Protein hydrolysis-targeted influenza virus, method of preparation thereof, and use

Abstract

The present application provides proteolytic targeting of influenza viruses, methods for their preparation and use. [Means] The proteolytic target influenza virus comprises a hydrolysis target M1 protein, and a TEVp recognition site and a proteolytic target molecule recognized by the ubiquitin-proteasome system are sequentially inserted into the C-terminus of the hydrolysis target M1 protein, and the proteolytic target molecule recognized by the ubiquitin-proteasome system comprises the amino acid sequence shown in SEQ ID No. 1-353. The hydrolysis target M1 protein can be recognized and hydrolyzed by the ubiquitin-proteasome system, which reduces the viral replication ability and ensures high safety. The proteolytic target influenza virus of the present application has a wide variety of subtypes, which plays an important role in the research and development of influenza virus vaccines.

Description

[Technical Field]

[0001] This application belongs to the field of biotechnology and specifically relates to protein hydrolysis target influenza viruses, methods for preparing them, and their use. [Background technology]

[0002] Influenza viruses are classified into three types: A, B, and C. Of these, outbreaks of influenza A virus are the most frequent and have the widest impact. The genome of influenza A virus consists of eight independent single-stranded RNA fragments and encodes 10 proteins, including hemagglutinin protein (HA), matrix protein (M), neuraminidase (NA), nucleocapsid protein (NP), non-structural protein (NS), and three polymerases: PB1, PB2, and PA. However, the matrix proteins include M1 and M2, and the non-structural proteins include NS1 and NEP. Matrix proteins M1 and M2 play important roles in maintaining the viral particle morphology and viral pathogenicity.

[0003] Influenza A viruses can seasonally infect humans and birds, and large-scale outbreaks can lead to extremely high morbidity and mortality rates, posing a serious threat to human health. Currently, vaccination is the primary means of preventing influenza and controlling its transmission.

[0004] CN102899294A discloses an H1N1 swine influenza virus vaccine strain and its use, wherein the H1N1 swine influenza virus vaccine strain is an inactivated vaccine for preventing swine influenza, provides good protection to pigs attacked by a highly virulent strain of the same species, has good immunogenicity, and can effectively prevent H1N1 swine influenza whether used as a single vaccine or a combination vaccine.

[0005] CN106075424A discloses an avian influenza virus vaccine in which the antigen is an inactivated H9 subtype avian influenza virus. The HA and EID50 of the novel virulence strain FJ11 of the H9 subtype avian influenza virus used in the avian influenza virus vaccine have high titers, good immunogenicity, and can protect against attacks from H9 subtype avian influenza viruses that have spread and been isolated in various locations. The vaccine has good safety, and analysis of characteristic shape, safety test, and efficacy test data in storage period tests shows that all indicators are stable and effective compared to similar products, and the inactivated H9 subtype avian influenza vaccine promotes rapid antibody production.

[0006] However, inactivated vaccines may have their effective antigenic components destroyed or altered during the inactivation process, potentially affecting their immune response. Furthermore, the duration of immunity from inactivated vaccines is short, requiring multiple doses for reinforcement, and they are generally costly. In addition, the aforementioned influenza virus vaccines can only protect against small amounts of several subtypes of the virus. Therefore, developing a safe, controllable, and highly bioavailable influenza virus library with a rich variety of subtypes through targeted degradation plays a crucial role in the research and development of influenza virus vaccines. [Overview of the project] [Problems that the invention aims to solve]

[0007] This application provides a protein hydrolysis-targeted influenza virus, a method for preparing the same, and its use. The protein hydrolysis-targeted influenza virus contains a hydrolysis-targeted M1 protein, which can be recognized by the ubiquitin-proteasome system and cleaved by tobacco etch virus protease (TEVp). It can be prepared in large quantities in a cell line that overexpresses TEVp. On the other hand, in a normal cell line, the ubiquitin-proteasome system recognizes the protein hydrolysis-target molecule fused with the viral protein, degrades the viral protein, weakens the virus's replication ability, and ultimately leads to the complete loss of replication ability. The resulting protein hydrolysis-targeted influenza virus has high safety. The protein hydrolysis-targeted influenza virus can be used as a live vaccine or a weakened vaccine for the prevention of influenza, and can also be applied to the treatment of tumors as an oncolytic virus. The variety of subtypes of the protein hydrolysis-targeted influenza virus according to this application is rich and plays an important role in the research and development of influenza virus vaccines. [Means for solving the problem]

[0008] In Embodiment 1, the present application is: A protein hydrolysis-targeted influenza virus containing the hydrolysis-targeted M1 protein, The C-terminus of the hydrolysis target M1 protein is sequentially inserted with a TEVp recognition site and a protein hydrolysis target molecule recognized by the ubiquitin-proteasome system, and the protein hydrolysis target molecule recognized by the ubiquitin-proteasome system includes the amino acid sequence indicated by SEQ ID No. 1 to 353. This provides an influenza virus that is a target of protein hydrolysis.

[0009] In this application, the design principle of the protein hydrolysis target virus is as follows.

[0010] (1) A proteolytic target molecule introduced into a specific site of a viral protein can be recognized by the ubiquitin - proteasome system in normal host cells, degrade the corresponding viral protein, and inactivate the virus.

[0011] (2) A proteolytic target molecule introduced into a specific site of a viral protein can be suppressed in a specific virus production system or selectively cleaved by a linking chain to separate from the viral protein, avoiding or reducing the degradation of the viral protein by the ubiquitin - proteasome system.

[0012] (3) A proteolytic target molecule introduced into a specific site of a viral protein cannot be suppressed in normal host cells, or the linking chain connecting the proteolytic target molecule and the viral protein cannot be cleaved in normal host cells. Therefore, the prepared virus can be recognized and degraded by the ubiquitin - proteasome system in host cells such as animals and humans, reducing its replication ability, and ultimately completely losing its replication and propagation ability, increasing the safety of the virus.

[0013] In the present application, 353 types of proteolytic target molecules that can be conditionally cleaved are introduced into the C - terminus of the hydrolysis target M1 protein. Since the ubiquitin - proteasome system exists in normal cells of humans and animals, it can recognize the proteolytic target molecule that is expressed by fusing with the viral protein, degrade the M1 protein of the virus by the ubiquitin - proteasome system, and thus degrade the viral protein. The hydrolytic target influenza virus containing the hydrolytic target M1 protein has weakened replication in animals and humans, and ultimately cannot replicate and propagate, increasing the safety of the virus. The obtained hydrolytic target influenza virus can be prepared into an influenza live virus vaccine.

[0014] The amino acid sequence of the protein hydrolysis target molecule is as follows: Among these, SEQ ID No. 1-4 and SEQ ID No. 146-167 correspond to Keap1-Cul3 E3 ubiquitin ligase; SEQ ID No. 5-7 correspond to SOCS2 E3 ubiquitin ligase; SEQ ID No. 8 corresponds to SOCS3 E3 ubiquitin ligase; SEQ ID No. 9 corresponds to SOCS6 E3 ubiquitin ligase; SEQ ID No. 10 corresponds to KLHL-12 E3 ubiquitin ligase; SEQ ID No. 11 corresponds to C-terminal Degrons-Cullin-RING E3 ubiquitin ligase; SEQ ID No. 12-17 correspond to KLHDC3 E3 ubiquitin ligase; SEQ ID No. 18-26 correspond to KLHDC2 E3 ubiquitin ligase; and SEQ ID No. 27 corresponds to APPBP2 SEQ IDs 28-31, 53-60, and 168-174 correspond to SPOP E3 ubiquitin ligase. SEQ IDs 32-34 and 175-176 correspond to KLHL3 E3 ubiquitin ligase. SEQ IDs 35-38 correspond to KLHL20 E3 ubiquitin ligase. SEQ IDs 39-40 correspond to SPSB E3 ubiquitin ligase. SEQ IDs 41-43 correspond to FBXO31 E3 ubiquitin ligase. SEQ IDs 44-46, 126-130, and 311-326 correspond to β-TrCP1 E3 ubiquitin ligase. SEQ IDs No. 47-50, SEQ ID No. 114-120, and SEQ ID No. 303-305 are SCF FBW7Corresponding to E3 ubiquitin ligase, SEQ ID No. 51-52 corresponds to ITCH E3 ubiquitin ligase, SEQ ID No. 61-64, SEQ ID No. 177-182 corresponds to MDM2 E3 ubiquitin ligase, SEQ ID No. 65-67 corresponds to CRBN E3 ubiquitin ligase, SEQ ID No. 68-75, SEQ ID No. 183-206 corresponds to SEQ ID No. 183 E3 ubiquitin ligase, SEQ ID No. 76-85, SEQ ID No. 207-260 corresponds to APC / C-Dbox E3 ubiquitin ligase, SEQ ID No. 86-90, SEQ ID No. 136-140, SEQ ID No. 261-278, SEQ ID No. 331-345 corresponds to APC / C-KENbox E3 ubiquitin ligase, SEQ ID No. 91-95, SEQ ID No. 294-298 corresponds to COP1 E3 ubiquitin ligase, SEQ ID No. 96-100, SEQ ID No. 299-300 corresponds to CRL4_CDT2_1 E3 ubiquitin ligase, SEQ ID No. 101-105, SEQ ID No. 301-302 corresponds to N-end UBR-box E3 ubiquitin ligase, SEQ ID No. 106-113 corresponds to VHL E3 ubiquitin ligase, SEQ ID No. 121-123 is SCF Skp2-Cks1 Corresponding to E3 ubiquitin ligase, SEQ ID No. 124-125, SEQ ID No. 306-310 is SCF TIR1 Corresponding to E3 ubiquitin ligase, SEQ ID No. 131-135, SEQ ID No. 327-330 corresponds to SIAH-1 E3 ubiquitin ligase, SEQ ID No. 141-145, SEQ ID No. 346-347 corresponds to APC / C-ABBA E3 ubiquitin ligase, SEQ ID No. 279-293 corresponds to APC / C-TPR1 E3 ubiquitin ligase.

