Degron-containing recombinant viruses and methods for their preparation and use

Abstract

The present application also provides a degron-containing recombinant virus and its preparation method and use, wherein at least one viral protein contains at least one degron that can be recognized by a host cell's proteolytic system, and the degron comprises any one or a combination of at least two of an amino acid sequence, a polypeptide, or a structural motif. The present application also provides a nucleic acid molecule, a recombinant vector, a method for preparing the degron-containing recombinant virus, a system for preparing the degron-containing recombinant virus, a vaccine, an oncolytic virus, and a pharmaceutical. [Means] The degron-containing recombinant virus of the present application can be recognized and degraded by the proteolytic system in host cells, and its replication ability is weakened or even eliminated. After being prepared into corresponding vaccines, oncolytic viruses or medicines, they have good efficacy and practical application value.

Description

[Technical Field]

[0001] The present application is in the field of biotechnology, and in particular relates to degron-containing recombinant viruses and methods for their preparation and use. [Background technology]

[0002] Viral infections pose serious threats to human health and socioeconomic development. Vaccines are one of the most effective means of preventing viral infections. Currently, viral vaccines are available in various forms, including inactivated vaccines, attenuated vaccines, subunit vaccines, viral vector vaccines, DNA vaccines, mRNA vaccines, and virus-like particles.

[0003] Although viral vaccine technology has made great progress over the past few decades, there are still limitations, such as the destruction of the natural structure of antigens due to virus inactivation treatment, resulting in reduced immunogenicity of the viral vaccine, or the safety risks of the vaccine due to residual replication activity of the viral vaccine due to insufficient virus attenuation, or the complicated preparation technology of viral vaccines, which has prevented their widespread use in other viral vaccines. Therefore, breaking through the traditional design concepts of viral vaccines and developing new vaccine technologies that are safe, highly efficient, simple, and versatile is an important development direction for the future of ideal viral vaccines, and has great scientific and clinical value.

[0004] How to provide a method for preparing a virus vaccine that is safer, more widely applicable, and more immunogenic has become an urgent issue that needs to be resolved. Summary of the Invention [Problem to be solved by the invention]

[0005] This application provides a degron-containing recombinant virus, and its preparation and use. The recombinant virus prepared by inserting at least one degron into at least one protein-encoding gene of the virus can be recognized by the proteolytic system of the host cell, where the viral protein can be degraded, weakening the replication ability of the virus and even completely eliminating its replication ability. The prepared vaccine or oncolytic virus is safer and has broader potential applications. [Means for solving the problem]

[0006] In a first aspect, the present application provides a degron-containing recombinant virus, wherein at least one viral protein contains at least one degron that can be recognized by a proteolytic system in a host cell, The degron comprises any one or a combination of at least two of an amino acid sequence, a polypeptide, or a structural motif.

[0007] In this application, by inserting at least one degron into at least one protein of a virus, the degron can be replicated along with the replication of the viral genome and fused to and expressed in the viral protein along with the translation of the viral protein, thereby obtaining a recombinant virus site-specifically modified by the degron. At the same time, the proteolytic system of the host cell is utilized to achieve stable degradation of the viral protein in the host cell, thereby artificially controlling the vital activities of the virus, such as replication in the host, and this can be used to prepare replication-controllable viral vaccines or oncolytic viruses, which has great application value.

[0008] As used herein, degrons refer to various molecules, such as specific amino acid sequences, polypeptides, structural motifs, or other molecules, that can be specifically recognized by proteolytic systems and mediate the degradation of matrix proteins. Degrons are typically short linear motifs with specific sequence models and strong evolutionary conservation of critically important amino acid residues.

[0009] Preferably, the proteolytic system includes any one or a combination of at least two of the ubiquitin-proteasome system, the lysosomal system, the organelle hydrolysis system, the cell membrane surface hydrolysis system, and the caspase protease system.

[0010] In this application, the normal execution of viral protein functions is a prerequisite for all life activities, such as viral replication in host cells. Multiple natural proteolytic systems exist in host cells and are crucial for maintaining intracellular protein stability and normal cellular function. These proteolytic systems can specifically identify degrons in matrix proteins. One important feature of degrons is their mobility, and after their attachment by genetic engineering, proteins with long life cycles become unstable. These proteolytic systems and corresponding degrons provide a biological basis for designing a "life switch" that regulates and stabilizes viral proteins in host cells.

[0011] Preferably, the degron is located in any one or a combination of at least two of the C-terminus, N-terminus, or middle coding region of the viral protein.

[0012] In this application, because the proteolytic systems of animals and humans can distinguish between multiple types of degrons, different types and numbers of degrons can be introduced into the N-terminus, C-terminus, or any other site of a viral protein. These different types and numbers of degrons can be combined arbitrarily to ensure the preparation of viral vaccines with different replication efficiencies and attenuation levels, which is extremely important for the production efficiency and immunogenicity of viral vaccines.

[0013] Preferably, the degron comprises any one of the amino acid sequences represented by SEQ ID Nos. 1 to 110.

