WO2023236041A1 - Mrna vaccine encoding pcrv and/or oprf-i protein - Google Patents
Mrna vaccine encoding pcrv and/or oprf-i protein Download PDFInfo
- Publication number
- WO2023236041A1 WO2023236041A1 PCT/CN2022/097390 CN2022097390W WO2023236041A1 WO 2023236041 A1 WO2023236041 A1 WO 2023236041A1 CN 2022097390 W CN2022097390 W CN 2022097390W WO 2023236041 A1 WO2023236041 A1 WO 2023236041A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- mrna
- protein
- acid sequence
- seq
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 92
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 91
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 86
- 229960005486 vaccine Drugs 0.000 title claims abstract description 44
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 35
- 201000010099 disease Diseases 0.000 claims abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 9
- 235000018102 proteins Nutrition 0.000 claims description 77
- 150000001413 amino acids Chemical group 0.000 claims description 67
- 150000002632 lipids Chemical class 0.000 claims description 25
- 239000013612 plasmid Substances 0.000 claims description 21
- 239000002773 nucleotide Substances 0.000 claims description 20
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- 108020004414 DNA Proteins 0.000 claims description 19
- 239000002105 nanoparticle Substances 0.000 claims description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 14
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 10
- 108020003589 5' Untranslated Regions Proteins 0.000 claims description 10
- 102000053602 DNA Human genes 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 8
- 235000014304 histidine Nutrition 0.000 claims description 6
- 101150077194 CAP1 gene Proteins 0.000 claims description 5
- 101100245221 Mus musculus Prss8 gene Proteins 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 239000001226 triphosphate Substances 0.000 claims description 3
- 102000002572 Alpha-Globulins Human genes 0.000 claims description 2
- 108010068307 Alpha-Globulins Proteins 0.000 claims description 2
- 102000006734 Beta-Globulins Human genes 0.000 claims description 2
- 108010087504 Beta-Globulins Proteins 0.000 claims description 2
- 101150014715 CAP2 gene Proteins 0.000 claims description 2
- 101710163595 Chaperone protein DnaK Proteins 0.000 claims description 2
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 claims description 2
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 claims description 2
- 102000002812 Heat-Shock Proteins Human genes 0.000 claims description 2
- 108010004889 Heat-Shock Proteins Proteins 0.000 claims description 2
- 101100260872 Mus musculus Tmprss4 gene Proteins 0.000 claims description 2
- 241001248479 Pseudomonadales Species 0.000 claims description 2
- 229930185560 Pseudouridine Natural products 0.000 claims description 2
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 claims description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 claims description 2
- OLRONOIBERDKRE-XUTVFYLZSA-N [[(2r,3s,4r,5s)-3,4-dihydroxy-5-(1-methyl-2,4-dioxopyrimidin-5-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 OLRONOIBERDKRE-XUTVFYLZSA-N 0.000 claims description 2
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 claims description 2
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 claims description 2
- 235000011178 triphosphate Nutrition 0.000 claims description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 150000002411 histidines Chemical class 0.000 claims 1
- 239000002777 nucleoside Substances 0.000 claims 1
- 125000003835 nucleoside group Chemical group 0.000 claims 1
- 229940126582 mRNA vaccine Drugs 0.000 abstract description 23
- 108700021021 mRNA Vaccine Proteins 0.000 abstract description 20
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 230000000069 prophylactic effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 28
- 241000699670 Mus sp. Species 0.000 description 24
- 238000002649 immunization Methods 0.000 description 19
- 230000003053 immunization Effects 0.000 description 19
- 238000000034 method Methods 0.000 description 16
- 238000010586 diagram Methods 0.000 description 14
- 108700026244 Open Reading Frames Proteins 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000002245 particle Substances 0.000 description 11
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
- 238000005538 encapsulation Methods 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 150000003904 phospholipids Chemical class 0.000 description 9
- 231100000111 LD50 Toxicity 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 101710116435 Outer membrane protein Proteins 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 230000001681 protective effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000002296 dynamic light scattering Methods 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 210000004379 membrane Anatomy 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 5
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 5
- 108010069584 Type III Secretion Systems Proteins 0.000 description 5
- NRLNQCOGCKAESA-KWXKLSQISA-N [(6z,9z,28z,31z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCC(OC(=O)CCCN(C)C)CCCCCCCC\C=C/C\C=C/CCCCC NRLNQCOGCKAESA-KWXKLSQISA-N 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 108020001507 fusion proteins Proteins 0.000 description 5
- 102000037865 fusion proteins Human genes 0.000 description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- -1 polyethylene Polymers 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 125000002091 cationic group Chemical group 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004945 emulsification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 3
- 101000941029 Homo sapiens Endoplasmic reticulum junction formation protein lunapark Proteins 0.000 description 3
- 108091092724 Noncoding DNA Proteins 0.000 description 3
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 3
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000004727 humoral immunity Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 101150106447 NUP gene Proteins 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 108010013381 Porins Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 125000000487 histidyl group Chemical class [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 102000007739 porin activity proteins Human genes 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940031626 subunit vaccine Drugs 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 229960000187 tissue plasminogen activator Drugs 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- JFBCSFJKETUREV-UHFFFAOYSA-N 1,2 ditetradecanoylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OCC(CO)OC(=O)CCCCCCCCCCCCC JFBCSFJKETUREV-UHFFFAOYSA-N 0.000 description 1
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical group O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 1
- 102000014133 Antimicrobial Cationic Peptides Human genes 0.000 description 1
- 108010050820 Antimicrobial Cationic Peptides Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108010060123 Conjugate Vaccines Proteins 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- ZRVUJXDFFKFLMG-UHFFFAOYSA-N Meloxicam Chemical compound OC=1C2=CC=CC=C2S(=O)(=O)N(C)C=1C(=O)NC1=NC=C(C)S1 ZRVUJXDFFKFLMG-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 208000032536 Pseudomonas Infections Diseases 0.000 description 1
- 241001240958 Pseudomonas aeruginosa PAO1 Species 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- CKNCMXJRFAUMEO-UHFFFAOYSA-N S(=O)(=O)(OC)OC.COC1=CC=CC2=[N+](C3=CC=CC=C3N=C12)C Chemical compound S(=O)(=O)(OC)OC.COC1=CC=CC2=[N+](C3=CC=CC=C3N=C12)C CKNCMXJRFAUMEO-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000907316 Zika virus Species 0.000 description 1
- 208000020329 Zika virus infectious disease Diseases 0.000 description 1
- 150000003869 acetamides Chemical class 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 229940031670 conjugate vaccine Drugs 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002951 depilatory effect Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 239000004815 dispersion polymer Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 231100000089 gene mutation induction Toxicity 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical group O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940124590 live attenuated vaccine Drugs 0.000 description 1
- 229940023012 live-attenuated vaccine Drugs 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960001929 meloxicam Drugs 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- JFFBOCCNFSWIPM-UHFFFAOYSA-N n-tetradecylacetamide Chemical compound CCCCCCCCCCCCCCNC(C)=O JFFBOCCNFSWIPM-UHFFFAOYSA-N 0.000 description 1
- 230000001937 non-anti-biotic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940023146 nucleic acid vaccine Drugs 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000009522 phase III clinical trial Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 229940126580 vector vaccine Drugs 0.000 description 1
- 229940023147 viral vector vaccine Drugs 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/04—Chelating agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
Definitions
- the present invention relates to the field of biomedicine, specifically to an mRNA vaccine encoding PcrV and/or OprF-I protein.
- Pseudomonas aeruginosa is a Gram-negative non-fermenting and aerobic opportunistic pathogen with diverse phenotypes, widely present in nature and human living environments, and often infects immune defense mechanisms Impaired patients such as cystic fibrosis (CF), sepsis and cancer patients.
- Pseudomonas aeruginosa is usually not pathogenic, but under certain conditions, it can cause chronic inflammation of secondary infection or mixed infection. It is one of the important pathogens of hospital infections and has the highest incidence rate in ICU wards, burns, and war wounds. One of the pathogenic bacteria. PA infection can occur in any tissue and part of the human body, and can also cause systemic infections such as endocarditis, pneumonia, and even sepsis. The systemic infection mortality rate exceeds 20%.
- PDR-PA Pan-drug-resistant Pseudomonas aeruginosa
- MDR-PA multi-drug-resistant Pseudomonas aeruginosa
- the vaccine targets include lipopolysaccharide (LPS), extracellular polysaccharide (EPS), flagella, outer membrane proteins ( outer membrane protein (OMP), bacterial toxins, outer membrane vesicles (outer membrane vesicle, OMV), etc.
- LPS lipopolysaccharide
- EPS extracellular polysaccharide
- OMV outer membrane vesicle
- mRNA is an intermediate messenger that transmits genetic information in DNA to proteins.
- mRNA vaccine treatment refers to synthesizing mRNA in vitro and introducing it into the body. After the mRNA enters the cells, it is translated and expressed into the antigen protein. The antigen stimulates the immune system in the body and activates humoral immunity and cellular immunity. When the body is exposed to the same antigen (cell/virus) again When it occurs, it can produce a rapid immune response and protect itself.
- the mRNA vaccine has achieved certain research results in various infectious diseases such as influenza virus, Ebola virus and Zika virus.
- the mRNA vaccine delivers the mRNA to cells and expresses it to produce proteins. This allows the body to gain immune protection.
- mRNA vaccines Compared with traditional vaccines (including inactivated vaccines, attenuated vaccines, subunit vaccines, viral vector vaccines), mRNA vaccines have the following main outstanding advantages: (1) mRNA vaccines can not only induce strong humoral immunity in host cells, but also activate Cellular immunity including cytotoxic T cells and specific recognition of pattern recognition receptors activate innate immunity; (2) mRNA vaccines have strong design, simple preparation process, can be quickly standardized and produced, and have a short development cycle; (3) mRNA vaccines The cost of vaccine research and development is highly controllable and the cost is relatively low; (4) The mRNA vaccine is highly safe and will undergo a natural degradation process after entering the body. Side effects can be more precisely controlled and there is no integration, induced gene mutation and exogenous viruses. Risk of infection.
- mRNA structural engineering such as 5' end cap structure, nucleotide modification, optimization of mRNA sequence, etc.
- the shortcomings of poor stability, low translation efficiency, and easy degradation of mRNA have been greatly improved, and the shortcomings of mRNA have been greatly improved.
- Application of therapy In recent years, mRNA has been widely used in cell programming and vaccine research, and has shown great application potential.
- mRNA vaccines are also used in the research of vaccines for various infectious diseases, such as the CMV vaccine and Zika vaccine of the American company Moderna, which have entered clinical research. There is currently no safe and effective vaccine against Pseudomonas aeruginosa.
- an mRNA molecule is provided, said mRNA molecule encoding at least one of PcrV protein and OprF-I protein;
- the PcrV protein contains the following amino acid sequence:
- the OprF protein contains the following amino acid sequence:
- the OprI protein contains the following amino acid sequence:
- lipid nanoparticles loaded with the mRNA molecules of the first aspect are provided.
- a protein comprising the amino acid sequence encoded by the mRNA described in the first aspect.
- a DNA molecule encoding the mRNA molecule described in the first aspect is provided.
- a recombinant plasmid containing the DNA molecule described in the fourth aspect is provided.
- a vaccine comprising the mRNA molecule described in the first aspect, the lipid nanoparticles described in the second aspect, the protein described in the third aspect, the protein described in the fourth aspect. DNA molecules or recombinant plasmids according to the fifth aspect.
- an antibody is provided, the antibody being induced and produced by the vaccine of the sixth aspect and isolated.
- the use of the mRNA molecule described in the first aspect in preparing a drug for treating and/or preventing diseases is provided.
- the mRNA vaccine encoding PcrV and/or OprF-I protein according to the above embodiments.
- the vaccine prepared from the mRNA designed in the present invention has excellent preventive and/or therapeutic effects on diseases caused by Pseudomonas aeruginosa.
- Figure 1 is a schematic diagram of the components of the mRNA vaccine
- Figure 2.1 is the map of plasmid pVAX1-PcrV in Example 1;
- Figure 2.2 is the map of plasmid pVAX1-OprF-I in Example 1;
- Figure 3 is the chemical structural formula of the Cap1 cap in Example 1;
- Figure 4 is a schematic diagram of T7 RNA polymerase transcription initiation in Example 1;
- Figures 5.1 and 5.2 are capillary electrophoresis analysis diagrams of the IVT transcript fragment in Example 1;
- Figure 6 is the structural formula of the compound raw materials for preparing mRNA-LNP in Example 1;
- Figure 7 is a diagram of the DLS test results of mRNA-pcrv-LNP in Example 1;
- Figure 8 is a diagram of the DLS test results of mRNA-OprF/I-LNP in Example 1;
- Figure 9 is a linear relationship diagram between mRNA concentration and fluorescence intensity in Example 1.
- Figure 10 is a diagram of the results of the mRNA-LNP cytotoxicity test in Example 1.
- Figure 11 is a photograph of the color development results of WB identification of cellular expression of mRNA in Example 1;
- Figure 12 is a diagram showing the expression and purification results of PcrV and OprF-I proteins in Example 1;
- Figure 13 is a schematic flow chart of mouse immunization and blood collection in Example 1;
- Figure 14.1 is a graph of total IgG antibody titers detected one week after the second immunization of mice with mRNA-PCRV-LNP (5, 25 ⁇ g) in Example 1;
- Figure 14.2 is a graph of total IgG antibody titers detected one week after the second immunization of mice with mRNA-OprF-I-LNP (5, 25 ⁇ g) in Example 1;
- Figure 14.3 is a diagram showing the IgM antibody titer detection results after 7 days of vaccine immunization in Example 1;
- Figure 14.4 is a graph showing the titer detection results of IgG subtypes (IgG1 and IgG2a) induced by mRNA-pcrv-LNP and mRNA-OprF-I-LNP in Example 1;
- Figure 15 is a schematic diagram of the construction process of the mouse burn model in Example 1;
- Figure 16 is a graph showing the results of the virus challenge experiment in Example 1.
- an open reading frame is the normal nucleotide sequence of a structural gene.
- the reading frame from the start codon to the stop codon can encode a complete polypeptide chain without interruption of translation. Stop codon.
- the amino acid sequence is from the N-terminus (ie, the end with a protruding amino group) to the C-terminus (ie, the end with a protruding carboxyl group).
- the N-terminus refers to the end of the amino acid sequence with a protruding amino group (-NH 2 )
- the C-terminus refers to the end of the amino acid sequence with a protruding carboxyl group (-COOH).
- nucleotide sequence is from the 5' end to the 3' end.
- the type III secretion system of Pseudomonas aeruginosa is a large syringe-like complex composed of more than 20 proteins. It can directly inject some virulence factors and other effector proteins into host cells, causing damage to host cells and thus leading to bacterial infectious diseases. play an important role in.
- PcrV is one of the important components of the type III secretion system of Pseudomonas aeruginosa. It can form homopolymers and form a "pipeline" for the secretion system to transport virulence factors and effector proteins. It is the type III secretion system of Pseudomonas aeruginosa. key components.
- this protein is an important target for therapeutic antibodies against P. aeruginosa infection.
- Anti-PcrV polyclonal antibodies and monoclonal antibodies have been proven to inhibit the secretion of the type III secretion system and protect the body from invasion by Pseudomonas aeruginosa.
- PcrV is also an important candidate vaccine molecule, but due to its ability to form homomultimers and other reasons, the expression and production of soluble recombinant proteins is limited.
- the present invention provides a nucleic acid vaccine encoding PcrV, especially an mRNA vaccine, which solves this problem well.
- the outer membrane protein of Pseudomonas aeruginosa is embedded in the outer membrane of PA lipopolysaccharide and phospholipid layer, and is a porin and other structural and functional components on the bacterial surface.
- OprF and OprI selected in the present invention are two important outer membrane proteins that are exposed on the PA surface and whose antigens are highly conserved.
- OprF is a non-specific channel pore on the surface of PA bacteria and is rich in ⁇ -helix structure. Participates in the virulence effect of PA by regulating the quorum sensing network.
- OprF can affect the formation of outer membrane vesicles by regulating Pseudomonas quinolone signaling (POS) levels.
- POS Pseudomonas quinolone signaling
- OprF can be used as a safe protective immunogen in all pathogenic and environmental strains of PA and is an important candidate for PA vaccine development.
- OprI is a lipoprotein in the inner monolayer of the outer membrane of PA cells and is rich in ⁇ -helical structure. OprI is a good carrier molecule for vaccination. It interacts with antigen-presenting cells (APC) and TLR2/4 of epithelial cells. The terminal lipid tail can trigger immune responses.
- APC antigen-presenting cells
- TLR2/4 antigen-presenting cells
- OprI is a new target of cationic antimicrobial peptides (AMP). It participates in the sensitivity of PA to hRNase7 and a-helical cationic AMP. It plays a role by inhibiting its antibacterial activity and increasing the permeability of bacterial cell membranes. It can be used for antibiotic resistance. Drug screening for drug-induced Pa infection is expected to be used in the development of new vaccines.
- AMP cationic antimicrobial peptides
- an mRNA molecule encoding at least one of the following proteins: 1) PcrV protein; 2) OprF protein and OprI protein;
- the PcrV protein contains the following amino acid sequence:
- the OprF protein contains the following amino acid sequence:
- the OprI protein contains the following amino acid sequence:
- the PcrV selected by the present invention is conservative and is a very ideal and important vaccine candidate antigen.
- amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are located in the same amino acid sequence. That is, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are located on the same amino acid sequence in a tandem manner.
- amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 can be expressed individually through independent nucleotides and expression vectors, or they can be expressed in the same sequence through tandem nucleotide sequences. expressed simultaneously in an expression vector. Simultaneous expression is preferred.
- amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are connected in series through a first linker sequence.
- the C-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the N-terminus of the first linker sequence
- the N-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the C-terminus of the first linker sequence.
- a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 8.
- the C-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the N-terminus of the first linker sequence, and the N-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the C-terminus of the first linker sequence.
- a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 9.
- the C-terminus of the amino acid sequence shown in SEQ ID No. 7 is sequentially connected in series with a second linker sequence and a tailing amino acid sequence.