[0015] SEQ ID No. 1: LDPETGEYL, SEQ ID No. 2: LDPETGEFL, SEQ ID No.3:LDEETGEFL、 SEQ ID No.4:AFFAQLQLDEETGEFL、 SEQ ID No.5:PVPDYTSIHIV、 SEQ ID No.6:NIDFYAQVSDI、 SEQ ID No.7:ASFEYTILDPS、 SEQ ID No.8:MPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRR、 SEQ ID No.9:NGNNYVYIDPT、 SEQ ID No.10:PGAPPGRDLA、 SEQ ID No.11:YYCFG、 SEQ ID No.12:YKKVGTMAAG、 SEQ ID No.13:RWGRRG、 SEQ ID No.14:RPCQRG、 SEQ ID No.15:RAPRQRSRDG、 SEQ ID No.16:SWRLTGFSGMKG、 SEQ ID No.17:RGPSSGG、 SEQ ID No.18:PPPMAGG、 SEQ ID No.19:RGPSSGG、 SEQ ID No.20:EAIGLLGG、 SEQ ID No.21:HLRGSPPPMAGG、 SEQ ID No.22:RGSPPPMAGG、 SEQ ID No.23:SPPPMAGG、 SEQ ID No.24:PPMAGG、 SEQ ID No.25:PMAGG、 SEQ ID No.26:RRGPSSGG、 SEQ ID No.27:VLIRVTYCGL、 SEQ ID No.28:KADTTTPTT、 SEQ ID No.29:PEQDCAVTSGE、 SEQ ID No.30:DEVTSTTSSSぁ SEQ ID No.31:KAASADSTTEGTPAD、 SEQ ID No.32:EPEEPEADQHQ� SEQ ID No.33:GPPSVFPPEPEEPEADQHQ、 SEQ ID No.34:ECEETEVDQHV� SEQ ID No.35:LPDLVぁ SEQ ID No.36:LPDMVぁ SEQ ID No.37:LGLPDLVAKYNぁ SEQ ID No.38:LGLPDMVAKHN、 SEQ ID No.39:ELNNNL、 SEQ ID No.40:DINNNNぁ SEQ ID No.41:PTDVRDVDIぁ SEQ ID No.42:PTDVRDIDLぁ SEQ ID No.43:PTDVTAIHLぁ SEQ ID No.44:KKERLLDDRHDSGLDS、 SEQ ID No.45:KAWQQQSYLDSGIHS、 SEQ ID No.46:LDSGIHSぁ SEQ ID No.47:SPLPSGLLTPPQSG、 SEQ ID No.48:CSLIPTPDKEぁ SEQ ID No.49:FELLPTPPLS、 SEQ ID No.50:PPTPPGSHぁ SEQ ID No.51:TPEAPPCYMDVI� SEQ ID No.52:KFMPPPTYTEVD� SEQ ID No.53:EVSIIQGADSTTぁ SEQ ID No.54:MVADSTTKIDぁ SEQ ID No.55:ADSTTぁ SEQ ID No.56:EVSIIQGADSTTぁ SEQ ID No.57:ASSSTぁ SEQ ID No.58:DEVTSTTSSSぁ SEQ ID No.59:ASSSSぁ SEQ ID No.60:PSSSS、 SEQ ID No.61:FSDLWKLLぁ SEQ ID No.62:FEHLWSSLぁ SEQ ID No.63:FEAQWAAL SEQ ID No.64:FQHIWDFLぁ SEQ ID No.65:LVDKKSGEKぁ SEQ ID No.66:FQCNQCGASぁ SEQ ID No.67:AINITNGEEぁ SEQ ID No.68:RAVENQYSFY、 SEQ ID No.69:RAINNQYSFVぁ SEQ ID No.70:GDYRR、 SEQ ID No.71:VDYRTぁ SEQ ID No.72:GDYREぁ SEQ ID No.73:YDYRGぁ SEQ ID No.74:RDYRD、 SEQ ID No.75:DDEGDSYSDNPぁ SEQ ID No.76:RKRLPVTぁ SEQ ID No.77:RMWLKITぁ SEQ ID No.78:RRTLKMI� SEQ ID No.79:RSGLQLS、 SEQ ID No.80:RKALETL、 SEQ ID No.81:PRRTLKVIQ� SEQ ID No.82:QRAALGNIS、 SEQ ID No.83:PRTALGDIGぁ SEQ ID No.84:HRKHLQEIPぁ SEQ ID No.85:SRKALGNVN、 SEQ ID No.86:NKENEぁ SEQ ID No.87:DKENVぁ SEQ ID No.88:DKENG� SEQ ID No.89:EKENEぁ SEQ ID No.90:KKENG� SEQ ID No.91:EEIRRVPEFぁ SEQ ID No.92:DEQFVPDFぁ SEQ ID No.93:SDQIVPEYぁ SEQ ID No.94:EEHFLVPDLぁ SEQ ID No.95:EEPQTVPEMぁ SEQ ID No.96:TPITDYFPKRKKI� SEQ ID No.97:RRVTDFFARRRPぁ SEQ ID No.98:MRVTDFFSQSKRGぁ SEQ ID No.99:RKLTDFYPVRRSぁ SEQ ID No.100:PLISDFFAKRKRS、 SEQ ID No.101:AKDASぁ SEQ ID No.102:RRFSPぁ SEQ ID No.103:MREGSAぁ SEQ ID No.104:VDNNTNぁ SEQ ID No.105:MCGGAぁ SEQ ID No.106:LAPFVDTYDMMQM、 SEQ ID No.107:IAPVNTKATIRLぁ SEQ ID No.108:LAPYISMDDDFQLぁ SEQ ID No.109:LAPAAGDTIISLDF、 SEQ ID No.110:LAPYIPMDDDDFQL、 SEQ ID No.111:LAPTPGDAIISLDF、 SEQ ID No.112:LAPYIPMDGEDFQL、 SEQ ID No.113:LAPVAPHSPFLL、 SEQ ID No.114: LLPTPPLS、 SEQ ID No.115:PGTPPSS、 SEQ ID No.116:LTPPPS、 SEQ ID No.117:ITPFTS、 SEQ ID No.118:PGETPPLS、 SEQ ID No.119:FPPSPPSS、 SEQ ID No.120:SLIPTPDK、 SEQ ID No.121: SVEQTPKK、 SEQ ID No.122:AVEQTPRK、 SEQ ID No.123:SVEQTPRK、 SEQ ID No.124:QIVGWPPVRSNRK、 SEQ ID No.125:QVVGWPPVCSYRK、 SEQ ID No.126:DSGIES、 SEQ ID No.127:DSGKGS、 SEQ ID No.128:DSGIIT、 SEQ ID No.129:DSGHES、 SEQ ID No.130:DSGNES、 SEQ ID No.131:PPVAQVMPG、 SEQ ID No.132:IPTACVRPT、 SEQ ID No.133:IPCAAVSPN、 SEQ ID No.134:APTAVVLPH、 SEQ ID No.135:RPVAMVRPT、 SEQ ID No.136:NKENC、 SEQ ID No.137:DKENV、 SEQ ID No.138:EKENH、 SEQ ID No.139:AKEND、 SEQ ID No.140:DKENG、 SEQ ID No.141:FDIYMD、 SEQ ID No.142:FHVFED、 SEQ ID No.143:ITVFDE、 SEQ ID No.144:FTPYVE、 SEQ ID No.145:FSIFDE、 SEQ ID No.146:NQETGE、 SEQ ID No.147:SEE、 SEQ ID No.148:SEEN、 SEQ ID No.149:DNETGE、 SEQ ID No.150:MONEY、 SEQ ID No.151:DHDTGE、 SEQ ID No.152:DPSTGE、 SEQ ID No.153:DPSTGE、 SEQ ID No.154:DGETGE、 SEQ ID No.155:SPETGE、 SEQ ID No.156:DEETGE、 SEQ ID No.157:DEETGE、 SEQ ID No.158:DEETGE、 SEQ ID No.159:NVESGE、 SEQ ID No.160:QDIDLGV、 SEQ ID No.161:QDIDLGV、 SEQ ID No.162:DPSTGE、 SEQ ID No.163:DGETGE、 SEQ ID No.164:SPETGE、 SEQ ID No.165:DEETGE、 SEQ ID No.166:NVESGE、 SEQ ID No.167:QDIDLGV、 SEQ ID No.168:ADSST、 SEQ ID No.169:ADSST、 SEQ ID No.170:PSSTS、 SEQ ID No.171:PSSTS、 SEQ ID No.172:VSSST、 SEQ ID No.173:VSSTS、 SEQ ID No.174:VTSTT、 SEQ ID No.175:LQLDEETGEFL、 SEQ ID