WO2021184884A1 - 基于水疱性口炎病毒载体的复制型重组新型冠状病毒及其制备方法与应用 - Google Patents

基于水疱性口炎病毒载体的复制型重组新型冠状病毒及其制备方法与应用 Download PDF

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WO2021184884A1
WO2021184884A1 PCT/CN2020/139866 CN2020139866W WO2021184884A1 WO 2021184884 A1 WO2021184884 A1 WO 2021184884A1 CN 2020139866 W CN2020139866 W CN 2020139866W WO 2021184884 A1 WO2021184884 A1 WO 2021184884A1
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sequence
ncov
envelope protein
virus
sars
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郑爱华
赵超越
李虹悦
张毓航
金万洙
赵建国
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中国科学院动物研究所
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Definitions

  • the invention relates to the technical field of biomedicine, in particular to a replication-type recombinant new coronavirus based on a vesicular stomatitis virus vector, and a preparation method and application thereof.
  • the new coronavirus 2019-nCoV also called SARS-CoV-2 or HCoV-19, is a new type of coronavirus of the genus p. Viral genome analysis shows that the virus is very close to the SARS-CoV (Severeacuterespiratorysyndrome-coronavirus) virus (89.1% nucleotide similarity), and infection with the new coronavirus 2019-nCoV can cause symptoms such as human pneumonia.
  • the new coronavirus 2019-nCoV (SARS-CoV-2) is a single-stranded positive-stranded RNA virus with an envelope.
  • the virus particles are round or oval with radial protrusions.
  • This radial protrusion is a characteristic structure of coronavirus, which is composed of the envelope protein Spike(S). S protein is an important structural protein of coronavirus, and it plays a key role in the process of virus invading host cells.
  • the source of infection currently seen is mainly patients with pneumonia infected by the new coronavirus 2019-nCoV (SARS-CoV-2); the main route of transmission is through respiratory droplets, and it can also be transmitted through contact; Generally susceptible, the elderly and those with underlying diseases are more ill after infection, and children and infants also have the disease. Based on current epidemiological investigations, the incubation period of patients is generally 3-7 days, and the longest is no more than 14 days. The clinical manifestations are mostly fever, fatigue, and dry cough. A small number of patients are accompanied by nasal congestion, runny nose, diarrhea and other symptoms. Critically ill patients present with dyspnea, acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis, and coagulation dysfunction.
  • SARS-CoV-2 new coronavirus 2019-nCoV
  • the new coronavirus 2019-nCoV (SARS-CoV-2) is in the stage of accumulative awareness. Symptomatic treatment and supportive treatment are mainly carried out for patients, and there is a lack of targeted specific drugs and treatment methods. In order to curb the new type of coronavirus infection, the development of the 2019-nCoV (SARS-CoV-2) vaccine cannot be delayed.
  • the purpose of the present invention is to provide a replication-type recombinant new coronavirus based on vesicular stomatitis virus vector and its preparation method and application.
  • the present invention first provides a recombinant virus.
  • the recombinant virus provided by the present invention is a virus obtained by replacing glycoprotein G of vesicular stomatitis virus with envelope protein S;
  • the envelope protein S includes the envelope protein S of 2019-nCoV (SARS-CoV-2) Extracellular region or part of its sequence;
  • amino acid sequence of the extracellular region of the envelope protein S of 2019-nCoV is a) or b) or c):
  • amino acid sequence is the protein shown at positions 1-210 of sequence 2 in the sequence listing;
  • the recombinant virus is as follows 1) or 2) or 3):
  • amino acid sequence of the envelope protein S of 2019-nCoV is sequence 2 in the sequence table;
  • the amino acid sequence of the envelope protein S formed by chimerizing the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV is: Sequence 4 in the sequence listing;
  • glycoprotein G of vesicular stomatitis virus with the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the transmembrane and intracellular region of glycoprotein G of vesicular stomatitis virus Virus obtained after chimeric envelope protein S;
  • the recombinant virus is a gene sequence that replaces the glycoprotein G coding gene sequence in the vesicular stomatitis virus genome sequence with the envelope protein S of 2019-nCoV (SARS-CoV-2) Virus obtained after coding gene sequence;
  • the recombinant virus is the replacement of the glycoprotein G coding gene sequence in the vesicular stomatitis virus genome sequence with the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and SARS-CoV envelope protein S transmembrane region and intracellular region are chimerized into a virus obtained after the envelope protein S coding gene sequence;
  • the recombinant virus is the replacement of the glycoprotein G coding gene sequence in the vesicular stomatitis virus genome sequence with the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and Vesicular stomatitis virus, the glycoprotein G transmembrane region and the intracellular region are chimerized by the envelope protein S coding gene sequence.
  • SARS-CoV-2 2019-nCoV
  • Vesicular stomatitis virus the glycoprotein G transmembrane region and the intracellular region are chimerized by the envelope protein S coding gene sequence.
  • the present invention provides a recombinant virus.
  • the recombinant virus provided by the present invention is obtained by transfecting a recombinant virus vector into a virus packaging cell and then performing cell culture; the recombinant virus vector is the following A) or B) or C) or D):
  • A) Replace the coding gene sequence of glycoprotein G in the vesicular stomatitis virus genome sequence in the vesicular stomatitis virus vector with the coding gene sequence of the envelope protein S of 2019-nCoV (SARS-CoV-2). a;
  • the coding gene sequence of the envelope protein S of the 2019-nCoV is sequence 1 in the sequence table;
  • the coding gene sequence of the envelope protein S formed by chimerizing the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV Is sequence 3 in the sequence listing;
  • the coding gene of the envelope protein S chimerized by the extracellular domain of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the glycoprotein G transmembrane domain and the intracellular domain of the vesicular stomatitis virus
  • the sequence is sequence 5 in the sequence listing.
  • the reporter gene may be a reporter gene commonly used in the prior art, such as the GFP gene (shown in sequence 14 in the sequence list).
  • the recombinant viral vector is a vector obtained by inserting the DNA molecule shown in sequence 1 between the restriction sites MluI and NotI of the rVSV ⁇ G vector.
  • the recombinant viral vector is a vector obtained by inserting the DNA molecule shown in sequence 3 between the restriction sites MluI and NotI of the rVSV ⁇ G vector.
  • the recombinant viral vector is a vector obtained by inserting the DNA molecule shown in sequence 5 between the restriction sites MluI and NotI of the rVSV ⁇ G vector.
  • the recombinant viral vector is the GFP gene shown in sequence 14 in the sequence list inserted between the 62-63 nucleotides in the nucleotide sequence of the recombinant viral vector in A) The vector obtained after the sequence.
  • the rVSV ⁇ G vector includes a T7 promoter sequence, a full genome sequence of the VSV Indiana strain lacking a glycoprotein G coding gene sequence, and an HDV terminator sequence, and its nucleotide sequence is sequence 7 in the sequence list.
  • the virus packaging cells may be cell lines commonly used for virus packaging in the prior art, such as 293T cells, Vero cells and BHK cells, and specifically may be Vero cells.
  • the recombinant virus is a combination of the recombinant viral vector, a plasmid expressing the N protein of VSV, a plasmid expressing the P protein of VSV, a plasmid expressing the L protein of VSV, a plasmid expressing the M protein of VSV, and a plasmid expressing the G protein of VSV.
  • the plasmid and the plasmid expressing the T7 RNA polymerase are co-transfected into the virus packaging cells, and then obtained after cell culture.
  • the plasmid expressing the N protein of VSV specifically is to clone the N protein coding gene sequence in the VSV genome (sequence 8 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site. The resulting plasmid.
  • the plasmid expressing the P protein of VSV is specifically to clone the P protein coding gene sequence in the VSV genome (sequence 9 in the sequence list) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site. The resulting plasmid.
  • the plasmid expressing the L protein of VSV specifically is to clone the L protein coding gene sequence in the VSV genome (sequence 10 in the sequence list) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site. The resulting plasmid.
  • the plasmid expressing the M protein of VSV is specifically to clone the M protein coding gene sequence in the VSV genome (sequence 11 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site. The resulting plasmid.
  • the plasmid expressing the G protein of VSV is specifically to clone the G protein coding gene sequence in the VSV genome (sequence 12 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site.
