SARS-CoV-2 small broad-spectrum vaccine immunogenic protein and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a novel small broad-spectrum vaccine immunogenic protein of coronavirus (SARS-CoV-2) and application of the novel small broad-spectrum vaccine immunogenic protein. The two proteins BLEP-1 and BLEP-2 have good immunogenicity, after the two proteins are matched with a Freund's adjuvant, a mouse can be induced to generate a high-titer anti-Wuhan-Hu-1 virus strain / Octhrough variant S protein specific IgG antibody, SARS-CoV-2Wuhan-Hu-1 strain S protein mediated cell-cell fusion can be effectively inhibited, and the effect of the BLEP-2 is superior to that of the BLEP-1. The amino acid sequences of the protein BLEP-1 and the protein BLEP-2 are respectively as shown in SEQ ID NO. 1 and SEQ ID NO. 2. The two candidate vaccines designed by the invention can induce an organism to generate specific Th1 / Th2 mixed immune response aiming at a Wuhan-Hu-1 virus strain and an Omicro variant strain, and have the potential of becoming a novel small broad-spectrum vaccine candidate antigen of coronavirus (SARS-CoV-2), and the design concept can provide a new scheme for the design of a vaccine candidate immunogen of a highly mutant virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a SARS-CoV-2 small broad-spectrum vaccine immunogenic protein and an application thereof. Background Art

[0002] Since the end of December 2019, the novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has been spreading for three years. It is a single-stranded positive-sense RNA virus with a very high broadcast rate and a wide range of transmission, causing pneumonia (Corona Virus Disease 2019, COVID-19), manifested as bronchitis, pneumonia or other severe respiratory diseases. The process of SARS-CoV-2 infecting the host can be divided into three stages: the virus attaches to the host cell and enters the shell, gene replication and transcription and translation of structural proteins, and viral particle assembly, maturation and release. The invasion of SARS-CoV-2 into the host cell is mainly mediated by the S protein on the surface of its viral envelope, which binds to the corresponding receptor on the surface of the host cell, promotes the membrane fusion between the host cell and the virus, and successfully releases the viral genetic material into the host cell.

[0003] The S protein mediates the entry of SARS-CoV-2 mainly through two pathways: first, when the virus is close to the host cell, the S protein receptor binding domain (RBD) binds to the ACE2 receptor on the host cell, and then the cell internalizes the virus to form a vesicle. Under the action of different environments inside the vesicle (such as lower pH) and different host proteases (such as furin protease), the S1 / S2 site is cleaved, resulting in S1 dissociation. The S protein undergoes conformational changes to expose the S2' cleavage site. Under the action of proteases (such as cathepsin L), the S2 protein conformational changes. The thrust of the unfolding peptide repeat sequence 1 (heptad repeat, HR1) pushes the fusion peptide (FP) to insert into the host cell membrane. Subsequently, HR2 folds backward to pull FP and the transmembrane segment (TM) to the same end. HR1 and HR2 interact to form a six-helix bundle (6-HB) fusion core, which shortens the distance between the virus and the cell membrane. This proximity causes the membranes they interact with to bend against each other, effectively leading to membrane fusion, thereby releasing the viral RNA into the cytoplasm and using the host cell machinery for subsequent replication. Another invasion mechanism is that after the S protein binds to ACE2, S1 dissociates and the S2' site can be directly cleaved by the transmembrane serine protease 2 expressed on the membrane surface by the host, directly inducing the fusion of the virus and the host cell membrane and releasing the viral genetic material into the cytoplasm.

[0004] After the COVID-19 outbreak, many vaccine manufacturers around the world have urgently invested in the research and development of a COVID-19 vaccine (referred to as the COVID-19 vaccine). As of March 30, 2023, 199 vaccines have entered the preclinical stage and 183 have entered the clinical trial stage. The World Health Organization (WHO) divides the current COVID-19 vaccines into 11 categories according to the research and development technology (data from the WHO website, https: / / www.who.int / publications / m / item / draft-landscape-of-covid-19-candidate-vacc ines). Currently, protein subunits account for the largest proportion of COVID-19 vaccine research technology, accounting for 32.3% of the types of vaccines entering clinical trials. The S protein of SARS-CoV-2 is the most immunogenic component of the virus and has been found to induce strong and protective humoral and cellular immunity. As the most important membrane protein on the surface of SARS-CoV-2, it has become a core target for vaccine and drug design. Yang J et al. compared four SARS-CoV-2 candidate vaccines based on viral protein structures: RBD, extracellular domain (ECD), S1 subunit, and S2 subunit, and found that the virus neutralization activity caused by RBD was much higher than that of ECD or S1 subunit, and no virus neutralization activity of S2 subunit was detected. These results show that RBD protein is the main target for the production of neutralizing antibodies caused by natural infection or vaccination of coronavirus, and is widely used in antibody detection and vaccine research. RBD is a highly potential candidate immunogen for the development of new crown vaccines.

[0005] RBD is located at the top of the S protein trimer. During infection, it undergoes structural fluctuations between the "down" and "up" conformations. Both conformations can induce antibodies against different epitopes when used as immunogens. According to the analysis of antigen epitope localization and functional characteristics, anti-RBD antibodies can be divided into four categories: 1-4: Category 1 is RBM-targeting antibodies; Category 2 is antibodies that simultaneously bind to the "up" and "down" conformations of RBD and contact adjacent RBDs; Category 3 is some non-ACE2 competitive antibodies that can interact with the "up" conformation; Category 4 antibodies can target the conserved proteoglycan epitopes at the distal end of the RBM, neutralize the virus through one or more IgG-specific bivalent mechanisms, and have broad cross-reactivity to related zoonotic coronaviruses. Among the four epitope types, categories 1 and 2 are highly variable, while categories 3 and 4 are more conserved, so inducing neutralizing antibodies against category 3 and 4 epitopes can enhance broad-spectrum protective immunity against current and future SARS-CoV-2 variants and emerging zoonotic viruses, but such epitopes are not easy to obtain.