- a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 7.
- a second linker sequence and a tailing amino acid sequence are sequentially concatenated at the C-terminal end of the amino acid sequence of the concatenated sequence.
- the first linker sequence contains glycine (G) and serine (S).
- the second linker sequence contains glycine and serine.
- the first linker sequence includes but is not limited to the following amino acid sequence: GSGSGSGSGS.
- the second linker sequence includes but is not limited to the following amino acid sequence: GGGS.
- the tailed amino acid sequence contains at least one histidine (H).
- the tailed amino acid sequence contains 6 to 12 histidines.
- the mRNA encoding the PcrV protein contains the following nucleotide sequence:
- the mRNA encoding OprF protein contains the following nucleotide sequence:
- the mRNA encoding the OprI protein contains the following nucleotide sequence:
- the mRNA molecule includes in order from the 5' end to the 3' end: a 5' cap structure, a 5'UTR (Untranslated Region, non-coding region) sequence, a nucleotide sequence encoding a signal peptide, and a PcrV encoding The nucleotide sequence, 3'UTR sequence, and polyadenylation sequence (PolyA) of the protein and/or OprF protein and OprI protein.
- a 5' cap structure a 5'UTR (Untranslated Region, non-coding region) sequence
- a nucleotide sequence encoding a signal peptide a signal peptide
- PcrV Polyadenylation sequence
- the 5' end cap structure includes but is not limited to any one of Cap0, Cap1, Cap2 and any other cap structure.
- the 5'UTR and 3'UTR sequences are independently derived from at least one of natural proteins and artificially synthesized proteins.
- natural proteins include, but are not limited to, any one of ⁇ -globulin, ⁇ -globulin, and heat shock protein HSP70.
- the mRNA molecule is unmodified or modified.
- the modification includes: at least one of pseudouridine triphosphate and N1-methylpseudouridine triphosphate modification.
- the PcrV protein includes the amino acid sequence shown in SEQ ID No. 1.
- the mRNA molecule encoding the PcrV protein includes the nucleotide sequence shown in SEQ ID No. 10.
- the OprF protein and OprI protein (i.e., OprF-I fusion protein) comprise the amino acid sequence shown in SEQ ID No. 6.
- the mRNA encoding the OprF protein and OprI protein includes the nucleotide sequence shown in SEQ ID No. 11.
- lipid nanoparticles (Lipid Nanoparticles, LNPs) loaded with the mRNA molecules described in the first aspect are provided.
- Lipid nanoparticles are a type of nanoparticle formed using lipids.
- the lipid nanoparticles include at least one of cationic lipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol-modified lipids.
- Ionized lipids are modified lipids. When LNP is endocytosed by cells, the ionized lipid will ionize in an acidic environment, allowing LNP to escape from the endosome. Ionizable cationic lipids are the most critical lipids and are a decisive factor in mRNA delivery and transfection efficiency.
- Neutral auxiliary phospholipids are generally saturated phospholipids, which can increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayer structures and stabilize their structural arrangement; cholesterol has strong membrane fusion properties and promotes intracellular uptake of mRNA. into the cytoplasm; PEG (polyethylene glycol) phospholipids are located on the surface of lipid nanoparticles to improve their hydrophilicity, avoid rapid clearance by the immune system, prevent particle aggregation, and increase stability.
- the cationic lipid includes but is not limited to 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester (DLin-MC3-DMA, CAS number: 1224606-06-7 ).
- neutral accessory phospholipids include, but are not limited to, distearoylphosphatidylcholine.
- polyethylene glycol-modified lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, 2-[(polyethylene glycol )-2000]-N,N tetradecyl acetamide, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG2000) or n-(carbonyl- At least one of methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine sodium salt, preferably 1,2-dimyristoyl-rac-glycerol -3-Methoxypolyethylene glycol 2000 (for the structural formula, see mPEG2000-DMG in Figure 6, CAS number: 160743-62-4), 2-[(polyethylene glycol)-2000]-N, N20 At least one of tetraalkyl acetamides.
- a protein comprising the amino acid sequence encoded by the mRNA described in the first aspect.
- a DNA molecule encoding the mRNA molecule described in the first aspect is provided.
- a recombinant plasmid containing the DNA molecule described in the fourth aspect is provided.
- a vaccine comprising the mRNA molecule described in the first aspect, the lipid nanoparticles described in the second aspect, the fusion protein described in the third aspect, the The DNA molecule or the recombinant plasmid of the fifth aspect.
- an antibody is provided, the antibody being induced and produced by the vaccine of the sixth aspect and isolated.
- the antibody includes but is not limited to at least one of IgG1 and IgG2a.
- the use of the mRNA molecule described in the first aspect in preparing a drug for treating and/or preventing diseases is provided.
- the disease includes a disease caused by bacteria.
- the bacteria include, but are not limited to, Pseudomonadales.
- the bacteria include, but are not limited to, Pseudomonas aeruginosa.
- the present invention provides an mRNA vaccine encoding PcrV, an important protein of the type III secretion system of Pseudomonas aeruginosa, and an OprF-I fusion protein in the outer membrane protein, delivered by lipid nanoparticles (LNP) for use in Pseudomonas aeruginosa Prevention and treatment of Pseudomonas infections.
- LNP lipid nanoparticles
- the present invention designs and synthesizes an mRNA encoding PcrV protein and OprF-I fusion protein, in which the cap of the mRNA is a Cap1 structure, obtained by the co-transcriptional capping method of the mRNA, and the natural guanine core in the mRNA All nucleotides are substituted with N1-methylpseudouridine.
- PcrV-mRNA and OprF-I-mRNA were packaged with LNP, three groups of blank, 10 ⁇ g, and 30 ⁇ g immunized Balb/c mice were set to evaluate the immune response of the vaccine.
- mice After the mice completed two immunizations, the Pseudomonas aeruginosa PAO1 strain was used. A challenge test was conducted on burnt mice to evaluate the protective effect of the vaccine. Experimental results show that mRNA can be transcribed with high quality and high-level protein expression can be achieved at the cellular level; the LNP encapsulating the mRNA has a uniform particle size and a high encapsulation rate, and immunized mice can induce high-titer antigen-specific antibodies; challenge the virus The test showed that the protective effect (i.e. mouse survival rate) of the PcrV-mRNA vaccine against the 50xLD 50 challenge dose was as high as 100%. The protective effect of OprF-I-mRNA vaccine can reach 50% to 66.7%.
- FIG. 1 is a schematic diagram of the constituent elements of the mRNA vaccine.
- the constituent elements of the mRNA drug and vaccine include in order from 5' to 3': 5' end cap structure, KOZAK sequence, 5' non-coding region (5 'UTR), open reading frame (Open Reading Frame, ORF), 3' non-coding region (3'UTR) and polyadenine tail (PolyA).
- 5' end cap structure 5' end cap structure
- KOZAK sequence 5' non-coding region
- 5 'UTR open reading frame
- 3'UTR open reading frame
- PolyA polyadenine tail
- Sequence 1 is the amino acid sequence encoded by the ORF of mRNA. Add 5' UTR and KOZAK sequence (sequence 2) to the 5' end of the ORF, and add 3' UTR and poly(A) sequence to the 3' end.
- the above gene fragment was synthesized by Jinweizhi Company and cloned into the pVAX1 vector to obtain the template plasmid pVAX1-PcrV.
- Figure 2.1 shows the map of plasmid pVAX1-PcrV
- Figure 2.2 shows the map of plasmid pVAX1-OprF-I.
- the signal peptide sequence is the signal peptide sequence of TPA protein (Sequence 3); the sequence of 5’UTR is shown in Sequence 4; the 3’UTR is shown in Sequence 5; the length of PolyA is 80 to 150.
- the sequence marked with thick underline is the signal peptide sequence.
- the "AUG” at the end of the Kozak sequence is the start codon of the ORF region.
- Tissue Plasminogen Activator (TPA) sequence (sequence 3) is as follows:
- the 5’UTR sequence (sequence 4) is as follows:
- the 3’UTR sequence (sequence 5) is as follows:
- amino acid sequence 6 encoded by the ORF region of mRNA-OprF-I is as follows:
- sequence 6 the bold part is the linker sequence (Linker).
- the sequence marked with bold underline is the signal peptide sequence.
- the mRNA sequence of PcrV is as follows:
- 5'UTR italics; Kozak sequence: ORF: bold; 3'UTR: double underline; PolyA: underline wavy line.
- mRNA sequence encoding signal peptide thick underline.
- mRNA sequence encoding histidine tag dashed underline.
- the mRNA sequence of OprF-I is as follows:
- 5'UTR italics; Kozak sequence: ORF: boldface; 3'UTR: double underline; PolyA: underlined wavy line; (GS)5 connection sequence: dotted underline.
- mRNA encoding signal peptide thick underline.
- mRNA sequence encoding histidine tag dashed underline.
- pVAX1-PcrV plasmid and pVAX1-OprF-I plasmid were transformed into DH5 ⁇ competent cells, spread on solid LB plates containing Kan+ (kanamycin) resistance, and cultured at 37°C for 12 to 15 hours. Then single colonies were picked and added to In Kan+-resistant liquid LB medium, the cells were cultured at 37°C and 250 rpm for 12 to 15 hours, then centrifuged to collect the cells, and a commercial plasmid extraction kit was used to extract the amplified plasmid.
- the linearized restriction site of the amplified plasmid was selected to be XhoI immediately adjacent to the PolyA tail.
- the degree of linearization was detected by DNA agarose gel electrophoresis.
- the linearized plasmid DNA was recovered by a PCR product recovery kit.
- the DNA concentration and quality were determined by NanoDrop ultra-micro volume.
- the values of OD260 and OD260/OD280 were determined by nucleic acid instrument and calculated.
- RNA In vitro synthesis of RNA (IVU) uses DNA (linearized plasmid or PCR product) containing the U7 promoter (UAAUACGACUCACUAUAGGG) or SP6 promoter (AUUUAGGUGACACUAUAG) sequence as a template, and uses NUP as the base under the action of U7 or SP6 RNA polymerase. It synthesizes an mRNA that is complementary to one strand of the template DNA, and enhances the stability of the mRNA by adding a cap structure to the 5' end and a polyA tail to the 3' end.
- U7 promoter UATACGACUCACUAUAGGG
- SP6 promoter AUUUAGGUGACACUAUAG
- the capping process of mRNA can use co-transcriptional capping or post-transcriptional capping, and the tailing process can use template transcription or post-transcriptional enzymatic tailing.
- the capping and tailing methods are co-transcriptional capping and template tailing respectively, and the 5' cap structure in this example is m7GpppN ( Figure 3).
- cotranscriptional capping cap analogs are added directly to the IVU, and their incorporation at the 5'-end by an RNA polymerase with relaxed substrate specificity directly produces the corresponding 5'-capped mRNA. Since the cap analogue lacks free 5'-triphosphate, no internal incorporation of the cap analogue occurs during IVU.
- Urilinker's ReagenU AG (3'OMe) is a cap structure ( Figure 3).
- Figure 4 shows a schematic diagram of the transcription initiation of U7 RNA polymerase. Under the action of U7 polymerase, transcription starts with 5'AG 3'.
- the specific transcription reaction is set according to the ratio of each component in Table 1.
- the steps are as follows: (1) Add RNase free waUer and NUPs; (2) Add Mix ReagenU AG (3'OMe) and centrifuge to collect the liquid; (3) Add 10X UranscripUion Buffer, mix and centrifuge to collect the liquid; (4) Add linearized DNA template; (5) Add Murine RNase InhibiUor, YeasU Inorganic PyrophosphaUase and U7 RNA Polymerase, mix well and centrifuge to collect the liquid; (6) React at 37°C for 2 to 3 hours. After the reaction, DNAaseI was added to remove the template DNA, and the transcript was purified using Monarch RNA cleanup kiU (NEB).
- the concentration of mRNA was calculated by the reading of Nano drop UV spectrophotometer, and the purity of mRNA was determined by capillary electrophoresis instrument 2100 Bioanalyzer.
- the results shown in Figures 5.1 and 5.2 show that the size of the in vitro transcribed mRNA molecules is in line with expectations, and the integrity and purity are high.
- the structural formula of the compound raw materials used in this step is shown in Figure 6.
- the lipid nanoparticles loaded with mRNA contain four components, namely ionizable cationic phospholipids (ionizable lipids), neutral auxiliary phospholipids, cholesterol (CholesUerol), and polyethylene glycol. Diol-modified phospholipid (PEGylaUed lipid).
- LNP LNP-containing lipid
- DLin-MC3-DMA ionizable lipid
- DSPC Distearoylphosphatidylcholine
- 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 for the structural formula, see mPEG2000-DMG in Figure 6, CAS number: 160743-62 -4)
- the particle size and uniformity of LNP were measured using dynamic light scattering (DLS).
- the specific operation is as follows: dilute the concentrated sample again in sterile PBS at a volume ratio of 1:100, use HORIBA-SZ100 equipment at dispersion angles of 25° and 90°, repeat three times, and obtain the particle size distribution and PDI value of LNP (Polymer dispersiUy index, polymer dispersion index). Particle size results are given as a ratio of particle size to intensity and also predict the stability of the LNP dispersion system.
- the DLS test results of mRNA-pcrv-LNP shown in Figure 7 show that the average particle size of LNP is 91.1nm and the PDI index is 0.237. This result shows that the prepared LNP has a uniform particle size distribution and a peak intensity of 91%, indicating that the surface is extremely Small amounts of nanoparticles aggregate.
- the DLS test results of mRNA-OprF-I-LNP shown in Figure 8 show that the average particle size of LNP is 110.9nm and the PDI index is 0.214. This result shows that the prepared LNP has a uniform particle size distribution and a peak intensity of 95.1%, indicating that A very small amount of nanoparticles aggregates on the surface.
- QuanU-iU TM RiboGreen TM RNA kit The encapsulation efficiency of mRNA was measured using QuanU-iU TM RiboGreen TM RNA kit.
- the measurement principle is: QuanU-iU TM RNA reagent is an ultra-sensitive fluorescent nucleic acid stain that can detect 1 to 200ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only free nucleic acids that are not encapsulated by LNP can be bound.
- UriUon-100 as a surfactant, is often used as a demulsifier. Using 1% UriUon-100 to treat the LNP-mRNA obtained can release the entrapped nucleic acid and obtain the total nucleic acid amount.
- the drug loading capacity can be obtained by calculating the difference in the amount of nucleic acid before and after emulsification, and then divided by the total nucleic acid amount to obtain the encapsulation rate.
- the formula is as follows:
- Encapsulation rate (%) (quantitative quantity after emulsification - quantitative quantity before emulsification)/quantitative quantity after emulsification.
- the specific operation method is as follows: Prepare rRNA standard solutions of different concentrations (i.e. 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0ng/mL) in UE (Uris-EDUA) buffer.
- the mRNA LNP to be measured is dissolved in UEbuffer to prepare a sample of approximately 250ng/mL mRNA. Similar samples were also prepared in UE buffer supplemented with UriUon-X100 surfactant (0.5%).
- mRNA-PcrV and mRNA-OprF-I were delivered by LNP and lip3000.
- the method of transfection of mRNA-LNP is to directly add LNPs encapsulating mRNA-PcrV and mRNA-OprF-I respectively to 293U cells that have grown to about 70%.
- the concentration of mRNA is about 0.5 ⁇ g/cm 2 .
- the lip3000 transfection method is carried out according to the instructions. 293U cells that can grow stably are selected and plated in six-well plates. Transfection is performed when the cells grow to 70-80%.
- solutions A and B first prepare solutions A and B; solution A: dilute 4 ⁇ g mRNA with 200 ⁇ L OpUi-MEM; solution B: dilute 10 ⁇ L lipo3000 (i.e. lipofeUamin3000) with 200 ⁇ L OpUi-MEM, and mix solutions A and B gently respectively. , let stand for 5 minutes, add liquid B to liquid A, mix gently, and let stand at room temperature for 20 minutes. While the mixture of solutions A and B is allowed to stand for 20 minutes, clean the six-well plate, aspirate the culture medium in the culture dish, and wash once with PBS or serum-free medium. After the mixture of A and B has been allowed to stand, add the transfection reagent to each well for 48 hours.
- solution A dilute 4 ⁇ g mRNA with 200 ⁇ L OpUi-MEM
- solution B dilute 10 ⁇ L lipo3000 (i.e. lipofeUamin3000) with 200 ⁇ L OpUi-MEM
- HEK 293U cells were cultured adherently and different doses of mRNA were added to observe the cytotoxicity of mRNA-LNPs. Briefly, 4 to 6 hours after cells were seeded in a 96-well plate, PBS, blank LNP, and mRNA-loaded LNPLNP-pcrV and LNP-OprF-I were added. After 72 hours of addition, 10 ⁇ LCCK8 was added to each well and cultured for 0.5 to 4 hours. CKK8 reagent contains WSU-8.
- WSU-8 The chemical name of WSU-8 is as follows: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 -Benzene disulfonate)-2H-tetrazole monosodium salt. It is reduced to a highly water-soluble yellow formazan product (Formazan) by dehydrogenase in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-MeUhoxy PMS). dye), the amount of formazan produced is proportional to the number of viable cells.
- the results in Figure 10 show that blank LNP has slight cytotoxicity, and the mRNA-loaded LNP is basically similar to blank LNP.
- the cell culture supernatant and cell lysate were added to 6 ⁇ loading buffer and then subjected to polyacrylamide gel electrophoresis in a boiling water bath for 5 minutes; gel electrophoresis was carried out to transfer the protein to PVDF; blocked with 1% BSA at room temperature for 60 minutes, washed 3 times with PBSU; The anUi-His-HRP antibody was incubated at room temperature for 2 hours, washed three times with UBSU, developed with an enhanced chemiluminescence kit (ECL), and scanned and photographed.
- ECL enhanced chemiluminescence kit
- Figure 11 shows a photograph of the color development results. It can be seen that whether PcrV-mRNA and mRNA-OprF-I are delivered using lipofeUamin3000 or LNP, they can express the target protein at high levels after transfection into cells and can secrete it efficiently. to the outside of the cell.