No.176:ECEETEVDQHV、 SEQ ID No.177:FEHLWSSL、 SEQ ID No.178:FSDLWKLL、 SEQ ID No.179:FEAQWAAL、 SEQ ID No.180:FEAQWAAL、 SEQ ID No.181:FQHIWDFL、 SEQ ID No.182: FSDLWKLL、 SEQ ID No.183: LLQPNNYQFC、 SEQ ID No.184:DYR、 SEQ ID No.185:RAVENQYSFY、 SEQ ID No.186:SHVENDYIDNPS、 SEQ ID No.187:LIEDNEYTARQG、 SEQ ID No.188:SIFNSLYTTLSD、 SEQ ID No.189:SIFDNLYTTLSD、 SEQ ID No.190:VSFNPYEPELA、 SEQ ID No.191:LLQPNNYQFC、 SEQ ID No.192:INGNNYVYIDP、 SEQ ID No.193:STNHIYSNLAN、 SEQ ID No.194:IPFLDYRTYAV、 SEQ ID No.195:NESVDYRATFP、 SEQ ID No.196:YSGSDYRSGLA、 SEQ ID No.197:ADYRE、 SEQ ID No.198:IPFLDYRTYAM、 SEQ ID No.199:RDYRD、 SEQ ID No.200:IRNTNEYTEGPT、 SEQ ID No.201:DSFLQRYSSDP、 SEQ ID No.202:KGDGGLYSSLPP、 SEQ ID No.203:TLNSDGYTPEPA、 SEQ ID No.204:RDNVYYYDEEG、 SEQ ID No.205:KEDPIYDEPEG、 SEQ ID No.206:AEDSTYDEYEN、 SEQ ID No.207:RLCLPQV、 SEQ ID No.208:RNSLRQT、 SEQ ID No.209:RAPLSTL、 SEQ ID No.210:RKALETL、 SEQ ID No.211:RMKLPSP、 SEQ ID No.212:RNFLTEQ、 SEQ ID No.213:RTPLSIV、 SEQ ID No.214:RYGLHPD、 SEQ ID No.215:RSAFEDL、 SEQ ID No.216:RHFLDRV、 SEQ ID No.217:RHCLPTL、 SEQ ID No.218:RKKLVLE、 SEQ ID No.219:RLPLRAV、 SEQ ID No.220:RSPLLEV、 SEQ ID No.221:RKALGTV、 SEQ ID No.222:RKALGTV、 SEQ ID No.223:RGSLEGA、 SEQ ID No.224:RSPLFIF、 SEQ ID No.225:RSSLLSR、 SEQ ID No.226:RSTLAEL、 SEQ ID No.227:RCQLAKK、 SEQ ID No.228:KTPLKEG、 SEQ ID No.229:RGTLKRQ、 SEQ ID No.230:RAALAVL、 SEQ ID No.231:RDELGGG、 SEQ ID No.232:RFALSQL、 SEQ ID No.233:RPELQDA、 SEQ ID No.234:RKFLSLA、 SEQ ID No.235:RLPLRAQ、 SEQ ID No.236:RRPLILK、 SEQ ID No.237:RRRLPLP、 SEQ ID No.238:RLPLPVQ、 SEQ ID No.239:RKSLSQK、 SEQ ID No.240:HLSLKDI、 SEQ ID No.241:RLPLVPE、 SEQ ID No.242:RARLCSS、 SEQ ID No.243:RVRLSWA、 SEQ ID No.244:RRFLENC、 SEQ ID No.245:RLPLAQV、 SEQ ID No.246:RSPLTIL、 SEQ ID No.247:RAALDRL、 SEQ ID No.248:RNLLHDN、 SEQ ID No.249:RKRLGDD、 SEQ ID No.250:RRGLILA、 SEQ ID No.251:RENLQRK、 SEQ ID No.252:RQPLSEA、 SEQ ID No.253:RASLNPRぁ SEQ ID No.254:RMKLKKMぁ SEQ ID No.255:RTPLASVぁ SEQ ID No.256:RTQLETKぁ SEQ ID No.257:RRSLSISぁ SEQ ID No.258:QRTVLGVIGぁ SEQ ID No.259:NRKPLTVLN、 SEQ ID No.260:SRTPLTTLN、 SEQ ID No.261:NKENCぁ SEQ ID No.262:EKENHぁ SEQ ID No.263:EKENQぁ SEQ ID No.264:AKEND、 SEQ ID No.265:DKENG� SEQ ID No.266:NKENK、 SEQ ID No.267:QKENG� SEQ ID No.268:NKENL、 SEQ ID No.269:EKENL、 SEQ ID No.270:SKENI、 SEQ ID No.271:DKENR、 SEQ ID No.272:EKENC、 SEQ ID No.273:QKENR、 SEQ ID No.274:YKENK、 SEQ ID No.275:LKENS SEQ ID No.276:LKENS SEQ ID No.277:DKENQぁ SEQ ID No.278:QKENVぁ SEQ ID No.279: QYLIR SEQ ID No.280: NKRIR、 SEQ ID No.281:FNQIRぁ SEQ ID No.282:VNHIRぁ SEQ ID No.283:RTTIR、 SEQ ID No.284:RTQIR、 SEQ ID No.285:KGNLR、 SEQ ID No.286:YKGIR、 SEQ ID No.287:LMTMR、 SEQ ID No.288:VNRIR、 SEQ ID No.289:VNRIR、 SEQ ID No.290:FTRIR、 SEQ ID No.291:YRSIR、 SEQ ID No.292:ILGMR、 SEQ ID No.293:HQGIR、 SEQ ID No.294:DEQFVPDY、 SEQ ID No.295:EELLMVPDM、 SEQ ID No.296:EEHFLVPDL、 SEQ ID No.297:EQTLQEVPTGL、 SEQ ID No.298:SLLDVQRVPSF、 SEQ ID No.299:TSMTDFYHSKRRL、 SEQ ID No.300:NDITNYYKVKRRP、 SEQ ID No.301:MCKGL、 SEQ ID No.302:MCRTL、 SEQ ID No.303:PPTPPPE、 SEQ ID No.304:PTPPLS、 SEQ ID No.305:PFLTPSPE、 SEQ ID No.306:QVVGWPPVRSYRK、 SEQ ID No.307:QIVGWPPVRSYRK、 SEQ ID No.308:QVVGWPPVRNYRK、 SEQ ID No.309:QVVGWPPIGLHRM、 SEQ ID No.310:PVVGWPPVRSSRR、 SEQ ID No.311:DSGRGS、 SEQ ID No.312:DSGNYS、 SEQ ID No.313:DSGICMS、 SEQ ID No.314:DSGVETS、 SEQ ID No.315:DSGLDS、 SEQ ID No.316:DSGIHS、 SEQ ID No.317:DSGIDS、 SEQ ID No.318:DSGIDS、 SEQ ID No.319:DSGLPS、 SEQ ID No.320:DSGLGS、 SEQ ID No.321:DSGRGDS、 SEQ ID No.322:DSGYNT、 SEQ ID No.323:DSGQGS、 SEQ ID No.324:DSGIGT、 SEQ ID No.325:DSGYSS、 SEQ ID No.326:SEGSDDSGL、 SEQ ID No.327:RPTAAVTPI、 SEQ ID No.328:PPRALVLPH、 SEQ ID No.329:KPAAVVAPI、 SEQ ID No.330:KPTAYVRPM、 SEQ ID No.331:NKENE、 SEQ ID No.332:EKENQ、 SEQ ID No.333:NKENK、 SEQ ID No.334:QKENG、 SEQ ID No.335:NKENL、 SEQ ID No.336:EKENL、 SEQ ID No.337:SKENI、 SEQ ID No.338:DKENR、 SEQ ID No.339: EKENC, SEQ ID No.340: QKENR, SEQ ID No.341: YKENK, SEQ ID No.342: LKENS, SEQ ID No.343: LKENS, SEQ ID No.344: DKENQ, SEQ ID No.345:QKENV, SEQ ID No.346:FTIHVD, SEQ ID No.347:FMLYEE, SEQ ID No.348:IARRNSLTRFLEKRKDRV, SEQ ID No.349:QARKASLARFLEKRKERV, SEQ ID No.350:QARKASLARFLEKRKERL, SEQ ID No.351:IARRASLHRFLEKRKDRV, SEQ ID No.352:LARKASLARFLEKRKERV, or SEQ ID No.353:IARRASLHRFLEKRKDRI.

