CN111228475A - Biological product for preventing novel coronavirus - Google Patents
Biological product for preventing novel coronavirus Download PDFInfo
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- CN111228475A CN111228475A CN202010107992.3A CN202010107992A CN111228475A CN 111228475 A CN111228475 A CN 111228475A CN 202010107992 A CN202010107992 A CN 202010107992A CN 111228475 A CN111228475 A CN 111228475A
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Abstract
A biological product for preventing new coronavirus (COVID-19) is provided. The biological product can be gene vaccine or gene medicine, the gene vaccine adopts human adenovirus 5 with E1 and E3 gene deleted as a vector, and carries and expresses S1 protein antigen of Spike S1 subunit of the novel coronavirus or simultaneously carries and expresses S1 protein antigen and N protein antigen. The immune response of the organism to the novel coronavirus is activated by the in vivo expression of the antigen protein, so that the effect of preventing the infection and infection of the novel coronavirus is achieved.
Description
Technical Field
The invention belongs to the technical field of biological pharmaceutical products, and relates to a biological product for preventing novel coronavirus, which is used for developing a gene vaccine product for preventing novel coronavirus (COVID-19) by utilizing gene synthesis, codon optimization and gene cloning to construct a recombinant adenovirus vector gene vaccine.
Background
The burst of the novel coronavirus (COVID-19) brings huge crisis to social public health, the infectivity is very strong, huge social health and economic influences are caused to human beings, the high attention of people is paid, and no effective treatment method and prevention vaccine exist at present. For the health of the people's public, the safety of the country and society, there is an urgent need to develop vaccines that can prevent the infection of novel coronaviruses.
Commonly used development methods for viral vaccines include 1) inactivated virus vaccines, 2) inactivated virus vaccines, 3) subunit vaccines, 4) vlp (virus like particle) vaccines, 5) DNA plasmid vaccines, and 6) recombinant viral vector vaccines. The high-tech recombinant virus vector gene vaccine is the latest technology for vaccine research and development, and the vaccine is safe and effective and has short research and development time. The FDA approved a vaccine product for the prevention of Ebola (Ebola) virus infection (Merck, ervbo, usa) that was marketed recently, 12/19/2019, as a recombinant viral vector gene vaccine (rVSV-ZEBOV) product.
The vaccine adopting the recombinant adenovirus vector has good clinical effect in preventing and treating MERS coronavirus infection similar to novel coronavirus. Provides scientific basis for developing novel coronavirus vaccines by adopting recombinant adenovirus vectors. In addition, recombinant adenoviral vector vaccines have several distinct advantages, including:
1) the vaccine has high safety. Adenovirus vectors are the most widely used viral vectors, and the safety is widely accepted. Clinical data of the marketed recombinant adenovirus products which are 'live today' for more than ten years can give full evidence;
2) the vaccine antigen protein after inoculation has fast expression speed and high expression amount. Can quickly and effectively trigger the preventive immunity against the novel coronavirus;
3) the vaccine is fast in research and development speed and mature in technology. After the novel coronavirus RNA gene sequence exists, the construction of vaccine seeds can be completed quickly within 3-4 weeks;
4) ready-made vaccine production technology;
5) the vaccine product is suitable for large-scale GMP production and wide public prevention and treatment application;
6) the vaccine product has high stability, can survive for more than 10 years at the temperature of minus 80 ℃, and is suitable for strategic storage for later use.
Recombinant virus vector gene vaccines are one of the latest vaccine development technologies recognized worldwide. At present, the prevalence of the next generation of rapid gene sequence detection technology ensures that scientists can classify and accurately sequence the gene sequence of the virus causing the disease in a short time. By adopting accurate gene cloning and modifying technology, the recombinant virus vector gene vaccine expressing the main virus antigen can be constructed accurately at the fastest speed. The expressed viral antigen maintains the structure and framework of the wild virus, ensuring that a powerful prophylactic immune response can be elicited in the patient. Conventional inactivated or inactivated vaccine preparations require live virus strains, and the inactivation or inactivation process also affects the integrity of important viral antigens and thus the ability to elicit an immune response in a vaccinated patient. In addition, live virus strains are not involved in the vaccine construction process, and the safety of the construction process is ensured.