[0006] Since the pandemic, the SARS-CoV-2 virus has continued to evolve, and many variants have appeared around the world. The WHO defines variants with wide transmission and strong pathogenicity as variants of concern (VOC), which now include five types: Alpha variant, Beta variant, Gamma variant, Delta variant, and Omicron variant. Currently, Omicron has become the dominant variant virus strain infected by SARS-CoV-2. Compared with the original SARS-CoV-2 virus strain Wuhan-Hu-1, these VOCs have greatly enhanced their ability to spread and are likely to increase the severity of the disease.

[0007] Significant mutations in variants often occur in the S protein. For example, the D614G mutation has been shown to be more efficient in infection and replication, while maintaining similar pathogenicity to the original strain; the N501Y mutation produced in the RBD region increases the affinity of SARS-CoV-2 for ACE2, and these mutations reduce the ability of neutralizing antibodies to bind to the mutation site. VOCs are less sensitive to vaccine-induced and infection-induced immune responses, and therefore have the ability to cause secondary infections in recovered individuals. A large number of reports have shown that VOCs have an adverse effect on the preventive effect of first-generation vaccines and the therapeutic effect of monoclonal antibodies. The emergence and spread of variants has brought great challenges to the prevention and control of the SARS-CoV-2 pandemic through vaccination.

[0008] In the design strategy for dealing with highly mutable viruses, "peptide vaccines" mainly use highly conserved immunodominant epitopes between different serotypes of pathogens, or construct long peptides containing multiple epitopes, which can induce broad immunity to multiple serotypes of viruses; "overlapping peptide vaccines" are designed based on the specific protein sequence of the virus, with a length of 15-20aa, and adjacent peptide segments repeated 10-11aa, covering the full length of the viral protein. They are connected using the cathepsin S cleavage site, which can simultaneously stimulate CD8 + and CD4 + The "mosaic vaccine" is a new protein composed of a combination of superior peptides selected by genetic algorithms. This protein is similar to natural viral proteins and can maximize the coverage of common potential T cell epitopes in the virus population, which can induce a greater T cell immune response and the production of neutralizing antibodies. The above peptide-based vaccine design strategies have shown great potential in providing safe and protective immune responses to highly mutable viral infections such as HIV and influenza.

[0009] The continuous emergence of SARS-CoV-2 variants enables it to evade immune defenses, including natural immunity acquired through infection or vaccination, as well as antibody-based treatments. Therefore, there is an urgent need to put on the market a small broad-spectrum vaccine with low production risk and high yield that can target multiple major circulating strains of SARS-CoV-2. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a SARS-CoV-2 small broad-spectrum vaccine immunogenic protein and its application, including BLEP-1 protein and BLEP-2 protein, both of which have good immunogenicity and immunoreactivity, and the induced antibodies have the ability to inhibit SARS-CoV-2 from entering infected cells.

[0011] The present invention provides a SARS-CoV-2 small broad-spectrum vaccine immunogenic protein, wherein the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein is any one of the following:

[0012] BLEP-1 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0013] BLEP-2 protein, the amino acid sequence is shown in SEQ ID NO.2.

[0014] The present invention also provides a gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein.

[0015] Furthermore, the immunogenic protein is BLEP-1 protein, and the nucleotide sequence of the gene encoding the BLEP-1 protein is shown in SEQ ID NO.3. The nucleotide sequence of the gene encoding the BLEP-1 protein is shown in SEQ ID NO.3.

[0016] Furthermore, the immunogenic protein is an immunogenic protein, and the gene nucleotide sequence encoding the BLEP-2 protein is shown in SEQ ID NO.4.

[0017] The present invention also provides a recombinant plasmid carrying the gene encoding the immunogenic protein of the SARS-CoV-2 small broad-spectrum vaccine.

[0018] Furthermore, the recombinant plasmid is obtained by inserting the gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein into the prokaryotic expression vector pET-21a.

[0019] The present invention also provides an application of the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein in the preparation of immune serum or antibodies against Wuhan-Hu-1 S, Wuhan-Hu-1 S1 and Omicron S1 proteins.

[0020] Furthermore, the dosage of the BLEP-1 protein is 10-40 μg.

[0021] Furthermore, the dosage of the BLEP-2 protein is 10-40 μg.

[0022] Furthermore, the BLEP-2 protein dosage is 20 μg.

[0023] The present invention also provides an application of the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein in the preparation of a new coronavirus small broad-spectrum vaccine.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention provides two SARS-CoV-2 small broad-spectrum vaccine immunogenic proteins, BLEP-1 protein and BLEP-2 protein; among them, BLEP-2 protein can be better cleaved by cathepsin S. Using mouse immunization experiments, it was found that after the first booster immunization of BLEP-1 and BLEP-2 proteins, the levels of three specific IgG antibodies against Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and Omicron S1 protein were all improved, and the optimal protein dose group was 20 μg.

[0026] This vaccine is produced based on the E. coli expression system, which has the advantages of low production cost, easy production scale expansion, high production stability, clear genetic background and small side effects. At the same time, the epitope of this vaccine comes from the RBD region of the S protein of different mutant strains of SARS-CoV-2, which can avoid the occurrence of ADE phenomenon and further increase the safety of the vaccine. The BLEP-1 protein and BLEP-2 protein prepared by the present invention have low production risk and high yield, and can target multiple major epidemic strains of SARS-CoV-2.