[0016] Preferably, the TEVp recognition site includes the amino acid sequence indicated by SEQ ID No. 354.

[0017] SEQ ID No.354: ENLYFQG.

[0018] Preferably, the C-terminus of the M1 protein and the TEVp recognition site are linked by a flexible linker 1.

[0019] Preferably, the flexible linker 1 contains the amino acid sequence indicated by SEQ ID No. 355.

[0020] SEQ ID No.355: GSGG.

[0021] Preferably, the TEVp recognition site and the protein hydrolysis target molecule are linked by a flexible linker 2.

[0022] Preferably, the flexible linker 2 contains the amino acid sequence of GSG.

[0023] Preferably, the protein hydrolysis target influenza virus is a virus of type A, type B, or type C.

[0024] In addition, the subtypes of the influenza virus are 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, H7N 7, including one or at least two combinations of 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, or H18N11.

[0025] In this application, the inventors introduce the nucleotide sequences of the TEVp recognition site and the protein hydrolysis target molecule into the genome of the influenza virus, thereby obtaining a virus that is site-specifically modified by the protein hydrolysis target molecule, i.e., a protein hydrolysis-targeting chimeric virus (PROTAC virus), in a cell line overexpressing TEVp, in which the influenza virus genome containing the protein hydrolysis target molecule can be replicated along with the replication of the viral genome and can be fused to and expressed with the viral protein during the translation of the viral protein.

[0026] In normal cells, the ubiquitin-proteasome system recognizes protein hydrolysis target molecules fused with the viral M1 protein, degrading the viral protein, weakening the virus's replication ability, and ultimately leading to its complete loss; therefore, PROTAC viruses possess high safety. Furthermore, because the PROTAC virus contains specific recognition sequences and sites of the ubiquitin-proteasome system, it can perform tumor lysis in the treatment of tumors by converting cold tumors into hot tumors.

[0027] In this application, the PROTAC virus can be efficiently replicated in a specific artificially modified cell line and produced and prepared on a large scale. Furthermore, the PROTAC virus may be further modified, for example, by introducing an immunostimulant into a specific region or specific amino acid of the viral protein to obtain a virus with improved performance and a PROTAC virus with enhanced immunogenicity.

[0028] In this application, the flexible linker and TEVp recognition site (linking chain) used, and the amino acid sequence structure of the flexible linker and protein hydrolysis target molecule may be applied to influenza B, or to other subtypes of influenza viruses, or may be used to modify one or at least two of other types of viruses, such as HIV virus, novel coronavirus, hand-foot-and-mouth virus, hepatitis D virus, or hepatitis E virus.

[0029] In Embodiment 2, the present application provides a nucleic acid molecule encoding the hydrolysis target M1 protein described in Embodiment 1.

[0030] In Embodiment 3, the present application provides a recombinant vector comprising at least one copied nucleic acid molecule according to Embodiment 2.

[0031] In aspect 4, the present application is: Step (1) Constructing an expression vector for preparing protein hydrolysis target influenza viruses, The method includes step (2) replacing the plasmid expressing the M gene of the influenza virus in the influenza virus rescue system with the expression vector obtained in step (1), cotransfecting it in a cell line, and obtaining the protein hydrolysis target influenza virus. The present invention provides a method for preparing a protein hydrolysis target influenza virus as described in Embodiment 1.

[0032] This invention provides 353 types of protein hydrolysis target viruses, wherein the C-terminus of the M1 protein of each protein hydrolysis target virus contains one protein hydrolysis target molecule recognized by the ubiquitin-proteasome system, the M1 protein and the protein hydrolysis target molecule are linked by a TEVp recognition site, the TEVp recognition site can be selectively cleaved, and the M1 protein and the TEVp recognition site, and the TEVp recognition site and the protein hydrolysis target molecule are linked by two flexible molecules, the operation of modifying the virus by the above method is simple, the resulting protein hydrolysis target influenza virus is safe and reliable and has high immunity.

[0033] Preferably, in step (1), the expression vector contains the coding sequence of the hydrolysis target M1 protein.

[0034] Preferably, in step (2), the cell line is an artificial cell line that overexpresses TEVp.

[0035] Preferably, in step (2), the influenza virus rescue system includes a 12 plasmid rescue system for the WSN influenza virus.

[0036] Preferably, the preparation method further includes the step of replicating the protein hydrolysis target influenza virus in an artificially modified cell line that overexpresses TEVp and producing it on a large scale.

[0037] In this application, the PROTAC virus can replicate only in cell lines that overexpress TEVp, and by utilizing the PROTAC virus's dependence on cell lines that overexpress TEVp, large-scale preparation of influenza virus can be carried out in cell lines that overexpress TEVp.

[0038] As a preferred technical proposal of the present invention, the preparation method includes the following steps:

[0039] (1) Construct an expression vector for preparing a protein hydrolysis target influenza virus, modify a plasmid expressing the influenza virus M gene in the WSN influenza virus 12 plasmid rescue system using genetic engineering methods, introduce an insertion sequence before the C-terminus and stop codon of the M1 protein gene sequence, obtain an expression vector, the expression vector containing the coding sequence of the hydrolysis target M1 protein.

[0040] (2) The plasmid expressing the M gene of the influenza virus in the influenza virus rescue system is replaced with the expression vector obtained in step (1), and the influenza virus rescue system after the replacement is co-transfected with an artificial cell system that overexpresses TEVp to obtain the protein hydrolysis target influenza virus.

[0041] The aforementioned protein hydrolysis target virus is replicated in an artificially modified cell line that overexpresses TEVp, thereby producing the protein hydrolysis target virus on a large scale.

[0042] In Embodiment 5, the present application provides an influenza virus vaccine comprising the protein hydrolysis-targeted influenza virus described in Embodiment 1.

[0043] Preferably, the influenza virus vaccine is one of the following: a weakened vaccine, a vaccine containing a replication-defective live virus, or a vaccine containing a replication-controllable live virus.

[0044] In Embodiment 6, the present invention provides the use of one or at least two of the following in the preparation of an agent for treating influenza and / or an oncolytic agent: the protein hydrolysis-targeted influenza virus described in Embodiment 1, the nucleic acid molecule described in Embodiment 2, the recombinant vector described in Embodiment 3, the method for preparing the protein hydrolysis-targeted influenza virus described in Embodiment 4, or the influenza virus vaccine described in Embodiment 5.

[0045] The numerical range relating to this application includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. For the sake of space and simplicity, this application does not comprehensively list the specific point values ​​included in the range. [Effects of the Invention]

[0046] Compared to the prior art, this invention offers the following beneficial effects.