Disclosure of Invention
The novel coronavirus is an enveloped positive-strand RNA virus with a genome length of 29.9 Kbp. A schematic of the structure of the novel coronavirus particle and the major viral structural proteins are shown in figure 1.
According to the mechanism of the novel coronavirus infected patient, the S1(Spike S1 subunit) protein and the N (nucleocapsid) protein of the virus are found to have strong immunity, can trigger antibody and T cell reaction in the patient body and are the optimal virus antigens for developing vaccines through research.
To infect a patient, the Spike protein (Spike, S protein) on the surface of the novel coronavirus first interacts with a receptor that infects cells of the patient and attaches to the cell surface. The S protein consists of two subunits, the S1 and S2 subunits. The S1 subunit includes the receptor binding domain functions of the virus, and the S2 subunit includes the functions of fusing the viral membrane and cell membrane. Cell surface peptidases are one of the major viral receptors. After the virus is attached to the cell surface, acid-dependent proteolytic cleavage of the cleavage site located between the S1 and S2 subunits is performed by cathepsin, the S protein is cleaved, fusion of the viral membrane and cell membrane is initiated, and the viral genome is transported into the cell, causing infection.
Wherein the S1 subunit of the ectodomain of the spike protein is the major part of the interaction with cell surface receptors, and the nucleocapsid N protein (nucleocapsid) of the virus is the major structural protein of the virus, except for the spike protein. According to the structure of the novel coronavirus and the mechanism of infecting patients, the S1(spike) protein and the N protein of the virus are the main viral antigens that elicit strong immune responses, and are the best viral antigens for developing vaccines.
The invention aims to construct a biological product for preventing novel coronavirus (COVID-19), wherein the biological product can be a gene vaccine or a gene medicine, and the recombinant adenovirus vector gene vaccine is constructed as an example in the following.
The recombinant adenovirus vector gene vaccine for preventing new type coronavirus is constructed with the antigen protein gene expression box of the new type coronavirus and through the expression of the antigen protein gene inside body, the immune reaction of organism to the new type coronavirus is activated to prevent the infection and infection of the new type coronavirus.
The structure of the antigen protein gene expression cassette comprises a CMV promoter, a Kozak sequence, an antigen protein gene and an SV40polyA sequence which are connected in sequence, namely the structure can be expressed as a CMV promoter-Kozak sequence-antigen protein gene-SV 40polyA sequence.
The antigen protein gene is S1 protein gene expressing S1 protein antigen, or S1 protein gene-T2A-N protein gene expressing S1 protein antigen and N protein antigen simultaneously.
According to the publicly published gene sequence (GenBank: MN908947.3) of the novel coronavirus, codon optimization was first performed, and gene fragments of the optimized S1 protein gene and N protein gene were synthesized by a conventional gene synthesis method.
Wherein, the S1 protein gene and/or the N protein gene is a gene sequence of the novel coronavirus which is artificially synthesized and optimized by a codon, and preferably, the nucleotide sequence of the S1 protein gene optimized by the codon is shown as SEQ ID NO. 1. The nucleotide sequence of the codon optimized N protein gene is shown as SEQ ID NO. 2.
By adopting a conventional gene cloning method, the invention constructs an S1 protein gene expression cassette for expressing S1 protein to achieve high expression of S1 protein, and the structure of the S1 protein gene expression cassette is shown in FIG. 2.
The nucleotide sequence of the S1 protein gene expression cassette is shown in SEQ ID NO. 3.
In order to improve the immune response to the maximum extent, the invention also constructs a gene expression cassette of the S1 protein and the N protein for simultaneously expressing the S1 protein and the N protein so as to achieve the simultaneous high expression of the S1 protein and the N protein. The structures of the S1 protein and N protein gene expression cassettes are shown in fig. 3.
The nucleotide sequences of the S1 protein and N protein gene expression cassettes are shown in SEQ ID NO. 4.
The constructed gene expression cassettes are respectively cloned into an expression shuttle plasmid of a pAdEasy system. The shuttle plasmid and the human adenovirus 5 plasmid with the deleted E1 and E3 genes of the pAdEasy system were simultaneously transduced into HEK293 cells. Through gene homologous recombination in HEK293 cells, recombinant adenovirus vector gene vaccine seeds expressing S1 protein antigen and expressing S1 and N protein antigen simultaneously are generated respectively.
