WO2021155846A1 - 一种新型冠状病毒肺炎双价疫苗的制备方法 - Google Patents

一种新型冠状病毒肺炎双价疫苗的制备方法 Download PDF

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WO2021155846A1
WO2021155846A1 PCT/CN2021/075588 CN2021075588W WO2021155846A1 WO 2021155846 A1 WO2021155846 A1 WO 2021155846A1 CN 2021075588 W CN2021075588 W CN 2021075588W WO 2021155846 A1 WO2021155846 A1 WO 2021155846A1
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ncov2019
gene
shrna
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cells
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翁炳焕
李兰娟
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翁炳焕
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    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to a preparation method of a novel coronavirus pneumonia bivalent vaccine used in the field of infectious disease prevention and treatment, and belongs to the technical field of new vaccine preparation methods.
  • Human coronaviruses can cause up to 30% of colds.
  • Animal coronaviruses such as porcine gastroenteritis coronavirus (TGEV), mouse hepatitis coronavirus (MHV), avian infectious bronchitis coronavirus (IBV), etc. can infect the respiratory tract, gastrointestinal tract, nervous system and liver of the corresponding host , Leading to corresponding symptoms.
  • the envelope of the coronavirus is petal-shaped protrusions, making the coronavirus look like a crown (Latin, corona), and its nucleocapsid is a variable long spiral.
  • the diameter of the virus particle of the coronavirus is 60nm ⁇ 140nm, spherical, the virus genome is 27kb ⁇ 32kb single-stranded sense RNA, which is the largest of all RNA virus genomes.
  • a variant of the coronavirus in 2003 caused the outbreak of severe acute respiratory syndrome (SARS).
  • the size of the SARS-CoV genome is 27 ⁇ 3lkb, with 14 open reading frames (ORF) and 1 s2m. Motif (s2m motif).
  • nCoV-2019 [Corrected according to Rule 26 01.03.2021] [Corrected in accordance with Rule 9.2 01.03.2021]
  • the World Health Organization named the coronavirus outbreak a new type of coronavirus, or "nCoV-2019". It has been reported that the whole genome of nCoV-2019 was basically the same from 5 patients. Among them, 79.5% of the sequence was consistent with SARS-CoV. By comparing its conserved 7 non-structural proteins, it was found that nCoV-2019 belongs to SARSr- CoV enveloped RNA virus.
  • nCoV-2019 contains 5'untranslated region (UTR), replicase complex (orf1ab), S gene, E gene, M gene, N gene, 3'UTR and several unknown non-structural open reading frames.
  • UTR 5'untranslated region
  • orf1ab replicase complex
  • S gene S gene
  • E gene E gene
  • M gene M gene
  • N gene 3'UTR
  • PCR genetic diagnosis
  • N nucleoprotein
  • Target 1 (ORF1ab): forward primer (F): CCCTGTGGGTTTTACACTTAA; reverse primer (R): ACGATTGTGCATCAGCTGA; probe (P): 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'.
  • Target 2 (N): Forward primer (F): GGGGAACTTCTCCTGCTAGAAT; Reverse primer (R): CAGACATTTTGCTCTCAAGCTG; Fluorescent probe (P): 5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ1-3'.
  • Vaccines can be divided into live attenuated vaccines, inactivated vaccines, subunit vaccines, DNA vaccines, recombinant vector vaccines, virus particle-like vaccines and peptide vaccines.
  • Traditional vaccines are mostly killed vaccines, live attenuated vaccines or recombinant subunit vaccines, while new vaccines are viral nucleic acids encoding antigen proteins or cellular vaccines that can stimulate specific immune responses.
  • the existing traditional vaccines mainly rely on the antigen proteins of pathogens to stimulate the body to produce protective antibodies.
  • the existing new vaccines can also stimulate specific cellular immune responses.
  • live vaccines are not safe and difficult to prepare. Inactivated vaccines may recombine with wild strains and should only be used as emergency reserves.
  • multi-linked vaccines such as DPT vaccine.
  • One vaccination can prevent diphtheria, tetanus and pertussis at the same time.
  • vaccines against different serotypes of the same pathogen are often mixed together, called bivalent or multivalent vaccines, such as multivalent HPV vaccines. But can it be possible to invent a multivalent vaccine against new coronavirus pneumonia that has nothing to do with antigen proteins, antibodies or cellular immunity, which is different from the existing vaccines?
  • RNA interference refers to a small RNA that can pair and bind to a target gene, specifically removing or shutting down the expression of the bound gene. That is, RNAi refers to the high-efficiency and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). It is reported in the literature that in 1995, Dr. Guo Shu from Fudan University injected antisense RNA into the body of C. ele-gans at Cornell University in an attempt to block the expression of par-1 gene.
  • dsRNA double-stranded RNA
  • RNA interference RNA interference
  • the silencing complex degrades mRNA that has a sequence homologous to the dsRNA, resulting in gene silencing at the post-transcriptional level and losing the ability to express proteins or polypeptides.
  • foreign genes such as viral genes, artificially transferred genes, and transposons are randomly integrated into the host cell genome, and when host cells are used for transcription, some dsRNA is often produced. The host cell responds quickly to these dsRNAs.
  • the endonuclease (Dicer) in the cytoplasm cuts the dsRNA into multiple small fragments of siRNA (approximately 21 to 23 bp) with a specific length and structure. The siRNA unwinds the RNA in the cell.
  • RNA-induced silencing complex RNA-induced silencing complex
  • RISC specifically binds to homologous regions of mRNA expressed by exogenous genes. RISC has the function of nuclease and cuts mRNA at the binding site. The cleavage site is the two ends that are complementary to the antisense strand of siRNA.
  • siRNA can not only guide RISC to cut homologous single-stranded mRNA, but also can be used as a primer to bind to target RNA and synthesize more new dsRNA under the action of RNA-dependent RNA polymerase (RdRP).
  • RdRP RNA-dependent RNA polymerase
  • the newly synthesized dsRNA is then Dicer
  • the cleavage produces a large amount of secondary siRNA, which further amplifies the effect of RNAi, and finally the target mRNA is completely degraded.
  • the effect of RNAi is similar to gene knockout, but it is much easier and safer than gene knockout. Because it is not really knocking out genes, it can avoid serious consequences caused by gene knockout. This provides a feasible mechanism for the development of a novel coronavirus dsRNA vaccine based on RNA interference.
  • RNAi Xia et a1.2004
  • the classic adenovirus packaging system includes HEK293 cells, a shuttle plasmid carrying foreign genes, and a skeleton plasmid containing the adenovirus genome.
  • Shuttle vectors generally have eukaryotic promoters and multiple cloning sites downstream of them. The multiple cloning sites into which foreign genes are inserted are called foreign gene expression cassettes.
  • HEK293 cells provide the E1 protein necessary for virus replication, so that the foreign genes in the shuttle vector are transferred to the backbone plasmid, so that the recombinant adenovirus particles are packaged.
  • the most clearly studied human adenoviruses include type 2 (Ad2) and type 5 (Ad5).
  • Ad5 The adenovirus vector developed by Ad5 has been commercialized in the field of disease treatment and scientific research. It has a broad spectrum of infection, non-integration, It is safe, easy to handle, high in vitro proliferation titer, and large non-essential fragments in the genome. It is currently the most widely used viral vector.
  • the immunogenicity is enhanced, and it may also act as an immune adjuvant for the immune effect.
  • respiratory epithelial cells are rich in adenovirus receptors (CAR).
  • CAR adenovirus receptors
  • the natural host of Ad5 adenovirus is human respiratory epithelial cells. Therefore, replication-deficient recombinant adenovirus vectors can efficiently infect respiratory epithelial cells. Immunization (spray or nasal drops) for the upper respiratory tract provides the possibility.
  • the immunogen expressed by the adenoviral vector basically maintains the natural conformation of the immunogen after being expressed, processed, folded, modified, and presented by the host cell, which is convenient to simulate the immune response in the natural state, and the new coronavirus is mainly infected through the respiratory tract.
  • Adenovirus vector vaccines can be conveniently made into oral or upper respiratory tract sprays. Aerosol inhalation is not only convenient, but also greatly reduces the cost of use. If adenovirus vector vaccines can be used to induce effective mucosal immune responses, it will have broad application prospects .
  • Atomized inhalation vaccination is to put drugs or vaccines into a specific aerosol generator, and inhale the drugs or vaccines into the respiratory tract to stimulate the respiratory tract mucosa to produce mucosal immunity and further enter the alveoli to take effect. Because the lungs have a huge alveolar surface area, abundant capillaries, and a very small transit distance, the inoculated drugs or vaccines can quickly exert their effects after being absorbed by the lungs. Foreign research on aerosol inhalation therapy can be traced back to the 1950s.
  • Aerosol immunization direct inoculation of the vaccine to the mucosa of the respiratory tract, not only provides a physiological and immunological advantage, but this route of vaccination also provides potential logistical advantages because it does not require well-trained personnel. Therefore, the aerosol immunization method is an immunization method worthy of steady promotion. In modern times, people have paid great attention to aerosol inhalation therapy, and more and more aerosol immune drugs have been developed.
  • Compressed air nebulizers are now widely used in clinical practice, and can be used for nebulized inhalation treatment and vaccination of measles, influenza, and Bacille Calmette-Guerin (BCG) vaccines.
  • BCG Bacille Calmette-Guerin
  • the purpose of the present invention is to solve the deficiencies in the literature reports and application of methods for preparing dsRNA vaccines based on the conservative genome of pathogens and generating gene silencing complexes based on dsRNA vaccination so that pathogen homologous mRNA is degraded in the prior art. , Provide a method for preparing a new coronavirus pneumonia bivalent vaccine that is different from the traditional technology.
  • the preparation method of Ad-nCoV2019dsRNA vaccine is to amplify the target interference gene shRNA sequence of nCoV2019, and the amplified shRNA sequence and the empty interference vector pSilencer4.1.CMV.neo are digested with BamH I and Hind III to construct interference
  • the vector pSilencer-shRNA, the interference vector is amplified by competent E.
  • coli DH5a and the shRNA is inserted correctly after being inserted into the empty shuttle vector pDC312 by Hind 11 and EcoR I to construct the shuttle vector pDC312-shRNA, the shuttle vector and adenovirus
  • the backbone plasmid pBHGloxAEl was co-transfected into HEK293 cells, and the recombinant adenovirus Ad-shRNA was obtained by homologous recombination in the cells, and then amplified by HEK293 multiple times to prepare the Ad-nCoV2019dsRNA vaccine of the recombinant adenovirus vector.
  • the targeted interference gene shRNA refers to a sequence complementary to the siRNA sequence with a length of 19 nt, and currently includes ORF1ab, 3'UTR, S, E, M, and N gene sequences.
  • the targeted interfering gene shRNA is a DNA template for expressing a hairpin structure, which is composed of two mostly complementary single-stranded DNAs, which can form sticky ends with BamH I and Hind III enzyme cleavage sites after annealing and complementation. DNA double-stranded.
  • the Ad-nCoV2019VdsRNA vaccine refers to a recombinant adenovirus vector carrying shRNA that enters the respiratory tract or digestive tract epithelial cells.
  • the shRNA can synthesize dsRNA in the cell, and the synthesized dsRNA can specifically degrade mRNA with homologous sequences. , So that it loses the ability to express proteins or polypeptides;
  • the Ad refers to a replication-deficient recombinant adenovirus vector;
  • the nCoV2019 refers to a conserved gene or a targeted interference sequence of a functional gene of the new coronavirus.
  • Ad-nCoV2019 DNA vaccine amplify the antibody expression gene of nCoV2019, the amplified sequence and the adenovirus shuttle plasmid pShuttle are digested with XbaI and KpnI to construct the recombinant adenovirus shuttle plasmid pShuttle-nCoV2019, the shuttle plasmid After being amplified by competent E.
  • the nCoV insertion is correct and co-transfected with the adenovirus backbone plasmid pAd-nCoV2019 into HEK293 cells, and homologous recombination is carried out in the cells to obtain the recombinant adenovirus Ad-nCoV2019
  • the recombinant adenovirus is amplified by HEK293 cells for multiple times to prepare Ad-nCoV2019 DNA vaccine of recombinant adenovirus vector.
  • the antibody expression gene refers to a gene that can express mRNA, mRNA express protein, and protein stimulates the body to produce antibodies.
  • nCoV2019 refers to the conserved gene and functional gene sequence of the new coronavirus; the conserved gene and functional gene currently refer to the ORF1ab, 3'UTR, S, E, M, N gene sequence.