  • the plasmid expressing T7RNA polymerase is specifically a plasmid obtained by cloning the T7RNA polymerase encoding gene sequence (sequence 13 in the sequence list) into the eukaryotic expression plasmid pCDNA3.1(+) through the BamHI-EcoRI restriction site .
  • the 2019-nCoV (SARS-CoV-2) is specifically the 2019-nCoV Wuhan-Hu-1 strain (GenBank: NC_045512.2).
  • the SARS-CoV is specifically the SARS-CoV BJ01 strain (GenBank: AY278488.2).
  • the vesicular stomatitis virus is specifically the vesicular stomatitis virus Indiana strain (GenBank: KF935251.1).
  • the above-mentioned recombinant virus vector also belongs to the protection scope of the present invention.
  • the present invention also provides a new use of the above-mentioned recombinant virus or the above-mentioned recombinant virus vector.
  • the present invention provides the application of the above-mentioned recombinant virus or the above-mentioned recombinant virus vector in any one of the following X1)-X3):
  • the screening of the new coronavirus invasion inhibitor is specifically embodied in the detection of the neutralizing antibody titers induced by the new coronavirus vaccine.
  • the present invention also provides a product for preventing and/or treating diseases caused by the new coronavirus.
  • the active ingredient of the product for preventing and/or treating diseases caused by the novel coronavirus provided by the present invention is the above-mentioned recombinant virus or the above-mentioned recombinant virus vector.
  • the product is a novel coronavirus vaccine.
  • the present invention finally provides a method for preventing and/or treating diseases caused by the new coronavirus.
  • the method for preventing and/or treating diseases caused by a novel coronavirus provided by the present invention includes the following steps: administering the above-mentioned product to a recipient animal for treatment or/and prevention of diseases caused by the novel coronavirus.
  • the subject animal includes a human.
  • the novel coronavirus is specifically 2019-nCoV (SARS-CoV-2).
  • the disease caused by the new coronavirus is specifically COVID-19 caused by the new coronavirus.
  • Vaccines are autoimmune preparations made of pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites through artificial attenuation, inactivation, or genetic engineering methods to prevent infectious diseases.
  • the vaccine retains the characteristics of pathogenic bacteria that stimulate the animal's immune system.
  • the immune system will produce certain protective substances, such as antibodies, etc.; when the animal comes into contact with this pathogen again, the animal’s immune system will follow its original Memory, create more protective substances to prevent the damage of pathogenic bacteria.
  • the new type of coronavirus belongs to the new type of coronavirus of the genus P. It has an envelope, round or elliptical particles, and a diameter of 60-140nm. Infected patients mainly manifested as fever, fatigue, and dry cough. In severe cases, they can progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and coagulation dysfunction, and some critically ill patients die.
  • VSV Vesicular Stomatitis Virus
  • N nucleocapsid protein
  • phosphoprotein P phosphoprotein
  • matrix protein M matrix protein
  • glycoprotein G glycoprotein G
  • Large polymerase L large protein
  • G protein is a type I integral membrane protein, which exists as a trimer on the surface of virus particles and performs the function of binding to target cell receptors and membrane fusion. In animals infected with VSV, most of the antibodies produced are directed against G protein.
  • VSV is a representative model virus of the Rhabdoviridae family and is widely used to study the mechanisms of enveloped viruses invading cells, replication and assembly.
  • Vesicular stomatitis caused by VSV virus is a contagious benign disease, which mainly infects rodents, cattle, pigs and horses, but can also infect humans and other animals. People are infected only occasionally, but they often show no symptoms or only mild fever.
  • the rate of VSV antibodies in the population is extremely low, and the seropositivity rate of people who are often exposed to VSV is high, such as some scientific researchers, farm workers of veterinarians who come into contact with sick animals.
  • the neutralizing antibody target of VSV is the G protein.
  • the recombinant virus will not be affected by the body's pre-existing immunity. Not being affected by pre-existing immunity and not having obvious pathogenicity are the prerequisites for VSV as a vaccine carrier.
  • VSV Vesicular Stomatitis Virus
  • the successful recovery of infectious VSV virus from DNA makes genetic manipulation of VSV possible.
  • the functional envelope protein of the foreign virus with a suitable cytoplasmic tail can be effectively packaged into the envelope of the virus to form a variety of chimeric recombinant VSV packaging heterologous envelope proteins.
  • the non-coding regions between the transcription units of the recombinant VSV can tolerate the insertion of up to 4.5 kb of foreign genes and obtain high-efficiency expression.
  • VSV has the following advantages: (1) Easy to culture: VSV can obtain high titer on most mammalian cells and is easy to prepare in large quantities. (2) Efficiency: immune response in a mouse model infected with VSV-HA, 10 infectious virus particles and immune induced 10 5 infectious particles as elicited significant. (3) Easy to use: It can be immunized through a variety of vaccination methods, and often a single vaccination can cause a strong immune response. (4) Produce a strong immune response: It can stimulate the body to produce a strong cellular immune response and humoral immune response, and can also cause a strong mucosal immune response. It is especially suitable for vaccine development of respiratory pathogens infected by mucosa.
  • VSV virus replicates completely in the cytoplasm, only from RNA to RNA, it will not be integrated into the host cell's DNA, and will eventually be cleared by the host immune system. Moreover, genome mutation and/or modification through reverse genetic manipulation can appropriately weaken the VSV virus and make it a safer recombinant vaccine vector.
  • the VSV recombinant virus prepared by the present invention is different from the VSV pseudovirus.
  • the biggest difference is that the VSV recombinant virus prepared by the present invention can replicate after infecting cells, while the VSV pseudovirus can only infect cells but cannot replicate.
  • the preparation methods and application ranges of the two are also different. Although both can be used for screening of viral inhibitors and basic research on viral infection mechanisms, the VSV recombinant virus prepared by the present invention can also be used for preparing vaccines, while VSV pseudoviruses cannot be used for preparing vaccines.
  • Figure 1 is a schematic diagram of the structure of the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2).
  • Figure 2 is a schematic diagram of the chimerization between the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV.
  • SARS-CoV-2 2019-nCoV
  • Figure 3 shows the replication curves of recombinant viruses rVSV-2019-nCoV (rVSV-SARS-CoV-2) and rVSV-2019-nCoV-SARS in Vero cells.
  • the control is VSV recombinant Ebola virus rVSV-EBOV.
  • Figure 4 shows the expression of S protein on recombinant viruses rVSV-2019-nCoV (rVSV-SARS-CoV-2) and rVSV-2019-nCoV-SARS.
  • Figure 5 shows the plaques formed by the recombinant viruses rVSV-2019-nCoV (rVSV-SARS-CoV-2) and rVSV-2019-nCoV-SARS infected with Vero cells.
  • the control is VSV recombinant Ebola virus rVSV-EBOV.
  • the scale bar represents 100 ⁇ m.
  • Figure 6 shows the neutralizing antibody stimulated by a single intranasal inoculation of the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) in cynomolgus monkeys.
  • Figure 7 shows the viral load of throat swabs inoculated with recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) and challenged with SARS-CoV-2.
  • Figure 8 shows the viral load of anal swabs inoculated with recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) and challenged with SARS-CoV-2.
  • Figure 9 shows the viral load in lung tissue of cynomolgus monkeys inoculated with recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) on the 7th day after challenge with SARS-CoV-2.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples, unless otherwise specified, are all conventional methods.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • control virus rVSV-EBOV in the following examples is described in the document "Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nature Med. (2005) 11,786-790", which can be obtained by the public from the Institute of Zoology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the plasmid expressing the N protein of VSV in the following examples is to clone the N protein coding gene sequence in the VSV genome (sequence 8 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1 through the BamHI-EcoRI restriction site (+)
  • the plasmid expressing the P protein of VSV in the following examples is to clone the P protein coding gene sequence in the VSV genome (sequence 9 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1 through the BamHI-EcoRI restriction site (+)
  • the plasmid expressing the L protein of VSV in the following examples is to clone the L protein coding gene sequence in the VSV genome (sequence 10 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1 through the BamHI-EcoRI restriction site (+)
  • the plasmid expressing the M protein of VSV in the following examples is to clone the M protein coding gene sequence in the VSV genome (sequence 11 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1 through the BamHI-EcoRI restriction site (+)
  • the plasmid expressing the G protein of VSV in the following examples is to clone the G protein coding gene sequence in the VSV genome (sequence 12 in the sequence list) into the eukaryotic expression plasmid pCDNA3.1 through the BamHI-EcoRI restriction site (+)
  • the plasmid expressing T7RNA polymerase in the following examples is the T7RNA polymerase encoding gene sequence (sequence 13 in the sequence list), cloned into the eukaryotic expression plasmid pCDNA3.1(+)( Beijing Shengyuan Kemeng Gene Biotechnology Co., Ltd.).
