WO2023151630A1 - 一种自噬靶向病毒及其制备方法和应用 - Google Patents

一种自噬靶向病毒及其制备方法和应用 Download PDF

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WO2023151630A1
WO2023151630A1 PCT/CN2023/075252 CN2023075252W WO2023151630A1 WO 2023151630 A1 WO2023151630 A1 WO 2023151630A1 CN 2023075252 W CN2023075252 W CN 2023075252W WO 2023151630 A1 WO2023151630 A1 WO 2023151630A1
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protein
virus
autophagy
targeting
targeting molecule
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French (fr)
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司龙龙
施小山
李静
李乐
沈金影
申权
陈丽
肖雪
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中国科学院深圳先进技术研究院
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Definitions

  • the application belongs to the field of biotechnology, and relates to an autophagy targeting virus and its preparation method and application.
  • Vaccines are one of the most effective means of preventing viral infections.
  • CN107537030A discloses a trivalent influenza virus subunit vaccine and its preparation method.
  • the cracked viral protein is further purified by using a lysing agent and a new purification method to make a quadrivalent influenza virus subunit vaccine, wherein each dose contains 1 Type (H1N1), type A3 (H3N2), and type B have three influenza hemagglutinin contents above 80%, no adjuvant, no preservatives such as thimerosal, and a preparation method of the influenza vaccine is also provided, including The following steps: virus inoculation, virus proliferation culture, allantoic fluid harvest, clarification, ultrafiltration concentration, inactivation, lysis and ultracentrifugation purification, gel filtration chromatography purification (ultrafiltration), mixing, filtration sterilization, aliquoting , packaging and other steps, the influenza virus subunit vaccine provided can improve the safety of influenza vaccines, eliminate the adverse reactions caused by adjuvants, and eliminate the toxic and side effects caused by thimerosal, but the preparation process
  • the present application provides an autophagy-targeting virus and its preparation method and application.
  • the autophagy-targeting virus contains molecules that can be recognized by the autophagy system.
  • the replication of the virus can be effectively controlled, and can be widely used in live vaccines, attenuation Virus vaccines, inactivated vaccines, virulent safety models, etc., are safe and controllable, and have good immunogenicity.
  • the preparation method is simple and versatile, and can be applied to the preparation of any virus vaccines; at the same time Autophagy-targeting viruses can be used as oncolytic viruses.
  • the present application provides an autophagy-targeting virus, which contains an autophagy-targeting molecule recognized by the autophagy system.
  • autophagy-targeting molecules refers to polypeptides, amino acid sequences, proteins or other molecules that can be recognized by the autophagy system.
  • the coding nucleotide sequence is introduced into the genome of the virus, and the autophagy targeting molecule can be replicated along with the replication of the viral genome, and can be fused and expressed in the viral protein along with the translation of the viral protein, thereby obtaining the autophagy target
  • a virus modified to a molecular point that is, an autophagy targeting virus, Autophagy-Targeting chimeric virus (AUTOTAC virus, AUTOTAC virus)
  • AUTOTAC virus Autophagy-Targeting chimeric virus
  • AUTOTAC virus Autophagy-Targeting chimeric virus
  • the autophagy targeting molecule is selected from a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.111 or a combination of at least two of them (as shown in Table 1).
  • the autophagy targeting molecule is linked to the viral protein of the autophagy targeting virus.
  • the autophagy targeting molecule can be linked to any position in the viral protein.
  • the phagocytic targeting molecule is linked to the C-terminal and/or N-terminal of the viral protein.
  • the autophagy targeting molecule and the viral protein are connected by a connecting chain.
  • the autophagy targeting molecule can be the same autophagy targeting molecule, or a combination of different autophagy targeting molecules, and the connecting chain can be the same connecting chain, or it can be different A combination of connecting chains.
  • said connecting strand comprises a cleaved molecule.
  • the autophagy system since the autophagy system widely exists in host cells, in order to avoid the degradation of the autophagy system in the cell during the preparation of the AUTOTAC virus, which would lead to a reduction in production efficiency, firstly, introduce A linker that can be selectively cleaved, which can be cleaved in a specific artificially engineered cell line, thereby separating the viral protein from the autophagy targeting molecule, and the viral protein will no longer be degraded by the autophagy system Therefore, the AUTOTAC virus can be efficiently replicated and produced in large quantities in the specific artificially modified cell line. In normal cells, the autophagy system will recognize autophagy targeting molecules fused with viral proteins, thereby degrading viral proteins, weakening or even completely losing the ability to replicate.
  • the AUTOTAC virus can be further modified, such as introducing some immune enhancers to specific regions or specific amino acids of the virus protein, so as to obtain a virus with improved performance and an AUTOTAC virus with enhanced immunogenicity.
  • cell lines with autophagy system knockout or functional deficiency can also be used to prepare AUTOTAC viruses. Such cell lines can not only prepare AUTOTAC viruses with autophagy targeting molecules introduced into the N-terminus and/or C-terminus of viral proteins, but also can It is used to prepare the AUTOTAC virus in which the autophagy targeting molecule is introduced into any other site of the virus protein.
  • Another purpose of the present application is to introduce the coding nucleotide sequence of the autophagy targeting molecule that can be conditionally cleaved or the coding nucleotide sequence of the autophagy targeting molecule that does not need to be cut into the influenza virus genome, so that the virus only has It can only be replicated in a specific virus production system that can cut off or inactivate autophagy targeting molecules.
  • a specific virus production system that can cut off or inactivate autophagy targeting molecules.
  • a large amount of AUTOTAC virus can be prepared in this system.
  • the prepared AUTOTAC virus has weakened or even defective replication and reproduction in animals and humans.
  • the safety of the virus is increased, so that the AUTOTAC virus has become a veritable influenza virus live vaccine.
  • autophagy systems in animals and humans can recognize a variety of autophagy targeting molecules, so different types and quantities of autophagy targeting molecules can be introduced into viral proteins; )
  • connecting chains can be introduced to realize the selective cutting of autophagy targeting molecules; these different types and numbers of autophagy targeting molecules and connecting chains can be combined in any way, and can be prepared Virus vaccines with different replication efficiencies and degrees of attenuation provide assurance, which is critical for the production efficiency and immunogenicity of influenza virus vaccines.
  • the principle of the autophagy-targeting virus of the present application lies in: (1) the autophagy-targeting molecule introduced into the specific site of the virus protein can be recognized by the autophagy system in the normal host cell, so that the relevant virus protein Degradation and inactivation; (2) Autophagy targeting molecules introduced into specific sites of viral proteins can be inhibited in specific viral production systems, or separated from viral proteins by selective cleavage of connecting chains, thereby avoiding Or reduce the degradation of viral proteins by the autophagy pathway; and (3) the autophagy targeting molecule introduced into the specific site of the viral protein cannot be inhibited in normal host cells, or the linking chain linking the autophagy targeting molecule and the viral protein Normal host cells cannot be cut, so the prepared virus can be recognized and degraded by the autophagy pathway in host cells such as animals and humans, and the replication ability is reduced or even completely lost, which increases the safety of the virus.
  • the cleaved molecule includes a molecule cleaved by tobacco plaque virus protease (Tobacco etch virus protease, TEVp), a molecule sensitively cleaved by thrombin (the cleavage site is LVPR*GS, such as SEQ ID NO.
  • the molecule cleaved by tobacco plaque virus protease includes a polypeptide, and the polypeptide has an amino acid sequence shown in general formula I: EX aa -X aa -YX aa -Q-$ general formula I;
  • X aa represents any amino acid
  • $ represents any one of amino acids G, S or M. Cutting occurs between Q and G, or between Q and S, or between Q and M.
  • the molecule cleaved by bacterial gelatinase comprises a polypeptide whose amino acid sequence is GPLGV (SEQ ID No.250).
  • said self-cleaving molecule comprises a 2A short peptide.
  • the 2A short peptide includes any of P2A of porcine teschovirus-1, E2A of equine rhinitis A virus, F2A or T2A of foot ans mouth disease virus One or a combination of at least two.
  • the connecting chain is selected from polypeptides having an amino acid sequence as shown in any one of SEQ ID NO.112 to SEQ ID NO.137 (as shown in Table 2).
  • a flexible linker is also included between the autophagy targeting molecule and the connecting chain.
  • the autophagy-targeting molecule, connecting chain and flexible linker have the following connection methods:
  • the autophagy targeting molecule, connecting chain and flexible linker are connected according to the connection mode of the flexible linker-connecting chain-flexible linker-autophagy targeting molecule and include the following sequence as SEQ ID NO.138-SEQ ID NO A polypeptide having an amino acid sequence as shown in any one of 248 (as shown in Table 3).
  • the autophagy-targeting virus is prepared from a wild virus through genetic modification.
  • the wild virus is selected from influenza virus, HIV, hand-foot-mouth virus, Coxsackie virus, hepatitis C virus, hepatitis B virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, Epstein-Barr virus, human papillomavirus , herpes simplex virus, cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, respiratory syncytial virus, dengue virus, Ebola virus, Marburg virus, Zika virus, Middle East respiratory syndrome virus, Rotavirus, rabies virus, measles virus, adenovirus, poliovirus, echovirus, Japanese encephalitis virus, forest encephalitis virus, hantavirus, novel enterovirus, rubella virus, mumps virus, para Influenza virus, PRRS virus, swine fever virus, foot-and-mouth disease virus, parvovirus, prions, smallpox virus, tobacco mosaic
  • the wild virus is influenza virus, HIV or SARS-CoV-2.
  • influenza virus includes any one or a combination of at least two of H1N1, H5N1, H7N9, H3N2 or B influenza viruses.
  • the autophagy-targeting virus is autophagy-targeting influenza virus.
  • the autophagy targets PA protein, PB1 protein, PB2 protein, NP protein, HA protein of influenza virus
  • PA protein PB1 protein, PB2 protein, NP protein, HA protein of influenza virus
  • One or more of protein, NA protein, M1 protein, M2 protein, NS1 protein or NEP protein is connected to the autophagy targeting molecule and the connecting chain.
  • both the PA protein and the PB2 protein are connected with the autophagy targeting molecule and the connecting chain.
  • both the PA protein and the PB1 protein are connected with the autophagy targeting molecule and the connecting chain.
  • both the PB2 protein and the PB1 protein are connected with the autophagy targeting molecule and the connecting chain.
  • the PA protein, the PB2 protein and the PB1 protein are all connected with the autophagy targeting molecule and the connecting chain.
  • the PA protein, PB2 protein, PB1 protein and M1 protein are all connected with the autophagy targeting molecule and the connecting chain.
  • the PA protein, PB2 protein, PB1 protein, M1 protein and NP protein are all connected with the autophagy targeting molecule and the connecting chain.
  • the autophagy targeting molecule and the connecting chain are all connected to the PB2 protein, PB1 protein and M1 protein.
  • both the PB1 protein and the M1 protein are connected with the autophagy targeting molecule and the connecting chain.
  • both the PB2 protein and the M1 protein are connected with the autophagy targeting molecule and the connecting chain.
  • the PB2 protein, PB1 protein, M1 protein and NS1 protein are all connected with the autophagy targeting molecule and the connecting chain.
  • the PB2 protein, PB1 protein, M1 protein and NEP protein are all connected with the autophagy targeting molecule and the connecting chain.
  • both the NS1 protein and the NEP protein are connected with the autophagy targeting molecule and the connecting chain.
  • any gene of the existing influenza virus model can be replaced by genes of other subtypes or strains, or the existing influenza virus model can be replaced by other subtypes Or strains, so as to prepare other subtypes or strains of influenza virus, which can be applied to any subtype or strain of influenza virus, and the prepared virus can be selectively excised in cell lines that target autophagy molecules A large number of replication, but in normal host cells will be recognized and degraded by the phagocytosis system.
  • the method can be applied to other subtypes or strains, including H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3 , H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H1 0N8, H10N9 , H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10,
  • multivalent influenza viruses such as multivalent influenza viruses containing surface antigens of H1N1, H3N2, and B influenza viruses. More importantly, the prepared mutant virulent and multivalent virus has very high safety and efficacy.
  • the autophagy-targeting virus is an autophagy-targeting coronavirus.
  • the autophagy targets spike protein, envelope glycoprotein, membrane glycoprotein, nucleocapsid protein, nonstructural protein 1, 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-ribose methyl transfer enzyme, endonuclease, 3'-to-5' exonuclease, helicase, RNA-dependent RNA polymerase, orf1a polyprotein, ORF10 protein, ORF8 protein, ORF7a protein, ORF6 protein or ORF3a protein
  • One or more of the autophagy targeting molecules and connecting chains are connected.
  • the autophagy targeting coronavirus is autophagy targeting the new coronavirus SARS-CoV-2.
  • the autophagy-targeting virus is autophagy-targeting HIV virus.
