DNA vaccine of SARS-CoV-2 as well as construction method and application of DNA vaccine

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a DNA vaccine of SARS-CoV-2 as well as a construction method and application of the DNA vaccine. According to the present invention, the Wuhan-Hu-1 strain RBD protein is adopted as the design target of the candidate DNA vaccine, and the B cell epitope prediction is performed; the recombinant DNA vaccine pcDNA3.1-LREP1 and pcDNA3.1-LREP2 is obtained by selecting a B epitope peptide with a proper length by combining with a binding site of a neutralizing antibody in an RBD region, connecting with minimum active sites' LMRK 'and' LRMK 'of cathepsin S to obtain LREP1 and LREP2, and inserting nucleotide sequences of the LREP1 and the LREP2 into a pcDNA3.1 eukaryotic expression vector. According to the present invention, the two DNA vaccines such as the pcDNA3.1-LREP1 and the pcDNA3.1-LREP2 prepared by the present invention have good immunogenicity, can induce the generation of the specific IgG antibody of the anti-Wuhan-Hu-1 virus strain S protein in the mouse, and can effectively inhibit the SARS-CoV-2Wuhan-Hu-1 strain S protein mediated cell-cell fusion, and the effect of the pcDNA3.1-LREP2 is superior to the effect of the pcDNA3.1-LREP2.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a DNA vaccine for SARS-CoV-2 and a construction method and application thereof. Background Art

[0002] The new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) poses a huge threat to global human health, economy and security.

[0003] The research team led by Professor Zhang Yongzhen sequenced the genome of the new virus and made it public. Genomic analysis of SARS-CoV-2 and related coronaviruses suggests that SARS-CoV and SARS-CoV-2 may have a common ancestor, belong to adjacent groups, and may have originated in bats and then spread to humans through an intermediate host.

[0004] SARS-CoV-2 is an enveloped, single-stranded, positive-strand RNA virus. It is spherical in shape, with a genome size of about 30kb. It contains 12 open reading frames encoding proteins, including four structural proteins: envelope protein E, membrane protein M, nucleocapsid protein N, and spike protein S. The host range of this virus may gradually expand to other mammals including primates.

[0005] SARS-CoV-2 invades host cells mainly through the S protein on the surface of its viral envelope, which allows the virus to attach to the corresponding receptors of the host cell, promotes the fusion of the host cell and the viral membrane, and releases the viral genetic material into the host cell. The S protein includes the S1 receptor binding unit and the S2 membrane fusion unit (such as Figure 1 As shown in the figure, HR2 of the S2 subunit is tightly anchored to the viral cell membrane. When the virus binds to the angiotensin-converting enzyme 2 (ACE2) on the cell surface through the receptor binding domain (RBD) protein, the fusion peptide at the top of HR1 will be inserted into the host cell phospholipid bilayer, and then HR1 will fold back to HR2, forming a highly stable six-helix structure, bringing the viral membrane and the host cell membrane into close proximity, and ultimately promoting membrane fusion, thereby promoting viral infection. Therefore, the binding of RBD protein to the receptor is a key step in SARS-CoV-2 infecting target cells, so its receptor binding domain (RBD) is the main target of neutralizing antibodies.

[0006] Antibody dependent enhancement (ADE) refers to the fact that when certain viruses invade the body, non-neutralizing antibodies present in the body promote viral infection. In the ADE effect, antibodies targeting regions outside the RBD can bind to the virus to form an antibody-virus complex. This complex binds to Fc receptor II, promoting the entry of the virus into host cells, thereby enhancing infection. A large number of non-neutralizing antibodies targeting regions outside the RBD can lead to an increased risk of ADE. Therefore, compared to vaccines with full-length S protein as the main antigen, using RBD as a vaccine target can limit the number of non-neutralizing epitopes, thereby reducing the risk of ADE, and is an important vaccine safety strategy.

[0007] Traditional vaccines have limited preventive effects on highly variable viruses such as SARS-CoV-2 and HIV. Many vaccination strategies are based on inducing the production of neutralizing antibodies, which in turn produce immune protection. However, due to the diversity of many viral surface proteins and the high mutability of the virus itself, the virus can escape the protection of antibodies produced by vaccination or infection and infect host cells.

[0008] In addition, vaccines that induce antibody production usually do not induce T cell responses well. However, T cells play an extremely important role in viral infection, so we need vaccines that can induce both neutralizing antibodies and T cell responses. As epitope vaccines of short peptides, MHC molecules can present them to CD4+T and CD8+T cells, thereby inducing humoral immune responses and specific T cell responses.

[0009] DNA is an isomer and does not integrate into the host cell DNA. Once DNA uptake is completed, it will be transferred to the nucleus, where the DNA is transcribed into mRNA and transported to the cytoplasm. It is transcribed and translated into endogenous antigens in the nucleus, and it is theoretically expected that it can induce CD8+T cell responses through the MHC class I processing pathway. In addition, some polypeptides also produce extracellular antigens through the exocytosis pathway along the endoplasmic reticulum / Golgi apparatus, which can be recognized by B cells and CD4+T cells. Therefore, the antigens encoded by the DNA vaccine will be presented and recognized by the two main pathways of the immune system, thereby stimulating humoral and cellular immune responses.

[0010] Nucleic acid vaccines are a promising alternative platform among all vaccines developed against COVID-19 infection. The spread of SARS-CoV-2 has stimulated research into the development of new vaccines, including those based on mRNA and DNA. mRNA was the first vaccine approved to fight the virus. In 2021, the DNA vaccine ZyCoV-D developed by Zydus Life Sciences Ltd. in India was approved for use.

[0011] Cathepsin S is an enzyme located in the lysosomes of APC endosomes. Its main physiological function is to cleave chaperone proteins from MHC class II molecules. Nicolas et al. designed substrates to specifically quantify the activity of cathepsin S in APCs, including B cells, macrophages, and dendritic cells, without using any enzyme inhibitors. They found that cathepsin S can more sensitively and effectively cleave substrates for APC presentation than cathepsin B or cathepsin L. The antigen was divided into overlapping peptides of 15 to 18 amino acids and connected using the cathepsin S cleavage site. After the recombinant antigen is internalized by the APC, it can be cut and presented at a specific point, thereby enhancing the effectiveness of APC cells in processing antigens and thereby improving the ability to present antigens.