  • the rVSV ⁇ G vector in the following examples is described in the document "Single dose of a rVSV-based vaccine elicits complete protection against severe disease with thrombocytopenia syndrome virus.NPJ Vaccines. 2019Jan Chinese Academy of Sciences; 4:5.” Obtained by the Animal Research Institute, the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the nucleotide sequence of the rVSV ⁇ G vector is sequence 7 in the sequence list, which includes the T7 promoter sequence, the full genome sequence of the VSV Indiana strain with the glycoprotein G coding gene sequence deleted, and the HDV terminator sequence.
  • the Vero cells in the following examples are products of ATCC (American type culture collection), and the product number is CCL-81.
  • Example 1 Preparation of recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2)
  • the sequence encoding the complete 2019-nCoV envelope protein S is humanized and optimized, and the optimized sequence is shown in sequence 1 in the sequence table.
  • the complete envelope protein S of 2019-nCoV is the envelope protein S of 2019-nCoV Wuhan-Hu-1 strain (GenBank: NC_045512.2) (GenBank: YP_009724390.1), and its amino acid sequence is as shown in the sequence table The sequence 2 is shown.
  • step 1 The optimized sequence in step 1 (sequence 1 in the sequence table) was inserted between the restriction sites MluI and NotI of the rVSV ⁇ G vector to obtain a recombinant vector, which was named the recombinant vector VSV-2019-nCoV.
  • step 2 After completing step 1, incubate 36 ⁇ L FuGENE 6 (Promaga, catalog number E2692) with Opti-MEM medium (Thermo Fisher, catalog number 51985091) for 5 minutes at room temperature, and then incubate with the recombinant vector VSV-2019-nCoV in step 1.
  • FuGENE 6 Promaga, catalog number E2692
  • Opti-MEM medium Thermo Fisher, catalog number 51985091
  • the plasmid expressing the N protein of VSV (1.59 ⁇ g), the plasmid expressing the N protein of VSV (1.286 ⁇ g), the plasmid expressing the P protein of VSV (639ng), the plasmid expressing the L protein of VSV (159.9ng), the plasmid expressing the M protein of VSV (159.9ng) ), the plasmid expressing the G protein of VSV (159.9ng), and the plasmid expressing T7 RNA polymerase (8.1 ⁇ g) are mixed, and incubated at room temperature for 15 minutes to obtain the post-incubation solution (total volume is 600 ⁇ L).
  • step 2 After completing step 2, add the solution incubated in step 2 to the Vero cell culture system in step 1, and change to fresh medium after 6 hours. Three days after transfection, the cell supernatant was collected, and the supernatant contained the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2). The obtained supernatant was used to infect new Vero cells to achieve the amplification of the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2).
  • the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) is to replace the glycoprotein G coding gene sequence in the vesicular stomatitis virus genome sequence with the envelope protein of 2019-nCoV (SARS-CoV-2) The virus obtained after the coding gene sequence of S.
  • the sequence encoding the envelope protein S which is chimeric from the extracellular region of the envelope protein S of 2019-nCoV and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV, was carried out.
  • Source optimization the optimized sequence is shown in sequence 3 in the sequence table.
  • the extracellular region of the envelope protein S of 2019-nCoV is the extracellular region of the envelope protein S of the 2019-nCoV Wuhan-Hu-1 strain (the amino acid sequence of the envelope protein S of the Wuhan-Hu-1 strain) 1-1210), the transmembrane and intracellular regions of the envelope protein S of SARS-CoV are the transmembrane envelope protein S of the SARS-CoV (Severe acute respiratory syndrome-coronavirus) strain BJ01 (GenBank: AY278488.2) Region and intracellular region (the 1202-1267th amino acid sequence of the envelope protein S of the BJ01 strain).
  • the amino acid sequence of the envelope protein S which is chimeric from the extracellular region of the envelope protein S of 2019-nCoV and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV, is as shown in sequence 4 in the sequence table. Show.
  • the chimeric schematic diagram of the extracellular region of the envelope protein S of 2019-nCoV and the transmembrane and intracellular regions of the envelope protein S of SARS-CoV is shown in Figure 2.
  • step 1 The optimized sequence in step 1 (sequence 3 in the sequence table) was inserted between the restriction sites MluI and NotI of the rVSV ⁇ G vector to obtain a recombinant vector, which was named the recombinant vector VSV-2019-nCoV-SARS.
  • the recombinant vector VSV-2019-nCoV-SARS in step 1 and the helper plasmid were co-transfected into Vero cells to prepare recombinant virus rVSV-2019-nCoV-SARS.
  • the structure diagram of the recombinant virus rVSV-2019-nCoV-SARS is shown in Figure 2.
  • the recombinant virus rVSV-2019-nCoV-SARS is to replace the glycoprotein G coding gene sequence in the vesicular stomatitis virus genome with the extracellular region of the envelope protein S of 2019-nCoV (SARS-CoV-2) and SARS- A virus obtained by chimerizing the transmembrane region and the intracellular region of the envelope protein S of CoV to encode the gene sequence of the envelope protein S.
  • the sequence encoding the envelope protein S which is chimeric from the extracellular domain of 2019-nCoV envelope protein S and the transmembrane and intracellular domains of glycoprotein G of VSV, is humanized and optimized.
  • the optimized sequence is shown in sequence 5 in the sequence table.
  • the envelope protein S extracellular region of 2019-nCoV is the envelope protein S extracellular region of 2019-nCOV Wuhan-Hu-1 strain.
  • the glycoprotein G transmembrane region and intracellular region of VSV are the glycoprotein G transmembrane region and intracellular region of the VSV Indiana strain.
  • the amino acid sequence of the envelope protein S formed by the chimerization of the extracellular domain of the envelope protein S of 2019-nCoV and the transmembrane and intracellular domains of glycoprotein G of VSV is shown in sequence 6 in the sequence table.
  • step 1 The optimized sequence in step 1 (sequence 5 in the sequence table) was inserted between the restriction sites MluI and NotI of the rVSV ⁇ G vector to obtain a recombinant vector, which was named the recombinant vector VSV-2019-nCoV'.
  • the recombinant vector VSV-2019-nCoV' in step 1 and the helper plasmid were co-transfected into Vero cells to prepare recombinant virus rVSV-2019-nCoV'.
  • the recombinant virus rVSV-2019-nCoV' is the replacement of the coding gene sequence of glycoprotein G in the vesicular stomatitis virus genome with the extracellular region of the envelope protein S and the vesicular mouth of 2019-nCoV (SARS-CoV-2).
  • the specific operation method of immunofluorescence to measure virus titer is as follows: Pass Vero cells into 96-well plates according to 10,000 cells per well. On the second day, when the cells have grown to a density of about 80%-90%, the supernatant of the virus to be tested is added. Perform a 10-fold gradient dilution of the virus supernatant, that is, 30 ⁇ l of virus stock solution and 270 ⁇ l of virus diluent are mixed, and so on, a total of 6-7 gradients are diluted.
  • the virus dilution is DMEM medium containing 2% (volume fraction) FBS. Add 100 ⁇ l of virus solution to each well, and set 3 wells for each dilution to repeat.
  • the virus titer (unit FFU/ml) was detected by staining with an antibody against S protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., rabbit anti-SARS-CoV S polyclonal antibody, item number 10150-RP01).
  • the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) prepared in Example 1 or the recombinant virus rVSV-2019-nCoV-SARS prepared in Example 2 was collected The supernatant of the infected Vero cells was centrifuged at 39,000 rpm for 3 hours to obtain the virus precipitate.
  • the results of the plaque experiment are shown in Figure 5.
  • the results showed that the two recombinant viruses (the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) prepared in Example 1 or the recombinant virus rVSV-2019-nCoV-SARS prepared in Example 2) infected Vero cells 6 After days, very small plaques can be formed ( Figure 5, see arrow). However, rVSV-EBOV can form larger plaques two days after infection of Vero cells.