  • the autophagy targets Gag polyprotein, pol polyprotein, gp160, HIV transcriptional transactivator, virion protein expression regulatory protein, viral negative factor, lentiviral protein R, viral infectivity of HIV virus
  • virion protein expression regulatory protein viral negative factor
  • lentiviral protein R viral infectivity of HIV virus
  • factor viral protein U, matrix protein, capsid protein, spacer peptide 1, nucleocapsid protein, spacer peptide 2, P6, reverse transcriptase, ribonuclease H, integrase, HIV protease, gp120, gp41 protein
  • the autophagy targeting molecule and the connecting chain are connected in one or more of them.
  • the present application provides a nucleic acid molecule, which contains a nucleic acid sequence encoding the autophagy-targeting virus as described in the first aspect.
  • the present application provides an expression vector, which contains the nucleic acid molecule as described in the second aspect.
  • the present application provides a recombinant cell, which includes a cell expressing a protease that cleaves the connecting chain or a cell with a defective autophagy system.
  • the protease comprises tobacco plaque virus protease.
  • the cells expressing the protease that selectively cleaves the connecting chain are prepared from HEK293T cells or MDCK cells.
  • the autophagy system-deficient cells lack any one or a combination of at least two of LC3A protein, LC3B protein, LC3C protein, GABARAP protein, GABARAPL1 protein or GABARAPL2 protein.
  • the recombinant cell contains the expression vector of the third aspect or the nucleic acid molecule of the second aspect.
  • the recombinant cells can be used for the preparation of the autophagy-targeting virus described in the first aspect.
  • the present application provides a method for preparing the autophagy targeting virus as described in the first aspect, the method comprising the following steps:
  • step (3) connecting the encoded nucleotide sequence of the viral protein obtained in step (2) to the vector to obtain an expression vector;
  • step (3) using reverse genetic technology, co-transfecting the expression vector in step (3) and the expression vector used for virus rescue into cells, and performing cell culture to obtain the autophagy targeting virus;
  • the vector in step (3) includes a plasmid
  • the cells in step (4) include cells expressing a protease that cleaves the connecting chain or cells with autophagy system defects;
  • the protease expressed by the cell expressing the protease that cleaves the connecting chain is tobacco plaque virus protease
  • any one or at least two of LC3A protein, LC3B protein, LC3C protein, GABARAP protein, GABARAPL1 protein or GABARAPL2 protein is missing in the cells with defective autophagy system;
  • step (1) the step of constructing a cell expressing a protease that selectively cleaves the connecting chain is also included;
  • the wild cell (referring to the initial cell for genetic modification) expressing the protease that cleaves the connecting chain includes a mammalian cell;
  • the mammalian cells are selected from CHO cells, Vero cells, MDCK.2 cells, HEK293T cells, MDCK cells, A549 cells, BHK cells, BHK-21/BRS cells, Sp2/0 cells, HEK293 cells, 293F cells , HeLa cells, TZM-bl cells, Sup-T1 cells, MRC-5 cells and VMK cells, LLC-MK2 cells, HCT-8 cells, Huh-7 cells or Caco2 cells, any one or a combination of at least two , preferably HEK293T cells and/or MDCK cells.
  • the medium for cell culture described in step (4) contains an autophagy inhibitor.
  • the autophagy inhibitor includes any one or at least two of Chloroquine, Hydroxychloroquine, Hydroxychloroquine, Leupeptin, 3-Methyladenine (3-MA), Wortmannin, Lys01 or LY294002 The combination.
  • the method comprises the steps of:
  • step (2') the nucleotide sequence encoded by the viral protein after the gene mutation obtained in step (2') is connected to the carrier to obtain an expression vector;
  • step (3') Using reverse genetic technology, the expression vector obtained in step (3'), the expression vector used for virus rescue and the expression vector obtained in step (4') are co-transfected into cells, and the cells are cultured to obtain the described Autophagy targets viruses.
  • the wild virus is influenza virus.
  • the method further comprises the step of detection, the detection comprising the determination of the autophagy-targeted virus in the cells expressing the protease that selectively cleaves the connecting chain and in the unmodified normal host cells Replication ability to determine whether the autophagy-targeting virus has been successfully engineered, wherein replication is performed in the cell expressing a protease that selectively cleaves the connecting chain, and replication ability is reduced or unable to be replicated in an unmodified normal host cell
  • the replicated autophagy-targeting virus is a successfully modified autophagy-targeting virus.
  • the method further includes using a successfully modified autophagy-targeting viral vector, repeating steps (1)-(4) or (1')-(5'), so that autophagy targets multiple viral proteins of the virus Autophagy-targeting molecules and connecting chains are introduced on each of the autophagy-targeting viruses; or multiple autophagy-targeting molecules and connecting chains are introduced on any viral protein of the autophagy-targeting virus.
  • a method for the preparation and large-scale production of autophagy targeting influenza virus comprising the following steps:
  • the mammalian cell lines are HEK293T cell lines and MDCK cell lines;
  • the stable cell lines constructed are HEK293T-TEVp and MDCK-TEVp; it should be noted that although in this example, tobacco plaque virus protease (Tobacco etch virus protease, TEVp) and its corresponding cleavable sequence are preferred as the research object , but those skilled in the art should understand that the principle of the application technology can be extended to any other molecules with cleavage (including proteins, polypeptides, nucleic acids, and chemical molecules);
  • one or more insertion sites are selected in gene segments encoding different proteins of influenza virus
  • Gene mutation use genetic engineering methods to introduce nucleotide sequences encoding autophagy targeting molecules and connecting chains at the identified influenza virus proteins and genes encoding selection sites;
  • the coding nucleotide sequence of the viral protein after the genetic mutation obtained in step (3") is connected to the plasmid to obtain the coding plasmid;
  • step (4" Using reverse genetic technology, co-transfect the plasmid in step (4") with the stable cells stably expressing TEVp obtained in step (1") with other plasmids used for influenza virus rescue to obtain autophagy targets to influenza virus;
  • the nucleotide sequence encoding the autophagy targeting molecule and the protease cleavage sequence of tobacco plaque virus is introduced into the genome of the autophagy targeting influenza virus, thereby introducing the autophagy targeting molecule and the cleavage sequence into the corresponding influenza virus protein.
  • the successfully transfected host cells are cultured in DMEM medium containing 0.5% FBS, 2 ⁇ g/mL TPCK-trypsin;
  • the obtained mutant sequence expression vector was co-transfected into host cells together with other plasmids required for virus rescue and plasmids overexpressing TEVp. Cultured in the DMEM medium of trypsin;
  • the supernatant is collected for infecting new MDCK-TEVp cells, and the culture medium is DMEM medium containing 0.5% FBS, 2 ⁇ g/mL TPCK-trypsin, after 4 days of infection, or when the infected virus has completely damaged the host cells in (1"), collect the supernatant to obtain autophagy targeting influenza virus .
  • the preparation method of the autophagy-targeted influenza virus further comprises a step (7") detection: the dependence of the autophagy-targeted influenza virus obtained in the determination step (5") on TEVp and the inactivation of its packaging product Dependence on the autophagy pathway to determine whether the autophagy-targeted influenza virus has been successfully transformed;
  • the so-called TEVp dependence of the packaging product refers to the fact that the virus packaged using the method can replicate and proliferate in a cell line with high TEVp expression, but cannot replicate in normal cells that do not express TEVp. decrease or defect.
  • the preparation method of the autophagy-targeting influenza virus further includes step (8") using the successfully transformed autophagy-targeting influenza virus vector to repeat steps (2") to (5"), so that the autophagy-targeting Autophagy targeting molecules and connecting chains are introduced into multiple viral proteins of influenza virus; or multiple autophagy targeting molecules and connecting chains are introduced on any viral protein of autophagy targeting influenza virus;
  • the autophagy-targeting influenza virus by measuring the dependence of the obtained autophagy-targeting influenza virus on TEVp and the dependence of the inactivation of its packaging product on the autophagy pathway, it is determined whether the autophagy-targeting virus has been successfully transformed; retention after long-term passage The autophagy-targeted virus that still maintains its dependence on TEVp is a successful candidate for transformation;
  • the method for preparing autophagy-targeting influenza virus also includes (9") selecting candidates for successful transformation, and purifying the product;
  • the safe influenza virus is the successfully modified influenza virus.
  • autophagy-targeting influenza virus gene into a vector (such as pHH21 plasmid)
  • transfecting the vector together with other vectors required for rescue of influenza virus can selectively AUTOTAC influenza virus can be obtained by excising stable cell lines of autophagy targeting molecules, preferably HEK293T-TEVp and MDCK-TEVp, which can selectively excise autophagy targeting molecules in stable cell lines, preferably HEK293T-TEVp and/or MDCK-TEVp for large scale amplification preparations.
  • a stable cell line (preferably HEK293T-TEVp and MDCK-TEVp) that can stably express tobacco plaque virus protease TEVp was established.
  • the mammalian stable cell line also solves the shortcomings of the traditional use of chicken embryos to propagate viruses and easily cause adverse reactions such as human allergies.
  • an appropriate amount of autophagy inhibitors can be added to the virus medium to inhibit the degradation of viral proteins by the autophagy system; or cell lines with defective autophagy systems can be used, such as LC3A, LC3B, LC3C, One or more of the key proteins of the autophagy system such as GABARAP, GABARAPL1 or GABARAPL2 are knocked out or knocked down in cell lines.
  • the inventors constructed a puromycin-resistant lentiviral overexpression vector, which carried the expression gene of TEVp, transduced HEK293T cells and MDCK cells by virus, and passed Puromycin screened to obtain stable cell lines stably expressing TEVp, these stable cell lines were monoclonalized and monoclonal cultured, and the stable cell line with the highest expression of TEVp was sorted out by Western Blot and RT-qPCR, which was the final The stable cell lines HEK 293T cells and MDCK cells.
  • the purified mutant AUTOTAC influenza virus can be obtained.
  • Preliminary in vivo and in vitro experiments have proved that AUTOTAC influenza virus has excellent safety and genetic Stability, and has better immune effect compared with inactivated virus.
  • the present application provides the autophagy-targeting virus described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. Application in drugs for preventing and/or treating viruses or tumors.
  • the present application provides a pharmaceutical composition comprising the autophagy-targeting virus described in the first aspect.
  • the pharmaceutical composition also contains excipients.
  • the auxiliary materials include pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilizers, osmotic pressure regulators, surface Any one or a combination of at least two of active agents, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
  • the pharmaceutical composition includes a vaccine.
  • the vaccine is an attenuated live vaccine, a replication-incompetent live vaccine (including a replication-attenuated or deficient live vaccine) or a replication-controllable live vaccine.
  • the present application provides an oncolytic virus, the oncolytic virus comprising the autophagy-targeting virus described in the first aspect.
  • This application provides the use of the AUTOTAC virus as an oncolytic virus, which can not only be used alone, but also directly kill tumor cells, activate the body's anti-tumor immunity, and transform "cold” tumors into “hot” tumors ; It can also be used in combination with antineoplastic drugs to produce other synergistic effects.
  • the present application provides a method for preparing attenuated live virus, replication incompetent live virus (including replication weakened or deficient live virus), replication controllable live virus and preparation of vaccines and drugs for the prevention and treatment of viral infection,
  • the method includes the preparation method of the autophagy-targeting virus described in the fifth aspect.
  • the present application provides a system for preparing the autophagy-targeting virus, the system comprising cells stably expressing a protease that selectively cleaves the connecting chain of the autophagy-targeting virus;
  • the cell is a cell line stably expressing tobacco plaque virus protease TEVp or a cell line with a defective autophagy system
  • the cell line is a HEK293T cell line stably expressing tobacco plaque virus protease TEVp or MDCK Cell lines
  • the cell lines with autophagy system defects include LC3A, LC3B, LC3C, GABARAP, GABARAPL1 or GABARAPL2 and other key autophagy system protein knockout or knockdown cell lines.
  • compositions, vaccines, and oncolytic viruses in this application can be prepared using conventional techniques in the field on the basis of site-directed mutation-modified AUTOTAC influenza viruses prepared in this application; they can be used to prevent or treat influenza virus infections, including humans and animals influenza virus infection; they can also be used to treat tumors.
  • Fig. 1 is the difference diagram of the replication ability of the AUTOTAC virus prepared by the present application in MDCK-TEVp cells and normal MDCK cells;
  • Fig. 2 is the western blot figure of viral M1 protein level expression after the AUTOTAC virus prepared by the present application is treated with an autophagy inhibitor;
  • Fig. 3 is the result figure of the safety evaluation of the AUTOTAC virus prepared by the present application at the animal level;
  • Fig. 4 is the result figure of the immunogenicity and protective evaluation of the AUTOTAC virus prepared by the present application at the animal level;
  • Fig. 5 is a graph showing the results of inhibition of tumor growth by the AUTOTAC virus prepared in the present application.