[0027] After the third immunization of the 20μg dose group of BLEP-1 protein, the titers of specific binding IgG antibodies to Wuhan-Hu-1 S protein, Wuhan-Hu-1S1 protein, and Ocmicron S1 protein reached 1:3,200, 1:12,800, and 1:800, respectively; after the third immunization of BLEP-2 protein, the titers of specific binding IgG antibodies to Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and Ocmicron S1 protein in the 10μg dose group reached 1:12,800, 1:102,400, and 1:6,400, respectively; in the 20μg dose group, they reached 1:6,400, 1:12,800, and 1:102,400, respectively; and in the 40μg dose group, they reached 1:102,400, 1:102,400, and 1:102,400, respectively.

[0028] BLEP-1 and BLEP-2 induced a Th1-biased cellular immune response, with BLEP-2 having a higher response level.

[0029] 2. The present invention uses bioinformatics methods to screen the RBD protein epitope sequences of SARS-CoV-2 and various VOC virus strains (peptide vaccine strategy), and uses the minimum active fragment of cathepsin S (LMRK) and (LRMK) to connect. Among them, the introduction of cathepsin S cleavage site will help APC to accurately process and present antigens, and improve the activation of the immune system (overlapping peptide vaccine, mosaic vaccine strategy). The protein covers the antigen peptides and protective B cell linear antigen epitopes in the RBD region of various VOC virus strains of SARS-CoV-2 that can produce highly effective neutralizing antibodies, and can then be directly recognized by B cell receptors, which can mediate humoral immune responses to produce effective neutralizing antibodies, and can enhance the ability to be processed and presented by APC, thereby efficiently stimulating the body's immune response level. The vaccine removes areas where non-neutralizing antibodies may be produced, reduces the possibility of ADE phenomenon, and enables the vaccine to exert the best preventive effect as much as possible to cope with the difficulties in the preparation of new crown vaccines caused by the high variation of SARS-CoV-2 and its VOCs.

[0030] 3. The main reason why the infection of the new coronavirus variant is difficult to prevent through the first-generation vaccine is the site of its continuous mutation, especially the Omicron virus strain. At present, the new coronavirus small broad-spectrum vaccine mostly adopts the method of adding the inactivated virus particles or S protein of the new variant to the previously developed vaccine to form a multivalent vaccine, which is costly and prone to ADE phenomenon. The present invention combines the conservative dominant epitopes in the RBD region of each VOC variant and the epitopes containing the main mutation sites, and has the advantages of relatively low production cost, safe production technology, good targeted immunity, and higher stability and safety, which will be more conducive to the promotion of the new coronavirus small broad-spectrum vaccine and the design of vaccines to deal with highly mutated viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The BLEP purification and Western Blot verification results of Example 2 of the present invention;

[0033] In the figure, A is the SDS-PAGE result of BLEP protein after purification by ultrafiltration and concentration, and B is the BLEP Western Blot result: lanes 1-4 are respectively the supernatant of the first ultrasonication of the bacterial solution after BLEP-1 protein induction, ultrasonic precipitation, protein solution after concentration, 80mM imidazole elution, and lane 5 is a marker; lanes 6-9 are the supernatant of the first ultrasonication of the bacterial solution after BLEP-2 protein induction, ultrasonic precipitation, 80mM imidazole elution, and protein solution after concentration;

[0034] Figure 2 The result of cathepsin S cleavage of BLEP protein in Example 2 of the present invention;

[0035] In the figure, A is the result of cathepsin S cleavage of BLEP-1 protein, and lanes 1-8 are BLEP-1 protein before cleavage, 0.5h cleavage, 1h cleavage, 2h cleavage, 4h cleavage, 6h cleavage, 8h cleavage, and 20KD small molecule marker, respectively;

[0036] B is the result of cathepsin S digestion of BLEP-2. Lanes 1-6 are BLEP-2 protein before digestion, 2h, 4h, 6h, 180KD marker, and 20KD small molecule marker, respectively.

[0037] Figure 3The P / N value of the mouse immune serum specific IgG antibody of Example 3 of the present invention is measured;

[0038] In the figure, A is the P / N value determination result of Wuhan-Hu-1 S protein specific IgG antibody in mouse immune serum of Example 3;

[0039] B is the P / N value determination result of Wuhan-Hu-1 S1 protein specific IgG antibody in mouse immune serum of Example 3;

[0040] C is the result of measuring the P / N value of the Omicron S1 protein-specific IgG antibody in the mouse immune serum of Example 3.

[0041] Figure 4 The specific IgG antibody titer of the mouse immune serum of Example 3 of the present invention is measured;

[0042] In the figure, A is the result of the determination of Wuhan-Hu-1 S protein-specific IgG antibody titer in the immune serum of mice immunized with BLEP-1 protein;

[0043] B is the result of the determination of Wuhan-Hu-1 S1 protein-specific IgG antibody titer in the immune serum of mice immunized with BLEP-1 protein;

[0044] C is the result of the determination of the titer of Omicron S1 protein-specific IgG antibody in the immune serum of mice after BLEP-1 protein immunization;

[0045] D is the result of the determination of Wuhan-Hu-1 S protein-specific IgG antibody titer in the immune serum of mice immunized with BLEP-2 protein;

[0046] E is the result of the determination of Wuhan-Hu-1 S1 protein-specific IgG antibody titer in the immune serum of mice immunized with BLEP-2 protein;

[0047] F is the result of the determination of the titer of Omicron S1 protein-specific IgG antibody in the immune serum of mice immunized with BLEP-2 protein.

[0048] Figure 5 The results of cytokine determination of spleen cells of immunized mice in Example 3 of the present invention are shown.

[0049] Figure 6 The results of constructing the cell-cell fusion model of Example 4 of the present invention;

[0050] In the figure, A shows the S protein-mediated cell-cell fusion at 2h, 4h, and 12h, B shows the cell-cell fusion and DAPI staining at 12h, and C shows the time-fusion curve.