[0047] (1) This invention utilizes the hydrolytic activity of the ubiquitin-proteasome system and the cleavage activity of TEVp to prepare large quantities of protein hydrolysis-targeted influenza virus in a cell line that overexpresses TEVp. On the other hand, in a normal cell line, the ubiquitin-proteasome system recognizes protein hydrolysis target molecules fused with viral proteins, degrades the viral proteins, weakens the virus's replication ability, and ultimately leads to the complete loss of replication ability. Therefore, the protein hydrolysis-targeted influenza virus has high safety, and the protein hydrolysis-targeted influenza virus is a live virus that can be attenuated, has replication defects, and whose replication can be controlled.

[0048] (2) The method for preparing the protein hydrolyzable target influenza virus described above is simple to operate, safe and reliable, and the protein hydrolyzable target influenza virus in this application is produced using a stable mammalian cell system, overcoming the shortcomings of conventional viral reproduction using fertilized chicken eggs (viral reproduction using fertilized chicken eggs is prone to causing adverse reactions such as human allergies). The obtained protein hydrolyzable target influenza virus has high immunogenicity and can be used in the preparation of vaccines and the development of drugs related to the treatment of viral infections, and has strong research and application value.

[0049] (3) The protein hydrolysis target influenza virus may be further modified, for example by introducing an immunoenhancing agent into a specific region or specific amino acid of the viral protein to obtain a virus with improved performance and an immunogenic protein hydrolysis target influenza virus.

[0050] (4) The protein hydrolysis-targeted influenza virus can activate the tumor microenvironment, transform a cold tumor into a hot tumor, and increase the tumor therapeutic effect. [Brief explanation of the drawing]

[0051] [Figure 1] This is a schematic diagram of the preparation process of the protein hydrolysis target influenza virus in Example 1. [Figure 2]These are growth curves of protein hydrolysis-targeted influenza viruses in MDCK-TEVp (+TEVp) cells and normal MDCK (-TEVp) cells. Since each E3 ubiquitin ligase selects one representative virulence strain for data display, each virulence strain in the figure is renamed using its corresponding E3 ubiquitin ligase. M1-PTDKLHDC2(M1-PTD22), M1-PTDKLHDC3(M1-PTD43), M1-PTDAPPBP2(M1-PTD71), M1-PTDKLHL20(M1-PTD84), M1-PTDFBXO31(M1-PTD91), M1-PTDβ-TrCP(M1-PTD94), M1-PTDSOCS2(M1-PTD57), M1-PTDFEM1C(M1-PTD63), M1-PTDITC H(M1-PTD101), M1-PTDSPOP(M1-PTD109), M1-PTDMDM2(M1-PTD110), M1-PTDCRBN(M1-PTD116), M1-PTDAPC / C-ABBA (M1-PTD205), M1-PTDCBL (M1-PTD119), M1-PTDAPC / C-KENbox (M1-PTD138), M1-PTDCOP1 (M1-PTD148), M1-PTDN-end UBR-box(M1-PTD165), M1-PTDFBW7(M1-PTD174), M1-PTDSkp2-Cks1(M1-PTD183), M1-PTD SIAH-1 (M1-PTD194), M1-PTDAPC / C-TPR1 (M1-PTD143), M1-PTDAPC / C-Dbox (M1-PTD132). [Figure 3] This study evaluates the safety of protein hydrolysis-targeted influenza viruses in mouse models, where *** indicates a significant difference compared to the wild-type (WT). Data is presented as representative for M1-PTDKLHDC2 (M1-PTD22), M1-PTDKLHDC3 (M1-PTD43), M1-PTDAPPBP2 (M1-PTD71), M1-PTDKLHL20 (M1-PTD84), M1-PTDFBXO31 (M1-PTD91), and M1-PTDβ-TrCP (M1-PTD94). [Figure 4]This study investigates the mechanism of action of protein hydrolysis-targeted influenza viruses. Since each E3 ubiquitin ligase selects one representative virulence strain for data display, each virulence strain in the figure is renamed using its corresponding E3 ubiquitin ligase. [Figure 5] This study evaluates the immunogenicity of protein hydrolysis-targeted influenza viruses in a mouse model, where (a) is the level of neutralizing (NT) and hemagglutination inhibitory (HI) antibodies, (b) is the level of Anti-HA IgG and Anti-NP IgG antibodies, (c) is the level of Anti-NP IgA antibody, (d) is the T cell immune response to influenza virus M1 antigen peptide (MGLIYNRM) in mouse lung tissue, (e) is the level of T cell immune response to influenza virus NP antigen peptide (ASNENMETME) (left figure) and M1 antigen peptide (MGLIYNRM) (right figure) in mouse spleen tissue, and (f) is the level of antigen presentation of protein hydrolysis-targeted influenza viruses in macrophage Raw264.7 cells. *, **, and *** indicate a significant difference compared to wild-type viruses (WT), #, ##, and ### indicate a significant difference compared to inactivated influenza vaccine (IIV), and +, ++, and +++ indicate a significant difference compared to cold-adapted attenuated influenza vaccine (CAIV). Data is displayed using M1-PTDKLHDC2 (M1-PTD22), M1-PTDKLHDC3 (M1-PTD43), M1-PTDAPPBP2 (M1-PTD71), M1-PTDKLHL20 (M1-PTD84), M1-PTDFBXO31 (M1-PTD91), and M1-PTDβ-TrCP (M1-PTD94) as representative examples. [Figure 6]This study evaluated the cross-immunoprotective effect of protein hydrolysis-targeted influenza virus vaccines in an adult (7-week-old) mouse model, where (a) is the titer of wild-type WSN influenza virus in lung tissue 3 days after infection with wild-type WSN influenza virus, (b) is the mouse body weight and survival rate within 2 weeks of infection with wild-type WSN influenza virus, and (c) is the titer of wild-type X-31 influenza virus in lung tissue 3 days after infection with wild-type influenza virus A / X-31(H3N2), and (b) is the mouse body weight and survival rate within 2 weeks of infection with wild-type influenza virus A / X-31(H3N2). Data is presented using M1-PTDβ-TrCP (M1-PTD94) as the representative model. [Figure 7] This study evaluated the immunoprotective effects of a protein hydrolyzable (protein hydrolyzable) targeted influenza virus vaccine in aged (15-month-old) mouse and ferret models, where (a) is the level of neutralizing (NT) antibody, hemagglutination inhibitory (HI) antibody, anti-HA IgG antibody, and anti-NP-IgG antibody induced by the protein hydrolyzable targeted influenza virus vaccine in aged mice; (b) is the level of T-cell immune response to influenza virus NP antigen peptide (ASNENMETME) induced by the protein hydrolyzable targeted influenza virus vaccine in the lung tissue (left) and spleen (right) of aged mice; (c) is the titer of wild-type WSN influenza virus in lung tissue 3 days after infection of immunized and unimmunized aged mice with wild-type WSN influenza virus; (d) is the body weight and survival rate of immunized and unimmunized aged mice within 2 weeks after infection with wild-type WSN influenza virus; and (e) is the titer of virus in nasal lavage 3 days after infection of immunized and unimmunized ferrets with wild-type WSN influenza virus. Data will be displayed using M1-PTDβ-TrCP (M1-PTD94) as a representative example. [Figure 8] This describes the tumor-suppressing effect of protein hydrolysis-targeted influenza viruses, with data presented using M1-PTDβ-TrCP (M1-PTD94) as a representative example. [Modes for carrying out the invention]

[0052] The technical proposal of this application will be further explained below with reference to specific embodiments. Those skilled in the art should understand that the above embodiments are merely for the purpose of understanding this application and should not be considered as specifically limiting it.

[0053] Unless specific techniques or conditions are described in the examples, the techniques or conditions described in the literature within this art or in accordance with the product instructions shall be followed. Unless the manufacturer of the reagents or equipment used is specified, they are all standard products available through legitimate channels. [Examples]

[0054] Example 1 This embodiment provides a series of protein hydrolysis-targeted influenza viruses, each containing a hydrolysis-targeted M1 protein, the C-terminus of which a TEVp recognition site and a protein hydrolysis-target molecule recognized by the ubiquitin-proteasome system are sequentially inserted, and the amino acid sequences of the protein hydrolysis-target molecules recognized by the ubiquitin-proteasome system are as shown in SEQ ID No. 1 to 353. The amino acid sequence of the TEVp recognition site is as shown in SEQ ID No. 354, the C-terminus of the M1 protein and the TEVp recognition site are linked by flexible linker 1, the amino acid sequence of flexible linker 1 is as shown in SEQ ID No. 355, the TEVp recognition site and the protein hydrolysis target molecule are linked by flexible linker 2, the amino acid sequence of flexible linker 2 is GSG, and the amino acid sequence structure from the C-terminus of the M1 protein to the stop codon is M1 protein C-terminus-flexible linker 1-TEVp recognition site-flexible linker 2-protein hydrolysis target molecule.