  • Test animals and methods The recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) prepared in Example 1 with 10 7 FFU was used to inoculate 6 2-6 year-old cynomolgus monkeys ( 3 males and 3 males, from Guangxi Xiongsen Primate Experimental Animal Breeding Development Co., Ltd.). The neutralizing antibody titer was detected by sera before immunization, 14 days after immunization, 21 days and 46 days after immunization.
  • the specific steps of the neutralizing antibody titer detection method are as follows: Pass the Vero cells into a 96-well plate according to 10,000 cells per well. The next day when the cells grow to about 80%-90%, the test is ready. First, the serum is diluted according to the 5-fold dilution method, such as 1:10, 1:50, 1:250, 1:1250. At the same time, the rVSV-GFP-SARS-CoV-2 virus was diluted to 2 ⁇ 10 3 FFU/ml, so that the final virus number per well was 100 FFU.
  • FRNT 50 [1/10] (logarithm of the dilution factor of the infection number of cells less than 50% + distance ratio x logarithm of the dilution factor) .
  • Distance ratio (50%-less than 50% cell infection number positive rate)/(more than 50% cell infection number positive rate-less than 50% cell infection number positive rate).
  • the dilution factor is the multiple dilution gradient.
  • rVSV-GFP-SARS-CoV-2 (rVSV-GFP-2019-nCoV) is as follows: 1) Between the 62-63 nucleotides in the nucleotide sequence of the recombinant vector VSV-2019-nCoV Insert the GFP gene sequence shown in sequence 14 in the sequence table to obtain the recombinant vector VSV-GFP-2019-nCoV. 2) According to the method in step 2 of Example 1, replace the recombinant vector VSV-2019-nCoV with the recombinant vector VSV-GFP-2019-nCoV, and prepare the recombinant virus rVSV-GFP-SARS-CoV-2 (rVSV-GFP- 2019-nCoV).
  • mice and methods 2-6 year-old cynomolgus monkeys (male, from Beijing Guierxin Institute of Biological Resources Co., Ltd.) were divided into 2 groups (experimental group and control group), with 2 in each group.
  • the experimental group (immune group) was inoculated with 5 ⁇ 10 6 FFU of the recombinant virus rVSV-2019-nCoV (rVSV-SARS-CoV-2) prepared in Example 1 through a single nasal drip, and the control group was inoculated with an equal volume of PBS .
  • the challenge dose was 1.0 ⁇ 10 7 TCID 50 , and the challenge method was 40% nasal drops (20%/nostril) +60% tracheal drip species.
  • Pharyngeal swab samples and anal swab samples were collected on day 0 before and on day 1, day 3, day 5, and day 7 after challenge, respectively.
  • the animals were euthanized on the 7th day after the challenge, and lung tissue samples (7 lung lobes) were taken.
  • N-F CGGAGGATTGACGACTAATGC
  • N-R ACCATCCGAGCCATTCGA.
  • the reaction program is as follows:
  • the present invention uses vesicular stomatitis virus VSV as a vector to construct three replicating recombinant viruses: 1. Replace the coding gene sequence of glycoprotein G of VSV virus with the envelope of 2019-nCoV (SARS-CoV-2) The coding gene sequence of protein S; 2. Replace the coding gene sequence of glycoprotein G of VSV virus with the extracellular domain of the envelope protein S of 2019-nCoV (SARS-CoV-2) and the envelope protein S of SARS-CoV The coding gene sequence of the envelope protein S formed by the chimerization of the transmembrane region and the intracellular region; 3.
  • Virus invasion and neutralizing antibody targets are mainly the extracellular region of S protein, so the infection mechanism and induced immune response of recombinant viruses are theoretically very similar to 2019-nCoV (SARS-CoV-2).
  • SARS-CoV-2 2019-nCoV
  • the recombinant virus can not only be developed into a vaccine, but also can be used as an ideal tool for studying the infection process of 2019-nCoV (SARS-CoV-2), and for screening inhibitors (such as antibodies, serum, small peptides, and small molecules) that inhibit virus invasion And the development of a new coronavirus vaccine is of great significance.

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Abstract

一种基于水疱性口炎病毒载体的重组新型冠状病毒及其制备方法与应用。所述重组新型冠状病毒为将水疱性口炎病毒的糖蛋白G替换为囊膜蛋白S后得到的病毒；所述囊膜蛋白S包含2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区或其部分序列。所述重组新型冠状病毒可以模拟2019-nCoV(SARS-CoV-2)入侵细胞的过程，并能激发机体产生针对2019-nCoV(SARS-CoV-2)的免疫反应，可用于研究2019-nCoV(SARS-CoV-2)感染过程，还可以开发成疫苗，可用于筛选抑制病毒入侵的抑制剂和疫苗研制。

Description

[根据细则26改正13.01.2021]　基于水疱性口炎病毒载体的复制型重组新型冠状病毒及其制备方法与应用 技术领域
本发明涉及生物医药技术领域，具体涉及基于水疱性口炎病毒载体的复制型重组新型冠状病毒及其制备方法与应用。
背景技术
[根据细则9.2改正18.03.2021]　
