WO2022105880A1 - 融合基因及一种重组新型冠状病毒高效免疫dna疫苗及其构建方法和应用 - Google Patents

融合基因及一种重组新型冠状病毒高效免疫dna疫苗及其构建方法和应用 Download PDF

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WO2022105880A1
WO2022105880A1 PCT/CN2021/131786 CN2021131786W WO2022105880A1 WO 2022105880 A1 WO2022105880 A1 WO 2022105880A1 CN 2021131786 W CN2021131786 W CN 2021131786W WO 2022105880 A1 WO2022105880 A1 WO 2022105880A1
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amino acid
gene
acid fragment
expressing
fusion
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PCT/CN2021/131786
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French (fr)
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于继云
王宇
宋卫卫
阎瑾琦
徐强
夏雨
刘亚超
高昆
邱创钧
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北京震旦鼎泰生物科技有限公司
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Priority to JP2023514153A priority Critical patent/JP2023550004A/ja
Priority to US18/013,663 priority patent/US20230355742A1/en
Priority to CN202180006035.4A priority patent/CN114829608B/zh
Priority to EP21894027.8A priority patent/EP4174183A1/en
Publication of WO2022105880A1 publication Critical patent/WO2022105880A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a fusion gene and a recombinant novel coronavirus high-efficiency immune DNA vaccine (ipDNA vaccine) (named as ZD-nCor19) and a construction method and application thereof.
  • ipDNA vaccine novel coronavirus high-efficiency immune DNA vaccine
  • Novel coronavirus is a new strain of coronavirus that has never been found in humans before.
  • Common signs of human infection with novel coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
  • the present invention aims to provide a fusion gene and a recombinant novel coronavirus high-efficiency immune DNA vaccine and its construction method and application.
  • a first aspect of the present invention provides a fusion gene comprising at least two of the following (1) to (4):
  • the fusion gene comprises at least three of the following (1) to (4):
  • the gene expressing the RBD amino acid fragment in the fusion gene is linked with the gene expressing the amino acid fragment at positions 301-538 of the S2 subunit to form a fusion fragment; in a preferred embodiment, the The nucleotide sequence of the fusion fragment comprises the sequence shown in SEQ ID NO:6; the nucleotide sequence of the gene expressing the 138-369 amino acid fragment of the N protein comprises the sequence shown in SEQ ID NO:10.
  • the nucleotide sequence of the gene expressing the CTB amino acid fragment is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the gene expressing the TT amino acid fragment is shown in SEQ ID NO: 3;
  • nucleotide sequence of the gene expressing the PADRE amino acid fragment is shown in SEQ ID NO: 4.
  • the gene expressing the Foldon amino acid fragment and the gene expressing the CPPCP amino acid fragment are connected into a synthetic fragment, and the nucleotide sequence of the synthetic fragment is as shown in SEQ ID NO:7;
  • the nucleotide sequence of the gene expressing the Furin2A amino acid fragment is shown in SEQ ID NO: 8;
  • the nucleotide sequence of the gene expressing the ERISS amino acid fragment is shown in SEQ ID NO: 9;
  • nucleotide sequence of the gene expressing the IRES amino acid fragment is shown in SEQ ID NO: 11; and/or
  • the nucleotide sequence of the gene expressing the OX40L amino acid fragment is shown in SEQ ID NO: 12.
  • the gene expressing the RBD amino acid fragment and the gene expressing the 301-538 amino acid fragment of the S2 subunit are connected to form a fusion fragment, and the upstream of the fusion fragment is sequentially
  • the gene expressing the amino acid fragment of CTB, TT and PADRE is connected, and the gene expressing the amino acid fragment of Foldon, CPPCP and Furin2A is sequentially connected to the downstream of the fusion fragment; and/or in the 138 expressing the N protein
  • the gene expressing the ERISS amino acid fragment is connected upstream of the gene of the amino acid fragment at position 369, and the genes expressing the IRES and OX40L amino acid fragments are sequentially connected downstream.
  • the gene expressing the RBD amino acid fragment and the gene expressing the 301-538 amino acid fragment of the S2 subunit are linked by a gene expressing a (G4S) 2 linker, the upstream of the fusion fragment It is linked with the gene expressing the PADRE amino acid fragment through the gene expressing the linker G6 whose nucleotide sequence is shown in SEQ ID NO:5.
  • nucleotide sequence of the fusion gene is shown in SEQ ID NO: 13.
  • the second aspect of the present invention provides a fusion protein obtained by expressing the above fusion gene.
  • the third aspect of the present invention provides a recombinant novel coronavirus high-efficiency immune DNA vaccine, named ZD-nCor19, which comprises the above-mentioned fusion gene and vector.
  • the vector is a pZDVac vector.
  • the fourth aspect of the present invention also provides a method for constructing the above-mentioned recombinant novel coronavirus high-efficiency immune DNA vaccine, which comprises the following steps:
  • the application of the above-mentioned recombinant novel coronavirus high-efficiency immune DNA vaccine in the preparation of a medicine for preventing and/or treating novel coronavirus infection also belongs to the content of the present invention.
  • the fusion gene precisely locks the genes expressing the RBD protein of the S1 subunit of the novel coronavirus, the 301-538aa segment of the S2 subunit, and the 138-369aa segment of the N protein.
  • the upstream and downstream of the target antigen gene are respectively connected with genes expressing immune potentiating molecules, and the Furin2A protease cleavage site is set between the genes expressing the target antigen, which can make
  • the recombinant novel coronavirus high-efficiency immune DNA vaccine (recombinant plasmid pZDVac-CRSNPO) constructed and obtained by the present invention can not only effectively avoid the ADE-related safety problems that may be generated by the full-length S protein and the full-length N protein, but also ensure the expression of Compared with using only S1 protein or only RBD or only using S1 protein (RBD) and N protein as target antigens, the fusion protein can cover more epitopes of the new coronavirus and achieve more comprehensive protection.
  • the vaccine uses only RBD, the 301-538aa segment of the S2 subunit and the 138-369aa segment of the N protein in the design of the target antigen, which have smaller fragments relative to the full-length S protein and N protein, which not only has It is beneficial to the construction of vaccines, and also makes it easier to enter cells, and has high expression efficiency and high immune effect.
  • the presence of the Furin2A protease cleavage site allows the secretory expression of two antigenic segments derived from the S protein (RBD and the 301-538aa segment of the S2 subunit), which in turn induces better humoral immune responses, while those derived from N An antigenic segment of the protein (138-369aa segment of the N protein) can be expressed intracellularly to induce T cell immune response, so the vaccine constructed and obtained by the present invention can efficiently induce humoral immune response and T cell immune response at the same time. Synergistically enhance the immune protection effect of the vaccine.
  • the present invention has the following beneficial effects:
  • the vaccine of the present invention belongs to the RNA vaccine in the form of DNA, and does not integrate into the chromosome, so it will not cause malignant transformation of cells. Moreover, after the vaccine enters cells, RNA transcription will be carried out on a large scale, resulting in rapid apoptosis of cells, further avoiding the possibility of malignant transformation.
  • the vaccine is a naked plasmid product, without additional liposomes, aluminum adjuvants, etc., and basically does not cause additional non-specific inflammatory reactions. Therefore, in terms of safety, it is the most advantageous and most guaranteed among all types of vaccines.
  • the vaccine of the present invention belongs to a class of high-efficiency immune DNA vaccines ((ipDNA vaccine) ZD-nCor19) that fuses the dual advantages of DNA vaccines and RNA vaccines, and the verified results show that the vaccine is vaccinated by intradermal injection and electrified pulses.
  • Auxiliary introduction two injections, 28 days apart, 1-4 weeks apart in emergencies, can efficiently induce mucosal-like surface immune protection effects, and induce systemic high-efficiency cellular immune responses, which can form a strong epidemiological Superficial immune protection effect, but also has a strong deep protection effect.
  • This kind of immune response belongs to the high-level immune response of the body.
  • systemic immune mobilization can be carried out quickly and efficiently, including the immune response at the surface mucosal level, as well as systemic innate immunity, humoral immunity and cellular immunity, to quickly kill and clear the new coronavirus that invades the body and prevent
  • the virus causes disease and the condition becomes severe, protects life, avoids various sequelae, and completes the efficient killing and removal of the virus when the body does not produce symptoms or only produces mild symptoms, and restores health quickly.
  • the high-efficiency immunity of the vaccine is also reflected in the ability to enhance the body's systemic immunity.
  • the vaccine is an excellent life-protecting vaccine, helping to protect the life and health of the elderly and immune-vulnerable groups.
  • the vaccine of the present invention not only has the characteristics of high-efficiency immune protection, the antigen-specific cellular immunity induced by the vaccine will retain the ability of immune memory for many years, so the vaccine is also a vaccine that can provide long-term immune protection. . Moreover, in the vaccine design, a partial fragment of the internal N protein of the virus is used as the target antigen, which is extremely stable and rarely produces clear mutation, so the vaccine of the present invention is also a vaccine that can effectively resist virus mutation. The vaccine of the present invention can still provide high-efficiency immune protection function after the significant mutation of the virus surface antigen makes some vaccines lose the immune protection effect.
  • the vaccine of the present invention can not only be used as a preventive vaccine, but also can be used for emergency immunotherapy for people with positive virus detection. Severe cases, help save lives and reduce the fatality rate of the virus. Therefore, the vaccine of the present invention will be a vaccine that can truly protect and save lives.
  • the vaccine of the present invention can utilize the Escherichia coli prokaryotic system to carry out biological fermentation production, so the existing biological fermentation industrial system can be used, and a new industrial system can be established, so the production of the vaccine can form a huge production capacity,
  • the cost is relatively low, and it is expected to meet the domestic and international demand for this vaccine variety.
  • DNA vaccines generally have strong stability and can be stored and transported at room temperature, while RNA vaccines, protein subunit and viral vector vaccines, inactivated vaccines, etc. require cold chain transportation, which is the phase of DNA vaccines.
  • the vaccine provided by the present invention is a vaccine, so it has a very great advantage in stability, and can become an excellent choice among the national new coronavirus vaccine foreign aid varieties.
  • the vaccine (ipDNA vaccine) ZD-nCor19 provided by the present invention is expected to have the characteristics and advantages of safety, high efficiency, long-term effect, anti-mutation, good stability and huge production capacity, and is a DNA with efficient immune effect.
  • Vaccines can be used for routine group prevention, specific immune protection of immune vulnerable groups such as the elderly, immune enhancement of specific groups, emergency vaccination after virus exposure, coordinated immune enhancement of different types of vaccines, and as a national foreign aid vaccine. An excellent vaccine for both prevention and treatment will be developed and applied in depth.