  • a gene vector of influenza virus WSN comprising a cleavable autophagy targeting molecule was constructed.
  • each viral protein PA, PB2, PB1, NP, HA, NA, M1, M2, NS1, NEP
  • PA, PB2, PB1, NEP corresponding to each viral protein of influenza virus WSN
  • the gene sequence of the autophagy targeting molecule that can be excised by TEVp and its expressed amino acid sequence are introduced into any protein of the virus as follows, and the gene sequence corresponding to the amino acid sequence used is humanized and optimized, and Inserted into the C-terminal of the coding region of the target protein gene, before the stop codon; and verified by sequencing that the mutation was successfully constructed.
  • the gene sequences that can encode the amino acid sequence of SEQ ID NO.138 ⁇ SEQ ID NO.248 were respectively introduced, and the successfully constructed viral vectors were named according to the inserted sequence number, Named M1-AUTOTAC-138, M1-AUTOTAC-139, M1-AUTOTAC-140, M1-AUTOTAC-141, ... (here are named according to the serial number, not listed one by one), M1-AUTOTAC-248 .
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, PB2-AUTOTAC-139, PB2-AUTOTAC-140, PB2-AUTOTAC-141, ..., PB2-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, PB1-AUTOTAC-139, PB1-AUTOTAC-140, PB1-AUTOTAC-141, ..., PB1-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, NP-AUTOTAC-139, NP-AUTOTAC-140, NP-AUTOTAC-141, ..., NP-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, HA-AUTOTAC-139, HA-AUTOTAC-140, HA-AUTOTAC-141, ..., HA-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, NA-AUTOTAC-139, NA-AUTOTAC-140, NA-AUTOTAC-141, ..., NA-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, M2-AUTOTAC-139, M2-AUTOTAC-140, M2-AUTOTAC-141, ..., M2-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, NS1-AUTOTAC-139, NS1-AUTOTAC-140, NS1-AUTOTAC-141, ..., NS1-AUTOTAC-248.
  • gene sequences that can encode the amino acid sequence of SEQ ID NO. 138, NEP-AUTOTAC-139, NEP-AUTOTAC-140, NEP-AUTOTAC-141, ..., NEP-AUTOTAC-248.
  • the 12 plasmids used for rescuing influenza virus were co-transfected into stable cell lines, and the plasmids transformed by site-directed mutagenesis in Example 1 were used to replace the corresponding plasmids among the 12 plasmids, corresponding to the six-well plate Add 0.2 ⁇ g of each plasmid to each well of each well. After transfection, observe the pathological changes of the cells, and screen out insertion sites that can rescue the virus and are dependent on TEVp, autophagy targeting molecules, and TEVp-cleavable Connection chain linker, and their combination. screen The selected strains were named according to the protein and the introduced cleavable autophagy targeting molecule.
  • this plasmid was combined with other plasmids Ben1 pPolI-WSN-PB2 to rescue influenza virus; Ben2 pPolI-WSN-PB1 ; Ben4 pPolI-WSN-HA; Ben5 pPolI-WSN-NP; Ben6 pPolI-WSN-NA; Ben7 pPolI-WSN-M; Ben8 pPolI-WSN-NS; Ben9 pcDNA 3(neo)-PB2; )-PB1; Ben11 pcDNA 3(neo)-PA; Ben13 pcAGGS/MCS-NP co-transfected a stable cell line expressing TEVp, thereby rescuing the mutant influenza virus that introduced SEQ ID NO.149 on the influenza virus PA gene fragment , named PA-AUTOTAC-149.
  • this plasmid was combined with other plasmids Ben1 pPolI-WSN-PB2 to rescue influenza virus; Ben2 pPolI-WSN-PB1; Ben3 pPolI-WSN-PA; Ben4 pPolI-WSN-HA; Ben5 pPolI-WSN-NP; Ben6 pPolI-WSN-NA; Ben8 pPolI-WSN-NS; Ben9 pcDNA 3(neo)-PB2; Ben10 pcDNA 3(neo)-PB1; Ben11 pcDNA 3(neo)-PA; Ben13 pcAGGS/MCS-NP co-transfected a stable cell line expressing TEVp to rescue the introduction of SEQ ID NO.149 on the influenza virus M1 gene fragment A mutant influenza
  • mutant influenza viruses with cleavable autophagy targeting molecules introduced into other sites can be obtained and named according to the same rules.
  • a mutation modification method such as M1-AUTOTAC-149 that has high efficiency in rescuing influenza virus, stable genetics, and the final replication ability in normal host cells is significantly reduced or even completely disappeared. and PA-AUTOTAC-150), combined to prepare influenza viruses containing multiple autophagy targeting molecules, and further select preferred strains therefrom.
  • the constructed recombinant plasmid is as follows:
  • PB2-N-autophagy targeting molecule PB2-R70-autophagy targeting molecule, PB2-I176-autophagy targeting molecule, PB2-V457-autophagy targeting molecule, PB2-N510-autophagy targeting molecule, PB2-Y531-autophagy targeting molecule, PB2-A623-autophagy targeting molecule, PB2-D680-autophagy targeting molecule, PB2-E700-autophagy targeting molecule, PB2-C-autophagy targeting molecule, PB1-N-autophagy targeting molecule, PB1-D70-autophagy targeting molecule, PB1-D295-autophagy targeting molecule, PB1-R327-autophagy targeting molecule, PB1-R430-autophagy targeting molecule, PB1-F490-autophagy targeting molecule, PB1-T566-autophagy targeting molecule, PB1-N626-autophagy targeting molecule, PB1-G710-autophag
  • amino acid sequence of the autophagy targeting molecule is shown in SEQ ID No.1 to SEQ ID No.111.
  • the above-mentioned recombinant vector is introduced into a cell line with defective autophagy system, and the recombinant virus containing the autophagy targeting molecule is obtained by packaging:
  • recombinant vector in this example to replace the corresponding wild-type plasmid, and co-transfect mammalian cell lines with autophagy system defects with 11 other plasmids (example: recombinant vector PB2-N-autophagy targeting molecule SEQ ID NO .1 replacement plasmid Ben1, co-transfection with 11 other plasmids), the amount of each plasmid added was 0.2 ⁇ g, and the cells after transfection were cultured in DMEM medium containing 0.5% FBS, 2 ⁇ g/mL TPCK-trypsin .
  • the medium was DMEM medium containing 0.5% FBS, 2 ⁇ g/mL TPCK-trypsin, Four days after infection, after the host cells were completely lesioned, the supernatant was collected.
  • the supernatant was centrifuged and passed through a 0.4 ⁇ m filter membrane to remove cell debris, and the autophagy system defect dependence of the packaging product was detected, as well as the dependence of the inactivation of the packaging product on the autophagy system, and the maintenance of the autophagy system defect dependence was retained. Mutants are recombinant viruses.
  • the prepared recombinant virus in the same manner as the recombinant vector.
  • the recombinant vector PB2-N-autophagy targeting molecule SEQ ID NO.1 is used to replace the plasmid Ben1, and the prepared virus is named PB2-N-autophagy targeting molecule SEQ ID NO.1;
  • the recombinant vector PB2-N -The autophagy targeting molecule SEQ ID NO.2 replaces the plasmid Ben1, and the prepared virus is named PB2-N-autophagy targeting molecule SEQ ID NO.2; and so on.
  • step (1) rescues the stable cells of the mutant AUTOTAC influenza virus and is completely diseased, or about 4 days after transfection, collect the cell supernatant, centrifuge at 5000 ⁇ g for 10 min, and use the new stable cell line for massive amplification , to be cell After about 4 days of complete lesion or expansion, the cell supernatant was collected and passed through a 0.45 ⁇ m filter membrane;
  • b Purify influenza virus by sucrose gradient density centrifugation. The specific steps are as follows: centrifuge the virus liquid in 1) at 105 ⁇ g for 2 hours in a 50 mL centrifuge tube (special for high speed), and resuspend the pellet in 1 mL of PBS;
  • step d Add the sucrose in step c to 50mL, drop the PBS resuspension in b on the sucrose solution, centrifuge at 11 ⁇ 10 4 ⁇ g for 2h;
  • step f Resuspend the pellet from step e with PBS.
  • the safety of the AUTOTAC virus was confirmed by investigating the dependence of the prepared mutant AUTOTAC influenza virus M1-AUTOTAC-141, 149, and 219 on the TEVp protein; long-term subculture was carried out to investigate the cleavable protein in the mutant virus Stability of autophagy targeting molecules.
  • Example 6 investigates whether the weakening of autophagy-targeted influenza virus replication ability is regulated by cellular autophagy
  • the results in Figure 2 show that after infecting MDCK cells, the wild-type virus can replicate in large quantities and produce a large amount of viral proteins.
  • the autophagy targeting molecules are modified on the viral protein M1 of the AUTOTAC virus, the viral M1 protein will be degraded 48h after infection; After Hydroxychloroquine Hydroxychloroquine (HCQ), Leupeptin, or LY294002, etc.), autophagy-mediated viral protein M1 degradation is inhibited.
  • the experimental results show that the autophagy targeting molecules introduced by the inventors can mediate the degradation of viral proteins; the reduction of the replication ability of AUTOTAC virus in normal cells is caused by the degradation of viral proteins mediated by autophagy, which is in line with the principle of this application .
  • inactivated influenza vaccine IIV
  • control inactivated influenza virus vaccine is prepared by the inventor with homologous influenza virus particles according to the method provided by the Chinese Pharmacopoeia
  • select M1-AUTOTAC-149 as the representative of AUTOTAC virus, and perform AUTOTAC Virus safety, immunogenicity and protective evaluation.
  • Virus inoculation the first group was inoculated with PBS by nasal drops; the second group was inoculated with 10 5 PFU M1-AUTOTAC-149 by nasal drops; the third group was inoculated with 10 5 PFU of wild-type WSN influenza virus;
  • mice were taken from each group, and their lung tissues were taken to detect the virus titer therein;
  • Virus inoculation the first group was inoculated with PBS by nasal drops; the second group was inoculated with 10 5 PFU M1-AUTOTAC-149 by nasal drops; the third group was inoculated with 10 5 PFU inactivated influenza vaccine;
  • mice were taken from each group, and their lung tissues and spleens were taken to detect the T cell immune response;
  • mice were taken from each group, and blood was taken for hemagglutination inhibition (HI) test, neutralizing (NT) antibody detection, and ELISA test to detect the antibody immune response therein;
  • HI hemagglutination inhibition
  • NT neutralizing
  • ELISA test to detect the antibody immune response therein;
  • mice Three weeks after inoculation, each group of mice was inoculated with 2 ⁇ 10 5 PFU of wild-type WSN influenza virus in the nasal cavity;
  • mice were taken from each group, and their lung tissues were taken to detect the virus titer therein;
  • the C57BL/c melanoma tumor-bearing model was used to evaluate the oncolytic effect of AUTOTAC influenza virus.
  • the specific experiment included the following steps:
  • this application creatively designed an autophagy-targeting virus, which can be efficiently replicated and mass-produced in a specific artificial cell line, but the replication ability of the virus is weakened or even completely lost in normal cells Replication ability, can be widely used in live vaccines, attenuated vaccines, inactivated vaccines, virulent safety models, etc. It is safe and controllable, and has good immunogenicity.
  • the autophagy targeting virus can be used as The oncolytic virus can not only directly kill the tumor, but also activate the immune effect of the body against the tumor.
  • the virus preparation method provided is simple and versatile.