- the PcrV and OprF-I protein coding genes were cloned into the pEU21a expression vector (InviUrogen) with a C-terminal 6 ⁇ His tag and transformed into E. coli BL 21 (DE 3 ) pLysS competent cells.
- the bacterial cells collected by centrifugation were crushed using a high-pressure homogenizer and centrifuged at high speed. The supernatant obtained was first crudely purified through a NUA-Ni affinity chromatography column, and then purified through size exclusion chromatography.
- Figure 12 shows the SDS-PAGE image of the protein. It can be seen that both PcrV and OprF-I proteins have been prepared with high purity and high yield.
- the schematic diagram of the mouse immunization and blood collection process is shown in Figure 13.
- PcrV-mRNA-LNP, mRNA-OprF-I-LNP and blank LNP were used to immunize balb/c mice through intramuscular injection.
- the immunization doses were set at 5 and 25 ⁇ g.
- the immunization volume to 100 ⁇ L.
- the second booster immunization is carried out 3 weeks after the first immunization.
- the dose, volume and method of immunization are the same as those of the first immunization.
- Blood was collected from the orbital venous plexus at 1, 3, and 5 weeks after the initial immunization, and serum samples were separated for testing.
- Enzyme-linked immunosorbent assay to determine mouse-specific antibody titer
- the specific antibody titer was measured using the indirect ELISA method.
- the specific steps are as follows: Coat the blank enzyme plate with PcrV and OprF expressed in E. coli, and coat overnight at 4°C. solution; wash with PBSU 3 times, add 1% BSA to each well and block for 2 hours at 37°C.
- Figures 14.1 and 14.2 respectively show the total IgG antibody titer detected one week after the second immunization of mice with mRNA-pcrv-LNP (5, 25 ⁇ g) and mRNA-OprF-I-LNP (5, 25 ⁇ g), each dose The group repeated the experiment 2 times.
- the abscissa is the dilution factor of mouse immune serum
- the ordinate is the reading of OD450.
- Each column in Figure 14.1 from left to right is mRNA-pcrv-LNP-5 ⁇ g-1, mRNA-pcrv- LNP--2, mRNA-pcrv-LNP-25 ⁇ g-1, mRNA-pcrv-LNP-25 ⁇ g-2, blank (blank control group) group
- each column in Figure 14.2 is mRNA-OprF from left to right.
- Figure 14.3 shows the IgM antibody detection results 7 days after vaccine immunization. On the basis of detecting the total IgG titer of the antibody, the subtypes of the antibodies were further analyzed.
- the results in Figure 14.4 show that the levels of IgG1 and IgG2a antibodies induced by mRNA-pcrv-LNP were significantly higher than those induced by mRNA-OprF-I-LNP. IgG1 and IgG2a antibody levels.
- experiments show that mice immunized with the mRNA vaccine prepared in this example can induce effective humoral immunity.
- FIG. 15 shows a schematic diagram of the construction process of mouse burn model.
- BALB/c mice were immunized twice and then separated by one month. All mice (immune group and unimmunized group, weight 20-30g) were first anesthetized with 250 ⁇ L 2.5% AverUin, and then used depilatory cream to remove hair on the right side of the waist and back of the body. Finally, the mice were 22mm in diameter, 100mm in length, A metal block weighing 165 g was heated to 104°C and burned on the animal's shaved area for 8 s to produce 3rd degree burns. Immediately thereafter, mice received intraperitoneal injections of 500 ⁇ L of 0.9% saline and 40 ⁇ L of meloxicam (1 mg/kg) every 24 h to prevent them from being irritated and feeling pain.
- the median lethal dose refers to the minimum number of bacteria that can cause the death of 50% of experimental animals under certain conditions.
- the determination method is as follows: first, measure the bacterial concentration by using a nanodrop micro-volume spectrophotometer to measure the OD600 reading, and then divide different concentrations (10 ⁇ 2, 10 ⁇ 3, 10 ⁇ 4, 5 ⁇ 10 ⁇ 4, 5x10 ⁇ 5, 10 ⁇ 5, 10 ⁇ 6, 10 ⁇ 7, 10 ⁇ 8) Pseudomonas aeruginosa strains (PAO1) were injected subcutaneously in The burn center of all mice was statistically analyzed for mouse death. Finally, SPSS software was used to analyze the results and determine the LD50, 2 ⁇ LD50, 5 ⁇ LD50, and 10 ⁇ LD50 doses. The test was repeated three times.
- the protective effect of the vaccine was evaluated through a challenge experiment with Pseudomonas aeruginosa.
- the specific implementation method is as follows: on the 14th day after the second injection of the mRNA vaccine, in mice (vaccine immunization group and control group ) In the burn center, Pseudomonas aeruginosa strain (PAO1) was subcutaneously injected at a dose of 50 ⁇ LD50, and the wounds and death of the mice were observed and recorded. For dead mice, the mice were dissected to remove various organs, and the amount of bacteria carried was measured.
- the results of the challenge experiment in Figure 16 show that compared with all the mice in the LNP blank immunization control group, none of the mice immunized with 5 ⁇ g and 25 ⁇ g of PcrV-mRNA vaccine in advance died, that is, the survival rate was 100%, showing that the vaccine has 100 % protective effect.
- the survival rate of mice in the 5 ⁇ g group of mice immunized in advance with the mRNA-OprF-I vaccine reached 50%, and the survival rate of mice in the 25 ⁇ g group of vaccine reached 66.67%.
Abstract
The present invention provides an mRNA vaccine encoding the protein(s) PcrV and/or OprF-I. The mRNA molecule encodes at least one of 1) the protein PcrV; and 2) the protein OprF and the protein OprI. The vaccine prepared from the mRNA designed by the present invention has an excellent prophylactic and/or therapeutic effect on diseases caused by Pseudomonas aeruginosa.
Description
本发明涉及生物医学领域，具体涉及编码PcrV和/或OprF-I蛋白的mRNA疫苗。The present invention relates to the field of biomedicine, specifically to an mRNA vaccine encoding PcrV and/or OprF-I protein.
铜绿假单胞菌(Pseudomonas aeruginosa，PA)是一种革兰氏阴性非发酵和需氧的机会性致病菌，其表型多样，广泛存在于自然界和人类生活环境中，常感染免疫防御机制受损的患者，如囊性纤维化(CF)、脓毒症和肿瘤病人。铜绿假单胞菌通常不致病，但在特定条件下，可引起继发感染或混合感染的慢性炎症，是医院感染的重要病原菌之一，也是ICU病房、烧伤、战创伤等发病率最高的病原菌之一。PA感染可以发生再人体任何组织和部位，也可引起心内膜炎、肺炎甚至败血症等全身感染，全身感染死亡率超过20％。Pseudomonas aeruginosa (PA) is a Gram-negative non-fermenting and aerobic opportunistic pathogen with diverse phenotypes, widely present in nature and human living environments, and often infects immune defense mechanisms Impaired patients such as cystic fibrosis (CF), sepsis and cancer patients. Pseudomonas aeruginosa is usually not pathogenic, but under certain conditions, it can cause chronic inflammation of secondary infection or mixed infection. It is one of the important pathogens of hospital infections and has the highest incidence rate in ICU wards, burns, and war wounds. One of the pathogenic bacteria. PA infection can occur in any tissue and part of the human body, and can also cause systemic infections such as endocarditis, pneumonia, and even sepsis. The systemic infection mortality rate exceeds 20%.
当前，由于抗生素滥用等原因，PA的耐药问题日益严重，出现了泛耐药铜绿假单胞(PDR-PA)和多重耐药铜绿假单胞菌(MDR-PA)，且耐药性PA的分离率逐年上升。由于抗生素耐药和新抗生素开发缓慢，寻找新的“非抗生素疗法”迫在眉睫，研制开发出安全有效的疫苗是一种重要的策略。从20世纪60年代起，至少有60种针对铜绿假单胞菌的疫苗在研，疫苗靶点包括脂多糖(lipopolysaccharide，LPS)、胞外多糖(extracellular polysaccharide，EPS)、鞭毛、外膜蛋白(outer membrane protein，OMP)、细菌毒素、外膜囊泡(outer membrane vesicle，OMV)等。根据技术路线不同，可分为组分疫苗、亚单位疫苗、减毒活疫苗、全菌体灭活疫苗、载体疫苗。迄今为止，有四个疫苗进入了Ⅲ期临床试验，分别是辉瑞公司开发的七价脂多糖疫苗、IMMUNO公司开发的A型和B型二价鞭毛疫苗、瑞士Serum and Vaccine Institute研发的八价脂多糖-内毒素A结合疫苗以及Valneva Austria GmbH公司研制的以铜绿假单胞菌外膜蛋白OprF190-342和OprI21-83融合蛋白为抗原的疫苗IC43，但这四个疫苗均宣告失败，目前尚无铜绿假单胞菌疫苗获批上市。At present, due to antibiotic abuse and other reasons, the drug resistance problem of PA is becoming increasingly serious. Pan-drug-resistant Pseudomonas aeruginosa (PDR-PA) and multi-drug-resistant Pseudomonas aeruginosa (MDR-PA) have emerged, and drug-resistant PA The separation rate is increasing year by year. Due to antibiotic resistance and the slow development of new antibiotics, it is urgent to find new "non-antibiotic treatments" and developing safe and effective vaccines is an important strategy. Since the 1960s, at least 60 vaccines against Pseudomonas aeruginosa have been developed. The vaccine targets include lipopolysaccharide (LPS), extracellular polysaccharide (EPS), flagella, outer membrane proteins ( outer membrane protein (OMP), bacterial toxins, outer membrane vesicles (outer membrane vesicle, OMV), etc. According to different technical routes, they can be divided into component vaccines, subunit vaccines, live attenuated vaccines, whole cell inactivated vaccines, and vector vaccines. So far, four vaccines have entered phase III clinical trials, namely the heptavalent lipopolysaccharide vaccine developed by Pfizer, the type A and type B bivalent flagellar vaccines developed by IMMUNO, and the eight-valent lipopolysaccharide vaccine developed by the Swiss Serum and Vaccine Institute. The polysaccharide-endotoxin A conjugate vaccine and the vaccine IC43 developed by Valneva Austria GmbH with Pseudomonas aeruginosa outer membrane protein OprF190-342 and OprI21-83 fusion protein as antigens, but these four vaccines have failed, and there is currently no Pseudomonas aeruginosa vaccine approved for marketing.
mRNA(Messenger RNA、信使RNA)是将DNA中的基因信息传递到蛋白的中间信使。mRNA疫苗治疗是指在体外合成mRNA，将其导入体内，mRNA进入细胞后翻译表达出抗原蛋白，抗原刺激体内免疫***，激活体液免疫和细胞免疫，当机体再次接触同种抗原(细胞/病毒)时，可产生快速免疫应答反应，自我保护。mRNA (Messenger RNA) is an intermediate messenger that transmits genetic information in DNA to proteins. mRNA vaccine treatment refers to synthesizing mRNA in vitro and introducing it into the body. After the mRNA enters the cells, it is translated and expressed into the antigen protein. The antigen stimulates the immune system in the body and activates humoral immunity and cellular immunity. When the body is exposed to the same antigen (cell/virus) again When it occurs, it can produce a rapid immune response and protect itself.
近年来RNA分子领域相关技术突破性进展，mRNA疫苗在流感病毒、埃博拉病毒和寨卡病毒等多种传染病上取得了一定的研究成果，mRNA疫苗将mRNA传递至细胞，表达产生蛋白，从而使机体获得免疫保护。mRNA疫苗相对于传统疫苗(包括灭活疫苗、减毒疫苗、亚单位疫苗、病毒载体疫苗)有如下主要突出的优点：(1)mRNA疫苗既能诱导宿主细胞产生强烈的体液免疫，还能激活细胞毒性T细胞在内的细胞免疫和通过模式识别受体的特异性识别来激活先天免疫；(2)mRNA疫苗设计性强，制备流程简便，可以快速标准化生产，研发周期短；(3)mRNA疫苗研发成本可控性强，成本相对较低；(4)mRNA疫苗安全性高，进入体内后会经历自然降解过程，副作用能得到更精确控制，不存在整合、诱导基因突变和外源性病毒感染的风险。此外，伴随着mRNA结构工程的发展，例如5'端帽子结构、核苷酸的修饰、mRNA序列的优化等，极大地改善了mRNA的稳定性差、翻译效率低、易降解的缺点，促进了mRNA疗法的应用。近年来，mRNA已被广泛应用于细胞编程和疫苗的研究中，并且显示出巨大的应用潜力。同时，mRNA疫苗也应用于多种传染病疫苗的研究，如美国Moderna公司的CMV疫苗、寨卡疫苗等均已进入临床研究。目前尚无安全有效的铜绿假单胞菌疫苗。In recent years, breakthroughs have been made in related technologies in the field of RNA molecules. The mRNA vaccine has achieved certain research results in various infectious diseases such as influenza virus, Ebola virus and Zika virus. The mRNA vaccine delivers the mRNA to cells and expresses it to produce proteins. This allows the body to gain immune protection. Compared with traditional vaccines (including inactivated vaccines, attenuated vaccines, subunit vaccines, viral vector vaccines), mRNA vaccines have the following main outstanding advantages: (1) mRNA vaccines can not only induce strong humoral immunity in host cells, but also activate Cellular immunity including cytotoxic T cells and specific recognition of pattern recognition receptors activate innate immunity; (2) mRNA vaccines have strong design, simple preparation process, can be quickly standardized and produced, and have a short development cycle; (3) mRNA vaccines The cost of vaccine research and development is highly controllable and the cost is relatively low; (4) The mRNA vaccine is highly safe and will undergo a natural degradation process after entering the body. Side effects can be more precisely controlled and there is no integration, induced gene mutation and exogenous viruses. Risk of infection. In addition, with the development of mRNA structural engineering, such as 5' end cap structure, nucleotide modification, optimization of mRNA sequence, etc., the shortcomings of poor stability, low translation efficiency, and easy degradation of mRNA have been greatly improved, and the shortcomings of mRNA have been greatly improved. Application of therapy. In recent years, mRNA has been widely used in cell programming and vaccine research, and has shown great application potential. At the same time, mRNA vaccines are also used in the research of vaccines for various infectious diseases, such as the CMV vaccine and Zika vaccine of the American company Moderna, which have entered clinical research. There is currently no safe and effective vaccine against Pseudomonas aeruginosa.
发明内容Contents of the invention
根据第一方面，在一实施例中，提供一种mRNA分子，所述mRNA分子编码PcrV蛋白、OprF-I蛋白中的至少一种；According to the first aspect, in one embodiment, an mRNA molecule is provided, said mRNA molecule encoding at least one of PcrV protein and OprF-I protein;
自N端至C端，所述PcrV蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the PcrV protein contains the following amino acid sequence:
自N端至C端，所述OprF蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprF protein contains the following amino acid sequence:
自N端至C端，所述OprI蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprI protein contains the following amino acid sequence:
根据第二方面，在一实施例中，提供负载有第一方面所述mRNA分子的脂质纳米颗粒。According to the second aspect, in one embodiment, lipid nanoparticles loaded with the mRNA molecules of the first aspect are provided.
根据第三方面，在一实施例中，提供一种蛋白，所述蛋白包含第一方面所述mRNA编码得到的氨基酸序列。According to the third aspect, in one embodiment, a protein is provided, the protein comprising the amino acid sequence encoded by the mRNA described in the first aspect.
根据第四方面，在一实施例中，提供一种编码第一方面所述mRNA分子的DNA分子。According to a fourth aspect, in one embodiment, a DNA molecule encoding the mRNA molecule described in the first aspect is provided.
根据第五方面，在一实施例中，提供含有第四方面所述DNA分子的重组质粒。According to the fifth aspect, in one embodiment, a recombinant plasmid containing the DNA molecule described in the fourth aspect is provided.
根据第六方面，在一实施例中，提供一种疫苗，所述疫苗包含第一方面所述mRNA分子、第二方面所述脂质纳米颗粒、第三方面所述蛋白、第四方面所述DNA分子或第五方面所述重组质粒。According to the sixth aspect, in one embodiment, a vaccine is provided, the vaccine comprising the mRNA molecule described in the first aspect, the lipid nanoparticles described in the second aspect, the protein described in the third aspect, the protein described in the fourth aspect. DNA molecules or recombinant plasmids according to the fifth aspect.
根据第七方面，在一实施例中，提供一种抗体，所述抗体是由第六方面所述疫苗诱导产生并分离获得的。According to the seventh aspect, in one embodiment, an antibody is provided, the antibody being induced and produced by the vaccine of the sixth aspect and isolated.
根据第八方面，在一实施例中，提供第一方面所述mRNA分子在制备治疗和/或预防疾病的药物中的用途。According to the eighth aspect, in one embodiment, the use of the mRNA molecule described in the first aspect in preparing a drug for treating and/or preventing diseases is provided.
依据上述实施例的编码PcrV和/或OprF-I蛋白的mRNA疫苗。本发明设计的mRNA制得的疫苗对铜绿假单胞菌引起的疾病具有优异的预防和/或治疗效果。The mRNA vaccine encoding PcrV and/or OprF-I protein according to the above embodiments. The vaccine prepared from the mRNA designed in the present invention has excellent preventive and/or therapeutic effects on diseases caused by Pseudomonas aeruginosa.