[0012] The heterologous Prime-Boost strategy offers several theoretical advantages: ① It changes the immune dominance hierarchy by focusing the immune response on the multiple inserted antigenic proteins; ② It increases the breadth and depth of the immune response due to the priming and enhancement of different inserted sequences; ③ The frequency of vaccine-induced effector memory T cells with unique phenotypes and functions that preferentially accumulate in non-lymphoid organs increases, and signals the immune response through the unique innate immune response induced by each vector. ④ The heterologous vector approach can also circumvent the consequences of pre-existing anti-vector immunity.

[0013] However, DNA vaccines can be developed quickly and safely, and are effective and inexpensive. However, their relatively weak immunogenicity needs to be further addressed. Because traditional SARS-CoV-2 vaccines have complex antigenic components, the non-neutralizing antibodies produced against regions other than RBD can mediate antibody-dependent enhancement (ADE) and promote viral infection. Summary of the invention

[0014] In view of the fact that antibodies produced by RBD epitopes can minimize the production of non-neutralizing antibodies and thus avoid the occurrence of ADE phenomenon, the present invention takes the RBD region of the S protein as the research object, conducts bioinformatics analysis, and searches for the optimal epitope; at the same time, it is connected with the smallest active fragment of cathepsin S, so that it can be cut at a fixed point in APC cells and presented efficiently. A DNA vaccine is constructed by inserting its encoding DNA sequence into the expression plasmid pcDNA3.1. The vaccine, combined with the Prime-Boost strategy, will produce RBD-specific antibodies while minimizing the production of non-neutralizing antibodies.

[0015] In order to prepare a highly targeted and immunocompetent COVID-19 vaccine, the present invention provides a SARS-CoV-2 DNA vaccine and a construction method and application thereof. The DNA vaccine has good immunogenicity and immunoreactivity, and the induced antibodies have the ability to inhibit SARS-CoV-2 from entering infected cells.

[0016] The object of the present invention is to provide an RBD multi-epitope protein selected from any one of the following:

[0017] LREP1, the amino acid sequence is shown in SEQ ID NO.1;

[0018] LREP2, the amino acid sequence is shown in SEQ ID NO.2.

[0019] The present invention also provides a gene fragment encoding the RBD multi-epitope protein.

[0020] Furthermore, the nucleotide sequence of the gene fragment encoding the RBD multi-epitope protein LREP1 is shown in SEQ ID NO.3.

[0021] Furthermore, the nucleotide sequence of the gene fragment encoding the RBD multi-epitope protein LREP2 is shown in SEQ ID NO.4.

[0022] The present invention also provides a recombinant DNA vaccine comprising the RBD multi-epitope protein gene fragment.

[0023] Furthermore, the recombinant DNA vaccine is obtained by inserting a gene fragment encoding the RBD multi-epitope protein into a pcDNA3.1 eukaryotic expression vector.

[0024] Furthermore, the recombinant DNA vaccine is any one of pcDNA3.1-LREP1 and pcDNA3.1-LREP2.

[0025] The present invention also provides a method for constructing a recombinant DNA vaccine of the RBD multi-epitope protein gene fragment, and inserting the gene fragment encoding the RBD multi-epitope protein into the pcDNA3.1 eukaryotic expression vector to obtain the recombinant DNA vaccine.

[0026] The present invention also provides an application of the recombinant DNA vaccine in the preparation of a product for preventing COVID-19.

[0027] Furthermore, the product can induce the production of specific IgG against Wuhan-Hu-1-S protein.

[0028] Furthermore, the product can induce and enhance the Th1 / Th2 mixed immune response in mice.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention provides two RBD multi-epitope proteins LREP1 and LREP2, and constructs pcDNA3.1-LREP1 and pcDNA3.1-LREP2 recombinant DNA vaccines respectively. The Wuhan-Hu-1 S-specific IgG level of the LREP1 (High) + S group is P / N = 2.38, and the P / N of the LREP2 (High) + S group is 12.67. The expression levels of the two candidate DNA vaccine immunization groups are higher than those of the N group (PBS group) and the Mock group (empty control group). Both LREP1 and LREP2 can induce enhanced Th1 / Th2 mixed immune responses in mice to a certain extent, among which LREP2 can induce Th1 type cellular immune responses better than LERP1.

[0031] The present invention combines the principle of polypeptide vaccines, takes the Wuhan-Hu-1 strain RBD protein as the design target of the candidate DNA vaccine, and predicts B cell epitopes. Combined with the binding site of the neutralizing antibody in the RBD region, the B epitope peptide of suitable length is selected and connected with the minimum active sites "LMRK" and "LRMK" of cathepsin S. After the company synthesizes the DNA coding sequence, it is cloned into the pcDNA3.1(+) vector as the candidate DNA vaccines LREP1 and LREP2. The DNA vaccine covers the B cell linear antigen epitopes of the antigen peptides in the SARS-CoV-2 RBD region that can produce highly effective neutralizing antibodies. This design can both induce humoral immunity to produce effective neutralizing antibodies and efficiently stimulate the body's immune response level.

[0032] 2. In the construction of the DNA vaccine provided by the present invention, due to the introduction of cathepsin S, the expressed recombinant protein is captured and endocytosed by APC, and then it will be accurately cut in the lysosome, processed into multiple epitope peptides, and presented to Th cells to activate the immune system, which can enhance the ability to be processed and presented by APC, thereby achieving the effect of enhancing the immune response. This design is expected to improve the common problems of traditional polypeptide vaccines, such as insufficient ability to activate the immune system and weak immune effect. The present invention combines the conservative dominant epitopes in the SARS-CoV-2 RBD region and the epitopes containing various mutation sites, and has good targeted immune ability. In addition, the synthesis cost of DNA vaccines is relatively low, the production speed is fast, and it shows great stability in storage. Recombinant DNA technology can quickly design plasmid DNA, which will be more conducive to the promotion of new coronavirus vaccines.