  • the pShuttle-nCoV2019 refers to a recombinant adenovirus shuttle plasmid containing nCoV2019;
  • the pAd-nCoV2019 refers to an adenovirus backbone plasmid containing nCoV2019;
  • the Ad-nCoV2019 refers to a replication-deficient recombinant adenovirus vector containing nCoV2019;
  • the Ad refers to a replication-deficient recombinant adenovirus vector.
  • Ad-nCoV2019 DNA vaccine refers to a recombinant adenovirus vector carrying antibody expression genes after entering the respiratory tract or digestive tract epithelial cells, the antibody expression genes therein can express mRNA, mRNA expression protein, and the protein stimulates the body to produce antibodies, and the antibodies neutralize The new type of coronavirus has lost its pathogenicity.
  • the bivalent vaccine of the present invention has the advantages of quick effect, good effect, safe use, convenient inoculation and the like.
  • the bivalent vaccine of the present invention is prepared by mixing Ad-nCoV dsRNA vaccine and Ad-nCoV 2019 DNA vaccine. After spray inoculation, the recombinant adenovirus vector is introduced into the respiratory tract infected cells, and the Ad-nCoV 2019 dsRNA vaccine immediately produces dsRNA through its shRNA.
  • the multivalent vaccine of the present invention has its own advantages and plays a complementary and lacking synergistic effect, especially for the nCoV2019dsRNA component.
  • the immune mechanism is different from the antigen-antibody reaction of the prior art to produce outstanding effects of emergency prevention, suggesting that in the preparation of new coronavirus vaccines, one target gene of one strain should be inserted into one expression vector, or one strain of virus should be inserted into one expression vector. Insert several target genes of several strains into one expression vector, or insert several target genes of several strains into one expression vector, prepare expression vectors for each strain accordingly, and then combine them into one polytype or multiple strains Multi-type Ad-nCoV2019dsRNA or/and Ad-nCoV2019DNA bivalent or multivalent vaccine for application.
  • the vectors for delivering the bivalent vaccine of the present invention are all replication-deficient adenoviruses, which have the advantages of safe use, stable expression, and easy operation. Because they can efficiently infect respiratory epithelial cells, they can provide immune spray vaccination through the oral cavity and upper respiratory tract. According to the theory, it is more suitable for the prevention of new coronavirus pneumonia.
  • telomere sequence (ORF1ab, S, E, M, N)
  • construct the expression vector pSilencer-shRNA pSilencer-ORF1ab/S/E/M/N
  • transfer the shRNA expression cassette to construct the adenovirus shuttle Plasmid pDC312-shRNA
  • co-transfected HEK293 cells with the adenoviral backbone plasmid pBHGloxAEl, obtained recombinant adenovirus Ad-shRNA by homologous recombination, and was amplified and purified by HEK293 cells for multiple times to prepare an immune enhancer as a medium Anti-new coronary pneumonia dsRNA vaccine, after spray vaccination, recombinant adenoviral vector (Ad-shRNA) introduces shRNA into respiratory epithelial cells and synthesizes dsRNA in the cells, and then specifically induces gene silencing or RNA interference response of nCoV mRNA degradation
  • This embodiment relates to a preparation method of nCoV2019 vaccine, involving but not limited to nCoV2019 ORF1ab, S, E, M, N genes and their primers, involving but not limited to the same experimental method, shRNA expression vector pSilencer, pDC312, pBHGloxAEl, pAd , PEGFP, HEK293 and other experimental materials, and also involve in vitro synthesis of nCoV2019-RNA interference therapeutic products.
  • RNAi target sites and the design of siRNA expression templates According to the sequenced new coronavirus ORF1ab, 3'UTR, S, E, M, N gene sequences, using Ambion's shRNA online design software ( http://www.ambion.com/techlib/misc/siRNAtools.html) Obtained multiple siRNA candidate sequences with a length of 19nt. According to the Tm value of RNA binding and specific comparison results, the siRNA sequence is preferred and the complementary one The genomic region was selected as the target site of interference, combined with the polyclonal restriction site of the pSilencer4.1.CMV.neo interference vector to design shRNA templates that can express the hairpin structure.
  • Each template consists of two 55bp that are mostly complementary It is composed of single-stranded DNA, which can be annealed and complemented to form a double-stranded DNA with sticky ends of BamH I and Hind III restriction sites, which is used to connect with the linearization interference vector pSilencer4.1.CMV.neo.
  • shRNA expression vector the above-mentioned oligonucleotide chain is annealed and complemented with the linearized shRNA expression vector pSilencer4.1.CMV.neo to be connected to construct a shRNA expression plasmid and transformed into competent cell DH5a.
  • the specific method of annealing complementary and carrier connection is as follows:
  • Annealing of oligo DNA dissolve the synthesized oligonucleotides to 100 ⁇ M with ddH 2 0, take 5 ⁇ L of each pair of complementary single strands to mix, and then place the 6 kinds of oligoDNA mixtures in a water bath at 98°C and heat for 5 minutes, then turn off Switch the water bath to cool naturally to room temperature to form double-stranded DNA.
  • the annealing system is as follows: 100 ⁇ M positive strand oligonucleotide: 5 ⁇ L; 100 ⁇ M negative strand oligonucleotide: 5 ⁇ L; 10xPCR buffer: 2 ⁇ L; ddH 2 0: 8 ⁇ L ; The total volume is 20 ⁇ L.
  • Vector connection further dilute the synthesized double-stranded DNA to 10nM and connect for 30min at 16°C.
  • the enzyme ligation system is as follows: pSilencer4.1.CMV.neo: 4 ⁇ L; 5xligation buffer: 2 ⁇ L; ds oligo (10nM): 4 ⁇ L; T4 DNA ligase (1U/ ⁇ L): 1 ⁇ L; ddH 2 0: 9 ⁇ L; total volume: 20 ⁇ L .
  • the present invention takes the ORF1ab, 3'UTR, S, E, M, and N genes of nCoV2019 as examples, and refers to the online primer design software, according to different vectors, restriction sites, and identification needs, and entrusts the biological company to design the institute as required
  • the 5'end of the upstream primer adds a start code to clone the amplified product into pEGFP-N1
  • the 5'end of the primer adds a homology arm for homologous recombination with the vector.
  • 3Linearization of pEGFP-N1 vector resuscitate the DH5a strain containing pEGFP-N1 plasmid, extract the plasmid according to the kit or literature, determine the concentration, and perform digestion.
  • the digestion system is as follows: 10xM Buffer: 5 ⁇ L; plasmid DNA: 20 ⁇ L; Hind III: 2 ⁇ L; ddH 2 0: 23 ⁇ L; total volume: 50 ⁇ L.
  • the mixture was mixed and placed in a 37°C water bath for digestion for 2 hours, identified by 0.8% agarose gel electrophoresis, and the linearized vector was recovered for use.
  • step (2) 3 times and then fully suspend the cells, and pipette the cells into a single.
  • test method 4 Take an appropriate number of cells to test on the machine.
  • the test method adopts Guavaexpress Plus method, and the number of passing cells per second is kept below 800.
  • the glass plate is washed and dried and then assembled to ensure a good airtightness. Prepare separation gel first.
  • Concentrated gel electrophoresis uses a voltage of 80V. After the sample enters the separation gel, adjust the voltage to 120V until the end of electrophoresis (bromophenol blue enters the electrophoresis solution).
  • Transfer membrane Put the unstained gel into the transfer buffer solution, and cut the filter paper and PVDF membrane to the size of the gel, but slightly smaller. Soak the PVDF membrane in anhydrous methanol for a short time and then put it into the transfer buffer solution. The filter paper is directly soaked in the transfer buffer solution.
  • Combined transfer membrane device (from bottom to top): filter paper (3 layers), PVDF membrane (1 layer), gel, filter paper (3 layers), after assembly, put it into the electrophoresis fixture and put it in the transfer tank , 150mA constant current transfer for 120min.
  • Primary antibody binding Put the membrane into 10 mL of primary antibody (anti-GFP monoclonal antibody, anti-IB-actin monoclonal antibody) diluted with 2.5 skimmed milk, and shake for 1 hour on a shaker.
  • Secondary antibody binding Put the PVDF membrane into the secondary antibody diluted 1:800, and shake it for 1 hour on a shaker.
  • the target gene and the B-actin internal reference gene copy number are converted from the CT value according to the standard curve equation.
  • shRNA interference vectors Routinely extract shRNA interference vectors (pSilencer-ORF1ab, pSilencer-3'UTR, pSilencer-S, pSilencer-E, pSilencer-M, pSilencer-N) and pDC312 or pShuttle adenovirus shuttle vector, while using restriction endonuclease Hind lII Enzyme digestion with EcoR I, cut out the shRNA expression cassette in the shRNA interference vector, and linearize the pDC312 vector.
  • the digestion system is as follows: 10xM Buffer: 5uL; plasmid DNA: 20uL; Hind III: 2uL; EcoRI: 2uL; ddH 2 0: 2luL; total volume 50uL.
  • the linearized vector and shRNA expression cassette were recovered and connected according to the following system: pDC312/pShuttle: 2uL; shRNA expression cassette: 4uL; 5xligation buffer: 4uL; T4 DNA ligase (1U/uL): 1uL; ddH 2 0: 9uL; total The volume is 20uL.
  • the ligation product was transformed into E. coli competent cell DH5a. After overnight culture, 4 clones were picked for sequencing and identification. After confirming that the inserted fragment was correct, the recombinant vector was named pDC312-nCoV2019(pDC312-ORF1ab, pDC312-3'UTR, pDC312 -S, pDC312-E, pDC312-M, pDC312-N), and save for future use.
  • Ad stands for recombinant adenovirus vector. Like 2019nCoV or nCoV2019, it represents the conservative or functional gene sites of the new coronavirus that can be used for targeted interference in Ad, such as ORF1ab, 3'UTR, S, E, M, N.
  • the present invention uses Admax's double plasmid transfection system to package recombinant adenovirus, and routinely transfect HEK293 cells.
  • the transfection ratio between the adenovirus backbone vector pBHGloxAEl and the adenovirus recombinant shuttle vector (pDC312-2019nCoV) is 1:3 by mass.
  • the harvested first-generation recombinant adenovirus (Ad-2019nCoV) was repeatedly infected with HEK293 cells to proliferate the virus, and the third-generation virus was extracted using a DNA extraction kit to extract DNA and perform PCR identification.
  • the titer of recombinant adenovirus was determined using a rapid adenovirus infectious titer (TCIDso) test kit. Infect 293 cells through a series of diluted virus samples, each dilution infects 8 cell wells. After about 10 days of culture, the presence of virus-induced cytopathic changes in each cell well is judged under a microscope, and the presence of phagocytosis The number of holes in the spots calculates the titer of adenovirus.
  • TCIDso rapid adenovirus infectious titer
  • Ad-nCoV2019 was inoculated into HEK293 cells cultured in 12-well plates at 10, 50, 100, 250, and 500 MOIs.
  • the control virus Ad-CMV of the CMV promoter that did not include the shRNA template sequence was inoculated as a control virus.
  • the 2019nCoV strain was inoculated (clinically isolated), and cells inoculated only with 2019nCoV were used as positive control cells. Cells were collected for detection 48h after 2019nCoV infection.
  • the relative expression levels of 2019nCoV genes (ORF1ab, 3'UTR, S, E, M, N) were detected on each group of cells to evaluate the interference effect.
  • Wistar rats are cultured in SPF-level animal laboratories, half male and half male, 6-8 weeks old, weighing 110+10g, and recording the animal production certificate number.
  • Ad-nCoV2019 (dsRNA) preparation Combining eukaryotic fermentation technology and adenovirus column chromatography purification technology, Ad-nCoV2019 (dsRNA) self-amplification, CsCl two centrifugal purification, titer of 5x10 10 pfu/ml.
  • Ad-nCoV2019 (dsRNA) intranasal instillation control group empty carrier control group
  • dsRNA intranasal instillation control group
  • pentobarbital intraperitoneal injection 5 minutes later, intranasal instillation, 0.5ml/head, dose 1 ⁇ 10 7 pfu/ dose/rat.
  • Ad-nCoV2019 (dsRNA) nasal drip group same anesthesia as above, nasal drip after 5 minutes, the dose is 1 ⁇ 10 7 pfu/dose/rat.
  • Ad-nCoV2019 (dsRNA) tail vein injection control group empty carrier control group
  • the dose is 1 ⁇ 10 7 pfu/dose/rat.
  • Ad-nCoV2019 (dsRNA) tail vein injection group administered by tail vein injection at a dose of 1 ⁇ 10 7 pfu/dose/rat.
  • Blank control group, PBS, 0.5ml/mouse was injected into the tail vein.
  • Each of the above groups has 10 animals in each group.
  • h.nCoV2019 diagnosis refer to the current commercially available PCR kit for detection and diagnosis.