[0051] Figure 7 This is the result of mouse immune serum inhibiting cell-cell fusion in Example 4 of the present invention. DETAILED DESCRIPTION

[0052] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0053] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0054] In the following examples, the concentration of PBS used was 0.1 M and the pH was 7.4.

[0055] The amino acid sequence of the protein provided in the embodiment of the present invention is shown in Table 3. The underlined part in Table 3 is the minimum active fragment of cathepsin S, and the introduction of this fragment can help APC to accurately process and present antigens and improve the activation of the immune system.

[0056] Example 1 Screening of dominant epitopes of RBD of VOC virus strain

[0057] 1. Comparison of RBD amino acid sequences

[0058] (1) The sequence of the standard strain Wuhan-Hu-1 (NC_45512.2) was downloaded from the NCBI Novel Coronavirus Database (https: / / www.ncbi.nlm.nih.gov / sars-cov-2 / ), and the high-quality complete amino acid sequences of the S proteins of the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) variants were screened and downloaded.

[0059] (2) The sequence was entered into the bioEdit software, and the S1 (including RBD, RBM, etc.) and S2 functional regions of the S protein sequences of all VOC virus strains were located with reference to the coding sequence information of each region of the Wuhan-Hu-1 strain, and the amino acid sequence of the RBD region of the VOC virus strain with the highest proportion was selected. The RBD amino acid sequence was entered into the EMBOSS Needle online software, and the differences in the sequences were compared. The results are shown in Table 1. It can be seen that compared with the Wuhan-Hu-1 strain, most of the mutation sites of the VOC virus strains are located in the RBM (aa438-508) region. The Omicron virus strain has the largest number of amino acid substitutions, up to 15, of which 10 are concentrated in the RBM region.

[0060] Table 1 Comparison results of RBD region sequences of VOC virus strains

[0061]

[0062] 2. Screening of dominant B cell epitopes

[0063] (1) The Bepipred Linear Epitope Prediction 2.0 online software from the IEDB website was used to predict the possible B cell linear epitope locations in the RBD region of each virus strain in Table 1. These epitopes were randomly divided into lengths of 10-14 aa, and repeated to cover all B cell epitopes.

[0064] (2) The antigenicity of each epitope peptide was determined using VaxiJen v2.0 online software, and the dominant epitope peptides were screened out by combining the antigenic peptides that produced highly efficient neutralizing antibodies. The results are shown in Table 2. The minimum active fragment of cathepsin S (LMRK) / (LRMK) was used to sequentially connect the peptides according to the position of the RBD sequence where the peptides were located to obtain the target protein amino acid sequences BLEP-1 and BLEP-2. The results are shown in Table 3.

[0065] Table 2 Dominant epitope peptides

[0066]

[0067]

[0068] Table 3 Target protein amino acid sequence

[0069]

[0070]

[0071] Example 2 Expression and purification of BLEP

[0072] 1. Construction of recombinant plasmid

[0073] The prokaryotic expression recombinant plasmids pET-21a-BLEP-1 and pET-21a-BLEP-2 were constructed.

[0074] The nucleotide sequence encoding the BLEP-1 protein is shown in SEQ ID NO. 3. The nucleotide sequence encoding the BLEP-2 protein is shown in SEQ ID NO. 4.

[0075] SEQ ID NO.3:

[0076] GGATCC CTCGAG

[0077] SEQ ID NO.4:

[0078] GGATCC CTCGAG

[0079] The underlined part is the base sequence of the restriction sites BamH I and Xho I added to the vector. The optimization was completed and synthesized by Shanghai Bioengineering Co., Ltd. and inserted into the prokaryotic expression vector pET-21a.

[0080] 2. BLEP protein expression

[0081] The recombinant plasmids pET-21a-BLEP-1 and pET-21a-BLEP-2 were transformed into competent Escherichia coli BL-21, respectively, and bacterial liquid containing BLEP-1 and bacterial liquid containing BLEP-2 were obtained by culture. The antibiotic used was ampicillin (Amp).

[0082] The specific steps are as follows:

[0083] (1) After the competent bacteria BL-21 was taken out from -80℃, it was placed on ice to slowly melt. 1μL of pET-21a-BLEP-1 and pET-21a-BLEP-2 plasmids were taken and slowly rotated into BL-21, and then placed on ice for 30 minutes. After being placed in a 42℃ hot water bath for 2 minutes, 900μL of LB liquid culture medium was slowly added along the tube wall, and the tube was placed at 37℃ for activation for 1 hour. After centrifugation at 5000rpm for 1 minute, the supernatant was poured out in a clean bench, 100μL of LB liquid culture medium was added, and the tube was gently resuspended by pipetting, and then spread on a plate containing Amp and cultured at 37℃ overnight. The colonies grown on the above plasmid plates were picked and activated in 3mL of LB liquid culture medium containing ampicillin resistance at 220rpm for 12 hours to obtain the expression bacterial solution containing BLEP-1 and the expression bacterial solution containing BLEP-2.

[0084] (2) The above 3 mL of bacterial solution containing BLEP-1 expression and BLEP-2 expression were added to 300 mL of LB liquid medium containing ampicillin resistance, respectively, and cultured at 37°C with shaking at 220 rpm for 5 h until the OD of the bacterial solution reached 0. 600 When the value reaches about 0.7, the recombinant protein is induced to express under the determined expression conditions (inducer IPTG = 0.5mmol / L, induced at 37℃ for 18h), and the bacteria are collected by centrifugation at 12000rmp for 5min. After resuspending and washing the bacteria twice with 0.1M PBS, centrifuge at 12000rmp for 5min, retain the precipitate, resuspend with 40mL 0.1M PBS, and break the bacteria with low temperature ultrasound (working 5s, interval 3s), after ultrasound break for 45min, centrifuge at 12000rmp for 10min, collect the supernatant and precipitate, take a small amount of the supernatant and precipitate for SDS-PAGE gel electrophoresis detection, and all experimental processes are operated on ice.