图1为mRNA疫苗的构成元件示意图；Figure 1 is a schematic diagram of the components of the mRNA vaccine;
图2.1为实施例1中的质粒pVAX1-PcrV图谱；Figure 2.1 is the map of plasmid pVAX1-PcrV in Example 1;
图2.2为实施例1中的质粒pVAX1-OprF-I图谱；Figure 2.2 is the map of plasmid pVAX1-OprF-I in Example 1;
图3为实施例1中Cap1帽子的化学结构式；Figure 3 is the chemical structural formula of the Cap1 cap in Example 1;
图4为实施例1中T7RNA聚合酶转录起始示意图；Figure 4 is a schematic diagram of T7 RNA polymerase transcription initiation in Example 1;
图5.1、5.2为实施例1中的IVT转录片段的毛细管电泳分析图；Figures 5.1 and 5.2 are capillary electrophoresis analysis diagrams of the IVT transcript fragment in Example 1;
图6为实施例1中制备mRNA-LNP的化合物原料的结构式；Figure 6 is the structural formula of the compound raw materials for preparing mRNA-LNP in Example 1;
图7为实施例1中mRNA-pcrv-LNP的DLS测试结果图；Figure 7 is a diagram of the DLS test results of mRNA-pcrv-LNP in Example 1;
图8为实施例1中mRNA-OprF/I-LNP的DLS测试结果图；Figure 8 is a diagram of the DLS test results of mRNA-OprF/I-LNP in Example 1;
图9为实施例1中mRNA浓度和荧光强度之间的线性关系图；Figure 9 is a linear relationship diagram between mRNA concentration and fluorescence intensity in Example 1;
图10为实施例1中mRNA-LNP细胞毒性测试结果图；Figure 10 is a diagram of the results of the mRNA-LNP cytotoxicity test in Example 1;
图11为实施例1中WB鉴定mRNA的细胞表达的显色结果拍照图；Figure 11 is a photograph of the color development results of WB identification of cellular expression of mRNA in Example 1;
图12为实施例1中PcrV和OprF-I蛋白的表达纯化结果图；Figure 12 is a diagram showing the expression and purification results of PcrV and OprF-I proteins in Example 1;
图13为实施例1中小鼠免疫和采血流程示意图；Figure 13 is a schematic flow chart of mouse immunization and blood collection in Example 1;
图14.1为实施例1中mRNA-pcrv-LNP(5，25μg)第二次免疫小鼠后一周检测的总IgG抗体滴度图；Figure 14.1 is a graph of total IgG antibody titers detected one week after the second immunization of mice with mRNA-PCRV-LNP (5, 25 μg) in Example 1;
图14.2为实施例1中mRNA-OprF-I-LNP(5，25μg)第二次免疫小鼠后一周检测的总IgG抗体滴度图；Figure 14.2 is a graph of total IgG antibody titers detected one week after the second immunization of mice with mRNA-OprF-I-LNP (5, 25 μg) in Example 1;
图14.3为实施例1中疫苗免疫7天后的IgM抗体效价检测结果图；Figure 14.3 is a diagram showing the IgM antibody titer detection results after 7 days of vaccine immunization in Example 1;
图14.4为实施例1中mRNA-pcrv-LNP、mRNA-OprF-I-LNP所诱导的IgG亚型(IgG1和IgG2a)效价检测结果图；Figure 14.4 is a graph showing the titer detection results of IgG subtypes (IgG1 and IgG2a) induced by mRNA-pcrv-LNP and mRNA-OprF-I-LNP in Example 1;
图15为实施例1中小鼠烧伤模型构建流程示意图；Figure 15 is a schematic diagram of the construction process of the mouse burn model in Example 1;
图16为实施例1中的攻毒实验结果图。Figure 16 is a graph showing the results of the virus challenge experiment in Example 1.
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本申请能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。在某些情况下，本申请相关的一些操作并没有在说明书中显示或者描述，这是为了避免本申请的核心部分被过多的描述所淹没，而对于本领域技术人员而言，详细描述这些 相关操作并不是必要的，他们根据说明书中的描述以及本领域的一般技术知识即可完整了解相关操作。The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many details are described in order to make the present application better understood. However, those skilled in the art can readily recognize that some of the features may be omitted in different situations, or may be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core part of the present application being overwhelmed by excessive descriptions. For those skilled in the art, it is difficult to describe these in detail. The relevant operations are not necessary, and they can fully understand the relevant operations based on the descriptions in the instructions and general technical knowledge in the field.
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。Additionally, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. At the same time, each step or action in the method description can also be sequentially exchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the description and drawings are only for clearly describing a certain embodiment, and do not imply a necessary sequence, unless otherwise stated that a certain sequence must be followed.
本文中为部件所编序号本身，例如“第一”、“第二”等，仅用于区分所描述的对象，不具有任何顺序或技术含义。The serial numbers assigned to components in this article, such as "first", "second", etc., are only used to distinguish the described objects and do not have any sequential or technical meaning.
如本文所用，开放阅读框(open reading frame，ORF)是结构基因的正常核苷酸序列，从起始密码子到终止密码子的阅读框可编码完整的多肽链，其间不存在使翻译中断的终止密码子。As used herein, an open reading frame (ORF) is the normal nucleotide sequence of a structural gene. The reading frame from the start codon to the stop codon can encode a complete polypeptide chain without interruption of translation. Stop codon.
本文中，如无特别说明，氨基酸序列均为自N端(即具有突出的氨基的末端)至C端(即具有突出的羧基的末端)。N端是指氨基酸序列上具有突出的氨基(-NH
2)的末端，C端是指氨基酸序列上具有突出的羧基(-COOH)的末端。
Herein, unless otherwise specified, the amino acid sequence is from the N-terminus (ie, the end with a protruding amino group) to the C-terminus (ie, the end with a protruding carboxyl group). The N-terminus refers to the end of the amino acid sequence with a protruding amino group (-NH 2 ), and the C-terminus refers to the end of the amino acid sequence with a protruding carboxyl group (-COOH).
本文中，如无特别说明，核苷酸序列均为自5’端至3’端。In this article, unless otherwise stated, the nucleotide sequence is from the 5' end to the 3' end.
铜绿假单胞菌的III型分泌***是由20多个蛋白质组成的注射器样的大复合物，能够将一些毒力因子及其他效应蛋白直接注入宿主细胞，引起宿主细胞损伤从而在细菌感染性疾病中发挥重要作用。PcrV是铜绿假单胞III型分泌***的重要组件之一，它能够形成同源多聚体，组成分泌***运输毒力因子和效应蛋白的“管道，”是铜绿假单胞菌III型分泌***的关键组件。鉴于PcrV在铜绿假单胞致病中的重要作用，该蛋白为铜绿假单胞菌感染治疗性抗体针对的重要靶标。抗PcrV的多克隆抗体、单克隆抗体证实能够抑制III型分泌***的分泌，保护机体免受铜绿假单胞菌的入侵。同样，PcrV也是重要的候选疫苗分子，但由于其本身能够形成同源多聚体等原因，限制了可溶性重组蛋白的表达生产。在一实施例中，本发明提供编码PcrV的核酸疫苗，尤其是mRNA疫苗，很好地解决了这一问题。The type III secretion system of Pseudomonas aeruginosa is a large syringe-like complex composed of more than 20 proteins. It can directly inject some virulence factors and other effector proteins into host cells, causing damage to host cells and thus leading to bacterial infectious diseases. play an important role in. PcrV is one of the important components of the type III secretion system of Pseudomonas aeruginosa. It can form homopolymers and form a "pipeline" for the secretion system to transport virulence factors and effector proteins. It is the type III secretion system of Pseudomonas aeruginosa. key components. In view of the important role of PcrV in the pathogenesis of P. aeruginosa, this protein is an important target for therapeutic antibodies against P. aeruginosa infection. Anti-PcrV polyclonal antibodies and monoclonal antibodies have been proven to inhibit the secretion of the type III secretion system and protect the body from invasion by Pseudomonas aeruginosa. Similarly, PcrV is also an important candidate vaccine molecule, but due to its ability to form homomultimers and other reasons, the expression and production of soluble recombinant proteins is limited. In one embodiment, the present invention provides a nucleic acid vaccine encoding PcrV, especially an mRNA vaccine, which solves this problem well.
铜绿假单胞菌的外膜蛋白是镶嵌在PA脂多糖和磷脂层外膜上，是细菌表面的孔蛋白和其他结构功能组分。本发明选择的OprF和OprI是PA表面暴露和抗原高度保守的2种重要的外膜蛋白。OprF是PA细菌表面非特异性通道孔，富含β螺旋结构。通过调节群体感应网络，参与PA的毒力作用。OprF可通过调节假单胞菌喹诺酮信号(POS)水平影响外膜囊泡的形成。采用无细胞表达***表达OprF孔蛋白，其功能表征能稳定结合在脂质双层膜中，有助于分析OprF的结构和功能。OprF在PA所有致病和环境菌株中可作为安全的保护性免疫原，是PA疫苗研制的重要候选物。OprI是PA细胞外膜的内单层中的一种脂蛋白，富含a螺旋结构。OprI是疫苗接种的良好载体分子，与抗原呈递细胞(APC)和上皮细胞的TLR2/4作用，末端脂质尾部可引发免疫反应，其与气管和小肠上皮的黏附有助于黏膜蛋白疫苗的开发。OprI是阳离子抗菌肽(cationic antimicrobial peptides,AMP)的新靶点，参与PA对hRNase7和a螺旋阳离子AMP的敏感性，通过抑制其抗菌活性，增大细菌细胞膜的渗透性而发挥作用，可用于耐药性Pa感染的药物筛选，有望用于新疫苗的开发。The outer membrane protein of Pseudomonas aeruginosa is embedded in the outer membrane of PA lipopolysaccharide and phospholipid layer, and is a porin and other structural and functional components on the bacterial surface. OprF and OprI selected in the present invention are two important outer membrane proteins that are exposed on the PA surface and whose antigens are highly conserved. OprF is a non-specific channel pore on the surface of PA bacteria and is rich in β-helix structure. Participates in the virulence effect of PA by regulating the quorum sensing network. OprF can affect the formation of outer membrane vesicles by regulating Pseudomonas quinolone signaling (POS) levels. A cell-free expression system was used to express the OprF porin, and its functional characterization can be stably incorporated into the lipid bilayer membrane, which is helpful for analyzing the structure and function of OprF. OprF can be used as a safe protective immunogen in all pathogenic and environmental strains of PA and is an important candidate for PA vaccine development. OprI is a lipoprotein in the inner monolayer of the outer membrane of PA cells and is rich in α-helical structure. OprI is a good carrier molecule for vaccination. It interacts with antigen-presenting cells (APC) and TLR2/4 of epithelial cells. The terminal lipid tail can trigger immune responses. Its adhesion to tracheal and small intestinal epithelium contributes to the development of mucosal protein vaccines. . OprI is a new target of cationic antimicrobial peptides (AMP). It participates in the sensitivity of PA to hRNase7 and a-helical cationic AMP. It plays a role by inhibiting its antibacterial activity and increasing the permeability of bacterial cell membranes. It can be used for antibiotic resistance. Drug screening for drug-induced Pa infection is expected to be used in the development of new vaccines.
根据第一方面，在一实施例中，提供一种mRNA分子，所述mRNA分子编码如下蛋白中的至少一种：1)PcrV蛋白；2)OprF蛋白以及OprI蛋白；According to the first aspect, in one embodiment, there is provided an mRNA molecule encoding at least one of the following proteins: 1) PcrV protein; 2) OprF protein and OprI protein;
自N端至C端，所述PcrV蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the PcrV protein contains the following amino acid sequence:
自N端至C端，所述OprF蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprF protein contains the following amino acid sequence:
自N端至C端，所述OprI蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprI protein contains the following amino acid sequence:
在一实施例中，本发明选择的PcrV是保守的，是非常理想和重要的疫苗候选抗原。In one embodiment, the PcrV selected by the present invention is conservative and is a very ideal and important vaccine candidate antigen.
在一实施例中，SEQ ID No.8所示氨基酸序列与SEQ ID No.9所示氨基酸序列位于同一氨基酸序列。即SEQ ID No.8所示氨基酸序列与SEQ ID No.9所示氨基酸序列通过串联的方式位于同一氨基酸序列上。In one embodiment, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are located in the same amino acid sequence. That is, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are located on the same amino acid sequence in a tandem manner.
在一实施例中，SEQ ID No.8所示氨基酸序列、SEQ ID No.9所示氨基酸序列可以通过独立的核苷酸以及表达载体单独表达，也可以通过串联的核苷酸序列，在同一表达载体中同时表达。优选为同时表达。In one embodiment, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 can be expressed individually through independent nucleotides and expression vectors, or they can be expressed in the same sequence through tandem nucleotide sequences. expressed simultaneously in an expression vector. Simultaneous expression is preferred.
在一实施例中，SEQ ID No.8所示氨基酸序列与SEQ ID No.9所示氨基酸序列通过第一接头序列串联。In one embodiment, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are connected in series through a first linker sequence.
在一实施例中，SEQ ID No.8所示氨基酸序列的C端串联至第一接头序列的N端，SEQ ID No.9所示氨基酸序列的N端串联至第一接头序列的C端。在一实施例中，SEQ ID No.8所示氨基酸序列的N端串联有信号肽序列。In one embodiment, the C-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the N-terminus of the first linker sequence, and the N-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the C-terminus of the first linker sequence. In one embodiment, a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 8.
在另一实施例中，SEQ ID No.9所示氨基酸序列的C端串联至第一接头序列的N端，SEQ ID No.8所示氨基酸序列的N端串联至第一接头序列的C端。在一实施例中，SEQ ID No.9所示氨基酸序列的N端串联有信号肽序列。In another embodiment, the C-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the N-terminus of the first linker sequence, and the N-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the C-terminus of the first linker sequence. . In one embodiment, a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 9.
在一实施例中，SEQ ID No.7所示氨基酸序列的C端依次串联有第二接头序列、加尾氨基酸序列。In one embodiment, the C-terminus of the amino acid sequence shown in SEQ ID No. 7 is sequentially connected in series with a second linker sequence and a tailing amino acid sequence.
在一实施例中，SEQ ID No.7所示氨基酸序列的N端串联有信号肽序列。In one embodiment, a signal peptide sequence is connected in series to the N-terminus of the amino acid sequence shown in SEQ ID No. 7.
在一实施例中，SEQ ID No.8、SEQ ID No.9所示氨基酸序列串联后，在串联序列的氨基酸序列C端依次串联有第二接头序列、加尾氨基酸序列。In one embodiment, after the amino acid sequences shown in SEQ ID No. 8 and SEQ ID No. 9 are concatenated, a second linker sequence and a tailing amino acid sequence are sequentially concatenated at the C-terminal end of the amino acid sequence of the concatenated sequence.
在一实施例中，所述第一接头序列含有甘氨酸(G)、丝氨酸(S)。In one embodiment, the first linker sequence contains glycine (G) and serine (S).
在一实施例中，所述第二述接头序列含有甘氨酸、丝氨酸。In one embodiment, the second linker sequence contains glycine and serine.
在一实施例中，所述第一接头序列包括但不限于如下氨基酸序列：GSGSGSGSGS。In one embodiment, the first linker sequence includes but is not limited to the following amino acid sequence: GSGSGSGSGS.
在一实施例中，所述第二接头序列包括但不限于如下氨基酸序列：GGGS。In one embodiment, the second linker sequence includes but is not limited to the following amino acid sequence: GGGS.
在一实施例中，所述加尾氨基酸序列含有至少一个组氨酸(H)。In one embodiment, the tailed amino acid sequence contains at least one histidine (H).
在一实施例中，所述加尾氨基酸序列含有6～12个组氨酸。In one embodiment, the tailed amino acid sequence contains 6 to 12 histidines.
在一实施例中，编码PcrV蛋白的mRNA含有如下核苷酸序列：In one embodiment, the mRNA encoding the PcrV protein contains the following nucleotide sequence:
在一实施例中，编码OprF蛋白的mRNA含有如下核苷酸序列：In one embodiment, the mRNA encoding OprF protein contains the following nucleotide sequence:
在一实施例中，编码OprI蛋白的mRNA含有如下核苷酸序列：In one embodiment, the mRNA encoding the OprI protein contains the following nucleotide sequence:
在一实施例中，所述mRNA分子从5’端到3’端依次包括：5’帽子结构、5’UTR(Untranslated Region，非编码区)序列、编码信号肽的核苷酸序列、编码PcrV蛋白和/或OprF蛋白以及OprI蛋白的核苷酸序列、3’UTR序列、多聚腺苷酸序列(PolyA)。In one embodiment, the mRNA molecule includes in order from the 5' end to the 3' end: a 5' cap structure, a 5'UTR (Untranslated Region, non-coding region) sequence, a nucleotide sequence encoding a signal peptide, and a PcrV encoding The nucleotide sequence, 3'UTR sequence, and polyadenylation sequence (PolyA) of the protein and/or OprF protein and OprI protein.
在一实施例中，5’端帽子结构包括但不限于Cap0、Cap1、Cap2和其他任何结构的帽子结构中的任意一种。In one embodiment, the 5' end cap structure includes but is not limited to any one of Cap0, Cap1, Cap2 and any other cap structure.
在一实施例中，所述5’UTR、3’UTR序列独立地来源于天然蛋白、人工合成蛋白中的至少一种。In one embodiment, the 5'UTR and 3'UTR sequences are independently derived from at least one of natural proteins and artificially synthesized proteins.
在一实施例中，天然蛋白包括但不限于α-球蛋白、β-球蛋白、热休克蛋白HSP70中的任意一种。In one embodiment, natural proteins include, but are not limited to, any one of α-globulin, β-globulin, and heat shock protein HSP70.
在一实施例中，所述mRNA分子为未修饰或经修饰的。In one embodiment, the mRNA molecule is unmodified or modified.
在一实施例中，所述修饰包括：假尿苷三磷酸、N1-甲基假尿苷三磷酸修饰中的至少一种。In one embodiment, the modification includes: at least one of pseudouridine triphosphate and N1-methylpseudouridine triphosphate modification.
在一实施例中，所述PcrV蛋白包含SEQ ID No.1所示氨基酸序列。In one embodiment, the PcrV protein includes the amino acid sequence shown in SEQ ID No. 1.
在一实施例中，编码所述PcrV蛋白的mRNA分子包含SEQ ID No.10所示核苷酸序列。In one embodiment, the mRNA molecule encoding the PcrV protein includes the nucleotide sequence shown in SEQ ID No. 10.
在一实施例中，OprF蛋白以及OprI蛋白(即OprF-I融合蛋白)包含SEQ ID No.6所示氨基酸序列。In one embodiment, the OprF protein and OprI protein (i.e., OprF-I fusion protein) comprise the amino acid sequence shown in SEQ ID No. 6.
在一实施例中，编码所述OprF蛋白以及OprI蛋白的mRNA包含SEQ ID No.11所示核苷酸序列。In one embodiment, the mRNA encoding the OprF protein and OprI protein includes the nucleotide sequence shown in SEQ ID No. 11.