[0014] SEQ ID No. 1: ALAPYIP, SEQ ID No.2:DRHDSGLDSM, SEQ ID No.3:SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV, SEQ ID No.4:FVNQHLCGSHLVEALYLVCGERGFFYTPKA, SEQ ID No. 5: DSGXXS (wherein X represents any amino acid), SEQ ID No. 6: DRHDSGXXSM (wherein X represents any amino acid), SEQ ID No. 7:LGSWRHWRGQEG, SEQ ID No. 8: SLYKKVVGTMAAG, SEQ ID No. 9:MLSESRNFPAQA, SEQ ID No. 10:ERAPTGRWGRRG, SEQ ID No. 11:KKVGRARPCQRG, SEQ ID No. 12:APRAPRQRSRDG, SEQ ID No. 13: SWRLTGSGMKG, SEQ ID No. 14:WRPGRRGPSSGG, SEQ ID No. 15:LRGPSPPPMAGG, SEQ ID No.16:WRPGRRGPSSGG、 SEQ ID No.17:MALAVRVVYCGA、 SEQ ID No.18:KKNVEAIGLLGG、 SEQ ID No.19:PPGPPLSSPRPR、 SEQ ID No.20:TMNNEEDLLRSL、 SEQ ID No.21:TMEPPGGRQKKR、 SEQ ID No.22:QERGPTWDKNLR、 SEQ ID No.23:GTMAVLRQRPGR、 SEQ ID No.24:LCTRSWDVTPNR、 SEQ ID No.25:ANQPAQCRKTRI、 SEQ ID No.26:EEARLKYDKSRI、 SEQ ID No.27:LNDGPKPGQSRF、 SEQ ID No.28:TSLYKKVGMGRK、 SEQ ID No.29:YKKVGTMRGRGL、 SEQ ID No.30:VGTMAAGRAPGK、 SEQ ID No.31:KKVGTMRGVGYP、 SEQ ID No.32:EGPLWHPRICGS、 SEQ ID No.33:EMALSPPRSWGQ、 SEQ ID No.34:RSGLRRRRHRGE、 SEQ ID No.35:VSGIMRRPWGMN、 SEQ ID No.36:KRVLIRVTYCGL、 SEQ ID No.37:MALAVRVVYCGA、 SEQ ID No.38:PSSPVQTTPLSQAVATPSRSSAAAAAALDLSGRRG、 SEQ ID No.39:HHGKGFFGSGHYTAYCYNTEGGACALLCGVGDTERG、 SEQ ID No.40:VAEITKQLPPVVPVSKPGALRRSLSRSMSQEAQRG、 SEQ ID No.41:KKRPPPGLDR、 SEQ ID No.42:SVNSLLKELR、 SEQ ID No.43:HEWVLREGEE、 SEQ ID No.44:EYLLKMATEE、 SEQ ID No.45:ELCKSYRRLQ、 SEQ ID No.46:VALKFKPRKH、 SEQ ID No.47:GLGCKVLRRH、 SEQ ID No.48:RHCGRT、 SEQ ID No.49:RSHGTL、 SEQ ID No.50:RFRGLR、 SEQ ID No.51:RNLGIR、 SEQ ID No.52:RGRLTRNKGP、 SEQ ID No.53:SLFRKRNKGK、 SEQ ID No.54:RTAASGRRWG、 SEQ ID No.55:GLLKRPCLRG、 SEQ ID No.56:QRKLQRTSRG、 SEQ ID No.57:PKSKVCQQRG、 SEQ ID No.58:KRLLKGSQYG、 SEQ ID No.59:PHKRLLKGSQYG、 SEQ ID No.60:KESNDCSCGG、 SEQ ID No.61:DCVCRGSTGG、 SEQ ID No.62、VAPRSRDERG、 SEQ ID No.63:AHQLQALRRG、 SEQ ID No.64:LTGKG、 SEQ ID No.65:YYCFFG、 SEQ ID No.66:LEKGG、 SEQ ID No.67:AAHKG、 SEQ ID No.68:ALRRG、 SEQ ID No.69:GGSGG、 SEQ ID No.70:RDERG、 SEQ ID No.71:SRVKG、 SEQ ID No.72:PASGG、 SEQ ID No.73:AIHGG、 SEQ ID No.74:GAEAG、 SEQ ID No.75:GSTGG、 SEQ ID No.76:RGMGG、 SEQ ID No.77:LVHAG、 SEQ ID No.78:SLQTG、 SEQ ID No.79:PVPGG、 SEQ ID No.80:NYKSG、 SEQ ID No.81:PRKQG、 SEQ ID No.82:TPRGG、 SEQ ID No.83:GCSGG、 SEQ ID No.84:EAQRG、 SEQ ID No.85:GKAWG、 SEQ ID No.86:PAGGG、 SEQ ID No.87:VVLYG、 SEQ ID No.88:LTLKG、 SEQ ID No.89:GFQSG、 SEQ ID No.90:RVQWG、 SEQ ID No.91:SRTEGQFGTTQSNGTFFNGASPGTPPAPSQHQQSLTSL、 SEQ ID No.92: YRPIPFQPEGAGEGTDEDKSNRIGNNGLRLNDGNGNGQLAPSPTPQGTEAVRA、 SEQ ID No.93:RKFSNNPQPNAISNGTSTSERPGEGATQGIVEEEVLQ、 SEQ ID No.94:IRTESAEEAEMASVPNGSPSWHPGASHVVNGAAGHSN、 SEQ ID No.95:WHEIEMESGEEAMEPANETGNTLNGSPSWHPSPSHVI、 SEQ ID No.96:NRTDFAGEEDEMDGVLNGSPSWHAATSHIVNGATVHQ、 SEQ ID No.97:NRTDTAAEAEMDSVLNGSPSWHPPAGHVVNGATVHRS、 SEQ ID No.98:NRTDTAAEAEMDSVLNGSPSWHPPAGHVVNGAAVHRS、 SEQ ID No.99:NRTEVLEGAEIPSTVNGSPSWHPADSRAVSGATGHSS、 SEQ ID No.100:NRTEAPEGTELPSTVNGSPSWHPADSRAGSGATGHSS、 SEQ ID No.101:NRTEAPEGTESERETPSAINGNPSWHLADSPAVNGAT、 SEQ ID No.102: NRTEAPEGTESEVETPSAINGNPSWQLADSPAINGATGHSSSLDAREVIPMAAVKQQAL、 SEQ ID No.103:NRTEAPEGTESDMETPSAINGNASWHLADSPAVNGAT、 SEQ ID No.104:NRTEAPEGTGPEMETPSAINGNPAWHPADSPAVNGAT、 SEQ ID No.105:NRTEAPEGTESDMETPSAINGNPSWHLADSPAVNGAT、 SEQ ID No.106:NRTEAPEGTESEAETPSAINGNPSWHLADSPAVNGAT、 SEQ ID No.107:NRTEAPEGTESEMETPSAINGNPSWHLADSPPANGAT、 SEQ ID No.108:NRTEAPEGTDSEMETPSAINGNPAWHLADSPAVNGAT、 SEQ ID No. 109:NRTEAPEGTDSEMETPSAINGNPSWHLADSPVVNGAT, or SEQ ID No.110:NRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGAT.

[0015] Preferably, the virus is influenza virus, AIDS virus, novel coronavirus (SARS-CoV-2), hand, foot and mouth virus, coxsackievirus, hepatitis C virus (HCV), hepatitis B virus (HBV), hepatitis A virus, hepatitis D virus, hepatitis E virus, human herpes virus (EB virus), human papillomavirus (HPV), herpes simplex virus (HSV), cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus (RSV), dengue virus, Ebola virus, Marburg virus, Zika virus, severe acute respiratory syndrome virus (SARS), Middle East respiratory virus (MRSA), or the like. The virus may be any one of the following: syndrome virus, rotavirus, rabies virus, measles virus, adenovirus, poliovirus, echovirus, Japanese encephalitis virus, shikoki encephalitis virus, hantavirus, new enteric virus, rubella virus, parotitis virus, parainfluenza virus, blue ear virus, swine fever virus, foot and mouth disease virus, minute virus, prion, smallpox virus, tobacco mosaic virus, phage, herpes virus, West Nile virus, norovirus, human bocavirus, or coronavirus, and is preferably any one of the influenza virus, AIDS virus, or novel coronavirus.

[0016] Preferably, any one or a combination of at least two of the influenza virus PA protein, PB1 protein, PB2 protein, NP protein, HA protein, NA protein, M1 protein, M2 protein, NS1 protein, or NEP protein comprises at least one degron.For example, the PA protein and PB2 protein of an influenza virus may both contain at least one degron, the PA protein and PB1 protein of an influenza virus may both contain at least one degron, the PB2 protein and PB1 protein of an influenza virus may both contain at least one degron, the PA protein, PB2 protein and PB1 protein of an influenza virus may both contain at least one degron, the PA protein, PB2 protein, PB1 protein and M1 protein of an influenza virus may both contain at least one degron, the PA protein, PB2 protein, PB1 protein, M1 protein and NP protein of an influenza virus may both contain at least one degron, and the PB2 protein and PB The PB1 protein and M1 protein of influenza virus may each comprise at least one degron, the PA protein and M1 protein of influenza virus may each comprise at least one degron, the PB1 protein and M1 protein of influenza virus may each comprise at least one degron, the PB2 protein and M1 protein of influenza virus may each comprise at least one degron, the PB2 protein, PB1 protein, M1 protein and NS1 protein of influenza virus may each comprise at least one degron, the PB2 protein, PB1 protein, M1 protein and NEP protein of influenza virus may each comprise at least one degron, and the NS1 protein and NEP protein of influenza virus may each comprise at least one degron.