[0085] 3. Washing and dissolving of inclusion body proteins

[0086] Resuspend the precipitate after ultrasonic disruption with 10 times the volume of PBS, centrifuge at 4°C, 12000 rpm for 10 min, collect the inclusion bodies, and repeat the washing once; use LE Buffer to dissolve the inclusion bodies (add 7.5 mL LE Buffer inclusion body dissolution solution for each mg of inclusion bodies), incubate at room temperature for 1 h, centrifuge at 4°C, 12000 rpm for 30 min, collect the supernatant after centrifugation and pass it through Ni-NTA affinity chromatography medium.

[0087] 4. Renaturation and purification

[0088] (1) Take 2mL Ni-NTA affinity chromatography medium (nickel column), add it to the chromatography column, drain the original preservation solution (PBS containing 20% ​​alcohol), add 5 column volumes of LE Buffer to balance for 5 minutes, and then circulate the supernatant collected in the previous step through the column at a constant flow rate of 1mL / min for 8h. (2) Use refolding solutions with urea concentrations from high to low to slowly circulate through the column, and finally use PBS to slowly rinse the column material. (3) Wash the filler with 10 column volumes of 30mM imidazole washing buffer. (4) Elute the protein with 80mM imidazole or above elution buffer. (5) Select a suitable ultrafiltration tube. Generally, the molecular weight cutoff should not be greater than 1 / 3 of the molecular weight of the target protein. In this example, the molecular weight of the target protein is about 38kDa, and an ultrafiltration tube with a molecular weight cutoff of 10kDa is selected to concentrate the protein. (6) Perform SDS-PAGE gel electrophoresis on samples at each stage.

[0089] Figure 1 A is the SDS-PAGE of the purified and concentrated BLEP proteins. The results show that after a large amount of induced expression of the recombinant proteins BLEP-1 and BLEP-2, purification and concentration, a target band appeared at a molecular weight of about 38 kDa, and the target band was relatively single and highly pure.

[0090] 5. Western Blot detection of purified protein samples

[0091] The protein bands in the gel were transferred by Western Blot after SDS-PAGE of the two groups of protein renaturation products. Figure 1B, lanes 1-4 are respectively the first ultrasonic supernatant, ultrasonic precipitation, concentrated protein solution, 80mM imidazole elution of the bacterial solution after BLEP-1 protein induction expression, lane 5 is a marker, and lanes 6-9 are the first ultrasonic supernatant, ultrasonic precipitation, 80mM imidazole elution, and concentrated protein solution of the bacterial solution after BLEP-2 protein induction expression. The results show that the inclusion body purification products of BELP-1 and BLEP-2 have a molecular weight of about 38kDa and bind to the monoclonal antibody His, proving that the protein expression has been successfully induced in this example.

[0092] 6. Cathepsin S cleavage of BLEP protein

[0093] (1) Replace the protein buffer with Mallvaine citrate-phosphate buffer.

[0094] (2) In vitro enzyme digestion: Referring to the experimental method of the reference (Ruckrich T, Brandenburg J, Cansier A, et al. Specificity of human cathepsin S determined by processing of peptidesubstrates and MHC class II-associated invariant chain [J]. Biol Chem, 2006, 387 (10-11): 1503-1511.), cathepsin S was mixed with BLEP-1 and BLEP-2 in proportion, and then digested in a water bath at 37°C. An appropriate amount of sample was taken every 2 hours, Tricine Buffer A was added in a ratio of 4:1, heated at 100°C for 10 min, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for use in Tricine SDS-PAGE.

[0095] Figure 2 The results of cathepsin S cleavage of BLEP protein in Example 2 of the present invention show that the amount of BELP-1 protein does not change significantly when cathepsin S is applied at different time periods ( Figure 2 A), while the amount of BELP-2 protein gradually decreased with the increase of exposure time, proving that BLEP-2 was successfully cleaved into small molecule peptides by cathepsin S ( Figure 2 B). This proves that the BLEP-2 protein designed by the present invention can be better cleaved by cathepsin S.

[0096] Example 3 Mouse Immunization and Serological Detection

[0097] The purified BLEP was combined with Freund's adjuvant for animal experiments: 6-8 week old female BALB / c mice were used, 4 mice in each group, and all were immunized by intradermal multi-point injection. The negative control group consisted of saline group (NS) and saline combined with adjuvant group (NS+adjuvant). The experimental groups were divided into 3 groups according to the dosage groups: 10μg protein combined with adjuvant group (10μg+adjuvant), 20μg protein combined with adjuvant group (20μg+adjuvant), and 40μg protein combined with adjuvant group (20μg+adjuvant). The positive control group was immunized with 20μg Wuhan-Hu-1 S protein combined with adjuvant. The immunization cycle was week 0, week 2, and week 4, with a total of three immunizations, and the total volume was 100μL. The adjuvant was Freund's adjuvant, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the next two boosts, mixed with equal volumes of protein solution or saline in a 1:1 ratio to form an oil-in-water state. 10 days after immunization, blood was collected from the orbital venous plexus. The immunization groups and process are shown in the table.

[0098] Table 4 Mouse immunization grouping and flow chart

[0099]

[0100]

[0101] 1. BLEP can induce the production of high titer specific binding antibodies

[0102] (1) Antigen coating: dilute the antigen with coating solution to make the final concentration of Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and Omicron S1 protein 1 μg / μL, 50 μL / well, and coat overnight at 4°C.

[0103] The Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and Omicron S1 protein were purchased from SinoBiological Company.

[0104] (2) Blocking: Remove the coating solution, wash three times with 1% PBST, 200 μL / well, 5 min each time. Block with 5% skim milk prepared with 1% PBST, 200 μL / well, and incubate at 37°C for 2 h.

