggttatttc acatcatcta tcagttctga aaaatttaat 60

gcgataaaag aaagcgcacg ccttccggaa ttaagtttat gggagaaaat caaagcatat 120

ttctttacca cccaccatgc agaggcgctc gaatgtatct ttaatcttta ccaccatcag 180

gaactgaatc taacaccggt acaggttcgc ggagcctaca tcaaacttcg agccttagcg 240

tctcagggat gtaaagaaca gtttattata gaatcacagg aacacgccga taagttgatt 300

attaaagatg ataatggtga aaatattttg tctattgagg ttgaatgtca tccggaagct 360

tttggtcttg caaaagaaat caataaatca catcccaagc ccaaaaatat ttctttgggt 420

gatattacca gactggtatt ttttggcgac agcttgtctg actccttagg gcgtatgttt 480

gaaaaaacac atcatatctt accctcctat ggtcaatact ttggcggaag gtttactaat 540

ggatttacct ggactgagtt tttatcatct ccacacttct taggtaaaga gatgcttaat 600

tttgctgaag ggggaagtac atcggcaagc tattcctgct ttaattgcat cggtgacttt 660

gtatcaaata cggacagaca agtcgcatct tacacccctt ctcaccagga cctggcgata 720

tttttattgg gggctaatga ctatatgaca ctacacaaag ataatgtaat aatggtcgtt 780

gagcaacaaa ttgatgatat tgaaaaaata atttccggtg gagttaataa tgttctggtc 840

atggggattc ccgatttgtc tttaacacct tatggcaaac attctgatga aaaaagaaag 900

cttaaggatg aaagcatcgc tcacaatgcc ctgttaaaaa ctaatgttga agaattaaaa 960

gaaaaatacc cccagcataa aatatgctat tacgagactg ccgatgcatt taaggtgata 1020

atggaggcgg ccagtaatat tggttatgat acggaaaacc cttatactca ccacggctat 1080

gtacatgttc ccggggctaa agaccctcag ctagatatat gtccgcaata cgtcttcaac 1140

gaccttgtcc atccaaccca ggaagtccat cattgttttg ccataatgtt agaaagtttt 1200

atagctcatc attattccac tgaa 1224

<210> 6

<211> 576

<212> DNA

<213> Artificial sequence (SARS-CoV-2 spike protein RBD Domain)

<400> 6

ccgaacatca ccaacctgtg cccgtttggc gaggttttca acgcgacccg tttcgcgagc 60

gtgtatgcgt ggaaccgtaa acgtatcagc aactgcgttg cggactatag cgtgctgtac 120

aacagcgcga gcttcagcac ctttaagtgc tatggtgtga gcccgaccaa actgaacgat 180

ctgtgcttta ccaacgttta cgcggatagc ttcgtgattc gtggcgacga ggttcgtcag 240

atcgcgccgg gtcaaaccgg caagattgcg gactacaact ataaactgcc ggacgatttc 300

accggctgcg ttatcgcgtg gaacagcaac aacctggata gcaaagtggg tggcaactac 360

aactatctgt accgtctgtt tcgtaagagc aacctgaaac cgttcgagcg tgacattagc 420

accgaaatct accaggcggg tagcaccccg tgcaacggtg ttgagggctt taactgctat 480

ttcccgctgc aaagctacgg tttccaaccg accaacggtg ttggttacca gccgtaccgt 540

gtggttgtgc tgagctttga actgctgcac gcgccg 576

<210> 7

<211> 45

<212> DNA

<213> Artificial sequence (Linker)

<400> 7

ggcggagggg gtagcggtgg tggcggcagc ggaggtggag gcagc 45

<210> 8

<211> 1997

<212> DNA

<213> Artificial sequence (AT element)