  • the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

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Abstract

提供了一种自噬靶向病毒及其制备方法和应用。该自噬靶向病毒含有被自噬系统识别的自噬靶向分子。该自噬靶向病毒的复制可被调控，可以在特定的细胞系中高效复制，但在正常的细胞中病毒的复制能力被减弱甚至完全失去复制能力，可应用于制备活疫苗、减毒疫苗、灭活疫苗或强毒的安全模型，具有安全可控性，且具有良好的免疫原性。该自噬靶向病毒还可作为溶瘤病毒。

Description

一种自噬靶向病毒及其制备方法和应用 技术领域
本申请属于生物技术领域，涉及一种自噬靶向病毒及其制备方法和应用。
背景技术
疫苗是预防病毒感染最有效的手段之一，然而目前仍存在明显局限性，包括安全性差、免疫效果差、或技术复杂不通用等，且难以实现疫苗个性化定制。
如CN107537030A公开一种三价流感病毒亚单位疫苗及其制备方法，采用裂解剂和新的纯化方法将裂解后的病毒蛋白进一步纯化，制成四价流感病毒亚单位疫苗，其中每剂含甲1型(H1N1)、甲3型(H3N2)、乙型共三种流感血凝素含量在80％以上，不含佐剂，不含硫柳汞等防腐剂，还提供了该流感疫苗的制备方法，包括以下步骤：病毒接种、病毒增殖培养、尿囊液收获、澄清、超滤浓缩、灭活、裂解和超速离心纯化、凝胶过滤层析纯化(超滤)、混配、过滤除菌、分装、包装等步骤，提供的流感病毒亚单位疫苗可以提高流感疫苗的安全性，消除了佐剂带来的不良反应，消除了硫柳汞引起的毒副作用，但制备工艺复杂，且不具备通用性，产品类型单一。
综上所述，探索并建立下一代疫苗创新的新理论、新方法和新策略是当今世界科技前沿的热点和难点(125 questions:Exploration and discovery.Science,doi:www.science.org/content/resource/125-questions-exploration-and-discovery(2021).)。
发明内容
本申请提供一种自噬靶向病毒及其制备方法和应用，所述自噬靶向病毒包含能被自噬系统识别的分子，病毒的复制可被有效控制，可广泛应用于活疫苗、减毒疫苗、灭活疫苗、强毒的安全模型等，具有安全的可控性，且具备良好的免疫原性，所述制备方法工艺简单且具备通用性，可应用于任意病毒疫苗的制备；同时自噬靶向病毒可以用作溶瘤病毒。
第一方面，本申请提供一种自噬靶向病毒，所述自噬靶向病毒含有被自噬系统识别的自噬靶向分子。
本申请中，术语“自噬靶向分子(Autophagy-Targeting molecules)”是指能被自噬系统识别的多肽、氨基酸序列、蛋白质或者其他分子等，本申请发现将所述自噬靶向分子的编码核苷酸序列引入到病毒的基因组中，该自噬靶向分子可以随着病毒基因组的复制而复制，并且可以随着病毒蛋白的翻译而融合表达于病毒蛋白中，从而得到被自噬靶向分子定点修饰的病毒，即自噬靶向病毒，Autophagy-Targeting chimeric virus(AUTOTAC virus，AUTOTAC病毒)，自噬系统会识别与病毒蛋白融合的自噬靶向分子，从而降解病毒蛋白，病毒的复制能力被减弱甚至完全失去复制能力，因此，AUTOTAC病毒具备高安全性。
所述自噬靶向分子选自具有如SEQ ID NO.1～SEQ ID NO.111任一项所示氨基酸序列的多肽或其中至少两种的组合(如表1所示)。
优选地，所述自噬靶向分子与所述自噬靶向病毒的病毒蛋白连接。
本申请中，所述自噬靶向分子可连接在病毒蛋白中的任意位点。
优选地，所述噬靶向分子连接在病毒蛋白的C端和/或N端。
优选地，所述自噬靶向分子和所述病毒蛋白之间通过连接链连接。
根据本申请，所述自噬靶向分子，可以是相同的自噬靶向分子，也可以是不同的自噬靶向分子的组合，所述连接链可以是相同的连接链，也可以是不同的连接链的组合。
优选地，所述连接链包括被切割的分子。
本申请中，由于自噬系统广泛存在于宿主细胞中，为了避免AUTOTAC病毒在制备过程中被细胞内的自噬系统降解导致生产效率降低，首先，在病毒蛋白与自噬靶向分子之间引入了可以被选择性切割的连接链(linker)，该连接链可以在特定的人工改造的细胞系中被切割，从而将病毒蛋白与自噬靶向分子分离，病毒蛋白不再会被自噬系统降解而得以保留，因此AUTOTAC病毒可以在该特定的人工改造的细胞系中高效复制、大量生产制备。而在正常的细胞中，自噬系统会识别与病毒蛋白融合的自噬靶向分子，从而降解病毒蛋白，病毒的复制能力被减弱甚至完全失去复制能力。此外，AUTOTAC病毒还可以进一步被修饰，例如引入一些免疫增强剂到病毒蛋白的特定区域或者特定氨基酸，从而得到性能改善的病毒，而得到免疫原性增强的AUTOTAC病毒。此外，还可采用自噬系统敲除或功能缺陷的细胞系，进行AUTOTAC病毒的制备，这类细胞系不仅可以制备自噬靶向分子引入病毒蛋白N端和/或C端的AUTOTAC病毒，而且可以用于制备自噬靶向分子引入病毒蛋白其他任意位点的AUTOTAC病毒。
本申请的另一个目的为在流感病毒基因组中引入了可被条件性切割的自噬靶向分子的编码核苷酸序列或者无需切割的自噬靶向分子的编码核苷酸序列，使得病毒只有在能够将自噬靶向分子切掉或者失活的特定的病毒生产体系中才可以复制，利用AUTOTAC病毒对该特定病毒生产体系的依赖，可以在该系统中进行AUTOTAC病毒的大量制备，由于人体和动物等的正常细胞中存在自噬系统，可以识别与病毒蛋白融合表达的自噬靶向分子，从而将病毒蛋白降解，因此制备出来的AUTOTAC病毒在动物和人体中复制繁殖能力减弱甚至缺陷，增加了病毒的安全性，从而使得该AUTOTAC病毒成了名副其实的流感病毒活疫苗。此外，动物和人体等的自噬系统可以识别多种自噬靶向分子，因此可以在病毒蛋白中引入不同种类、不同数量的自噬靶向分子；而可以被选择性切割的连接链(linker)也有多种，因此可以引入不同种类、不同数量的连接链来实现自噬靶向分子的选择性切割；这些不同种类和数量的自噬靶向分子和连接链可以进行任意组合，可以为制备不同复制效率、不同减毒程度的病毒疫苗提供保证，这对于流感病毒疫苗的生产效率和免疫原性至关重要。
综上所述，本申请自噬靶向病毒的原理在于：(1)引入到病毒蛋白特定位点的自噬靶向分子可以被正常宿主细胞中的自噬系统识别，从而将相关的病毒蛋白降解、失活；(2)引入到病毒蛋白特定位点的自噬靶向分子可以在特定的病毒生产系统中被抑制，或者通过连接链被选择性地切割，而与病毒蛋白分离，从而避免或减少病毒蛋白被自噬途径降解；以及(3)引入到病毒蛋白特定位点的自噬靶向分子在正常宿主细胞中不能被抑制，或者链接自噬靶向分子与病毒蛋白的连接链在正常宿主细胞中不能被切割，因此制备出来的病毒在动物和人体等的宿主细胞中可以被自噬途径识别、降解，而复制能力降低甚至完全失去复制繁殖能力，增加了病毒的安全性。
表1



优选地，所述被切割的分子包括被烟草蚀斑病毒蛋白酶(Tobacco etch virus protease，TEVp)切割的分子、被凝血酶敏切割的分子(酶切位点为LVPR*GS，如SEQ ID NO.249LEAGCKNFFPRSFTSCGSLE，*表示切割发生的位置)、被凝血因子Xa切割的分子(酶切位点为IDGR*)、被肠激酶切割的分子(酶切位点为DDDDK*)、被3C蛋白酶切割的分子(酶切位点为ETLFQ*GP)、被SUMO蛋白酶切割的分子、被细菌明胶酶切割的分子(如GPLGV(SEQ ID No.250))或自剪切的分子中的任意一种或至少两种的组合。
优选地，所述被烟草蚀斑病毒蛋白酶切割的分子包括多肽，所述多肽具有通式I所示的氨基酸序列：
E-Xaa-Xaa-Y-Xaa-Q-$   通式I；
其中，Xaa代表任意氨基酸，$代表氨基酸G、S或M中的任意一种。切割发生在Q和G之间，或者Q和S之间，或者Q和M之间。
优选地，所述被细菌明胶酶切割的分子包括氨基酸序列为GPLGV(SEQ ID No.250)的多肽。
优选地，所述自剪切的分子包括2A短肽。
优选地，所述2A短肽包括猪捷申病毒(porcine teschovirus-1)的P2A、马鼻炎病毒(equine rhinitis A virus)的E2A、口蹄疫病毒(foot ans mouth disease virus)的F2A或T2A中的任意一种或至少两种的组合。
优选地，所述连接链选自具有如SEQ ID NO.112～SEQ ID NO.137中任一项所示氨基酸序列的多肽(如表2所示)。
表2
优选地，所述自噬靶向分子和连接链之间还包含柔性接头。
优选地，所述自噬靶向分子、连接链和柔性接头具有如下连接方式：
柔性接头-连接链-柔性接头-自噬靶向分子。
优选地，所述自噬靶向分子、连接链和柔性接头按所述柔性接头-连接链-柔性接头-自噬靶向分子的连接方式连接后包括具有如SEQ ID NO.138-SEQ ID NO.248中任一项所示氨基酸序列的多肽(如表3所示)。
表3



优选地，所述自噬靶向病毒由野生病毒经过遗传改造制备得到。
优选地，所述野生病毒选自流感病毒、艾滋病毒、手足口病毒、柯萨奇病毒、丙肝病毒、乙肝病毒、甲肝病毒、丁型肝炎病毒、戊型肝炎病毒、EB病毒、人乳头瘤病毒、单纯疱疹病毒、巨细胞病毒、水痘-带状疱疹病毒、水泡性口炎病毒、呼吸道合胞病毒、登革病毒、埃博拉病毒、马尔堡病毒、寨卡病毒、中东呼吸综合征病毒、轮状病毒、狂犬病毒、麻疹病毒、腺病毒、脊髓灰质炎病毒、埃可病毒、乙型脑炎病毒、森林脑炎病毒、汉坦病毒、新型肠道病毒、风疹病毒、腮腺炎病毒、副流感病毒、蓝耳病毒、猪瘟病毒、口蹄疫病毒、细小病毒、朊病毒、天花病毒、烟草花叶病毒、腺相关病毒、噬菌体、疱疹病毒、西尼罗河病毒、诺如病毒、人博卡病毒或冠状病毒中的任意一种或至少两种的组合。
优选地，所述野生病毒为流感病毒、艾滋病毒或新冠病毒SARS-CoV-2。
优选地，所述流感病毒包括H1N1、H5N1、H7N9、H3N2或B型流感病毒中的任意一种或至少两种的组合。
优选地，所述自噬靶向病毒为自噬靶向流感病毒。
优选地，所述自噬靶向流感病毒的PA蛋白、PB1蛋白、PB2蛋白、NP蛋白、HA蛋 白、NA蛋白、M1蛋白、M2蛋白、NS1蛋白或NEP蛋白中的一个或多个连接所述自噬靶向分子和连接链。
优选地，所述PA蛋白和PB2蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PA蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB2蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PA蛋白、PB2蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PA蛋白、PB2蛋白、PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PA蛋白、PB2蛋白、PB1蛋白、M1蛋白和NP蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB2蛋白、PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB2蛋白和M1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB2蛋白、PB1蛋白、M1蛋白和NS1蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述PB2蛋白、PB1蛋白、M1蛋白和NEP蛋白中均连接所述自噬靶向分子和连接链。
优选地，所述NS1蛋白和NEP蛋白中均连接所述自噬靶向分子和连接链。