  • FIG. 1 is a schematic diagram of the connection structure of a target antigen and an immune-enhancing molecule in a recombinant novel coronavirus high-efficiency immune DNA vaccine provided by an embodiment of the present invention
  • Fig. 2 is the gel electrophoresis photo of pZDVac-CCCPO plasmid double digestion
  • Fig. 3 is the gel electrophoresis photograph of the double digestion of pUC57-CRSNP plasmid
  • Fig. 4 is the recovery identification gel electrophoresis photograph of the target gene fragment of recombinant plasmid pZDVac-CRSNPO and backbone vector;
  • Fig. 5 is the construction schematic diagram of recombinant plasmid pZDVac-CRSNPO;
  • Fig. 6 is the gel electrophoresis photograph of colony PCR identification positive clone
  • Fig. 7 is the gel electrophoresis photo of positive clone mini-plastid digestion identification
  • Figure 8 is a bar graph of the secretion frequency of cytokines IFN- ⁇ and IL-4 in mouse spleen cells with N protein as a stimulus after immunizing mice with recombinant plasmid pZDVac-CRSNPO;
  • Figure 9 is a bar graph of the secretion frequency of cytokines IFN- ⁇ and IL-4 in mouse spleen cells using RBD protein as a stimulus after immunizing mice with recombinant plasmid pZDVac-CRSNPO;
  • Figure 10 is a scatter plot of the type bias analysis of T cell responses induced by recombinant plasmid pZDVac-CRSNPO in a mouse model
  • Figure 11 is a scatter plot of the secretion of cytokine IFN- ⁇ in human PBMCs with N protein and RBD protein as stimulators before and after the recombinant plasmid pZDVac-CRSNPO immunizes human subjects;
  • Figure 12 is a scatter plot of the secretion of cytokine IFN- ⁇ in human PBMCs with N protein and RBD protein as stimulators before and after immunization of human subjects with recombinant plasmid pZDVac-CRSNPO at different doses;
  • Figure 13 is a scatter diagram of the detection results of antibodies induced by using the recombinant plasmid pZDVac-CRSNPO to immunize mice under different immunization routes and different electroporation conditions;
  • Figure 14 is a bar chart of the Elispot detection results of N antigen, RBD antigen and S2 antigen induced after immunizing mice with recombinant plasmid pZDVac-CRSNPO under different immunization routes and different electrotransduction conditions.
  • the present invention aims to provide a recombinant novel coronavirus high-efficiency immune DNA vaccine (ipDNA vaccine) ZD-nCor19 with both prevention and treatment, and a construction method and application thereof, which are mainly realized based on the following technical means.
  • ipDNA vaccine novel coronavirus high-efficiency immune DNA vaccine
  • the present invention uses a replicon DNA vaccine vector pZDVac based on alphavirus transformation in the construction of a recombinant novel coronavirus high-efficiency immune DNA vaccine.
  • the vector is based on the existing pSFV1 plasmid and replaces the original SP6 promoter with the human cytomegalovirus CMV promoter.
  • pZDVac vector namely the pSFVK1 disclosed in the patent document CN105343874A
  • the alpha virus vector can induce the production of type I IFN, provide the adjuvant effect for the expressed antigen, and has the effect of enhancing immunogenicity.
  • the present invention fully considers the following factors in antigen design and proposes an optimized design scheme:
  • S1 subunit and its receptor binding region RBD Two subunits or truncated S protein (S1 subunit and its receptor binding region RBD) form, and the highly conserved nucleocapsid protein N (SARS-CoV-2 Nucleocapsid) in the new coronavirus. It is generally believed that it is safer to choose the full-length S protein, which contains most of the antigenic epitopes and has a wider coverage; RBD is selected because it contains a critical neutralizing region (CND), which can induce potent neutralizing antibodies. reaction and possible cross-protection against mutant strains; N protein was chosen because it is a highly immunogenic and conserved phosphoprotein among coronaviruses, which can not only induce specific antibodies, but also specific cytotoxic cell activity , and the conservation is better.
  • CND critical neutralizing region
  • S protein is an antigen that is more likely to mutate in the virus. If the virus mutates, it is likely to Affect the immune effect of the vaccine) and cause the risk of vaccine failure.
  • S1 subunit and N protein of the new coronavirus are the main viral antigens that trigger strong immune responses, and are the best viral antigens for vaccine development.
  • the RBD of the S1 subunit contains the key neutralizing region, which is the key to vaccine development; the 301-538aa segment of the S2 subunit contains most of the T cell epitopes, and the 138-369aa segment of the N protein also contains Multiple T cell antigen epitopes, such a target antigen design can not only effectively avoid the ADE-related safety problems that may be caused by the full-length S protein and the full-length N protein, but also ensure that the designed fusion protein is compared to using only S1.
  • the protein or only RBD or only the fusion protein of S1 protein (RBD) and N protein can cover more antigenic epitopes of the new coronavirus to achieve more comprehensive protection, and only RBD,
  • the 301-538aa segment of the S2 subunit and the 138-369aa segment of the N protein have smaller fragments relative to the full-length S protein and N protein, which are not only beneficial for the construction of vaccines, but also easier to enter into cells, And it has high expression efficiency and high immune effect.
  • the two antigen segments derived from the S protein are designed for secretory expression, which can induce better humoral immune responses;
  • the source An antigenic segment of the N protein (138-369aa segment of the N protein) is designed for intracellular expression, which can further enhance the induced T cell immune response, so the vaccine constructed based on this design can efficiently induce humoral immunity at the same time.
  • Response and T cell immune response the two synergistically enhance the immune protection effect of the vaccine.
  • the inventor also considers that the immunogenicity of the DNA vaccine or viral vector vaccine may be insufficient if the antigen is used alone. Therefore, in order to further improve the immunogenicity of the recombinant novel coronavirus DNA vaccine obtained by construction, and then improve its immunity Protective efficacy, also screened and determined the immune potentiating molecules suitable for the target antigen in the vaccine constructed by the present invention, including:
  • Cholera toxin B subunit it can initiate innate immunity, activate DC, enhance immune response, help transfer antigen into cells, induce TH1 and TH2 immune pathways, and act as mucosal immune adjuvant ;
  • Tetanus toxin helper T cell epitopes TT and pan-DR helper T cell epitopes (PARDE): they can enhance CD4+ T cell responses;
  • Foldon-CPPCP Foldon is a domain that can cause non-covalent oligomerization of target proteins. It is derived from the C-terminus of T4 phage fibrin and consists of 27 amino acids. The oligomerization is formed by non-covalent force. The chemical structure has high stability, and it will depolymerize when the temperature is higher than 75°C or in SDS of more than 2% at normal temperature. After depolymerization, the trimer can still be formed again in a suitable environment.
  • CPPCP cyste-proline-proline-cysteine-proline
  • This type of immune potentiating molecule can make the antigen expressed by the vaccine form multimers and granules, which can further enhance its immunogenicity;
  • ERISS sequence it is the adenovirus E3 leader sequence (adenovirus E3 leader sequence), which is the endoplasmic reticulum insertion signal sequence, namely ER insertion signal sequence (ERISS), and ERISS can bind to the endoplasmic reticulum signal recognition particle (SRP).
  • SRP endoplasmic reticulum signal recognition particle
  • OX40L It can provide a costimulatory signal for T cell activation.
  • Fusion genes include various combinations (the following description is based on the protein structure after fusion gene expression), such as:
  • Combination 1 The RBD protein and the S2 subunit 301-538aa segment are connected through the (G4S) 2 linker, and then the cholera toxin B subunit and the tetanus toxin auxiliary T are sequentially introduced at the amino terminus (N terminus) of the RBD protein.
  • pan-DR helper T-cell epitopes wherein the pan-DR helper T-cell epitopes and RBD proteins are connected with G6 flexible linker (linker)
  • the sequence of attachment is relative to other attachment sequences (e.g., tetanus toxin helper T cell epitope, pan-DR helper T cell epitope, and cholera toxin B subunit, or tetanus toxin helper T cell epitope, cholera toxin B subunit, in that order) base and pan-DR helper T cell epitopes, etc.) are more conducive to display the target antigen and enhance the immune response induced by the target antigen.
  • attachment sequences e.g., tetanus toxin helper T cell epitope, pan-DR helper T cell epitope, and cholera toxin B subunit, or tetanus toxin helper T cell epitope, cholera toxin B subunit, in that order
  • Combination 2 Introduce Foldon-CPPCP at the carboxy-terminus (C-terminus) of the 301-538aa segment of the S2 subunit, in which the function of Foldon is to easily trimerize the antigen, and CPPCP is to further granulate the trimeric antigen
  • C-terminus carboxy-terminus
  • CPPCP is to further granulate the trimeric antigen
  • the combination of the two makes it easier to multimerize and granulate the target antigen after expression. Compared with soluble antigens, it is easier to be phagocytosed by APC. It can activate Th cells, CTL cells, and B cells at the same time. It can stimulate cells Immunity can also stimulate humoral immunity, so it can produce a comprehensive immune response and greatly enhance the immune protection effect of the vaccine.
  • the inventors unexpectedly found that the two adjuvant molecules, Foldon and CPPCP, were used in combination, and were placed relative to the fusion fragment of the RBD protein and the 301-538aa segment of the S2 subunit in the ligation sequence of Foldon-CPPCP.
  • Other ligation sequences such as CPPCP-Foldon
  • other positions are more conducive to the display of Foldon and CPPCP after expression; and if the adjuvant molecule Foldon-CPPCP is placed in other positions of the fusion fragment, it is likely to cause these two adjuvant molecules.
  • Combination of Combination 2 and Combination 1 can produce a stacking effect or synergy.
  • Combination 3 Based on combination 2 at the carboxy terminus of CPPCP or based on combination 1 at the carboxy terminus of the 301-538aa segment of the S2 subunit, the segments 138-369aa of Furin2A, ERISS and N proteins were sequentially linked. Among them, ERISS can enhance the processing of antigens by APC and the presentation of MHC class I molecules, thereby enhancing the T cell immune response of the N protein segment expressed in cells. Furin2A is a protease cleavage site, which can make the N protein 138-369aa segment independent Expressed intracellularly, induces T cell immune responses.
  • Combination 4 Based on Combination 3, an internal ribosome entry site (IRES) sequence was attached to the C-terminus of the 138-369aa segment of the N protein, and downstream of the IRES sequence, an OX40L molecule was attached, which can deliver a second for T cell activation. Signals that can further stimulate the immune response and induce a shift in the immune response towards a TH1-type high-level immune response.
  • IRES internal ribosome entry site
  • a Kozak sequence and a signal peptide sequence (which can also be added to the backbone vector in advance) can be sequentially introduced upstream of the above-mentioned combined fusion genes, and the signal peptide can be selected from a signal peptide suitable for eukaryotic expression.
  • Figure 1 shows an example of the connection structure of the target antigen and immune potentiator molecules in the recombinant novel coronavirus high-efficiency immune DNA vaccine, including the fusion gene integrating the above combination 1-combination 4 and the Kozak sequence upstream of the fusion gene and signal peptide sequences.