根据第二方面，在一实施例中，提供负载有第一方面所述mRNA分子的脂质纳米颗粒(Lipid Nanoparticle，LNP)。脂质纳米颗粒是使用脂质形成的一种纳米微粒。According to the second aspect, in one embodiment, lipid nanoparticles (Lipid Nanoparticles, LNPs) loaded with the mRNA molecules described in the first aspect are provided. Lipid nanoparticles are a type of nanoparticle formed using lipids.
在一实施例中，所述脂质纳米颗粒包括阳离子脂质、中性辅助磷脂、胆固醇、聚乙二醇修饰的脂质中的至少一种。离子化脂质是修饰化的脂质，当LNP被细胞内吞后，离子化脂质会在酸性环境下离子化，从而让LNP逃离内体。可电离阳离子脂质为最关键的脂质，它是mRNA递送和转染效率的决定性因素。中性辅助磷脂一般为饱和磷脂，可提高阳离子脂质体的相变温度，支持层状脂质双层结构的形成并稳定其结构排列；胆固醇有较强的膜融合性，促进mRNA胞内摄入和胞质进入；PEG(聚乙二醇)化磷脂位于脂质纳米粒表面，改善其亲水性，避免被免疫***快速清除，防止颗粒聚集，增加稳定性。In one embodiment, the lipid nanoparticles include at least one of cationic lipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol-modified lipids. Ionized lipids are modified lipids. When LNP is endocytosed by cells, the ionized lipid will ionize in an acidic environment, allowing LNP to escape from the endosome. Ionizable cationic lipids are the most critical lipids and are a decisive factor in mRNA delivery and transfection efficiency. Neutral auxiliary phospholipids are generally saturated phospholipids, which can increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayer structures and stabilize their structural arrangement; cholesterol has strong membrane fusion properties and promotes intracellular uptake of mRNA. into the cytoplasm; PEG (polyethylene glycol) phospholipids are located on the surface of lipid nanoparticles to improve their hydrophilicity, avoid rapid clearance by the immune system, prevent particle aggregation, and increase stability.
在一实施例中，阳离子脂质包括但不限于4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯(DLin-MC3-DMA，CAS号：1224606-06-7)。In one embodiment, the cationic lipid includes but is not limited to 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester (DLin-MC3-DMA, CAS number: 1224606-06-7 ).
在一实施例中，中性辅助磷脂包括但不限于二硬脂酰磷脂酰胆碱。In one embodiment, neutral accessory phospholipids include, but are not limited to, distearoylphosphatidylcholine.
在一实施例中，聚乙二醇修饰的脂质包括但不限于1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000、2-[(聚乙二醇)-2000]-N,N二十四烷基乙酰胺、1,2-二硬脂酰-rac-甘油-3-甲氧基聚乙二醇2000(DSG-PEG2000)或n-(羰基-甲氧基聚乙二醇2000)-1,2-二硬脂酰-sn-甘油-3-磷脂酰乙醇胺钠盐中的至少一种，优选为1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(结构式见图6中的mPEG2000-DMG，CAS号：160743-62-4)、2-[(聚乙二醇)-2000]-N,N二十四烷基乙酰胺中的至少一种。In one embodiment, polyethylene glycol-modified lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, 2-[(polyethylene glycol )-2000]-N,N tetradecyl acetamide, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG2000) or n-(carbonyl- At least one of methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine sodium salt, preferably 1,2-dimyristoyl-rac-glycerol -3-Methoxypolyethylene glycol 2000 (for the structural formula, see mPEG2000-DMG in Figure 6, CAS number: 160743-62-4), 2-[(polyethylene glycol)-2000]-N, N20 At least one of tetraalkyl acetamides.
在一实施例中，按摩尔量计，阳离子脂质：中性辅助磷脂：胆固醇：聚乙二醇修饰的脂质＝50：38.5：10：1.5。In one embodiment, on a molar basis, cationic lipid: neutral auxiliary phospholipid: cholesterol: polyethylene glycol modified lipid = 50: 38.5: 10: 1.5.
根据第三方面，在一实施例中，提供一种蛋白，所述蛋白包含第一方面所述mRNA编码得到的氨基酸序列。According to the third aspect, in one embodiment, a protein is provided, the protein comprising the amino acid sequence encoded by the mRNA described in the first aspect.
根据第四方面，在一实施例中，提供一种编码第一方面所述mRNA分子的DNA分子。According to a fourth aspect, in one embodiment, a DNA molecule encoding the mRNA molecule described in the first aspect is provided.
根据第五方面，在一实施例中，提供含有第四方面所述DNA分子的重组质粒。According to the fifth aspect, in one embodiment, a recombinant plasmid containing the DNA molecule described in the fourth aspect is provided.
根据第六方面，在一实施例中，提供一种疫苗，所述疫苗包含第一方面所述mRNA分子、第二方面所述脂质纳米颗粒、第三方面所述融合蛋白、第四方面所述DNA分子或第五方面所述重组质粒。According to the sixth aspect, in one embodiment, a vaccine is provided, the vaccine comprising the mRNA molecule described in the first aspect, the lipid nanoparticles described in the second aspect, the fusion protein described in the third aspect, the The DNA molecule or the recombinant plasmid of the fifth aspect.
根据第七方面，在一实施例中，提供一种抗体，所述抗体是由第六方面所述疫苗诱导产生并分离获得的。According to the seventh aspect, in one embodiment, an antibody is provided, the antibody being induced and produced by the vaccine of the sixth aspect and isolated.
在一实施例中，所述抗体包括但不限于IgG1、IgG2a中的至少一种。In one embodiment, the antibody includes but is not limited to at least one of IgG1 and IgG2a.
根据第八方面，在一实施例中，提供第一方面所述mRNA分子在制备治疗和/或预防疾病的药物中的用途。According to the eighth aspect, in one embodiment, the use of the mRNA molecule described in the first aspect in preparing a drug for treating and/or preventing diseases is provided.
在一实施例中，所述疾病包括细菌引起的疾病。In one embodiment, the disease includes a disease caused by bacteria.
在一实施例中，所述所述细菌包括但不限于假单胞菌目。In one embodiment, the bacteria include, but are not limited to, Pseudomonadales.
在一实施例中，所述细菌包括但不限于铜绿假单胞菌。In one embodiment, the bacteria include, but are not limited to, Pseudomonas aeruginosa.
在一实施例中，本发明提供一种编码铜绿假单胞菌三型分泌***重要蛋白PcrV和外膜蛋白中OprF-I融合蛋白，以脂质纳米颗粒(LNP)递送的mRNA疫苗用于铜绿假单胞菌感染的预防和治疗。In one embodiment, the present invention provides an mRNA vaccine encoding PcrV, an important protein of the type III secretion system of Pseudomonas aeruginosa, and an OprF-I fusion protein in the outer membrane protein, delivered by lipid nanoparticles (LNP) for use in Pseudomonas aeruginosa Prevention and treatment of Pseudomonas infections.
在一实施例中，本发明设计合成了带有编码PcrV蛋白和OprF-I融合蛋白的mRNA，其中mRNA的帽子为Cap1结构，经由mRNA共转录加帽方法所得，且mRNA中天然的鸟嘌呤核苷酸全部取代为N1-甲基假尿苷。PcrV-mRNA、OprF-I-mRNA经LNP包裹后设置空白、10μg、30μg三组免疫Balb/c小鼠评价疫苗的免疫应答，并在小鼠完成两次免疫后采用铜绿假单胞杆菌PAO1菌株对烧伤小鼠进行攻毒试验来评价疫苗的保护效果。实验结果表明mRNA可以高质量转录，在细胞水平上可实现高水平的蛋白表达；包裹mRNA的LNP粒径均一，包封率高，免疫小鼠可以诱导高滴度的抗原特异性抗体；攻毒试验显示PcrV-mRNA疫苗对50xLD
50攻毒剂量的保护效果(即小鼠存活率)高达100％。OprF-I-mRNA疫苗保护效果可达50％～66.7％。
In one embodiment, the present invention designs and synthesizes an mRNA encoding PcrV protein and OprF-I fusion protein, in which the cap of the mRNA is a Cap1 structure, obtained by the co-transcriptional capping method of the mRNA, and the natural guanine core in the mRNA All nucleotides are substituted with N1-methylpseudouridine. After PcrV-mRNA and OprF-I-mRNA were packaged with LNP, three groups of blank, 10 μg, and 30 μg immunized Balb/c mice were set to evaluate the immune response of the vaccine. After the mice completed two immunizations, the Pseudomonas aeruginosa PAO1 strain was used. A challenge test was conducted on burnt mice to evaluate the protective effect of the vaccine. Experimental results show that mRNA can be transcribed with high quality and high-level protein expression can be achieved at the cellular level; the LNP encapsulating the mRNA has a uniform particle size and a high encapsulation rate, and immunized mice can induce high-titer antigen-specific antibodies; challenge the virus The test showed that the protective effect (i.e. mouse survival rate) of the PcrV-mRNA vaccine against the 50xLD 50 challenge dose was as high as 100%. The protective effect of OprF-I-mRNA vaccine can reach 50% to 66.7%.
在一实施例中，如图1所示为mRNA疫苗的构成元件示意图，mRNA药物和疫苗构成元件从5’到3’依次包括：5’端帽子结构、KOZAK序列、5’非编码区(5’UTR)、开放阅读框(Open Reading Frame，ORF)、3’非编码区(3’UTR)和多聚腺嘌呤尾(PolyA)。mRNA能否高效表达外源基因，与上述各个元件的组成和核苷酸修饰密切相关，因此，要获得可高效表达外源目的基因的mRNA疫苗，需要多方面考虑并进行不断的筛选和优化。In one embodiment, as shown in Figure 1 is a schematic diagram of the constituent elements of the mRNA vaccine. The constituent elements of the mRNA drug and vaccine include in order from 5' to 3': 5' end cap structure, KOZAK sequence, 5' non-coding region (5 'UTR), open reading frame (Open Reading Frame, ORF), 3' non-coding region (3'UTR) and polyadenine tail (PolyA). Whether mRNA can efficiently express foreign genes is closely related to the composition and nucleotide modification of each of the above components. Therefore, to obtain an mRNA vaccine that can efficiently express foreign genes of interest requires multiple considerations and continuous screening and optimization.
实施例1Example 1
本实施例的具体设计流程如下：The specific design process of this embodiment is as follows:
1、模板基因的构建1. Construction of template genes
参照PcrV蛋白序列和OprF、OprI(Uniport：PA1706)蛋白序列，并在N端和C端添加分泌信号肽和6*His标签，根据前述氨基酸序列，设计编码序列。下文的序列1为mRNA的ORF编码的氨基酸序列。在ORF的5’端添加5’UTR和KOZAK序列(序列2)，在3’端添加3’UTR和聚腺苷酸(PolyA)序列。上述基因片段由金唯智公司合成并克隆入pVAX1载体得到模板质粒pVAX1-PcrV。图2.1为质粒pVAX1-PcrV图谱，图2.2为质粒pVAX1-OprF-I图谱。Refer to the PcrV protein sequence and the OprF and OprI (Uniport: PA1706) protein sequences, and add the secretion signal peptide and 6*His tag at the N-terminus and C-terminus, and design the coding sequence based on the aforementioned amino acid sequence. Sequence 1 below is the amino acid sequence encoded by the ORF of mRNA. Add 5' UTR and KOZAK sequence (sequence 2) to the 5' end of the ORF, and add 3' UTR and poly(A) sequence to the 3' end. The above gene fragment was synthesized by Jinweizhi Company and cloned into the pVAX1 vector to obtain the template plasmid pVAX1-PcrV. Figure 2.1 shows the map of plasmid pVAX1-PcrV, and Figure 2.2 shows the map of plasmid pVAX1-OprF-I.
本实施例中，信号肽序列为TPA蛋白的信号肽序列(序列3)；5’UTR的序列为序列4所示；3’UTR为序列5所示；PolyA的长度为80～150个。In this example, the signal peptide sequence is the signal peptide sequence of TPA protein (Sequence 3); the sequence of 5’UTR is shown in Sequence 4; the 3’UTR is shown in Sequence 5; the length of PolyA is 80 to 150.
mRNA-PcrV ORF编码的氨基酸序列(序列1)：Amino acid sequence encoded by mRNA-PcrV ORF (sequence 1):
其中，粗下划线标示的序列为信号肽序列。Among them, the sequence marked with thick underline is the signal peptide sequence.
Kozak序列(序列2)：Kozak sequence (sequence 2):
GCCACCAUGG(SEQ ID No.2)GCCACCAUGG(SEQ ID No.2)
Kozak序列末尾的“AUG”即为ORF区的起始密码子。The "AUG" at the end of the Kozak sequence is the start codon of the ORF region.
分泌信号肽Tissue Plasminogen Activator(TPA)序列(序列3)如下：The secretion signal peptide Tissue Plasminogen Activator (TPA) sequence (sequence 3) is as follows:
5’UTR序列(序列4)如下：The 5’UTR sequence (sequence 4) is as follows:
3’UTR序列(序列5)如下：The 3’UTR sequence (sequence 5) is as follows:
mRNA-OprF-I的ORF区编码的氨基酸序列(序列6)如下：The amino acid sequence (sequence 6) encoded by the ORF region of mRNA-OprF-I is as follows:
序列6中，加粗部分为接头序列(Linker)。粗下划线标示的序列为信号肽序列。In sequence 6, the bold part is the linker sequence (Linker). The sequence marked with bold underline is the signal peptide sequence.
PcrV的mRNA序列如下：The mRNA sequence of PcrV is as follows:
其中，各区域说明如下：Among them, each area is explained as follows:
5’UTR：斜体；Kozak序列：
ORF：加粗；3’UTR：双下划线；PolyA：下划波浪线。编码信号肽的mRNA序列：粗下划线。编码组氨酸标签的mRNA序列：虚下划线。
5'UTR: italics; Kozak sequence: ORF: bold; 3'UTR: double underline; PolyA: underline wavy line. mRNA sequence encoding signal peptide: thick underline. mRNA sequence encoding histidine tag: dashed underline.
OprF-I的mRNA序列如下：The mRNA sequence of OprF-I is as follows:
其中，各区域说明如下：Among them, each area is explained as follows:
5’UTR：斜体；Kozak序列：
ORF：黑体加粗；3’UTR：双下划线；PolyA：下划波浪线；(GS)5连接序列：点式下划线。编码信号肽的mRNA：粗下划线。编码组氨酸标签的mRNA序列：虚下划线。
5'UTR: italics; Kozak sequence: ORF: boldface; 3'UTR: double underline; PolyA: underlined wavy line; (GS)5 connection sequence: dotted underline. mRNA encoding signal peptide: thick underline. mRNA sequence encoding histidine tag: dashed underline.
2、质粒模板扩增和线性化2. Plasmid template amplification and linearization
pVAX1-PcrV质粒和pVAX1-OprF-I质粒转化DH5α感受态细胞，涂布在含Kan+(卡那霉素)抗性的固体LB平板上，37℃培养12～15h后挑取单菌落加至含Kan+抗性的液体LB培养基中，37℃、250rpm培养12～15h后离心收集细胞，并利用商业化的质粒提取试剂盒提取扩增的质粒。扩增质粒的线性化酶切位点选择紧邻PolyA尾的XhoI，线性化程度通过DNA琼脂糖凝胶电泳检测，线性化的质粒DNA通过PCR产物回收试剂盒回收，DNA浓度和质量通过NanoDrop超微量核酸仪测定OD260和OD260/OD280的值计算得到。pVAX1-PcrV plasmid and pVAX1-OprF-I plasmid were transformed into DH5α competent cells, spread on solid LB plates containing Kan+ (kanamycin) resistance, and cultured at 37°C for 12 to 15 hours. Then single colonies were picked and added to In Kan+-resistant liquid LB medium, the cells were cultured at 37°C and 250 rpm for 12 to 15 hours, then centrifuged to collect the cells, and a commercial plasmid extraction kit was used to extract the amplified plasmid. The linearized restriction site of the amplified plasmid was selected to be XhoI immediately adjacent to the PolyA tail. The degree of linearization was detected by DNA agarose gel electrophoresis. The linearized plasmid DNA was recovered by a PCR product recovery kit. The DNA concentration and quality were determined by NanoDrop ultra-micro volume. The values of OD260 and OD260/OD280 were determined by nucleic acid instrument and calculated.
3、mRNA体外转录和纯化3. In vitro transcription and purification of mRNA
体外合成RNA(IVU)是以含有U7启动子(UAAUACGACUCACUAUAGGG)或SP6启动子(AUUUAGGUGACACUAUAG)序列的DNA(线性化质粒或PCR产物)为模板，在U7或SP6RNA聚合酶的作用下，以NUP为底物合成与模板DNA中一条链互补的mRNA，并通过在5’端加上帽子结构和3’端加ployA尾加强mRNA的稳定性。In vitro synthesis of RNA (IVU) uses DNA (linearized plasmid or PCR product) containing the U7 promoter (UAAUACGACUCACUAUAGGG) or SP6 promoter (AUUUAGGUGACACUAUAG) sequence as a template, and uses NUP as the base under the action of U7 or SP6 RNA polymerase. It synthesizes an mRNA that is complementary to one strand of the template DNA, and enhances the stability of the mRNA by adding a cap structure to the 5' end and a polyA tail to the 3' end.
mRNA的加帽工艺可采用共转录加帽或转录后加帽，加尾工艺可采用模板转录加尾或转录后酶法加尾。在本实施例中，加帽和加尾方法分别为共转录加帽和模板加尾，其中5’帽结构，本实施例为m7GpppN(图3)。在共转录加帽中，将帽类似物直接添加到IVU中，它们通过具有松弛底物特异性的RNA聚合酶在5'-末端掺入直接产生相应的5'-加帽的mRNA。由于帽类似物缺乏游离的5'-三磷酸，因此在IVU期间不会发生帽类似物的内部掺入。本实施例中采用Urilinker公司的
ReagenU AG(3'OMe)为帽子结构(图3)，如图4所示为U7RNA聚合酶转录起始示意图，在U7聚合酶作用下，以5’AG 3’为起始开始转录。
The capping process of mRNA can use co-transcriptional capping or post-transcriptional capping, and the tailing process can use template transcription or post-transcriptional enzymatic tailing. In this example, the capping and tailing methods are co-transcriptional capping and template tailing respectively, and the 5' cap structure in this example is m7GpppN (Figure 3). In cotranscriptional capping, cap analogs are added directly to the IVU, and their incorporation at the 5'-end by an RNA polymerase with relaxed substrate specificity directly produces the corresponding 5'-capped mRNA. Since the cap analogue lacks free 5'-triphosphate, no internal incorporation of the cap analogue occurs during IVU. In this embodiment, Urilinker's ReagenU AG (3'OMe) is a cap structure (Figure 3). Figure 4 shows a schematic diagram of the transcription initiation of U7 RNA polymerase. Under the action of U7 polymerase, transcription starts with 5'AG 3'.