[0017] Preferably, the antibody is selected from the group consisting of Gag polyprotein, pol polyprotein, gp160, HIV trans-activator of transcription (Tat), regulator of expression of virion proteins (Rev), negative factor (Nef), lentivirus protein R (Vpr), viral infectivity factor (Vif), viral protein U (Vpu), matrix protein (MA, p17), capsid protein (CA, p24), spacer peptide 1 (SP1, p2), nucleocapsid protein (NC, p7), spacer peptide 2 (SP2, p1), P6, and reverse transcriptase. Any one or a combination of at least two of the following proteins comprises at least one degron: transcriptase (RT), ribonuclease H (RNase H), integrase (IN), HIV protease (PR), gp120, or gp41.

[0018] Preferably, the antibody is a spike protein (S protein), envelope glycoprotein (E glycoprotein), membrane glycoprotein (M glycoprotein), nucleocapsid protein (N protein), nonstructural protein 1 (nsp1), nonstructural protein 2, nonstructural protein 3, nonstructural protein 4, nonstructural protein 5, nonstructural protein 6, nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, nonstructural protein 13, nonstructural protein 14, nonstructural protein 15, nonstructural protein 16, 3a protein, 3b protein, 6 protein, 7a protein, 7b protein, 8a protein, 8b protein, 9b protein, 3C-like protease, leader protein, 2'-O-ribosylmethyltransferase (2'-O-ribosylmethyltransferase), or the like of the novel coronavirus. At least one degron is comprised of any one or a combination of at least two of the following proteins: 3'- to 5' exonuclease, endoRNAse, 3'- to 5' exonuclease, helicase, RNA-dependent RNA polymerase, orf1a polyprotein, ORF10 protein, ORF8 protein, ORF7a protein, ORF6 protein, or ORF3a protein.

[0019] In the present application, the recombinant virus may be further modified, for example, by introducing some immunopotentiators into specific regions or specific amino acids of viral proteins to obtain a recombinant virus with improved performance and enhanced immunogenicity.

[0020] In a second aspect, the present application provides a nucleic acid molecule encoding the degron-containing recombinant virus according to the first aspect.

[0021] In a third aspect, the present application provides a recombinant vector comprising the nucleic acid molecule according to the second aspect.

[0022] Preferably, the recombinant vector expresses the degron-containing recombinant virus according to the first aspect.

[0023] In a fourth aspect, the present application provides a method for preparing a degron-containing recombinant virus according to the first aspect, comprising: constructing a cell line deficient in proteolytic systems; introducing a nucleotide sequence encoding a degron into a viral protein-encoding gene; and constructing a recombinant vector according to the third aspect, introducing the vector into the cell line lacking a proteolytic system, and packaging the vector to obtain the degron-containing recombinant virus.

[0024] The present application provides an artificially modified cell line that is deficient in a proteolytic system, to avoid a decrease in production efficiency due to the degradation of recombinant viruses by the cellular proteolytic system during the preparation process, since proteolytic systems are widely distributed in host cells. In this artificially modified cell line, degrons are not recognized, and viral proteins are retained without being degraded by the proteolytic system, allowing the recombinant virus to replicate efficiently in this specific artificially modified cell line and produce large quantities. In normal cells, the proteolytic system recognizes the degron fused to the viral protein and degrades the viral protein, weakening the viral replication ability and eventually completely eliminating the viral replication ability, making the recombinant virus extremely safe.

[0025] The dependency of recombinant viruses on this specific virus production system allows for the preparation of large quantities of recombinant viruses in this system. Normal cells in humans and animals have a proteolytic system that can recognize the degron fused to the viral protein and degrade the viral protein. Therefore, the prepared recombinant virus has reduced or no replication ability in the animal or human body, improving the safety of the virus and making it truly an attenuated live virus vaccine.

[0026] The principles of recombinant virus preparation in this application are as follows: (1) A degron introduced into a specific site of a viral protein can be recognized by the proteolytic system in host cells, inducing the degradation and inactivation of the relevant viral protein. (2) A degron introduced into a specific site of a viral protein can be unrecognized or inhibited in a specific virus production system, avoiding or reducing the degradation of the viral protein by the proteolytic system in host cells. (3) Because a degron introduced into a specific site of a viral protein is recognized by the proteolytic system in normal host cells, the prepared virus can be recognized and degraded by the proteolytic system in host cells, such as animal or human cells, thereby reducing its replication ability and ultimately completely eliminating its replication and reproduction ability, thereby improving the safety of the virus.

[0027] Preferably, the protein degradation system deficiency includes a ubiquitin-proteasome system deficiency including any one or a combination of at least two of E3 ligase knockout or knockdown and proteasome knockout or knockdown.

[0028] Preferably, the cell line comprises a mammalian cell line comprising any one of a CHO cell line, a Vero cell line, an MDCK cell line, a HEK293T cell line, an MDCK cell line, an A549 cell line, a BHK cell line, a BHK-21 / BRS cell line, an Sp2 / 0 cell line, a HEK293 cell line, a 293F cell line, a HeLa cell line, a TZM-bl cell line, a Sup-T1 cell line, an MRC-5 cell line, a VMK cell line, a LLC-MK2 cell line, an HCT-8 cell line, a Huh-7 cell line or a Caco2 cell line, and preferably any one of a HEK293T cell line, an MDCK cell line or an A549 cell line.

[0029] Furthermore, by using mammalian cell lines in the present invention, the drawback of the conventional method of propagating viruses using chicken embryos, which is likely to cause adverse reactions such as allergies in the human body, has been overcome.

[0030] Preferably, a proteolytic inhibitor is added to the degron-containing recombinant virus during the preparation process.

[0031] Preferably, the protein degradation inhibitor includes a proteasome inhibitor including any one or a combination of at least two of MG132, MG-341, and lactacystin.

[0032] Preferably, the preparation method further comprises the step of detecting the replication competence, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient cell line and an unmodified cell line.

[0033] In this application, the successful modification of the degron-containing recombinant virus is determined by detecting the replication ability of the degron-containing recombinant virus in the proteolytic system-deficient cell line and in an unmodified cell line, and a recombinant virus that replicates normally in the proteolytic system-deficient cell line and has reduced or no replication ability in unmodified normal host cells is a successfully modified recombinant virus.

[0034] Preferably, the preparation method further comprises the step of large scale production.

[0035] In a preferred technical embodiment, the method for preparing a degron-containing recombinant virus described in the present application comprises the following steps: Step (1), constructing a proteolytic system-deficient mammalian cell line, Using gene editing techniques, key components of the protein degradation system are knocked out in cellular systems. Step (2): The viral protein into which the degron has been introduced and the type, number, and insertion site of the degron are determined, and a nucleotide sequence encoding the degron is introduced into the gene encoding the viral protein to obtain a recombinant viral sequence. Step (3), constructing a recombinant vector, The recombinant viral sequence in step (2) is ligated with the plasmid to obtain the recombinant vector. Step (4), introducing the recombinant vector into the cell line lacking the proteolytic system and packaging it to obtain the degron-containing recombinant virus, Using reverse genetics techniques, the recombinant vector in step (3) and a viral rescue plasmid are co-transfected into the cell line lacking the proteolytic system, followed by adding a proteolytic inhibitor and packaging to obtain the degron-containing recombinant virus. The replication competence, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient and unmodified cell lines will be determined and produced on a large scale.

[0036] In step (2) of the present application, bioinformatics analysis and protein structure prediction are performed on influenza viruses to predict and analyze the protein structures of viruses in which degron sequences have been introduced into each viral protein, thereby selecting viral proteins in which degrons have been introduced and determining the type, number, and specific site of the introduced degrons.