[0055] The amino acid sequence of SEQ ID No. 354 is ENLYFQG.

[0056] The amino acid sequence of SEQ ID No. 355 is GSGG.

[0057] The amino acid sequences of the protein hydrolysis target molecules recognized by the ubiquitin-proteasome system are shown in Table 1.

[0058] [Table 1] TIFF0007832348000002.tif250159 TIFF0007832348000003.tif250156 TIFF0007832348000004.tif250156 TIFF0007832348000005.tif250161 TIFF0007832348000006.tif250157 TIFF0007832348000007.tif250157 TIFF0007832348000008.tif250157 TIFF0007832348000009.tif250157 TIFF0007832348000010.tif250157 TIFF0007832348000011.tif250157 TIFF0007832348000012.tif250157 TIFF0007832348000013.tif250157 TIFF0007832348000014.tif250157 TIFF0007832348000015.tif250157 TIFF0007832348000016.tif250157 TIFF0007832348000017.tif250157 TIFF0007832348000018.tif250157 TIFF0007832348000019.tif250157 TIFF0007832348000020.tif250157 TIFF0007832348000021.tif52169

[0059] The influenza viruses among the aforementioned protein hydrolysis target influenza viruses are type A, type B, and type C viruses, and the subtypes of the influenza viruses are H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H Includes 6N4, 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, and H18N11.

[0060] As shown in Figure 1, a schematic diagram of the preparation process of the protein hydrolysis-targeted influenza virus, the method for preparing the protein hydrolysis-targeted influenza virus includes the following steps.

[0061] (1) An expression vector was constructed to prepare an influenza virus targeted by protein hydrolysis.

[0062] Using a plasmid expressing the M gene of the influenza virus in the influenza virus rescue system as a template, a gene sequence capable of expressing the amino acid sequence "flexible linker 1 (SEQ ID No. 355: GSGG)-TEVp recognition site (SEQ ID No. 354: ENLYFQG)-flexible linker 2 (GSG)-protein hydrolysis target molecule" is inserted before the stop codon at the C-terminus of the gene sequence expressing the M1 protein using a site-directed mutation method. If the amino acid sequence of the protein hydrolysis target molecule is PTD3, the constructed vector is named M1-PTD3, and if the amino acid sequence of the protein hydrolysis target molecule is PTD4, the constructed vector is named M1-PTD4. The vector naming convention is thus inferred. The obtained target vector underwent sequencing validation, and the construction of an expression vector was successful.

[0063] (2) The plasmid expressing the M gene of the influenza virus in the influenza virus rescue system was replaced with the expression vector obtained in step (1), and the influenza virus rescue system after the replacement was co-transfected with the cell line to obtain the protein hydrolysis target influenza virus.

[0064] We performed rescue of WSN influenza viruses using the 12-plasmid rescue system for WSN influenza viruses. When rescuing protein hydrolysis-targeted influenza viruses, the plasmid for expressing the M gene in the 12-plasmid rescue system is replaced with the expression vector constructed in step (1), and the virus strain obtained by rescue was named after the corresponding vector in step (1).

[0065] Cells expressing TEVp were inoculated into a 6-well plate. The following day, the M1-PTD3 vector from step (1) and 11 other plasmids from the WSN influenza virus rescue system were co-transfected into a cell line expressing the TEVp protein (HEK293T cell line), with 0.2 μg of each plasmid added to each well of the 6-well plate. Six hours after transfection, the medium was replaced with fresh DMEM medium containing 0.5% FBS, 1 μg / mL TPCK-trypsin, and bispecific antibodies. Subsequently, the lesion status of the cells was observed daily, and when 80% of the cells were lesioned, the viral supernatant was collected to obtain a protein hydrolysis target virus, which was named M1-PTD3.

[0066] Following a similar method, other protein hydrolysis-targeted influenza viruses can be obtained and named according to similar rules. The obtained protein hydrolysis-targeted influenza viruses are shown in Table 2.

[0067] [Table 2] TIFF0007832348000023.tif250157 TIFF0007832348000024.tif250158 TIFF0007832348000025.tif215170

[0068] The obtained viral supernatant was used to infect cell lines expressing a new TEVp protein, and the constructed protein hydrolysis target viruses were examined according to a standard for determining whether or not they could induce cellular lesions.

[0069] If it is possible to induce cell lineage lesions expressing the TEVp protein (e.g., HEK293T cells expressing TEVp and / or MDCK cells expressing TEVp), it means that the rescue of the protein hydrolysis target virus has been successful.

[0070] If cells expressing TEVp (e.g., HEK293T cells and / or MDCK cells expressing TEVp) are unable to induce disease, it means that the rescue of the protein hydrolysis target virus has failed.

[0071] The results showed that the rescue of all protein hydrolysis-targeted influenza viruses was successful.

[0072] Test Example 1 The preparation efficiency and safety of the aforementioned protein hydrolysis target influenza virus were evaluated.

[0073] (1) Evaluation of the efficiency and safety of preparing protein hydrolysis target influenza viruses at the cellular level.

[0074] We investigated the circumstances and growth curves under which influenza virus strains M1-PTD3~M1-PTD413 caused cellular lesions in MDCK cell lines expressing TEVp (MDCK-TEVp cell lines) and normal MDCK cell lines, and examined the efficiency and safety of strain preparation. Wild-type influenza virus was used as a control.

[0075] The criteria for determining the safety of toxic strains are as follows:

[0076] Observation confirmation based on cellular lesions: Wild-type influenza virus can cause complete cellular lesions (100%) in both MDCK-TEVp cells and MDCK cells. If a virulent strain can cause significant cellular lesions (50-100% cellular lesions) in the MDCK-TEVp cell line, it means the preparation efficiency of the virulent strain is high. If a virulent strain cannot cause cellular lesions, or causes less cellular lesions (lower than 100% cellular lesions caused by wild-type virus), compared to the wild-type virus, it means the virulent strain is safe.

[0077] Analysis based on growth curves: If a toxic strain can replicate highly in the MDCK-TEVp cell system compared to the wild-type virus (viral titer is higher than, equivalent to, or more than 1 / 1000th of the wild-type virus), it means that the preparation efficiency of the toxic strain is high. If the replication ability of a toxic strain is reduced, and consequently it cannot replicate, in a normal MDCK cell system compared to the wild-type virus (viral titer is lower than that of the wild-type virus), it means that the toxic strain is safe.

[0078] (a) The steps for detection by cellular lesions are as follows:

[0079] Prepared protein hydrolysis-targeted influenza viruses and wild-type influenza viruses were used to infect MDCK-TEVp cell lines and normal MDCK cell lines at a ratio of MOI = 0.01, respectively. The disease status of the cells was observed and recorded daily for 4 days.

[0080] According to the test results, in the MDCK-TEVp cell line, all protein hydrolysis-targeted influenza viruses and wild-type influenza viruses were able to induce significant cellular lesions. On the other hand, in a normal MDCK cell line, only wild-type influenza viruses were able to induce significant cellular lesions, and lesions caused by protein hydrolysis-targeted influenza viruses were reduced, and eventually eliminated. These results indicate that the prepared protein hydrolysis-targeted influenza viruses are safe at the cellular level.

[0081] (b) The steps for detection using growth curves are as follows:

[0082] Prepared protein hydrolysis-targeted influenza viruses and wild-type influenza viruses were used to infect MDCK-TEVp cell lines and normal MDCK cell lines, respectively, at a ratio of MOI = 0.001. Cell culture supernatants were collected 24, 48, 72, and 96 hours after infection, and TCID was applied to each cell. 50By detecting the virus titer in the supernatant in the experiment and plaque experiment, the replication ability of the virus in two types of cells could be known.