具体转录反应按照表1的各成分配比设置，步骤依次如下：(1)加入RNase free waUer和NUPs；(2)加入
ReagenU AG(3'OMe)混匀后离心收集液体；(3)加入10X UranscripUion Buffer，混匀后离心收集液体；(4)加入线性化的DNA模板；(5)加入Murine RNase InhibiUor、YeasU Inorganic PyrophosphaUase和U7 RNA Polymerase，混匀后离心收集液体；(6)37℃反应2～3hours。反应结束后，加入DNAaseI去除模板DNA，转录产物采用Monarch RNA cleanup kiU(NEB)纯化。mRNA的浓度通过Nano drop紫外分光光度计的读值来计算，mRNA的纯度通过毛细管电泳仪2100Bioanalyzer测定。如图5.1、5.2所示的结果显示体外转录的mRNA分子大小符合预期，且完整度和纯度高。
The specific transcription reaction is set according to the ratio of each component in Table 1. The steps are as follows: (1) Add RNase free waUer and NUPs; (2) Add Mix ReagenU AG (3'OMe) and centrifuge to collect the liquid; (3) Add 10X UranscripUion Buffer, mix and centrifuge to collect the liquid; (4) Add linearized DNA template; (5) Add Murine RNase InhibiUor, YeasU Inorganic PyrophosphaUase and U7 RNA Polymerase, mix well and centrifuge to collect the liquid; (6) React at 37°C for 2 to 3 hours. After the reaction, DNAaseI was added to remove the template DNA, and the transcript was purified using Monarch RNA cleanup kiU (NEB). The concentration of mRNA was calculated by the reading of Nano drop UV spectrophotometer, and the purity of mRNA was determined by capillary electrophoresis instrument 2100 Bioanalyzer. The results shown in Figures 5.1 and 5.2 show that the size of the in vitro transcribed mRNA molecules is in line with expectations, and the integrity and purity are high.
表1 mRNA体外转录反应各成分配比表Table 1 Ratio of components of mRNA in vitro transcription reaction
| Nuclease-free waUerNuclease-free waUer | 补足至20μLMake up to 20μL | 补足至20μLMake up to 20μL |
| 10X ReacUion Buffer10X ReacUion Buffer | 2μL2μL | 1X1X |
| AUP(100mM)AUP(100mM) | 1.5μL1.5μL | 7.5mM final7.5mM final |
| GUP(100mM)GUP(100mM) | 1.5μL1.5μL | 7.5mM final7.5mM final |
| UUP or N1-pUUP(100mM)UUP or N1-pUUP(100mM) | 1.5μL1.5μL | 7.5mM final7.5mM final |
| CUP(100mM)CUP(100mM) | 1.5μL1.5μL | 7.5mM final7.5mM final |
| Cleancap AG(Cap1)Cleancap AG(Cap1) | 1.2μL1.2μL | 6mM final6mM final |
| UemplaUe DNAUemplaUe DNA | XμLXμL | 1μg1μg |
| U7 RNA Polymerase(50U/μL)U7 RNA Polymerase(50U/μL) | 2μL2μL | 5U/μL5U/μL |
| RNA polymerase inhibiUor(40U/μL)RNA polymerase inhibiUor(40U/μL) | 1μL1μL | 4U/μL4U/μL |
| Inorganic pyrophosphaUase(0.1U/μL)Inorganic pyrophosphaUase(0.1U/μL) | 1μL1μL | 0.005U/μL0.005U/μL |
| UoUal reacUion volumeUoUal reacUion volume | 20μL20μL | 20μL reacUion20μL reacUion |
4、mRNA-LNP制备4. Preparation of mRNA-LNP
该步骤使用的化合物原料的结构式如图6所示，负载mRNA的脂质纳米粒包含四种成分，分别为可电离的阳离子磷脂(ionizable lipids)、中性辅助磷脂、胆固醇(CholesUerol)、聚乙二醇修饰的磷脂(PEGylaUed lipid)。具体LNP的制备过程为：将可电离脂质(DLin-MC3-DMA，结构式见图6中的sm-102，CAS号：1224606-06-7)、胆固醇(CholesUerol)、辅助脂质(DSPC，二硬脂酰磷脂酰胆碱)、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(结构式见图6中的mPEG2000-DMG，CAS号：160743-62-4)按照50：38.5：10：1.5的摩尔比溶于无水乙醇中，总浓度为10mg/mL，构成有机相；将编码抗原蛋白的mRNA以0.1mg/mL的总浓度溶于柠檬酸钠缓冲液(50mM，pH为4)中，构成水相；将有机相与水相按1：3的体积比，以12mL/min的速度使用微流控设备混匀得到LNP-mRNA混合液；将LNP-mRNA混合液使用无菌PBS(10mM，pH为7.2)将混合液稀释40倍，并转移至预灭菌的
离心过滤器(截止值＝100KDa)。为了实现缓冲液交换和产品浓缩，在4000×g条件下，在15～30分钟内完成离心，并在添加新鲜PBS后重复三次，浓缩得到浓度为2mg/mL的mRNA-LNP。最终产品在4℃下储存，直至使用。
The structural formula of the compound raw materials used in this step is shown in Figure 6. The lipid nanoparticles loaded with mRNA contain four components, namely ionizable cationic phospholipids (ionizable lipids), neutral auxiliary phospholipids, cholesterol (CholesUerol), and polyethylene glycol. Diol-modified phospholipid (PEGylaUed lipid). The specific preparation process of LNP is as follows: ionizable lipid (DLin-MC3-DMA, structural formula is shown as sm-102 in Figure 6, CAS number: 1224606-06-7), cholesterol (CholesUerol), auxiliary lipid (DSPC, Distearoylphosphatidylcholine), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (for the structural formula, see mPEG2000-DMG in Figure 6, CAS number: 160743-62 -4) Dissolve in absolute ethanol according to the molar ratio of 50:38.5:10:1.5, with a total concentration of 10mg/mL to form an organic phase; dissolve the mRNA encoding the antigen protein in citric acid with a total concentration of 0.1mg/mL In sodium buffer (50mM, pH 4), form an aqueous phase; mix the organic phase and aqueous phase at a volume ratio of 1:3 using a microfluidic device at a speed of 12mL/min to obtain an LNP-mRNA mixture; Dilute the LNP-mRNA mixture 40 times with sterile PBS (10mM, pH 7.2) and transfer it to a pre-sterilized Centrifugal filter (cutoff = 100KDa). In order to achieve buffer exchange and product concentration, complete centrifugation within 15 to 30 minutes at 4000 × g, repeat three times after adding fresh PBS, and concentrate to obtain mRNA-LNP with a concentration of 2 mg/mL. The final product was stored at 4°C until use.
5、mRNA-LNP粒径和均一度测定5. Determination of mRNA-LNP particle size and uniformity
LNP的粒径和均一度采用动态光散射仪(DLS)测定。具体操作如下：在无菌PBS中以1:100的体积比再次稀释浓缩样品，使用HORIBA-SZ100设备在25°和90°的分散角下进行，重复三次，得到LNP的粒径分布和PDI值(Polymer dispersiUy index，聚合物分散性指数)。粒径结果以颗粒大小与强度的比值形式给出，同时预测LNP分散***的稳定性。The particle size and uniformity of LNP were measured using dynamic light scattering (DLS). The specific operation is as follows: dilute the concentrated sample again in sterile PBS at a volume ratio of 1:100, use HORIBA-SZ100 equipment at dispersion angles of 25° and 90°, repeat three times, and obtain the particle size distribution and PDI value of LNP (Polymer dispersiUy index, polymer dispersion index). Particle size results are given as a ratio of particle size to intensity and also predict the stability of the LNP dispersion system.
图7所示的mRNA-pcrv-LNP的DLS测试结果显示，LNP的平均粒径为91.1nm，PDI指数为0.237，该结果表明制备的LNP粒径分布均一，91％的峰强度，说明表面极少量的纳米颗粒聚集。The DLS test results of mRNA-pcrv-LNP shown in Figure 7 show that the average particle size of LNP is 91.1nm and the PDI index is 0.237. This result shows that the prepared LNP has a uniform particle size distribution and a peak intensity of 91%, indicating that the surface is extremely Small amounts of nanoparticles aggregate.
图8所示的mRNA-OprF-I-LNP的DLS测试结果显示，LNP的平均粒径为110.9nm，PDI指数为0.214，该结果表明制备的LNP粒径分布均一，95.1％的峰强度，说明表面极少量的纳米颗粒聚集。The DLS test results of mRNA-OprF-I-LNP shown in Figure 8 show that the average particle size of LNP is 110.9nm and the PDI index is 0.214. This result shows that the prepared LNP has a uniform particle size distribution and a peak intensity of 95.1%, indicating that A very small amount of nanoparticles aggregates on the surface.
6、mRNA-LNP包封率测定6. Determination of mRNA-LNP encapsulation efficiency
mRNA的包封率采用QuanU-iU
TM RiboGreen
TM RNA试剂盒测定，测定原理为：QuanU-iU
TM
RNA试剂是一种超灵敏的荧光核酸染色剂，可检测溶液中1～200ng的核酸，这种核酸染料无法透过LNP，因此只有游离的未被LNP包载的核酸可以被结合。UriUon-100作为一种表面活性剂常被用做破乳剂，使用1％的UriUon-100处理获得的LNP-mRNA可以使包载的核酸释放，得到总核酸量。通过计算破乳前后核酸量的差异得到载药量，再除以总核酸量即可得到包封率，公式如下：
The encapsulation efficiency of mRNA was measured using QuanU-iU TM RiboGreen TM RNA kit. The measurement principle is: QuanU-iU TM RNA reagent is an ultra-sensitive fluorescent nucleic acid stain that can detect 1 to 200ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only free nucleic acids that are not encapsulated by LNP can be bound. UriUon-100, as a surfactant, is often used as a demulsifier. Using 1% UriUon-100 to treat the LNP-mRNA obtained can release the entrapped nucleic acid and obtain the total nucleic acid amount. The drug loading capacity can be obtained by calculating the difference in the amount of nucleic acid before and after emulsification, and then divided by the total nucleic acid amount to obtain the encapsulation rate. The formula is as follows:
包封率(％)＝(破乳后定量-破乳前定量)/破乳后定量。Encapsulation rate (%) = (quantitative quantity after emulsification - quantitative quantity before emulsification)/quantitative quantity after emulsification.
具体操作方法如下：在UE(Uris-EDUA)缓冲液中制备不同浓度(即1000、500、250、125、62.5、31.25、15.625和0ng/mL)的rRNA标准溶液。待测mRNA LNP溶于UEbuffer制成约250ng/mL mRNA的样品。类似的样品也在补充有UriUon-X100表面活性剂(0.5％)的UE缓冲液中制备。将100μL每个样品(包括标准溶 液和mRNA LNP样品)加入96孔板的微孔中。随后，添加100μL 1：200稀释的Ribogreen试剂。在黑暗和室温下培养5分钟后，使用CyUaUion 3(BioUek，Winooski，VU，USA)记录荧光强度，分别在485和528nm处应用激发和发射。从分散在UE和UE/UriUon-X100中的mRNA LNP样品中获得的荧光理论上分别归因于游离(未包封)和总mRNA。尽管如此，即使在没有UriUon-X100的情况下，Ribogreen试剂也能轻微穿透LNP。因此，从分散在UE中的过滤样品中获得的荧光与UE中类似未过滤样品发出的荧光相减。使用标准溶液绘制的标准曲线用于将荧光强度转换为浓度，最后，根据公式计算封装效率。两种mRNA分别包封，使用同一标准曲线计算包封率。图9所示的标准曲线表示了mRNA浓度和荧光强度之间的线性关系(R
2＝0.9993)，利用标准曲线计算包封率，获得约95％的包封率。
The specific operation method is as follows: Prepare rRNA standard solutions of different concentrations (i.e. 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0ng/mL) in UE (Uris-EDUA) buffer. The mRNA LNP to be measured is dissolved in UEbuffer to prepare a sample of approximately 250ng/mL mRNA. Similar samples were also prepared in UE buffer supplemented with UriUon-X100 surfactant (0.5%). Add 100 μL of each sample (including standard solution and mRNA LNP sample) into the microwells of a 96-well plate. Subsequently, add 100 μL of 1:200 diluted Ribogreen reagent. After 5 min of incubation in the dark and room temperature, fluorescence intensity was recorded using CyUaUion 3 (BioUek, Winooski, VU, USA), applying excitation and emission at 485 and 528 nm, respectively. The fluorescence obtained from mRNA LNP samples dispersed in UE and UE/UriUon-X100 is theoretically attributed to free (unencapsulated) and total mRNA, respectively. Nonetheless, Ribogreen reagent slightly penetrates LNPs even in the absence of UriUon-X100. Therefore, the fluorescence obtained from a filtered sample dispersed in the UE is subtracted from the fluorescence emitted by a similar unfiltered sample in the UE. A standard curve drawn using standard solutions was used to convert fluorescence intensity into concentration, and finally, the encapsulation efficiency was calculated according to the formula. The two kinds of mRNA were encapsulated separately, and the encapsulation efficiency was calculated using the same standard curve. The standard curve shown in Figure 9 represents the linear relationship between mRNA concentration and fluorescence intensity (R 2 =0.9993). The encapsulation efficiency was calculated using the standard curve, and an encapsulation efficiency of approximately 95% was obtained.
7、mRNA-PcrV和mRNA-OprF-I细胞转染7. Transfection of mRNA-PcrV and mRNA-OprF-I cells
mRNA-PcrV和mRNA-OprF-I的细胞转染通过LNP和lip3000两种方式递送。mRNA-LNP的转染方法为直接将分别包裹mRNA-PcrV和mRNA-OprF-I的LNP加入到生长至70％左右的293U细胞，mRNA的浓度约为0.5μg/cm
2。lip3000转染方式按照说明书进行，将可以稳定生长的293U细胞选择六孔板进行铺板，待细胞生长至70-80％时进行转染。根据说明书，首先配制A液和B液；A液：200μL的OpUi-MEM稀释4μg mRNA；B液:用200μL OpUi-MEM稀释10μL lipo3000(即lipofeUamin3000)，分别将A液、B液轻轻混匀，静置5min，吸取B液加入至A液中，轻轻混匀，室温静置20min。在A、B液混合液静置的这20min时，对六孔板进行清洗，吸去培养皿中的培养基，用PBS或者无血清培养基清洗一次。待A、B混合液静置完成后，加入转染试剂到每个孔中48h。
Cell transfection of mRNA-PcrV and mRNA-OprF-I was delivered by LNP and lip3000. The method of transfection of mRNA-LNP is to directly add LNPs encapsulating mRNA-PcrV and mRNA-OprF-I respectively to 293U cells that have grown to about 70%. The concentration of mRNA is about 0.5 μg/cm 2 . The lip3000 transfection method is carried out according to the instructions. 293U cells that can grow stably are selected and plated in six-well plates. Transfection is performed when the cells grow to 70-80%. According to the instructions, first prepare solutions A and B; solution A: dilute 4 μg mRNA with 200 μL OpUi-MEM; solution B: dilute 10 μL lipo3000 (i.e. lipofeUamin3000) with 200 μL OpUi-MEM, and mix solutions A and B gently respectively. , let stand for 5 minutes, add liquid B to liquid A, mix gently, and let stand at room temperature for 20 minutes. While the mixture of solutions A and B is allowed to stand for 20 minutes, clean the six-well plate, aspirate the culture medium in the culture dish, and wash once with PBS or serum-free medium. After the mixture of A and B has been allowed to stand, add the transfection reagent to each well for 48 hours.
8、mRNA-LNPs细胞毒性测定8. Cytotoxicity assay of mRNA-LNPs
为了研究mRNA-LNPs可能的细胞毒性，采用HEK 293U细胞贴壁培养并加入不同剂量的mRNA来观察mRNA-LNP的细胞毒性。简言之，在96孔板中接种细胞4～6小时后，加入PBS、空白LNP和负载mRNA的LNPLNP-pcrV和LNP-OprF-I。在添加72小时后，每孔加入10μLCCK8后培养0.5～4h。CKK8试剂中含有WSU-8，WSU-8的化学名如下：2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐。它在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-MeUhoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物(Formazan dye)，生成的甲瓒物的数量与活细胞的数量成正比，用酶标仪测定450nm处的吸光度(OD)来定量每孔细胞数。图10结果显示空白LNP有轻微细胞毒性，负载mRNA的LNP基本上和空白LNP类似。In order to study the possible cytotoxicity of mRNA-LNPs, HEK 293U cells were cultured adherently and different doses of mRNA were added to observe the cytotoxicity of mRNA-LNPs. Briefly, 4 to 6 hours after cells were seeded in a 96-well plate, PBS, blank LNP, and mRNA-loaded LNPLNP-pcrV and LNP-OprF-I were added. After 72 hours of addition, 10 μLCCK8 was added to each well and cultured for 0.5 to 4 hours. CKK8 reagent contains WSU-8. The chemical name of WSU-8 is as follows: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 -Benzene disulfonate)-2H-tetrazole monosodium salt. It is reduced to a highly water-soluble yellow formazan product (Formazan) by dehydrogenase in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-MeUhoxy PMS). dye), the amount of formazan produced is proportional to the number of viable cells. Use a microplate reader to measure the absorbance (OD) at 450 nm to quantify the number of cells in each well. The results in Figure 10 show that blank LNP has slight cytotoxicity, and the mRNA-loaded LNP is basically similar to blank LNP.