新型冠状病毒2019-nCoV又叫SARS-CoV-2或者HCoV-19，是一种新出现的p属冠状病毒。病毒基因组分析显示，该病毒与SARS-CoV(Severeacuterespiratorysyndrome-coronavirus)病毒非常接近(89.1%的核苷酸相似性)，感染新型冠状病毒2019-nCoV后会引起人类肺炎等症状。新型冠状病毒2019-nCoV(SARS-CoV-2)属于单股正链RNA病毒，具有囊膜。在电镜下观察，病毒颗粒呈圆形或椭圆形，具有放射状突起。这种放射状突起是冠状病毒的特征结构，它由囊膜蛋白Spike(S)构成。S蛋白是冠状病毒的重要结构蛋白，在病毒侵入宿主细胞的过程中起着关键作用。
[根据细则9.2改正18.03.2021]　
目前由新型冠状病毒2019-nCoV(SARS-CoV-2)导致的新型冠状病毒肺炎(COVID-19)己纳入《中华人民共和国传染病防治法》规定的“乙类”传染病，并采取“甲类”传染病的预防、控制措施。其流行病学特点为：目前所见的传染源主要是新型冠状病毒2019-nCoV(SARS-CoV-2)感染的肺炎患者；主要传播途径为经呼吸道飞沫传播，亦可通过接触传播；人群普遍易感，老年人及有基础疾病者感染后病情较重，儿童及婴幼儿也有发病。基于目前的流行病学调查，患者潜伏期一般为3-7天，最长不超过14天。临床表现多为发热、乏力、干咳。少数患者伴有鼻塞、流涕、腹泻等症状。重症患者表现为呼吸困难、急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍。
目前对新型冠状病毒2019-nCoV(SARS-CoV-2)处于累积认识阶段，对患者主要开展对症治疗和支持治疗，缺乏针对性的的特效药物和治疗手段。为了阻遏新型冠状病毒感染，2019-nCoV(SARS-CoV-2)疫苗的研制刻不容缓。
发明公开
本发明的目的是提供一种基于水疱性口炎病毒载体的复制型重组新型冠状病毒及其制备方法与应用。
为了实现上述目的，本发明首先提供了一种重组病毒。
本发明提供的重组病毒为将水疱性口炎病毒的糖蛋白G替换为囊膜蛋白S后得到的病毒；所述囊膜蛋白S包含2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区或其部分序列；
所述2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区的氨基酸序列为a)或 b)或c)：
a)氨基酸序列是序列表中的序列2第1-1210位所示的蛋白质；
b)将序列表中的序列2第1-1210位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
c)与序列表中的序列2第1-1210位所示的氨基酸序列具有85％或85％以上的同源性且具有相同功能的蛋白质。
进一步的，所述重组病毒为如下1)或2)或3)：
1)将水疱性口炎病毒的糖蛋白G替换为2019-nCoV(SARS-CoV-2)的囊膜蛋白S后得到的病毒；
所述2019-nCoV(SARS-CoV-2)的囊膜蛋白S的氨基酸序列为序列表中的序列2；
2)将水疱性口炎病毒的糖蛋白G替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S后得到的病毒；
所述由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列为序列表中的序列4；
3)将水疱性口炎病毒的糖蛋白G替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S后得到的病毒；
所述由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列为序列表中的序列6。
更进一步的，所述1)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为2019-nCoV(SARS-CoV-2)的囊膜蛋白S的编码基因序列后得到的病毒；
所述2)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒；
所述3)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒。
为了实现上述目的，本发明又提供了一种重组病毒。
本发明提供的重组病毒是将重组病毒载体转染病毒包装细胞，然后进行细胞培养后得到的；所述重组病毒载体为如下A)或B)或C)或D)：
A)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为2019-nCoV(SARS-CoV-2)的囊膜蛋白S的编码基因序列后得到的载体；
B)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的载体；
C)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的载体。
D)在A)或B)或C)所述的重组病毒载体中插入报告基因后得到的载体。
进一步的，所述2019-nCoV(SARS-CoV-2)的囊膜蛋白S的编码基因序列为序列表中的序列1；
所述由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的序列3；
所述由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的序列5。
所述报告基因可为现有技术中常用的报告基因，如GFP基因(序列表中的序列14所示)。
更进一步的，所述A)中，所述重组病毒载体为将序列1所示的DNA分子插入rVSVΔG载体的酶切位点MluI和NotI之间后得到的载体。
所述B)中，所述重组病毒载体为将序列3所示的DNA分子插入rVSVΔG载体的酶切位点MluI和NotI之间后得到的载体。
所述C)中，所述重组病毒载体为将序列5所示的DNA分子插入rVSVΔG载体的酶切位点MluI和NotI之间后得到的载体。
所述D)中，所述重组病毒载体为在A)所述重组病毒载体的核苷酸序列中的第62-63位核苷酸之间插入了序列表中的序列14所示的GFP基因序列后得到的载体。
所述rVSVΔG载体包括T7启动子序列、缺失糖蛋白G编码基因序列的VSV Indiana毒株全基因组序列和HDV终止子序列，其核苷酸序列为序列表中的序列7。
所述病毒包装细胞可为现有技术中常见的用于病毒包装的细胞系，如293T细胞、Vero细胞和BHK细胞，具体可为Vero细胞。
所述重组病毒是将所述重组病毒载体、表达VSV的N蛋白的质粒、表达VSV的 P蛋白的质粒、表达VSV的L蛋白的质粒、表达VSV的M蛋白的质粒、表达VSV的G蛋白的质粒及表达T7RNA聚合酶的质粒共同转染所述病毒包装细胞，然后进行细胞培养后得到的。
所述表达VSV的N蛋白的质粒具体为将VSV基因组中的N蛋白编码基因序列(序列表中的序列8)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。所述表达VSV的P蛋白的质粒具体为将VSV基因组中的P蛋白编码基因序列(序列表中的序列9)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。所述表达VSV的L蛋白的质粒具体为将VSV基因组中的L蛋白编码基因序列(序列表中的序列10)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。所述表达VSV的M蛋白的质粒具体为将VSV基因组中的M蛋白编码基因序列(序列表中的序列11)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。所述表达VSV的G蛋白的质粒具体为将VSV基因组中的G蛋白编码基因序列(序列表中的序列12)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。所述表达T7RNA聚合酶的质粒具体为将T7RNA聚合酶编码基因序列(序列表中的序列13)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)后得到的质粒。
所述2019-nCoV(SARS-CoV-2)具体为2019-nCoV Wuhan-Hu-1毒株(GenBank：NC_045512.2)。
所述SARS-CoV具体为SARS-CoV BJ01毒株(GenBank：AY278488.2)。
所述水疱性口炎病毒具体为水疱性口炎病毒Indiana毒株(GenBank：KF935251.1)。
上述重组病毒载体也属于本发明的保护范围。
为了实现上述目的，本发明还提供了上述重组病毒或上述重组病毒载体的新用途。
本发明提供了上述重组病毒或上述重组病毒载体在如下X1)-X3)中任一种中的应用：
X1)制备新型冠状病毒疫苗；
X2)制备预防和/或治疗新型冠状病毒导致的疾病的产品；
X3)筛选新型冠状病毒入侵抑制剂。
上述应用中，所述筛选新型冠状病毒入侵抑制剂具体体现在用于检测新型冠状病毒疫苗诱导产生的中和抗体滴度。
为了实现上述目的，本发明还提供了一种用于预防和/或治疗新型冠状病毒导致的疾病的产品。
本发明提供的用于预防和/或治疗新型冠状病毒导致的疾病的产品的活性成分为上述重组病毒或上述重组病毒载体。
上述任一所述应用或产品中，所述产品为新型冠状病毒疫苗。
为了实现上述目的，本发明最后提供了一种预防和/或治疗新型冠状病毒导致的疾病的方法。
本发明提供的预防和/或治疗新型冠状病毒导致的疾病的方法包括如下步骤：给受体动物施用上述产品进行治疗或/和预防新型冠状病毒导致的疾病。
上述方法中，所述受试动物包括人。
上述任一所述应用或产品或方法中，所述新型冠状病毒具体为2019-nCoV(SARS-CoV-2)。
所述新型冠状病毒导致的疾病具体为新型冠状病毒导致的COVID-19。
本发明所涉及的概念如下：
疫苗是将病原微生物(如细菌、立克次氏体、病毒等)及其代谢产物，经过人工减毒、灭活或利用基因工程等方法制成的用于预防传染病的自动免疫制剂。疫苗保留了病原菌刺激动物体免疫系统的特性。当动物体接触到这种不具伤害力的病原菌后，免疫系统便会产生一定的保护物质，如抗体等；当动物再次接触到这种病原菌时，动物体的免疫系统便会依循其原有的记忆，制造更多的保护物质来阻止病原菌的伤害。
[根据细则9.2改正18.03.2021]　
新型冠状病毒属于P属的新型冠状病毒，有囊膜，颗粒呈圆形或椭圆形，直径60-140nm。感染者主要表现为发热、乏力、干咳，严重者可进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍，部分重症患者死亡。
