A recombinant strain and its uses

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a recombinant bacterial strain and its uses. The invention provides a recombinant plasmid comprising a nucleic acid molecule encoding a Spike protein immunoepitope polypeptide, a backbone plasmid, and acceptable expression elements; the Spike protein immunoepitope polypeptide has the amino acid sequence shown in SEQ ID NO:1. The invention also provides a recombinant bacterial strain whose surface stably displays the Spike protein immunoepitope polypeptide, which can be used to prepare oral vaccines for the prevention and / or treatment of coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant bacterial strain and its uses. Background Technology

[0002] Coronaviruses are a type of RNA virus that spreads through the respiratory tract or by contact. Common clinical manifestations after infection include fever, cough, shortness of breath, and respiratory symptoms. Severe cases can lead to pneumonia, acute respiratory syndrome, kidney failure, and even death. Due to the continuous mutation of coronaviruses, many variant strains have emerged, increasing the workload of vaccination and consuming a large amount of human, material, and financial resources.

[0003] The spike protein (S) is the main antigenic protein of coronavirus structural proteins. It contains important information about the natural evolution of coronaviruses and plays a crucial role in viral recognition and invasion of host cells. It is also an important candidate protein for the preparation of coronavirus vaccines and the development of antibody diagnostic reagents. The spike protein is a pathogenic protein on the surface of coronaviruses. Its main function is to bind to receptors on the host cell membrane, causing a conformational change that allows the hydrophobic fusion peptide to fuse nearly with the cell membrane, mediating viral invasion of normal human cells and attacking the body's immune cells. The relative molecular mass of the spike protein is approximately 2 × 10⁻⁶. 5 Composed of 1200 to 1500 amino acid residues, it contains both a receptor-binding domain (RBD) and a fusion-related domain, and is a key protein for coronavirus CoV infection of the host.

[0004] Coronaviruses can adapt to new environments through mutation or recombination, making the development of suitable vaccines an urgent priority for the effective prevention and control of their spread and the infectious diseases they cause. Oral vaccination offers advantages such as convenience, reduced hazardous waste, and the generation of mucosal immunity, making it a promising new option.

[0005] Therefore, the present invention provides a product that stably expresses Spike protein immune epitope peptides, which has great potential in the preparation of oral vaccines for the prevention and / or treatment of coronavirus infection. Summary of the Invention

[0006] This invention aims to provide an oral vaccine based on the effective immunogenic epitope of the Spike protein. A gene with an enzyme cleavage site was designed and synthesized, named SAE (Spike Antigenic epitope), and a recombinant plasmid pJS700-SAE was constructed. Competent cells of Bacillus subtilis were prepared, and the recombinant plasmid was transformed into Bacillus subtilis. Successfully transformed recombinant Bacillus subtilis was screened by PCR and amylase assays, displaying the Spike protein polypeptide on the spore surface for further synthesis of an oral vaccine and prevention of coronavirus invasion.

[0007] One objective of this invention is to provide a recombinant plasmid comprising a nucleic acid molecule encoding a Spike protein immunoepitope polypeptide, a backbone plasmid, and an acceptable expression element. The Spike protein immunoepitope polypeptide has the amino acid sequence shown in SEQ ID NO: 1.

[0008] SEQ ID NO: 1: NKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF.

[0009] In some embodiments, the nucleic acid molecule encoding the Spike protein immunoepitope polypeptide has a nucleotide sequence as shown in SEQ ID NO: 2.

[0010] SEQ ID NO: 2: .

[0011] In some embodiments, the backbone plasmid includes shuttle plasmids and integrative plasmids.

[0012] In some preferred embodiments, the backbone plasmid is an integrative plasmid.

[0013] In some preferred embodiments, the backbone plasmid is pJS700.

[0014] In some implementations, the acceptable expression elements include, but are not limited to, one or more of promoters, enhancers, and terminators.

[0015] A second objective of this invention is to provide a recombinant strain, which is transformed and / or transfected with the aforementioned recombinant plasmid.

[0016] In some embodiments, the chassis strains transformed and / or transfected include one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris.

[0017] In some preferred embodiments, the chassis strain being transformed and / or transfected is Bacillus subtilis.

[0018] A third objective of this invention is to provide a method for preparing a recombinant bacterial strain, the method comprising: (1) Integrate the nucleic acid molecule encoding the Spike protein immunoepitope polypeptide into the integration site of the backbone plasmid to obtain a recombinant plasmid; (2) Transform and / or transfect the recombinant plasmid into the chassis strain to obtain the recombinant strain.