[0033] 3. Some key advantages of epitope vaccines are the ability to induce both B cell and T cell immune responses in the absence of any infectious source, and its highly stable high storage capacity, the ability to encode several viral proteins in a single vaccine, the need to extract proteins from pathogens, and the need to avoid contact with pathogens during production, and high safety. Compared with traditional inactivated vaccines, it is also a good starter vaccine because it can focus the immune response on a narrow set of highly immunogenic epitopes. In addition, peptide vaccines have improved preparation safety due to their high purity and controllable extraction and production process, while traditional protein and live attenuated vaccines have a higher risk of contamination by other preparations and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the SARS-CoV-2S protein structure and the membrane fusion mechanism it mediates.

[0035] Figure 2 This is a schematic diagram of the design of the candidate DNA vaccine of the present invention;

[0036] In the figure, A is a schematic diagram of the selection of B epitope peptides in the RBD protein of Wuhan-Hu-1 strain;

[0037] B is a schematic diagram of the connection of epitope polypeptides.

[0038] Figure 3 Schematic diagram of screening for dominant epitope peptides.

[0039] Figure 4 PCR amplification of the target fragment constructed for the candidate DNA vaccine in Example 2 of the present invention;

[0040] In the figure, A is PCR amplification of LREP1 fragment, M: D2000; B is PCR amplification of LREP2 fragment, M: D2000.

[0041] Figure 5 Colony PCR identification of the candidate DNA vaccine constructed in Example 2 of the present invention;

[0042] Note: Lanes 1 to 5 are LREP1; Lanes 6 to 9 are LREP2, M: AL2000.

[0043] Figure 6 Double enzyme digestion identification of the candidate DNA vaccine constructed in Example 2 of the present invention;

[0044] In the figure, A is the double enzyme digestion identification of LREP1; B is the double enzyme digestion identification of LREP2.

[0045] Figure 7 This is the WB expression verification result of the candidate DNA vaccine in Example 2 of the present invention;

[0046] In the figure, A is the WB verification of LREP1, where lane 1: culture supernatant of LREP1 transfection, lane 2: cell lysate of LREP1 transfection, Mock: culture supernatant of pcDNA3.1(+) vector transfection, M: 180kD protein marker;

[0047] B is WB verification of LREP2, wherein lane 1: culture supernatant of LREP2 transfection, lane 2: cell lysate of LREP2 transfection, Mock: culture supernatant of pcDNA3.1(+) vector transfection, M: 180kD protein Marker.

[0048] Figure 8 The indirect immunofluorescence detection result of the candidate DNA vaccine in Example 2 of the present invention;

[0049] In the figure, A is the indirect immunofluorescence detection result of the empty plasmid transfected with pcDNA3.1(+);

[0050] B is the indirect immunofluorescence detection result of pcDNA3.1-LREP1 vaccine;

[0051] C is the indirect immunofluorescence detection result of pcDNA3.1-LREP2 vaccine.

[0052] Fig. 9 The P / N value of the mouse immune serum specific IgG antibody of Example 3 of the present invention is determined, Prime: the first immunization, Boost 1: the first booster immunization, Boost 2: the second booster immunization.

[0053] Fig.10 The results of Example 4 show that different groups of mice were induced to produce high levels of specific IgG antibodies after heterologous immunization.

[0054] Fig.11 The results of cytokine detection induced by different groups of treatments in Example 4 of the present invention are shown;

[0055] In the figure, A is the detection of IL-2 cytokines induced under different treatments; B is the detection of IL-4 cytokines induced under different treatments; C is the detection of IFN-γ cytokines induced under different treatments;

[0056] Fig.12 The results of constructing the Wuhan-Hu-1-S mediated cell fusion model in Example 5;

[0057] In the figure, A shows the cell fusion status at 4 h after the Wuhan-Hu-1-S-mediated cell fusion model was constructed;

[0058] B shows the cell fusion status of the Wuhan-Hu-1-S mediated cell fusion model at 24 hours.

[0059] Fig.13 These are the results of the inhibition of cell-cell fusion detection by mouse immune sera from different groups in Example 5. DETAILED DESCRIPTION

[0060] 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.

[0061] In the description of the present invention, unless otherwise specified, the reagents or methods are conventional reagents or methods in the art. In the following examples, the concentration of PBS used is 0.1 M and the pH is 7.4.

[0062] Example 1 Screening of B cell epitopes in the dominant RBD region of Wuhan-Hu-1 strain

[0063] The present invention combines the principle of polypeptide vaccine. The present invention uses the Wuhan-Hu-1 strain RBD protein as the design target of the candidate DNA vaccine and predicts the B cell epitope. Combined with the binding site of the neutralizing antibody in the RBD region, the B epitope peptide (or epitope polypeptide) of suitable length is selected and connected with the minimum active site "LMRK" and "LRMK" of cathepsin S. After the company synthesizes the DNA coding sequence, it is cloned into the pcDNA3.1(+) vector as the candidate DNA vaccine LREP1 (LMRK-linked RBDmulti-epitope Protein) and LREP2 (LRMK-linked RBD multi-epitope Protein) ( Figure 2 ), Figure 2 In the figure, panel A shows the PCR amplification results of LREP1; Figure 2 Figure B shows the PCR amplification result of LREP1.

[0064] 1. Prediction of linear B cell antigens in the RBD region

[0065] (1) The target S protein sequence was compared using BioEdit software, and the amino acid sequence of the RBD region was screened to be 319aa to 541aa, and the RBM region was 438aa to 508aa. We used the RBD protein as the target and used the IEBD website (http: / / tools.iedb.org / bcell / ) to predict possible sites of B lymphocyte linear epitopes, and selected the predicted epitopes with a length of 10 to 15 amino acids to cover all predicted B epitopes as much as possible.

[0066] (2) The online software Bepipred Linear Epitope Prediction 2.0 was used to predict the B cell linear epitopes in the RBD region of the Wuhan-Hu-1 strain, with the threshold set at >0.5. We screened for dominant epitope peptides based on the reported neutralizing antibody concentration sites. The results are shown in Figure 3 As shown, we found that the B cell linear epitopes in the RBD region of the Wuhan-Hu-1 strain were basically concentrated in the RBM region (438aa~508aa).