在本申请的一个实施方案中，利用反向遗传技术，可以将现有的流感病毒模型的任意基因替换成其他亚型或者毒株的基因，或者将现有的流感病毒模型替换成其他亚型或者毒株，从而制备出其他亚型或者毒株的流感病毒，可以应用于任意亚型或毒株的流感病毒，而且制备出的病毒在可以选择性切掉自噬靶向分子的细胞株中大量复制，而在正常的宿主细胞中会被噬系统识别降解。此外，该方法可以适用于其他亚型或者毒株，包括H1N1、H1N2、H1N3、H1N8、H1N9、H2N2、H2N3、H2N8、H3N1、H3N2、H3N8、H4N2、H4N4、H4N6、H4N8、H5N1、H5N2、H5N3、H5N6、H5N8、H5N9、H6N1、H6N2、H6N4、H6N5、H6N6、H6N8、H7N1、H7N2、H7N3、H7N7、H7N8、H7N9、H8N4、H9N1、H9N2、H9N5、H9N8、H10N3、H10N4、H10N7、H10N8、H10N9、H11N2、H11N6、H11N9、H12N1、H12N3、H12N5、H13N6、H13N8、H14N5、H15N2、H15N8、H16N3、H17N10和H18N11。此外，利用反向遗传技术，还可以制备多价流感病毒，如含有H1N1、H3N2、B型流感病毒表面抗原的多价流感病毒。更重要的是，制备出的突变型强毒和多价病毒具有非常高的安全性和有效性。
优选地，所述自噬靶向病毒为自噬靶向冠状病毒。
优选地，所述自噬靶向冠状病毒中刺突蛋白、包膜糖蛋白、膜糖蛋白、核衣壳蛋白、非结构蛋白1、非结构蛋白2、非结构蛋白3、非结构蛋白4、非结构蛋白5、非结构蛋白6、非结构蛋白7、非结构蛋白8、非结构蛋白9、非结构蛋白10、非结构蛋白11、非结构蛋白12、非结构蛋白13、非结构蛋白14、非结构蛋白15、非结构蛋白16、3a蛋白、3b蛋白、6蛋白、7a蛋白、7b蛋白、8a蛋白、8b蛋白、9b蛋白、3C样蛋白酶、前导蛋白、2’-O-核糖甲基转移酶、核酸内切酶、3’-至-5’核酸外切酶、解旋酶、RNA依赖的RNA聚合酶、orf1a多聚蛋白、ORF10蛋白、ORF8蛋白、ORF7a蛋白、ORF6蛋白或ORF3a蛋白中的一个或多个连接所述自噬靶向分子和连接链。
优选地，所述自噬靶向冠状病毒为自噬靶向新冠病毒SARS-CoV-2。
优选地，所述自噬靶向病毒为自噬靶向HIV病毒。
优选地，所述自噬靶向HIV病毒的Gag多聚蛋白、pol多聚蛋白、gp160、HIV转录反式激活子、病毒体蛋白表达调节蛋白、病毒负因子、慢病毒蛋白R、病毒感染性因子、病毒蛋白U、基质蛋白、衣壳蛋白、间隔肽1、核衣壳蛋白、间隔肽2、P6、逆转录酶、核糖核酸酶H、整合酶、HIV蛋白酶、gp120、gp41蛋白中的一个或多个中连接所述自噬靶向分子和连接链。
第二方面，本申请提供一种核酸分子，所述核酸分子含有编码如第一方面所述的自噬靶向病毒的核酸序列。
第三方面，本申请提供一种表达载体，所述表达载体含有如第二方面所述的核酸分子。
第四方面，本申请提供一种重组细胞，所述重组细胞包括表达切割所述连接链的蛋白酶的细胞或自噬系统缺陷的细胞。
优选地，所述蛋白酶包括烟草蚀斑病毒蛋白酶。
优选地，所述表达选择性切割所述连接链的蛋白酶的细胞由HEK293T细胞或MDCK细胞制备得到。
优选地，所述自噬系统缺陷的细胞缺失LC3A蛋白、LC3B蛋白、LC3C蛋白、GABARAP蛋白、GABARAPL1蛋白或GABARAPL2蛋白中的任意一种或至少两种的组合。
优选地，所述重组细胞含有第三方面所述的表达载体或第二方面所述核酸分子。
本申请中，所述重组细胞可以用于第一方面所述自噬靶向病毒的制备。
第五方面，本申请提供一种制备如第一方面所述的自噬靶向病毒的方法，所述方法包括以下步骤：
(1)统计分析野生病毒的宿主细胞内的自噬系统的表达分布，利用生物信息学分析和病毒蛋白质结构预测，选择引入自噬靶向分子和连接链的病毒蛋白及其中的引入位点；
(2)在所述引入位点的编码基因处插入编码自噬靶向分子和连接链的核苷酸序列；
(3)将步骤(2)得到的基因突变后的病毒蛋白的编码核苷酸序列与载体连接，得到表达载体；
(4)利用反向遗传技术，将步骤(3)中的表达载体与用于病毒拯救的表达载体共转染到细胞中，进行细胞培养，得到所述自噬靶向病毒；
优选地，步骤(3)所述载体包括质粒；
优选地，步骤(4)所述细胞包括表达切割所述连接链的蛋白酶的细胞或自噬系统缺陷的细胞；
优选地，所述表达切割所述连接链的蛋白酶的细胞表达的蛋白酶为烟草蚀斑病毒蛋白酶；
优选地，所述自噬系统缺陷的细胞中缺失LC3A蛋白、LC3B蛋白、LC3C蛋白、GABARAP蛋白、GABARAPL1蛋白或GABARAPL2蛋白中的任意一种或至少两种；
优选地，步骤(1)之前还包括构建表达选择性切割所述连接链的蛋白酶的细胞的步骤；
优选地，所述表达切割所述连接链的蛋白酶的细胞的野生细胞(指进行遗传改造的初始细胞)包括哺乳动物细胞；
优选地，所述哺乳动物细胞选自CHO细胞、Vero细胞、MDCK.2细胞、HEK293T细胞、MDCK细胞、A549细胞、BHK细胞、BHK-21/BRS细胞、Sp2/0细胞、HEK293细胞、293F细胞、HeLa细胞、TZM-bl细胞、Sup-T1细胞、MRC-5细胞和VMK细胞、LLC-MK2细胞、HCT-8细胞、Huh-7细胞或Caco2细胞中的任意一种或至少两种的组合，优选为HEK293T 细胞和/或MDCK细胞。
优选地，步骤(4)所述细胞培养的培养基中含有自噬抑制剂。
优选地，所述自噬抑制剂包括氯喹Chloroquine、羟氯喹Hydroxychloroquine、亮肽素Leupeptin、3-Methyladenine(3-MA)、渥曼青霉素(Wortmannin)、Lys01或LY294002中的任意一种或至少两种的组合。
优选地，所述方法包括以下步骤：
(1’)统计分析野生病毒的宿主细胞内的自噬系统的表达分布，利用生物信息学分析和病毒蛋白质结构预测，选择引入自噬靶向分子和连接链的病毒蛋白及其中的引入位点；
(2’)使用基因工程的方法，在所述引入位点的编码基因处引入编码自噬靶向分子和连接链的核苷酸序列；
(3’)将步骤(2’)得到的基因突变后的病毒蛋白的编码核苷酸序列与载体连接，得到表达载体；
(4’)构建表达切割所述连接链的蛋白酶的表达载体；
(5’)利用反向遗传技术，将步骤(3’)得到的表达载体、用于病毒拯救的表达载体以及步骤(4’)得到的表达载体共同转染细胞，进行细胞培养，得到所述自噬靶向病毒。
优选地，所述野生病毒为流感病毒。
优选地，所述方法还包括检测的步骤，所述检测包括通过测定得到的自噬靶向病毒在所述表达选择性切割所述连接链的蛋白酶的细胞与未经改造的正常宿主细胞中的复制能力，来确定所述自噬靶向病毒是否改造成功，其中，在所述表达选择性切割所述连接链的蛋白酶的细胞中复制，在未经改造的正常宿主细胞中复制能力降低或不能复制的自噬靶向病毒为改造成功的自噬靶向病毒。
优选地，所述方法还包括使用改造成功的自噬靶向病毒载体，重复步骤(1)～(4)或(1’)～(5’)，使得自噬靶向病毒的多个病毒蛋白上均引入自噬靶向分子和连接链；或者在自噬靶向病毒的任一病毒蛋白上引入多个自噬靶向分子和连接链。
根据本申请的具体的实施例，提供了一种自噬靶向流感病毒的制备和大规模生产的方法，包括以下步骤：
(1”)构建细胞系：将烟草蚀斑病毒蛋白酶TEVp转导至哺乳动物细胞系中，构建可以稳定表达烟草蚀斑病毒蛋白酶TEVp的细胞系；
优选地，所述哺乳动物细胞系为HEK293T细胞系和MDCK细胞系；
构建的稳定细胞系为HEK293T-TEVp和MDCK-TEVp；需要说明的是，虽然在本实施例中，优选烟草蚀斑病毒蛋白酶(Tobacco etch virus protease，TEVp)及其对应的可切割序列作为研究对象，但本领域技术人员应理解的是，该申请技术的原理可以拓展至其他任何具有切割作用的分子(包括蛋白、多肽、核酸、及化学分子)；
(2”)位点选择：通过对宿主体内的自噬体系的表达分布进行统计分析、对流感病毒进行生物信息学和蛋白质结构预测，预测分析在流感病毒的各个蛋白中引入自噬靶向分子及连接链烟草蚀斑病毒蛋白酶切割序列后的流感病毒的蛋白结构，以确定引入自噬靶向分子和连接链的基因片段及位点；
优选地，在编码流感病毒的不同蛋白的基因片段中选择一个或多个插入位点；
(3”)基因突变：使用基因工程的方法，在所确定的流感病毒蛋白及选择位点的编码基因处引入编码自噬靶向分子和连接链的核苷酸序列；
(4”)构建质粒：
将步骤(3”)得到的基因突变后的病毒蛋白的编码核苷酸序列与质粒连接，得到编码质粒；
(5”)利用反向遗传技术，将步骤(4”)中的质粒与用于流感病毒拯救的其他质粒在步骤(1”)得到的稳定表达TEVp的稳定细胞中共转染，获得自噬靶向流感病毒；
其中，在自噬靶向流感病毒的基因组中引入了自噬靶向分子及烟草蚀斑病毒蛋白酶切割序列的编码核苷酸序列，从而在对应的流感病毒的蛋白中引入自噬靶向分子及烟草蚀斑病毒蛋白酶切割序列得到自噬靶向分子修饰的流感病毒(AUTOTAC流感病毒)；
优选地，将转染成功后的宿主细胞在含有0.5％FBS、2μg/mL TPCK-trypsin的DMEM培养基中培养；
简言之，将得到的突变序列表达载体与其他病毒拯救所需的质粒以及过表达TEVp的质粒共同转染宿主细胞，将转染成功后的宿主细胞在含有0.5％FBS、2μg/mL TPCK-trypsin的DMEM培养基中培养；
任选地，(6”)将该自噬靶向流感病毒在稳定表达TEVp的稳定细胞系中进行大规模生产；
优选地，转染约4天后，或者当包装出的病毒使(1”)中的宿主细胞完全病变或者90％以上病变后，收集上清，用于感染新的MDCK-TEVp细胞，培养基为含有0.5％FBS、2μg/mL TPCK-trypsin的DMEM培养基，感染4天后，或当感染的病毒使(1”)中的宿主细胞完全病变后，收集上清，即得到自噬靶向流感病毒。
优选地，所述自噬靶向流感病毒的制备方法还包括步骤(7”)检测：通过测定步骤(5”)得到的自噬靶向流感病毒对TEVp的依赖性以及其包装产物的失活对自噬途径的依赖性，来确定所述自噬靶向流感病毒是否改造成功；
具体地，将上清离心并过0.45μm滤膜去除细胞碎片，对包装产物进行TEVp依赖性的检测，以及包装产物的失活对自噬途径的依赖性，保留维持着TEVp依赖性的突变体设定为改造成功的候选物，所谓包装产物的TEVp依赖性，指的是使用所述方法包装的病毒在TEVp高表达的细胞系中可以复制增殖，而在不表达TEVp的正常细胞中复制能力降低或者缺陷。
任选地，所述自噬靶向流感病毒的制备方法还包括步骤(8”)使用改造成功的自噬靶向流感病毒载体重复步骤(2”)～(5”)，使得自噬靶向流感病毒的多个病毒蛋白上均引入了自噬靶向分子和连接链；或者在自噬靶向流感病毒的任一病毒蛋白上引入多个自噬靶向分子和连接链；
优选地，通过测定得到的自噬靶向流感病毒对TEVp的依赖性以及其包装产物的失活对自噬途径的依赖性，来确定所述自噬靶向病毒是否改造成功；保留经过长期传代仍旧维持着对TEVp的依赖性的自噬靶向病毒为改造成功候选物；
任选地，所述自噬靶向流感病毒的制备方法方法还包括(9”)选择改造成功的候选物，并对产物进行纯化；
(10”)对(9”)中的自噬靶向流感病毒进行安全性或免疫原性检测，相比野生型病毒，安全的流感病毒为改造成功的流感病毒。
在本申请的一个具体的实施方案中，通过将自噬靶向流感病毒基因插入到载体(例如pHH21质粒)中，将所述的载体与拯救流感病毒所需的其他载体一起转染可以选择性 切掉自噬靶向分子的稳定细胞系，优选HEK293T-TEVp和MDCK-TEVp，即可获得AUTOTAC流感病毒，该病毒在可以选择性切掉自噬靶向分子的稳定细胞系，优选HEK293T-TEVp和/或MDCK-TEVp中进行大量扩增制备。
在本申请的一个实施方案中，为了提高流感病毒的产出效率，以及将来的工业化生产，建立了可以稳定表达烟草蚀斑病毒蛋白酶TEVp的稳定细胞系(优选HEK293T-TEVp和MDCK-TEVp)，该哺乳动物稳定细胞系还解决了传统使用鸡胚繁殖病毒易引起人体过敏等不良反应的缺点。为了进一步提高产出效率，可以在病毒培养基中加入适量的自噬抑制剂用于抑制自噬系统对病毒蛋白的降解；或者可以使用自噬系统缺陷的细胞系，如LC3A、LC3B、LC3C、GABARAP、GABARAPL1或GABARAPL2等自噬系统关键蛋白中的一个或者多个蛋白敲除或者敲低的细胞系。
为了建立稳定表达TEVp的哺乳动物细胞细胞系，发明人构建了带有嘌呤霉素抗性的慢病毒过表达载体，其携带TEVp的表达基因，通过病毒分别转导HEK293T细胞和MDCK细胞，并经过嘌呤霉素筛选，得到稳定表达TEVp的稳定细胞株，将这些稳定细胞株单克隆化，进行单克隆培养，利用Western Blot和RT-qPCR分选出TEVp表达量最高的稳定细胞株，即为最终的稳定细胞系HEK 293T细胞和MDCK细胞。
经中空纤维柱和凝胶层析方法、蔗糖梯度密度离心方法或者PEG沉淀方法，即可得到纯化后的突变型AUTOTAC流感病毒，经体内外实验初步证明，AUTOTAC流感病毒具有优良的安全性和遗传稳定性，并且与灭活病毒相比具有更好的免疫效果。