[0019] The fourth objective of this invention is to provide a culture obtained by culturing the aforementioned recombinant strain or the recombinant strain obtained by the aforementioned preparation method under conditions suitable for growth and / or proliferation.

[0020] In some embodiments, the culture contains the Spike protein immunoepitope polypeptide, which has biological function.

[0021] The fifth objective of this invention is to provide the use of the aforementioned recombinant plasmids, recombinant strains, recombinant strains obtained by the aforementioned preparation methods, and / or the aforementioned cultures in the preparation of oral vaccines for the prevention and / or treatment of coronaviruses.

[0022] In some implementations, the coronavirus is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0023] In some embodiments, the oral vaccine further includes a pharmaceutically acceptable vaccine adjuvant and / or a pharmaceutically acceptable excipient.

[0024] In some embodiments, the oral vaccine further includes a second antigen for inducing and / or activating a host immune response.

[0025] Bacillus subtilis is a Gram-positive, non-pathogenic probiotic. Because Bacillus subtilis spores have extremely strong resistance and can survive for a long time under extreme adverse conditions, Bacillus subtilis can become an effective carrier for transporting exogenous proteins in extreme environments.

[0026] This invention constructs a recombinant plasmid containing a nucleic acid molecule encoding a Spike protein immunoepitope polypeptide. Utilizing the characteristics of Bacillus subtilis, the Spike protein immunoepitope polypeptide is displayed on the surface of Bacillus subtilis using Bacillus subtilis surface display technology to construct recombinant Bacillus subtilis. Subsequently, successfully transformed recombinant Bacillus subtilis is screened out and used as an immunogen to prepare an oral vaccine for the prevention and / or treatment of coronaviruses.

[0027] Compared to existing technologies, this invention uses the non-pathogenic Gram-positive probiotic Bacillus subtilis as a carrier, leveraging the strong stress resistance of its spores to ensure stable survival in extreme environments and harsh gastrointestinal conditions without requiring stringent cold chain storage and transportation. Through surface display technology, the coronavirus Spike protein immunoepitaph peptide is presented on the bacterial surface, resulting in sufficient antigenic epitope exposure and strong immunogenicity, effectively activating the body's mucosal, humoral, and cellular immune responses. This recombinant strain is highly safe, has no toxic side effects, and can be formulated into an oral vaccine, offering convenient administration and good patient compliance. Furthermore, the strain's culture process is simple and easily industrialized for large-scale fermentation. It can also transiently colonize the intestine and continuously induce specific immune responses, providing long-lasting immune protection, demonstrating promising application prospects in the prevention and treatment of coronaviruses. Attached Figure Description

[0028] Figure 1 The results of agarose gel electrophoresis of the SAE gene cloned using pUC57-SAE plasmid as a template are shown.

[0029] Figure 2 The agarose gel electrophoresis results during the construction of the recombinant plasmid pJS700-SAE are shown. A represents the agarose gel electrophoresis results of the recombinant plasmid pET30a-SAE; B represents the agarose gel electrophoresis results of the enzyme-digested pJS700 plasmid; C represents the agarose gel electrophoresis results of the ligation of the enzyme-digested pJS700 and SAE fragments into the recombinant plasmid pJS700-SAE; and D represents the agarose gel electrophoresis results of colony PCR identification of the recombinant plasmid pJS700-SAE.

[0030] Figure 3 A flowchart for constructing the recombinant plasmid pJS700-SAE is shown.

[0031] Figure 4 The results of amylase activity analysis of wild-type and recombinant Bacillus subtilis strains are shown. In the figure, A represents wild-type Bacillus subtilis on LB starch plates (…). Bacillus subtilis ) and recombinant Bacillus subtilis ( Transgenic Bacillus subtilis B represents wild-type Bacillus subtilis stained with iodine-potassium iodide solution on LB starch plates. Bacillus subtilis ) and recombinant Bacillus subtilis ( Bacillus subtilis ). Detailed Implementation

[0032] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0033] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0034] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0035] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0036] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0037] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0038] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0039] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0040] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0041] Experimental Materials and Methods : Experimental materials: Bacillus subtilis, Escherichia coli DH5α, BL21, plasmid pET-30a, plasmid pJS700, PCR reagents (TaKaRa LA Taq, 10×LA Taq Buffer, 25 mM MgCl2, dNTP Mixture), DNA ligase, and T4 DNA ligase were all obtained from the School of Biological and Food Engineering, Suzhou University. Specific primers for PCR amplification, stab-forming bacteria containing the SAE gene, and pUC57 plasmid were all purchased from General Biotechnology (Anhui) Co., Ltd.