[0067] 2. Screening of dominant linear B epitope peptides

[0068] (1) After obtaining all possible B epitopes, the VaxiJen v2.0 server (http: / / www.ddg-pHarmfac.net / vaxijen / VaxiJen / VaxiJen.html) was used to predict the antigenicity of all candidate epitope peptides. Based on the reported RBD neutralizing antibody concentration region, the epitope peptide length was set to the length that can be co-presented by MHC class I and MHC class II molecules: 10 to 13 aa, and the overlapping part of the peptide sequence was repeated for 5 to 7 aa to cover the predicted linear B cell epitope in the RBD region. The VaxiJen v2.0 server was used for antigenicity prediction to select the dominant epitope peptides (Table 1).

[0069] Table 1 Information of dominant epitope peptides

[0070]

[0071]

[0072] (2) The artificial sequence LREP1 was designed using LMRK (Protease cleavage site 1) The amino acid sequence of the fusion protein connected to the predicted epitope. The sequence content is "IL-2 signal peptide" ( ATMYRMQLLSCIALSLALVTSS)+ P1 + P2+P2+P2+P3+P3+P3+P5+P5+P6+P6+P7+P8+P8+P8+P9+P9+6×His”.

[0073] The amino acid sequence of LREP1 is shown in SEQ ID NO.1:

[0074] SEQ ID NO.1:

[0075] ATMYRMQLLSCIALSLALVTSSLMRK GDEVRQIAPG LMR PGQTGKIADYNYK LMR PGQTGKIADYNYK LMR PGQTGKIADYNYK LMR ADYNYKLPDDFT LMR ADYNYKLPDDFT LMR ADYNYKLPDDFT LMR LDSKVGGNYNY LMR LDSKVGGNYNY LMR NYLYRLFRKSNL LMR NYLYRLFRKSNL LMR KSNLKPFERD LMR STEIYQAGSTP LMR STEIYQAGSTP LMR STEIYQAGSTP LMR GVEGFNCYFPLQ LMR GVEGFNCYFPLQ LMR GVEGFNCYFPLQ LMR SYGFQPTNGVGYQ LMR SYGFQPTNGVGYQ LMR SYGFQPTNGVGYQ LMR EFHHHHHH.

[0076] The artificial sequence LREP2 was designed using LRMK (Protease cleavage site 2) The amino acid sequence of the fusion protein connected to the predicted epitope. The sequence content is "IL-2 signal peptide" ( ATMYRMQLLSCIALSLALVTSS)+ P1 + P2+P2+P2+P3+P3+P3+P5+P5+P6+P6+P7+P8+P8+P8+P9+P9+6×His

[0077] The amino acid sequence of LREP2 is shown in SEQ ID NO.2:

[0078] SEQ ID NO.2:

[0079] ATMYRMQLLSCIALSLALVTSSLRMK GDEVRQIAPG LRMK PGQTGKIADYNYK LRMK PGQTGKIADYNYK LRMK PGQTGKIADYNYK LRMK ADYNYKLPDDFT LRMK ADYNYKLPDDFT LRMK ADYNYKLPDDFT LRMK LDSKVGGNYNY LRMK LDSKVGGNYNY LRMK NYLYRLFRKSNL LRMKNYLYRLFRKSNL LRMK KSNLKPFERD LRMK STEIYQAGSTP LRMK STEIYQAGSTP LRMK STEIYQAGSTP LRMK GVEGFNCYFPLQ LRMK GVEGFNCYFPLQ LRMK GVEGFNCYFPLQ LRMK SYGFQPTNGVGYQ LRMK SYGFQPTNGVGYQ LRMK SYGFQPTNGVGYQ LRMK EFHHHHHH.

[0080] Example 2 Construction and expression of candidate DNA vaccines

[0081] 1. Construction of recombinant plasmid

[0082] Through PCR and the restriction sites BamH I and Xho I of the vector, the CDS sequences encoding LREP1 protein and LREP2 protein optimized and synthesized by Shanghai Bioengineering Co., Ltd. were inserted into the pcDNA3.1 eukaryotic expression vector to obtain recombinant DNA vaccines pcDNA3.1-LREP1 and pcDNA3.1-LREP2.

[0083] Wherein, the nucleotide sequence of the gene fragment encoding LREP1 protein is shown in SEQ ID NO.3;

[0084] SEQ ID NO.3:

[0085]

[0086] The nucleotide sequence of the gene fragment encoding the LERP2 protein is shown in SEQ ID NO.4;

[0087] SEQ ID NO.4:

[0088]

[0089] The underlined part is the base sequence of the restriction sites BamH I and Xho I added to the vector, the double wavy line is the Kozak sequence, and the single wavy line is the signal peptide sequence, which was optimized and synthesized by Shanghai Bioengineering Co., Ltd. and subsequently connected to the pcDNA3.1(+) vector to obtain the recombinant DNA vaccine.

[0090] Figure 4 The target fragment is amplified by PCR using specific primers for the target fragment. The obtained product is Figure 4A is the PCR amplification of the LREP1 gene fragment; Figure 4 B is PCR amplification of LREP2 gene fragment;

[0091] Figure 5 For suspected positive colonies, single colonies were picked for colony PCR identification; Figure 6 To isolate the positive colonies with the correct bands, the plasmids were extracted for double enzyme digestion identification. A is the LREP1 gene fragment and B is the LREP2 gene fragment. The above results show that the recombinant DNA vaccines pcDNA3.1-LREP1 and pcDNA3.1-LREP2 were successfully constructed.

[0092] 2. WB verification of target protein expression

[0093] The recombinant plasmids pcDNA3.1-LREP1 and pcDNA3.1-LREP2 were transfected into 293T cells and their expression was verified by Western Blot.

[0094] The specific steps are as follows:

[0095] (1) Transfection

[0096] 1) Medium change: 1 h before transfection, discard the complete medium in the 6-well plate, wash once with PBS, and add 1 mL of pre-warmed serum-free DEME.

[0097] 2) Preparation of EZ Trans-DNA complex: Equilibrate the transfection reagent EZ and recombinant plasmid at room temperature for about 10 minutes; prepare two clean EP tubes, add 9 μL EZ into 125 μL DMEM in one tube, and slowly add 3000 ng recombinant plasmid into 125 μL DMEM in the other tube, vortex and oscillate to mix thoroughly, then add the diluted EZ Trans reagent into the plasmid diluted in DMEM at one time; prepare 6 tubes of EZ Trans-DNA complex according to the above steps, vortex and oscillate to mix well, and let stand for 15 minutes.