<400> 8

gcccggggga tccgatactc tctcctttca tcgaaataac cctatgaaaa atatggctgc 60

aatcatcttg aatagtagac tcacaagggc taatacaaag cgccaaaaga ccaaacaaaa 120

taccagtatt gccaaaaata ctacgaagtt taaatgcaca gctgcagcat ggcgcataag 180

tatttcgcca acaaatagga ctgacaatag aggaatgatt ttattcatat ggatctcctt 240

ctttctctta ggttatcggg tgttgcccgc ttatttcgca ccacctaagc ggcgggggcg 300

gttggagcga ttggtgcgaa ataagaacca ttcccttagt tactaggttt tttgtcctta 360

tttcacacat tctcactttt cggtataaat gattattgcc ttttttcctc tcaaatcgtt 420

gatatggcat tcgtttacgg tcaagttcag gatccataag cgactaatgc caattgcaat 480

aggaaataaa cggttacccg ttacgttgga tgaaaagaga caaaaagaat tgcagcaact 540

aaagcagaag tacggcaaaa gtgaatccag gattatgtgt attgcgttag atttattgat 600

tgcccaagaa aaagcaggat ttgaggtacc agcactcaaa aagtgacgtc accttttatc 660

ctaaaaacta aaagtgatag cacttttaat tataagaagt tagaatatta atcatttgct 720

taattgtaca atataatgta caattgtttt atagaaataa ataaggggtg aaaggaatgg 780

aagcagtagc ttattcaaat ttccgccaaa atttacgtag ttatatgaaa caagttaatg 840

aggatgctga aacacttatt gtaacaagta aagatgtaga agatacagtt gttgtattat 900

caaaaagaga ttatgattct atgcaagaaa cgttgagaac actttctaat aattacgtca 960

tggaaaaaat tcgtcgagga gatgaacaat tctccaaagg tgcatttaaa acacatgact 1020

taatcgaggt tgaatctgat gattaaggct tggtctgatg atgcttggga tgattatctt 1080

tattggcatg agcaaggaaa caaaagcaat ataaaaaaga ttaacaagtt aataaaagat 1140

atcgatcgtt ccccctttgc tggattagga aaacctgagc cattaaagca tgatttatct 1200

ggaaaatggt ccagaagaat tacagatgaa catagactga tatatagagt tgaaaatgaa 1260

acgatattta tttattctgc aaaagatcac tattaaccaa tcggaagtaa ggaaagggtc 1320

agaaacttaa aagtttttga tccttatttt atttacccta gtcatttaaa aagctaatat 1380

agcttagtgt tgattgttat taatgaatgt gtttgttacg cgtattacgg atataaggtt 1440

agtaaaatca tttctaaagt tgaggaaaag taaatataaa tggcttaaat ttcaacaatt 1500

tgaagttgaa tagatatgtt ataatactat tgtagtgtgg gatgttagtt actaaaggat 1560

gacgcttata tatatgactg aatagaataa gcaataggtt taataatcta ttttaaattt 1620

tttgtactag ttttagtcaa ttagcaaaaa caacaaaaat aaacttctca tagaatttag 1680

ctaaaaatta atgatttatt tacatattaa atttggatac agttaagtaa tttttatata 1740

ttggaggaga agtaatggaa tataaattta acttgaattt gaaagaagta tcgagctcgg 1800

aagcttggca gcggccgctg gcgggtgtgt cgagtggatg gtaggatcga caaagatctg 1860

gctacactcg atcagcagtt agataataaa atcgctatcc atcgaagatg gatgtgtgtt 1920

ggttttttgt gtgtgtaacg caacgattga tagcataacc ccttggggcc tctaaacggg 1980

tcttgagggg ttttttg 1997

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence (PNirB P1 primer)

<400> 9

gcaaaatccc ttataagaat tgagggttac cggcccgatc 40

<210> 10

<211> 40

<212> DNA

<213> Artificial sequence (PNirB P2 primer)

<400> 10

cctctttctc tagtatctag accgcctacc ttaacgattc 40

<210> 11

<211> 40

<212> DNA

<213> Artificial sequence (PSseA P1 primer)

<400> 11

caaaatccct tataagaatt agaagagaac aacggcaagt 40

<210> 12

<211> 40

<212> DNA

<213> Artificial sequence (PSseA P2 primer)

<400> 12

tgtccaacac tcaatggcat acgatagata attaacgtgc 40

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence (PsifB P1 primer)

<400> 13

caaaatccct tataagaatt ctgccctacc gctaaacatc 40

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence (PsifB P2 primer)

<400> 14

tgtccaacac tcaatggcat ccacaagtga ttatatgata 40

<210> 15

<211> 40

<212> DNA

<213> Artificial sequence (sseJ P1 primer)

<400> 15

tatcatataa tcacttgtgg atgccattga gtgttggaca 40

<210> 16

<211> 40

<212> DNA

<213> Artificial sequence (sseJ P2 primer)