  • Ad-nCoV2019 (dsRNA) can effectively interfere with the replication of nCoV2019, then Ad-nCoV2019 (dsRNA) will be named "new coronavirus dsRNA vaccine” or "nCoV2019-dsRNA vaccine”
  • Bottle-Top Filter Unit into the ultra-clean table, open the lid, and place the Pre-filter Disc on the 0.45micron filter.
  • the vacuum pump is set to vacuum off, and 10 ml of 100 mM Tris-HCI (pH 7.4) is added to the Pre-Filter to make the Pre-Filter and 0.45 micron filter close to each other.
  • the vacuum pump is set to vacuum on. When the collection vessel is full, disconnect the vacuum pump and transfer the filtrate to the sterilization bottle. If the Bottle-TopFilter Unit is blocked, change to another Bottle-Top FiRerUnit.
  • T Turn on the vacuum pump slowly, and use a water stop clip to control the flow rate at about 20ml/min. After filtering, clamp the TubingAssembly tube and remove the vacuum pump tube.
  • adenovirus Connect a 20ml syringe to the inlet of BD Adeno-X Purification Filter. At the same time, connect the Tubing Assembly to the outlet end, and then put it into a centrifuge tube containing 20m1 1 ⁇ Elution Buffer. Extract l ⁇ Elution Buffer, the purified adenovirus is in the syringe, and transfer it to a 50ml centrifuge tube.
  • Recombinant adenovirus (nCoV2019-Ad-dsRNA vaccine) preservation store at -70°C for later use.
  • the prepared recombinant adenovirus (Ad) carries the new coronavirus (nCoV2019) targeted interfering gene (ORF1ab, 3'UTR, S, E, M, N) shRNA sequence, which is called Ad-nCoV dsRNA vaccine, and the shRNA can Produce dsRNA, which in turn produces RNA interference.
  • the new coronavirus nCoV that is, the antibody-producing gene
  • Ad-ORF1ab/N a recombinant adenovirus Ad-nCoV (Ad-ORF1ab/N) that is non-toxic to humans, easily enters respiratory epithelial cells and can express nCoV-mRNA in the cell, and then prepared It can stimulate the body to produce anti-nCoV antibodies to produce specific immune effects of respiratory tract spray vaccination DNA vaccine, which is used for emergency prevention of new coronavirus pneumonia.
  • This embodiment relates to a preparation method of nCoV vaccine, involving but not limited to nCoV ORF1ab, S, E, M, N genes and their primers, involving but not limited to the same experimental methods, cloning vectors and other experimental materials, involving but not limited to Used to synthesize nCoV protein antigen or antibody in vivo.
  • Primer design At present, the Chinese Center for Disease Control and Prevention recommends the selection of primers for the open reading frame 1ab (open reading frame, ORF1ab) and nucleoprotein (N) gene region of nCoV-2019.
  • ORF1ab open reading frame
  • N nucleoprotein
  • the open reading frame ORF1ab R: CCCTGTGGGTTTTACACTTAA
  • F ACGATTGTGCATCAGCTGA
  • Nucleocapsid protein N F: GGGGAACTTCTCCTGCTAGAAT
  • R CAGACATTTTGCTCTCAAGCTG.
  • Vector preparation Transform pShuttle plasmid into DH5II bacteria, pick positive clones, amplify a small amount with LB solution containing ampicillin, extract pShuttle plasmid with PLASMID MINIPREP KIT (QIAGEN), and use a large amount of Xba I after the restriction enzyme digestion is correct. +Kpn I digestion, separate large fragments by gel electrophoresis, recover vector fragments with GELEXTRACTION (QIAGEN), and dissolve in sterilized double-distilled water.
  • Insert fragment preparation Use PCR Purification KIT (QIAGEN) to recover the Sw gene fragment amplified in the previous step, and elute the DNA with sterilized double distilled water.
  • Xba I and Kpn I were digested to identify the pShuttle-ORF1ab/N plasmid.
  • Vector preparation Use I-Ceu I and PI-Sce I to cut the DNA transcribed by nCoV2019 in large quantities, separate large fragments by gel electrophoresis, and recover the vector fragments with GEL EXTRACTION (QIAGEN), and dissolve them in sterilized double-distilled water.
  • 2Reaction system 10 ⁇ PCRBuffer: 5u1; primers: 50pmol each; Taq enzyme (Gibco): 2.0U; viral DNA: 5ng, make up to a total volume of 50ul with deionized water.
  • 3Reaction conditions amplification in a PE9600 thermal cycler, pre-denaturation at 95°C for 8 minutes, cycle parameters: 94°C for 1 minute, 55°C for 1 minute, 72°C for 60 seconds, 30 cycles.
  • the plasmid pAd- SN containing the SN expression framework is digested and linearized with PacI: disinfected double-distilled water: 20ul; pAd- SN DNA (500ng/ul): 10ul; 10X Pac I Digestion Buffer: 4ul; 10XBSA: 4ul ; Pac I (1unit/ul): 2ul, total volume 40ul.
  • 2Prepare DNA-Lipofectamin TM 2000 complex a. Dilute 0.8ug of pAd- SN DNA with 50ul serum-free RPMll640, and mix gently. b. Mix Lipofectamin TM 2000 gently before use, dilute 2ul of Lipofectamin TM 2000 with 50u 1 serum-free RPMI 1640, mix gently, and incubate at room temperature for 5 minutes. c. Mix the diluted Lipofectamin TM 2000 and the diluted pAd- SN DNA in a total volume of 100ul, mix gently, and incubate at room temperature for 20 minutes.
  • CPE cytopathic effect
  • CPE appears after a week. Gently pipette the cells and collect the cells into a 15ml centrifuge tube.
  • the cells were repeatedly frozen and thawed in a 37°C water bath-liquid nitrogen for 3 times. After each thawing, vortex and vibrate to promote cell lysis.
  • the vacuum pump is set to vacuum off, and 10 ml of 100 mM Tris-HCI (pH 7.4) is added to the Pre-Filter to make the Pre-Filter and the 0.45 micron filter close to each other.
  • the vacuum pump is set to vacuum on. When the collection vessel is full, disconnect the vacuum pump and transfer the filtrate to the sterilization bottle. If the Bottle-TopFilter Unit is blocked, change to another Bottle-Top FiRerUnit.
  • Eluting adenovirus Connect a 20ml syringe to the inlet of BD Adeno-X Purification Filter. At the same time, connect the Tubing Assembly to the outlet end, and then put it into a centrifuge tube containing 20m1 1 ⁇ Elution Buffer. Extract l ⁇ Elution Buffer, the purified adenovirus is in the syringe, and transfer it to a 50ml centrifuge tube.
  • a. 500pl virus solution was digested with 50ul of 20mg/ml proteinase K for 60 minutes, and extracted twice.
  • the strong anion exchange pre-packed column is Q Sepharose XL (Amersharn Pharmacia, Sweden), the column matrix is cross-linked agarose, the diameter of the filler is 45-165um, and the column bed volume (CV>1ml, stored in 20% ethanol, The charge is -N+(CH3), and the ion exchange capacity is 0.18 ⁇ 0.26mmolC1-/ml glue.
  • the Vcro-E6 cell line is an African green monkey kidney epithelial cell (ATCC CRL 1586), from ATCC, using 1640 culture medium containing 10% FBS, 2mM glutamine, 100U/ml penicillin and 10099/ml streptomycin Incubate at 37°C, 5% CO 2 and saturated humidity.
  • 1Vem-E6 cells were seeded into a 6-well plate, 2x10 5 cells/well.
  • Vero-E6 cells were infected with different doses of Ad-ORF1ab or Ad-N (20MOI) for 48h.
  • RNA 6Reverse transcription of mRNA with AMV reverse transcription system (Invitrogen)
  • cDNA RNA: 4ug; 10 ⁇ AMVBuffer: 2ul; AMV reverse transcriptase (Promega): 20U; RNAsin: 20U; dNTP: 0.5ul; OligodT (Promega) : 30pmol, make up to 20ul of the total species with deionized water.
  • the amplified fragment is ORF1ab or N primer of 624bp.
  • Reaction system the above reversed product: 20ul; 10 ⁇ PCR Buffer: 5ul; primer: 30pmol each; pfuDNA polymerase (Gibco): 2.5U, make up to the total volume of 50ul with deionized water.
  • Vero-E6 cells were infected with different doses of Ad-ORF1ab/N (20MOI) for 48h.
  • Ad-ORF1ab or Ad-N Combining eukaryotic fermentation technology and adenovirus column chromatography purification technology, Ad-ORF1ab or Ad-N self-amplification, CsCl two centrifugal purification, titer of 5x10 10 pfu/ml .
  • nCoV-2019 antibody (IgG) detection system purchased from a company that may produce it in the future, and HRP-conjugated anti-rat IgG can be purchased from Santa Cruz.
  • 2Ad-ORF1ab/N nasal drip group same as the anesthesia above, nasal drip after 5 minutes, the dose is 1 ⁇ 10 7 pfu/dose/rat.
  • 3Ad- SN tail vein injection control group empty carrier control group
  • the dose is 1 ⁇ 10 7 pfu/dose/rat.
  • Each of the above groups has 10 animals in each group.
  • nCoV-2019 specific IgG antibody HRP-conjugated anti-rat IgG (use concentration 1:5000) is used as the secondary antibody, and the human nCoV-2019 antibody (IgG) detection system is used to detect the anti-nCoV-2019 IgG antibody in rat serum Horizontally, each sample has three multiple holes. Measure OD450 with a microplate reader, and the reference wavelength is 630. The titration endpoint is defined as the natural logarithm of the highest dilution that OD450 is at least 0.16 higher than the negative control group.
  • the neutralization titer is defined as the highest serum dilution that can completely inhibit Vero.E6 cells.
  • Ad-nCoV2019 The recombinant adenovirus Ad-nCoV2019 is prepared in batches according to the above-mentioned experimental method of the present invention, and stored at -70°C for later use. Among them, Ad stands for recombinant adenovirus vector, and nCoV2019 stands for a functional gene capable of expressing protein, such as ORF1ab or N gene. In the present invention, Ad-nCoV2019 is also called Ad-nCoVDNA.