[0105] (3) Primary antibody incubation (mouse serum): Remove skim milk and wash three times with 1% PBST, 200 μL / well, 5 min each time. Add 1% PBST diluted mouse serum, 50 μL / well, and incubate at 37°C for 2 h.

[0106] (4) Secondary antibody incubation (goat anti-mouse IgG-HRP): Remove the primary antibody, wash three times with 1% PBST, 200 μL / well, 5 min each time. Add goat anti-mouse IgG antibody diluted 1:3000 with 1% PBST, 50 μL / well, and incubate at 37°C for 2 h.

[0107] (5) Color development: Remove the secondary antibody, wash three times with 1% PBST, 200 μL / well, 5 min each time. Add 50 μL / well of color development substrate, and color development for 30 min at room temperature in the dark.

[0108] (6) Stop reading: Add 50 μL / well of 10% dilute sulfuric acid to stop color development, and measure the absorbance at 450 nm using a multifunctional microplate reader. The blank well is the one without incubation with primary antibody, and the cutoff value is 2.1 times the OD value of the blank well. The highest serum dilution greater than the cutoff value is the titer.

[0109] (7) GraphadPrism 7.0 was used for statistical analysis and drawing. The unpaired Mann-Whitoey test was used to compare the means of two groups of data. One-way ANOVA was used to compare the means of multiple groups of data. P ≤ 0.05 was considered statistically significant. The conditions indicated in the charts are "*": P ≤ 0.05; "**": P ≤ 0.01; "***": P ≤ 0.001; "****": P ≤ 0.0001. The geometric mean method was used to calculate the mean of the specific IgG antibody titer determined by ELISA, and the error bars represent the standard deviation within the group.

[0110] Figure 3 The P / N value of the specific IgG antibody of the mouse immune serum of Example 3 of the present invention was determined. The results showed that: with the increase in the number of immunizations, the level of serum specific IgG antibodies gradually increased. Compared with the negative control group, the levels of three specific IgG antibodies against Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and Omicron S1 protein were all improved after the first booster immunization of BLEP-1 and BLEP-2 proteins, and significantly increased after the second booster (P / N value> 2.1 is the effective value), and the immune effects of different dose groups were different. BLEP-1 and BLEP-2 proteins were used to immunize mice in different dose groups, and their specific IgG antibody production levels were different. Except for the BLEP-1 protein 10μg and 40μg dose groups, the P / N values ​​of the other experimental groups were higher than the positive control group. The optimal dose groups of both proteins were 20μg.

[0111] Figure 4The specific IgG antibody titer of immune serum of mice with higher P / N than the positive control group was determined. The results showed that after the third immunization of the BLEP-1 protein 20μg dose group, the specific binding IgG antibody titers of Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and OcmicronS1 protein could reach 1:3,200, 1:12,800, and 1:800, respectively;

[0112] After the third immunization with BLEP-2 protein, the specific binding IgG antibody titers of Wuhan-Hu-1 S protein, Wuhan-Hu-1 S1 protein, and OcmicronS1 protein in the 10μg dose group reached 1:12,800, 1:102,400, and 1:6,400, respectively; in the 20μg dose group, they reached 1:6,400, 1:12,800, and 1:102,400, respectively; and in the 40μg dose group, they reached 1:102,400, 1:102,400, and 1:102,400, respectively.

[0113] Compared with the 20 μg dose groups of BLEP-1 protein and BLEP-2 protein, the titers of the three specific IgG binding antibodies of BLEP-2 were higher than those of BLEP-1.

[0114] 2. BLEP induces a mixed Th1 / Th2 immune response

[0115] To explore the immune type and response induced by the two proteins, we took spleen cells from mice in the negative control group, BLEP-1 protein 20 μg dosage group, and BLEP-2 protein 10, 20, and 40 μg immunization groups 10 days after each immunization, and used Wuhan-Hu-1 S and Omicron S1 proteins as specific stimuli, respectively, to determine the expression levels of IFN-γ, IL-2, and IL-4 cytokines by ELISA.

[0116] (1) Isolation of mouse spleen cells: 10 days after the immunization, the mice were killed by dislocation of the neck, and soaked in 75% alcohol for 10 min. The spleen was dissected and isolated under aseptic conditions in a biosafety cabinet to obtain spleen cells. The specific steps are as follows:

[0117] (2) Spleen grinding: The mouse spleen was removed and placed in a 35 mm cell culture dish. After washing twice with PBS, the spleen was placed in 2 mL of 1640 cell culture medium and chopped into small pieces. The chopped tissue was placed in a 100 μM mesh cell filter and ground into a 50 mL centrifuge tube. The residual cells on the filter were rinsed with 2 mL of 1640 cell culture medium.

[0118] (3) Cell washing: Centrifuge the cells at 1500 rpm for 5 min at 4°C, discard the supernatant, add 5 mL of 1640 cell culture medium, gently pipette to resuspend, and wash the cells twice.

[0119] (4) Red blood cell lysis: Resuspend the cells in 1 mL of 1640 cell culture medium, add 4 mL of red blood cell lysis solution, lyse on ice for 4 min, and then add an equal volume of 1640 complete culture medium containing 10% fetal bovine serum to terminate the lysis. (If the lysis is not sufficient, repeat the lysis once).

[0120] (5) Cell culture: Centrifuge the cells at 1500 rpm for 5 min at 4°C, discard the supernatant, add 10 mL of 1640 complete culture medium, gently pipette to resuspend, and wash the cells twice. Resuspend in 5 mL of 1640 complete culture medium and transfer to a T25 culture flask for culture until use.

[0121] (6) Splenocyte stimulation: 24 h before the measurement, spleen cells were centrifuged at 1500 rpm for 5 min, resuspended in 1640% cell culture medium and plated (5 × 10 6 cells / mL), Wuhan-Hu-1 S and Omicron S1 protein peptide pools (4 ng / mL) were added to the spleen cell suspension and cultured in an incubator at 37°C and 5% CO2 for 24 h.