  • the design of the structure of the recombinant novel coronavirus DNA vaccine provided by a preferred embodiment of the present invention can make synergistic synergy between target antigens, between immune-enhancing molecules, and between target antigens and immune-enhancing molecules
  • the DNA vaccine is designed to express the segment of S protein (301-538aa segment of RBD and S2 subunit) and the segment of N protein (138-369aa segment), respectively , the segment of S protein is secreted and expressed, and the segment of N protein is expressed intracellularly, so it can induce good humoral immune response and T cell immune response at the same time, which can further improve the immune protection effect of the DNA vaccine.
  • Example 1 Construction of recombinant novel coronavirus high-efficiency immune DNA vaccine ZD-nCor19
  • the pZDVac vector was used as the carrier of the DNA vaccine, the RBD of the novel coronavirus, the 301-538aa segment of the S2 subunit and the 138-369aa segment of the N protein were used as the target antigens, and cholera toxin B, disruptor Cold toxin helper T cell epitopes, pan-DR helper T cell epitopes, Foldon-CPPCP, Furin2A, ERISS, IRES and OX40L (the respective corresponding nucleotide sequences are shown in Table 1 below, wherein the RBD and S2 subunits are The 301-538aa segment is given by (G4S) 2 ligation (RBD-(G4S) 2 -S2), and Linker G6 in Table 1 is indicated as the linker sequence between RBD and PARDE, the 138-369aa segment of the N protein.
  • the nucleotide sequence is given by NP in Table 1) as an immune boosting molecule to construct a
  • the pZDVac-CCCPO plasmid (which is the synthetic gene sequence CTB-TT-CTLA-4+PD-L1 extracellular region-PADRE-IRES-OX40L was cloned into the pZDVac vector, the structure is pZDVac-CTB-TT-CTLA-4+ PD-L1 extracellular region-PADRE-IRES-OX40L, the corresponding nucleotide sequence is SEQ ID NO: 1) Double-enzyme digestion with AclI and XbaI, the photo of 0.45% gel electrophoresis is shown in Figure 2, wherein swimming lanes 1 and 2 are the results of the pZDVac-CCCPO plasmid after double digestion with AclI and XbaI.
  • the purified and recovered large fragment of about 12 kb is pZDVac-IRES-OX40L.
  • the recovered fragment was identified by gel electrophoresis again.
  • the results are shown in Figure 4 As shown in lane 2, it can be used as an expression vector backbone for constructing a novel coronavirus high-efficiency immune DNA vaccine.
  • CRSNP CTB-TT-PARDE-G6-RBD-(G4S) 2 -S2-Foldon-CPPCP-Furin2A-ERISS-NP
  • the pUC57-CRSNP recombinant plasmid was double digested with AclI and XbaI (purchased from New England Biolabs (NEB) company), and the photo of 0.45% agarose DNA gel electrophoresis (Regular Agarose G-10 from Biowest company) is shown in Figure 3
  • Lane 1 shows the electrophoresis results of the pUC57-CRSNP recombinant plasmid
  • lane 2 shows the electrophoresis results of the pUC57-CRSNP recombinant plasmid after double digestion with AclI and XbaI, and the 3.6kb target fragment was recovered (Large Agarose Gel DNA Recovery Kit).
  • the inventors also synthesized two other target gene fragments: RBD-(G4S) 2 -S2-NP (named as the second target gene fragment, which differs from the first target gene fragment only in that there is no Connect immune potentiating molecules) and Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2 -S2-CTB-Furin2A-ERISS-NP (named as the third target gene fragment, which is the same as the first target gene fragment The difference lies in the different positions of the immune adjuvant molecules Foldon-CPPCP and CTB).
  • Table 1 Nucleotide sequence information of each structure in the recombinant novel coronavirus high-efficiency immune DNA vaccine
  • the expression vector backbone pZDVac-IRES-OX40L obtained in the above step 1.1 is connected with the first target gene CRSNP obtained in the step 1.2 by T4 ligase, and then the positive clone and colony PCR identification can be obtained.
  • the recombinant expression vector of the first target gene fragment CRSNP pZDVac-CTB-TT-PARDE-G6-RBD-(G4S) 2 -S2-Foldon-CPPCP-Furin2A-ERISS-NP-IRES-OX40L (named pZDVac-CRSNPO recombinant Plasmid, wherein the nucleotide sequence corresponding to fusion gene CTB-TT-PARDE-G6-RBD-(G4S) 2 -S2-Foldon-CPPCP-Furin2A-ERISS-NP-IRES-OX40L is as shown in SEQ ID NO:13, In addition to the nucleotide sequence shown in Table 1 above, it also contains an enzyme cleavage site), which is the recombinant novel coronavirus high-efficiency immune DNA vaccine ZD-nCor19, and the specific construction steps include:
  • the expression vector skeleton pZDVac-IRES-OX40L (about 12kb) obtained by the gel recovery in the above step 1.1 was connected with the first target gene fragment CRSNP (about 3.6kb) obtained by the gel recovery in the step 1.2, and the ligation product was obtained overnight at 16°C.
  • the system is as follows 2 shows:
  • Upstream primer F CCCAGGAGACCCGGTTCTAGAGACGGACATT (SEQ ID NO: 14);
  • Downstream primer R AAGCGGCTTCGGCCAGTAACGTTAGGGGGGG (SEQ ID NO: 15);
  • the PCR system (25 ⁇ L) was: 12.5 ⁇ L of 2 ⁇ PCR mix (purchased from Beijing Biomed); 11.5 ⁇ L of ddH 2 O; 0.5 ⁇ L of F (10 ⁇ M) and 0.5 ⁇ L of R (10 ⁇ M) ), PCR amplification was performed on the transformed colonies of the ligation products.
  • PCR reaction conditions were 1 cycle at 95°C for 5 min; 35 cycles of amplification at 94°C for 30s, 55°C for 30s, and 72°C for 50s; and 72°C for 7 min.
  • the PCR products were analyzed by 0.45% agarose DNA gel electrophoresis, and the results are shown in Figure 6, wherein lanes 1-5 are the PCR results of different single colonies.
  • Colonies identified as positive by colony PCR were streaked on solid medium containing kanamycin (50 ⁇ g/mL) and incubated overnight at 37°C inversion. Then pick a single clone and inoculate it in 1.5 mL LB medium containing kanamycin (50 ⁇ g/mL), after overnight culture at 37°C and 200 rpm, inoculate it in 20 mL containing kanamycin (50 ⁇ g/mL) at a ratio of 1:50. /mL) of LB medium, 37 ° C, 200 rpm overnight culture.
  • a recombinant plasmid pZDVac-RBD-(G4S ) 2 -S2 -NP ( The empty vector pZDVac was used as the backbone vector, named as control plasmid 1), and the third target gene (Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2 -S2-CTB-Furin2A-ERISS-NP was carried) ) of the recombinant plasmid pZDVac-Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2 -S2-CTB-Furin2A-ERISS-NP-IRES-OX40L (designated as control plasmid 2).
  • This example uses the pZDVac-CRSNPO recombinant plasmid constructed and obtained in the above Example 1 to verify the immunogenic effect induced by the pZDVac-CRSNPO in the mouse model, which specifically includes the following steps.
  • Table 5 Grouping of pZDVac-CRSNPO recombinant plasmids in mouse model cellular immune response detection experiments
  • control plasmid 1 and control plasmid 2 The immunogenic effect induced by the control plasmid 2 was not as good as that induced by the pZDVac-CRSNPO recombinant plasmid.
  • the pZDVac-CRSNPO recombinant plasmid constructed and obtained in Example 1 is used to immunize human subjects, and the immunogenicity effect induced in the human body is verified, which specifically includes the following steps.
  • Table 6 Grouping of pZDVac-CRSNPO recombinant plasmids in the detection of cellular immune response in human subjects
  • Figure 11 shows that compared with before immunization, strong cellular immune responses can be induced after secondary and tertiary immunization.
  • N protein and RBD protein are used as stimulators, respectively, the secretion frequency of cytokine IFN- ⁇ in PBMC has Very significant increase; and when the N protein was used as a stimulus, the secretion frequency of the cytokine IFN- ⁇ after the third immunity was higher than that after the second immunity, and when the RBD protein was used as the stimulus, the cytokine IFN- ⁇ after the second immunity was used.
  • the secretion frequency is higher than the three immunity.
  • the 15 volunteers screened in this example had regular follow-up visits and physical examinations (at least 3 months) after the vaccine injection, and all volunteers did not show any discomfort, which preliminarily shows that the pZDVac-CRSNPO recombinant plasmid provided by the present invention It has a good safety profile when used as a vaccine.
  • This example uses the pZDVac-CRSNPO recombinant plasmid constructed and obtained in Example 1 to immunize mice through different immunization routes (intramuscular injection or intradermal injection) and different electroporation conditions to evaluate different immunization routes and different electroporation conditions.
  • the intensity of the immune response induced under the introduction conditions specifically includes the following steps.
  • mice 40 healthy 6-8 week female Balb/c mice (purchased from Viton Lever) were randomly divided into 5 groups, 8 mice in each group, respectively by intramuscular injection or The mice in each group were immunized by single-point administration on the left hind limb by intradermal injection and electro-induction (1 Hz ⁇ 6 times, electrode needle spacing 0.5 cm). A blank control group was set without treatment, and mice in other groups were given 1 ⁇ g/mice of pSFVK1-CRSNPO recombinant plasmid. The administration day was counted as 0d, and 5 groups of mice were immunized twice on 0d and 21d respectively.
  • mice in each group were sacrificed, and ELISPOT detection (IFN- ⁇ ) was performed according to the instructions of MabTech's IFN- ⁇ ELISPOT board.
  • the antibody level induced by the intramuscular injection group (60V, 50ms, 1Hz ⁇ 6 times, the electrode needle spacing was 0.5cm, and the field strength was 120V/cm) was statistically significantly different; the intradermal injection group a (60V, 50ms, 1Hz ⁇ 6 times, electrode needle spacing 0.5cm, field strength 120V/cm), the level of antibody induced by the control group was also statistically significantly different; while the intradermal injection group b ( Compared with the blank control group, the antibody levels induced by 36V, 50ms) and c (36V, 10ms) have no statistically significant difference; it shows that under the conditions of intramuscular injection and subcutaneous injection, when the electro-induction conditions are voltage 60V, pulse width 50ms When compared with the voltage of 36V, the electro-introduction condition with a pulse width of 50ms has a relatively better immune effect.
  • the level of antibody induced by intradermal injection group a was not statistically different from that of intramuscular injection group, but in general, immunization with pSFVK1-CRSNPO recombinant plasmid via intramuscular injection would have Relatively better humoral immunity effect.
  • the ELISPOT detection results are shown in Figure 14. It can be seen that the pSFVK1-CRSNPO recombinant plasmid can induce the specificity of N antigen, RBD antigen and S2 antigen after immunizing mice by different routes (intramuscular injection and intradermal injection) and under different electroporation conditions.