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Abstract

提供了一种新型冠状病毒肺炎双价疫苗的制备方法，具体为扩增2019-nCoV靶向干扰基因shRNA，酶切构建干扰载体pSilencer-shRNA，经DH5a转化后构建穿梭载体pDC312-shRNA，与腺病毒骨架质粒pBHGloxAEl共转染HEK293而制成Ad-nCoVdsRNA；扩增2019-nCoV抗体表达基因，酶切构建穿梭质粒pShuttle-nCoV，经DH5a转化后与腺病毒骨架质粒pAd-nCoV共转染HEK293而制成重组腺病毒Ad-nCoVDNA；其中，以Ad-nCoVdsRNA：Ad-nCoVDNA：H20＝1：1：19的比例配制双价疫苗。该双价疫苗经呼吸道喷雾接种后，腺病毒载体将nCoVdsRNA和nCoVDNA导入细胞， nCoVdsRNA立即通过shRNA产生dsRNA，即时降解同源的病毒mRNA，nCoVDNA则通过编码mRNA表达蛋白，随后产生抗体中和病毒，两者有互补作用。

Description

一种新型冠状病毒肺炎双价疫苗的制备方法 技术领域
本发明涉及一种用于传染病防治领域的新型冠状病毒肺炎双价疫苗的制备方法，属于疫苗制备新方法技术领域。
背景技术
人类冠状病毒(HcoV 229E和HcoV OC43)可引起达30％的感冒。动物冠状病毒，如猪胃肠炎冠状病毒(TGEV)、小鼠肝炎冠状病毒(MHV)、鸟传染性气管炎冠状病毒(IBV)等可感染相应宿主的呼吸道、胃肠道、神经系统和肝脏，导致相应症状。冠状病毒的包膜呈花瓣形突起，使冠状病毒看起来像王冠(拉丁语，corona)，其核衣壳是可变的长螺旋形。冠状病毒的病毒粒子直径为60nm～140nm，球形，病毒基因组是27kb～32kb的单链正义RNA，是所有RNA病毒基因组中最大的。2003年冠状病毒的一个变种引起了严重急性呼吸综合征即非典(SARS)的爆发，SARS-CoV基因组大小为27～3lkb，有14个开放读码框(open reading frame，ORF)和1个s2m模序(s2m motif)。
[根据细则26改正01.03.2021]　
[根据细则9.2改正01.03.2021]　
2020年1月12日，世界卫生组织将爆发的冠状病毒命名为新型冠状病毒，即“nCoV-2019”。有报道从5名病人体内获得了基本一致的nCoV-2019全基因组，其中有79.5％的序列与SARS-CoV一致，通过对其保守的7个非结构蛋白进行对比，发现nCoV-2019属于SARSr-CoV的包膜RNA病毒。nCoV-2019含有5'非翻译区(UTR)、复制酶复合体(orf1ab)、S基因、E基因、M基因、N基因、3'UTR以及几个未知的非结构性开放式阅读框架。对于nCoV-2019的基因诊断(PCR)，目前中国疾病预防控制中心推荐了针对新型冠状病毒nCoV-2019的开放读码框1ab(ORF1ab)、核壳蛋白(nucleoprotein，N)基因区域的引物和探针序列，其中：Target 1(ORF1ab)：正向引物(F)：CCCTGTGGGTTTTACACTTAA；反向引物(R)：为ACGATTGTGCATCAGCTGA；探针(P)：5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'。Target 2(N)：正向引物(F)：GGGGAACTTCTCCTGCTAGAAT；反向引物(R)：CAGACATTTTGCTCTCAAGCTG；荧光探针(P)：5'-FAM-TTGCTGCTGCTTGACAGATT-BHQ1-3'。nCoV-2019全基因组序列和功能的分析(A Novel Coronavirus from Patients with Pneumonia in China,2019.The New England Journal of Medicine,2020,January 24)也为nCoV-2019的进一步研究奠定了良好的基础，但目前尚缺少有关nCoV-2019的更确切信息。
[根据细则26改正01.03.2021]　
[根据细则9.2改正01.03.2021]　
随着新型冠状病毒肺炎的爆发和蔓延，治疗药物和生物制品的研究已成为当务 之急。国务院国有资产监督管理委员会于2020年1月22日印发紧急通知，要求中央企业积极开展新型冠状病毒抗血清和疫苗的研制。国际流行病防范创新联盟(CEPI)首席执行官理查德·哈切特(Richard Hatchett)于2020年1月23日表示，他所在的机构已首轮投资1500万美元进行新型冠状病毒疫苗的研制，若进展顺利，有望6个月后开始临床测试。2020年1月26日，中国疾病控制中心表示，该中心开始启动新型冠状病毒疫苗的研发，目前已经成功分离病毒，正在筛选种子毒株。2020年1月26日，浙江大学传染病诊治国家重点实验室李兰娟院士也表示分离到3株新型冠状病毒，为下一步的疫苗研究奠定了基础。疫苗可分为减毒活疫苗、灭活疫苗、亚单位疫苗、DNA疫苗、重组载体疫苗、病毒颗粒样疫苗和多肽疫苗等。传统疫苗多为死疫苗、减毒活疫苗或重组亚单位疫苗，而新型疫苗则为编码抗原蛋白的病毒核酸或能激发特异性免疫应答的细胞疫苗。现有的传统疫苗主要靠病原体的抗原蛋白刺激机体产生保护性抗体，现有的新型疫苗除此之外还能激发特异性细胞免疫应答。鉴于2019-nCoV的生物学特性还不太明了，传染性很强，应该注意到，活疫苗不具有安全性而且制备困难，灭活疫苗可能会与野毒株重组而回复，只宜作为应急储备。相比之下，基于现代生物技术的亚单位疫苗、DNA疫苗和多肽疫苗的安全性、实用性和可操作性更强。为了减少疫苗运输、存放、接种的成本以及方便接种，常将数种不同病原体的疫苗混合在一起，称为多联疫苗，如百白破疫苗，一次接种可同时预防白喉、破伤风和百日咳。此外，为克服因毒株多型性而造成免疫效果差的缺点，常将针对同种病原体不同血清型的疫苗混合在一起，称为双价或多价疫苗，如多价HPV疫苗。但能否发明一种与抗原蛋白、抗体或细胞免疫无关的不同于现有疫苗的抗新型冠状病毒肺炎多价疫苗呢？
在新冠肺炎爆发的非常时期，本发明人拟尽早公开一种基于RNA干扰的新型冠状病毒双价疫苗的制备方法。RNA干扰(RNA interference,RNAi)是指某种小RNA可以与目的基因配对结合，特异性地剔除或关闭被结合基因的表达。即RNAi是指由双链RNA(double-stranded RNA，dsRNA)诱发的同源mRNA的高效特异性降解。文献报道，1995年来自复旦大学的Guo Shu博士在美国康奈尔大学将反义RNA注入秀丽新小杆线虫(C·ele-gans)体内，试图阻断par-1基因的表达。同时她还将正义RNA注入对照组线虫，以期观察到par-1基因表达增强的效果。然而对照组的par-1基因表达不仅未增强，反而与实验组一样被阻断。这种结果无法用传统的反义RNA技术做出解释，但她们还是将研究结果如实地投寄给了Cell杂志，并被发表。这一悬疑引起了华盛顿卡内基研究院Fire A博士的注意，他采用凝胶电泳将正义RNA和反义RNA纯化，同时故意将纯化后的正、反义RNA混合在一起，制成dsRNA杂合体，分别注入线虫。结果发现纯化后的反义RNA基因阻抑作用显著减弱，而dsRNA杂合体却高效、特异地阻断了同源mRNA表达，其阻断作用超过反义RNA至少100倍以上。这一结果意外地证实了dsRNA 在基因阻抑中发挥着重大作用。Hre A将dsRNA的这一作用称为RNA干扰(RNA interference，RNAi)。1998年，Hre A将研究结果发表于Nature杂志，以致迅速在全球掀起了RNA干扰研究的热潮，并证实RNA干扰是因dsRNA注入真核细胞后，激发真核细胞内防御反应，生成dsRNA诱导的沉默复合体，将与dsRNA具有同源序列的mRNA降解，导致基因在转录后水平沉默，丧失表达蛋白或多肽的能力。进一步的研究表明，病毒基因、人工转入基因、转座子等外源性基因随机整合到宿主细胞基因组内，并利用宿主细胞进行转录时，常产生一些dsRNA。宿主细胞对这些dsRNA迅即产生反应，其胞质中的核酸内切酶(Dicer)将dsRNA切割成多个具有特定长度和结构的小片段siRNA(大约21～23bp)，siRNA在细胞内RNA解旋酶的作用下解链成正义链和反义链，由反义siRNA再与体内一些酶(包括内切酶、外切酶、解旋酶等)结合形成RNA诱导的沉默复合物(RNA-induced silencing complex，RISC)。RISC与外源性基因表达的mRNA的同源区进行特异性结合，RISC具有核酸酶的功能，在结合部位切割mRNA，切割位点即是与siRNA中反义链互补结合的两端。被切割后的断裂mRNA随即降解，从而诱发宿主细胞针对这些mRNA的降解反应。siRNA不仅能引导RISC切割同源单链mRNA，而且可作为引物与靶RNA结合并在RNA聚合酶(RNA-dependent RNA polymerase，RdRP)作用下合成更多新的dsRNA，新合成的dsRNA再由Dicer切割产生大量的次级siRNA，从而使RNAi的作用进一步放大，最终靶mRNA被完全降解。RNAi作用类似于基因剔除，但远比基因剔除简单易行，安全性高，因不是真正剔除基因，可以避免基因误剔而带来的严重后果。这为研制基于RNA干扰的新型冠状病毒dsRNA疫苗提供了可行的机制。
文献报道，RNAi抗病毒复制的体外试验较易成功，但siRNA在体内血清中易被RNA酶(Rnase)降解，这就需要一种理想的载体将shRNA递送到细胞内表达。腺病毒重组载体技术于2004年首次应用于RNAi(Xia et a1.2004)，经典的腺病毒包装系统包括HEK293细胞、携带外源基因的穿梭质粒、包含腺病毒基因组的骨架质粒。穿梭载体一般具有真核启动子和位于其下游的多克隆位点，插入了外源基因的多克隆位点称为外源基因表达盒。穿梭载体与骨架质粒共转染HEK293细胞后，因HEK293细胞提供了病毒复制所必须的E1蛋白，使穿梭载体中的外源基因转移到骨架质粒，从而使重组腺病毒颗粒被包装出来。目前研究最明确的人腺病毒包括2型(Ad2)和5型(Ad5),由Ad5研制的腺病毒载体，已有商品化产品应用于疾病治疗及科研领域，具有感染谱广、不整合、安全、易操作、体外增殖滴度高、基因组中非必需片段大等特点，是目前应用最广泛的病毒载体，而且当E3区缺失后免疫原性增强，可能还充当免疫佐剂的免疫效应。最重要的是呼吸道上皮细胞含有丰富的腺病毒受体(CAR)，Ad5腺病毒的天然宿主是人体呼吸道上皮细胞，故复制缺陷型重组腺病毒载体能高效地感染呼吸道上皮细胞，为通过口腔、上呼吸道进行免疫接种(喷雾剂或鼻腔滴剂)提供了可能。另外，腺 病毒载体表达的免疫原经宿主细胞表达、加工、折叠、修饰、提呈后基本保持了免疫原的天然构象，便于模拟自然状态下的免疫反应，而且新型冠状病毒主要通过呼吸道感染，腺病毒载体疫苗可方便地制成口腔或上呼吸道喷雾剂，雾化吸入接种不仅方便，而且大大降低了使用成本，若能用腺病毒载体疫苗诱导有效的粘膜免疫反应，将具有广阔的应用前景。
雾化吸入接种是将药物或疫苗放入特定的气溶胶发生器中，将药物或疫苗雾化吸入呼吸道内，刺激呼吸道黏膜产生粘膜免疫并进一步进入肺泡发生作用。由于肺部具有巨大的肺泡表面积、丰富的毛细血管以及极小的转运距离，接种的药物或疫苗经肺吸收后可迅速的发挥药效。国外对雾化吸入治疗的研究可以追溯到上世纪五十年代，Riker实验室早在1956年研发上市了首个压力定量吸入器Medihaler-Epi TM含肾上腺素，Medihaler-Iso TM含异丙肾上腺素。气雾免疫，直接将疫苗接种于呼吸道粘膜，不仅能提供一种生理和免疫学的优势，而且这一路线的疫苗接种也提供了潜在的后勤优势，因为它不需要训练有素的人员。因此，气雾免疫法是一种值得稳步推广的免疫方法。近代人们对雾化吸入治疗给予了巨大的关注，并且越来越多的气雾免疫药物被开发出来，在人的气雾免疫研究方面麻疹疫苗的气溶胶接种是研究最多并被证实有确切的疗效。随着科学技术的不断进步，雾化接种将会越来越完善并成为一种常规的免疫治疗途径。雾化吸入接种或给药的重点始终离不开雾化器，压缩空气雾化器需要空气压缩机来驱动雾化。压缩的高速气流通过文丘里氏管，产生文丘里效应，从而在喷嘴周围产生一个负压环境，使药液杯里的药物被高速气流带出来，通过特殊的喷嘴高速撞击挡板从而破碎成为直径为5μm的雾滴。现在压缩空气雾化器在临床上的应用比较广泛，可对麻疹、流行性感冒和卡介苗(BCG)疫苗等药物进行雾化吸入治疗和接种。
总上，虽然基于病原体抗原制备抗原性疫苗的方法以及基于抗原性疫苗接种产生免疫性抗体从而使病原体被清除的现有疫苗的制备方法已得到广泛应用，但未见基于病原体保守基因组制备dsRNA疫苗的方法以及基于dsRNA疫苗接种产生基因沉默复合物从而使病原体同源mRNA被降解的dsRNA疫苗制备方法的文献报道和应用。
发明内容