[0122] (7) Cytokine indirect ELISA assay: Cytokines are expressed in the supernatant. After stimulation, the cell suspension is centrifuged at 1500 rpm for 5 min to obtain the supernatant. The cytokine assay kit provided by Beijing Sizhengbai Biotechnology Co., Ltd. is used to detect the secretion of splenic lymphocyte-specific INF-γ and IL-2, IL-4 cytokines, and to evaluate the type and level of immune response induced by BLEP-1 and BLEP-2.

[0123] Figure 5 The results are for the determination of cytokines in mouse immune spleen cells. The results showed that after stimulation with Wuhan-Hu-1 S and OmicronS1 proteins, the levels of IL-4 cytokines expressed by spleen cells of mice immunized with BLEP-1 and BLEP-2 were higher than those in the negative control group (NS, NS+adjuvant), and the difference was statistically significant. This indicates that BLEP-1 and BLEP-2 induce a certain Th2-biased immune response in the body, and the level of BLEP-2 is higher. Different dose groups of BLEP-2 induce different levels of Th2-biased responses to Omicron S1 protein, with the 20μg dose group being the highest ( Figure 5 B), the difference was statistically significant, and the level of Th2-biased response induced against Wuhan-Hu-1S protein was not significantly different ( Figure 5A); after stimulation with Wuhan-Hu-1 S and Omicron S1 proteins, the expression levels of IFN-γ and IL-2 in the spleen cells of mice immunized with BLEP-1 and BLEP-2 were higher than those in the negative control group (NS, NS+adjuvant), and the difference was statistically significant, indicating that BLEP-1 and BLEP-2 induced the body to produce a Th1-biased cellular immune response, and the BLEP-2 level was higher. Different dose groups of BLEP-2 induced different levels of Th1-biased responses to Wuhan-Hu-1 S and Omicron S1 proteins, with the 40μg dose group being the highest ( Figure 5 C. Figure 5 D. Figure 5 E. Figure 5 F), the difference was statistically significant; however, there was no significant difference in the level of Th1-biased response induced against Wuhan-Hu-1 S protein ( Figure 5 C and Figure 5 E).

[0124] Example 4 Evaluation of the protective effect of immune serum

[0125] Studies have shown that the protective effect of immune serum can be detected by cell-cell fusion model (reference: Liu Z, Zhou J, Xu W, et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs [J]. Cell Res, 2022, 32 (3): 269-287.). Compared with other detection models, this model is fast to build and has low laboratory requirements. General laboratories can perform related experimental operations and can evaluate the inhibitory effect of drugs or immune serum on virus entry into cells.

[0126] Based on this, we constructed a cell-cell fusion model to evaluate the immune protective effect of immune serum.

[0127] 1. Construction of SARS-CoV-2 cell-cell fusion detection model

[0128] The cloning vector plasmid pAAV-IRES-EGFP expressing Enhanced Green Fluorescent Protein (EGFP) and the recombinant plasmid pAAV-IRES-EGFP-SARS-2-S co-expressing Wuhan-Hu-1 S protein and EGFP were successfully constructed and preserved by our laboratory team (reference: Shan Jing, Yang Manqing, Shang Fangjian, et al. Construction of non-infectious cell-cell fusion model of SARS-CoV-2 [J]. Chinese Journal of Pathogenic Biology, 2023, 18(06): 625-629. DOI: 10.13350 / j.cjpb.230602.). 12 hours before transfection, 293T cells were cultured at 8×10 5 The cells were plated in six wells, and when the cell density reached about 80%, pAAV-IRES-EGFP and pAAV-IRES-EGFP-SARS-2-S plasmid transfection was performed according to the following steps.

[0129] (1) Preparation of EZ Trans transfection complex: 1-2 hours before transfection, replace the complete cell culture medium (DMEM containing 10% FBS) with DMEM. Mix 4 μg of plasmid + 125 μL DMEM to form complex A, and 9 μL EZ Trans transfection reagent + 125 μL DMEM to form complex B. Add complex B to complex A and mix by pipetting (this process is irreversible), and let it stand for 15 minutes to form EZ Trans transfection complex.

[0130] (2) Cell transfection: The EZ Trans transfection complex was evenly dropped into a six-well plate, and cultured for 12 h at 37° C. and 5% CO2. The liquid was then removed and replaced with 2 mL of complete culture medium. Culture was continued for 36-48 h, and the number of fluorescent cells and transfection efficiency were observed under a fluorescence microscope to obtain effector cells 293T / SARS-2 / EGFP and negative control cells 293T / EGFP.

[0131] (3) Target cell plating: One day before cell fusion, plate approximately 3×10 target Huh-7 cells per well. 4 The cells were plated in 96-well plates and cultured at 37°C, 5% CO2 for 12 h.

[0132] (4) Cell-cell co-culture: Remove the culture medium of target Huh-7 cells and add 100 μL of effector 293T / SARS-2 / EGFP cells (1×10 5 The co-culture of 293T / EGFP cell suspension and Huh-7 was used as the negative control. The cell-cell fusion was observed under a fluorescence microscope at different time periods, and the time-fusion relationship curve was drawn.

