CN113754749A - 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 - Google Patents
一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 Download PDFInfo
- Publication number
- CN113754749A CN113754749A CN202111179827.XA CN202111179827A CN113754749A CN 113754749 A CN113754749 A CN 113754749A CN 202111179827 A CN202111179827 A CN 202111179827A CN 113754749 A CN113754749 A CN 113754749A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- cryptosporidium parvum
- protein
- vaccine
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 97
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 91
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 91
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 65
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 63
- 241000223936 Cryptosporidium parvum Species 0.000 title claims abstract description 53
- 101000600770 Canis lupus familiaris Podoplanin Proteins 0.000 title claims abstract description 33
- 101000742428 Mycobacterium phage L5 Gene 40 protein Proteins 0.000 title claims abstract description 33
- 229940126580 vector vaccine Drugs 0.000 title abstract description 11
- 241000701161 unidentified adenovirus Species 0.000 title abstract description 10
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 20
- 229960005486 vaccine Drugs 0.000 claims abstract description 16
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 201000010099 disease Diseases 0.000 claims abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 210000004027 cell Anatomy 0.000 claims description 48
- 108020004707 nucleic acids Proteins 0.000 claims description 18
- 102000039446 nucleic acids Human genes 0.000 claims description 18
- 150000007523 nucleic acids Chemical class 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 230000009261 transgenic effect Effects 0.000 claims description 8
- 238000003259 recombinant expression Methods 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 230000035755 proliferation Effects 0.000 claims description 5
- 239000013603 viral vector Substances 0.000 claims description 5
- 208000008953 Cryptosporidiosis Diseases 0.000 claims description 3
- 206010011502 Cryptosporidiosis infection Diseases 0.000 claims description 2
- 229940021704 adenovirus vaccine Drugs 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 2
- 239000013642 negative control Substances 0.000 abstract description 11
- 238000000338 in vitro Methods 0.000 abstract description 10
- 102100037850 Interferon gamma Human genes 0.000 abstract description 8
- 108010074328 Interferon-gamma Proteins 0.000 abstract description 8
- 241000699670 Mus sp. Species 0.000 abstract description 7
- 241000700605 Viruses Species 0.000 abstract description 7
- 230000028993 immune response Effects 0.000 abstract description 6
- 230000036039 immunity Effects 0.000 abstract description 3
- 230000036737 immune function Effects 0.000 abstract description 2
- 101800000385 Transmembrane protein Proteins 0.000 abstract 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 abstract 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 abstract 1
- 238000004153 renaturation Methods 0.000 description 18
- 150000001413 amino acids Chemical class 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 108010090804 Streptavidin Proteins 0.000 description 11
- 239000002808 molecular sieve Substances 0.000 description 11
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 11
- 230000006287 biotinylation Effects 0.000 description 10
- 238000007413 biotinylation Methods 0.000 description 10
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 10
- 210000003000 inclusion body Anatomy 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 10
- 238000001890 transfection Methods 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 230000003053 immunization Effects 0.000 description 9
- 238000010186 staining Methods 0.000 description 9
- 238000004587 chromatography analysis Methods 0.000 description 8
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 8
- 238000002649 immunization Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 238000005342 ion exchange Methods 0.000 description 6
- 210000004989 spleen cell Anatomy 0.000 description 6
- 241000223935 Cryptosporidium Species 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000004255 ion exchange chromatography Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000012137 double-staining Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000003046 sporozoite Anatomy 0.000 description 4
- 238000011814 C57BL/6N mouse Methods 0.000 description 3
- 101710094581 Distal tail protein Proteins 0.000 description 3