[0042] Restriction endonucleases ( Nco I. Sac I. Kpn I), DNA Ladder Marker, sterile water, 70% ethanol, 75% ethanol, CaCl2 solution, 10% glycerol, 30% glycerol, basic plasmid extraction buffer (Solution I: Tris-HCl 25 mM, glucose 50 mM, EDTA 10 mM; Solution II: 1% SDS, NaOH 200 mM, mix well before use; Solution III: glacial acetic acid, potassium acetate 5 M, distilled water), ethidium bromide (EB), CTAB extraction buffer (1 mol / L NaCl, 100 mmol / L Tris-HCl pH 8.0, 1% CTAB), lysozyme (10 mg / mL), DNA extraction reagent, sodium acetate, ampicillin 100 μg / mL, erythromycin, 1% agarose gel, TE buffer, 2 mg / mL L-Trp solution, GM I solution, GM II solution, TBE buffer, LB liquid medium, LB solid medium, LB starch solid medium, etc.

[0043] PCR amplification instrument (T100 Thermal Cycler 621BR44402), high-speed refrigerated centrifuge (D-37520Osterode am Harz), electrophoresis apparatus (DYY-10C type), ultrapure water purifier (Heal Force PWVF 0808026), gel imaging equipment (Mfg Serial C19ADB00603), micro spectrophotometer (Thermo Science NanoDrop2000), constant temperature water bath (Water Bath HH-4), ice maker (IMS-100), autoclave (ZEALWAY G1100T), shaker (ZQTY-70N), constant temperature incubator (GHP-9270N), 4℃ refrigerator (SC-450G), ultra-clean workbench (SW-CJ-2D), electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.), electronic balance (Ohaus Corporation), etc.

[0044] Example 1: Construction of the integrative recombinant plasmid pJS700-SAE 1.1 Origin of SAE gene sequence and PCR amplification The gene sequence encoding the Spike protein was retrieved from NCBI (NCBI accession number: YP_009724390.1). A suitable Spike protein immunoepitope polypeptide was selected, and its amino acid sequence is shown in SEQ ID NO: 1. Based on the immunoepitope sequence, a gene sequence with restriction enzyme sites was designed and synthesized, named SAE (Spike Antigenicepitope), and its nucleotide sequence is shown in SEQ ID NO: 2. Specific PCR amplification primers were designed using Primer Premier 5.0 software (underlined are restriction endonuclease sites):

[0045] SAE-F (SEQ ID NO: 3): 5'-CATG CCATGG AACAAGTGCGTGAACTTCAACT-3' Nco Ⅰ; SAE-R (SEQ ID NO: 4): 5'-C GAGCTC GAAGCTGCAGGGGGTGATGTC-3' Sca I.

[0046] DNA primers (SAE-F and SAE-R) containing the SAE gene and the pUC57 plasmid were purchased from General Biotechnology (Anhui) Co., Ltd. The primers were 500 ng each, dissolved in 20 μL of sterile double-distilled water, resulting in a plasmid concentration of approximately 25 ng / μL. The SAE gene was amplified using the specific primers SAE-F and SAE-R. It was then cloned into the vector BL21-30a to obtain the pET30a-SAE plasmid, which was subsequently transformed into *E. coli*. The plasmid was extracted from the *E. coli* bacteria, and its concentration was measured.

[0047] The PCR reaction system is shown in Table 1.

[0048] Table 1 PCR reaction system PCR conditions were: 95℃, 3 min; 94℃, 30 s; 58℃, 30 s; 72℃, 2 min; 30 cycles.

[0049] PCR amplification results as follows Figure 1As shown, using the plasmid pUC57 containing the SAE gene, i.e., pUC57-SAE plasmid, as a template, the SAE gene was amplified using specific primers SAE-F and SAE-R, resulting in a specifically amplified fragment.

[0050] 1.2 Construction of the integrative recombinant plasmid pJS700-SAE The workflow for constructing the integrative recombinant plasmid pJS700-SAE is as follows: Figure 3 As shown.