本发明的目的在于解决现有技术未见基于病原体保守基因组制备dsRNA疫苗的方法以及基于dsRNA疫苗接种产生基因沉默复合物从而使病原体同源mRNA被降解的dsRNA疫苗制备方法的文献报道和应用的不足，提供一种不同于传统工艺的新型冠状病毒肺炎双价疫苗的制备方法。
本发明的目的通过以下技术方案实现：
(1)Ad-nCoV2019dsRNA疫苗的制备方法，扩增nCoV2019的靶向干扰基因shRNA序列， 所扩增的shRNA序列与空干扰载体pSilencer4.1.CMV.neo经BamH I和Hind III酶切而构建干扰载体pSilencer-shRNA，该干扰载体经感受态大肠杆菌DH5a扩增并经鉴定shRNA插入无误后与空穿梭载体pDC312经Hind lII和EcoR I酶切而构建穿梭载体pDC312-shRNA，该穿梭载体与腺病毒骨架质粒pBHGloxAEl共转染HEK293细胞，在该细胞内进行同源重组而获得重组腺病毒Ad-shRNA，再经HEK293多次扩增后制备重组腺病毒载体的Ad-nCoV2019dsRNA疫苗。
进一步的，所述靶向干扰基因shRNA指长度为19nt的与siRNA序列互补的序列，目前包括ORF1ab、3'UTR、S、E、M、N基因序列。
进一步的，所述靶向干扰基因shRNA为表达发夹结构的DNA模板，由两条大部分互补的单链DNA构成，经退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链。
进一步的，所述Ad-nCoV2019VdsRNA疫苗指携带shRNA的重组腺病毒载体进入呼吸道或消化道上皮细胞后，其中的shRNA能在细胞内合成dsRNA，所合成的dsRNA能特异性降解具有同源序列的mRNA，使之丧失表达蛋白或多肽的能力；所述Ad指复制缺陷型重组腺病毒载体；所述nCoV2019指新型冠状病毒的保守基因或功能基因的靶向干扰序列。
(2)Ad-nCoV2019DNA疫苗的制备方法，扩增nCoV2019的抗体表达基因，所扩增的序列与腺病毒穿梭质粒pShuttle经XbaI和KpnI酶切后构建重组腺病毒穿梭质粒pShuttle-nCoV2019，该穿梭质粒经感受态大肠杆菌DH5a扩增并经XbaI和KpnI酶切鉴定nCoV插入无误后和腺病毒骨架质粒pAd-nCoV2019共转染HEK293细胞，在该细胞内进行同源重组而获得重组腺病毒Ad-nCoV2019，该重组腺病毒经HEK293细胞多次扩增后制备重组腺病毒载体的Ad-nCoV2019DNA疫苗。
进一步的，所述抗体表达基因指能表达mRNA，mRNA表达蛋白，蛋白刺激机体产生抗体的基因。
进一步的，所述nCoV2019指新型冠状病毒的保守基因和功能基因序列；所述保守基因和功能基因目前指ORF1ab、3'UTR、S、E、M、N基因序列。
进一步的，所述pShuttle-nCoV2019指含有nCoV2019的重组腺病毒穿梭质粒；所述pAd-nCoV2019指含有nCoV2019的腺病毒骨架质粒；所述Ad-nCoV2019指含有nCoV2019的复制缺陷型重组腺病毒载体；所述Ad指复制缺陷型重组腺病毒载体。
进一步的，所述Ad-nCoV2019DNA疫苗指携带抗体表达基因的重组腺病毒载体进入呼吸道或消化道上皮细胞后，其中的抗体表达基因能表达mRNA，mRNA表达蛋白，蛋白刺激机体产生抗体，抗体中和新型冠状病毒，使之丧失致病力。
(3)按Ad-nCoV2019dsRNA：Ad-nCoV2019DNA：H 20体积比＝1：1：5～19配制成本发明的 新型冠状病毒肺炎双价疫苗。
本发明的有益效果在于：本发明的双价疫苗具有见效快、效果好、使用安全、接种方便等优点。首先，本发明的双价疫苗由Ad-nCoVdsRNA疫苗和Ad-nCoV2019DNA疫苗混合配制而成，经喷雾接种后被重组腺病毒载体导入呼吸道感染细胞后，其中Ad-nCoV2019dsRNA疫苗立即通过其shRNA产生dsRNA，激活核酸酶活性，即时降解同源的病毒mRNA，很快使病毒失去致病作用；而Ad-nCoV2019DNA疫苗则表达蛋白，产生抗体而较迟起中和病毒作用；因各组分的免疫作用机制和时间点不同，所以适合于不同病程，可避免因单价疫苗免疫失败而引起的严重后果，所以本发明的多价疫苗各具优势，起到相互补缺的增效作用，尤其是nCoV2019dsRNA组分的免疫机制不同于现有技术的抗原抗体反应而产生紧急预防的突出效果，提示在新型冠状病毒疫苗制备中，应将1株毒株的1个目的基因插入1个表达载体，或将1株毒株的数个目的基因插入1个表达载体，或将数株毒株的数个目的基因插入1个表达载体，依此制备各株毒株的表达载体，然后组合成1株多型或多株多型的Ad-nCoV2019dsRNA或/和Ad-nCoV2019DNA的双价或多价疫苗进行应用。其次，传递本发明双价疫苗的载体均为复制缺陷型腺病毒，具有使用安全、稳定表达和易操作等优点，因能高效感染呼吸道上皮细胞而为通过口腔、上呼吸道进行免疫喷雾接种提供了依据，理论上更适合于新型冠状病毒肺炎的预防。
具体实施方式
下面对本发明的具体实施方法作详细的描述。
一、实施例1
筛选靶向干扰基因shRNA序列(ORF1ab、S、E、M、N)，构建表达载体pSilencer-shRNA(pSilencer-ORF1ab/S/E/M/N)，转移其中的shRNA表达盒用构建腺病毒穿梭质粒pDC312-shRNA，并使其与腺病毒骨架质粒pBHGloxAEl共转染HEK293细胞，经同源重组获得重组腺病毒Ad-shRNA，经HEK293细胞多次扩增、纯化而制备以免疫增强剂为介质的抗新冠肺炎dsRNA疫苗，喷雾接种后由重组腺病毒载体(Ad-shRNA)把shRNA导入呼吸道上皮细胞并在细胞内合成dsRNA，继之特异性诱导nCoV mRNA降解的基因沉默或RNA干扰反应。
本实施例涉及一种nCoV2019疫苗的制备方法，涉及但不限于nCoV2019 ORF1ab、S、E、M、N基因及其引物，涉及但不限于相同的实验方法、shRNA表达载体pSilencer、pDC312、pBHGloxAEl、pAd、pEGFP、HEK293等实验材料，还涉及体外合成nCoV2019-RNA干扰治疗制品。
1、新型冠状病毒RNAi靶位点的选择及其shRNA干扰载体的构建
(1)RNAi靶位点的选择及siRNA表达模板的设计：根据已测序得知的新型冠状病毒 ORF1ab、3'UTR、S、E、M、N基因序列，利用Ambion公司的shRNA在线设计软件(http://www.ambion.com/techlib/misc/siRNAtools.html)获得长度为19nt的多个siRNA备选序列，根据RNA结合的Tm值及特异性比对结果，优选siRNA序列，与其互补的基因组区域被选为干扰的靶位点，结合pSilencer4.1.CMV.neo干扰载体的多克隆酶切位点设计出可以表达发夹结构的shRNA模板，每个模板由两条大部分互补的55bp的单链DNA构成，退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链，用于与线性化干扰载体pSilencer4.1.CMV.neo的连接。
(2)shRNA表达载体的构建：将上述寡核苷酸链进行退火互补与线性化的shRNA表达载体pSilencer4.1.CMV.neo进行连接，构建shRNA表达质粒，并转化至感受态细胞DH5a。退火互补和载体连接的具体方法如下：
①寡聚DNA退火：将合成的寡核苷酸用ddH 20溶解成100μM，两两互补单链各取5μL进行混合，然后将6种oligoDNA混合液放置在98℃水浴锅中加热5min，关闭水浴锅开关使其自然冷却到室温，使双链DNA形成，退火体系如下：100μM正链寡核苷酸:5μL；100μM负链寡核苷酸:5μL；10xPCR buffer:2μL；ddH 20:8μL；总体积20μL。
②载体连接：将合成的双链DNA进一步稀释成10nM，16℃连接30min。酶连接体系如下：pSilencer4.1.CMV.neo：4μL；5xligation buffer：2μL；ds oligo(10nM)：4μL；T4 DNA连接酶(1U/μL):1μL；ddH 20:9μL；总体积:20μL。构建pSilencer-ORF1ab、pSilencer-3'UTR、pSilencer-S、pSilencer-E、pSilencer-M、pSilencer-N载体。
③载体的鉴定:将连接产物转化大肠杆菌感受态细胞DH5a，每个重组载体平板挑选6个克隆进行测序鉴定，确定插入片段正确后，保存备用。
2、shRNA干扰载体的效果鉴定
通过构建荧光标签载体并与shRNA干扰载体共转染293T细胞进行鉴定。
(1)ORF1ab、3'UTR、S、E、M、N基因荧光标签载体的构建
①ORF1ab、3'UTR、S、E、M、N基因引物设计：根据中国国家基因组科学数据中心(NGDC)发布的新型冠状病毒(nCoV-2019)基因组序列(序列号为GWHABKF00000000、GWHABKG00000000、GWHABKH00000000、GWHABKI00000000、GWHABKJ00000000)，选择保守区域设计所需的下、下游引物，或根据新发新型冠状病毒毒株的基因测序结果设计所需引物。本发明以nCoV2019的ORF1ab、3'UTR、S、E、M、N基因为实施例，参照网上在线的引物设计软件，根据不同载体、酶切位点、鉴定需要，委托生物公司按要求设计所需的引物，上游引物5'端添加起始密码，为将扩增产物克隆到pEGFP-N1中，引物的5'端添加用于与载体发生同源重组的同源臂。
②ORF1ab、3'UTR、S、E、M、N基因的扩增：基因扩增反应体系及反应条件按上海生工 提供的试剂盒进行，基因扩增产物经回收纯化备用。
③pEGFP-N1载体的线性化：复苏含有pEGFP-N1质粒的DH5a菌种，按试剂盒或文献提取质粒，测定浓度后进行酶切，酶切体系如下：10xM Buffer：5μL；质粒DNA：20μL；Hind III：2μL；ddH 20：23μL；总体积：50μL。将混合液混匀后置于37℃水浴锅中酶切2h，0.8％琼脂糖凝胶电泳鉴定并回收线性化的载体备用。
④pEGFP-ORF1ab、pEGFP-3'UTR、pEGFP-S、pEGFP-E、pEGFP-M、pEGFP-N载体的构建：使用金斯瑞公司的同源重组试剂盒进行连接，体系及条件如下：线性化载体(100-200ng/μL):6μL；纯化的PCR产物：8μL；10xCloneEZ buffer：2μL；CloneEZ Enzyme:2μL；ddH20：2μL；总体积：20μL。混合物配制好后轻轻混匀，25℃保持30min，之后在冰上保持5min。连接结束后可保存在-20℃备用或马上进行转化。
(2)共转染方法：分别将干扰载体pSilencer-ORF1ab、pSilencer-3'UTR、pSilencer-S、pSilencer-E、pSilencer-M、pSilencer-N与对应的荧光标签载体pEGFP-ORF1ab、pEGFP-3'UTR、pEGFP-S、pEGFP-E、pEGFP-M、pEGFP-N共转染293T细胞。干扰载体与标签载体的质量比为1：2，同时设立无关干扰孔和未干扰孔作为对照，转染后48h观察细胞内GFP蛋白的融合表达情况，根据荧光强度评价干扰效果。
(3)共转染后的流式细胞检测：为将不同干扰载体的干扰效果进行定量分析，用流式细胞术检测的细胞，分析表达荧光蛋白的细胞在总细胞数中的比例。流式细胞分析方法如下：
①将试验的细胞用胰蛋白酶从细胞板上消化下来并吹打为单个细胞，转入1.5mL离心管。
②将细胞悬液细胞40C 300Xg离心10min，弃上清，加入预冷PBS进行洗涤。
③重复步骤(2)3次后充分悬浮细胞，并将细胞吹打为单个。
④取适当数量细胞上机检测，检测方法采用Guavaexpress Plus方法，每秒钟通过细胞数保持在800个以下。
⑤检测数据保存后用Flowjo流式分析软件进行结果的分析与统计。
(4)ORF1ab、3'UTR、S、E、M、N蛋白的Westernbolt分析
①细胞的收集与裂解：采用RIPA组织裂解液裂解细胞，具体步骤如下：
a.将细胞用PBS洗涤1次备用；以10μL的PMSF与lmL RIPA混合的比例将裂解液配好。
b.每孔按加入150-250μL的裂解液，用移液器吹打数次，使裂解液与细胞充分接触。
c.裂解后的样品12000xg离心3.5min，取上清备用。
②蛋白样品的SDS-PAGE电泳：
将样品加入等体积的2xSDS上样缓冲液，沸水煮5min，冰浴2min，12000xg，10min，SDS-PAGE胶的制备如下：
a.按照垂直电泳装置的使用方法，将玻璃板洗净晾干后开始装配，保证良好的密封性。先配制分离胶。
b.按下列成分配制10ml15％分离胶：30％丙烯酰胺混合液:5.0mL；1.5M Tris(pH8.8):2.5mL；10％过硫酸铵:O.1mL；10％SDS：0.1mL；TEMED：0.004mL；ddH 20 2.3mL。最后加入TEMED，分离胶配好后迅速混匀，用移液器将配好的凝胶溶液在玻璃板的一端注入到间隙中，当胶面距玻璃板边缘3cm左右时停止加液。并在胶的顶端加入ddH20，当凝胶完全凝集后将水倒掉，用滤纸小心吸干凝胶上的残余的液体。