水疱性口炎病毒(Vesicular Stomatitis Virus，VSV)为单股负链不分节段RNA病毒，基因组长约11Kb，结构简单。从3'到5'依次转录出5条mRNA，分别编码5种蛋白：核衣壳蛋白N(nucleocapsid protein)、磷蛋白P(phosphoprotein)、基质蛋白M(matrix protein)、糖蛋白G(glycoprotein)、大聚合酶L(large protein)。G蛋白是I型整合膜蛋白，在病毒粒子表面以三聚体形式存在，行使与靶细胞受体结合及膜融合的功能。在感染了VSV的动物体内，所产生的抗体绝大部分针对G蛋白。VSV是弹状病毒科的代表模式毒种，被广泛用作研究囊膜病毒入侵细胞、复制和装配等机制。VSV病毒引起的水疱性口炎是接触性传染的良性疾病，主要感染啮齿类动物牛、猪和马，也能感染人类和其它动物。人只偶然感染，但常不显症状或仅轻微发热。人群中具有VSV抗体的比率极低，只有经常暴露接触VSV的人群血清阳性率较高，比如一些科研人员，接触病畜的兽医的农场工人。VSV的中和抗体靶点是G蛋白，一旦G蛋白被替换成外源病毒的囊膜蛋白，重组病毒将不受人体预存免疫(pre-existing immunity)的影响。不受预存免疫影响和不具明显致病性是VSV作为疫苗载体的前提条件。
在现有的各种活病毒疫苗载体中，水疱性口炎病毒(Vesicular Stomatitis Virus，VSV)具有人群中血清阳性率低；能诱导粘膜免疫；基因组简单，易于规模化生产等突出的优点，被认为是最具有开发潜力的病毒疫苗载体之一。成 功的从DNA恢复出有感染性的VSV病毒，使得对VSV进行遗传操作变为可能。带有合适胞质尾的外源病毒功能囊膜蛋白可以有效包装进病毒的囊膜，形成各种包装异源囊膜蛋白的嵌合重组VSV。重组VSV各转录单元之间的非编码区可耐受长达4.5kb外源基因的插入并获得高效表达。这些特性赋予了VSV作为活病毒疫苗载体的应用潜力。
作为一种新兴的活载体疫苗，VSV具有以下优点：(1)易于培养：VSV在大多数哺乳动物细胞上都可获得很高滴度，容易大量制备。(2)高效性：在VSV-HA感染的小鼠模型中，10个感染性病毒颗粒诱导的免疫应答和10 5个感染性颗粒引起的免疫应答一样显著。(3)易使用：可通过多种接种途径进行免疫，往往一次接种即可引起强烈的免疫应答反应。(4)产生较强免疫反应：能刺激机体产生强烈的细胞免疫反应和体液免疫反应，还能引起较强的粘膜免疫反应，特别适合于通过粘膜感染的呼吸道病原体的疫苗研制。(5)安全性好：由于VSV病毒完全在细胞质中复制，仅从RNA→RNA，不会整合到宿主细胞的DNA中，最终会被宿主免疫系统清除。而且通过反向遗传操作进行基因组突变和/或修饰，可对VSV病毒进行适当的致弱，使之成为更安全的重组疫苗载体。
本发明制备的VSV重组病毒不同于VSV假病毒，其最大区别在于本发明制备的VSV重组病毒感染细胞后可以复制，而VSV假病毒只能感染细胞却不能复制。另外，两者制备方法及应用范围也不同。虽然二者都可以用于病毒抑制剂的筛选和病毒感染机制的基础研究，但本发明制备的VSV重组病毒还可以用于制备疫苗，而VSV假病毒不能用于制备疫苗。
附图说明
图1为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)的结构示意图。
图2为2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区与SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合示意图。
图3为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)和rVSV-2019-nCoV-SARS在Vero细胞中的复制曲线。对照为VSV重组埃博拉病毒rVSV-EBOV。
图4为S蛋白在重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)和rVSV-2019-nCoV-SARS上的表达情况。
图5为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)和rVSV-2019-nCoV-SARS感染Vero细胞形成的噬斑。对照为VSV重组埃博拉病毒rVSV-EBOV。比例尺代表100μm。
图6为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)单次滴鼻接种食蟹猴刺激产生的中和抗体。
图7为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)接种的食蟹猴，用SARS-CoV-2攻毒后咽拭子病毒载量。
图8为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)接种的食蟹猴，用 SARS-CoV-2攻毒后肛拭子病毒载量。
图9为重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)接种的食蟹猴，用SARS-CoV-2攻毒后第7天肺脏组织病毒载量。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的对照病毒rVSV-EBOV记载于文献“Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.Nature Med.(2005)11,786–790”中，公众可从中国科学院动物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的表达VSV的N蛋白的质粒为将VSV基因组中的N蛋白编码基因序列(序列表中的序列8)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的表达VSV的P蛋白的质粒为将VSV基因组中的P蛋白编码基因序列(序列表中的序列9)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的表达VSV的L蛋白的质粒为将VSV基因组中的L蛋白编码基因序列(序列表中的序列10)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的表达VSV的M蛋白的质粒为将VSV基因组中的M蛋白编码基因序列(序列表中的序列11)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的表达VSV的G蛋白的质粒为将VSV基因组中的G蛋白编码基因序列(序列表中的序列12)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的表达T7RNA聚合酶的质粒为将T7RNA聚合酶编码基因序列(序列表中的序列13)，通过BamHI-EcoRI酶切位点克隆入真核表达质粒pCDNA3.1(+)(北京盛元科萌基因生物科技有限公司)后得到的质粒。
下述实施例中的rVSVΔG载体记载于文献“Single dose of a rVSV-based vaccine elicits complete protection against severe fever with thrombocytopenia syndrome virus.NPJ Vaccines.2019 Jan 25；4:5.”中，公众可从中国科学院动物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。rVSVΔG载体的核苷酸序列为序列表中的序列7，其包括T7启动子序列、缺失糖蛋白G编码基因序列的VSV Indiana毒株全基因组序列和HDV终止子序列。
下述实施例中的Vero细胞是ATCC(American type culture collection)的产品，货号为CCL-81。
实施例1、重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)的制备
一、重组载体VSV-2019-nCoV的制备
1、囊膜蛋白S序列的优化
为了研制2019-nCoV疫苗，将编码完整的2019-nCoV的囊膜蛋白S的序列进行人源化优化，优化后的序列如序列表中的序列1所示。所述完整的2019-nCoV的囊膜蛋白S为2019-nCoV Wuhan-Hu-1毒株(GenBank：NC_045512.2)的囊膜蛋白S(GenBank：YP_009724390.1)，其氨基酸序列如序列表中的序列2所示。
2、重组载体VSV-2019-nCoV的构建
将步骤1中优化后的序列(序列表中的序列1)插入rVSVΔG载体的酶切位点MluI和NotI之间，得到重组载体，将其命名为重组载体VSV-2019-nCoV。
二、重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)的制备
将步骤一中的重组载体VSV-2019-nCoV以及辅助质粒(表达VSV的N蛋白的质粒、表达VSV的P蛋白的质粒、表达VSV的L蛋白的质粒、表达VSV的M蛋白的质粒、表达VSV的G蛋白的质粒、表达T7RNA聚合酶的质粒)共同转染Vero细胞，制备得到重组病毒rVSV-2019-nCoV。重组病毒rVSV-2019-nCoV的结构示意图如图1所示。具体步骤如下：
1、将Vero细胞传代至培养皿中，第二天细胞密度达到70％-80％时，把完全培养基换成含有2％(体积分数)胎牛血清(FBS，Thermo Fisher，货号为10091)的DMEM培养基(Thermo Fisher，货号为SH30243.01B)，得到Vero细胞培养体系。
2、完成步骤1后，将36μL FuGENE 6(Promaga公司，货号为E2692)与Opti-MEM培养基(Thermo Fisher，货号为51985091)室温孵育5分钟，与步骤一中的重组载体VSV-2019-nCoV(1.59μg)、表达VSV的N蛋白的质粒(1.286μg)、表达VSV的P蛋白的质粒(639ng)、表达VSV的L蛋白的质粒(159.9ng)、表达VSV的M蛋白的质粒(159.9ng)、表达VSV的G蛋白的质粒(159.9ng)、表达T7RNA聚合酶的质粒(8.1μg)混匀，室温孵育15分钟，得到孵育后溶液(总体积为600μL)。
3、完成步骤2后，将步骤2中孵育后的溶液加入步骤1中的Vero细胞培养体系中，6小时后换新鲜培养基。转染3天之后收取细胞上清，该上清中含有重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)。用所获得的上清感染新的Vero细胞，实现重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)的扩增。重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为2019-nCoV(SARS-CoV-2)的囊膜蛋白S的编码基因序列后得到的病毒。