[0051] First, plasmids were extracted from DH5α-30a and the stab bacteria containing the SAE gene, respectively. To obtain a recombinant plasmid with erythromycin resistance, [the following was used]. Sac I and Nco After digesting the two plasmids with enzymes, ligation was performed using T4 DNA ligase at 16℃. The double digestion system is shown in Table 2, and the ligation system is shown in Table 3. The digested 30a and SAE fragments were recovered by agarose gel electrophoresis. These fragments were then transformed into BL21 bacteria. Single colonies were picked and cultured on LB agar medium at 37℃ for 12 h. The next day, single colonies were picked and cultured on a shaker, plasmids were extracted, and PCR amplification was performed. The agarose gel electrophoresis results in the recombinant plasmid pET30a-SAE. Figure 2 As shown in A.

[0052] Table 2 Double enzyme digestion system Table 3 Connection System Extract pJS700 and pET30a-SAE plasmids respectively, and use Sac I and Kpn I. The two plasmids were digested with enzymes, and the digestion system is shown in Table 4. Then, the digested pJS700 and SAE fragments were detected by agarose gel electrophoresis and recovered from the gel. The results are as follows: Figure 2 As shown in B. The ligation system in Table 5 was ligated overnight at 16°C, and then detected by agarose gel electrophoresis. The results are as follows. Figure 2 As shown in C. Next, it was transformed into DH5α *E. coli* and cultured. Single colonies were transferred to LB agar and incubated upside down at 37°C. After 12 h, single colonies were selected for amplification, plasmid was extracted and PCR amplified, and the results were detected by agarose gel electrophoresis. The obtained plasmid was the integrated recombinant plasmid pJS700-SAE, as shown in Figure C. Figure 2 As shown in D.

[0053] Table 4 Connection System Table 5 Connection System Example 2 Transformation of Bacillus subtilis First, competent Bacillus subtilis cells were prepared as follows: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C with shaking at 180 rpm for 12 h. The next day, an appropriate amount of bacterial culture was spread onto LB solid medium and cultured at 37°C for 12 h. On the third day, 8 to 10 single strains were picked and inoculated into 3 mL of GM I medium with shaking at 125 rpm for 12 h at 30°C. Subsequently, the bacterial culture from GM I was inoculated into sterile GM I medium at a ratio of 1:1000 and cultured at 37°C with shaking at 250 rpm for 3.5 h. After the culture was completed, the obtained bacterial solution was inoculated into 30 mL of sterile GM II solution and cultured at 125 rpm and 37℃ for 1.5 h with shaking. After the culture was completed, the obtained bacterial solution was dispensed into EP tubes, centrifuged at 5000 rpm for 10 min, leaving 1 / 10 of the precipitate. Finally, the bacterial cells were suspended in the liquid to obtain competent Bacillus subtilis cells, which were then stored in a -80℃ refrigerator with 30% glycerol.

[0054] After obtaining competent Bacillus subtilis cells, efficient transformation experiments can be performed. The method is as follows: The recombinant plasmid pJS700-SAE was extracted, and its concentration was measured using a micro-spectrophotometer. The cells were then stored at -40℃. Frozen competent Bacillus subtilis cells were heat-shocked at 45℃ for 30-40 seconds. 70 ng of plasmid was added to the competent cells, and after transformation, the cells were directly cultured on a shaker for 1 h, resulting in the optimal transformation rate. Then, 2 μL of the recombinant plasmid pJS700-SAE was added to the competent cells, and the cells were incubated at 37℃ for 1 h, followed by shaking culture at 37℃ and 200 rpm for 2 h. The cells were then centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the bacterial culture was retained. After thorough mixing, the culture was spread onto LB agar plates containing erythromycin in the dark and incubated overnight at 37℃ in the dark.

[0055] Example 3 Identification and Screening of Recombinant Bacillus subtilis Because the integration of the SAE gene at the amyE locus interrupts the expression of amylase, the starch produced by colonies growing on LB solid medium containing 1% starch is not broken down by amylase and will produce a blue reaction with iodine-potassium iodide (I-KI) solution.

[0056] The recombinant plasmid carries an ampicillin resistance gene. pJS700-SAE was successfully transformed into *Bacillus subtilis*, and the recombinant *Bacillus subtilis* expressed ampicillin resistance. Therefore, the recombinant bacteria can be inoculated into a medium containing ampicillin for initial screening. After initial screening, single colonies from the medium are picked and cultured. The resulting bacterial solution is inoculated onto LB agar plates containing 1% starch and incubated at 37°C and 180 rpm for 12 h. The next day, an appropriate amount of I-KI solution is evenly dropped onto the plate. If no clear zone appears around the colony, the recombinant *Bacillus subtilis* has been successfully constructed.