The vaccine seeds were confirmed by DNA sequencing to carry the correct S1 protein and N protein gene sequences and the correct gene structure. Further expansion in HEK293 cells by cell culture establishes a vaccine seed bank for large scale production and clinical application.
Drawings
FIG. 1 is a schematic diagram of the structure of a novel coronavirus particle.
FIG. 2 is a schematic structural diagram of an S1 protein gene expression cassette.
FIG. 3 is a schematic structural diagram of gene expression cassettes of S1 protein and N protein.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the biochemical reagents and materials used in the examples are commercially available.
Example 1 construction of recombinant adenovirus vector Gene vaccine seeds expressing S1 protein antigen
The gene sequence of the S1 protein (GenBank: MN908947.3) of the novel coronavirus was optimized using codon optimization software of IDT to increase the protein expression rate in human body. The optimized nucleotide sequence is shown as SEQ ID NO. 1. The gene fragment of the S1 protein was synthesized from the order gene of IDT corporation in usa. Gene sequencing verified the correctness of the gene sequence.
In order to realize rapid and high-quantity expression of the S1 protein, a CMV promoter is adopted in an expression cassette of the S1 protein gene, and a Kozak sequence is added. The SV40polyA sequence was used to ensure correct protein expression. The complete S1 protein gene expression cassette is cloned and constructed by adopting the conventional molecular biological method and reagent. A schematic of the S1 protein gene expression cassette is shown in fig. 2.
The nucleotide sequence of the S1 protein gene expression cassette is shown in SEQ ID NO.3, and the length is 2703 bp.
The constructed S1 protein gene expression cassette was cloned into a commercially available expression shuttle plasmid of the pAdEasy system according to the method provided by the supplier. The shuttle plasmid and the human adenovirus 5 plasmid with the deleted E1 and E3 genes of the pAdEasy system were simultaneously transduced into HEK293 cells. Through gene homologous recombination in HEK293 cells, recombinant adenovirus vector gene vaccine seeds expressing S1 protein antigens are generated.
The vaccine seeds are confirmed to carry the correct S1 protein gene expression cassette sequence through DNA sequencing, and the nucleotide sequence is shown as SEQ ID NO. 3.
The constructed vaccine seeds can be further amplified in HEK293 cells through cell culture, and a vaccine seed bank is established for the production and clinical application of large-scale vaccine products.
Example 2 construction of recombinant adenovirus vector Gene vaccine seed expressing both S1 protein antigen and N protein antigen
The gene sequence of S1 protein and the gene sequence of N protein (GenBank: MN908947.3) of the novel coronavirus were optimized using codon optimization software of IDT corporation to increase the protein expression rate in human body. The optimized S1 nucleotide sequence is shown as SEQ ID NO.1, and the N nucleotide sequence is shown as SEQ ID NO. 2. In order to realize the expression of S1 protein antigen and N protein antigen in equivalent quantity at the same time, a gene for expressing a T2A cleavable polypeptide is added between an S1 protein gene and an N protein gene, and a compound gene segment for expressing S1 protein and N protein is formed. Gene fragments expressing the S1 protein and the N protein were synthesized from the order genes of IDT, USA. Gene sequencing verified the correctness of the gene sequence.
In order to realize rapid and high-quantity simultaneous expression of the S1 protein and the N protein, a CMV promoter and a Kozak sequence are adopted in an expression cassette of the S1 protein and the N protein. The SV40polyA sequence was used to ensure correct protein expression. The complete S1 protein and N protein compound gene expression cassette, namely the S1 protein and N protein gene expression cassette, is cloned and constructed by adopting the conventional molecular biological method and reagent. A schematic of the S1 protein and N protein gene expression cassettes are shown in fig. 3.
The nucleotide sequence of the S1 protein and N protein gene expression cassette is shown in SEQ ID NO.4, and the length is 4032 bp.