[0133] (5) DAPI staining: After the cells are fused, remove the culture medium and add 100 μL 4% paraformaldehyde at room temperature for 30 minutes to fix the cells and terminate the fusion. After gently washing the cells twice with PBS, add 20 μL of 10 μg / mL DAPI staining solution (just cover the bottom of the 96 wells) and stain in the dark for 10 minutes. Wash twice with PBS, add 100 μL DMEM, and observe the results under a fluorescence microscope. Reference (Xia S, Liu M, Wang C, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacit-y to mediate membrane fusion [J]. Cell Res, 2020, 30 (4): 343-355.) records that when the target cell Huh-7 is co-cultured with the transfected 293T cells, the effector cells can recognize the receptors of the target cells, so that the two cells fuse. In this example, Huh-7 cells were selected as target cells, pAAV-IRES-EGFP-SARS-2-S and pAAV-IRES-EGFP were transfected into 293T cells respectively, and then the two cells were incubated together. The effector cells specifically recognized the receptors on the Huh-7 cells, and the two cells fused. At the same time, since the transfected cells also expressed EGFP, the early fusion of cells in this model had the advantage of visualization.

[0134] Figure 6 This is the result of constructing the cell-cell fusion model. After 2 hours, obvious cell-cell fusion phenomenon can be observed under a fluorescence microscope ( Figure 6 (As shown by the white arrow in A), at the same fluorescence intensity, the fluorescence intensity of the fused cells is weaker and the shape is irregular compared with the non-fused cells, while the negative control 293T / EGFP has no fusion phenomenon; as time goes by, the proportion of cell-cell fusion gradually increases and the area gradually increases.

[0135] After 12 hours, there was a large area of ​​fusion under the fluorescent field of view, and after DAPI staining, multiple cell nuclei could be seen in the fusion area ( Figure 6 The results of the cell-cell fusion rate curve at different times were observed under a fluorescence microscope and the results showed that after 4-6 hours, the fusion rate reached 50% ( Figure 6 C), which provides the best observation result time for the subsequent inhibition model.

[0136] 2. Candidate vaccine immune serum can effectively inhibit cell-cell fusion

[0137] The cell fusion inhibition experiment was performed according to the reference (Liu Z, Zhou J, Xu W, et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs [J]. Cell Res, 2022, 32 (3): 269-287.). The steps are as follows:

[0138] (1) Obtaining mouse serum: 300 μL of whole blood from the venous plexus of immunized mice was obtained by the orbital venous plexus blood sampling method. The blood was allowed to stand at room temperature for 3 h, centrifuged at 5000 rpm for 10 min, and the upper layer of serum was obtained. The serum was incubated in a water bath at 56°C for 30 min to inactivate complement.

[0139] (2) Serum cell-cell fusion inhibition activity detection: Mouse serum was diluted in DMEM gradient, 293T / SARS-2 / EGFP was co-cultured with Huh-7 as the positive control group, and 293T / EGFP was co-cultured with Huh-7 as the negative control group; gradient diluted mouse immune serum and 293T / SARS-2 / EGFP were incubated at 37°C and 5% CO2 for 1 hour and then added to Huh-7 as the experimental group. The rest of the experimental conditions were the same, the reaction system was 100 μL, and 3 replicate test wells were set for each group. After the cell-cell fusion rate of the positive control group reached 50%, an equal volume of 4% paraformaldehyde was added to terminate the fusion, the fusion of the experimental group was observed and the inhibition rate was calculated, and the inhibition fitting curve was drawn with the serum dilution as the horizontal axis and the fusion inhibition rate as the vertical axis. Inhibition rate = [1-(EN) / (PN)] × 100%, where "E" represents the cell-cell fusion rate of the experimental group; "P" and "N" represent the cell-cell fusion rates of the positive control group and the negative group, respectively. The half maximal inhibitory concentration (IC50) was calculated using Calsusyn software.

[0140] Figure 7The results showed that the third immune serum of the negative control group (NS, NS+adjuvant) had no inhibitory effect on Wuhan-Hu-1 S-mediated cell-cell fusion, while the immune serum of the experimental group BLEP-1 and BLEP-2 had an inhibitory effect. The inhibitory effects of the four groups of sera were different, and the BLEP-2 20μg dose group had the best inhibitory effect, and the serum still had a 50% inhibitory effect when diluted 1:110.

[0141] This example preliminarily proves that the recombinant protein BLEP can induce the body to produce neutralizing antibodies against SARS-CoV-2, can effectively inhibit S protein-mediated cell entry, and the effect of BLEP-2 protein is better than BLEP-1, so it is speculated that it has the effect of inhibiting viral infection.

[0142] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiment, in order to avoid redundancy, the present invention describes a preferred embodiment.

[0143] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0144] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A SARS-CoV-2 small broad-spectrum vaccine immunogenic protein, It is characterized in that The SARS-CoV-2 small broad-spectrum vaccine immunogenic protein is any one of the following: BLEP-1 protein, the amino acid sequence of which is shown in SEQ ID NO.1; BLEP-2 protein, the amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein of claim 1.

3. The gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein according to claim 2, It is characterized in that The immunogenic protein is BLEP-1 protein, and the gene nucleotide sequence encoding the BLEP-1 protein is shown in SEQ ID NO.

3.

4. The gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein according to claim 2, It is characterized in that The immunogenic protein is BLEP-2 protein, and the gene nucleotide sequence encoding the BLEP-2 protein is shown in SEQ ID NO.

4.

5. A recombinant plasmid carrying the gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein of claim 2, It is characterized in that The recombinant plasmid is obtained by inserting the gene encoding the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein into the prokaryotic expression vector pET-21a.

6. Use of the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein according to claim 1 in the preparation of immune serum or antibodies against Wuhan-Hu-1S, Wuhan-Hu-1 S1 and Omicron S1 proteins.

7. The use according to claim 6, It is characterized in that The BLEP-1 protein dosage is 10-40 μg.

8. The use according to claim 6, It is characterized in that The BLEP-2 protein dosage is 10-40 μg.

9. The use according to claim 7, It is characterized in that The BLEP-2 protein dosage was 20 μg.

10. Use of the SARS-CoV-2 small broad-spectrum vaccine immunogenic protein according to claim 1 in the preparation of a new coronavirus small broad-spectrum vaccine.

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