[0083] According to the results in Figure 2, in the MDCK-TEVp cell line, all proteolytic target influenza viruses and wild-type influenza viruses had good replication ability. On the other hand, in the normal MDCK cell line, only the wild-type influenza virus showed good replication ability, and the replication ability of the proteolytic target influenza virus decreased, and it was found that there were defects in replication. The results indicated that the prepared proteolytic target influenza virus had safety at the cellular level.

[0084] (2) Evaluation of the safety of proteolytic target influenza virus at the animal level

[0085] The safety of the proteolytic target influenza virus at the animal level was evaluated using BALB / c and C-57BL / 6J mice. Six proteolytic target influenza virus strains (M1-PTD KLHDC2 , M1-PTD KLHDC3 , M1-PTD APPBP2 , M1-PTD KLHL20 , M1-PTD FBXO31 , M1-PTD β-TrCP ) were selected as representatives of the proteolytic target influenza virus, and the safety of the virus was evaluated.

[0086] The specific steps for the safety evaluation are as follows.

[0087] (a) Eighty 7-week-old female BALB / c mice or C57BL / 6J mice were divided into 8 groups, with 10 mice in each group.

[0088] (b) Each mouse in the first group was inoculated intranasally with PBS (Vehicle), and each mouse in the second group was inoculated with 1×10 5 TCID 50Wild-type WSN influenza virus (WT) was administered via nasal spray, and each of the 3rd to 8th groups received 1 x 10⁶ doses. 5 TCID 50 M1-PTD KLHDC2 M1-PTD KLHDC3 M1-PTD APPBP2 M1-PTD KLHL20 M1-PTD FBXO31 , or M1-PTD β-TrCP It was administered via nasal spray.

[0089] (c) Three days after vaccination, five mice were taken from each group, their lung tissue was collected, and the viral titer was detected.

[0090] (d) The weight and mortality status of the remaining 5 mice in each group were observed and monitored for 14 days.

[0091] As shown in Figure 3, wild-type WSN influenza virus replicates highly in the lungs of mice, causing a significant decrease in mouse body weight and mouse death. On the other hand, protein hydrolysis-targeted influenza viruses showed weak replication ability in the lungs of mice (below the detection limit), did not cause a decrease in mouse body weight, and did not cause mouse death. Therefore, the protein hydrolysis-targeted virus vaccine has good safety.

[0092] Test Example 2 We investigated the replication ability of protein hydrolysis-targeted influenza viruses.

[0093] (1) The expression level of the M1 protein of the protein hydrolysis-targeted influenza virus was detected using Western Blot, and five representative virulent strains of the protein hydrolysis-targeted influenza virus were selected. The replication ability of the protein hydrolysis-targeted influenza virus in normal cells was then investigated.

[0094] Protein hydrolysis-targeted influenza virus and wild-type influenza virus were used to infect normal MDCK cell lines (MOI=0.1), respectively. The culture medium was supplemented with 25 nM, 50 nM, and 100 nM of the proteasome inhibitor MG-132, and a mixed solution of DMSO (at the same dilution ratio as the virus) and normal MDCK cell lines was used as a control. Cell samples were collected 24, 48, and 72 hours after infection, and the expression level of the viral M1 protein was detected using Western Blot.

[0095] (2) Immunofluorescence experiments were used to detect the expression level of the protein hydrolysis-targeted influenza virus M1 protein, and the replication ability of the protein hydrolysis-targeted influenza virus was examined.

[0096] The aforementioned protein hydrolysis-targeted influenza virus and wild-type influenza virus infected MDCK-TEVp cell lines and normal MDCK cell lines (MOI=0.01), respectively. The culture media were supplemented with 0nM, 25nM, 50nM, and 100nM of the proteasome inhibitor MG-132, respectively. A mixed solution of DMSO (at the same dilution ratio as the virus) and normal MDCK cell lines was used as a control. 48 hours after infection, the cells were fixed with 4% PFA, and the expression level of the protein hydrolysis-targeted influenza virus M1 protein was detected by immunofluorescence.

[0097] According to the experimental results in Figure 4, it was found that after infecting MDCK-TEVp cells with the protein hydrolysis-targeted influenza virus, it could replicate in large quantities and synthesize a large amount of viral protein. On the other hand, after infecting normal MDCK cells with the protein hydrolysis-targeted influenza virus, it could not replicate in large quantities, so a small amount of viral protein M1 signal was detected. This indicated that the proteasome system of the cell was suppressed, followed by an increase in the viral protein M1 signal, meaning that the viral replication capacity was enhanced after the proteasome system was suppressed. The detection results were consistent with the Western Blot detection results in Test Example 2, further demonstrating that the introduction of the protein hydrolysis-targeted molecule can mediate the degradation of viral proteins by the cell's proteasome, and thus suppress the viral replication capacity. After the proteasome system of the cell was suppressed, the viral replication capacity was restored, which is consistent with the design principle of the protein hydrolysis-targeted influenza virus.

[0098] Test Example 3 Considerations on the immunogenicity and protective effects of protein hydrolysis-targeted influenza viruses at the animal level.

[0099] The immunogenicity and protective properties of the aforementioned protein hydrolysis-targeted influenza viruses were evaluated at the animal level. An inactivated influenza vaccine (IIV) was used as a control (the inactivated influenza virus vaccine was prepared by the inventor using the same type of influenza virus particles based on the method of the Chinese Pharmacopoeia), and a clinically used low-temperature attenuated vaccine was also used as a control. Six protein hydrolysis-targeted influenza virus virulence strains (M1-PTD) were selected as representative protein hydrolysis-targeted influenza viruses. KLHDC2 M1-PTD KLHDC3 M1-PTD APPBP2 M1-PTD KLHL20 M1-PTD FBXO31 M1-PTD β-TrCP We selected the appropriate virus and evaluated the immunogenicity and protective properties of the protein hydrolysis-targeted influenza virus.

[0100] The specific steps for considering immunogenicity and protective properties are as follows:

[0101] (1) 180 seven-week-old female BALB / c or C57BL / 6J mice were divided into 9 groups of 20 mice each.

[0102] (2) Each mouse in the first group was inoculated with PBS (Vehicle) via nasal drops, and each mouse in the second group was given 1 × 10 5 TCID 50 The cold-adapted attenuated influenza vaccine (CAIV) was administered nasally, and 1 × 10⁶ mice were given to each of the third group of mice. 5 TCID 50 Inactivated influenza vaccine (IIV) was administered by intramuscular injection, and 10 doses were given to each of the 4th to 9th groups. 5 TCID 50 M1-PTD KLHDC2 M1-PTD KLHDC3 M1-PTD APPBP2 M1-PTD KLHL20 M1-PTD FBXO31 , or M1-PTD β-TrCP I received the vaccine.

[0103] (3) One week after vaccination, five mice were taken from each group, and their lung tissue and spleen were collected, and the immune response of T cells in them was detected.

[0104] (4) Three weeks after vaccination, five mice were taken from each group, blood was collected, and these were used for hemagglutination inhibition (HI) test, neutralizing (NT) antibody detection, and ELISA detection, respectively, to detect the antibody immune response.

[0105] (5) Three weeks after vaccination, 2 × 10⁶ mice in each group 5 PFU administered either the same type of wild-type WSN influenza virus or a different type of wild-type influenza virus A / X-31(H3N2) via nasal inoculation.

[0106] (6) Three days after inoculation with wild-type influenza virus, five mice were taken from each group, their lung tissue was collected, and the viral titer was detected.

[0107] (7) The weight and mortality status of the remaining 5 mice in each group were observed and monitored for 14 days.

[0108] As shown in Figure 5, all protein hydrolyzable target influenza viruses were able to induce high levels of hemagglutination inhibitory (HI) antibody titers, neutralizing (NT) antibody titers, anti-HA IgG, anti-NP IgG, anti-NP IgA, and T-cell immune responses in animals. Furthermore, the levels of induced hemagglutination inhibitory antibodies, neutralizing antibodies, anti-NP IgG, anti-NP IgA, and T-cell immune responses were significantly higher than those induced by inactivated influenza vaccines, and the level of immune responses induced by some virulent strains was higher than that of cold-adapted attenuated vaccines. As shown in Figure 6, vaccination with protein hydrolyzable target influenza virus vaccines was able to significantly reduce wild-type WSN influenza virus titers and X-31(H3N2) virus titers in animal lung tissue and protect the survival of all animals. Therefore, protein hydrolyzable target influenza virus vaccines can provide cross-immunoprotective effects, and the protective effect provided by protein hydrolyzable target influenza virus vaccines is significantly superior to that of inactivated influenza vaccines. Therefore, the aforementioned protein hydrolysis-targeted influenza virus vaccine exhibits superior immunogenicity and protective effects. Furthermore, as shown in Figure 7, the protein hydrolysis-targeted influenza virus vaccine was found to exert high levels of immunoprotective effects in aged (15-month-old) mouse and ferret models.