The constructed complex gene expression cassette of S1 protein and N protein was cloned into a commercially available expression shuttle plasmid of the pAdEasy system according to the method provided by the supplier. The shuttle plasmid and the human adenovirus 5 plasmid with the deleted E1 and E3 genes of the pAdEasy system were simultaneously transduced into HEK293 cells. Through gene homologous recombination in HEK293 cells, recombinant adenovirus vector gene vaccine seeds capable of expressing S1 protein antigens and N protein antigens simultaneously are generated.
The DNA sequencing confirms that the vaccine seed carries the correct sequence of the composite gene expression cassette of the S1 protein and the N protein, and the nucleotide sequence is shown as SEQ ID NO. 4.
The constructed vaccine seeds can be further amplified in HEK293 cells through cell culture, and a vaccine seed bank is established for the production and clinical application of large-scale vaccine products.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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<120> biological product for preventing novel coronavirus
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cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag acacttgaga ttcttgacat 1760
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gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa atgaaagatc tcagtccaag 320
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cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt caagcctctt ctcgttcctc 560
atcacgtagt cgcaacagtt caagaaattc aactccaggc agcagtaggg gaacttctcc tgctagaatg gctggcaatg 640
gcggtgatgc tgctcttgct ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa aaacgtactg ccactaaagc 800
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aaggaactga ttacaaacat tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
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catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 320
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 400
ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480
acggtgggag gtctatataa gcagagctgc caccatgttt gtttttcttg ttttattgcc actagtctct agtcagtgtg 560
ttaatcttac aaccagaact caattacccc ctgcatacac taattctttc acacgtggtg tttattaccc tgacaaagtt 640
ttcagatcct cagttttaca ttcaactcag gacttgttct tacctttctt ttccaatgtt acttggttcc atgctataca720
tgtctctggg accaatggta ctaagaggtt tgataaccct gtcctaccat ttaatgatgg tgtttatttt gcttccactg 800
agaagtctaa cataataaga ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat tgttaataac 880
gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc atttttgggt gtttattacc acaaaaacaa 960
caaaagttgg atggaaagtg agttcagagt ttattctagt gcgaataatt gcacttttga atatgtctct cagccttttc 1040
ttatggacct tgaaggaaaa cagggtaatt tcaaaaatct tagggaattt gtgtttaaga atattgatgg ttattttaaa 1120
atatattcta agcacacgcc tattaattta gtgcgtgatc tccctcaggg tttttcggct ttagaaccat tggtagattt 1200
gccaataggt attaacatca ctaggtttca aactttactt gctttacata gaagttattt gactcctggt gattcttctt 1280
caggttggac agctggtgct gcagcttatt atgtgggtta tcttcaacct aggacttttc tattaaaata taatgaaaat 1360
ggaaccatta cagatgctgt agactgtgca cttgaccctc tctcagaaac aaagtgtacg ttgaaatcct tcactgtaga 1440
aaaaggaatc tatcaaactt ctaactttag agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt 1520
gcccttttgg tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag caactgtgtt 1600
gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg ttatggagtg tctcctacta aattaaatga 1680
tctctgcttt actaatgtct atgcagattc atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg 1760
gaaagattgc tgattataat tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa caatcttgat 1840
tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc taatctcaaa ccttttgaga gagatatttc 1920
aactgaaatc tatcaggccg gtagcacacc ttgtaatggt gttgaaggtt ttaattgtta ctttccttta caatcatatg 2000
gtttccaacc cactaatggt gttggttacc aaccatacag agtagtagta ctttcttttg aacttctaca tgcaccagca 2080
actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt caatttcaac ttcaatggtt taacaggcac 2160
aggtgttctt actgagtcta acaaaaagtt tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg 2240
tccgtgatcc acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca gtgttataac accaggaaca 2320