  • the T cell immune response, the experimental group (intramuscular injection group, subcutaneous injection group a, b, c) and the blank control group were all statistically significantly different. There was no significant difference between the N antigen, RBD antigen and S2 antigen-specific T cell immune responses induced after immunization with the N antigen, RBD antigen and S2 antigen-specific T cell immune responses induced by intramuscular immunization.
  • the results show that the pSFVK1-CRSNPO recombinant plasmid vaccine provided by the present invention can induce strong specific T cell immune responses in mice stimulated by different immunization routes and different electroporation conditions, and the intramuscular injection group and the intradermal injection group There is no significant difference in the induced cellular immune response; the pSFVK1-CRSNPO recombinant plasmid vaccine provided by the present invention has a relatively better induction effect in terms of inducing humoral immune response under the conditions of voltage 60V and pulse width 50ms. Injections produced a relatively better humoral immune response.
  • the pSFVK1-CRSNPO recombinant plasmid provided by the present invention can induce a good cellular immune response and humoral immune response when used as a vaccine, and can exert a good protective effect of vaccination, which is consistent with the results of Examples 2 and 3 above.
  • pSFVK1- The CRSNPO recombinant plasmid was used to immunize hACE2 transgenic mice or rhesus monkeys, and a negative control group was set up and given normal saline. The dosing day was counted as 0d, the animals in each group were immunized twice on 0d and 21d, respectively, and the animals in each group were challenged (new coronavirus liquid) 2 weeks after the last immunization.
  • the results of the above examples show that the recombinant novel coronavirus high-efficiency immune DNA vaccine (that is, the pZDVac-CRSNPO recombinant plasmid) constructed and obtained by the present invention can effectively induce high-efficiency immune responses in the immunological function test at the animal level, including natural immune responses and antigens.
  • the specific cellular immune response proved to be a new type of coronavirus DNA vaccine with great development potential.
  • three dose groups 100 ⁇ g, 300 ⁇ g, 500 ⁇ g
  • the present invention provides a fusion gene and a recombinant novel coronavirus high-efficiency immune DNA vaccine based on the fusion gene and a construction method and application thereof, wherein the recombinant novel coronavirus high-efficiency immune DNA vaccine has the dual effects of preventing and treating novel coronavirus infection , suitable for industrial applications.

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Abstract

一种融合基因及一种重组新型冠状病毒高效免疫DNA疫苗及其构建方法和应用，其中提供的免疫DNA疫苗ZD-nCor19以新型冠状病毒RBD蛋白、S2亚基的301-538aa区段和N蛋白的138-369aa区段作为靶抗原，并在适当位置引入特定的免疫增效类分子，可以同时高效诱导体液免疫和细胞免疫，并且可以避免由全长S蛋白和全长N蛋白可能产生的ADE相关的安全性问题，兼具有预防和治疗的双重效果。

Description

融合基因及一种重组新型冠状病毒高效免疫DNA疫苗及其构建方法和应用
相关申请的交叉引用
本申请要求于2020年11月20日提交的中国专利申请号为202011310160.8的权益，其全部内容通过引用并入本文。
技术领域
本发明属于生物医学技术领域，具体涉及一种融合基因及一种重组新型冠状病毒高效免疫DNA疫苗(ipDNA vaccine)(命名为ZD-nCor19)及其构建方法和应用。
背景技术
新型冠状病毒(COVID-19)是以前从未在人体中发现的冠状病毒新毒株，人感染新型冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。
目前，全球累计确诊的新型冠状病毒感染病例已经达到数千万例，累计死亡病例已超过百万人，给全人类的生命健康安全造成巨大的威胁。然而COVID-19疫情仍在全球多个地区迅速蔓延，已经明确该病毒的传播性和致病性强、危害性大，全球有78亿人面临感染以及发病和死亡的风险，国际公认接种安全有效的疫苗是保护人类安全健康的重要措施和有效手段，因此迫切需要开发出有效且安全的疫苗来遏制这场新型冠状病毒大流行。
目前在全球范围内，已有近200项新型冠状病毒疫苗处于不同的开发阶段，其中10种新型冠状病毒疫苗正在进行Ⅲ期临床试验(截止至2020年10月13日)，主要包括新型冠状病毒灭活疫苗、重组亚单位疫苗、腺病毒载体疫苗、减毒流感病毒载体疫苗、核酸疫苗等，进行新型冠状病毒疫苗的研发，虽已经取得了阶段性的成果，但对开发出防治兼用型的新型冠状病毒疫苗品种仍有迫切需求。
发明内容
本发明旨在提供一种融合基因及一种重组新型冠状病毒高效免疫DNA疫苗及其构建方法和应用。
本发明的第一方面提供一种融合基因，其包含以下(1)至(4)中的至少两种：
(1)表达新型冠状病毒COVID-19的RBD氨基酸片段的基因；
(2)表达新型冠状病毒COVID-19的S2亚基或其部分氨基酸片段的基因；
(3)表达新型冠状病毒COVID-19的N蛋白或其部分氨基酸片段的基因；
(4)表达选自以下中的任一种或其组合的氨基酸片段的基因：CTB、TT、PADRE、Foldon、CPPCP、Furin2A、ERISS、IRES和OX40L。
在一个实施方案中，所述融合基因包含以下(1)至(4)中的至少三种：
(1)表达新型冠状病毒COVID-19的RBD氨基酸片段的基因；
(2)表达新型冠状病毒COVID-19的S2亚基的301-538位氨基酸片段的基因；
(3)表达新型冠状病毒COVID-19的N蛋白的138-369位氨基酸片段的基因；
(4)表达以下的氨基酸片段的基因：CTB、TT、PADRE、Foldon、CPPCP、Furin2A、ERISS、IRES和OX40L。
在一个实施方案中，所述融合基因中表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因连接成融合片段；在一个优选的实施方案中，所述融合片段的核苷酸序列包含SEQ ID NO:6所示的序列；表达所述N蛋白的138-369位氨基酸片段的基因的核苷酸序列包含SEQ ID NO:10所示的序列。
在另一个实施方案中，所述融合基因中：
表达所述CTB氨基酸片段的基因的核苷酸序列如SEQ ID NO:2所示；
表达所述TT氨基酸片段的基因的核苷酸序列如SEQ ID NO:3所示；
表达所述PADRE氨基酸片段的基因的核苷酸序列如SEQ ID NO:4所示；
表达所述Foldon氨基酸片段的基因与表达所述CPPCP氨基酸片段的基因连接成一合成片段，该合成片段的核苷酸序列如SEQ ID NO:7所示；
表达所述Furin2A氨基酸片段的基因的核苷酸序列如SEQ ID NO:8所示；
表达所述ERISS氨基酸片段的基因的核苷酸序列如SEQ ID NO:9所示；
表达所述IRES氨基酸片段的基因的核苷酸序列如SEQ ID NO:11所示；和/或