[0098] 3) Transfection: Add 250 μL of the prepared EZ Trans-DNA complex evenly to a 6-well plate, and gently shake the six-well plate up and down and left and right for about 80 times to mix; after culturing for about 14 hours, discard the supernatant of the culture medium containing the transfection reagent (if the cell state is not good, add 1 mL of fresh 30% FBS complete culture medium 3 hours after transfection), replace with fresh 10% FBS complete culture medium and continue culturing for 12 hours, discard the supernatant of the culture medium, wash twice with 1 mL / well PBS, and add 3 mL / well DMEM to culture until the cells are not dead.

[0099] (2) SDS gel electrophoresis and membrane transfer

[0100] 1) Gel electrophoresis: Take out the gel plate, fasten it with the electrophoresis tank, add the inner and outer tank solutions, slowly pull out the comb, and gently flush the electrophoresis wells with the gun tip to blow out the residual colloid and air; take 20 μL of sample for loading, and perform electrophoresis at 80V until the bromophenol blue runs into the separation gel and the marker bands begin to separate, then switch to 120V and continue electrophoresis until the bromophenol blue reaches the bottom of the gel and stops the electrophoresis.

[0101] 2) Transfer: Cut the gel of the target protein size along the marker band, and cut out a 0.22μm PVDF membrane of similar size to the gel block; place the transfer sandwich in the order of thick sponge, filter paper board, PVDF membrane, gel block, filter paper board, and thick sponge, and be careful to avoid bubbles between the gel block and the PVDF membrane, and place them in the transfer tank in the order of positive membrane, negative gel, and transfer at 270mA for 1h.

[0102] 3) Blocking: Weigh an appropriate amount of skim milk powder and dissolve it in TBST to prepare a 5% blocking agent. Incubate at room temperature at 60 rpm for 2 h, and wash three times with TBST at 100 rpm, 15 min each time.

[0103] 4) Primary antibody incubation: Prepare an appropriate amount of anti-His antibody at a ratio of 1:2500, incubate the PVDF membrane at room temperature at 60 rpm for 30 min, then transfer it to a 4°C refrigerator for incubation for 18 h, and wash it three times with TBST at 100 rpm, 15 min each time.

[0104] 5) Secondary antibody incubation: Prepare an appropriate amount of goat anti-mouse secondary antibody at a ratio of 1:5000, incubate the PVDF membrane at room temperature at 60 rpm for 1 h, and then wash it 4 times with 100 rpm TBST, 15 min / time.

[0105] 6) Development: According to the size of the PVDF membrane, mix appropriate amounts of ultrasensitive chemiluminescent detection reagent A solution and B solution in a 1:1 ratio under light-proof conditions (prepare immediately before use), place in ImageQuant LAS 4000 for exposure and development, check and save the results.

[0106] WB results are as follows Figure 7 , A is the WB verification of LREP1, and B is the WB verification of LREP2. Lane 1 in A and B is the culture supernatant of transfected LREP1 and LREP2; Lane 2 is the cell lysate of transfected LREP1 and LREP2; Mock is the culture supernatant of pcDNA3.1(+) vector; M is the 180kD protein marker. The results show that the proteins in the supernatant and cell lysate of 293T transfected with pcDNA3.1-LREP1 and pcDNA3.1-LREP2 are consistent with the expected size.

[0107] 3. Indirect immunofluorescence detection of intracellular expression of candidate DNA vaccines

[0108] 293T cells were passaged and cultured at 80 × 10 4 Each well was plated into a six-well plate. When the cell confluence reached about 70%, pcDNA3.1-LREP1 and pcDNA3.1-LREP2 were transfected into 293T cells. The indirect immunofluorescence procedure was as follows:

[0109] (1) Cell fixation: 48 h after transfection, discard the culture supernatant, wash the cells slowly and gently three times with PBS (pH = 7.4) (be careful not to wash away the cells), fix the cells with 2 mL of 4% paraformaldehyde for 30 min, discard the liquid, wash the cells gently three times with PBS (5 min / time), and leave the lid open at room temperature for 15 min to evaporate all the paraformaldehyde.

[0110] (2) Perforation and blocking: 2 mL of 0.25% Triton X-100 was added to the six-well plate and perforated for 5 min. The plate was washed three times with PBS, 5 min each time. The plate was blocked with 5% skim milk for 2 h and washed three times with PBS, 5 min each time.

[0111] (3) Primary antibody incubation: dilute mouse anti-6×His tag monoclonal antibody 1:100 with PBS, incubate at 37°C for 1 h, and wash three times with PBS, 5 min each time.

[0112] (4) Secondary antibody incubation: dilute goat anti-mouse FITC-labeled secondary antibody at 1:100 with PBS, incubate at 37°C for 1 h, wash four times with PBS, 5 min each time, and dry at room temperature in the dark for about 15 min.

[0113] (5) Nuclear staining: DAPI was diluted to 1 μg / mL with PBS, and the cells were stained for 10 min at room temperature in the dark. The cells were then washed three times with PBS, 3 min each time.

[0114] (6) Observation and photography: Use a CKX53 optical microscope to observe DAPI and FITC staining at 20x magnification and take photos for preservation.

[0115] Figure 8 This is an indirect immunofluorescence result. Under the same conditions, 293T cells transfected with pcDNA3.1(+) empty plasmid (Mock) were not specifically labeled with FITC fluorescence, while 293T cells transfected with candidate DNA vaccine pcDNA3.1-LREP1 and pcDNA3.1-LREP2 plasmids showed obvious fluorescence expression. However, the fluorescence expression level of the cells is not necessarily proportional to the immune effect of subsequent animal experiments and can only be used as a result to identify that the candidate DNA vaccine can induce protein expression.