[0037] In step (4) of the present application, reverse genetics techniques can be used to replace any gene in a conventional virus model with a gene from another subtype or virulent strain, or an existing virus model can be replaced with another subtype or virulent strain to prepare a recombinant virus of another subtype or virulent strain, and this method can be used for viruses of any subtype or virulent strain.Furthermore, the prepared virus replicates in large quantities in a cell line lacking a proteolytic system and is recognized and degraded by the proteolytic system in normal host cells.

[0038] In the present application, a successfully modified recombinant virus can be obtained by repeating steps (2) to (4) to introduce degrons into multiple viral proteins of the recombinant virus, or by introducing multiple degrons into any one viral protein of the recombinant virus.

[0039] Furthermore, reverse genetics technology allows the preparation of more polyvalent viruses, and more importantly, the prepared mutant highly virulent and polyvalent viruses have extremely high safety and efficacy.

[0040] In a fifth aspect, the present application provides a preparation system for the degron-containing recombinant virus according to the first aspect, the preparation system comprising a cell line deficient in a proteolytic system, a recombinant vector, and a viral rescue plasmid.

[0041] In a sixth aspect, the present application provides the use of any one or a combination of at least two of the degron-containing recombinant virus described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the method for preparing a degron-containing recombinant virus described in the fourth aspect, or the system for preparing a degron-containing recombinant virus described in the fifth aspect, in the preparation of a vaccine and / or a pharmaceutical.

[0042] In a seventh aspect, the present application provides a vaccine comprising the degron-containing recombinant virus according to the first aspect.

[0043] Preferably, the vaccine comprises any one of a live attenuated vaccine, a live replication-incompetent vaccine, a live replication-regulatable vaccine, or an oncolytic virus vaccine.

[0044] Preferably, the vaccine further comprises an adjuvant and auxiliary materials.

[0045] In an eighth aspect, the present application provides an oncolytic virus comprising the degron-containing recombinant virus according to the first aspect.

[0046] In a ninth aspect, the present application provides a pharmaceutical product comprising the degron-containing recombinant virus according to the first aspect.

[0047] Preferably, the pharmaceutical product further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient. [Effects of the Invention]

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] (1) Regarding versatility, viral replication can be controlled simply by introducing a degron into a viral protein. Therefore, the technical solution of the present application can modify all viruses and can be used to prepare all viral vaccines. There are multiple selectable proteolytic systems and corresponding degrons, which means there are few limitations and strong applicability.

[0050] (2) The operation is simple. Only simple viral vector construction and viral packaging technology are required, and the technology is mature with a high success rate, facilitating the preparation and popularization of related products.

[0051] (3) Good efficacy: The technical solution of this application makes it possible to design vaccines with different inactivation levels, which has the potential for safety control. The prepared vaccines have excellent immunogenicity and broad future potential. [Brief explanation of the drawings]

[0052] [Figure 1A] FIG. 1 shows the efficiency of preparing a virulent strain containing Degron 1 in Example 3. [Figure 1B] FIG. 10 shows the efficiency of preparing a virulent strain containing Degron 2 in Example 3. [Figure 1C] FIG. 10 shows the efficiency of preparing a recombinant toxin strain simultaneously containing Degron 1 and Degron 2 in Example 3. [Figure 2A] 1 shows the growth curves of toxic strains containing Degron 1 in MDCK cells in Example 3. [Figure 2B] 1 shows the growth curves of toxic strains containing Degron 2 in MDCK cells in Example 3. [Figure 2C] 1 shows the growth curves in MDCK cells of recombinant toxin strains containing multiple Degrons 1 and / or 2 in Example 3. [Figure 3] FIG. 1 shows viral protein expression levels detected by Western blotting in Example 4. [Figure 4A] FIG. 1 shows changes in body weight after inoculation of mice with recombinant influenza viruses in Example 5. [Figure 4B] FIG. 1 shows the survival rate of mice after inoculation with recombinant influenza viruses in Example 5. [Figure 4C] FIG. 1 shows virus titers in the lungs after inoculation of mice with recombinant influenza viruses in Example 5. [Figure 5A] FIG. 1 shows the neutralizing antibody titer and the coagulation-inhibiting antibody titer after inoculation with recombinant influenza viruses in Example 6. [Figure 5B] FIG. 10 shows the titers of anti-NP IgG, anti-HA IgG, and anti-NP IgA after inoculation with recombinant influenza viruses in Example 6. [Figure 5C] FIG. 1 shows T cell responses in the spleen and lungs after inoculation with recombinant influenza viruses in Example 6. [Figure 5D] In Example 6, the graph shows changes in body weight of mice after inoculation with a recombinant influenza virus and then challenged with a wild-type homologous influenza virulent strain (WSN). [Figure 5E] In Example 6, the graph shows the survival rate of mice inoculated with recombinant influenza viruses and then challenged with a wild-type homologous influenza virulence strain (WSN). [Figure 5F]In Example 6, mice were inoculated with recombinant influenza viruses and then challenged with a wild-type homologous influenza virulent strain (WSN), showing virus titers in the lungs 3 days later. [Figure 6A] In Example 7, the graph shows changes in body weight of mice after inoculation with a recombinant influenza virus and then challenged with a wild-type heterologous influenza virulent strain (H3N2). [Figure 6B] In Example 7, this figure shows the survival rate of mice inoculated with a recombinant influenza virus and then challenged with a wild-type heterologous influenza virulent strain (H3N2). [Figure 6C] In Example 7, the graph shows virus titers in the lungs of mice 3 days after inoculation with recombinant influenza viruses and subsequent challenge with a wild-type heterologous influenza virulent strain (H3N2). [Figure 7] This diagram illustrates the working principle of degron-containing viruses. In normal cells, degrons covalently bind to viral proteins and induce E3 ubiquitin ligase to recognize the viral proteins, ubiquitinating them. The proteasome then degrades the ubiquitinated viral proteins, ultimately attenuating the virus and making it into a vaccine. In cells with E3 ubiquitin ligase knockout, degrons are unable to induce ubiquitination of viral proteins, so the viral proteins are retained without being degraded by the proteasome. Finally, the virus replicates efficiently and can be mass-produced like wild-type viruses. DETAILED DESCRIPTION OF THE INVENTION

[0053] In order to further explain the technical means used in the present application and their effects, the present application will be further described below with reference to examples. It can be understood that the specific embodiments described herein are only for the purpose of interpreting the present application, and are not intended to limit the present application.

[0054] Unless specific techniques or conditions are specified in the examples, they are carried out in accordance with the techniques or conditions described in the literature in the field or in the product manuals. Unless the manufacturer is specified, the reagents or equipment used are all conventional and can be purchased from authorized wholesalers.

[0055] material: pHH21, pCDNA3(neo) and pcAAGGS / MCS vectors were purchased from Beijing Zhongke Yubo Biotechnology Co., Ltd.

[0056] Example 1 This example provides a recombinant vector expressing a degron-containing recombinant influenza virus, which is prepared by the following method.

[0057] (1) Construction of viral rescue plasmids Based on the genetic sequence of influenza virus A / WSN / 1933 published in pubmed.

[0058] https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+PB2, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+PB1, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+PA, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+HA, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+NA, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+NP, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+M, https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=WSN+NS.