表达所述OX40L氨基酸片段的基因的核苷酸序列如SEQ ID NO:12所示。
在一个实施方案中，所述融合基因中：表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因连接成融合片段，在所述的融合片段的上游依次连接有表达所述CTB、TT和PADRE氨基酸片段的基因，在所述的融合片段的下游依次连接有表达所述Foldon、CPPCP和Furin2A氨基酸片段的基因；和/或在表达所述N蛋白的138-369位氨基酸片段的基因的上游连接有表达所述ERISS氨基酸片段的基因，下游依次连接有表达所述IRES和OX40L氨基酸片段的基因。
在一个优选的实施方案中，表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因通过表达(G4S) 2连接臂的基因连接，所述融合片段的上游与表达所述PADRE氨基酸片段的基因通过核苷酸序列如SEQ ID NO:5所示的表达连接 臂linker G6的基因连接。
在进一步优选的实施方案中，所述融合基因的核苷酸序列如SEQ ID NO:13所示。
本发明的第二方面提供一种融合蛋白，其由上述的融合基因表达获得。
本发明的第三方面提供一种重组新型冠状病毒高效免疫DNA疫苗，命名为ZD-nCor19，其包含上述的融合基因和载体。
在一个实施方案中，所述载体为pZDVac载体。
本发明的第四方面还提供了上述的重组新型冠状病毒高效免疫DNA疫苗的构建方法，其包括以下步骤：
1)合成上述的融合基因；
2)将所述融合基因插入pZDVac载体中，获得所述重组新型冠状病毒高效免疫DNA疫苗。
在本发明的第五方面中，上述的重组新型冠状病毒高效免疫DNA疫苗在制备预防和/或治疗新型冠状病毒感染的药物中的应用也属于本发明的内容。
在本发明提供的重组新型冠状病毒高效免疫DNA疫苗中，融合基因精准锁定表达新型冠状病毒S1亚基的RBD蛋白、S2亚基的301-538aa区段和N蛋白的138-369aa区段的基因作为表达靶抗原的基因，另外结合其它特别设计，如在靶抗原基因的上下游分别连接有表达免疫增效类分子的基因，在表达靶抗原的基因之间设置Furin2A蛋白酶切割位点，可以使得本发明构建获得的重组新型冠状病毒高效免疫DNA疫苗(重组质粒pZDVac-CRSNPO)既可以有效避免由全长S蛋白和全长N蛋白可能产生的ADE相关的安全性问题，还可以保证由其表达的融合蛋白相对于仅使用S1蛋白或仅使用RBD或者仅使用S1蛋白(RBD)与N蛋白作为靶抗原时能够覆盖新型冠状病毒更多的抗原表位，达到较为全面的保护。该疫苗在靶抗原的设计中仅使用RBD、S2亚基的301-538aa区段和N蛋白的138-369aa区段，其相对于全长S蛋白和N蛋白具有较小的片段，这不仅有利于疫苗的构建，还使其更容易进入细胞中，且具有高效的表达效率和高效的免疫效果。另外，Furin2A蛋白酶切割位点的存在使得来源于S蛋白的两个抗原区段(RBD和S2亚基的301-538aa区段)可分泌表达，进而诱导更好的体液免疫反应，而来源于N蛋白的一个抗原区段(N蛋白的138-369aa区段)可细胞内表达，进而诱导T细胞免疫反应，因此本发明构建获得的疫苗可以同时高效诱导体液免疫反应和T细胞免疫反应，两者协同增强疫苗的免疫保护效果。
与现有技术相比，本发明具有以下有益效果：
1)安全性强：本发明疫苗属于DNA形式的RNA疫苗，不整合到染色体，所以不会 引起细胞的恶性转化。而且该疫苗进入细胞后，会大规模进行RNA的转录，导致细胞的快速凋亡，进一步避免了恶性转化的可能性。另外，该疫苗为裸质粒产品，没有额外添加脂质体、铝佐剂等，基本不引起额外的非特异性炎症反应。所以安全性方面，在各类疫苗中是最具优势最有保障的。
2)高效免疫：本发明疫苗属于一类融合了DNA疫苗和RNA疫苗双重优势的高效免疫DNA疫苗((ipDNA vaccine)ZD-nCor19)，经验证结果表明，其通过采用皮内注射接种加电脉冲辅助导入(两针接种，间隔28天，紧急情况可间隔1-4周不等)，可高效诱导类粘膜表层免疫保护效应，并诱导全身系统性的高效细胞免疫应答，既能形成强大的表浅免疫保护效力，又具备强大的深层保护作用。这种免疫应答，属于身体高等级的免疫响应，具备快速免疫决策动员的、集团军运动歼灭战式的、深度纵深主动防御清除的特点，为人体提供高效持久的免疫保护。一旦遇到病毒袭击，可以快速高效进行系统性的免疫动员，包括表层类粘膜层面的免疫应答以及全身系统性的天然免疫、体液免疫和细胞免疫，快速杀灭清除侵入体内的新型冠状病毒，防止病毒致病和病情重症化，保护生命，避免出现各类后遗症，在身体不产生症状或仅仅产生轻微症状的情况下，完成对病毒的高效杀灭清除，快速恢复健康。
另外，该疫苗的高效免疫还体现在能够提升人体全身免疫力方面。对于免疫力比较弱的老年人群体和有严重基础性疾病的人群，该疫苗是一款极为优秀的生命保护疫苗，帮助守护老年人和免疫弱势群体的生命和健康。
3)长效，抗变异：本发明疫苗除了具备高效免疫保护的特点，该疫苗诱导的抗原特异性细胞免疫，多年都会保留免疫记忆能力，所以该疫苗也是一款可以提供长效免疫保护的疫苗。而且在疫苗设计中，使用了病毒内部N蛋白部分片段作为靶抗原，极其稳定，很少产生明确变异，所以本发明疫苗也是可以有效抗病毒变异的疫苗。在病毒表面抗原产生显著变异使某些疫苗失去免疫保护效力之后，本发明疫苗仍然能够提供高效的免疫保护功能。
4)防治兼用：本发明疫苗不但可以作为预防疫苗来用，同时对于病毒检测阳性的人群，也可以进行紧急免疫治疗使用，该疫苗可以快速诱导高效细胞免疫应答，及时高效清理体内病毒，防止病情重症化，帮助挽救生命，降低病毒致死率。因此，本发明疫苗将是是一款可以真正保护生命、救助生命的疫苗。
5)产能巨大，成本低廉：本发明疫苗可以利用大肠杆菌原核系统进行生物发酵生产，因此可以借助已有的生物发酵工业体系，并建立新的工业体系，因此该疫苗的生产可以形成巨大产能，成本相对低廉，可望满足国内和国际对该疫苗品种的需求。
6)稳定性好：DNA疫苗一般具有稳定性较强，可以常温储存和运输的特点，而RNA疫苗、蛋白质亚基和病毒载体疫苗、灭活疫苗等则需要冷链运输，这是DNA疫苗相较于其他类型疫苗品种的一个特别优势。本发明提供的疫苗是一款疫苗，因此在稳定性方面具有非常大的优势，可以成为国家新型冠状病毒疫苗援外品种中的上乘之选。
7)市场广阔：目前还没有任何一种新型冠状病毒疫苗上市，因此目前临床或在研的疫苗的效果无法判断，这就不得不需要运用多款研发中的疫苗打出一套“组合拳”，形成疫苗互补，不但可以单独使用本申请疫苗以提供高效保护，对于其他类型的疫苗，也可以将本申请疫苗用于加强免疫，与其他类型的疫苗协同应用，提供更加卓越的免疫保护能力。另外，本发明提供的疫苗甚至可以帮助某些可能有ADE效应(antibody-dependent enhancement抗体依赖增强)隐患的疫苗，克服可能的ADE效应，避免ADE效应加重病情的严重问题。
综上所述，本发明提供的疫苗(ipDNA vaccine)ZD-nCor19，有望具备安全、高效、长效、抗变异、稳定性好、产能巨大的特点和优点，是一款具有高效免疫效果的DNA疫苗，可用于常规群体预防、老年人等免疫弱势群体特异性免疫保护、特定人群免疫加强、病毒暴露后紧急接种免疫治疗、不同类型疫苗免疫协同强化以及作为国家对外援助疫苗等，可望作为一种防治兼用的优秀疫苗，进行深度开发和应用。
附图说明
图1为本发明一个实施例提供的重组新型冠状病毒高效免疫DNA疫苗中靶抗原和免疫增效类分子的连接结构示意图；
图2为pZDVac-CCCPO质粒双酶切的凝胶电泳照片；
图3为pUC57-CRSNP质粒双酶切的凝胶电泳照片；
图4为重组质粒pZDVac-CRSNPO的目的基因片段及骨架载体的回收鉴定凝胶电泳照片；
图5为重组质粒pZDVac-CRSNPO的构建示意图；
图6为菌落PCR鉴定阳性克隆的凝胶电泳照片；
图7为阳性克隆小提质粒酶切鉴定的凝胶电泳照片；
图8为重组质粒pZDVac-CRSNPO免疫小鼠后以N蛋白为刺激物的小鼠脾脏细胞中细胞因子IFN-γ和IL-4的分泌频率柱状图；
图9为重组质粒pZDVac-CRSNPO免疫小鼠后以RBD蛋白为刺激物的小鼠脾脏细胞中细胞因子IFN-γ和IL-4的分泌频率柱状图；
图10为重组质粒pZDVac-CRSNPO诱导小鼠模型T细胞反应类型偏向分析散点图；
图11为重组质粒pZDVac-CRSNPO对人受试者免疫前和免疫后以N蛋白和RBD蛋白为刺激物的人PBMC中细胞因子IFN-γ的分泌散点图；
图12为重组质粒pZDVac-CRSNPO以不同剂量对人受试者免疫前和免疫后以N蛋白和RBD蛋白为刺激物的人PBMC中细胞因子IFN-γ的分泌散点图；
图13为不同免疫途径及不同电导入条件下使用重组质粒pZDVac-CRSNPO对小鼠进行免疫后诱导的抗体检测结果散点图；
图14为不同免疫途径及不同电导入条件下使用重组质粒pZDVac-CRSNPO对小鼠进行免疫后诱导的N抗原、RBD抗原和S2抗原的Elispot检测结果柱状图。
具体实施方式
本发明旨在提供一种防治兼具的重组新型冠状病毒高效免疫DNA疫苗(ipDNA vaccine)ZD-nCor19及其构建方法和应用，主要基于以下技术手段实现。
本发明在构建重组新型冠状病毒高效免疫DNA疫苗中使用基于甲病毒改造的复制子DNA疫苗载体pZDVac，该载体是在现有的pSFV1质粒基础上，用人巨细胞病毒CMV启动子替换原SP6启动子、添加多聚腺苷酸尾SV40poly(A)，并将pSFV1质粒的抗性基因替换为临床上安全性更好的卡那霉素抗性基因，最终将改造成功后的复制子真核表达载体，命名为pZDVac载体(即专利文献CN105343874A中公开的pSFVK1)，该载体可以避免体外制备RNA的繁琐过程，可以以DNA质粒形式直接转染人和动物细胞进行外源蛋白的高水平表达，并承袭了甲病毒载体可诱导产生I型IFN，为表达的抗原提供佐剂效果的功能，具有增强免疫原性的功效。
本发明在抗原设计方面充分考虑了以下因素并提出优化的设计方案：
(1)由于目前对新型冠状病毒的基础研究还较少，而且还没有任何一种新型冠状病毒疫苗上市，因此针对新型冠状病毒疫苗用靶抗原的选择没有确切标准，即没有任何依据可以确定关于新型冠状病毒的哪一段抗原作为疫苗靶抗原时更加有效。因此研究者们普遍以SARS病毒疫苗的开发工作为选择新型冠状病毒的抗原依据，在靶抗原选择上一般采用新型冠状病毒的全长刺突糖蛋白(Spike Protein，S蛋白，其由S1和S2两个亚基组成)或截短型S蛋白(S1亚基及其受体结合区RBD)形式，以及新型冠状病毒中保守性较高的核衣壳蛋白N(SARS-CoV-2 Nucleocapsid)。普遍认为选择全长的S蛋白比较保险，其包含了大部分的抗原表位，覆盖面较广；选择RBD，则是因为其含有关键中和区(CND)，CND可以诱发强效的中和抗体反应以及可能产生对变异株的交叉保护；选择N蛋白则是因为其是冠状病毒中具有高度免疫原性和保守性的磷蛋白，不仅能诱导特异的抗体，还能诱导特异的细胞毒细胞活性，且保守性更好，在多种冠状 病毒中保守性极高，相对不易产生变异，能够避免因为病毒变异(S蛋白是病毒中更容易产生变异的抗原，如果病毒发生了变异，很可能会影响到疫苗的免疫效果)而造成疫苗失效的风险。其中，根据新型冠状病毒的结构和感染患者的机制，普遍认为新型冠状病毒的S1亚基和N蛋白是引发强免疫反应的主要病毒抗原，是研发疫苗的最佳病毒抗原。