[0116] Example 3 Mouse Immunization and Serological Detection

[0117] The purchased 6-8 week old female mice were randomly divided into 6 groups, 4 mice in each group. They were divided into a blank group (Negative control, N group), an empty load control group (Mock group) and two candidate DNA vaccine experimental groups (pcDNA3.1-LREP1 and pcDNA3.1-LREP2). The immunization cycle was week 0, week 3, and week 5, with a total of three immunizations. The immunization method was intramuscular injection of the mouse calf, and the total volume was 100 μL. Ten days after each immunization, blood was collected from the orbital venous plexus for standby use. Ten days after the end of the booster immunization, the eyeballs were removed for blood collection and the spleen was removed by cervical dislocation. The specific immunization groups and abbreviations during this period are shown in the following table:

[0118] Table 2 Mouse immunization grouping scheme

[0119]

[0120]

[0121] Recombinant DNA vaccine can induce the production of specific IgG antibodies

[0122] (1) Antigen coating: Based on the optimal coating concentration determined under the above conditions, the protein antigen Wuhan-Hu-1-S was coated into a 96-well plate at 50 μL / well according to the required number of wells. The plate was coated overnight at 4°C and washed three times with PBST for 5 min each time.

[0123] (2) Blocking: Weigh the required amount of skim milk and dissolve it in PBST to prepare a 5% blocking agent. Add 200 μL / well to a 96-well plate and block at 37°C for 2 h. Wash three times with PBST, 5 min each time.

[0124] (3) Primary antibody incubation: Dilute mouse DNA vaccine immune serum 1:200, 50 μL / well, add each serum to one well in a 96-well plate, incubate at 37°C for 2 h, and wash three times with PBST, 5 min each time.

[0125] (4) Secondary antibody incubation: dilute the required amount of HRP-labeled goat anti-mouse secondary antibody at 1:3000, 50 μL / well, incubate at 37°C for 2 h, and wash three times with PBST, 5 min each time.

[0126] (5) Color development, termination, and plate reading: First, prepare the required amount of color development solution and add 50 μL / well to a clean 96-well ELISA plate. Develop the color at room temperature in the dark for 15 to 20 min. After adding 10% sulfuric acid to terminate the reaction, immediately read the OD450 using an ELISA reader and save and organize the statistical data.

[0127] Fig. 9The 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 in the mouse immune serum of the candidate DNA vaccine (pcDNA3.1-LREP1 and pcDNA3.1-LREP2), after the primary immunization and two booster immunizations, the LREP1 high-dose group was able to obtain a higher specific antibody level than the low-dose group at the primary immunization and the first booster immunization; and for LREP2, whether it was the primary immunization or the booster immunization, the high-dose group (High) immune level was always higher than the low-dose group (Low). In addition, in terms of inducing the specific IgG level against the Wuhan-Hu-1-S protein, the effect of LREP2 (High) was always better than that of LREP1 (High).

[0128] Example 4 DNA vaccine candidate prime S protein boost induces high level immune response

[0129] After a simple three-dose candidate DNA vaccine immunization, we designed a new DNA vaccine booster immunization strategy based on the heterologous Prime-Boost strategy to further explore whether the antigen presentation enhanced vaccine design of the present invention can achieve the desired desired purpose.

[0130] The three-dose DNA vaccine immunization was changed to two-dose DNA vaccine plus one-dose Wuhan-Hu-1 S protein, hoping to induce a higher level of immune response based on the heterologous priming-enhancing immunization strategy. The positive control group (Postive control) was immunized with 10μg of Wuhan-Hu-l S protein, and the immunization cycle was week 0, week 2, and week 4, with a total of three immunizations, and the total volume was 100μL. In the positive control group, Wuhan-Hu-1-S protein used Freund's adjuvant, Freund's complete adjuvant was used for primary immunization, and Freund's incomplete adjuvant was used for booster immunization. Equal volumes of Wuhan-Hu-1-S protein dissolved in PBS were mixed in a 1:1 ratio until an oil-in-water state was formed. Blood was collected from the orbital venous plexus 10 days after each immunization. The immunization groups are as follows:

[0131] Table 3 Mouse immunization groups

[0132]

[0133] 1. The present invention induces mice to produce high levels of specific IgG antibodies after heterologous immunization

[0134] The mice were bled from the eye sockets according to different groups, placed at room temperature for 1-2 hours, centrifuged at 5000rpm for 10 minutes, and the serum was carefully removed, inactivated at 60°C for 30 minutes, and then stored at -20°C to avoid repeated freezing and thawing. Mouse serum-specific IgG detection was performed according to the ELISA steps in Example 3. On the basis of Example 3, we used the LREP1 (High / Low) + S / LREP2 (High / Low) + S strategy to immunize mice, and took the mouse serum 10 days after booster immunization to detect the specific IgG antibody level against Wuhan-Hu-1 S.

[0135] Fig.10 The results of the invention inducing mice to produce high levels of specific IgG antibodies after heterologous immunization (the figure shows the P / N value of the mouse serum ELISA result 10 days after booster immunization; whether in the high-dose group or the low-dose group, the LREP1 group or the LREP2 group, the specific IgG antibody level is always two injections of DNA vaccine plus one injection of S protein booster immunization, that is, the immunization strategy of LREP1 (High / Low) + S and LREP2 (High / Low) + S is better than the immunization strategy of simple three injections of DNA vaccine (LREP1 and LREP2), and the high-dose group is always better than the low-dose group. In addition, the Wuhan-Hu-1 S specific IgG level of the LREP1 (High) + S group P / N = 2.38, and the P / N of the LREP2 (High) + S group is 12.67; the specific IgG antibody level of the positive group (P group) is P / N = 5.39, and the specific IgG antibody level induced by the LREP2 + S group is about twice that of the P group (P < 0.01). And the LREP2 + S immunization group against Wuhan-Hu-1 The level of specific IgG antibody of S was also significantly increased. In the figure, * represents P<0.05 when the two groups were tested for hypothesis, ** represents P<0.01 when the two groups were tested for hypothesis, and *** represents P<0.001 when the two groups were tested for hypothesis.

[0136] 2. LREP1 and LREP2 induce a mixed Th1 / Th2 immune response

[0137] To explore the immune type and response induced by the two proteins, we took the spleen cells of mice in the negative control group, empty control group, LREP1 (High) + S group and LREP2 (High) + S group after primary and booster immunization, and performed ELSA detection of IL-2, IL-4 and IFN-γ cytokines in the supernatant after stimulation with SARS-CoV-2S protein. The steps are as follows:

[0138] (1) According to the grouping, the mice were eyeballed and blood was collected (the blood serum was centrifuged and stored for later use), then killed by dislocating the neck, and placed in 75% alcohol for 15 minutes for disinfection.