[0059] The genes for each gene segment of the influenza virus were synthesized by whole gene synthesis. They were then ligated into pHH21, pCDNA3(neo), and pcAAGGS / MCS vectors, respectively, to obtain wild-type influenza virus rescue plasmids. The names and structures of the resulting plasmids are shown in Table 1. [Table 1]

[0060] (2) Construction of recombinant vectors The degron coding sequence was introduced into the viral protein and ligated into a plasmid to construct a series of recombinant plasmids, which was completed in collaboration with Beijing Tsingke Biotech Co., Ltd. Sequencing was performed to verify the success of the mutation construction.

[0061] The naming rules for recombinant vectors are as follows:

[0062] 1. The viral vector constructed by introducing a degron into the N-terminus of the viral protein was named "viral protein name-N-degron name."

[0063] 2. The viral vector constructed by introducing a degron into the C-terminus of the viral protein was named "viral protein name-C-degron name."

[0064] 3. Viral vectors constructed by introducing a degron into the coding region of a viral protein were named as follows: "viral protein name - name and amino acid number of the upstream amino acid adjacent to the introduction site - degron name."

[0065] The recombinant plasmids constructed are as follows:

[0066] PB2-N-Degron1, PB2-R70-Degron1, PB2-I176-Degron1, PB2-V457-Degron1, PB2-N510-Degron1, PB2-Y531-Degron1, PB2-A623-Degron1, PB2-D680-Degron1, PB2-E700-Degron1, PB2-C-Degron1, PB1-N-Degron1, PB1-D70-Degron1, PB1-D295-Degron1, PB1-R327-Degron1, PB1-R430-Degron1, PB1-F490-Degron1, PB1-T566 Degron1, PB1-N626-Degron1, PB1-G710-Degron1, PB1-C-Degron1, PA-N-Degron1, PA-D294-Degron1, PA-N350-Degron1, PA-E372-Degron1, PA-L425-Degron1, PA-H510-Degron1, PA-A553-Degron1, PA-E604-Degron1, PA-S624-Degron1, PA-C-Degron1, NP-N-Degron1, NP-G126-Degron1, NP-N247-Degron1, NP-R317-Degron1, NP-V353-Degron1, NP-A366-Degron1、NP-Q409-Degron1、NP-E465-Degron1、NP-M481-Degron1、NP-C-Degron1、M1-N-Degron1 、M1-A33-Degron1、M1-V68-Degron1、M1-D89-Degron1、M1-R105-Degron1、M1-M135-Degron1、M1-Q164-De gron1、M1-H222-Degron1、M1-A239-Degron1、M1-C-Degron1、M2-C-Degron1、NEP-C-Degron1、NS1-N-Degron on1、NS1-A76-Degron1、NS1-A82-Degron1、NS1-H101-Degron1、NS1-A122-Degron1、NS1-T151-Degron1、N S1-L163-Degron1, NS1-C-Degron1, HA-N-Degron1, HA-C-Degron1, NA-N-Degron1, NA-C-Degron1, PB2-N -Degron2、PB2-R70-Degron2、PB2-I176-Degron2、PB2-V457-Degron2、PB2-N510-Degron2、PB2-Y531-Deg ron2、PB2-A623-Degron2、PB2-D680-Degron2、PB2-E700-Degron2、PB2-C-Degron2、PB1-N-Degron2、PB1- D70-Degron2、PB1-D295-Degron2、PB1-R327-Degron2、PB1-R430-Degron2、PB1-F490-Degron2、PB1-T566Degron2, PB1-N626-Degron2, PB1-G710-Degron2, PB1-C-Degron2, PA-N-Degron2, PA-D294-Degron2, PA-N3 50-Degron2, PA-E372-Degron2, PA-L425-Degron2, PA-H510-Degron2, PA-A553-Degron2, PA-E604-Degron2, PA-S624-Degron2, PA-C-Degron2, NP-N-Degron2, NP-G126-Degron2, NP-N247-Degron2, NP-R317-Degron2, NP-V353-Degron2, NP-A366-Degron2, NP-Q409-Degron2, NP-E465-Degron2, NP-M481-Degron2, NP-C-Degron 2, M1-N-Degron2, M1-A33-Degron2, M1-V68-Degron2, M1-D89-Degron2, M1-R105-Degron2, M1-M135-Degron 2, M1-Q164-Degron2, M1-H222-Degron2, M1-A239-Degron2, M1-C-Degron2, M2-C-Degron2, NEP-C-Degron2, N S1-N-Degron2, NS1-A76-Degron2, NS1-A82-Degron2, NS1-H101-Degron2, NS1-A122-Degron2, NS1-T151-De gron2, NS1-L163-Degron2, NS1-C-Degron2, HA-N-Degron2, HA-C-Degron2, NA-N-Degron2 and NA-C-Degron2.

[0067] The sequence of Degron 1 is shown as SEQ ID No. 1, the coding nucleotide sequence as SEQ ID No. 111, and the sequence of Degron 2 is shown as SEQ ID No. 2, the coding nucleotide sequence as SEQ ID No. 112.

[0068] SEQ ID No.111:GCATTGGCCCCCTACATTCCA, SEQ ID No.112:GATCGCCACGATTCAGGGCTCGATTCCATG.

[0069] Example 2 This example provides a degron-containing recombinant influenza virus, which is prepared by the following method.

[0070] (1) We constructed a mammalian cell line deficient in protein degradation systems.

[0071] CRISPR / Cas9 gene knockout technology was used to knock out components of the protein degradation system in cell lines.

[0072] (2) The recombinant vector was introduced into the cell line lacking the protein degradation system and packaged to obtain the degron-containing recombinant virus.

[0073] The recombinant vector in Example 1 was used to replace the corresponding wild-type plasmid and co-transfected with 11 other plasmids into a mammalian cell line lacking a proteolytic system (e.g., the recombinant vector PB2-N-Degron1 was used to replace the plasmid Ben1 and co-transfected with the other 11 plasmids). 0.2 μg of each plasmid was added, and the transfected cells were cultured in DMEM medium containing 0.5% FBS and 2 μg / mL TPCK-trypsin.

[0074] Four days after transfection, the host cells were either completely or 90% or more infected, and the supernatant was collected and used to infect new proteolytically deficient cells. The medium was DMEM containing 0.5% FBS and 2 μg / mL TPCK-trypsin. Proteolytic inhibitors were added, and four days after infection, the host cells were completely infected, and the supernatant was collected.

[0075] The supernatant is centrifuged and passed through a 0.4 μm filter to remove cell debris, and the dependency of the packaging product on the proteolytic system deficiency and the dependency of the inactivation of the packaging product on the proteolytic system are detected. Mutants that maintain the dependency on the proteolytic system deficiency are retained and used as recombinant viruses.

[0076] The recombinant viruses prepared were named according to the same scheme as the recombinant vectors.