(2)发明人则考虑到新型冠状病毒的全长S蛋白和全长N蛋白可能会产生ADE相关的安全性问题，因此本发明人通过优化设计并最终确定选择S蛋白的S1亚基的RBD、S2亚基的301-538aa区段和N蛋白的138-369aa区段进行靶抗原设计。其中S1亚基的RBD含有关键中和区，是疫苗研发的关键；S2亚基的301-538aa区段中则含有大部分的T细胞抗原表位，N蛋白的138-369aa区段中也含有多个T细胞抗原表位，这样的靶抗原设计既可以有效避免由全长S蛋白和全长N蛋白可能产生的ADE相关的安全性问题，还可以保证设计得到的融合蛋白相对于仅使用S1蛋白或仅使用RBD或者仅使用S1蛋白(RBD)与N蛋白的融合蛋白能够覆盖新型冠状病毒更多的抗原表位，达到较为全面的保护，同时本发明的靶抗原的设计中仅使用RBD、S2亚基的301-538aa区段和N蛋白的138-369aa区段，其相对于全长S蛋白和N蛋白具有较小的片段，这不仅有利于疫苗的构建，还更容易进入细胞中，且具有高效的表达效率和高效的免疫效果。另外，在靶抗原设计中，将来源于S蛋白的两个抗原区段(RBD和S2亚基的301-538aa区段)设计成可分泌表达，其可诱导更好的体液免疫反应；将来源于N蛋白的一个抗原区段(N蛋白的138-369aa区段)设计成可细胞内表达，其可以进一步增强诱导的T细胞免疫反应，因此基于该设计构建获得的疫苗可以同时高效诱导体液免疫反应和T细胞免疫反应，两者协同增强疫苗的免疫保护效果。
(3)发明人还考虑到单纯用抗原做DNA疫苗或者病毒载体类疫苗，其免疫原性可能不足，因此为了使构建获得的重组新型冠状病毒DNA疫苗的免疫原性进一步提高，进而提高其免疫保护效力，还筛选并确定了适用于本发明构建的疫苗中靶抗原的免疫增效类分子，其包括：
(i)霍乱毒素B亚基(CTB)：其能够启动天然免疫、活化DC，增强免疫反应，有助于将抗原转入细胞内，诱导产生TH1和TH2免疫通路，起粘膜免疫佐剂的功效；
(ii)破伤风毒素辅助性T细胞表位(TT)和泛DR辅助T细胞表位(pan-DR helper T cell epitopes，PARDE)：其能够增强CD4+T细胞反应；
(iii)Foldon-CPPCP：Foldon是一种可以使目标蛋白发生非共价寡聚的结构域，它来源于T4噬菌体纤维蛋白C端，由27个氨基酸组成，非共价作用力所形成寡聚化结构稳 定性较高，在温度高于75℃或常温下在2％以上的SDS中才会解聚，解聚后在适宜环境中仍可重新形成三聚体。CPPCP(半胱氨酸-脯氨酸-脯氨酸-半胱氨酸-脯氨酸)类似于抗体铰链区，适用于双特异性抗体和融合蛋白，易于三聚体蛋白形成颗粒化。该类免疫增效分子可以使疫苗表达的抗原形成多聚体和颗粒化，可进一步增强其免疫原性；
(iv)ERISS序列：其为腺病毒E3引导序列(adenovirus E3 leader sequence)，是内质网插入信号序列，即ER insertion signal sequence(ERISS)，ERISS可结合内织网信号识别粒(SRP)。通过SRP能够结合ER膜表面的SRP受体的特性，将融合的目的抗原有效地转运到ER内，加强APC对抗原的加工与MHCI类分子的提呈，从而增强诱导有效的保护性CTL产生；
(v)OX40L：其能够为T细胞活化提供共刺激信号。
将表达这些免疫增效类分子的部分或全部的基因整合至表达RBD、S2亚基的301-538aa区段和N蛋白的138-369aa区段的基因中的任一种或其组合组成的基因片段中，能获得系列融合基因。将这些融合基因插入所确定的pZDVac载体中，构建获得本发明的重组新型冠状病毒DNA疫苗。
融合基因包括多种组合形式(以下基于融合基因表达后的蛋白结构进行描述)，例如：
组合1：将RBD蛋白和S2亚基301-538aa区段通过(G4S) 2连接臂进行连接，然后在RBD蛋白的氨基端(N端)依次引入霍乱毒素B亚基、破伤风毒素辅助性T细胞表位、泛DR辅助T细胞表位(其中将泛DR辅助T细胞表位与RBD蛋白以G6柔性linker(连接头)连接)，本发明人惊讶发现，这三种免疫增效类分子的连接顺序相对于其他连接顺序(例如依次为破伤风毒素辅助性T细胞表位、泛DR辅助T细胞表位和霍乱毒素B亚基，或破伤风毒素辅助性T细胞表位、霍乱毒素B亚基和泛DR辅助T细胞表位等)更有利于展示靶抗原，并增强靶抗原诱导的免疫反应。
组合2：在S2亚基的301-538aa区段的羧基端(C端)引入Foldon-CPPCP，其中Foldon的作用是易于让抗原三聚体化，CPPCP则是让三聚体的抗原进一步颗粒化，两者的联用协同使靶抗原表达后更容易进行多聚体化和颗粒化，相对于可溶性的抗原更容易被APC吞噬，能同时活化Th细胞、CTL细胞、B细胞，既能激发细胞免疫，又可激发体液免疫，因此可产生全面的免疫反应，大大增强疫苗的免疫保护效果。在该组合中，本发明人意外发现，将该两种佐剂分子Foldon和CPPCP联用，并以Foldon-CPPCP的连接顺序放置于RBD蛋白和S2亚基301-538aa区段的融合片段之后相对于其他连接顺序(例如CPPCP-Foldon)和其他位置更有利于Foldon和CPPCP在表达后展示出来；而如果将佐剂 分子Foldon-CPPCP放置在融合片段的其他位置，很可能会导致这两种佐剂分子在表达后，由于其分子量太小容易被包裹在抗原蛋白中间，因此两者的空间结构就展示不出来，发挥不了使靶抗原表达后进行多聚体化和颗粒化的作用。组合2与组合1联合能发挥叠加效果或协同增效。
组合3：基于组合2在CPPCP的羧基端或基于组合1在S2亚基的301-538aa区段的羧基端依次连入Furin2A，ERISS和N蛋白的138-369aa区段。其中ERISS可加强APC对抗原的加工与MHCI类分子的提呈，从而增强细胞内表达的N蛋白区段的T细胞免疫反应，Furin2A是蛋白酶切割位点，可以使N蛋白138-369aa区段独立在细胞内表达，诱导T细胞免疫反应。
组合4：基于组合3在N蛋白138-369aa区段的C端连接了内部核糖体进入位点(IRES)序列，在IRES序列的下游，连接了OX40L分子，其能为T细胞活化传递第二信号，可以进一步刺激免疫应答，并诱导免疫反应向TH1类型高级别免疫响应偏移。
另外，可在上述各组合融合基因的上游依次引入Kozak序列和信号肽序列(也可事先添加在骨架载体中)，信号肽可以选用适合于真核表达的信号肽。
图1给出了重组新型冠状病毒高效免疫DNA疫苗中靶抗原和免疫增效类分子的一种连接结构示例，包含了整合了以上组合1-组合4的融合基因以及该融合基因上游的Kozak序列和信号肽序列。
本发明的一个优选实施例提供的重组新型冠状病毒DNA疫苗的结构的设计可以使得各靶抗原之间、各免疫增效分子之间、靶抗原与各免疫增效类分子之间产生协同增效作用，从而提高DNA疫苗的免疫保护效果；同时该DNA疫苗设计成S蛋白的区段(RBD和S2亚基的301-538aa区段)和N蛋白的区段(138-369aa区段)分别表达，其中S蛋白的区段分泌表达，N蛋白的区段细胞内表达，因此其可以同时诱导良好的体液免疫反应和T细胞免疫反应，可进一步提高该DNA疫苗的免疫保护效果。
以下结合具体实施例和附图详细说明本发明的内容。
在下文中，仅简单地描述了某些示例性实施例。正如本领域技术人员可认识到的那样，在不脱离本发明的精神或范围的情况下，可通过各种不同方式修改所描述的实施例。因此，附图和描述被认为本质上是示例性的而非限制性的。
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。
实施例中描述到的各种生物材料的取得途径仅是提供一种试验获取的途径以达到 具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
本发明提及的所有引物和序列用已有技术合成。
实施例1：重组新型冠状病毒高效免疫DNA疫苗ZD-nCor19的构建
在该实施例中使用pZDVac载体作为DNA疫苗的载体，使用新型冠状病毒的RBD、S2亚基的301-538aa区段和N蛋白的138-369aa区段作为靶抗原，并使用霍乱毒素B、破伤风毒素辅助性T细胞表位、泛DR辅助T细胞表位、Foldon-CPPCP、Furin2A、ERISS、IRES和OX40L(各自所对应的核苷酸序列如下表1所示，其中RBD和S2亚基的301-538aa区段由(G4S) 2连接给出(RBD-(G4S) 2-S2)，表1中Linker G6表示为RBD和PARDE之间的连接头序列，N蛋白的138-369aa区段的核苷酸序列在表1中以NP给出)作为免疫增效分子来构建重组新型冠状病毒高效免疫DNA疫苗，具体包括以下步骤。
1.1、表达载体骨架的制备和鉴定
将pZDVac-CCCPO质粒(其为将合成基因序列CTB-TT-CTLA-4+PD-L1胞外区-PADRE-IRES-OX40L克隆至pZDVac载体中，结构为pZDVac-CTB-TT-CTLA-4+PD-L1胞外区-PADRE-IRES-OX40L，对应的核苷酸序列为SEQ ID NO:1)用AclI和XbaI进行双酶切，经0.45％凝胶电泳的照片如图2所示，其中泳道1和2均为pZDVac-CCCPO质粒经过AclI和XbaI双酶切之后的结果，纯化回收12kb左右大片段即为pZDVac-IRES-OX40L，对回收片段经再次凝胶电泳鉴定的结果如图4中泳道2所示，其可以作为构建新型冠状病毒高效免疫DNA疫苗的表达载体骨架。
1.2、基因片段的合成和鉴定
如图1所示，按照CTB-TT-PARDE-G6-RBD-(G4S) 2-S2-Foldon-CPPCP-Furin2A-ERISS-NP(命名为第一目的基因，简称CRSNP)的基因连接顺序进行全基因合成，并在目的基因的上游引入酶切位点XbaI、NruI、PmeI，下游引入AclI单酶切位点，获得了携带有目的基因片段CRSNP的重组质粒，命名为pUC57-CRSNP重组质粒，该步骤由北京博迈德基因有限公司(Biomed)完成；
将pUC57-CRSNP重组质粒用AclI与XbaI双酶切(购自New England Biolabs(NEB)公司)，经0.45％琼脂糖DNA凝胶电泳(Regular Agarose G-10来自Biowest公司)的照片如图3所示，其中泳道1表示pUC57-CRSNP重组质粒的电泳结果，泳道2表示pUC57-CRSNP重组质粒经AclI与XbaI双酶切之后的电泳结果，回收3.6kb目的片段(大 量琼脂糖凝胶DNA回收试剂盒(离心柱型)来自TIANGEN公司)即可获得目的基因片段CRSNP(如图3中箭头所示，命名为第一目的基因片段)，回收的第一目的基因片段经再次凝胶电泳鉴定的结果如图4中泳道1所示。