c.浓缩胶的制备t按下列成分配制2ml 5％的浓缩：30％丙烯酰胺混合液:O.33mL；1.0M Tris(pH6.8)0.25mL；10％过硫酸铵:O.02mL；10％SDS 0.02mL；TEMED 0.002mL；H 20 1.4mL。浓缩胶配好后迅速混匀，TEMED最后加入，用移液器将配好的凝胶溶液在玻璃板的一端注入到间隙中，加完后轻轻插入梳子防止产生气泡，除去流出的多余凝胶。
d.浓缩胶凝固后拔下梳子。
e.将凝胶在制胶架上拆下，装在电泳装槽内，加入1xTris-甘氨酸电泳液，先外后内，最后点样，每孔20此。
f.浓缩胶电泳使用80V电压，待样品进入分离胶后，调整电压为120V直到电泳结束(溴酚蓝进入到电泳液中)。
g.小心的取下凝胶，用考马斯亮蓝R-250染色液在水平摇床上进行染色中，更换脱色液后再在水平摇床上过夜脱色，其间更换脱色液，直至观察到的凝胶上出现清晰条带为止。
③Western blot检测。
a.转膜：将未染色凝胶放入转膜缓冲液，将滤纸和PVDF膜剪至与凝胶大小相近，但略小一些。用无水甲醇将PVDF膜短暂浸泡后再放入转膜缓冲液，滤纸直接在转膜缓冲液中浸透。组合转膜的装置(从下至上的顺序)：滤纸(3层)、PVDF膜(1层)、凝胶、滤纸(3层)，组装好后放入电泳夹具中并放到转印槽内，150mA恒流转印120min。
b.封闭：将膜取出，放入10mL 2.5％PBST稀释的脱脂奶中，摇床振摇lh。
c.一抗结合：将膜放入装有10mL用2.5脱脂奶中稀释的一抗中(抗GFP单克隆抗体、抗IB-actin单克隆抗体)，摇床振摇1h。
d.洗涤：用PBST洗涤3遍。
e.二抗结合：将PVDF膜放入装稀释1：800稀释的二抗中，摇床振摇1h。
f.洗涤：用PBST洗涤3遍。
g.显色：将膜使用天根HRP-DAB底物显色试剂盒进行显色。
h.用去离子水洗涤终止显色，观察结果。
(5)转染细胞ORF1ab、3'UTR、S、E、M、N的相对表达量检测。
a.检测转染细胞中ORF1ab、3'UTR、S、E、M、N基因的相对表达量，定量评价不同干扰载体的干扰效果，采用相对荧光定量RT-PCR检测方法。
b.对样品的目的基因转录进行相对定量检测时，分别根据标准曲线方程，由CT值换算出目的基因以及B-actin内参基因拷贝数。以B-actin内参基因校正病毒基因mRNA相对表达量(目的基因拷贝数/B-actin拷贝数)。
3、干扰2019nCoV复制的重组腺病毒穿梭载体(pDC312-2019nCoV)构建
(1)shRNA干扰载体中shRNA表达盒的转移
常规提取shRNA干扰载体(pSilencer-ORF1ab、pSilencer-3'UTR、pSilencer-S、pSilencer-E、pSilencer-M、pSilencer-N)和pDC312或pShuttle腺病毒穿梭载体，同时使用限制性内切酶Hind lII和EcoR I进行酶切，将shRNA干扰载体中的shRNA表达盒切下，并将pDC312载体进行线性化。
酶切体系如下：10xM Buffer：5uL；质粒DNA：20uL；Hind III：2uL；EcoR I：2uL；ddH 20：2luL；总体积50uL。
回收线性化载体及shRNA表达盒,按如下体系连接：pDC312/pShuttle：2uL；shRNA表达盒：4uL；5xligation buffer：4uL；T4 DNA连接酶(1U/uL):1uL；ddH 20：9uL；总体积20uL。
将连接产物转化大肠杆菌感受态细胞DH5a，隔夜培养后挑取4个克隆进行测序鉴定，确定插入片段正确后，将该重组载体命名为pDC312-nCoV2019(pDC312-ORF1ab、pDC312-3'UTR、pDC312-S、pDC312-E、pDC312-M、pDC312-N)，并保存备用。
(2)重组腺病毒穿梭载体(pDC312-nCoV2019)鉴定引物的设计
根据Genbank发布的Ad5序列以及中国国家基因组科学数据中心发布的nCoV-2019基因组序列，由上海生工公司设计可以扩增ORF1ab、3'UTR、S、E、M、N区域880bp片段的引物(如ORF1ab：F：CCCTGTGGGTTTTACACTTAA；R：ACGATTGTGCATCAGCTGA。N：F：GGGGAACTTCTCCTGCTAGAAT；R：CAGACATTTTGCTCTCAAGCTG)，用于重组腺病毒(pDC312/pShuttle)的鉴定。
(3)重组腺病毒(Ad-2019nCoV)的包装与扩增
标题中Ad代表重组腺病毒载体，2019nCoV或nCoV2019一样，代表Ad中可用于靶向干扰的新型冠状病毒的保守或功能基因位点，如ORF1ab、3'UTR、S、E、M、N。本发明采用Admax的双质粒转染系统进行重组腺病毒的包装，常规进行HEK293细胞的转染。腺病毒骨架载体pBHGloxAEl与腺病毒重组穿梭载体(pDC312-2019nCoV)的转染比例为质量比1：3。转染后每天观察细胞病变情况，大约8d左右待，80％细胞出现CPE时准备收毒。将待收毒的细胞瓶 /培养板冻融3次，使细胞破裂崩解，细胞内的病毒得到充分释放。将冻融液离心并收集含病毒的上清液，3000xg 4℃离心lOmin后取上清保存备用。将收获的第一代重组腺病毒(Ad-2019nCoV)反复感染HEK293细胞增殖病毒，提取第三代病毒应用DNA提取试剂盒提取DNA，进行PCR鉴定。
(4)Ad-2019nCoV滴度测定
重组腺病毒滴度的测定采用快速腺病毒感染性滴度(TCIDso)检测试剂盒。通过一系列稀释的病毒样品感染293细胞，每个稀释度感染8个细胞孔，经过大约10d的培养后，在显微镜下对每个细胞孔内是否存在病毒引起的细胞病变进行判断，通过出现噬斑的孔数计算出腺病毒的滴度。
4、Ad-2019nCoV(dsRNA)干扰2019nCoV复制的验证
①体外验证：为研究重组腺病毒Ad-2019nCoV对2019nCoV复制的干扰效果，将Ad-nCoV2019以lO、50、100、250、500个MOI接种于12孔板培养的HEK293细胞中，同时使用仅含有CMV启动子的未包括shRNA模板序列的对照病毒Ad-CMV作为对照病毒接种。感染24h后再接种2019nCoV株(临床分离)，将只接种2019nCoV的细胞作为阳性对照细胞。2019nCoV感染48h后收集细胞进行检测。对各组细胞进行2019nCoV基因(ORF1ab、3'UTR、S、E、M、N)相对表达量的检测，评价干扰效果。
②动物试验：
A.实验动物：Wistar大鼠在SPF级的动物实验室中培养，雌雄各半，6～8周龄，体重110+10g，记录动物生产合格证号。
B.Ad-nCoV2019(dsRNA)准备：结合真核发酵技术和腺病毒柱层析纯化技术，Ad-nCoV2019(dsRNA)自行扩增、CsCl两次离心纯化，滴度为5x10 10pfu/ml。
C.拟定实验方法
a.Ad-nCoV2019(dsRNA)滴鼻对照组(空载体对照组)，先用3％戊巴比妥腹腔注射麻醉，5分钟后滴鼻，O.5ml/只，剂量1×10 7pfu/dose/rat。
b.Ad-nCoV2019(dsRNA)滴鼻组，同上麻醉，5分钟后滴鼻，剂量1×10 7pfu/dose/rat。
c.Ad-nCoV2019(dsRNA)尾静脉注射对照组(空载体对照组)，剂量1×10 7pfu/dose/rat。
d.Ad-nCoV2019(dsRNA)尾静脉注射组，尾静脉注射给药，剂量1×10 7pfu/dose/rat。
e.空白对照组，尾静脉注射PBS，O.5ml/只鼠。
f.饲养方法：上述各组大鼠同等暴露于已确诊的nCoV2019患者的分泌物中或含nCoV2019毒株的环境中。
以上各组，每组10只动物。
g.观察和取样：观察大鼠的发病情况，检测第0、1、2、3、4周样本的nCoV2019。
h.nCoV2019诊断:参照目前市售PCR试剂盒检测诊断。
i.根据上述实验结果判断Ad-nCoV2019(dsRNA)干扰nCoV2019复制的效果。
j.命名：如果上述结果证明Ad-nCoV2019(dsRNA)能有效干扰nCoV2019复制，则将Ad-nCoV2019(dsRNA)命名为“新型冠状病毒dsRNA疫苗”或“nCoV2019-dsRNA疫苗”
5、nCoV2019-dsRNA疫苗的批量制备
(1)取上述制备的重组腺病毒包装HEK293细胞，37℃水浴-液氮冻融细胞3次。每次融解后涡旋振荡，利于细胞裂解(如用已制备的nCoV2019-dsRNA疫苗作进一步的批量制备，则从下述第7步开始操作)。
(2)离心，去除细胞碎片。
(3)在60mm培养板(24孔板)内接种293细胞，1×10 6细胞/250ul/孔，用无血清RPMI 1640过夜培养至75％融合。
(4)加入细胞裂解上清，250ul/孔。
(5)37℃、5％C02条件下培养，每日观察CPE(cytopathiceffect)，一周内应出现CPE。
(6)当50％以上的细胞脱落时，收获细胞。
(7)参照上述步骤，反复用病变293细胞的裂解上清液感染293细胞，用150cm2培养瓶扩增细胞、制备重组腺病毒(nCoV2019-dsRNA疫苗)：
A.将全部病毒溶解到培养液中制成储存液，以保证每瓶感染的病毒总数相同。
B.常规方法培养293细胞，长至90％～100％汇合时按150cm2培养面积接种病毒颗粒l×10 10个计算，用腺病毒感染293细胞。
C.37℃培养36小时左右，在此期间观察腺病毒的扩增情况，随细胞病变的发展，细胞变圆，折光度改变，开始从培养瓶的表面脱落。
(8)重组腺病毒(nCoV2019-dsRNA疫苗)纯化、浓度检测及贮存
A.吹打293细胞，将细胞转移至离心管。
B.4℃，1500g离心20分钟。
C.收集上清(Supematant#1)，储存于4℃离心管。
D.用25ml无菌100mM Tris-HCI(pH7.4)重悬细胞沉淀。
E.在37℃水浴-液氮中反复冻融细胞3次。每次融解后旋涡振荡，促进细胞裂解。
F.4℃，1500g离心20分钟，收集上清(Supematant#2)。
G.混合Supematant#1和Supematant#2。
H.将Bottle-Top Filter Unit，放入超净台内，开盖，把Pre-filter Disc放在0.45micron  filter上面。
I.将Bottle-TopFilterUnit与真空泵连接。
J.真空泵置vacuum off档，往Pre-Filter加lO ml的100mM Tris-HCI(pH7.4)，使Pre-Filter与0.45micron filter紧贴。
K.小心将细胞裂解上清倒入Bottle-Top Filter Unit。
H.真空泵置vacuuln on档，当collectionvessel接满后，断开真空泵，将滤过液转至消毒瓶内。若Bottle-TopFilter Unit堵塞，换另一个Bottle-Top FiRerUnit。
L.在滤过液内加入终浓度为10unitsBenzonase/ml的BenzonaseNuclease(Novagen)，37℃孵育30min。
M.用无菌Milli-QH20稀释5×Dilution buffers和5×Wash bufferS至1×浓度。
N.将等体积的滤过液和1×Dilution Buffer混合。
O.纯化腺病毒。
P准备好以下物品：Tubing Assembly和BD Adeno-X Purification Filter：1×WashBuffer、1×Elution Butter；无菌PBS：1×Formulation Buffer；5-ml、20-ml、60-ml BD Luer-Lok TM Tip注射器；无菌15-m1、50-ml离心管。
Q.连接BD Adeno-X Purification Filter和Tubing Assembly。
R.将纯化系统与真空泵连接，把Tubing Assembly A一端放入无菌PBS中，缓慢打开真空泵，抽取10～20ml无菌PBS使之通过BD Adeno-X Purification Filter和Tubing Assembly，关闭止水夹，关闭真空泵，以去除BD Adeno-x Purification Filter和Tubing Assembly中的气泡。
S.把TubingAssembly管放入病毒液中。
T.缓慢开启真空泵，用止水夹将流速控制在20ml/min左右。过滤后，夹紧TubingAssembly管，卸下真空泵管。
U.从接收瓶卸下TubingAssembly，放入1×WashBuffer。
V.将WashBuffer用BDAdeno-xPurificationFilter滤过，流速20ml/min。