[0057] The amylase plate experiment showed that no clear zone was formed around the recombinant colonies, confirming that the SAE gene had been successfully transferred into Bacillus subtilis. Figure 4 As shown.

[0058] Example 4: Uses of Recombinant Bacillus subtilis As shown in Examples 1-3, the present invention successfully constructed a recombinant Bacillus subtilis oral vaccine strain displaying the coronavirus Spike protein immunoepitope polypeptide.

[0059] The construction of the humanized ACE2 mouse model is shown in ZHANG H, LIU S, CHEN Y, et al. Development and characterization of a fully humanized ACE2 mouse model[J]. SignalTransduction and Targeted Therapy, 2025, 10(1): 89.

[0060] Adenovirus-mediated transfection of the human ACE2 gene into normal mice was constructed as shown in KAZACHEK S, KOVÁČOVÁ E, ŠVABENOVÁ I, et al. Evaluation of immune response to mucosal immunization with an oral probiotic-based vaccine in mice: potential for prime-boost immunization against SARS-CoV-2[J]. International Journal of Molecular Sciences, 2024, 25(1): 215.

[0061] Immunological evaluation experiments were conducted in humanized ACE2 mouse models and normal mice transfected with adenovirus-mediated human ACE2 gene. The results showed that the recombinant strain oral vaccine, administered via the gastrointestinal route, could tolerate the extreme acid-base environment of gastric and intestinal fluids and survive stably, effectively presenting Spike protein immunoepitope peptides on the bacterial surface; it could effectively stimulate the intestinal mucosal immune system of model mice, while activating the body's mucosal immunity, humoral immunity and cellular immunity responses, inducing mice to produce high levels of anti-coronavirus specific antibodies and specific immune memory cells; it could significantly enhance the anti-infection ability of model mice against coronavirus, reduce the severity of viral infection and organ damage, and has a good preventive and protective effect.

[0062] Because conventional mice have different ACE2 receptors than humans, they are not naturally susceptible to coronaviruses, making it difficult to objectively evaluate the protective efficacy of vaccines. However, this invention, by using a humanized ACE2 mouse model and adenovirus-delivered human ACE2 gene-modified mice, can effectively avoid the experimental limitations caused by species receptor differences and objectively verify the immunogenicity and potential application value of the recombinant Bacillus subtilis oral vaccine.

[0063] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A recombinant plasmid, characterized in that, The recombinant plasmid contains a nucleic acid molecule encoding a Spike protein immunoepitope polypeptide, a backbone plasmid, and acceptable expression elements; The Spike protein immunoepitope polypeptide has the amino acid sequence shown in SEQ ID NO:

1.

2. The recombinant plasmid according to claim 1, characterized in that, The backbone plasmid is an integrative plasmid.

3. The recombinant plasmid according to claim 2, characterized in that, The backbone plasmid is pJS700.

4. The recombinant plasmid according to claim 1, characterized in that, The acceptable expression elements include one or more of promoters, enhancers, and terminators.

5. A recombinant bacterial strain, characterized in that, The recombinant strain is transformed and / or transfected with any of the recombinant plasmids described in claims 1 to 4.

6. The recombinant strain according to claim 5, characterized in that, The chassis strain used for transformation and / or transfection is Bacillus subtilis.

7. The method for preparing the recombinant strain according to claim 6, characterized in that, The preparation method includes: (1) Integrate the nucleic acid molecule encoding the Spike protein immunoepitope polypeptide into the integration site of the backbone plasmid to obtain a recombinant plasmid; (2) Transform and / or transfect the recombinant plasmid into the chassis strain to obtain the recombinant strain.

8. A culture, characterized in that, The culture is obtained by culturing the recombinant strain of claim 6 or the recombinant strain obtained by the preparation method of claim 7 under suitable growth and / or proliferation conditions.

9. Use of the recombinant plasmid according to claim 4, the recombinant strain according to claim 6, the recombinant strain obtained by the preparation method according to claim 7, or the culture according to claim 8 in the preparation of an oral vaccine for the prevention and / or treatment of coronaviruses.

10. The use according to claim 9, characterized in that, The coronavirus in question is Severe Acute Respiratory Syndrome Coronavirus 2.

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