在该实施例中，本发明人还合成了另外两条目的基因片段：RBD-(G4S) 2-S2-NP(命名为第二目的基因片段，其与第一目的基因片段的区别仅在于没有连接免疫增效类分子)以及Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2-S2-CTB-Furin2A-ERISS-NP(命名为第三目的基因片段，其与第一目的基因片段的区别在于免疫佐剂分子Foldon-CPPCP和CTB的位置不同)。
表1：重组新型冠状病毒高效免疫DNA疫苗中各结构的核苷酸序列信息
Figure PCTCN2021131786-appb-000001
Figure PCTCN2021131786-appb-000002
Figure PCTCN2021131786-appb-000003
Figure PCTCN2021131786-appb-000004
1.3、重组新型冠状病毒高效免疫DNA疫苗的构建
如图5所示，将上述步骤1.1获得的表达载体骨架pZDVac-IRES-OX40L与步骤1.2获得的第一目的基因CRSNP通过T4连接酶连接，随后经阳性克隆和菌落PCR鉴定后即可获得携带有第一目的基因片段CRSNP的重组表达载体：pZDVac-CTB-TT-PARDE-G6-RBD-(G4S) 2-S2-Foldon-CPPCP-Furin2A-ERISS-NP-IRES-OX40L(命名为pZDVac-CRSNPO重组质粒，其中融合基因CTB-TT-PARDE-G6-RBD-(G4S) 2-S2-Foldon-CPPCP-Furin2A-ERISS-NP-IRES-OX40L对应的核苷酸序列如SEQ ID NO:13所示，其中除包含有上表1所示的核苷酸序列外，还包含有酶切位点)，即为重组新型冠状病毒高效免疫DNA疫苗ZD-nCor19，具体构建步骤包括：
1.3.1、T4连接
将上述步骤1.1胶回收获得的表达载体骨架pZDVac-IRES-OX40L(约12kb)与步骤1.2胶回收获得的第一目的基因片段CRSNP(约3.6kb)连接，16℃过夜获得连接产物，体系如下表2所示：
表2：T4连接体系
Figure PCTCN2021131786-appb-000005
1.3.2、阳性克隆筛选
取100μL stable感受态细胞置于冰浴中，待感受态细胞融化后，向感受态细胞悬液中加入10μL上述步骤1.3.1获得的连接产物，轻轻混匀，冰浴30min，立即转移到42℃水浴锅中热击90s，迅速转移到冰浴中2min。向感受态细胞中加入500μL无菌的不含抗生素的LB培养基，混匀后置于37℃中，150rpm震荡培养60min。12000rpm离心1min，弃 去500μL上清。取100μL已转化的感受态细胞均匀涂布于含有卡那霉素(50μg/mL)的固体培养基上，37℃倒置培养48小时。
挑取上述转化的单克隆菌体，进行菌落PCR，鉴定阳性克隆。设计上游引物F与下游引物R进行PCR扩增(引物序列由北京博迈德基因有限公司(Biomed)合成)：
上游引物F：CCCAGGAGACCCGGTTCTAGAGACGGACATT(SEQ ID NO:14)；
下游引物R：AAGCGGCTTCGGCCAGTAACGTTAGGGGGGG(SEQ ID NO:15)；
PCR体系(25μL)为：12.5μL的2×PCR mix(购自北京博迈德基因有限公司(Biomed))；11.5μL的ddH 2O；0.5μL的F(10μM)和0.5μL的R(10μM)，对连接产物转化菌落进行PCR扩增。PCR反应条件为95℃5min 1个循环；94℃30s，55℃30s，72℃50s，扩增35个循环；72℃7min。PCR产物用0.45％琼脂糖DNA凝胶电泳分析，结果如图6所示，其中泳道1-5为不同单菌落的PCR结果。
1.3.3、阳性克隆提取质粒和酶切鉴定
菌落PCR鉴定为阳性的克隆，在含有卡那霉素(50μg/mL)的固体培养基上划线，37℃，倒置过夜培养。而后挑取单克隆，接种于1.5mL含有卡那霉素(50μg/mL)的LB培养基中，37℃，200rpm过夜培养后，按1:50的比例接种于20mL含有卡那霉素(50μg/mL)的LB培养基，37℃，200rpm过夜培养。无菌环境下，取其中12mL菌液，按1.2mL菌液+0.6mL60％甘油菌的比例，制备成甘油菌。剩余菌液离心后提取质粒，分别用AclI和XbaI进行单酶切及双酶切鉴定，单酶切和双酶切体系如下表3和表4所示。
表3：单酶切体系
Figure PCTCN2021131786-appb-000006
表4：双酶切体系
Figure PCTCN2021131786-appb-000007
Figure PCTCN2021131786-appb-000008
按照上表3或4的酶切体系混匀样品，并短暂离心，使样品及酶沉于管底。将离心管放置于37℃水浴锅中，孵育2h。用0.45％琼脂糖DNA凝胶电泳对酶切结果进行鉴定，鉴定结果如图7所示，其中泳道1-3分别为图6中经PCR鉴定的阳性克隆1、2、4；泳道4-6分别为阳性克隆菌落1、2、4质粒的AclI单酶切结果；泳道7-9分别为阳性克隆菌落1、2、4质粒的XbaI单酶切结果；泳道10-12分别为阳性克隆菌落1、2、4质粒的AclI和XbaI双酶切结果，其中包括大小约12kb的条带和大小约为3.6kb的条带，证明最终构建成功重组质粒pZDVac-CRSNPO，即为重组新型冠状病毒高效免疫DNA疫苗。
该实施例中还利用与上述步骤1.3类似的方法构建获得了携带有第二目的基因(RBD-(G4S) 2-S2-NP)的重组质粒pZDVac-RBD-(G4S) 2-S2-NP(其中利用空载体pZDVac作为骨架载体，命名为对照质粒1)，和携带有第三目的基因(Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2-S2-CTB-Furin2A-ERISS-NP)的重组质粒pZDVac-Foldon-CPPCP-TT-PARDE-G6-RBD-(G4S) 2-S2-CTB-Furin2A-ERISS-NP-IRES-OX40L(命名为对照质粒2)。
实施例2：pZDVac-CRSNPO重组质粒在小鼠模型中诱导的免疫原性
该实施例利用上述实施例1构建获得的pZDVac-CRSNPO重组质粒，验证其在小鼠模型中诱导的免疫原性效果，具体包括以下步骤。
2.1、将20只6-8周龄SPF级雌性BALB/c小鼠(购自北京维通利华实验动物技术有限公司)随机分为4组，每组5只，分别通过肌肉注射+电脉冲刺激的方式进行3次免疫，免疫间隔为7天。实验分组情况及剂量情况如表5所示。
表5：pZDVac-CRSNPO重组质粒在小鼠模型细胞免疫反应检测实验中的分组情况
Figure PCTCN2021131786-appb-000009
2.2、末次免疫后2周，处死小鼠，分离脾脏淋巴细胞，分别使用新型冠状病毒N蛋白和RBD蛋白刺激培养20小时后，按MabTech公司Mouse IFN-γ与IL-4ELISPOT板说明书进行免疫学检测。不同蛋白刺激物下细胞因子IFN-γ和IL-4的分泌频率检测统计结果分别如图8和图9所示(其中p＜0.05为统计学差异具有显著性)，抗原特异性T细胞免疫反应偏向性统计结果如图10所示。
图8至图10的结果显示，不同剂量的pZDVac-CRSNPO重组质粒免疫小鼠后均能诱 导N抗原与RBD抗原特异性T细胞免疫反应，且既分泌代表Th1型反应的IFN-γ，也能分泌代表Th2型反应的IL-4；而Th1型细胞因子IFN-γ的分泌频率要明显高于Th2型细胞因子IL-4。表明该pZDVac-CRSNPO重组质粒诱导的T细胞免疫反应偏向Th1，且相对0.3μg组和10μg组而言，1μg为最佳免疫剂量。
按照上述步骤2.1和2.2同样的方法，分别对6-8周龄SPF级雌性BALB/c小鼠免疫对照质粒1和对照质粒2后所诱导的免疫原性进行检测，结果表明：对照质粒1和对照质粒2所诱导的免疫原性效果均不及pZDVac-CRSNPO重组质粒所诱导的免疫原性效果。
实施例3：pZDVac-CRSNPO重组质粒在人体中的免疫原性检测
该实施例利用实施例1构建获得的pZDVac-CRSNPO重组质粒对人受试者进行免疫，验证其在人体内诱导的免疫原性效果，具体包括以下步骤。
3.1、将经过筛选的志愿者15人(申请单位内部人员，已签署志愿参与试验)，随机分为3组，每组5人，免疫间隔为28天，进行三次免疫。分别在第一次免疫前、第二次免疫后14天及第三次免疫后21天，取志愿者PBMC进行ELISPOT检测。前两次免疫方式为肌肉注射+电脉冲刺激，第三次免疫为皮内注射+电脉冲刺激。实验分组及剂量情况如下表6所示。
表6：pZDVac-CRSNPO重组质粒在人受试者细胞免疫反应检测实验中的分组情况
Figure PCTCN2021131786-appb-000010
3.2、取从志愿者体内获得的新鲜的PBMC，分别使用新型冠状病毒N蛋白和RBD蛋白刺激培养20小时后，按MabTech公司Human IFN-γELISPOT板说明书进行免疫学检测。三次免疫后不同蛋白刺激物下细胞因子IFN-γ的分泌频率检测统计结果如图11所示(其中p＜0.05为统计学差异具有显著性；p＜0.01为统计学差异具有非常显著性)，三次免疫不同剂量反应统计结果如图12所示(其中p＜0.05为统计学差异具有显著性；p＜0.01为统计学差异具有非常显著性)。
图11结果显示，与免疫前相比，进行二免和三免后均能够诱导强烈的细胞免疫反应，分别使用N蛋白和RBD蛋白作为刺激物时，PBMC中细胞因子IFN-γ的分泌频率具有非常显著的提高；并且当使用N蛋白作为刺激物时，三免后细胞因子IFN-γ的分泌频率高于二免后，而当使用RBD蛋白作为刺激物时，二免后细胞因子IFN-γ的分泌频率高于三免。图12结果显示，不同剂量组免疫后与免疫前相比，斑点数均有显著增加，说明各剂量下均能有效诱导特异性细胞免疫；而使用N蛋白和RBD蛋白作为刺激物时，二免 后各剂量组之间斑点数的统计学差异具有显著性(p＝0.0343，为低中高三个剂量组之间的方差分析结果)。可见，相对于高剂量组和低剂量组，中剂量组免疫效果更佳，且三免后斑点数并未增加太多，因此该pZDVac-CRSNPO重组质粒作为新型冠状病毒DNA疫苗时，采用中剂量免疫2针(2针之间例如间隔28天)即可。
另外，对该实施例筛选的15名志愿者在疫苗注射后进行定期随访和体检(至少3个月)，所有志愿者均没有表现出任何不适状况，初步表明本发明提供的pZDVac-CRSNPO重组质粒作为疫苗使用时具有良好的安全性。
实施例4：pZDVac-CRSNPO重组质粒的免疫途径及电导入条件
该实施例利用实施例1构建获得的pZDVac-CRSNPO重组质粒通过不同的免疫途径(肌肉注射或皮内注射)以及不同的电导入条件对小鼠进行免疫，以评价不同的免疫途径和不同的电导入条件下诱导产生的免疫反应强弱，具体包括以下步骤。