[0109] Test Example 4 We investigated the potential of protein hydrolysis-targeted influenza viruses to be used as oncolytic viruses.

[0110] The tumor-lytic effect of the aforementioned protein hydrolysis-targeted influenza virus was evaluated using a C57BL / c melanoma carcinoma model.

[0111] The specific testing steps are as follows:

[0112] (1) Mice were injected with melanoma subcutaneously into the back of their bodies and kept for 9 days, and the tumor volume was approximately 100 mm. 3 Then, continue the experimental procedure.

[0113] (2) 50 μL of protein hydrolysis-targeted influenza virus M1-PTD in the tumor β-TrCP (M1-PTD94) and PBS were injected separately, once every other day, for a total of four injections.

[0114] (3) The volume of the tumor was detected daily.

[0115] As shown in Figure 8, the protein hydrolysis-targeted influenza virus was found to effectively suppress the increase in tumor volume, demonstrating that the protein hydrolysis-targeted influenza virus has the potential to become an oncolytic virus.

[0116] In summary, the protein hydrolyzable influenza virus according to this application can be recognized and hydrolyzed by the ubiquitin-proteasome system, has weak replication ability, possesses high safety, and can be used for influenza prevention as a live vaccine or attenuated vaccine. The protein hydrolyzable influenza virus can also be used as an oncolytic virus in tumor treatment. The protein hydrolyzable influenza virus according to this application has a rich variety of subtypes and plays an important role in the research and development of influenza virus vaccines and in tumor treatment.

[0117] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. The applicant states that any modification or substitution that a person skilled in the art would readily conceive of within the technical scope disclosed herein is included within the scope of protection and disclosure of the present application. This disclosure relates, for example, to the following: [1] A protein hydrolysis-targeted influenza virus containing the hydrolysis-targeted M1 protein, The C-terminus of the hydrolysis target M1 protein is sequentially inserted with a TEVp recognition site and a protein hydrolysis target molecule recognized by the ubiquitin-proteasome system, and the protein hydrolysis target molecule recognized by the ubiquitin-proteasome system includes the amino acid sequence indicated by SEQ ID No. 1 to 353. Protein hydrolysis-targeted influenza virus. [2] The TEVp recognition site includes the amino acid sequence indicated by SEQ ID No. 354. The protein hydrolysis target influenza virus described in [1] above. [3] The C-terminus of the M1 protein and the TEVp recognition site are linked by a flexible linker 1. Preferably, the flexible linker 1 includes the amino acid sequence indicated by SEQ ID No. 355, Preferably, the TEVp recognition site and the protein hydrolysis target molecule are linked by a flexible linker 2. Preferably, the flexible linker 2 includes the amino acid sequence indicated by SEQ ID No. 356. The protein hydrolysis target influenza virus described in [1] or [2] above. [4] The aforementioned protein hydrolysis target influenza viruses are viruses of type A, type B, and type C. Preferably, the subtypes of the protein hydrolysis target influenza virus are 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, H7N 3, including one or at least two combinations of 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, or H18N11, The protein hydrolysis target influenza virus described in any one of the above items [1] to [3]. [5] Encoding the hydrolysis target M1 protein described in any one of the above [1] to [4], Nucleic acid molecule. [6] A nucleic acid molecule comprising at least one copy of the nucleic acid molecule described in [5] above, Recombinant vector. [7] A method for preparing a protein hydrolysis target influenza virus according to any one of the above items [1] to [4], Step (1) Constructing an expression vector for preparing protein hydrolysis target influenza viruses, The method includes step (2) replacing the plasmid expressing the M gene of the influenza virus in the influenza virus rescue system with the expression vector obtained in step (1), cotransfecting it in a cell line, and obtaining the protein hydrolysis target influenza virus. A method for preparing protein hydrolysis-targeted influenza viruses. [8] In step (1), the expression vector contains the coding sequence of the hydrolysis target M1 protein, Preferably, in step (2), the cell line is an artificial cell line that overexpresses TEVp, Preferably, in step (2), the influenza virus rescue system includes a 12 plasmid rescue system for the WSN influenza virus. The method for preparing protein hydrolysis target influenza viruses as described in [7] above. [9] The process further includes replicating the aforementioned protein hydrolysis-targeted influenza virus in an artificially modified cell line that overexpresses TEVp and producing it on a large scale. A method for preparing protein hydrolysis target influenza viruses as described in [7] or [8] above.

[10] An influenza virus vaccine comprising a protein hydrolysis-targeted influenza virus as described in any one of the above items [1] to [4], Preferably, the influenza virus vaccine is one of the following: a weakened vaccine, a vaccine containing a replication-defective live virus, or a vaccine containing a replication-controllable live virus. Influenza virus vaccine.

[11] Use of one or at least two of the following in the preparation of drugs for treating influenza and / or oncolytic agents: a protein hydrolyzable target influenza virus as described in any one of items [1] to [4] above, a nucleic acid molecule as described in item [5] above, a recombinant vector as described in item [6] above, a method for preparing a protein hydrolyzable target influenza virus as described in any one of items [7] to [9] above, or an influenza virus vaccine as described in item

[10] above.

Claims

1. A protein hydrolysis target influenza virus containing the hydrolysis target M1 protein, The C-terminus of the hydrolysis target M1 protein is sequentially inserted with a TEVp recognition site and a protein hydrolysis target molecule recognized by the ubiquitin-proteasome system, and the protein hydrolysis target molecule recognized by the ubiquitin-proteasome system contains an amino acid sequence represented by one of SEQ ID Nos. 1-4, 146, 149, 154-158, or 163-165. Protein hydrolysis-targeted influenza virus.

2. The TEVp recognition site includes the amino acid sequence indicated by SEQ ID No.

354. The protein hydrolysis target influenza virus according to claim 1.

3. The C-terminus of the M1 protein and the TEVp recognition site are linked by a flexible linker 1. The flexible linker 1 contains the amino acid sequence indicated by SEQ ID No. 355, The TEVp recognition site and the protein hydrolysis target molecule are linked by a flexible linker 2. The flexible linker 2 includes the amino acid sequence indicated by SEQ ID No.

356. The protein hydrolysis target influenza virus according to claim 1.

4. The aforementioned protein hydrolysis target influenza viruses are type A, type B, and type C viruses. The aforementioned protein hydrolysis target influenza virus subtypes are 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, H Includes one or at least two combinations of 7N7, 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, or H18N11. The protein hydrolysis target influenza virus according to claim 1.

5. Encoding the hydrolysis target M1 protein according to any one of claims 1 to 4, Nucleic acid molecule.

6. A nucleic acid molecule comprising at least one copied claim 5, Recombinant vector.

7. A method for preparing a protein hydrolysis target influenza virus according to any one of claims 1 to 4, Step (1) to construct an expression vector for preparing protein hydrolysis target influenza viruses, The influenza virus rescue system includes step (2), which involves replacing the plasmid expressing the M gene of the influenza virus with the expression vector obtained in step (1), co-transfecting it in a cell line, and obtaining the protein hydrolysis target influenza virus. A method for preparing protein hydrolysis-targeted influenza viruses.

8. In step (1), the expression vector contains the coding sequence of the hydrolysis target M1 protein, In step (2), the cell line is an artificial cell line that overexpresses TEVp, In step (2), the influenza virus rescue system includes a 12 plasmid rescue system for the WSN influenza virus. A method for preparing a protein hydrolysis target influenza virus according to claim 7.

9. The process further includes replicating the aforementioned protein hydrolysis-targeted influenza virus in an artificially modified cell line that overexpresses TEVp and producing it on a large scale. A method for preparing a protein hydrolysis target influenza virus according to claim 7.

10. An influenza virus vaccine comprising a protein hydrolysis-targeted influenza virus according to any one of claims 1 to 4, The aforementioned influenza virus vaccine is one of the following: a weakened vaccine, a vaccine containing a live virus with replication defects, or a vaccine containing a live virus with controllable replication. Influenza virus vaccine.

11. A pharmaceutical composition for treating influenza and / or lysing tumors, comprising a protein hydrolysis-targeted influenza virus according to any one of claims 1 to 4.

Images