[0077] Example 3 The degron-containing recombinant influenza viruses (shown in Table 2 below) were evaluated for preparation efficiency and safety at the cellular level. [Table 2-1] [Table 2-2]

[0078] (1) Preparation efficiency of degron-containing recombinant influenza viruses Degron-containing recombinant influenza viruses and wild-type influenza viruses were infected into E3 ubiquitin ligase knockout cell lines (MOI = 0.01), respectively, and after 48 hours of culture, the cell supernatants were collected and the virus titers were measured. The efficiency of degron-containing recombinant influenza virus preparation was determined by comparing the titers of the degron-containing recombinant influenza viruses with those of the wild-type virus. In this example, the efficiency of degron-containing recombinant influenza virus preparation was demonstrated using virulent strains containing Degron 1, Degron 2, and a combination thereof as examples. As shown in Figure 1, in HEK293T cells in which the VHL E3 ubiquitin ligase was knocked out, the titer of the recombinant influenza toxin strain containing all Degrons 1 was comparable to that of the wild-type influenza virus WSN. In HEK293T cells in which the β-TrCP E3 ubiquitin ligase was knocked out, the titer of the recombinant influenza toxin strain containing all Degrons 2 was comparable to that of the wild-type influenza virus WSN. At the same time, the titer of the recombinant influenza strain containing multiple Degrons 1 and / or 2 was comparable to that of the wild-type influenza virus WSN. These results demonstrate the high efficiency of the preparation of degron-containing recombinant influenza viruses.

[0079] (2) Safety of degron-containing recombinant influenza viruses at the cellular level The safety of virulent strains was determined by examining the growth curves of degron-containing recombinant influenza viruses and wild-type influenza viruses in normal MDCK cells. The criteria for determining the safety of virulent strains are as follows: If a virulent strain has a weakened replication ability in a normal MDCK cell system compared to wild-type viruses, or even does not replicate (the virus titer is lower than that of the wild-type virus), the virulent strain is considered safe. In this example, the safety of degron-containing recombinant influenza viruses was demonstrated using virulent strains containing Degron 1, Degron 2, and a combination thereof as examples. The steps for detection based on the growth curves are as follows:

[0080] The prepared degron-containing recombinant influenza virus and wild-type influenza virus were infected into normal MDCK cell lines at an MOI of 0.001. The viral replication ability was determined by detecting the viral titer in the cells using qPCR at 24, 48, 72, and 96 hours postinfection. As can be seen from the results (Figure 2), the degron-containing recombinant influenza virus had a weakened replication ability compared to the wild-type influenza virus in normal MDCK cell lines, and was therefore replication-deficient. This result indicated that the prepared degron-containing recombinant influenza virus was safe at the cellular level.

[0081] Example 4 Verification of the attenuation mechanism of degron-containing recombinant influenza viruses Normal MDCK cell lines were infected with degron-containing recombinant influenza viruses and wild-type influenza viruses at an MOI of 0.1. The medium was supplemented with 50 nM and 100 nM of the proteasome inhibitor MG-132, respectively, and DMSO (at the same dilution ratio as the virus) served as a control. 48 h after infection, cell samples were collected and viral protein expression levels were detected by Western blotting.

[0082] As can be seen from the test results (Figure 3), after infection of normal MDCK cells with the degron-containing recombinant influenza virus, only a small signal for the viral protein M1 was detected, as the virus was unable to replicate in large quantities. After the cellular proteasome system was inhibited, the signal for the viral protein M1 increased. This indicated that the viral replication ability was enhanced after the proteasome system was inhibited. This demonstrated that the introduction of a proteolytic targeting molecule could mediate the degradation of viral proteins by the cellular proteasome, thereby inhibiting the viral replication ability. This is consistent with the design principles of the degron-containing recombinant influenza virus.

[0083] Example 5 Safety evaluation of degron-containing recombinant influenza viruses in animals The safety of the degron-containing recombinant influenza viruses was evaluated in C57BL / 6J mice, and the safety of the recombinant toxin strain containing Degron 1 and Degron 2 (designated the Degrons recombinant toxin strain) was also evaluated.

[0084] The specific steps for safety evaluation are as follows:

[0085] (a) Thirty 7-week-old female C57BL / 6J mice were divided into three groups, with 10 mice in each group.

[0086] (b) In the first group, each mouse was inoculated with DMEM by nasal instillation, and in the second group, each mouse was inoculated with 10 5 TCID 50 The third group was inoculated with 10 Degrons recombinant venom strains by nasal instillation. 5 TCID 50 Wild-type WSN influenza virus was inoculated via nasal instillation.

[0087] (c) Three days after inoculation, five mice were selected from each group, and their lung tissues were collected to detect the virus titer.

[0088] (d) The remaining 5 mice in each group were then observed and monitored for weight and mortality for 14 days.

[0089] As can be seen from the results (Figure 4), the wild-type WSN influenza virus replicated at a high level in the lungs of mice, causing significant weight loss and death in the mice. However, the titer of the degron-containing recombinant influenza virus in the lungs of mice was below the detection limit, causing neither weight loss nor death in the mice. Therefore, the degron-containing recombinant influenza virus vaccine has good safety.

[0090] Example 6 Immunogenicity and protection of degron-containing recombinant influenza virus vaccines in animals The immunogenicity and protective properties of the degron-containing recombinant influenza virus were evaluated in animals. An inactivated influenza vaccine (IIV) was used as a control (the inactivated influenza virus vaccine was prepared by the inventors using the same influenza virus particles according to the method provided in the Chinese Pharmacopoeia), and a recombinant toxin strain containing Degron 1 and Degron 2 (designated the Degrons recombinant toxin strain) was used as a representative to evaluate the immunogenicity and protective properties of the degron-containing recombinant influenza virus vaccine.

[0091] Specific steps for considering immunogenicity and protection are as follows:

[0092] (1) Sixty 7-week-old female C57BL / 6J mice were divided into three groups, each with 20 mice.

[0093] (2) In the first group, each mouse was inoculated with DMEM by nasal instillation, and in the second group, each mouse was inoculated with 10 5 TCID 50 The third group was inoculated with 10 Degrons recombinant venom strains by nasal instillation. 5 PFU inactivated influenza vaccine IIV was administered by nasal instillation.

[0094] (3) One week after inoculation, five mice were selected from each group, and their lung tissues and spleens were collected to detect the immune response of T cells.

[0095] (4) Three weeks after inoculation, five mice from each group were selected, and blood samples were collected and used for hemoglobin inhibition (HI) test, neutralizing (NT) antibody detection, and ELISA detection, respectively, to detect the antibody immune response.

[0096] (5) Three weeks after inoculation, each group received 2 × 10 5 TCID 50 The wild-type WSN influenza virus was administered by nasal inoculation.

[0097] (6) Three days after inoculation with wild-type WSN influenza virus, five mice were selected from each group, and their lung tissues were collected to detect the virus titer.

[0098] (7) The remaining five mice in each group were then monitored for weight and mortality for 14 days.

[0099] As can be seen from these results (Figure 5), the Degrons recombinant toxin strain can induce high levels of anticoagulant antibodies, neutralizing antibodies, anti-NP IgG, anti-HA IgG, anti-NP IgA, etc. in animals. The levels of anticoagulant antibodies, neutralizing antibodies, anti-NP IgG, anti-HA IgG, and anti-NP IgA induced by the Degrons recombinant toxin strain were significantly higher than those induced by inactivated influenza vaccines. Inoculation with the Degrons recombinant toxin strain provided complete immune protection; for example, viral titers in the lungs of mice were suppressed below the detection limit, and all mice survived with unchanged weight. In contrast, inoculation with the inactivated vaccine provided only limited immune protection; for example, high levels of wild-type influenza virus replication remained in the lungs of mice, some mice survived, and weight loss was evident. These data demonstrate that the protection provided by the Degrons recombinant toxin strain vaccine is significantly superior to that provided by inactivated influenza vaccines. Therefore, the degron-containing recombinant influenza virus vaccine has better immunogenicity and protective effect.