实施例2、重组病毒rVSV-2019-nCoV-SARS的制备
一、重组载体VSV-2019-nCoV-SARS的制备
1、嵌合S序列的优化
为了研制2019-nCoV疫苗，将编码由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的序列进行人源化优化，优化后的序列如序列表中的序列3所示。所述2019-nCoV的囊膜蛋白S胞外区是2019-nCoV Wuhan-Hu-1毒株的囊膜蛋白S胞外区(Wuhan-Hu-1毒株的囊膜蛋白S的氨基酸序列的第1-1210位)，SARS-CoV的囊膜蛋白S跨膜区和胞内区是SARS-CoV(Severe acute respiratory syndrome-coronavirus)BJ01毒株(GenBank：AY278488.2)的囊膜蛋白S跨膜区及胞内区(BJ01毒株的囊膜蛋白S的氨基酸序列的第1202-1267位)。所述由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列如序列表中的序列4所示。2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合示意图如图2所示。
2、重组载体VSV-2019-nCoV-SARS的构建
将步骤1中优化后的序列(序列表中的序列3)插入rVSVΔG载体的酶切位点MluI和NotI之间，得到重组载体，并将其命名为重组载体VSV-2019-nCoV-SARS。
二、重组病毒rVSV-2019-nCoV-SARS的制备
按照实施例1步骤二中的方法，将步骤一中的重组载体VSV-2019-nCoV-SARS以及辅助质粒共同转染Vero细胞，制备得到重组病毒rVSV-2019-nCoV-SARS。重组病毒rVSV-2019-nCoV-SARS的结构示意图如图2所示。重组病毒rVSV-2019-nCoV-SARS为将水疱性口炎病毒基因组中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒。
实施例3、重组病毒rVSV-2019-nCoV′的制备
一、重组载体VSV-2019-nCoV′的制备
1、囊膜蛋白S序列的优化
为了研制2019-nCoV疫苗，将编码由2019-nCoV的囊膜蛋白S胞外区和VSV的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的序列进行人源化优化，优化后的序列如序列表中的序列5所示。所述2019-nCoV的囊膜蛋白S胞外区是2019-nCOV Wuhan-Hu-1毒株的囊膜蛋白S胞外区。所述VSV的糖蛋白G跨膜区和胞内区是VSV Indiana毒株的糖蛋白G跨膜区和胞内区。所述由2019-nCoV的囊膜蛋白S胞外区和VSV的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列如序列表中的序列6所示。
2、重组载体VSV-2019-nCoV′的构建
将步骤1中优化后的序列(序列表中的序列5)插入rVSVΔG载体的酶切位点MluI和NotI之间，得到重组载体，将其命名为重组载体VSV-2019-nCoV′。
二、重组病毒rVSV-2019-nCoV′的恢复
按照实施例1步骤二中的方法，将步骤一中的重组载体VSV-2019-nCoV′以及辅助质粒共同转染Vero细胞，制备得到重组病毒rVSV-2019-nCoV′。重组病毒 rVSV-2019-nCoV′为将水疱性口炎病毒基因组中的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒。
实施例4、重组病毒的生长曲线
将Vero细胞按照1:3的比例传代于10cm细胞培养皿中。第二天细胞长到80％左右密度时加入实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)或实施例2制备的重组病毒rVSV-2019-nCoV-SARS或对照病毒rVSV-EBOV，M.O.I.＝0.01，感染2个小时后换液成含有2％(体积分数)FBS的DMEM培养基。感染后每隔12小时取样，一直取到感染后144个小时。通过免疫荧光法测定不同感染时间上清液中的病毒滴度。
免疫荧光法测病毒滴度的操作方法具体如下：将Vero细胞按照每孔1.0万个细胞传代于96孔板中。第二天细胞长到80％-90％左右密度时加入待测病毒上清。对病毒上清进行10倍梯度稀释，即30μl病毒原液与270μl病毒稀释液混匀，以此类推，共稀释6-7个梯度。病毒稀释液为含有2％(体积分数)FBS的DMEM培养基。每个孔中加入100μl病毒液，每个稀释度设3孔重复。将细胞放置于37℃细胞培养箱中，感染2小时后换液为含有20mM NH 4Cl和2％(体积分数)FBS的DMEM培养基，将细胞放置于28℃细胞培养箱中。感染24小时后通过用抗S蛋白的抗体(北京义翘神州科技有限公司，兔抗SARS-CoV S多抗，货号10150-RP01)染色检测病毒滴度(单位FFU/ml)。选择96孔板中的荧光数在10 2左右的孔进行计数，计算方法为：三个孔中的平均荧光数×稀释倍数×10。例如稀释倍数为10 4的孔中荧光为21个，病毒滴度为21×10 4×10＝2.1×10 6FFU/ml。
结果如图3所示。结果显示：感染60小时后，rVSV-2019-nCoV(rVSV-SARS-CoV-2)的扩增快于rVSV-2019-nCoV-SARS。rVSV-2019-nCoV(rVSV-SARS-CoV-2)最高滴度可达1.0×10 6FFU/ml，而rVSV-2019-nCoV-SARS的最高滴度(1.0×10 5FFU/ml)比rVSV-2019-nCoV低10倍。对照病毒rVSV-EBOV滴度在感染后48小时达到峰值，接近1.0×10 7FFU/ml。
实施例5、重组病毒的鉴定
为了鉴定S蛋白在重组病毒颗粒上的表达情况，收集实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)或实施例2制备的重组病毒rVSV-2019-nCoV-SARS感染的Vero细胞上清，采用超速离心方法，39,000转离心3小时，获得病毒沉淀，重悬病毒沉淀后得到浓缩的病毒；然后采用Western Blot(北京义翘神州科技有限公司，兔抗SARS-CoV S多抗，货号为10150-RP01)检测S蛋白在重组病毒上的表达情况。以未转染的Vero细胞作为对照(CON)。
结果如图4所示。结果显示：重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)及rVSV-2019-nCoV-SARS浓缩后均可检测到S蛋白的表达，S蛋白的表达产物为大小约180KD和100KD的两条带。该结果说明S蛋白成功的包装进了重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)和rVSV-2019-nCoV-SARS中。
实施例6、噬斑实验
用10倍梯度稀释的重组病毒(实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)或实施例2制备的重组病毒rVSV-2019-nCoV-SARS)感染Vero细胞，通过噬斑实验检测噬斑形成情况。噬斑实验的具体操作方法参照文献“A VSV-based Zika virus vaccine protects mice from lethal challenge.Sci Rep.(2018)8,11043”中的方法。Vero细胞病毒感染六天后进行结晶紫染色，观察重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)及rVSV-2019-nCoV-SARS感染Vero细胞形成的噬斑。同时以埃博拉重组病毒rVSV-EBOV作为对照，病毒感染两天后进行结晶紫染色观察。
噬斑实验结果如图5所示。结果显示：两种重组病毒(实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)或实施例2制备的重组病毒rVSV-2019-nCoV-SARS)在感染Vero细胞六天后，均可以形成很小的噬斑(图5，见箭头)。而rVSV-EBOV在感染Vero细胞两天后可形成较大噬斑。
实施例7、食蟹猴免疫实验
试验动物与方法：用10 7FFU的实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)通过单次滴鼻的方式接种6只2-6岁的食蟹猴(雌雄各3只，来源于广西雄森灵长类实验动物养殖开发有限公司)。分别取免疫前、免疫后第14天、21天和46天的血清检测中和抗体滴度。
中和抗体滴度检测方法的具体步骤如下：将Vero细胞按照每孔1.0万个细胞传代于96孔板中。第二天细胞长到80％-90％左右时准备试验。首先将血清按照5倍稀释法进行稀释，例如1:10、1:50、1:250、1:1250。同时把rVSV-GFP-SARS-CoV-2病毒稀释至2×10 3FFU/ml，使得最终每孔病毒数为100FFU。在空的96孔板中，每孔加入等量的上述已稀释好的病毒液后，按照相应的稀释比例将血清以相同量逐一加入，然后混匀，混匀后血清稀释倍数相应地变为原始稀释数的二倍。室温孵育30min后，取100μl混合液覆盖到Vero细胞层。2小时后更换含20mM NH 4Cl的培养基，24小时后在荧光显微镜下读取各孔的GFP阳性细胞数。根据Reed Muench方法计算中和抗体滴度(FRNT 50)。计算公式：FRNT 50＝【1/10】 (小于50％细胞感染数稀释倍数的对数+距离比例×稀释系数的对数)。距离比例＝(50％-小于50％细胞感染数阳性率)/(大于50％细胞感染数阳性率-小于50％细胞感染数阳性率)。稀释系数即为倍比稀释梯度。
rVSV-GFP-SARS-CoV-2(rVSV-GFP-2019-nCoV)的制备方法如下：1)在重组载体VSV-2019-nCoV的核苷酸序列中的第62-63位核苷酸之间插入序列表中的序列14所示的GFP基因序列，得到重组载体VSV-GFP-2019-nCoV。2)按照实施例1步骤二中的方法，将重组载体VSV-2019-nCoV替换为重组载体VSV-GFP-2019-nCoV，制备得到重组病毒rVSV-GFP-SARS-CoV-2(rVSV-GFP-2019-nCoV)。