4.1、按照下表7所示的组别将40只健康的6-8周雌性Balb/c小鼠(购自维通利华)随机分为5组，每组8只，分别通过肌肉注射或皮内注射&电导入(1Hz×6次，电极针间距0.5cm)的方式对各组小鼠左后肢单点给药进行免疫。设置空白对照组不做处理，其他各组小鼠给予pSFVK1-CRSNPO重组质粒1μg/只。给药日计为0d，分别于0d、21d对5组小鼠进行两次免疫。
表7：pZDVac-CRSNPO重组质粒不同免疫途径及电导入条件下的小鼠分组情况
Figure PCTCN2021131786-appb-000011
4.2、于末次免疫后2周取血清进行抗体检测，具体为使用ACRO Biosystems抗体检测试剂盒进行血清抗体(RBD IgG)检测，小鼠血清按1:100稀释，阳性判定为OD450nm处吸光值＞0.1。并于末次免疫2周后，处死各组小鼠，按MabTech公司IFN-γELISPOT板说明书进行ELISPOT检测(IFN-γ)，其中ELISPOT实验的斑点计数表达方式为：spot-forming units(SFU)/10 6splenocytes；实验孔阳性的判断标准为：实验孔斑点数＞阴性对照孔斑点均值+2SD；每只小鼠阳性斑点数计算方法为：1只小鼠的脾细胞做5个复孔，至少3个孔为阳性，则该小鼠的数据有效，该小鼠斑点数＝实验孔平均斑点数-阴性对照孔斑点数；每组小鼠的斑点数计算方法为：将该组每只有效小鼠数据求平均值， 以平均值±标准差表示。数据的统计学分析用GraphPad Prism 8软件进行，其中两组间差异用Student's t test检验进行分析，多组间差异比较用单因素方差分析。p<0.05为统计学上差异具有显著性，P<0.01为统计学上差异具有非常显著性。
抗体检测的结果如图13所示，可见pSFVK1-CRSNPO重组质粒经不同途径(肌肉注射和皮内注射)或不同电导入条件免疫小鼠后，均能诱导抗体反应。肌肉注射组(60V，50ms，1Hz×6次，电极针间距0.5cm，场强120V/cm)诱导产生的抗体水平与空白对照组相比，统计学上差异具有显著性；皮内注射组a(60V，50ms，1Hz×6次，电极针间距0.5cm，场强120V/cm)诱导产生的抗体水平与空白对照组相比，统计学上差异也具有显著性；而皮内注射组b(36V，50ms)和c(36V，10ms)诱导产生的抗体水平与空白对照组相比，统计学上差异不显著；表明在肌肉注射和皮下注射条件下当电导入条件为电压60V，脉宽50ms时，相对于电压36V，脉宽50ms的电导入条件具有相对更好的免疫效果。另外，在相同的电导入条件下，皮内注射组a诱导产生的抗体水平与肌肉注射组相比，统计学上差异虽不显著，但总体上经肌肉注射途径免疫pSFVK1-CRSNPO重组质粒会有相对更好的体液免疫效果。
ELISPOT检测检测结果如图14所示，可见pSFVK1-CRSNPO重组质粒经不同途径(肌肉注射和皮内注射)和不同电导入条件下免疫小鼠后均能诱导N抗原、RBD抗原和S2抗原特异性T细胞免疫反应，实验组(肌肉注射组、皮下注射组a、b、c)与空白对照组相比，在统计学上差异均具有显著性，其中各电导入条件下经皮内注射途径免疫后诱导的N抗原、RBD抗原和S2抗原特异性T细胞免疫反应与经肌肉注射途径免疫后诱导的N抗原、RBD抗原和S2抗原特异性T细胞免疫反应均无明显差异。
综上结果表明，本发明提供的pSFVK1-CRSNPO重组质粒疫苗经不同免疫途径及不同电导入条件刺激小鼠，均能够诱导较强的特异性T细胞免疫反应，并且肌肉注射组与皮内注射组诱导的细胞免疫反应无明显差异；本发明提供的pSFVK1-CRSNPO重组质粒疫苗在诱导体液免疫反应方面，电压60V，脉宽50ms的电导入条件具有相对更好的诱导效果，且总体上疫苗经肌肉注射能产生相对更好的体液免疫反应。因此本发明提供的pSFVK1-CRSNPO重组质粒在作为疫苗使用时能够诱导良好的细胞免疫反应和体液免疫反应，可以发挥良好的疫苗接种保护效果，这与上述实施例2和实施例3的结果一致。
实施例5：免疫攻毒试验
该实施例按照上述实施例4中的疫苗免疫接种方式(肌肉注射或皮下注射&电导入(60V，50ms，1Hz×6次，电极针间距0.5cm，场强120V/cm))，将pSFVK1-CRSNPO重组质粒免疫hACE2转基因小鼠或恒河猴，设置阴性对照组，给予生理盐水。给药日计 为0d，分别于0d、21d对各组动物进行两次免疫，于末次免疫后2周分别对各组动物进行攻毒(新型冠状病毒液)，攻毒后观察各组动物，并考察各组动物肺部病毒载量下降和肺部的病理情况，以肺部病毒载量下降(≥2个log)和肺部病理改善为有效性评价的基本要求。结果表明：阴性对照组动物在攻毒后均表现出感染新型冠状病毒的典型临床症状(例如发热、精神萎靡、体重下降、甚至死亡等)，而免疫组至少4/5的动物获得保护，未表现出感染新型冠状病毒的典型临床症状，且肺部病毒载量下降(≥2个log)和肺部病理明显改善。表明本发明提供的重组新型冠状病毒高效免疫DNA疫苗ZD-nCor19具有有效性和安全性。
综上实施例结果显示，本发明构建获得的重组新型冠状病毒高效免疫DNA疫苗(即pZDVac-CRSNPO重组质粒)在动物水平的免疫学功能测试中能够有效诱导高效免疫应答，包括天然免疫反应和抗原特异性细胞免疫应答，证实是一款有较大发展潜力的新型冠状病毒DNA疫苗品种。使用该疫苗对人受试者进行免疫后，发现3个剂量组(100μg、300μg、500μg)均可以诱导出较高水平的天然免疫应答和抗原特异性细胞免疫应答，其中300μg剂量组诱导的免疫应答水平最高。所有参与测试的人受试者，在定期的随访和体检期间均没有出现发热、过敏、头痛、全身无力等明显不良反应。通过比较不同的免疫途径及不同电导入条件诱导产生的免疫反应强弱，表明本发明提供的重组新型冠状病毒高效免疫DNA疫苗可以通过肌肉注射和皮下注射的方式接种，均可以诱导良好的特异性T细胞免疫反应和体液免疫反应。免疫攻毒试验结果也表明本发明的重组新型冠状病毒高效免疫DNA疫苗具有良好的安全性和免疫保护效果。以上结果均初步证实是本发明构建的疫苗是一款既能诱导高级别免疫响应同时又非常温和安全的疫苗品种。
此处描述的实施例只用于说明(作为例证)，技术人员所做的各种修改或变更也应包括在专利申请的实质范围内。
工业应用性
本发明提供了融合基因及基于该融合基因的重组新型冠状病毒高效免疫DNA疫苗及其构建方法和应用，其中该重组新型冠状病毒高效免疫DNA疫苗兼具有预防和治疗新型冠状病毒感染的双重效果，适于工业应用。

Claims (10)

  1. 一种融合基因，其包含以下(1)至(4)中的至少两种：
    (1)表达新型冠状病毒COVID-19的RBD氨基酸片段的基因；
    (2)表达新型冠状病毒COVID-19的S2亚基或其部分氨基酸片段的基因；
    (3)表达新型冠状病毒COVID-19的N蛋白或其部分氨基酸片段的基因；
    (4)表达选自以下中的任一种或其组合的氨基酸片段的基因：CTB、TT、PADRE、Foldon、CPPCP、Furin2A、ERISS、IRES和OX40L。
  2. 根据权利要求1所述的融合基因，其包含以下(1)至(4)中的至少三种：
    (1)表达新型冠状病毒COVID-19的RBD氨基酸片段的基因；
    (2)表达新型冠状病毒COVID-19的S2亚基的301-538位氨基酸片段的基因；
    (3)表达新型冠状病毒COVID-19的N蛋白的138-369位氨基酸片段的基因；
    (4)表达以下的氨基酸片段的基因：CTB、TT、PADRE、Foldon、CPPCP、Furin2A、ERISS、IRES和OX40L。
  3. 根据权利要求2所述的融合基因，其中表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因连接成融合片段；
    优选地，所述融合片段的核苷酸序列包含SEQ ID NO:6所示的序列；表达所述N蛋白的138-369位氨基酸片段的基因的核苷酸序列包含SEQ ID NO:10所示的序列。
  4. 根据权利要求1-3中任一项所述的融合基因，其中：
    表达所述CTB氨基酸片段的基因的核苷酸序列如SEQ ID NO:2所示；
    表达所述TT氨基酸片段的基因的核苷酸序列如SEQ ID NO:3所示；
    表达所述PADRE氨基酸片段的基因的核苷酸序列如SEQ ID NO:4所示；
    表达所述Foldon氨基酸片段的基因与表达所述CPPCP氨基酸片段的基因连接成一合成片段，该合成片段的核苷酸序列如SEQ ID NO:7所示；
    表达所述Furin2A氨基酸片段的基因的核苷酸序列如SEQ ID NO:8所示；
    表达所述ERISS氨基酸片段的基因的核苷酸序列如SEQ ID NO:9所示；
    表达所述IRES氨基酸片段的基因的核苷酸序列如SEQ ID NO:11所示；和/或
    表达所述OX40L氨基酸片段的基因的核苷酸序列如SEQ ID NO:12所示。
  5. 根据权利要求2-4中任一项所述的融合基因，其中：
    表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因连接成融合片段，在所述的融合片段的上游依次连接有表达所述CTB、TT和PADRE氨基酸片段的基因，在所述的融合片段的下游依次连接有表达所述Foldon、 CPPCP和Furin2A氨基酸片段的基因；和/或
    在表达所述N蛋白的138-369位氨基酸片段的基因的上游连接有表达所述ERISS氨基酸片段的基因，下游依次连接有表达所述IRES和OX40L氨基酸片段的基因；
    优选地，表达所述RBD氨基酸片段的基因与表达所述S2亚基的301-538位氨基酸片段的基因通过表达(G4S) 2连接臂的基因连接，所述融合片段的上游与表达所述PADRE氨基酸片段的基因通过核苷酸序列如SEQ ID NO:5所示的表达连接臂linker G6的基因连接；
    进一步优选地，所述融合基因的核苷酸序列如SEQ ID NO:13所示。
  6. 一种融合蛋白，其由权利要求1-5中任一项所述的融合基因表达获得。
  7. 一种重组新型冠状病毒高效免疫DNA疫苗，命名为ZD-nCor19，其包含权利要求1-5中任一项所述的融合基因和载体。
  8. 根据权利要求7所述的重组新型冠状病毒高效免疫DNA疫苗，其中所述载体为pZDVac载体。
  9. 权利要求7或8所述的重组新型冠状病毒高效免疫DNA疫苗的构建方法，其包括以下步骤：
    1)合成权利要求1-5中任一项所述的融合基因；
    2)将所述融合基因插入pZDVac载体中，获得所述重组新型冠状病毒高效免疫DNA疫苗。
  10. 权利要求7或8所述的重组新型冠状病毒高效免疫DNA疫苗在制备预防和/或治疗新型冠状病毒感染的药物中的应用。
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