第六方面，本申请提供第一方面所述的所述的自噬靶向病毒、第二方面所述的核酸分子、第三方面所述的表达载体或第四方面所述的重组细胞在制备预防和/或治疗病毒或肿瘤的药物中的应用。
第七方面，本申请提供一种药物组合物，所述药物组合物包含第一方面所述的自噬靶向病毒。
优选地，所述药物组合物还含有辅料。
优选地，所述辅料包括药学可接受的载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
优选地，所述药物组合物包括疫苗。
优选地，所述疫苗为减毒活疫苗、复制无能活疫苗(包括复制减弱或者缺陷活疫苗)或复制可控活疫苗。
第八方面，本申请提供一种溶瘤病毒，所述溶瘤病毒包含第一方面所述的自噬靶向病毒。
本申请中提供了所述AUTOTAC病毒作为溶瘤病毒的用途，其不仅可以单独使用，一方面直接杀伤肿瘤细胞，一方面可以激活机体的抗肿瘤免疫，将“冷”肿瘤转化为“热”肿瘤；其还可以与抗肿瘤药物联用，产生其他协同效应。
第九方面，本申请提供一种制备减毒活病毒、复制无能活病毒(包括复制减弱或者缺陷活病毒)、复制可控活病毒以及制备用于预防和治疗病毒感染相关疫苗和药物的方法，所述方法包括第五方面所述的自噬靶向病毒的制备方法。
第十方面，本申请提供了一种制备所述的自噬靶向病毒的系统，所述系统包括稳定表达对自噬靶向病毒的连接链具有选择性切割作用的蛋白酶的细胞；
优选地，所述细胞为稳定表达烟草蚀斑病毒蛋白酶TEVp的细胞系或者自噬系统缺陷的细胞系，更优选地，所述细胞系为稳定表达烟草蚀斑病毒蛋白酶TEVp的HEK293T细胞系或MDCK细胞系，所述自噬系统缺陷的细胞系包括LC3A、LC3B、LC3C、GABARAP、GABARAPL1或GABARAPL2等自噬系统关键蛋白中的一个或者多个蛋白敲除或者敲低的细胞系。
本申请中上述药物组合物、疫苗、溶瘤病毒可以在本申请制备定点突变修饰的AUTOTAC流感病毒的基础上采用本领域常规技术制备；它们可以用于预防或治疗流感病毒感染，包括人和动物的流感病毒感染；它们还可以用于治疗肿瘤。
与现有技术相比，本申请具有以下有益效果：
(1)通用性：蛋白质是所有病毒的共性生命物质，只需要在想要降解的病毒蛋白中引入“可切割的或者无需切割的自噬靶向分子”，就可以控制病毒的复制。因此该技术可用于所有病毒疫苗的制备。
(2)可供选择的自噬靶向分子有成千上万种，这些分子都可以被开发利用来制备这种病毒疫苗或者溶瘤病毒，因此有丰富的后备资源可供选择。
(3)操作简单：只需要简单的病毒载体的构建和病毒的制备技术，生产者不需要太多的病毒生物学知识就可以制备这类疫苗。
(4)实验证明，该技术可以用于设计成具有不同失活程度的疫苗，具有安全的可控性，且具有良好的免疫原性。
(5)实验证明，自噬靶向病毒可以作为溶瘤病毒。
附图说明
图1为本申请制备的AUTOTAC病毒在MDCK-TEVp细胞和正常MDCK细胞中的复制能力差异图；
图2为本申请制备的AUTOTAC病毒用自噬抑制剂处理后病毒M1蛋白水平表达的western blot图；
图3为本申请制备的AUTOTAC病毒在动物水平的安全性评价结果图；
图4为本申请制备的AUTOTAC病毒在动物水平的免疫原性和保护性评价结果图；
图5为本申请制备的AUTOTAC病毒抑制肿瘤生长的结果图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1构建包含可切割的自噬靶向分子的流感病毒WSN的基因载体
本实施例构建包含可切割的自噬靶向分子的流感病毒WSN的基因载体。
(1)获取拯救野生型流感病毒WSN的质粒
根据pubmed公布的流感病毒A/WSN/1933的基因序列，相关网址包括：
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；
经全基因合成，获得该流感病毒各个基因片段的基因，然后将其分别连接在pHH21、pCDNA 3(neo)、pcAAGGS/MCS载体上(获自北京中科裕博生物技术有限公司)，获得拯救野生型流感病毒WSN的质粒。获得的质粒的命名及构成如表4所示。
表4
(2)构建引入可切割的自噬靶向分子的病毒载体
在流感病毒WSN的各个病毒蛋白(PA，PB2，PB1，NP，HA，NA，M1，M2，NS1，NEP)对应的基因编码区的C端、终止密码子之前，分别引入可被TEVp切割的自噬靶向分子的基因序列，并构建了如下的病毒载体。
具体地，在病毒的任一蛋白上引入所述可被TEVp切掉的自噬靶向分子的基因序列及其表达的氨基酸序列如下，将所用氨基酸序列对应的基因序列进行人源化优化，并插入到目的蛋白基因的编码区C端、终止密码子之前；并经测序验证突变构建成功。
发明人构建的病毒载体示例：
在M1蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别根据插入序列编号命名，命名为M1-AUTOTAC-138，M1-AUTOTAC-139，M1-AUTOTAC-140，M1-AUTOTAC-141，……(此处为根据序列编号依次命名，不再一一列举)，M1-AUTOTAC-248。
在PA蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID  NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为PA-AUTOTAC-138，PA-AUTOTAC-139，PA-AUTOTAC-140，PA-AUTOTAC-141，……，PA-AUTOTAC-248。
在PB2蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为PB2-AUTOTAC-138，PB2-AUTOTAC-139，PB2-AUTOTAC-140，PB2-AUTOTAC-141，……，PB2-AUTOTAC-248。
在PB1蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为PB1-AUTOTAC-138，PB1-AUTOTAC-139，PB1-AUTOTAC-140，PB1-AUTOTAC-141，……，PB1-AUTOTAC-248。
在NP蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为NP-AUTOTAC-138，NP-AUTOTAC-139，NP-AUTOTAC-140，NP-AUTOTAC-141，……，NP-AUTOTAC-248。
在HA蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为HA-AUTOTAC-138，HA-AUTOTAC-139，HA-AUTOTAC-140，HA-AUTOTAC-141，……，HA-AUTOTAC-248。
在NA蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为NA-AUTOTAC-138，NA-AUTOTAC-139，NA-AUTOTAC-140,NA-AUTOTAC-141，……，NA-AUTOTAC-248。
在M2蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为M2-AUTOTAC-138,M2-AUTOTAC-139，M2-AUTOTAC-140，M2-AUTOTAC-141，……，M2-AUTOTAC-248。
在NS1蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为NS1-AUTOTAC-138，NS1-AUTOTAC-139，NS1-AUTOTAC-140，NS1-AUTOTAC-141，……，NS1-AUTOTAC-248。
在NEP蛋白对应的基因编码区的C端、终止密码子之前，分别引入可以编码SEQ ID NO.138～SEQ ID NO.248氨基酸序列的基因序列，构建成功的病毒载体分别命名为NEP-AUTOTAC-138，NEP-AUTOTAC-139，NEP-AUTOTAC-140，NEP-AUTOTAC-141，……，NEP-AUTOTAC-248。
实施例2定点突变修饰后的AUTOTAC流感病毒的拯救
按照常规的拯救流感病毒方法，将拯救流感病毒所用的12个质粒共转染稳定细胞系，并使用实施例1中定点突变改造的质粒替换这12个质粒中相应的质粒，对应于六孔板的每个孔，每种质粒加0.2μg，转染后，观察细胞的病变情况，筛选出可以拯救出病毒并且对TEVp具有依赖性的插入位点、自噬靶向分子、可被TEVp切割的连接链linker、以及它们的组合。筛 选出的毒株根据蛋白及引入的可切割的自噬靶向分子进行命名。
示例说明，将Ben3 pPolI-WSN-PA质粒上引入可切割自噬靶向分子SEQ ID NO.149之后，将该质粒与拯救流感病毒的其他质粒Ben1 pPolI-WSN-PB2；Ben2 pPolI-WSN-PB1；Ben4 pPolI-WSN-HA；Ben5 pPolI-WSN-NP；Ben6 pPolI-WSN-NA；Ben7 pPolI-WSN-M；Ben8 pPolI-WSN-NS；Ben9 pcDNA 3(neo)-PB2；Ben10 pcDNA 3(neo)-PB1；Ben11 pcDNA 3(neo)-PA；Ben13 pcAGGS/MCS-NP共同转染表达TEVp的稳定细胞系，从而拯救出在流感病毒PA基因片段上引入SEQ ID NO.149的突变型流感病毒，命名为PA-AUTOTAC-149。在Ben7 pPolI-WSN-M质粒上的M1蛋白的编码区C端引入可切割自噬靶向分子SEQ ID NO.149之后，将该质粒与拯救流感病毒的其他质粒Ben1 pPolI-WSN-PB2；Ben2 pPolI-WSN-PB1；Ben3 pPolI-WSN-PA；Ben4 pPolI-WSN-HA；Ben5 pPolI-WSN-NP；Ben6 pPolI-WSN-NA；Ben8 pPolI-WSN-NS；Ben9 pcDNA 3(neo)-PB2；Ben10 pcDNA 3(neo)-PB1；Ben11 pcDNA 3(neo)-PA；Ben13 pcAGGS/MCS-NP共同转染表达TEVp的稳定细胞系，从而拯救出在流感病毒M1基因片段上引入SEQ ID NO.149的突变型流感病毒，命名为M1-AUTOTAC-149。
依照同样的方法，可获取其他位点引入可切割的自噬靶向分子的突变型流感病毒，并按照同样的规则进行命名。
按照能否引起细胞病变的标准对构建的所有AUTOTAC病毒载体进行考察：如果可以引起HEK293T-TEVp和/或MDCK-TEVp细胞病变，说明该AUTOTAC病毒拯救成功；如果不能引起HEK293T-TEVp和/或MDCK-TEVp细胞病变，说明该AUTOTAC病毒拯救失败。部分的突变型流感病毒的拯救情况作为示例，可见表5，表5中总结了可以引起MDCK-TEVp细胞发生病变的部分AUTOTAC流感病毒毒株。所述结果表明在流感病毒的8个基因片段上均可引入可选择性切割的自噬靶向分子，并且均可以拯救出AUTOTAC流感病毒。
表5
从上述突变位点、可切割的自噬靶向分子中，选择拯救流感病毒效率高、遗传稳定、且在正常宿主细胞最终复制能力明显下降甚至完全消失的突变修饰方式(如M1-AUTOTAC-149和PA-AUTOTAC-150)，进行组合，制备含有多个自噬靶向分子的流感病毒，并从中进一步选择优选的毒株。
实施例3包含(不需要切割的)自噬靶向分子的流感病毒WSN的基因载体的构建以及相应AUTOTAC病毒的拯救
重组载体的命名原则如下：
1.在病毒蛋白N端引入自噬靶向分子，构建的病毒载体命名为“病毒蛋白名称-N-自噬靶向分子”；
2.在病毒蛋白C端引入自噬靶向分子，构建的病毒载体命名为“病毒蛋白名称-C-自噬靶向分子”；
3.在病毒蛋白的编码区内部引入自噬靶向分子，构建的病毒载体，命名为“病毒蛋白名称-与引入位点相邻的上游氨基酸的名称和氨基酸编号-自噬靶向分子”。
构建的重组质粒如下：