结果如图6所示，单次免疫后14天，6只猴中有3只在接种14天之后中和 抗体滴度达到1:1000以上。其余3只猴子的血清也具有一定的中和活性，滴度为1:327-1:73。免疫后46天，血清的中和活性有所下降但仍维持相对较高的中和抗体滴度。这些结果说明重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)在猴中能刺激高水平的SARS-CoV-2中和抗体。
实施例8、攻毒实验
试验动物与方法：2-6岁的食蟹猴(雄性，来源于北京协尔鑫生物资源研究所有限责任公司)分为2组(实验组和对照组)，每组2只。实验组(免疫组)用5×10 6FFU的实施例1制备的重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)通过单次滴鼻的方式接种，对照组接种等体积的PBS。免疫后6个月在中国科学院昆明动物研究所P3实验室开展SARS-CoV-2攻毒实验，攻毒剂量为1.0×10 7TCID 50，攻毒方式为40％滴鼻(20％/鼻孔)+60％气管滴种。分别于攻毒前第0天及攻毒后第1天、3天、5天、7天采咽拭子样本和肛拭子样本。攻毒后第7天对动物实施安乐死，取肺脏组织样本(7个肺叶)。
采用qRT-PCR方法使用One Step TB
Figure PCTCN2020139866-appb-000001
PrimeScript TM RT-PCR Kit(货号：RR066A，厂家：TaKaRa)试剂盒检测各样本中的病毒载量。引物信息如下：
N-F：CGGAGGATTGACGACTAATGC；
N-R：ACCATCCGAGCCATTCGA。
反应体系如表1所示。
表1反应体系
试剂 体积
One Step TB Green RT-PCR BufferⅢ 5μl
TaKaRa Ex Taq HS(5U/μl) 0.2μl
PrimeScript RT enzyme MixⅡ 0.2μl
PCR Forward Primer(10μM) 0.4μl
PCR Reverse Primer(10μM) 0.4μl
Total RNA 1μl
RNase Free dH 2O 2.8μl
反应程序如下所示：
(1)反转录反应：42℃ 5min，95℃ 10sec。
(2)PCR反应：循环数：40，95℃ 5sec，60℃ 30sec。
试验结果如图7-9所示，实验组攻毒后咽拭子的病毒载量比对照组低1-2个数量级(图7)。肛拭子的结果也类似，攻毒后实验组第1天、5天和7天的病毒载量比对照组降低2个数量级(图8)。攻毒后第7天，实验组相比对照组肺部组织中的病毒载量显著下降(图9)，实验组在肺中基本检测不到SARS-CoV-2病毒RNA。这些结果说明重组病毒rVSV-2019-nCoV(rVSV-SARS-CoV-2)免疫能 有效保护SARS-CoV-2感染食蟹猴。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
工业应用
本发明以水疱性口炎病毒VSV为载体，构建了三个复制型的重组病毒：一、将VSV病毒的糖蛋白G的编码基因序列替换为2019-nCoV(SARS-CoV-2)的囊膜蛋白S的编码基因序列；二、将VSV病毒的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列；三、将VSV病毒的糖蛋白G的编码基因序列替换为由2019-nCoV(SARS-CoV-2)的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列。通过实验证明：三种重组病毒均可在Vero细胞中复制，不具备2019-nCoV(SARS-CoV-2)的致病性，但可以模拟2019-nCoV入侵细胞的过程，并能激发机体产生针对2019-nCoV(SARS-CoV-2)的免疫反应。病毒的入侵以及中和抗体靶点主要是S蛋白的胞外区，因此重组病毒的感染机制和诱发的免疫反应理论上与2019-nCoV(SARS-CoV-2)非常相似。该重组病毒不仅可以开发成疫苗，还可以作为研究2019-nCoV(SARS-CoV-2)感染过程的理想工具，对筛选抑制病毒入侵的抑制剂(如抗体、血清、小肽和小分子等)和新型冠状病毒疫苗的研制具有重要意义。

Claims (18)

  1. 一种重组病毒，为将水疱性口炎病毒的糖蛋白G替换为囊膜蛋白S后得到的病毒；所述囊膜蛋白S包含2019-nCoV的囊膜蛋白S胞外区或其部分序列；
    所述2019-nCoV的囊膜蛋白S胞外区的氨基酸序列为a)或b)或c)：
    a)氨基酸序列是序列表中的序列2第1-1210位所示的蛋白质；
    b)将序列表中的序列2第1-1210位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    c)与序列表中的序列2第1-1210位所示的氨基酸序列具有85％或85％以上的同源性且具有相同功能的蛋白质。
  2. 根据权利要求1所述的重组病毒，其特征在于：所述重组病毒为如下1)或2)或3)：
    1)将水疱性口炎病毒的糖蛋白G替换为2019-nCoV的囊膜蛋白S后得到的病毒；
    所述2019-nCoV的囊膜蛋白S的氨基酸序列为序列表中的序列2；
    2)将水疱性口炎病毒的糖蛋白G替换为由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S后得到的病毒；
    所述由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列为序列表中的序列4；
    3)将水疱性口炎病毒的糖蛋白G替换为由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S后得到的病毒；
    所述由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的氨基酸序列为序列表中的序列6。
  3. 根据权利要求2所述的重组病毒，其特征在于：所述1)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为2019-nCoV的囊膜蛋白S的编码基因序列后得到的病毒；
    或，所述2)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒；
    或，所述3)中，所述重组病毒为将水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的病毒。
  4. 根据权利要求3所述的重组病毒，其特征在于：所述1)中，所述2019-nCoV的囊膜蛋白S的编码基因序列为序列表中的序列1；
    或，所述2)中，所述由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的 序列3；
    或，所述3)中，所述由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的序列5。
  5. 根据权利要求1-4任一所述的重组病毒，其特征在于：所述2019-nCoV为2019-nCoV Wuhan-Hu-1毒株；
    或，所述SARS-CoV为SARS-CoV BJ01毒株；
    或，所述水疱性口炎病毒为水疱性口炎病毒Indiana毒株。
  6. 一种重组病毒，是将重组病毒载体转染病毒包装细胞，然后进行细胞培养后得到的；所述重组病毒载体为如下A)或B)或C)或D)：
    A)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为2019-nCoV的囊膜蛋白S的编码基因序列后得到的载体；
    B)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的载体；
    C)将水疱性口炎病毒载体中的水疱性口炎病毒基因组序列中的糖蛋白G的编码基因序列替换为由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列后得到的载体；
    D)在A)或B)或C)所述的重组病毒载体中插入报告基因后得到的载体。
  7. 根据权利要求6所述的重组病毒，其特征在于：所述A)中，所述2019-nCoV的囊膜蛋白S的编码基因序列为序列表中的序列1；
    或，所述B)中，所述由2019-nCoV的囊膜蛋白S胞外区和SARS-CoV的囊膜蛋白S跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的序列3；
    或，所述C)中，所述由2019-nCoV的囊膜蛋白S胞外区和水疱性口炎病毒的糖蛋白G跨膜区和胞内区嵌合而成的囊膜蛋白S的编码基因序列为序列表中的序列5；
    或，所述D)中，所述报告基因为GFP基因。
  8. 根据权利要求6或7所述的重组病毒，其特征在于：所述2019-nCoV为2019-nCoV Wuhan-Hu-1毒株；
    或，所述SARS-CoV为SARS-CoV BJ01毒株；
    或，所述水疱性口炎病毒为水疱性口炎病毒Indiana毒株。
  9. 权利要求6中所述的重组病毒载体。
  10. 权利要求1-8任一所述的重组病毒或权利要求9所述的重组病毒载体在如下X1)-X3)中任一种中的应用：
    X1)制备新型冠状病毒疫苗；
    X2)制备预防和/或治疗新型冠状病毒导致的疾病的产品；
    X3)筛选新型冠状病毒入侵抑制剂。
  11. 根据权利要求10所述的应用，其特征在于：所述新型冠状病毒为2019-nCoV。
  12. 根据权利要求10所述的应用，其特征在于：所述新型冠状病毒导致的疾病为新型冠状病毒导致的COVID-19。
  13. 一种用于预防和/或治疗新型冠状病毒导致的疾病的产品，其活性成分为权利要求1-8任一所述的重组病毒或权利要求9所述的重组病毒载体。
  14. 根据权利要求13所述的产品，其特征在于：所述新型冠状病毒为2019-nCoV。
  15. 根据权利要求13所述的产品，其特征在于：所述新型冠状病毒导致的疾病为新型冠状病毒导致的COVID-19。
  16. 一种预防和/或治疗新型冠状病毒导致的疾病的方法，包括如下步骤：给受体动物施用权利要求13所述的产品进行治疗或/和预防新型冠状病毒导致的疾病。
  17. 根据权利要求16所述的方法，其特征在于：所述新型冠状病毒为2019-nCoV。
  18. 根据权利要求16所述的方法，其特征在于：所述新型冠状病毒导致的疾病为新型冠状病毒导致的COVID-19。
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