W.卸下Tubing Assembly B，Tubing Assembly A留作下步洗脱用。
X.洗脱腺病毒：将20ml注射器接到BD Adeno-X Purification Filter入口端。同时将Tubing Assembly接到出口端，然后放入装有20m1 1×Elution Buffer的离心管中。抽取l×Elution Buffer，注射器内即为纯化的腺病毒，将之转入50ml离心管。
Y.用BD Adeno-X TMRapid Titer Kit测定腺病毒滴度。
Z.重组腺病毒(nCoV2019-Ad-dsRNA疫苗)保藏：置-70℃保存备用。所制重组腺病毒(Ad) 中携带了新型冠状病毒(nCoV2019)靶向干扰基因(ORF1ab、3'UTR、S、E、M、N)shRNA序列，称为Ad-nCoVdsRNA疫苗，其中的shRNA能产生dsRNA，进而产生RNA干扰。
二、实施例2
筛选新型冠状病毒nCoV的蛋白表达即抗体产生基因，构建腺病毒穿梭质粒pShuttle-nCoV(pShuttle-ORF1ab/N)和重组腺病毒骨架质粒pAd-nCoV(pAd-ORF1ab/N)，包装成重组腺病毒Ad-nCoV(Ad-ORF1ab/N)，在体外制备对人体无毒、易进入呼吸道上皮细胞并能在细胞内表达nCoV-mRNA的重组腺病毒Ad-nCoV(Ad-ORF1ab/N)，进而制成能刺激机体产生抗nCoV抗体从而产生特异性免疫作用的呼吸道喷雾接种DNA疫苗，用于新型冠状病毒肺炎的紧急预防。
本实施例涉及一种nCoV疫苗的制备方法，涉及但不限于nCoV ORF1ab、S、E、M、N基因及其引物，涉及但不限于相同的实验方法、克隆载体等实验材料，涉及但不限于用于体内合成nCoV蛋白抗原或抗体。
1、扩增nCoV2019的ORF1ab或N基因
引物设计：目前中国疾病预防控制中心推荐选用针对nCoV-2019的开放读码框1ab(open reading frame,ORF1ab)、核壳蛋白(nucleoprotein，N)基因区域的引物，其中：开放读码框ORF1ab：R：CCCTGTGGGTTTTACACTTAA；F：ACGATTGTGCATCAGCTGA。核壳蛋白N：F：GGGGAACTTCTCCTGCTAGAAT；R：CAGACATTTTGCTCTCAAGCTG。委托公司设计引物，在5'端和3'端分别引入XbaI和KpnI酶切位点，按PCR试剂盒说明书操作。
2、构建腺病毒穿梭质粒(pShuttle-ORF1ab/N)
(1)载体准备：将pShuttle质粒转化DH5Ⅱ细菌，挑取阳性克隆，用含氨苄青霉素的LB溶液小量扩增，用PLASMID MINIPREP KIT(QIAGEN)提取pShuttle质粒，酶切鉴定正确后用大量Xba I+Kpn I酶切，用凝胶电泳分离大片段，用GELEXTRACTION(QIAGEN)回收载体片段，溶于消毒双蒸水。
(2)插入片段准备：用PCR PURIFICATION KIT(QIAGEN)回收上步扩增的Sw基因片段，用消毒双蒸水洗脱DNA。
(3)连接反应：插入片段：5u1；载体：2ul；solution I：7ul；总体积14u1。16℃过夜连接，载体片段和插入片段的摩尔数之比约为1：3。
(4)将连接反应产物10ul转化DH5a感受态细菌。
(5)从转化平板挑取抗性克隆，用含氨苄青霉素的LB溶液小量扩增。
(6)用PLASMIDMINIPREPKIT(QIAGEN)提取pShuttle-SN质粒。
(7)Xba I和Kpn I酶切鉴定pShuttle-ORF1ab/N质粒。
(8)pShuttle-ORF1ab/N质粒的序列分析由上海博亚生物技术有限公司用测序引物进行序列分析。
(9)大量扩增DH5a/pShuttle-ORF1ab/N，用PLASMID MtDIPREP KIT(QIAGEN)提取pShuttle-ORF1ab/N质粒备用。
3、构建重组腺病毒骨架质粒(pAd-ORF1ab/N)
(1)载体准备用I-Ceu I和PI-Sce I大量酶切nCoV2019转录的DNA，用凝胶电泳分离大片段，用GEL EXTRACTION(QIAGEN)回收载体片段，溶于消毒双蒸水。
(2)插入片段准备：
①pShuttle-ORF1ab/N用I-Ceu I和PI—Sce I双酶切：消毒双蒸水：19.5ul；10×DoubleDigestionBuffer：3.0ul；pShuttle-Stq DNA(500ng/ul)：2.0ul；PI-Sce I(1unit/ul)：2.0ul；I—Ceu I(5units/ul)：O.5ul；10×BSA：3.0ul，总体积30ul。
②用凝胶电泳分离大片段。
③用GEL EXTRACTION(QIAGEN)回收载体片段，溶于双蒸水，得到ORF1ab/N表达框架。
(3)连接反应：
①将载体片段和插入片段和等体积ligation solution I混合，进行体外连接，16℃过夜，载体片段和插入片段的摩尔数之比约为1：3。
②将连接反应产物连接产物用Swa I酶切去除自身环化的连接体。
③酚氯仿抽提，乙醇沉淀DNA。
(4)取10ul纯化的DNA转化DH5a感受态细菌。
(5)从转化平板挑取抗性克隆，用台氨苄青霉素的LB溶液小量扩增。
(6)用PLASMID MINIPREPKIT(QIAGEN)提取阳性克隆质粒。
(7)酶切鉴定阳性克隆质粒。
(8)阳性克隆质粒的PCR鉴定。
①设计引物，扩增片段505bp。
②反应体系：10×PCRBuffer：5u1；引物：各50pmol；Taq酶(Gibco)：2.0U；病毒DNA：5ng，用去离子水补至总体积50ul。
③反应条件：在PE9600热循环仪扩增，95℃预变性8分钟，循环参数：94℃1分钟，55℃1分钟，72℃60秒，30个循环。
④结果分析：用1％琼脂糖凝胶电泳分析，紫外灯下观察结果。
(9)大量扩增DH5a/pAd-ORF1ab/N，用PLASMID MIDIPKEPKIT(QIAGEN)提取 pAd-ORF1ab/N质粒，备用。
4、包装重组腺病毒(Ad-ORF1ab/N)
(1)pAd-S N线性化
①含有S N表达框架的质粒pAd-S N用PacI酶切线性化：消毒双蒸水：20ul；pAd-S N DNA(500ng/ul):10ul；10X Pac I Digestion Buffer:4ul；10XBSA：4ul；Pac I(1unit/ul):2ul,总体积40ul。
②37℃消化2小时，加入60ul TE Buffer(pH8.0)以及100ul酚：氯仿：异戊醇(25：24：I)，稍涡旋。
③用1/10体积3mol/L NaAc(pH5.2)和2倍体积无水乙醇、1ul glycogen(20mg/m1)沉淀DNA。
④用lO ul消毒TE(pH8.O)TE溶解DNA。
(2)转染293细胞
由Lipofectamin TM2000(Inviuogen)介导，用线性化pAd-S N转染293细胞，观察CPE(细胞病变)的形成。
①于转染前一天在60mm培养板(24孔板)内接种293细胞，1×106细胞/孔，用无血清RPMI 1640过夜培养至75％融合。
②准备DNA-Lipofectamin TM2000复合物：a.用50ul无血清RPMll640稀释O.8ug的pAd-S NDNA，轻轻混匀。b.使用前轻轻混匀Lipofectamin TM2000，用50u 1无血清RPMI 1640稀释2ul的Lipofectamin TM2000，轻轻混匀，室温下孵育5分钟。c.混合稀释后的Lipofectamin TM2000和稀释后的pAd-S NDNA，总体积100ul，轻轻混匀，室温下孵育20分钟。
③将上步制备的DNA Lipofectamin TM2000复合物加到24孔板，在前后方向轻轻摇动培养板，使DNA-Lipofectamin TM2000复合物在培养孔内分布均匀。
④在C02培养箱内37℃、5％C02条件下培养36小时。
⑤观察有无细胞病变(cytopathiceffect，CPE)。
(3)一周后出现CPE，轻轻吹打细胞，收集细胞至15ml离心管。
(4)室温下1500g离心5分钟。
(5)用500ul消毒PBS重悬细胞。
(6)37℃水浴-液氮冻融细胞，3次。每次融解后涡旋振荡，利于细胞裂解。
(7)离心，去除细胞碎片。
(8)在60mm培养板(24孔板)内接种293细胞，1×106细胞/250ul/孔，用无血清RPMI 1640过夜培养至75％融合。
(9)加入细胞裂解上清，250ul/孔。
(10)37℃、5％C02条件下培养，每日观察CPE，一周内应出现CPE。
(11)当50％以上的细胞脱落时，收获细胞。
(12)参照步骤(6)～(11)，用病变293细胞的裂解上清液反复感染293细胞，用150cm2培养瓶扩增重组腺病毒：
①将所需的全部病毒溶解到培养液中制成储存液，以保证每瓶感染的病毒总数相同。
②常规方法培养扩增293细胞，长至90％～100％汇合时按150cm2培养面积接种病毒颗粒l×10 10个计算，用腺病毒感染293细胞。
③37℃培养36小时左右，在此期间观察腺病毒的扩增情况，随细胞病变的发展，细胞变圆，折光度改变，开始从培养瓶的表面脱落。
5、重组腺病毒(Ad-ORF1ab/N)纯化及浓度检测
(1)吹打293细胞，将细胞转移至离心管。
(2)4℃，1500g离心20分钟。
(3)收集上清(Supematant#1)，储存于4℃离心管。
(4)用25ml无菌100mM Tris-HCI(pH7.4)重悬细胞沉淀。
(5)在37℃水浴-液氮中反复冻融细胞，3次。每次融解后旋涡振荡，促进细胞裂解。
(6)4℃，1500g离心20分钟，收集上清(Supematant#2)。
(7)混合Supematant#1和Supematant#2。
(8)将Bottle-Top Filter Unit，放入超净台内，开盖，把Pre-filter Disc放在0.45micron filter上面。
(9)将Bottle-TopFilterUnit与真空泵连接。
(10)真空泵置vacuum off档，往Pre-Filter加lO ml的100mM Tris-HCI(pH7.4)，使Pre-Filter与0.45micron filter紧贴。
(11)小心将细胞裂解上清倒入Bottle-Top Filter Unit。
(12)真空泵置vacuuln on档，当collectionvessel接满后，断开真空泵，将滤过液转移至消毒瓶内。若Bottle-TopFilter Unit堵塞，换另一个Bottle-Top FiRerUnit。
(13)在滤过液内加入终浓度为10unitsBenzonase/ml的BenzonaseNuclease(Novagen)，37℃孵育30min。
(14)用无菌Milli-QH20稀释5×Dilution buffers和5×Wash bufferS至1×浓度。
(15)将等体积的滤过液和1×Dilution Buffer混合。
(16)纯化腺病毒。
(17)准备好以下物品：Tubing Assembly和BD Adeno-X Purification Filter：1×WashBuffer、1×Elution Butter；无菌PBS：1×Formulation Buffer；5-ml、20-ml、60-ml BD Luer-Lok TM Tip注射器；无菌15-m1、50-ml离心管。
(18)连接BD Adeno-X Purification Filter和Tubing Assembly。
(19)将纯化系统与真空泵连接，把Tubing Assembly A一端放入无菌PBS中，缓慢打开真空泵，抽取10～20ml无菌PBS使之通过BD Adeno-X Purification Filter和Tubing Assembly，关闭止水夹，关闭真空泵，以去除BD Adeno-x Purification Filter和Tubing Assembly中的气泡。
(20)把TubingAssembly管放入病毒液中。
(21)缓慢开启真空泵，用止水夹控制流速在20ml/min左右。滤完后夹紧TubingAssembly管，卸下真空泵管。
(22)从接收瓶卸下TubingAssembly，放入1×WashBuffer。
(23)将WashBuffer用BDAdeno-xPurificationFilter滤过，流速20ml/min。
(24)卸下Tubing Assembly B，Tubing Assembly A留作下步洗脱用。
(25)洗脱腺病毒：将20ml注射器接到BD Adeno-X Purification Filter入口端。同时将Tubing Assembly接到出口端，然后放入装有20m1 1×Elution Buffer的离心管中。抽取l×Elution Buffer，注射器内即为纯化的腺病毒，将之转入50ml离心管。