PB2-N-自噬靶向分子、PB2-R70-自噬靶向分子、PB2-I176-自噬靶向分子、PB2-V457-自噬靶向分子、PB2-N510-自噬靶向分子、PB2-Y531-自噬靶向分子、PB2-A623-自噬靶向分子、PB2-D680-自噬靶向分子、PB2-E700-自噬靶向分子、PB2-C-自噬靶向分子、PB1-N-自噬靶向分子、PB1-D70-自噬靶向分子、PB1-D295-自噬靶向分子、PB1-R327-自噬靶向分子、PB1-R430-自噬靶向分子、PB1-F490-自噬靶向分子、PB1-T566-自噬靶向分子、PB1-N626-自噬靶向分子、PB1-G710-自噬靶向分子、PB1-C-自噬靶向分子、PA-N-自噬靶向分子、PA-D294-自噬靶向分子、PA-N350-自噬靶向分子、PA-E372-自噬靶向分子、PA-L425-自噬靶向分子、PA-H510-自噬靶向分子、PA-A553-自噬靶向分子、PA-E604-自噬靶向分子、PA-S624-自噬靶向分子、PA-C-自噬靶向分子、NP-N-自噬靶向分子、NP-G126-自噬靶向分子、NP-N247-自噬靶向分子、NP-R317-自噬靶向分子、NP-V353-自噬靶向分子、NP-A366-自噬靶向分子、NP-Q409-自噬靶向分子、NP-E465-自噬靶向分子、NP-M481-自噬靶向分子、NP-C-自噬靶向分子、M1-N-自噬靶向分子、M1-A33-自噬靶向分子、M1-V68-自噬靶向分子、M1-D89-自噬靶向分子、M1-R105-自噬靶向分子、M1-M135-自噬靶向分子、M1-Q164-自噬靶向分子、M1-H222-自噬靶向分子、M1-A239-自噬靶向分子、M1-C-自噬靶向分子、M2-C-自噬靶向分子、NEP-C-自噬靶向分子、NS1-N-自噬靶向分子、NS1-A76-自噬靶向分子、NS1-A82-自噬靶向分子、NS1-H101-自噬靶向分子、NS1-A122-自噬靶向分子、NS1-T151-自噬靶向分子、NS1-L163-自噬靶向分子、NS1-C-自噬靶向分子、HA-N-自噬靶向分子、HA-C-自噬靶向分子、NA-N-自噬靶向分子、NA-C-自噬靶向分子、PB2-N-自噬靶向分子、PB2-R70-自噬靶向分子、PB2-I176-自噬靶向分子、PB2-V457-自噬靶向分子、PB2-N510-自噬靶向分子、PB2-Y531-自噬靶向分子、PB2-A623-自噬靶向分子、PB2-D680-自噬靶向分子、PB2-E700-自噬靶向分子、PB2-C-自噬靶向分子、PB1-N-自噬靶向分子、PB1-D70-自噬靶向分子、PB1-D295-自噬靶向分子、PB1-R327-自噬靶向分子、PB1-R430-自噬靶向分子、PB1-F490-自噬靶向分子、PB1-T566自噬靶向分子、PB1-N626-自噬靶向分子、PB1-G710-自噬靶向分子、PB1-C-自噬靶向分子、PA-N-自噬靶向分子、PA-D294-自噬靶向分子、PA-N350-自噬靶向分子、PA-E372-自噬靶向分子、PA-L425-自噬靶向分子、PA-H510-自噬靶向分子、PA-A553-自噬靶向分子、PA-E604-自噬靶向分子、PA-S624-自噬靶向分子、PA-C-自噬靶向分子、NP-N-自噬靶向分子、NP-G126- 自噬靶向分子、NP-N247-自噬靶向分子、NP-R317-自噬靶向分子、NP-V353-自噬靶向分子、NP-A366-自噬靶向分子、NP-Q409-自噬靶向分子、NP-E465-自噬靶向分子、NP-M481-自噬靶向分子、NP-C-自噬靶向分子、M1-N-自噬靶向分子、M1-A33-自噬靶向分子、M1-V68-自噬靶向分子、M1-D89-自噬靶向分子、M1-R105-自噬靶向分子、M1-M135-自噬靶向分子、M1-Q164-自噬靶向分子、M1-H222-自噬靶向分子、M1-A239-自噬靶向分子、M1-C-自噬靶向分子、M2-C-自噬靶向分子、NEP-C-自噬靶向分子、NS1-N-自噬靶向分子、NS1-A76-自噬靶向分子、NS1-A82-自噬靶向分子、NS1-H101-自噬靶向分子、NS1-A122-自噬靶向分子、NS1-T151-自噬靶向分子、NS1-L163-自噬靶向分子、NS1-C-自噬靶向分子、HA-N-自噬靶向分子、HA-C-自噬靶向分子、NA-N-自噬靶向分子和NA-C-自噬靶向分子。
其中，自噬靶向分子的氨基酸序列如SEQ ID No.1～SEQ ID No.111所示。
将上述重组载体导入自噬系统缺陷的细胞系，包装得到所述含有自噬靶向分子的重组病毒：
使用本实施例中的重组载体替换对应的野生型质粒，并与其他11种质粒共转染自噬系统缺陷的哺乳动物细胞系(示例：重组载体PB2-N-自噬靶向分子SEQ ID NO.1替代质粒Ben1，与其他11种质粒共转染)，每种质粒的加入量为0.2μg，转染后的细胞在含有0.5％FBS、2μg/mL TPCK-胰蛋白酶的DMEM培养基中培养。
转染4天后，宿主细胞完全病变或者90％以上病变后，收集上清，感染新的自噬系统缺陷的细胞，培养基为含有0.5％FBS、2μg/mL TPCK-胰蛋白酶的DMEM培养基，感染4天后，宿主细胞完全病变后，收集上清。
上清离心并过0.4μm滤膜去除细胞碎片，对包装产物进行自噬系统缺陷依赖性的检测，以及包装产物的失活对自噬系统的依赖性，保留维持着自噬系统缺陷依赖性的突变体即为重组病毒。
按照与重组载体相同的方式，对制备得到的重组病毒进行命名。示例，用重组载体PB2-N-自噬靶向分子SEQ ID NO.1替代质粒Ben1，制备出来的病毒命名为PB2-N-自噬靶向分子SEQ ID NO.1；用重组载体PB2-N-自噬靶向分子SEQ ID NO.2替代质粒Ben1，制备出来的病毒命名为PB2-N-自噬靶向分子SEQ ID NO.2；以此类推。
结果表明在流感病毒的8个基因片段上均可引入(不需要切割的)自噬靶向分子，并且均可以拯救出AUTOTAC流感病毒。
实施例4定点突变的流感病毒的拯救和纯化
(1)含有可切割的自噬靶向分子的AUTOTAC病毒的拯救
将实施例2的步骤中定点突变改造后的流感病毒的拯救中获得的突变型流感病毒包装质粒共转染表达TEVp的稳定细胞系，6小时候换成新的培养基，培养基中含有1％的FBS和2μg/mL的TPCK-trypsin，并以正常细胞作为对照。此拯救实验采用的阳性对照为野生型流感病毒WSN，除了拯救病毒的质粒不同之外，其余条件均与突变型流感病毒的拯救条件相同。转染完成后，每天观察细胞的状态，用TEVp稳定细胞系中出现病变，而正常细胞不出现病变或者病变较少的突变体为阳性突变体。而野生型流感病毒在TEVp稳定细胞系和正常细胞系中均出现病变。
(2)AUTOTAC流感病毒的纯化
a.当步骤(1)拯救突变型AUTOTAC流感病毒的稳定系细胞完全病变时，或者转染大约4天后，收集细胞上清，于5000×g离心10min，用新的稳定细胞系进行大量扩增，待细胞 完全病变或者扩增大约4天后，收集细胞上清，过0.45μm的滤膜；
b.使用蔗糖梯度梯度密度离心的方法纯化流感病毒。具体步骤如下：将1)中的病毒液用50mL离心管(高速专用)于105×g离心2h，沉淀用1mL PBS重悬；
c.用NTE Buffer(100mM NaCl，10mM Tris-Cl，pH7.4，1mM EDTA)溶解蔗糖，配成20％蔗糖溶液，过0.45μm滤膜；
d.将步骤c的蔗糖加入到50mL，将b中的PBS重悬液滴在蔗糖溶液上，11×104×g，离心2h；
e.沉淀加约15mL NTE buffer，11×104×g，继续离心2h；
f.将步骤e中的沉淀用PBS重悬。
实施例5自噬靶向流感病毒在细胞水平的安全性考察
通过对制备出的突变型AUTOTAC流感病毒M1-AUTOTAC-141、149、219进行TEVp蛋白的依赖性考察来确认AUTOTAC病毒的安全性；进行长期的传代培养，来考察该突变型病毒中可切割的自噬靶向分子的稳定性。
具体实验1：将制备的突变型AUTOTAC流感病毒按照MOI＝0.01的比例感染MDCK-TEVp细胞和正常的MDCK细胞，4天后取上清检测病毒的滴度，将AUTOTAC病毒在MDCK-TEVp细胞中的病毒滴度定为100％，比较AUTOTAC病毒在MDCK-TEVp细胞和正常MDCK细胞中的相对病毒滴度，可知病毒在两种细胞中的复制能力差异。
具体实验2：将新制备出的突变型AUTOTAC流感病毒按照MOI＝0.01的比例接种在新的培养基中并感染稳定系细胞，培养基中含有1％FBS、2μg/mL的TPCK-trypsin，并用正常的细胞系作为对照。待TEVp稳定细胞系完全病变后时，取出上清，过0.45μm的滤膜，再按照MOI＝0.01的比例接种在新的培养基中并感染稳定系细胞，同样以正常的细胞系作为对照。如此重复，进行长期的病毒传代。通过基因测序检测引入的自噬靶向分子是否发生了突变。
图1结果表明，AUTOTAC病毒在MDCK-TEVp细胞中可以复制增殖，而在正常的MDCK细胞中的复制能力显著降低甚至不能复制，即具有对TEVp的依赖性，说明该病毒是安全的。另外，经过长期传代，突变型AUTOTAC病毒毒株中引入的自噬靶向分子没有发生突变，说明在流感病毒基因中引入的可切割的自噬靶向分子是稳定存在的，进而说明在遗传上是稳定的。
实施例6考察自噬靶向流感病毒复制能力的减弱是否是由细胞的自噬调控的
以M1-AUTOTAC-141、149、219、232、235、236、238、240、241、242为代表性毒株，考察了设计的AUTOTAC流感病毒在正常细胞中的复制能力的减弱是否是由细胞内的自噬系统介导的。
具体实验1：将制备的突变型AUTOTAC流感病毒或野生型病毒感染正常的MDCK细胞(MOI＝0.1)，培养基中补充100nM自噬抑制剂(氯喹Chloroquine(CQ)、羟氯喹Hydroxychloroquine(HCQ)、亮肽素Leupeptin、或LY294002等)或者相同稀释比例的DMSO作为对照。分别在感染后48h，收集细胞样品，用Western Blot检测病毒M1蛋白水平。
图2结果表明，野生型病毒在感染MDCK细胞后，可以大量复制，产生大量的病毒蛋白。相比之下，AUTOTAC病毒的病毒蛋白M1上修饰了自噬靶向分子后，在感染后48h后病毒的M1蛋白会被降解；而培养基中加入自噬抑制剂(氯喹Chloroquine(CQ)、羟氯喹Hydroxychloroquine(HCQ)、亮肽素Leupeptin、或LY294002等)后，自噬介导的病毒蛋白M1 的降解被抑制。该实验结果说明，发明人引入的自噬靶向分子可以介导病毒蛋白的降解；AUTOTAC病毒在正常细胞中复制能力的降低是由自噬介导的病毒蛋白的降解引起的，符合本申请原理。
以上结果证明，AUTOTAC病毒的复制能力的降低或者缺陷是由细胞的自噬系统介导的；符合发明人对AUTOTAC病毒的设计预期。
实施例7自噬靶向流感病毒(AUTOTAC流感病毒)在动物水平的安全性、免疫原性和保护性考察
本实施例中使用BALB/c和C57BL/6J小鼠对AUTOTAC病毒在动物水平的安全性和免疫原性及保护性进行评价。以灭活流感疫苗(IIV)为对照(灭活流感病毒疫苗为发明人根据中国药典提供的方法用同源的流感病毒颗粒制备的)，选择M1-AUTOTAC-149作为AUTOTAC病毒的代表，进行AUTOTAC病毒的安全性、免疫原性和保护性评价。
安全性评价具体实验：
(1)将30只6-8周的雌性BALB/c小鼠或C57BL/6J小鼠，分成3组，每组10只；
(2)接种病毒：第一组滴鼻接种PBS；第二组滴鼻接种105PFU M1-AUTOTAC-149；第三组接种105PFU野生型WSN流感病毒；
(3)接种三天后，每组取5只小鼠，取其肺组织，检测其中的病毒滴度；
(4)继续观察监测每组剩余5只小鼠的体重和死亡情况，持续14天。
图3结果表明：野生型病毒可以在小鼠的肺中高度复制，并引起小鼠体重明显下降和死亡。而AUTOTAC病毒在小鼠肺中的复制能力很弱，并且不会引起小鼠体重下降，也不会引起小鼠死亡。因此AUTOTAC病毒疫苗具有良好的安全性。
免疫原性和保护性评价：
(1)将60只6-8周的雌性BALB/c小鼠或C57BL/6J小鼠，分成3组，每组20只；
(2)接种病毒：第一组滴鼻接种PBS；第二组滴鼻接种105PFU M1-AUTOTAC-149；第三组接种105PFU灭活流感疫苗；
(3)接种一周后，每组取5只小鼠，取其肺组织和脾脏，检测其中的T细胞免疫反应；
(4)接种三周后，每组取5只小鼠，取血，用于血凝抑制(HI)试验、中和(NT)抗体检测、ELISA实验检测其中的抗体免疫反应；