9、WB鉴定mRNA的细胞表达9. WB identification of cellular expression of mRNA
通过WB鉴定PcrV-mRNA、PcrV-mRNA-LNP和mRNA-OprF/I、mRNA-OprF-I-LNP转染细胞所得的细胞培养上清液和细胞裂解液中目标蛋白是否按照预期表达。细胞培养上清和细胞裂解液加入6×loading buffer后沸水浴5min进行聚丙烯酰胺凝胶电泳；凝胶电转移，使蛋白转移到PVDF上；用1％BSA室温封闭60min，PBSU洗涤3次；用anUi-His-HRP抗体室温孵育2h，UBSU洗涤3次后用增强的化学发光试剂盒(ECL)显色并扫描拍照。Use WB to identify whether the target protein in the cell culture supernatant and cell lysate obtained from transfected cells with PcrV-mRNA, PcrV-mRNA-LNP and mRNA-OprF/I, and mRNA-OprF-I-LNP is expressed as expected. The cell culture supernatant and cell lysate were added to 6× loading buffer and then subjected to polyacrylamide gel electrophoresis in a boiling water bath for 5 minutes; gel electrophoresis was carried out to transfer the protein to PVDF; blocked with 1% BSA at room temperature for 60 minutes, washed 3 times with PBSU; The anUi-His-HRP antibody was incubated at room temperature for 2 hours, washed three times with UBSU, developed with an enhanced chemiluminescence kit (ECL), and scanned and photographed.
图11所示为显色结果拍照图，可见，PcrV-mRNA和mRNA-OprF-I无论以lipofeUamin3000还是以LNP为递送方式，都可以在转染细胞后高水平的表达目的蛋白，并能高效分泌到细胞外。Figure 11 shows a photograph of the color development results. It can be seen that whether PcrV-mRNA and mRNA-OprF-I are delivered using lipofeUamin3000 or LNP, they can express the target protein at high levels after transfection into cells and can secrete it efficiently. to the outside of the cell.
10、PcrV和OprF-I蛋白的表达纯化10. Expression and purification of PcrV and OprF-I proteins
PcrV和OprF-I蛋白编码基因克隆到带有C末端6×His标签的pEU21a表达载体(InviUrogen)并转化大肠杆菌BL
21(DE
3)pLysS感受态细胞。携带有目标蛋白质粒的单菌落在37℃，IPUG诱导下表达携带有6个连续组氨酸残基的重组PcrV和OprF-I蛋白。离心收集的菌体，使用高压均质机破碎、高速离心后得到的上清首先通过NUA-Ni亲和色谱柱粗纯，然后通过分子排阻色谱精纯。
The PcrV and OprF-I protein coding genes were cloned into the pEU21a expression vector (InviUrogen) with a C-terminal 6×His tag and transformed into E. coli BL 21 (DE 3 ) pLysS competent cells. A single colony carrying the target protein plasmid expressed recombinant PcrV and OprF-I proteins carrying 6 consecutive histidine residues under IPUG induction at 37°C. The bacterial cells collected by centrifugation were crushed using a high-pressure homogenizer and centrifuged at high speed. The supernatant obtained was first crudely purified through a NUA-Ni affinity chromatography column, and then purified through size exclusion chromatography.
图12所示为蛋白的SDS-PAGE图，可见，PcrV和OprF-I蛋白都已高纯度、高产量制备。Figure 12 shows the SDS-PAGE image of the protein. It can be seen that both PcrV and OprF-I proteins have been prepared with high purity and high yield.
11、小鼠免疫与采血11. Mouse immunization and blood collection
小鼠免疫和采血流程示意图见图13，PcrV-mRNA-LNP、mRNA-OprF-I-LNP和空白LNP通过肌肉注射方式免疫balb/c小鼠，免疫剂量设5和25μg两个剂量，免疫体积为100μL。在首次免疫3周后进行第二次加强免 疫，免疫剂量、体积和免疫方式与第一次相同。在初次免疫的第1、3、5周从眼眶静脉丛采血，分离血清留样检测。The schematic diagram of the mouse immunization and blood collection process is shown in Figure 13. PcrV-mRNA-LNP, mRNA-OprF-I-LNP and blank LNP were used to immunize balb/c mice through intramuscular injection. The immunization doses were set at 5 and 25 μg. The immunization volume to 100μL. The second booster immunization is carried out 3 weeks after the first immunization. The dose, volume and method of immunization are the same as those of the first immunization. Blood was collected from the orbital venous plexus at 1, 3, and 5 weeks after the initial immunization, and serum samples were separated for testing.
12、酶联免疫吸附实验(ELISA)测定小鼠特异性抗体效价12. Enzyme-linked immunosorbent assay (ELISA) to determine mouse-specific antibody titer
对于免疫前和免疫后各次分离的血清，采用间接ELISA的方法进行特异性抗体效价测定，具体操作步骤如下：将大肠杆菌表达的PcrV和OprF包被空白酶标板，4℃过夜包被液；PBSU洗涤3次，每孔加入1％BSA37℃封闭2h，PBSU洗涤3次，每孔加入100μL梯度稀释的小鼠血清，37℃作用60min，轻轻震荡；PBSU洗涤3次，加入100μL HRP标记的兔抗鼠IgG或IgG1、IgG2A(1：1000稀释)，37℃作用60min，轻轻震荡；PBSU洗涤3次，加入HRP显色底物UMB 100μL，室温15min避光反应；加入100μL2％的硫酸终止液；OD450检测吸光度。图14.1～14.4为ELISA结果。其中，图14.1和14.2分别为mRNA-pcrv-LNP(5，25μg)和mRNA-OprF-I-LNP(5，25μg)第二次免疫小鼠后一周检测的总IgG抗体滴度，每个剂量组重复实验2次。图中，横坐标为小鼠免疫血清的稀释倍数，纵坐标为OD450的读数，其中在图14.1中的每一栏从左到右依次为mRNA-pcrv-LNP-5μg-1、mRNA-pcrv-LNP--2、mRNA-pcrv-LNP-25μg-1、mRNA-pcrv-LNP-25μg-2、blank(空白对照组)组，在图14.2中的每一栏从左到右依次为mRNA-OprF-I-LNP-5μg-1、mRNA-OprF-I-LNP--2、mRNA-OprF-I-LNP-25μg-1、mRNA-OprF-I-LNP-25μg-2、blank(空白对照组)组。图14.3为疫苗免疫7天后的IgM抗体检测结果。在检测抗体总IgG滴度的基础上，进一步对抗体的亚型进行了分析，图14.4的结果显示mRNA-pcrv-LNP诱导的IgG1和IgG2a抗体水平明显高于mRNA-OprF-I-LNP所诱导的IgG1和IgG2a抗体水平。总之，实验表明，本实施例制备的mRNA疫苗免疫小鼠能够诱导到效的体液免疫。For the serum separated before and after immunization, the specific antibody titer was measured using the indirect ELISA method. The specific steps are as follows: Coat the blank enzyme plate with PcrV and OprF expressed in E. coli, and coat overnight at 4°C. solution; wash with PBSU 3 times, add 1% BSA to each well and block for 2 hours at 37°C. Wash with PBSU 3 times, add 100 μL of gradient diluted mouse serum to each well, incubate at 37°C for 60 min, shake gently; wash with PBSU 3 times, add 100 μL HRP Labeled rabbit anti-mouse IgG or IgG1, IgG2A (1:1000 dilution), incubate at 37°C for 60 minutes, shake gently; wash with PBSU 3 times, add 100μL of HRP chromogenic substrate UMB, react at room temperature for 15min in the dark; add 100μL of 2% Sulfuric acid stop solution; detect absorbance with OD450. Figures 14.1 to 14.4 show the ELISA results. Among them, Figures 14.1 and 14.2 respectively show the total IgG antibody titer detected one week after the second immunization of mice with mRNA-pcrv-LNP (5, 25 μg) and mRNA-OprF-I-LNP (5, 25 μg), each dose The group repeated the experiment 2 times. In the figure, the abscissa is the dilution factor of mouse immune serum, and the ordinate is the reading of OD450. Each column in Figure 14.1 from left to right is mRNA-pcrv-LNP-5μg-1, mRNA-pcrv- LNP--2, mRNA-pcrv-LNP-25μg-1, mRNA-pcrv-LNP-25μg-2, blank (blank control group) group, each column in Figure 14.2 is mRNA-OprF from left to right. -I-LNP-5μg-1, mRNA-OprF-I-LNP--2, mRNA-OprF-I-LNP-25μg-1, mRNA-OprF-I-LNP-25μg-2, blank (blank control group) Group. Figure 14.3 shows the IgM antibody detection results 7 days after vaccine immunization. On the basis of detecting the total IgG titer of the antibody, the subtypes of the antibodies were further analyzed. The results in Figure 14.4 show that the levels of IgG1 and IgG2a antibodies induced by mRNA-pcrv-LNP were significantly higher than those induced by mRNA-OprF-I-LNP. IgG1 and IgG2a antibody levels. In summary, experiments show that mice immunized with the mRNA vaccine prepared in this example can induce effective humoral immunity.
13、小鼠烧伤模型的建立13. Establishment of mouse burn model
图15所示为小鼠烧伤模型构建流程示意图，BALB/c小鼠，免疫两次后再间隔一个月。所有小鼠(免疫组和未免疫组，体重20～30g)首先用250μL 2.5％AverUin麻醉，然后用脱毛膏对小鼠腰部右侧和身体背部进行脱毛，最后将直径为22mm、长度为100mm、重量为165g的金属块加热到104℃，并在动物的剃毛部位上施加8s的灼伤，以产生3级烧伤。之后，小鼠立即接受腹膜注射500μL的0.9％盐水和40μL美洛昔康(1mg/kg)，每24h一次，以防止它们受到刺激和感到疼痛。Figure 15 shows a schematic diagram of the construction process of mouse burn model. BALB/c mice were immunized twice and then separated by one month. All mice (immune group and unimmunized group, weight 20-30g) were first anesthetized with 250μL 2.5% AverUin, and then used depilatory cream to remove hair on the right side of the waist and back of the body. Finally, the mice were 22mm in diameter, 100mm in length, A metal block weighing 165 g was heated to 104°C and burned on the animal's shaved area for 8 s to produce 3rd degree burns. Immediately thereafter, mice received intraperitoneal injections of 500 μL of 0.9% saline and 40 μL of meloxicam (1 mg/kg) every 24 h to prevent them from being irritated and feeling pain.
14、铜绿假单胞菌感染小鼠的半数致死量(LD 50)测定14. Determination of the median lethal dose (LD 50) of mice infected with Pseudomonas aeruginosa
半数致死量是指在一定条件下能引起50％实验动物死亡的最小细菌数，其测定方法具体如下：首先通过使用nanodrop微量分光光度计测定OD600的读数来测量细菌浓度，然后将不同浓度(10^2、10^3、10^4、5×10^4、5x10^5、10^5、10^6、10^7、10^8)的铜绿假单胞菌菌株(PAO1)皮下注射于所有小鼠的烧伤中心，通过统计学分析小鼠死亡情况，最后使用SPSS软件对结果进行分析，并确定LD50、2×LD50、5×LD50和10×LD50剂量，该测试重复进行3次。The median lethal dose refers to the minimum number of bacteria that can cause the death of 50% of experimental animals under certain conditions. The determination method is as follows: first, measure the bacterial concentration by using a nanodrop micro-volume spectrophotometer to measure the OD600 reading, and then divide different concentrations (10 ^2, 10^3, 10^4, 5×10^4, 5x10^5, 10^5, 10^6, 10^7, 10^8) Pseudomonas aeruginosa strains (PAO1) were injected subcutaneously in The burn center of all mice was statistically analyzed for mouse death. Finally, SPSS software was used to analyze the results and determine the LD50, 2 × LD50, 5 × LD50, and 10 × LD50 doses. The test was repeated three times.
15、小鼠攻毒评价mRNA疫苗保护效果15. Evaluate the protective effect of mRNA vaccine by challenging mice
本实施例中，通过铜绿假单胞菌的攻毒实验来评价疫苗的保护效果，具体实施方法如下：在mRNA疫苗第二次注射后的第14天，在小鼠(疫苗免疫组和对照组)烧伤中心以50×LD50的剂量皮下注射铜绿假单胞菌菌株(PAO1)，并观察记录小鼠伤口，死亡情况。对于死亡小鼠，解剖小鼠取各脏器，并测定带菌量。图16的攻毒实验结果显示，相比于LNP空白免疫对照组小鼠全部死亡，提前免疫PcrV-mRNA疫苗的5μg和25μg的小鼠无一死亡，即存活率为100％，显示疫苗具有100％的保护效果。提前免疫mRNA-OprF-I疫苗的5μg组小鼠存活率可达50％，25μg组疫苗的小鼠存活率达到66.67％。In this example, the protective effect of the vaccine was evaluated through a challenge experiment with Pseudomonas aeruginosa. The specific implementation method is as follows: on the 14th day after the second injection of the mRNA vaccine, in mice (vaccine immunization group and control group ) In the burn center, Pseudomonas aeruginosa strain (PAO1) was subcutaneously injected at a dose of 50×LD50, and the wounds and death of the mice were observed and recorded. For dead mice, the mice were dissected to remove various organs, and the amount of bacteria carried was measured. The results of the challenge experiment in Figure 16 show that compared with all the mice in the LNP blank immunization control group, none of the mice immunized with 5 μg and 25 μg of PcrV-mRNA vaccine in advance died, that is, the survival rate was 100%, showing that the vaccine has 100 % protective effect. The survival rate of mice in the 5 μg group of mice immunized in advance with the mRNA-OprF-I vaccine reached 50%, and the survival rate of mice in the 25 μg group of vaccine reached 66.67%.
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。The above specific examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. For those skilled in the technical field to which the present invention belongs, several simple deductions, modifications or substitutions can be made based on the ideas of the present invention.
Claims (10)
- 一种mRNA分子，所述mRNA分子编码如下蛋白中的至少一种：1)PcrV蛋白；2)OprF蛋白以及OprI蛋白；An mRNA molecule encoding at least one of the following proteins: 1) PcrV protein; 2) OprF protein and OprI protein;自N端至C端，所述PcrV蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the PcrV protein contains the following amino acid sequence:MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI(SEQ ID No.7)；MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSI KDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI(SEQ ID No.7);自N端至C端，所述OprF蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprF protein contains the following amino acid sequence:MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE(SEQ ID No.8)；MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE(SEQ ID No.8);自N端至C端，所述OprI蛋白含有如下氨基酸序列：From N-terminus to C-terminus, the OprI protein contains the following amino acid sequence:HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK(SEQ ID No.9)。HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK(SEQ ID No.9).