aatacttcta accaggttgc tgttctttat caggatgtta actgcacaga agtccctgtt gctattcatg cagatcaact 2400
tactcctact tggcgtgttt attctacagg ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg 2480
tcaacaactc atatgagtgt gacataccca ttggtgcagg tatatgcgct agttatcaga ctcagactaa ttctcctcgg 2560
cgggcacgtt aaaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 2640
atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctg 2703
<210>4
<211>4032
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<223> nucleotide sequence of gene expression cassette of S1 protein and N protein
<400>4
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 80
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 160
catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 320
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 400
ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480
acggtgggag gtctatataa gcagagctgc caccatgttt gtttttcttg ttttattgcc actagtctct agtcagtgtg 560
ttaatcttac aaccagaact caattacccc ctgcatacac taattctttc acacgtggtg tttattaccc tgacaaagtt 640
ttcagatcct cagttttaca ttcaactcag gacttgttct tacctttctt ttccaatgtt acttggttcc atgctataca 720
tgtctctggg accaatggta ctaagaggtt tgataaccct gtcctaccat ttaatgatgg tgtttatttt gcttccactg 800
agaagtctaa cataataaga ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat tgttaataac 880
gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc atttttgggt gtttattacc acaaaaacaa 960
caaaagttgg atggaaagtg agttcagagt ttattctagt gcgaataatt gcacttttga atatgtctct cagccttttc 1040
ttatggacct tgaaggaaaa cagggtaatt tcaaaaatct tagggaattt gtgtttaaga atattgatgg ttattttaaa 1120
atatattcta agcacacgcc tattaattta gtgcgtgatc tccctcaggg tttttcggct ttagaaccat tggtagattt 1200
gccaataggt attaacatca ctaggtttca aactttactt gctttacata gaagttattt gactcctggt gattcttctt 1280
caggttggac agctggtgct gcagcttattatgtgggtta tcttcaacct aggacttttc tattaaaata taatgaaaat 1360
ggaaccatta cagatgctgt agactgtgca cttgaccctc tctcagaaac aaagtgtacg ttgaaatcct tcactgtaga 1440
aaaaggaatc tatcaaactt ctaactttag agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt 1520
gcccttttgg tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag caactgtgtt 1600
gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg ttatggagtg tctcctacta aattaaatga 1680
tctctgcttt actaatgtct atgcagattc atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg 1760
gaaagattgc tgattataat tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa caatcttgat 1840
tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc taatctcaaa ccttttgaga gagatatttc 1920
aactgaaatc tatcaggccg gtagcacacc ttgtaatggt gttgaaggtt ttaattgtta ctttccttta caatcatatg 2000
gtttccaacc cactaatggt gttggttacc aaccatacag agtagtagta ctttcttttg aacttctaca tgcaccagca 2080
actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt caatttcaac ttcaatggtt taacaggcac 2160
aggtgttctt actgagtcta acaaaaagtt tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg 2240
tccgtgatcc acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca gtgttataac accaggaaca 2320
aatacttcta accaggttgc tgttctttat caggatgtta actgcacaga agtccctgtt gctattcatg cagatcaact 2400
tactcctact tggcgtgttt attctacagg ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg 2480
tcaacaactc atatgagtgt gacataccca ttggtgcagg tatatgcgct agttatcaga ctcagactaa ttctcctcgg 2560
cgggcacgtc tcgagggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctat 2640
gtctgataat ggaccccaaa atcagcgaaa tgcaccccgc attacgtttg gtggaccctc agattcaact ggcagtaacc 2720
agaatggaga acgcagtggg gcgcgatcaa aacaacgtcg gccccaaggt ttacccaata atactgcgtc ttggttcacc 2800
gctctcactc aacatggcaa ggaagacctt aaattccctc gaggacaagg cgttccaatt aacaccaata gcagtccaga 2880
tgaccaaatt ggctactacc gaagagctac cagacgaatt cgtggtggtg acggtaaaat gaaagatctc agtccaagat 2960
ggtatttcta ctacctagga actgggccag aagctggact tccctatggt gctaacaaag acggcatcat atgggttgca 3040
actgagggag ccttgaatac accaaaagat cacattggca cccgcaatcc tgctaacaat gctgcaatcg tgctacaact 3120
tcctcaagga acaacattgc caaaaggctt ctacgcagaa gggagcagag gcggcagtca agcctcttct cgttcctcat 3200
cacgtagtcg caacagttca agaaattcaa ctccaggcag cagtagggga acttctcctg ctagaatggc tggcaatggc 3280
ggtgatgctg ctcttgcttt gctgctgctt gacagattga accagcttga gagcaaaatg tctggtaaag gccaacaaca 3360
acaaggccaa actgtcacta agaaatctgc tgctgaggct tctaagaagc ctcggcaaaa acgtactgcc actaaagcat 3440