[0139] (2) Use autoclaved tweezers and small scissors to remove the spleen from the right side of the mouse and place it in a sterile cell culture dish. Wash it three times with sterile PBS and discard the liquid. Add 1 mL of pre-cooled 1640 culture medium and use small scissors to cut the spleen into small pieces as much as possible. Add it to the sterile 70 μm pore size nylon mesh prepared in advance. Use the piston push rod of a disposable sterile syringe to grind the chopped spleen pieces until there are no obvious tissue fragments visible to the naked eye. Repeat the grinding 2 to 3 times.

[0140] (3) Transfer the ground mouse spleen cell tissue homogenate to a clean and sterile 50 mL centrifuge tube, add 3 mL of 1640 culture medium, pipette and mix, centrifuge at 2500 rpm for 5 min, discard the supernatant; add 4 mL of 1640 culture medium to resuspend, centrifuge at 2500 rpm for 5 min, and repeat the washing once.

[0141] (4) After washing, resuspend with 1 mL of 1640 and add 3 mL of red blood cell lysis buffer and place on ice for 30 seconds to 1 minute. Immediately add 6 mL of 1% dual antibody 1640 medium to terminate the reaction and centrifuge at 2500 rpm for 5 minutes.

[0142] (5) Add 10 mL of 1% double antibody-containing 1640 culture medium to resuspend and wash. Repeat the operation once, resuspend with 2 mL of culture medium, add to a 6-well plate pre-added with 3 mL of 1% double antibody-containing 1640 culture medium, and place in a 37°C carbon dioxide incubator for culture.

[0143] (6) Cytokine ELISA detection: Cytokines are expressed in the supernatant. After the stimulation, the cell suspension was centrifuged at 1500 rpm for 5 min to obtain the supernatant. The splenic lymphocyte-specific INF-γ and IL-2, IL-4 cytokine secretion was detected according to the instructions of the cytokine assay kit of Beijing Sizhengbai Biotechnology Co., Ltd. to evaluate the type and level of immune response induced by pcDNA3.1-LREP1 and pcDNA3.1-LREP2.

[0144] Fig.11 The results of the cytokine detection induced by the present invention (A: IL-2 cytokine detection; B: IL-4 cytokine detection; C: IFN-γ cytokine detection), the IL-2 level after three immunizations ( Fig.11A), the expression levels of the two candidate DNA vaccine immunization groups were higher than those of the N group (PBS group) and the Mock group (empty control group). The IL-2 of the LREP2 (High) + S group was 136.39 ± 16.78 pg / mL, which was higher than the IL-2 of the LREP1 (High) + S group (25.42 ± 1.19 pg / mL), the difference was significant (P < 0.001), and LREP2 was 5.36 times that of LREP1. In the figure, * represents P < 0.05 when the two groups are tested for hypothesis, ** represents P < 0.01 when the two groups are tested for hypothesis, and *** represents P < 0.001 when the two groups are tested for hypothesis.

[0145] In terms of IL-4 cytokine expression level ( Fig.11 B), IL-4 in the LREP1(High)+S group was 33.64±2.11pg / mL, and IL-4 in the LREP2(High)+S group was 38.09±5.17pg / mL. However, the difference between the LREP1(High)+S group and the LREP2(High)+S group was not significant (P=0.352>0.05).

[0146] In addition, for IFN-γ ( Fig.11 C), the IFN-γ level of LREP2(High)+S group was 1195.57±8.46pg / mL, which was higher than that of LREP1(High)+S group (224.59±9.13pg / mL), and the difference was significant (P<0.001). LREP2 was 5.32 times that of LREP1.

[0147] In general, compared with the LREP1(High)+S group, the cytokine IFN-γ, which induces Th1 immune response, and the cytokine IL-4, which induces Th2 immune response, in the LREP2(High)+S group were both up-regulated, with IFN-γ being particularly significant. In addition, IL-2, which can stimulate T cell proliferation, also increased significantly. It was judged that both LREP1 and LREP2 can induce and enhance the mixed Th1 / Th2 immune response of mice to a certain extent, and LREP2 can induce Th1 cellular immune response better than LERP1.

[0148] Example 5 Evaluation of the protective effect of immune serum

[0149] 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.

[0150] Based on this, we constructed a cell-cell fusion model to evaluate the immune protective effect of immune serum.

[0151] 1. Construction of Wuhan-Hu-1-S mediated cell fusion model

[0152] The cloning vector plasmid pAAV-IRES-EGFP expressing Enhanced Green Fluorescent Protein (EGFP) and the recombinant plasmid pAAV-GFP-Wuhan-Hu-1 S co-expressing Wuhan-Hu-1 S protein and EGFP were successfully constructed and preserved by our laboratory team in the early stage (for specific construction methods, see references: Shan Jing, Yang Manqing, Shang Fangjian, et al. Construction of a 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 Each well was plated in six wells. When the cell density reached about 80% to 85%, pAAV-IRES-EGFP and pAAV-GFP-Wuhan-Hu-1 S plasmids were transfected and the fusion model was constructed according to the following steps:

[0153] (1) Preparation of EZ Trans-DNA complex: Equilibrate the required EZ transfection reagent and target plasmids pAAV-GFP-Wuhan-Hu-1 S (positive control) and pAAV-IRES-EGFP to room temperature in advance; prepare two clean EP tubes, add 9 μL EZ to 125 μL DMEM in one EP tube (to dilute the EZ Trans reagent), and slowly add 3000 ng pAAV-GFP-Wuhan-Hu-1 and 3000 ng pAAV-IRES-EGFP plasmids to the marked EP tubes containing 125 μL DMEM in the other tube (to dilute the plasmid); mix them by pipetting, and use a vortex mixer to ensure sufficient mixing when necessary; add the diluted EZ Trans reagent to the plasmid diluted in DMEM at one time; mix them by pipetting thoroughly and let stand for 15 min.

[0154] (2) Transfection: Change the medium at least 1 h before transfection (change the complete medium to serum-free DMEM medium); gently blow the EZ Trans-DNA complex prepared in advance with a total volume of 250 μL using a pipette and evenly add it to the 6-well plate that has been changed in advance, and gently shake the 6-well plate to evenly distribute the EZ Trans-DNA complex; place it in a CO2 incubator at 37°C and culture it for about 12 h, then discard the culture medium supernatant containing the EZ Trans-DNA complex, replace it with fresh 10% FBS complete medium and continue to culture it for about 48 h, during which time observe the fluorescence transfection efficiency for subsequent experiments.