- 如权利要求1所述的mRNA分子，其特征在于，SEQ ID No.8所示氨基酸序列与SEQ ID No.9所示氨基酸序列位于同一氨基酸序列；The mRNA molecule of claim 1, wherein the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are located in the same amino acid sequence;优选地，SEQ ID No.8所示氨基酸序列与SEQ ID No.9所示氨基酸序列通过第一接头序列串联；Preferably, the amino acid sequence shown in SEQ ID No. 8 and the amino acid sequence shown in SEQ ID No. 9 are connected in series through the first linker sequence;优选地，SEQ ID No.8所示氨基酸序列的C端串联至所述第一接头序列的N端，SEQ ID No.9所示氨基酸序列的N端串联至第一接头序列的C端；Preferably, the C-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the N-terminus of the first linker sequence, and the N-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the C-terminus of the first linker sequence;优选地，SEQ ID No.9所示氨基酸序列的C端串联至所述第一接头序列的N端，SEQ ID No.8所示氨基酸序列的N端串联至第一接头序列的C端；Preferably, the C-terminus of the amino acid sequence shown in SEQ ID No. 9 is connected in series to the N-terminus of the first linker sequence, and the N-terminus of the amino acid sequence shown in SEQ ID No. 8 is connected in series to the C-terminus of the first linker sequence;优选地，SEQ ID No.7所示氨基酸序列的C端依次串联有第二接头序列、加尾氨基酸序列；Preferably, the C-terminus of the amino acid sequence shown in SEQ ID No. 7 is sequentially connected in series with a second linker sequence and a tailed amino acid sequence;优选地，SEQ ID No.8、SEQ ID No.9所示氨基酸序列串联后，在串联序列的氨基酸序列C端依次串联有第二接头序列、加尾氨基酸序列；Preferably, after the amino acid sequences shown in SEQ ID No. 8 and SEQ ID No. 9 are concatenated, a second linker sequence and a tailed amino acid sequence are sequentially concatenated at the C-terminal end of the amino acid sequence of the concatenated sequence;优选地，所述第一接头序列含有甘氨酸、丝氨酸；Preferably, the first linker sequence contains glycine and serine;优选地，所述第二述接头序列含有甘氨酸、丝氨酸；Preferably, the second linker sequence contains glycine and serine;优选地，所述第一接头序列包括如下氨基酸序列：GSGSGSGSGS；Preferably, the first linker sequence includes the following amino acid sequence: GSGSGSGSGS;优选地，所述第二接头序列包括如下氨基酸序列：GGGS；Preferably, the second linker sequence includes the following amino acid sequence: GGGS;优选地，所述加尾氨基酸序列含有至少一个组氨酸；Preferably, the tailed amino acid sequence contains at least one histidine;优选地，所述加尾氨基酸序列含有6～12个组氨酸；Preferably, the tailed amino acid sequence contains 6 to 12 histidines;优选地，编码PcrV蛋白的mRNA含有如下核苷酸序列：Preferably, the mRNA encoding the PcrV protein contains the following nucleotide sequence:AUGGAGGUGAGAAACCUGAACGCCGCCCGGGAGCUGUUCCUGGACGAGCUCCUGGCCGCUUCCGCCGCCCCCGCCUCCGCUGAGCAAGAGGAACUGCUGGCUCUGCUGAGAAGCGAGAGAAUCGUCCUGGCCCACGCCGGCCAACCCCUGUCCGAGGCCCAAGUCCUGAAAGCUCUGGCCUGGCUGCUGGCUGCCAAUCCUAGCGCCCCUCCCGGCCAAGGCCUGGAGGUGCUGAGAGAGGUGCUGCAAGCUAGAAGACAGCCCGGCGCUCAGUGGGACCUGAGAGAGUUCCUGGUGAGCGCCUACUUCAGCCUGCACGGCAGACUGGACGAGGACGUGAUCGGCGUGUACAAGGACGUGCUGCAGACCCAAGACGGCAAGAGAAAGGCCCUGCUGGACGAGCUCAAGGCCCUCACCGCCGAGCUGAAGGUGUACAGCGUGAUUCAGAGCCAAAUCAACGCCGCCCUGAGCGCCAAGCAAGGCAUCAGAAUCGACGCCGGCGGCAUCGACCUGGUGGACCCCACCCUGUACGGCUACGCCGUGGGCGACCCUAGAUGGAAGGACAGCCCCGAGUACGCCCUGCUGAGCAACCUGGACACCUUCAGCGGCAAGCUGAGCAUCAAGGACUUCCUGAGCGGCAGCCCCAAGCAGAGCGGCGAGCUGAAGGGCCUGAGCGACGAGUACCCCUUCGAGAAGGACAACAACCCCGUGGGCAACUUCGCCACCACCGUGAGCGACAGAAGCAGACCCCUGAACGACAAGGUGAACGAGAAGAC CACCCUGCUGAACGACACAAGCAGCAGAUACAACAGCGCCGUGGAGGCCCUGAACAGAUUCAUUCAGAAGUACGACAGCGUGCUGAGAGACAUCCUGAGCGCCAUC(SEQ ID No.12)；AUGGAGGUGAGAAACCUGAACGCCGCCCGGGAGCUGUUCCUGGACGAGCUCCUGCCCGCUUCCGCCGCCCCCGCCUCCGCUGAGCAAGAGGAACUGCUGGCUCUGCUGAGAAGCGAGAGAAUCGUCCUGGCCCACGCCGGCCAACCCCUGUCCGAGGCCCAAGUCCUGAAAGCUCUGGCCUGGCUGCUGGCUGCCAAUCCUAGCGCCCCUCCCGGCCAAGGCCUGGAGGUGCUGAGAGAGGUGCUGCAA GCUAGAAGACAGCCCGGCGCUCAGUGGGACCUGAGAGUUCCUGGUGAGCGCCUACUUCAGCCUGCACGGCAGACUGGACGAGGACGUGAUCGGCGUGUACAAGGACGUGCUGCAGACCCAAGACGGCAAGAAAGGCCCUGCUGGACGAGCUCAAGGCCCUCACCGCCGAGCUGAAGGUGUACAGCGUGAUUCAGAGCCAAAUCAACGCCGCCCUGAGCGCCAAGCAAGGCAUCAGAAUCGACGCCGGCG GCAUCGACCUGGUGGACCCCACCCUGUACGGCUACGCCGUGGGCGACCCUAGAUGGAAGGACAGCCCCGAGUACGCCCUGCUGAGCAACCUGGACACCUUCAGCGGCAAGCUGAGCAUCAAGGACUUCCUGAGCGGCAGCCCCAAGCAGAGCGGCGAGCUGAAGGGCCUGAGCGACGAGUACCCCUUCGAGAAGGACAACAACCCCGUGGGCAACUUCGCCACCACCGUGAGCGACAGAAGCAGACCCCUGAACGACAA GGUGAACGAGAAGAC CACCCUGCUGAACGACACAAGCAGCAGAUACAACAGCGCCGUGGAGGCCCUGAACAGAUUCAUUCAGAAGUACGACAGCGUGCUGAGAGACAUCCUGAGCGCCAUC(SEQ ID No.12);优选地，编码OprF蛋白的mRNA含有如下核苷酸序列：Preferably, the mRNA encoding the OprF protein contains the following nucleotide sequence:AUGAACGCUUUCGCUGCUCCCGCCCCCGAGCCCGUGGCCGAUGUGUGUAGCGACAGCGACAACGACGGCGUGUGCGACAACGUGGACAAAUGUCCUGACACCCCUGCUAACGUGACCGUGGACGCCAACGGCUGCCCUGCCGUGGCCGAGGUGGUGAGAGUGCAGCUGGACGUGAAGUUCGACUUCGACAAGAGCAAGGUGAAGGAGAACAGCUACGCCGACAUCAAGAACCUGGCCGACUUCAUGAAGCAGUACCCUAGCACAAGCACCACCGUGGAGGGCCACACCGACAGCGUGGGCACCGACGCCUACAAUCAGAAGCUGAGCGAGAGAAGAGCCAACGCCGUGAGAGACGUGCUGGUGAACGAGUACGGCGUGGAGGGCGGCAGAGUGAACGCCGUGGGCUACGGCGAGAGCAGACCCGUGGCUGACAACGCCACCGCCGAGGGCAGAGCCAUCAACAGAAGAGUGGAG(SEQ ID No.13)；AUGAACGCUUUCGCUGCUCCCGCCCCGAGCCCGUGGCCGAUGUGUAGCGACAGCGACAACGACGGCGUGUGCGACAACGUGGACAAAUGUCCUGACACCCCUAGCUAACGUGACCGUGGACGCCAACGGCUGCCCUGCCGUGGCCGAGGUGGUGAGAGUGCAGCUGGACGUGAAGUUCGACUUCGACAAGAGCAAGGUGAAGGAGAACAGCUACGCCGACAUCAAGAACCUGGCCGACUUCAUGAAGCA GUACCCUAGCACAAGCACCACCGUGGAGGGCCACACCGACAGCGUGGGCACCGACGCCUACAAUCAGAAGCUGAGCGAGAGAAGAGCCAACGCCGUGAGACGUGCUGGUGAACGAGUACGGCGUGGAGGGCGGCAGAGUGAACGCCGUGGGCUACGGCGAGAGCAGACCCGUGGCUGACAACGCCACCGCCGAGGGCAGAGCCAUCAACAGAAGAGUGGGAG (SEQ ID No. 13);优选地，编码OprI蛋白的mRNA含有如下核苷酸序列：Preferably, the mRNA encoding the OprI protein contains the following nucleotide sequence:CACUCCAAGGAGACCGAGGCUAGACUGACCGCCACAGAGGACGCCGCCGCUAGAGCCCAAGCUAGAGCUGACGAGGCCUACAGAAAGGCCGACGAGGCCCUGGGCGCCGCUCAGAAGGCUCAGCAGACCGCCGACGAGGCUAAUGAGAGAGCCCUGAGAAUGCUGGAGAAGGCCUCCCGGAAG(SEQ ID No.14)；CACUCCAAGGAGACCGAGGCUAGACUGACCGCCACAGAGGACGCCGCCGCUAGAGCCCAAGCUAGAGCUGACGAGGCCUACAGAAAGGCCGACGAGGCCCUGGGCGCCGCUCAGAAGGCUCAGCAGACCGCCGACGAGGCUAAUGAGAGCCCUGAGAAUGCUGGAGAAGGCCUCCCGGAAG(SEQ ID No.14);优选地，所述mRNA分子从5’端到3’端依次包括：5’帽子结构、5’UTR序列、编码信号肽的核苷酸序列、编码PcrV蛋白和/或OprF、OprI蛋白的核苷酸序列、3’UTR序列、多聚腺苷酸序列；Preferably, the mRNA molecule includes in sequence from the 5' end to the 3' end: a 5' cap structure, a 5'UTR sequence, a nucleotide sequence encoding a signal peptide, and nucleosides encoding PcrV protein and/or OprF and OprI proteins. Acid sequence, 3'UTR sequence, poly(A) sequence;优选地，5’端帽子结构包括Cap0、Cap1、Cap2；Preferably, the 5' end cap structure includes Cap0, Cap1, and Cap2;优选地，所述5’UTR、3’UTR序列独立地来源于天然蛋白、人工合成蛋白中的至少一种；Preferably, the 5’UTR and 3’UTR sequences are independently derived from at least one of natural proteins and artificially synthesized proteins;优选地，所述天然蛋白包括α-球蛋白、β-球蛋白、热休克蛋白HSP70中的任意一种；Preferably, the natural protein includes any one of α-globulin, β-globulin, and heat shock protein HSP70;优选地，所述5’UTR序列与所述编码信号肽的核苷酸序列之间还含有Kozak序列；Preferably, the Kozak sequence is also contained between the 5' UTR sequence and the nucleotide sequence encoding the signal peptide;优选地，所述mRNA分子为未修饰或经修饰的；Preferably, the mRNA molecule is unmodified or modified;优选地，所述修饰包括：假尿苷三磷酸修饰、N1-甲基假尿苷三磷酸修饰；Preferably, the modification includes: pseudouridine triphosphate modification, N1-methylpseudouridine triphosphate modification;优选地，所述PcrV蛋白包含SEQ ID No.1所示氨基酸序列；Preferably, the PcrV protein contains the amino acid sequence shown in SEQ ID No. 1;优选地，编码所述PcrV蛋白的mRNA分子包含SEQ ID No.10所示核苷酸序列；Preferably, the mRNA molecule encoding the PcrV protein includes the nucleotide sequence shown in SEQ ID No. 10;优选地，OprF蛋白以及OprI蛋白包含SEQ ID No.6所示氨基酸序列；Preferably, the OprF protein and OprI protein comprise the amino acid sequence shown in SEQ ID No. 6;优选地，编码所述OprF蛋白以及OprI蛋白的mRNA包含SEQ ID No.11所示核苷酸序列。Preferably, the mRNA encoding the OprF protein and OprI protein includes the nucleotide sequence shown in SEQ ID No. 11.
- 负载有权利要求1～2任意一项所述mRNA分子的脂质纳米颗粒。Lipid nanoparticles loaded with the mRNA molecule according to any one of claims 1 to 2.
- 一种蛋白，其特征在于，所述蛋白包含权利要求1～2任意一项所述mRNA编码得到的氨基酸序列。A protein, characterized in that the protein includes the amino acid sequence encoded by the mRNA of any one of claims 1 to 2.
- 一种编码权利要求1～2任意一项所述mRNA分子的DNA分子。A DNA molecule encoding the mRNA molecule according to any one of claims 1 to 2.
- 含有权利要求5所述DNA分子的重组质粒。A recombinant plasmid containing the DNA molecule of claim 5.
- 一种疫苗，其特征在于，所述疫苗包含权利要求1～2任意一项所述mRNA分子、权利要求3所述脂质纳米颗粒、权利要求4所述蛋白、权利要求5所述DNA分子或权利要求6所述重组质粒。A vaccine, characterized in that the vaccine contains the mRNA molecule described in any one of claims 1 to 2, the lipid nanoparticle described in claim 3, the protein described in claim 4, the DNA molecule described in claim 5, or The recombinant plasmid of claim 6.
- 一种抗体，其特征在于，所述抗体是由权利要求7所述疫苗诱导产生并分离获得。An antibody, characterized in that the antibody is induced and produced by the vaccine according to claim 7 and is isolated.
- 权利要求1～2任意一项所述mRNA分子，或权利要求4所述蛋白，或权利要求5所述DNA分子，或权利要求6所述重组质粒在制备治疗和/或预防疾病的药物中的用途。The mRNA molecule of any one of claims 1 to 2, or the protein of claim 4, or the DNA molecule of claim 5, or the recombinant plasmid of claim 6 in the preparation of drugs for treating and/or preventing diseases. use.
- 如权利要求9所述的用途，其特征在于，所述疾病包括细菌引起的疾病；The use according to claim 9, wherein the disease includes a disease caused by bacteria;优选地，所述细菌包括假单胞菌目；Preferably, the bacteria comprise the order Pseudomonadales;优选地，所述细菌包括铜绿假单胞菌。Preferably, the bacterium includes Pseudomonas aeruginosa.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202280001765.XA CN117222740A (en) | 2022-06-07 | 2022-06-07 | mRNA vaccines encoding PcrV and/or OprF-I proteins |
| PCT/CN2022/097390 WO2023236041A1 (en) | 2022-06-07 | 2022-06-07 | Mrna vaccine encoding pcrv and/or oprf-i protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2022/097390 WO2023236041A1 (en) | 2022-06-07 | 2022-06-07 | Mrna vaccine encoding pcrv and/or oprf-i protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023236041A1 true WO2023236041A1 (en) | 2023-12-14 |
Family
ID=89039404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/097390 WO2023236041A1 (en) | 2022-06-07 | 2022-06-07 | Mrna vaccine encoding pcrv and/or oprf-i protein |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN117222740A (en) |
| WO (1) | WO2023236041A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103270047A (en) * | 2010-12-23 | 2013-08-28 | 因特塞尔奥地利股份公司 | Oprf/i agents and their use in hospitalized and other patients |
| CN108778322A (en) * | 2015-10-21 | 2018-11-09 | 葛兰素史密丝克莱恩生物有限公司 | The antigen vaccine of pseudomonas aeruginosa PCRV connections |
| CN112237623A (en) * | 2020-10-30 | 2021-01-19 | 中国人民解放军陆军军医大学第二附属医院 | Pseudomonas aeruginosa III type secretory protein PcrV and application thereof in induced polarization macrophage |
| CN113966343A (en) * | 2019-06-11 | 2022-01-21 | 瑞泽恩制药公司 | anti-PcrV antibodies that bind PcrV, compositions comprising anti-PcrV antibodies, and methods of use thereof |
| CN114480442A (en) * | 2020-10-27 | 2022-05-13 | 深圳吉诺因生物科技有限公司 | mRNA and novel coronavirus mRNA vaccine containing same |
-
2022
- 2022-06-07 CN CN202280001765.XA patent/CN117222740A/en active Pending
- 2022-06-07 WO PCT/CN2022/097390 patent/WO2023236041A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103270047A (en) * | 2010-12-23 | 2013-08-28 | 因特塞尔奥地利股份公司 | Oprf/i agents and their use in hospitalized and other patients |
| CN108778322A (en) * | 2015-10-21 | 2018-11-09 | 葛兰素史密丝克莱恩生物有限公司 | The antigen vaccine of pseudomonas aeruginosa PCRV connections |
| CN113966343A (en) * | 2019-06-11 | 2022-01-21 | 瑞泽恩制药公司 | anti-PcrV antibodies that bind PcrV, compositions comprising anti-PcrV antibodies, and methods of use thereof |
| CN114480442A (en) * | 2020-10-27 | 2022-05-13 | 深圳吉诺因生物科技有限公司 | mRNA and novel coronavirus mRNA vaccine containing same |
| CN112237623A (en) * | 2020-10-30 | 2021-01-19 | 中国人民解放军陆军军医大学第二附属医院 | Pseudomonas aeruginosa III type secretory protein PcrV and application thereof in induced polarization macrophage |
Non-Patent Citations (1)
| Title |
|---|
| FAKOOR MOHAMMAD HADI, MOUSAVI GARGARI SEYED LATIF, OWLIA PARVIZ, SABOKBAR AZAR: "Protective Efficacy of the OprF/OprI/PcrV Recombinant Chimeric Protein Against Pseudomonas aeruginosa in the Burned BALB/c Mouse Model", INFECTION AND DRUG RESISTANCE, vol. 13, pages 1651 - 1661, XP093114277, ISSN: 1178-6973, DOI: 10.2147/IDR.S244081 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117222740A (en) | 2023-12-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tiwari et al. | Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection | |
| JP2014518620A (en) | Liposome preparation | |
| JP2024514183A (en) | Epstein-Barr virus mRNA vaccine | |
| CN115297892A (en) | Compositions and methods for mRNA vaccines against novel coronavirus infections | |
| JP2009500037A5 (en) | ||
| EP2910634B1 (en) | Vaccine for preventing porcine edema disease | |
| TW202222821A (en) | Compositions and methods for the prevention and/or treatment of covid-19 | |
| US20160000901A1 (en) | Compositions and Methods for the Production of Virus-Like Particles | |
| KR20160022919A (en) | Modified matrix proteins of vesicular stomatitis virus | |
| US20230218746A1 (en) | Compositions and methods relating to antiviral therapeutics | |
| WO2021095838A1 (en) | Nucleic acid lipid particle vaccine encapsulating hpv mrna | |
| JP2023523423A (en) | Vaccine against SARS-CoV-2 and its preparation | |
| WO2023236041A1 (en) | Mrna vaccine encoding pcrv and/or oprf-i protein | |
| WO2023160654A1 (en) | Preparation and use of recombinant multicomponent sars-cov-2 trimeric protein vaccine capable of inducing broad-spectrum neutralizing activity | |
| CN116410992A (en) | mRNA and vaccine for preventing and/or treating novel coronavirus, and preparation method and application thereof | |
| Nazarian et al. | Preclinical study of formulated recombinant nucleocapsid protein, the receptor binding domain of the spike protein, and truncated spike (S1) protein as vaccine candidates against COVID-19 in animal models | |
| CA3020313A1 (en) | Novel pneumococcal vaccine formulations | |
| WO2022233287A1 (en) | Vaccine reagent and vaccination method | |
| TW202330922A (en) | Compositions and methods of ribonucleic acid respiratory syncytial virus (rsv) vaccines | |
| WO2021235503A1 (en) | Conjugated protein monomer supporting coronavirus protein, aggregate of said monomers, and component vaccine comprising said aggregate as active component | |
| WO2021253807A1 (en) | Anti-novel coronavirus complex and preparation method therefor | |
| CN117205309B (en) | Influenza immunogen composition, preparation method and application thereof | |
| WO2023215828A1 (en) | Nanoparticles for inducing a th1 t cell immune response | |
| WO2023202711A1 (en) | Mrna vaccine based on novel coronavirus | |
| RU2781294C1 (en) | CONJUGATE OF THE RECEPTOR-BINDING DOMAIN (RBD) PROTEIN OF SARS-CoV-2 SURFACE GLYCOPROTEIN S WITH POLYGLUCIN-SPERMIDINE (PGS) POLYMER AND VACCINE COMPLEX AGAINST CORONAVIRUS INFECTION COVID-19 BASED ON THE SPECIFIED CONJUGATE AND PLASMID DNA PVAX-RBD |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22945189 Country of ref document: EP Kind code of ref document: A1 |