acaatgtaac acaagctttc ggcagacgtg gtccagaaca aacccaagga aattttgggg accaggaact aatcagacaa 3520
ggaactgatt acaaacattg gccgcaaatt gcacaatttg cccccagcgc ttcagcgttc ttcggaatgt cgcgcattgg 3600
catggaagtc acaccttcgg gaacgtggtt gacctacaca ggtgccatca aattggatga caaagatcca aatttcaaag 3680
atcaagtcat tttgctgaat aagcatattg acgcatacaa aacattccca ccaacagagc ctaaaaagga caaaaagaag 3760
aaggctgatg aaactcaagc cttaccgcag agacagaaga aacagcaaac tgtgactctt cttcctgctg cagatttgga 3840
tgatttctcc aaacaattgc aacaatccat gagcagtgct gactcaactc aggcctaatg aaacttgttt attgcagctt 3920
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 4000
tccaaactca tcaatgtatc ttatcatgtc tg 4032
Claims (10)
1. The biological product for preventing the novel coronavirus is characterized in that an expression vector is constructed by carrying an antigen protein gene expression cassette of the novel coronavirus, and the immune response of a human body to the novel coronavirus is activated by the in-vivo expression of the antigen protein gene, so that the infection and infection of the novel coronavirus are prevented.
2. The biological preparation for preventing a novel coronavirus according to claim 1, wherein the antigen protein gene expression cassette has a structure comprising a CMV promoter, a Kozak sequence, an antigen protein gene and an SV40polyA sequence, which are sequentially linked.
3. The biological preparation for preventing a novel coronavirus according to claim 2, wherein the antigenic protein gene is an S1 protein gene expressing an S1 protein antigen, or an S1 protein gene-T2A-N protein gene expressing both an S1 protein antigen and an N protein antigen.
4. The biological preparation for preventing a novel coronavirus according to claim 3, wherein the S1 protein gene and/or the N protein gene are artificially synthesized and codon-optimized gene sequences of the novel coronavirus.
5. The biological preparation for the prevention of a novel coronavirus according to claim 4, wherein the nucleotide sequence of the S1 protein gene is represented by SEQ ID No. 1.
6. The biological preparation for the prevention of a novel coronavirus according to claim 4, wherein the nucleotide sequence of the N protein gene is represented by SEQ ID No. 2.
7. The biological preparation for the prevention of a novel coronavirus according to claim 3, wherein the nucleotide sequence of the antigenic protein gene expression cassette is represented by SEQ ID No. 3.
8. The biological preparation for the prevention of a novel coronavirus according to claim 3, wherein the nucleotide sequence of the antigenic protein gene expression cassette is represented by SEQ ID No. 4.
9. The biological preparation for the prevention of a novel coronavirus according to claim 1, wherein said expression vector is a human adenovirus type 5 or a plasmid in which genes E1 and E3 are deleted.
10. The biological preparation according to any one of claims 1 to 9 for use in the prevention of a novel coronavirus which biological preparation is a genetic vaccine or a genetic drug.
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| CN202010107992.3A CN111228475A (en) | 2020-02-21 | 2020-02-21 | Biological product for preventing novel coronavirus |
| PCT/CN2020/080861 WO2021164097A1 (en) | 2020-02-21 | 2020-03-24 | Biological product for preventing novel coronavirus |
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| CN202010107992.3A CN111228475A (en) | 2020-02-21 | 2020-02-21 | Biological product for preventing novel coronavirus |
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| CN111569058A (en) * | 2020-06-18 | 2020-08-25 | 武汉生物制品研究所有限责任公司 | SARS-CoV-2 inactivated vaccine and its preparation method |
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| CN112618707A (en) * | 2020-10-15 | 2021-04-09 | 广州达博生物制品有限公司 | SARS-CoV-2 coronavirus vaccine and its preparation method |
| CN112641937A (en) * | 2020-10-15 | 2021-04-13 | 广州达博生物制品有限公司 | Application of recombinant adenovirus in preparing medicine for preventing virus |
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| CN113336831A (en) * | 2020-07-24 | 2021-09-03 | 上海延立药业有限公司 | Long-acting recombinant glycoprotein for developing new coronavirus vaccine |
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