[0155] (3) Cell subculture and plating of target Huh-7 cells: Digest with 1 mL of EDTA trypsin for about 5 min, terminate digestion with 3 mL of complete culture medium containing 10% FBS, and beat into a single cell suspension visible under a microscope. Centrifuge at 800 rpm for 3 min and discard the culture supernatant.

[0156] (4) Cell-cell co-culture: Use 1 mL of fresh complete culture medium to resuspend the cell pellet and pipette to form a single cell suspension. After standing for 15 to 30 min, take 10 μL of the middle layer cell suspension and flush the abalone counting plate. Place the plate under an ordinary optical microscope to count the cells. 1.5 to 3 × 10 4 The required cell volume was calculated based on the number of cells / mL, and Huh-7 cells were plated into a 96-well plate at 200 μL / well within 24 hours after 293T cell transfection. When the cell confluence reached about 85% to 90%, a cell fusion inhibition experiment was performed.

[0157] (5) DAPI staining: After the cells are fused, remove the culture medium and add 100 μL 4% paraformaldehyde at room temperature for 30 min 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 min. 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, causing the two cells to fuse. In this example, Huh-7 cells were selected as target cells, pAAV-GFP-Wuhan-Hu-1 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.

[0158] Fig.12 The results of constructing the Wuhan-Hu-1-S mediated cell fusion model: At 4 hours, under a 20x microscope, we observed that the 293T cells expressing only GFP fluorescent protein did not fuse with Huh-7, while the 293T cells expressing S protein had undergone obvious cell fusion with the target cells. After DAPI staining, multiple cell nuclei could be seen in the fusion area (white wireframe area), and at 24 hours, it had begun to develop towards a large fusion direction.

[0159] 2. Candidate vaccine immune serum can effectively inhibit cell-cell fusion

[0160] The cell fusion inhibition experiment was performed according to the reference (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:

[0161] (1) Dilution of immune serum: The sera of the three immune mice in the LREP1(High)+S group, LREP1(Low)+S group, LREP2(High)+S group, LREP2(Low)+S group, N group and Mock group were diluted in serum-free 1% double-antibody DMEM using high-pressure sterilized EP tubes. The dilution ratio was 1:10-1:1280, and the total dilution volume was 50 μL.

[0162] (2) Co-incubation: After 48 h, the 293T cells transfected with pAAV-GFP-Wuhan-Hu-1 S and pAAV-GFP-IRES plasmids on six-well plates were digested with EDTA trypsin and digested with fresh complete culture medium. After centrifugation at 800 rpm, the cells were resuspended in serum-free DMEM. After the fluorescent cells were adjusted to the appropriate density under a 10x microscope, 50 μL of 293T fluorescent cells were added to each dilution ratio of the serum in each group, and the cells were mixed by pipetting and incubated at 37°C for 30 min.

[0163] (3) Cell-cell fusion: After 30 minutes of co-incubation, add the cells to a 96-well plate with target cells Huh-7 pre-plated, observe the inhibitory effect for 4 hours, take 3 photos / well of the control well and all experimental wells, save and count the number of fusions and single cells, calculate the fusion rate and inhibition rate, and judge the inhibitory effect of the candidate DNA vaccine immune serum. Draw the inhibition fitting curve 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) is calculated using Calsusyn software.

[0164] Fig.13The mouse immune serum was used to detect the inhibition of cell-cell fusion. The results showed that the candidate DNA vaccine immune serum, whether LREP1(High)+W group or LERP2(High)+W group, had an inhibitory effect on Wuhan-Hu-1-S-mediated cell-cell fusion. The IC50 of the LREP2(High)+W group was the highest, with a serum dilution of 1:92.557±0.564, and that of the LREP1(High)+W group was 1:86.351±73.021. This result was consistent with the specific IgG antibody induction result.

[0165] This example preliminarily proves that the recombinant DNA vaccines pcDNA3.1-LREP1 and pcDNA3.1-LREP2 can induce the body to produce neutralizing antibodies against Wuhan-Hu-1-S, which can effectively inhibit the S protein-mediated cell entry, and the immune effect of pcDNA3.1-LREP2 is better than that of pcDNA3.1-LREP1. Therefore, we infer that the present invention has the effect of inhibiting viral infection.

[0166] 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 value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. 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 know the basic creative concept. Therefore, the attached 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.

[0167] 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 RBD multi-epitope protein, characterized in that: Choose from any of the following: LREP1, the amino acid sequence is shown in SEQ ID NO.1; LREP2, the amino acid sequence is shown in SEQ ID NO.

2.

2. A gene fragment encoding the RBD multi-epitope protein according to claim 1.

3. The gene fragment encoding the RBD multi-epitope protein according to claim 2, characterized in that: The nucleotide sequence of the gene fragment encoding the RBD multi-epitope protein LREP1 is shown in SEQ ID NO.

3.

4. The gene fragment encoding the RBD multi-epitope protein according to claim 2, characterized in that: The nucleotide sequence of the gene fragment encoding the RBD multi-epitope protein LREP2 is shown in SEQ ID NO.

4.

5. A recombinant DNA vaccine comprising a gene fragment of the RBD multi-epitope protein according to claim 2.

6. The recombinant DNA vaccine comprising a gene fragment of an RBD multi-epitope protein according to claim 5, characterized in that: The recombinant DNA vaccine is obtained by inserting a gene fragment encoding the RBD multi-epitope protein into a pcDNA3.1 eukaryotic expression vector.

7. The recombinant DNA vaccine of the gene fragment of the RBD multi-epitope protein according to claim 6, characterized in that: The recombinant DNA vaccine is any one of pcDNA3.1-LREP1 and pcDNA3.1-LREP2.

8. Use of the recombinant DNA vaccine according to claim 5 in the preparation of a product for preventing COVID-19.

9. The use according to claim 8, characterized in that: The product can induce the production of specific IgG against Wuhan-Hu-1-S protein.

10. The use according to claim 9, characterized in that: The product can induce and enhance the Th1 / Th2 mixed immune response in mice.

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