<400> 16

gccttcagtg gaataatgat gagctataaa actttctaac 40

<210> 17

<211> 40

<212> DNA

<213> Artificial sequence (PsseJ-sseJ P1 primer)

<400> 17

caaaatccct tataagaatt tcacataaaa cactagcact 40

<210> 18

<211> 40

<212> DNA

<213> Artificial sequence (PsseJ-sseJ P2 primer)

<400> 18

gccttcagtg gaataatgat gagctataaa actttctaac 40

<210> 19

<211> 36

<212> DNA

<213> Artificial sequence (RBD P1)

<400> 19

agcggaggtg gaggcagccc gaacatcacc aacctg 36

<210> 20

<211> 38

<212> DNA

<213> Artificial sequence (RBD P2 primer)

<400> 20

tctggaacat cgtatgggta cggcgcgtgc agcagttc 38

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Vec P1 primer)

<400> 21

tacccatacg atgttccaga ttacg 25

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Vec P2 primer)

<400> 22

gctgcctcca cctccgctgc 20

Claims

1.A novel coronavirus vaccine antigen presentation system for the secretion and expression of an RBD domain protein from attenuated Salmonella, comprising: the novel coronavirus vaccine antigen presentation system for the attenuated salmonella secretory expression RBD structural protein comprises a III type secretion system promoter and a signal peptide sequence; the secretory expression of the novel coronavirus antigen in an antigen presenting cell can be realized by attenuated salmonella, and an organism is induced to generate high titer antibodies in a mouse model.

2. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the antigen of the secretory expression of the bacterial secretion signal is regulated and controlled by using the intracellular inducible promoter of the antigen presenting cell, the antigen can be secreted by using a salmonella secretory expression system, and the plasmid loss prevention element is added to improve the plasmid stability of the expression vector in salmonella, so that the efficient and stable antigen presenting cell intracellular regulated and controlled antigen presenting system of the secretory expression of the attenuated salmonella is obtained.

3. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the novel coronavirus vaccine antigen is a screened novel coronavirus antigen epitope combination capable of inducing optimal immune response, and specifically, the novel coronavirus antigen epitope combination capable of inducing optimal immune response is a SARS-CoV-2 spike protein RBD structural domain, the gene sequence of the RBD structural domain is a nucleotide sequence shown as SEQ ID No.6, and is positioned at 319-541 bits amino acid of the whole amino acid sequence of S protein.

4. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the antigen presenting cell intracellular inducible promoter is a salmonella sifB promoter; the gene sequence of the salmonella sifB promoter is a nucleotide sequence shown in SEQ ID No. 4.

5. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the antigen presenting cell intracellular inducible promoter is an escherichia coli NirB promoter, or a salmonella SseA promoter, or a salmonella SseJ promoter; the gene sequence of the escherichia coli sifB promoter is a nucleotide sequence shown by SEQ ID No.1, the gene sequence of the salmonella SseA promoter is a nucleotide sequence shown by SEQ ID No.2, and the gene sequence of the salmonella SseJ promoter is a nucleotide sequence shown by SEQ ID No. 3.

6. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the bacterial secretion signal secretion expression system is a salmonella III type secretion expression system, the III type secretion signal is a salmonella virulence island 2 effector protein SseJ signal peptide, and the gene sequence of the salmonella virulence island 2 effector protein SseJ signal peptide is a nucleotide sequence shown in SEQ ID No. 5.

7. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the plasmid anti-loss element is an AT element sequence, and the gene sequence of the AT element sequence is a nucleotide sequence shown in SEQ ID No. 8.

8. The attenuated salmonella secreted novel coronavirus vaccine antigen presentation system expressing an RBD domain protein of claim 1, wherein: the expression of the vaccine antigen is realized by attenuated salmonella, wherein the attenuated salmonella is VNP20009, and htrA gene defect attenuated salmonella VNP 20009.

9. Use of the attenuated salmonella secreting a novel coronavirus vaccine antigen presentation system expressing an RBD domain protein according to any one of claims 1 to 8, characterized in that: the body is induced to produce high titer antibodies in a mouse model by orally administering attenuated salmonella containing a novel coronavirus vaccine antigen presentation system in which the attenuated salmonella secretes a protein expressing the RBD domain.

10. Use of the attenuated salmonella secreting a novel coronavirus vaccine antigen presentation system expressing an RBD domain protein according to any one of claims 1 to 8 as a medicament for the preparation of a novel coronavirus vaccine for the prevention of a novel coronavirus.