[0100] Example 7 Cross-protective effects of degron-containing recombinant influenza virus vaccines in animals The specific steps are as follows:

[0101] (1) Ten 7-week-old female C57BL / 6J mice were divided into three groups, each with 10 mice.

[0102] (2) In the first group, each mouse was inoculated with DMEM by nasal instillation, and in the second group, each mouse was inoculated with 10 5 TCID 50 The third group was inoculated with 10 Degrons recombinant venom strains by nasal instillation. 5 PFU inactivated influenza vaccine IIV was administered by nasal instillation.

[0103] (3) Three weeks after inoculation, each group received 103 TCID 50 wild-type H3N2 influenza virus was inoculated by nasal instillation.

[0104] (4) Three days after inoculation with wild-type H3N2 influenza virus, five mice were selected from each group, and their lung tissues were collected to detect the virus titer.

[0105] (5) The remaining five mice in each group were then monitored for weight and mortality for 14 days.

[0106] As can be seen from these results (Figure 6), vaccination with the Degrons recombinant toxin strain provided cross-immune protection; for example, virus titers in the lungs of mice were suppressed below the detection limit, and all mice survived with unchanged body weight. In contrast, vaccination with an inactivated vaccine failed to provide cross-immune protection; for example, H3N2 influenza virus levels in the lungs of mice were comparable to those of unvaccinated mice, all mice died, and their body weights significantly decreased. These data demonstrate that the cross-immune protection provided by the Degrons recombinant toxin strain vaccine is significantly superior to that of an inactivated influenza vaccine. Therefore, the degron-containing recombinant influenza virus vaccine has superior cross-immunogenicity and protective effect.

[0107] In summary, by introducing a degron into a viral protein, the prepared recombinant virus can be recognized and degraded by the proteolytic system of the host cell, weakening the recombinant virus's replication ability and ultimately eliminating it. However, the recombinant virus can replicate in cells lacking the proteolytic system, making large-scale production possible. After being prepared into the corresponding vaccine or pharmaceutical, the recombinant virus has excellent safety and immunogenicity and is expected to be widely used. The method for preparing the recombinant virus is highly applicable and simple to operate, facilitating the use and widespread use of the product.

[0108] The present application describes the detailed method of the present application through the above examples, but the applicant declares that the present application is not limited to the above detailed method, that is, it does not mean that the present application must be carried out depending on the above detailed method. Those skilled in the art should understand that any improvements to the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific forms, etc. are all within the protection scope and disclosure scope of the present application. Further aspects of the present invention are described below: [Section 1] 1. A degron-containing recombinant virus, wherein at least one viral protein comprises at least one degron that is recognizable by a host cell proteolytic system, A degron-containing recombinant virus, wherein the degron comprises any one or a combination of at least two of an amino acid sequence, a polypeptide, or a structural motif. [Section 2] Item 1. The degron-containing recombinant virus according to Item 1, wherein the protein degradation system comprises any one or a combination of at least two of the following: a ubiquitin-proteasome system, a lysosome system, an organelle hydrolysis system, a cell membrane surface hydrolysis system, or a caspase protease system. [Section 3] Item 3. The degron-containing recombinant virus according to Item 1 or 2, wherein the degron is located in any one or a combination of at least two of the C-terminus, N-terminus, or intermediate coding region of the viral protein. [Section 4] Item 4. The degron-containing recombinant virus according to any one of Items 1 to 3, wherein the degron comprises any one of the amino acid sequences represented by SEQ ID Nos. 1 to 110. [Section 5] The viruses include influenza virus, AIDS virus, novel coronavirus, hand, foot and mouth virus, coxsackievirus, hepatitis C virus, hepatitis B virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, human herpes virus, human papillomavirus, herpes simplex virus, giant cell virus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus, dengue virus, Ebola virus, Marburg virus, Zika virus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus, rotavirus, rabies virus, measles virus, adenovirus, and polio. Item 5. The degron-containing recombinant virus according to any one of Items 1 to 4, which comprises any one of a virus, echovirus, Japanese encephalitis virus, Forest Colchicine encephalitis virus, hantavirus, new enteric virus, rubella virus, parotitis virus, parainfluenza virus, blue ear virus, swine fever virus, foot-and-mouth disease virus, minute virus, prion, smallpox virus, tobacco mosaic virus, phage, herpesvirus, West Nile virus, norovirus, human bocavirus, and coronavirus, and is preferably any one of influenza virus, AIDS virus, and novel coronavirus. [Section 6] A nucleic acid molecule encoding the degron-containing recombinant virus according to any one of Items 1 to 5. [Section 7] A recombinant vector comprising the nucleic acid molecule according to item 6, Preferably, the recombinant vector expresses the degron-containing recombinant virus according to any one of Items 1 to 5. [Section 8] A method for preparing the degron-containing recombinant virus according to any one of Items 1 to 5, comprising: constructing a cell line deficient in proteolytic systems; introducing a nucleotide sequence encoding a degron into a viral protein-encoding gene; Constructing the recombinant vector according to Item 7, introducing it into the cell line lacking a proteolytic system, and packaging it to obtain the degron-containing recombinant virus; Preferably, the protein degradation system deficiency includes a ubiquitin-proteasome system deficiency including any one or a combination of at least two of E3 ligase knockout or knockdown, and proteasome knockout or knockdown; Preferably, the cell line comprises a mammalian cell line comprising any one of a CHO cell line, a Vero cell line, an MDCK cell line, a HEK293T cell line, an MDCK cell line, an A549 cell line, a BHK cell line, a BHK-21 / BRS cell line, an Sp2 / 0 cell line, a HEK293 cell line, a 293F cell line, a HeLa cell line, a TZM-bl cell line, a Sup-T1 cell line, an MRC-5 cell line, a VMK cell line, a LLC-MK2 cell line, an HCT-8 cell line, a Huh-7 cell line or a Caco2 cell line, and preferably any one of a HEK293T cell line, an MDCK cell line or an A549 cell line; Preferably, a proteolytic inhibitor is added to the degron-containing recombinant virus during the preparation process; Preferably, the proteolytic inhibitor comprises a proteasome inhibitor comprising any one or a combination of at least two of MG132, MG-341, and lactacystin. [Section 9] The preparation method further comprises detecting the replication competence, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient cell line and an unmodified cell line; Preferably, the preparation method further comprises the step of producing on a large scale, Preferably, the preparation method comprises: Step (1) of constructing a proteolytically deficient mammalian cell line, (1) knocking out key components of the protein degradation system in a cell line using gene editing technology; (2) determining a viral protein into which a degron has been introduced, as well as the type, number, and introduction site of the degron, and introducing a nucleotide sequence encoding the degron into a gene encoding the viral protein to obtain a recombinant viral sequence; Step (3) of constructing a recombinant vector, Step (3) of ligating the recombinant viral sequence in step (2) with the plasmid to obtain the recombinant vector; (4) introducing the recombinant vector into the cell line lacking a proteolytic system and packaging it to obtain the degron-containing recombinant virus; (4) co-transfecting the recombinant vector in step (3) and the viral rescue plasmid into the cell line lacking a proteolytic system by reverse genetics, adding a proteolytic inhibitor, and packaging the resulting virus to obtain the degron-containing recombinant virus; The preparation method described in Item 8, comprising the steps of detecting the replication ability, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient cell line and an unmodified cell line, and producing it on a large scale. [Section 10] A system for preparing a degron-containing recombinant virus according to any one of Items 1 to 5, A preparation system comprising a proteolytically deficient cell line, a recombinant vector and a viral rescue plasmid. [Section 11] Use of any one or a combination of at least two of the degron-containing recombinant virus of any one of Items 1 to 5, the nucleic acid molecule of Item 6, the recombinant vector of Item 7, the method for preparing a degron-containing recombinant virus of Item 8 or 9, or the system for preparing a degron-containing recombinant virus of Item 10 in the production of a vaccine, an oncolytic virus, and / or a pharmaceutical. [Section 12] A vaccine comprising the degron-containing recombinant virus according to any one of Items 1 to 5, Preferably, the vaccine comprises any one of a live attenuated vaccine, a live replication-incompetent vaccine, a live replication-regulatable vaccine, or an oncolytic virus vaccine; Preferably, the vaccine further comprises an adjuvant and auxiliary materials. [Section 13] Item 6. An oncolytic virus comprising the degron-containing recombinant virus according to any one of Items 1 to 5. [Section 14] A pharmaceutical comprising the degron-containing recombinant virus according to any one of Items 1 to 5, Preferably, the pharmaceutical product further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