(26)用BD Adeno-X TMRapid Titer Kit测定腺病毒滴度。
(27)腺病毒置-70℃保存。
6、重组腺病毒(Ad-ORF1ab/N)基因的鉴定
(1)酶切鉴定
①纯化病毒DNA
a.500pl病毒液用50ul的20mg/ml蛋白酶K消化60分钟,抽提2次。
b.用2倍体积无水乙醇和1/10体积乙酸钠(pH5.2)沉淀DNA。
c.用70％乙醇洗涤DNA2次；用消毒双蒸水50ul溶解DNA。
d.取10ul病毒DNA稀释20倍，在BeckmanUV640紫外分光光度计测定OD260、0D280、OD260/0D280值，计算DNA浓度。
②用l-Ceu I/PI-SeeI、HindIIl、XhoI、KpnI和KpnI/XbaI进行病毒DNA限制性酶切分析，用O.8％琼脂糖凝胶电泳分析酶切反应。
(2)滴度测定
①感染细胞
a.消化对数生长期的293细胞，重悬于含10％FBS的RPMI 1640，接种至12孔板中，每孔1ml，含有5×10 5个细胞。
b.用PBS连续IO倍稀释病毒样品，稀释梯度10 -2～10 -5。
c.将稀释后的病毒样品加至12孔板，10ul/孔。
d.5％C02、37℃培养48小时。
e.吸尽培养液，在超净台内吹5分钟。
②细胞固定和标记一抗
a.往每孔加入lml冰冷的l00％甲醇,-20℃孵育10分钟。
b.吸尽甲醇,用lml含有1％BSA的PBS轻轻洗板3次。
c.用含有1％BSA的PBS稀释小鼠抗Hexon抗体至1：1000。
d.吸尽洗涤液,加入1：1000稀释的小鼠抗Hexon抗体0.5ml/孔，37℃振荡孵育1小时。
e.吸去小鼠抗Hexon抗体，用lml含有1％BSA的PBS轻轻洗板3次。
f.用含有1％BSA的PBS稀释HRP标记的大鼠抗小鼠抗体至1：500。
g.吸去洗涤液，加入1：500稀释的大鼠抗小鼠抗体O.5ml/孔，37℃振荡孵育1小时。
h.用1xStablePeroxidaseBuffer稀释10×DAB Substrate至1×工作液，液平衡至室温。
i.吸去HRP标记的大鼠抗小鼠抗体，用lml含有1％BSA的PBS轻轻洗板，3次。
③显色
a.吸去含有1％BSA的PBS，加入DAB工作液，500u1/孔，室温下孵育10分钟。
b.吸去DAB工作液，每孔加入Iml PBS。
c.用20倍物镜计数阳性染色细胞(黑/褐色)，至少观察3个视野，计算每孔的平均阳性细胞数。
d.感染单位：ifu＝每个视野的阳性细胞数×每孔视野数÷病毒液体积(m1)÷稀释倍数。
(3)纯度鉴定
①仪器准备
a.高效液相色谱仪(HPll00型，美国惠普公司产品)。
b.强阴离子交换预装柱为Q Sepharose XL(Amersharn pharmacia公司，瑞典)，柱基质为交连琼脂糖，填料直径45～165um，柱床体积(CV>1ml，保存于20％的乙醇中，电荷为-N+(CH3)，离子交换能力为0.18～0.26mmolC1-/ml胶。
②实验方法
a.用A液(20mM Tris-HCI，pH7.5)柱平衡，加样量为150ul，流速为1ml/min,压力为26bar，温度为26℃,监测OD260。
b.用4.5CV的A液淋洗。
c.用25.5CV 0～70％梯度的B液(1M NaCl+20mMTris-HCl，pH7.5)淋洗。
d.再用第二梯度3CV 70-100％B液洗脱。以上所有样本及流动相均用0.45um滤膜过滤,样本重复测试。
7、重组腺病毒(Ad-ORF1ab/N)基因的表达检测
(1)细胞培养
Vcro-E6细胞株是一种非洲绿猴肾的上皮细胞(ATCC CRL 1586)，来自ATCC，用含10％FBS、2mM谷氨酰胺、100U/ml青霉素和10099/ml链霉素的1640培养液在37℃、5％C02和饱和湿度下培养。
(2)RT-PCR检测
①Vem-E6细胞种入6孔板中，2x10 5个细胞/孔。
②16h以后，以不同剂量Ad-ORF1ab或Ad-N(20MOI)感染Vero-E6细胞48h。
③收集感染细胞。
④用TRlZOL试剂抽提(Invitrogen)细胞总RNA。
⑤用RQl RNase-free DNase I(Promega)去除残留的DNA。
⑥用AMV反转录系统(Invitrogen)将mRNA反转录cDNA：RNA:4ug；10×AMVBuffer：2ul；AMV逆转录酶(Promega):20U；RNAsin:20U；dNTP:0.5ul；OligodT(Promega):30pmol,用去离子水补至总体种20ul。
⑦反应条件：混匀，37℃反应60分钟。95℃10分钟灭活逆转录酶。
⑧PCR扩增cDNA。
a.据前述设计扩增片段为624bp的ORF1ab或N引物。
b.反应体系：上述逆转产物:20ul；10×PCR Buffer:5ul；引物:各30pmol；pfuDNA聚合酶(Gibco):2.5U,用去离子水补至总体积50ul。
c.在PE 9600热循环仪扩增，95℃预变性8分钟，循环参数：94℃1分钟，55℃1分钟，72℃90秒，35个循环。
d.用1％琼脂糖凝胶电泳分析，紫外灯下观察。
(3)Western blot鉴定
①Vero-E6细胞种入6孔板中，2x10 15个细胞/孔。
②16h以后，以不同剂量Ad-ORF1ab/N(20MOI)感染Vero-E6细胞48h。
③收集培养上清，制备蛋白样品。
④SDS-PAGE电泳。
⑤在4℃条件下将电泳结果转印到硝酸纤维膜上。
⑥封闭。
⑦加入1：1000的一抗，充分作用，洗涤。
⑧加入HRP偶联的抗兔lgG(1：1000)作用，洗涤。
⑨经LumiGLO TM(NEB)处理、胶片感光，观察实验结果。
8、重组腺病毒(Ad-ORF1ab/N)的免疫功能检测
(1)实验动物：按文献报道进行，Wistar大鼠在SPF级的动物实验室中培养，雌雄各半，6～8周龄，体重110+10g，记录动物生产合格证号。
(2)Ad-ORF1ab或Ad-N：结合真核发酵技术和腺病毒柱层析纯化技术，Ad-ORF1ab或Ad-N自行扩增、CsCl两次离心纯化，滴度为5x10 10pfu/ml。
(3)nCoV-2019抗体(IgG)检测系统：从以后可能生产的公司购买，HRP偶联的抗大鼠IgG可购自Santa Cruiz公司。
(4)拟定实验方法
①Ad-S N滴鼻对照组(空载体对照组)，先用3％戊巴比妥腹腔注射麻醉，5分钟后滴鼻，O.5ml/只，剂量1×10 7pfu/dose/rat。
②Ad-ORF1ab/N滴鼻组，同上麻醉，5分钟后滴鼻，剂量1×10 7pfu/dose/rat。
③Ad-S N尾静脉注射对照组(空载体对照组)，剂量1×10 7pfu/dose/rat。
④Ad-ORF1ab/N尾静脉注射组，尾静脉注射给药，剂量1×10 7pfu/dose/rat。
⑤空白对照组，尾静脉注射PBS，O.5ml/只鼠。
以上各组，每组10只动物。
(5)免疫程序：大鼠在第0、1、2周免疫动物共三次。收集第0、1、2、3、4周的血清。
(6)检测指标
①nCoV-2019特异IgG抗体的检测：以HRP偶联的抗大鼠IgG(使用浓度1：5000)为二抗，用人nCoV-2019抗体(IgG)检测系统检测大鼠血清的抗nCoV-2019 IgG抗体水平，每个样品均设三复孔。用酶标仪测定OD450，参考波长为630。滴定终点定义为OD450比阴性对照组至少高0.16的最高稀释度的自然对数。
②免疫保护实验：用Vero-E6为模型，测定动物血清保护细胞免受nCoV-2019感染的作用。该部分实验在3级安全(3P)实验室完成。
中和滴度定义为可以完全抑制Vero.E6细胞的最高血清稀释度。
9、重组腺病毒(Ad-nCoV2019)的批量制备
按本发明上述实验方法批量制备重组腺病毒Ad-nCoV2019,置-70℃保存备用。其中Ad 代表重组腺病毒载体，nCoV2019代表能表达蛋白的功能基因，例如ORF1ab或N基因。本发明中Ad-nCoV2019也称为Ad-nCoVDNA。
三、双价疫苗的配制
将实施例1制备的Ad-nCoV2019dsRNA和实施例2制备的Ad-nCoV2019DNA与H 20按Ad-nCoV2019dsRNA：Ad-nCoV2019DNA：H 20的体积比＝1：1：5～19配制成本发明的新型冠状病毒肺炎双价疫苗。

Claims (9)

  1. 一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，由Ad-nCoVdsRNA疫苗和Ad-nCoVDNA疫苗按比例配制组成，其中Ad-nCoVdsRNA：Ad-nCoVDNA：H 20的体积比＝1：1：19；所述Ad-nCoVdsRNA的制备方法为：扩增nCoV2019的靶向干扰基因shRNA序列，所得产物与空干扰载体pSilencer经BamH I和Hind III酶切而构建干扰载体pSilencer-shRNA，该干扰载体pSilencer-shRNA经感受态大肠杆菌DH5a扩增并经鉴定shRNA插入无误后与空穿梭载体pDC312经Hind lII和EcoR I酶切而构建穿梭载体pDC312-shRNA，该穿梭载体与腺病毒骨架质粒pBHGloxAEl共转染HEK293细胞，在该细胞内进行同源重组而获得重组腺病毒Ad-shRNA，再经HEK293细胞多次扩增后制备Ad-nCoV2019dsRNA；所述Ad-nCoV2019DNA的制备方法为：扩增nCoV2019的抗体表达基因，所扩增的抗体表达基因序列与腺病毒穿梭质粒pShuttle经XbaI和KpnI酶切后构建重组腺病毒穿梭质粒pShuttle-nCoV2019，该穿梭质粒经感受态大肠杆菌DH5a扩增并经XbaI和KpnI酶切鉴定nCoV2019无误后和腺病毒骨架质粒pAd-nCoV2019共转染HEK293细胞，在HEK293细胞内进行同源重组而获得重组腺病毒Ad-nCoV2019，该重组腺病毒经HEK293细胞多次扩增后制备Ad-nCoV2019DNA。
  2. 根据权利要求1所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述靶向干扰基因shRNA序列指长度为19nt的与siRNA序列互补的RNA序列。
  3. 根据权利要求1所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述靶向干扰基因shRNA序列为表达发夹结构的模板，由两条大部分互补的单链DNA构成，经退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链。
  4. 根据权利要求1所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述nCoV2019指新型冠状病毒的保守基因或功能基因序列；所述保守基因或功能基因序列包括ORF1ab、3'UTR、S、E、M、N基因序列。
  5. 根据权利要求1所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述pShuttle-nCoV2019指含有nCoV2019的重组腺病毒穿梭质粒；所述pAd-nCoV2019指含有nCoV2019的腺病毒骨架质粒；所述Ad-nCoV2019指含有nCoV2019的复制缺陷型重组腺病毒载体；所述Ad指复制缺陷型重组腺病毒载体。
  6. 根据权利要求1所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述Ad-nCoV2019DNA指携带抗体表达基因的重组腺病毒。
  7. 根据权利要求1所述的新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述双价疫苗接种后，Ad将nCoV2019dsRNA和nCoV2019DNA导入细胞内，其中nCoV2019dsRNA立即通过其shRNA产生dsRNA，即时降解同源的病毒mRNA，nCoVDNA则通过编码mRNA而表达蛋白，随后产生抗体而中和病毒。
  8. 根据权利要求7所述的新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述双价疫苗通过喷雾接种，Ad将nCoV2019dsRNA和nCoV2019DNA导入呼吸道上皮细胞，nCoV2019dsRNA在细胞内合成dsRNA，继之特异性诱导同源的nCoV2019 mRNA降解，产生抗nCoV2019的转录后基因沉默或RNA干扰，nCoV2019DNA则通过编码mRNA而表达蛋白，随后产生抗体而中和病毒。
  9. 根据权利要求1或6所述的一种新型冠状病毒肺炎双价疫苗的制备方法，其特征在于，所述抗体表达基因指能表达mRNA，mRNA表达蛋白，蛋白刺激机体产生抗体的基因。
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