(5)接种三周后，每组小鼠鼻腔接种2×105PFU的野生型WSN流感病毒；
(6)接种野生型病毒三天后，每组取5只小鼠，取其肺组织，检测其中的病毒滴度；
(7)继续观察监测每组剩余5只小鼠的体重和死亡情况，持续14天；
图4结果表明，AUTOTAC病毒就可以在动物体内诱导高水平的血凝抑制抗体滴度、中和抗体滴度、anti-NP IgG、anti-NP IgA等。AUTOTAC病毒诱导的血凝抑制抗体、中和抗体、anti-NP IgG、anti-NP IgA水平，显著高于由灭活疫苗诱导的抗体水平。AUTOTAC病毒疫苗的接种可以显著减少动物肺组织中的野生型病毒滴度；AUTOTAC病毒疫苗提供的保护性显著地优于灭活疫苗。
实施例8 AUTOTAC病毒作为溶瘤病毒的评价
本实施例中使用C57BL/c的黑色素瘤荷瘤模型，对AUTOTAC流感病毒的溶瘤效果进行评价，具体实验包括以下步骤：
(1)在小鼠(购自维通利华，5-8周龄，雌性)的背部皮下注射黑色素瘤，饲养8天，当瘤体体积达到约100mm3时，开始下面的实验操作；
(2)向瘤体内注射50μL105TCID50AUTOTAC流感病毒(M1-AUTOTAC-149)，以注射等量PBS作为对照组，每间隔1天注射1次，共注射4次；
(3)每天检测瘤体的体积。
图5结果表明，自噬靶向流感病毒可以有效抑制肿瘤体积的增加，证明自噬靶向流感病毒具备成为溶瘤病毒的潜能。
综上所述，本申请创造性地设计了一种自噬靶向病毒，其可以在特定的人工细胞系中高效复制、大量生产制备，但在正常的细胞中病毒的复制能力被减弱甚至完全失去复制能力，可广泛应用于活疫苗、减毒疫苗、灭活疫苗、强毒的安全模型等，具有安全可控性，且具有良好的免疫原性，此外，所述自噬靶向病毒可以作为溶瘤病毒，不仅可以直接杀伤肿瘤，而且可以激活机体对肿瘤免疫效果，提供的病毒制备方法工艺简单且具备通用性。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 一种自噬靶向病毒，其含有被自噬系统识别的自噬靶向分子。
  2. 根据权利要求1所述的自噬靶向病毒，其中，所述自噬靶向分子选自具有如SEQ ID NO.1～SEQ ID NO.111任一项所示氨基酸序列的多肽或其中至少两种的组合；
    优选地，所述自噬靶向分子与所述自噬靶向病毒的病毒蛋白连接；
    优选地，所述自噬靶向分子和所述病毒蛋白之间通过连接链连接；
    优选地，所述连接链包括被切割的分子；
    优选地，所述被切割的分子包括被烟草蚀斑病毒蛋白酶切割的分子、被凝血酶敏切割的分子、被凝血因子Xa切割的分子、被肠激酶切割的分子、被3C蛋白酶切割的分子、被SUMO蛋白酶切割的分子、被细菌明胶酶切割的分子或自剪切的分子中的任意一种或至少两种的组合；
    优选地，所述被烟草蚀斑病毒蛋白酶切割的分子包括多肽，所述多肽具有通式I所示的氨基酸序列：E-Xaa-Xaa-Y-Xaa-Q-$  通式I；
    其中，Xaa代表任意氨基酸，$代表氨基酸G、S或M中的任意一种；
    优选地，所述被细菌明胶酶切割的分子包括氨基酸序列为SEQ ID No.250：GPLGV的多肽；
    优选地，所述自剪切的分子包括2A短肽；
    优选地，所述2A短肽包括猪捷申病毒的P2A、马鼻炎病毒的E2A、口蹄疫病毒的F2A或T2A中的任意一种或至少两种的组合；
    优选地，所述连接链选自具有如SEQ ID NO.112～137中任一项所示氨基酸序列的多肽；
    优选地，所述自噬靶向分子和连接链之间还包含柔性接头；
    优选地，所述自噬靶向分子、连接链和柔性接头具有如下连接方式：
    柔性接头-连接链-柔性接头-自噬靶向分子；
    所述自噬靶向分子、连接链和柔性接头按所述柔性接头-连接链-柔性接头-自噬靶向分子的连接方式连接后包括具有如SEQ ID NO.138～248中任一项所示氨基酸序列的多肽。
  3. 根据权利要求1或2所述的自噬靶向病毒，其中，所述自噬靶向病毒由野生病毒经过遗传改造制备得到；
    优选地，所述野生病毒选自流感病毒、艾滋病毒、手足口病毒、柯萨奇病毒、丙肝病毒、乙肝病毒、甲肝病毒、丁型肝炎病毒、戊型肝炎病毒、EB病毒、人乳头瘤病毒、单纯疱疹病毒、巨细胞病毒、水痘-带状疱疹病毒、水泡性口炎病毒、呼吸道合胞病毒、登革病毒、埃博拉病毒、马尔堡病毒、寨卡病毒、中东呼吸综合征病毒、轮状病毒、狂犬病毒、麻疹病毒、腺病毒、脊髓灰质炎病毒、埃可病毒、乙型脑炎病毒、森林脑炎病毒、汉坦病毒、新型肠道病毒、风疹病毒、腮腺炎病毒、副流感病毒、蓝耳病毒、猪瘟病毒、口蹄疫病毒、细小病毒、朊病毒、天花病毒、烟草花叶病毒、腺相关病毒、噬菌体、疱疹病毒、西尼罗河病毒、诺如病毒、人博卡病毒或冠状病毒中的任意一种或至少两种的组合；
    优选地，所述野生病毒为流感病毒、艾滋病毒或新冠病毒SARS-CoV-2；
    优选地，所述流感病毒包括H1N1、H5N1、H7N9、H3N2或B型流感病毒中的任意一种或至少两种的组合；
    优选地，所述自噬靶向病毒为自噬靶向流感病毒；
    优选地，所述自噬靶向流感病毒的PA蛋白、PB1蛋白、PB2蛋白、NP蛋白、HA蛋白、 NA蛋白、M1蛋白、M2蛋白、NS1蛋白或NEP蛋白中的一个或多个连接所述自噬靶向分子和连接链；
    优选地，所述PA蛋白和PB2蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PA蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB2蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PA蛋白、PB2蛋白和PB1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PA蛋白、PB2蛋白、PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PA蛋白、PB2蛋白、PB1蛋白、M1蛋白和NP蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB2蛋白、PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB1蛋白和M1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB2蛋白和M1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB2蛋白、PB1蛋白、M1蛋白和NS1蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述PB2蛋白、PB1蛋白、M1蛋白和NEP蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述NS1蛋白和NEP蛋白中均连接所述自噬靶向分子和连接链；
    优选地，所述自噬靶向病毒为自噬靶向冠状病毒；
    优选地，所述自噬靶向冠状病毒中刺突蛋白、包膜糖蛋白、膜糖蛋白、核衣壳蛋白、非结构蛋白1、非结构蛋白2、非结构蛋白3、非结构蛋白4、非结构蛋白5、非结构蛋白6、非结构蛋白7、非结构蛋白8、非结构蛋白9、非结构蛋白10、非结构蛋白11、非结构蛋白12、非结构蛋白13、非结构蛋白14、非结构蛋白15、非结构蛋白16、3a蛋白、3b蛋白、6蛋白、7a蛋白、7b蛋白、8a蛋白、8b蛋白、9b蛋白、3C样蛋白酶、前导蛋白、2’-O-核糖甲基转移酶、核酸内切酶、3’-至-5’核酸外切酶、解旋酶、RNA依赖的RNA聚合酶、orf1a多聚蛋白、ORF10蛋白、ORF8蛋白、ORF7a蛋白、ORF6蛋白或ORF3a蛋白中的一个或多个连接所述自噬靶向分子和连接链；
    优选地，所述自噬靶向冠状病毒为自噬靶向新冠病毒SARS-CoV-2；
    优选地，所述自噬靶向病毒为自噬靶向HIV病毒；
    优选地，所述自噬靶向HIV病毒的Gag多聚蛋白、pol多聚蛋白、gp160、HIV转录反式激活子、病毒体蛋白表达调节蛋白、病毒负因子、慢病毒蛋白R、病毒感染性因子、病毒蛋白U、基质蛋白、衣壳蛋白、间隔肽1、核衣壳蛋白、间隔肽2、P6、逆转录酶、核糖核酸酶H、整合酶、HIV蛋白酶、gp120、gp41蛋白中的一个或多个中连接所述自噬靶向分子和连接链。
  4. 一种核酸分子，其含有编码如权利要求1-3中任一项所述的自噬靶向病毒的核酸序列。
  5. 一种表达载体，其含有如权利要求4所述的核酸分子。
  6. 一种重组细胞，其包括表达切割所述连接链的蛋白酶的细胞或自噬系统缺陷的细胞；
    优选地，所述蛋白酶包括烟草蚀斑病毒蛋白酶；
    优选地，所述表达切割所述连接链的蛋白酶的细胞由HEK293T细胞或MDCK细胞制备得到；
    优选地，所述自噬系统缺陷的细胞缺失LC3A蛋白、LC3B蛋白、LC3C蛋白、GABARAP蛋白、GABARAPL1蛋白或GABARAPL2蛋白中的任意一种或至少两种的组合；
    优选地，所述重组细胞含有权利要求5所述表达载体或权利要求4所述核酸分子。
  7. 一种制备如权利要求1-3中任一项所述的自噬靶向病毒的方法，其包括以下步骤：
    (1)统计分析野生病毒的宿主细胞内的自噬系统的表达分布，利用生物信息学分析和病毒蛋白质结构预测，选择引入自噬靶向分子和连接链的病毒蛋白及其中的引入位点；
    (2)在所述引入位点的编码基因处插入编码自噬靶向分子和连接链的核苷酸序列；
    (3)将步骤(2)得到的基因突变后的病毒蛋白的编码核苷酸序列与载体连接，得到表达载体；以及
    (4)利用反向遗传技术，将步骤(3)中的表达载体与用于病毒拯救的表达载体共转染到细胞中，进行细胞培养，得到所述自噬靶向病毒；
    优选地，步骤(3)所述载体包括质粒；
    优选地，步骤(4)所述细胞包括表达切割所述连接链的蛋白酶的细胞或自噬系统缺陷的细胞；
    优选地，所述表达切割所述连接链的蛋白酶的细胞表达的蛋白酶为烟草蚀斑病毒蛋白酶；
    优选地，所述自噬系统缺陷的细胞中缺失LC3A蛋白、LC3B蛋白、LC3C蛋白、GABARAP蛋白、GABARAPL1蛋白或GABARAPL2蛋白中的任意一种或至少两种；
    优选地，所述方法包括以下步骤：
    (1’)统计分析野生病毒的宿主细胞内的自噬系统的表达分布，利用生物信息学分析和病毒蛋白质结构预测，选择引入自噬靶向分子和连接链的病毒蛋白及其中的引入位点；
    (2’)使用基因工程的方法，在所述引入位点的编码基因处引入编码自噬靶向分子和连接链的核苷酸序列；
    (3’)将步骤(2’)得到的基因突变后的病毒蛋白的编码核苷酸序列与载体连接，得到表达载体；
    (4’)构建表达切割所述连接链的蛋白酶的表达载体；以及
    (5’)利用反向遗传技术，将步骤(3’)得到的表达载体、用于病毒拯救的表达载体以及步骤(4’)得到的表达载体共同转染细胞，进行细胞培养，得到所述自噬靶向病毒；
    优选地，所述野生病毒为流感病毒。
  8. 权利要求1-3中任一项所述的所述的自噬靶向病毒、权利要求4所述的核酸分子、权利要求5所述的表达载体或权利要求6所述的重组细胞在制备预防和/或治疗病毒或肿瘤的药物中的应用。
  9. 一种药物组合物，其包含权利要求1-3中任一项所述的自噬靶向病毒；
    优选地，所述药物组合物还含有辅料；
    优选地，所述辅料包括药学可接受的载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合；
    优选地，所述药物组合物包括疫苗；
    优选地，所述疫苗为减毒活疫苗、复制无能活疫苗或复制可控活疫苗。
  10. 一种溶瘤病毒，其包含权利要求1-3中任一项所述的自噬靶向病毒。
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CN112175914A (zh) * 2019-07-05 2021-01-05 司龙龙 蛋白水解靶向病毒、其活疫苗及其制备方法和用途
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