Claims

1. 1. A degron-containing recombinant virus, wherein at least one viral protein comprises at least one degron that is recognizable by a host cell proteolytic system, The degron comprises any one or a combination of at least two of an amino acid sequence, a polypeptide, or a structural motif; The degron comprises any one of the amino acid sequences represented by SEQ ID Nos. 1 to 110; The location of the degron is in the intermediate coding region of a viral protein. Degron-containing recombinant viruses.

2. The degron-containing recombinant virus of claim 1, wherein the proteolytic system comprises any one or a combination of at least two of a ubiquitin-proteasome system, a lysosomal system, an organelle hydrolysis system, a cell membrane surface hydrolysis system, or a caspase protease system.

3. The viruses include influenza virus, AIDS virus, novel coronavirus, hand, foot and mouth virus, coxsackie virus, hepatitis C virus, hepatitis B virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, human herpes virus, human papilloma virus, herpes simplex virus, giant cell virus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus, dengue virus, Ebola virus, Marburg virus, Zika virus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus, Rotau virus, 2. The degron-containing recombinant virus of claim 1, comprising any one of the following viruses: rabies virus, measles virus, adenovirus, poliovirus, echovirus, Japanese encephalitis virus, hymenopteran encephalitis virus, hantavirus, new enteric virus, rubella virus, parotitis virus, parainfluenza virus, blue ear virus, swine fever virus, foot-and-mouth disease virus, minute virus, prion, smallpox virus, tobacco mosaic virus, phage, herpesvirus, West Nile virus, norovirus, human bocavirus, or coronavirus.

4. A nucleic acid molecule encoding the degron-containing recombinant virus of any one of claims 1 to 3.

5. A recombinant vector comprising a nucleic acid molecule, The nucleic acid molecule encodes the degron-containing recombinant virus of any one of claims 1 to 3, A recombinant vector that expresses the degron-containing recombinant virus of any one of claims 1 to 3.

6. A method for preparing the degron-containing recombinant virus according to any one of claims 1 to 3, comprising: constructing a cell line deficient in proteolytic systems; introducing a nucleotide sequence encoding a degron into the intermediate coding region of a viral protein-encoding gene; constructing a recombinant vector, introducing it into the proteolytic system-deficient cell line, and packaging it to obtain the degron-containing recombinant virus, wherein the recombinant vector comprises a nucleic acid molecule, and the nucleic acid molecule encodes the degron-containing recombinant virus of any one of claims 1 to 3; The cell line includes a mammalian cell line including any one of a CHO cell line, a Vero cell line, an MDCK cell line, a HEK293T cell line, an MDCK cell line, an A549 cell line, a BHK cell line, a BHK-21 / BRS cell line, an Sp2 / 0 cell line, a HEK293 cell line, a 293F cell line, a HeLa cell line, a TZM-bl cell line, a Sup-T1 cell line, an MRC-5 cell line, a VMK cell line, an LLC-MK2 cell line, an HCT-8 cell line, a Huh-7 cell line, or a Caco2 cell line; The degron-containing recombinant virus is prepared by adding a proteolytic inhibitor during the preparation process; The method for preparing the composition, wherein the protein degradation inhibitor includes a proteasome inhibitor including any one or a combination of at least two of MG132, MG-341, and lactacystin.

7. The preparation method described in claim 6, wherein the protein degradation system deficiency includes a ubiquitin-proteasome system deficiency including any one or a combination of at least two of E3 ligase knockout or knockdown, and proteasome knockout or knockdown.

8. The preparation method further comprises detecting the replication competence, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient cell line and an unmodified cell line; The method of claim 6, further comprising the step of producing on a large scale.

9. The preparation method comprises: Step (1) of constructing a proteolytically deficient mammalian cell line, (1) knocking out key components of the protein degradation system in a cell line using gene editing technology; (2) determining a viral protein into which a degron has been introduced, as well as the type, number, and introduction site of the degron, and introducing a nucleotide sequence encoding the degron into a gene encoding the viral protein to obtain a recombinant viral sequence; Step (3) of constructing a recombinant vector, Step (3) of ligating the recombinant viral sequence in step (2) with the plasmid to obtain the recombinant vector; (4) introducing the recombinant vector into the cell line lacking a proteolytic system and packaging it to obtain the degron-containing recombinant virus; (4) co-transfecting the recombinant vector in step (3) and a viral rescue plasmid into the cell line lacking a proteolytic system by reverse genetics, adding a proteolytic inhibitor, and packaging the resulting virus to obtain the degron-containing recombinant virus; and detecting the replication competence, safety, and immunogenicity of the degron-containing recombinant virus in the proteolytic system-deficient cell line and an unmodified cell line, and producing the virus on a large scale.

10. A system for preparing a degron-containing recombinant virus according to any one of claims 1 to 3, comprising: A preparation system comprising a proteolytically deficient cell line, a recombinant vector and a viral rescue plasmid.

11. Use of any one or a combination of at least two of the degron-containing recombinant virus, nucleic acid molecule, recombinant vector, method for preparing a degron-containing recombinant virus, or system for preparing a degron-containing recombinant virus according to any one of claims 1 to 3 in the production of a vaccine, an oncolytic virus, and / or a pharmaceutical, comprising: wherein the nucleic acid molecule encodes the degron-containing recombinant virus of any one of claims 1 to 3; wherein the recombinant vector comprises the nucleic acid molecule; The method for preparing the degron-containing recombinant virus includes constructing a cell line deficient in a proteolytic system; introducing a nucleotide sequence encoding a degron into a viral protein-encoding gene; constructing the recombinant vector, introducing it into the proteolytic system-deficient cell line, and packaging it to obtain the degron-containing recombinant virus; The degron-containing recombinant virus preparation system includes a proteolytic system-deficient cell line, a recombinant vector, and a viral rescue plasmid.

12. A vaccine comprising the degron-containing recombinant virus of any one of claims 1 to 3, the vaccine comprises any one of a live attenuated vaccine, a live replication-incompetent vaccine, a live replication-regulatable vaccine, or an oncolytic virus vaccine; The vaccine further comprises an adjuvant and auxiliary materials.

13. An oncolytic virus comprising the degron-containing recombinant virus of any one of claims 1 to 3.

14. A pharmaceutical comprising the degron-containing recombinant virus of any one of claims 1 to 3, The pharmaceutical product further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent or an excipient.

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