- 101710195944 Gene 15 protein Proteins 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 101150069153 Cp15 gene Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 101000805533 Mycobacterium phage L5 Gene 60 protein Proteins 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000003250 oocyst Anatomy 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- PCDUALPXEOKZPE-DXCABUDRSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoic acid Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O PCDUALPXEOKZPE-DXCABUDRSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 1
- QOIGKCBMXUCDQU-KDXUFGMBSA-N Ala-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N)O QOIGKCBMXUCDQU-KDXUFGMBSA-N 0.000 description 1
- 241000224482 Apicomplexa Species 0.000 description 1
- UISQLSIBJKEJSS-GUBZILKMSA-N Arg-Arg-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(O)=O UISQLSIBJKEJSS-GUBZILKMSA-N 0.000 description 1
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 1
- IICZCLFBILYRCU-WHFBIAKZSA-N Asn-Gly-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IICZCLFBILYRCU-WHFBIAKZSA-N 0.000 description 1
- WONGRTVAMHFGBE-WDSKDSINSA-N Asn-Gly-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N WONGRTVAMHFGBE-WDSKDSINSA-N 0.000 description 1
- SNYCNNPOFYBCEK-ZLUOBGJFSA-N Asn-Ser-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O SNYCNNPOFYBCEK-ZLUOBGJFSA-N 0.000 description 1
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 1
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 1
- XACXDSRQIXRMNS-OLHMAJIHSA-N Asp-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)O XACXDSRQIXRMNS-OLHMAJIHSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- NZWDWXSWUQCNMG-GARJFASQSA-N Asp-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)N)C(=O)O NZWDWXSWUQCNMG-GARJFASQSA-N 0.000 description 1
- RPUYTJJZXQBWDT-SRVKXCTJSA-N Asp-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N RPUYTJJZXQBWDT-SRVKXCTJSA-N 0.000 description 1
- MGSVBZIBCCKGCY-ZLUOBGJFSA-N Asp-Ser-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MGSVBZIBCCKGCY-ZLUOBGJFSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- OXOQBEVULIBOSH-ZDLURKLDSA-N Cys-Gly-Thr Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O OXOQBEVULIBOSH-ZDLURKLDSA-N 0.000 description 1
- 101000662675 Enterobacteria phage T4 DNA topoisomerase small subunit Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- SHERTACNJPYHAR-ACZMJKKPSA-N Gln-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O SHERTACNJPYHAR-ACZMJKKPSA-N 0.000 description 1
- SMLDOQHTOAAFJQ-WDSKDSINSA-N Gln-Gly-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SMLDOQHTOAAFJQ-WDSKDSINSA-N 0.000 description 1
- VOUSELYGTNGEPB-NUMRIWBASA-N Gln-Thr-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O VOUSELYGTNGEPB-NUMRIWBASA-N 0.000 description 1
- CKRUHITYRFNUKW-WDSKDSINSA-N Glu-Asn-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CKRUHITYRFNUKW-WDSKDSINSA-N 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- MXJYXYDREQWUMS-XKBZYTNZSA-N Glu-Thr-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O MXJYXYDREQWUMS-XKBZYTNZSA-N 0.000 description 1
- BRFJMRSRMOMIMU-WHFBIAKZSA-N Gly-Ala-Asn Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O BRFJMRSRMOMIMU-WHFBIAKZSA-N 0.000 description 1
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 1
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 1
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 1
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- RMNMUUCYTMLWNA-ZPFDUUQYSA-N Ile-Lys-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N RMNMUUCYTMLWNA-ZPFDUUQYSA-N 0.000 description 1
- UDBPXJNOEWDBDF-XUXIUFHCSA-N Ile-Lys-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)O)N UDBPXJNOEWDBDF-XUXIUFHCSA-N 0.000 description 1
- UYODHPPSCXBNCS-XUXIUFHCSA-N Ile-Val-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C UYODHPPSCXBNCS-XUXIUFHCSA-N 0.000 description 1
- JZBVBOKASHNXAD-NAKRPEOUSA-N Ile-Val-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N JZBVBOKASHNXAD-NAKRPEOUSA-N 0.000 description 1
- KKXDHFKZWKLYGB-GUBZILKMSA-N Leu-Asn-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKXDHFKZWKLYGB-GUBZILKMSA-N 0.000 description 1
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- RVOMPSJXSRPFJT-DCAQKATOSA-N Lys-Ala-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RVOMPSJXSRPFJT-DCAQKATOSA-N 0.000 description 1
- PBIPLDMFHAICIP-DCAQKATOSA-N Lys-Glu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PBIPLDMFHAICIP-DCAQKATOSA-N 0.000 description 1
- SQRLLZAQNOQCEG-KKUMJFAQSA-N Lys-Tyr-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 SQRLLZAQNOQCEG-KKUMJFAQSA-N 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- WDTLNWHPIPCMMP-AVGNSLFASA-N Met-Arg-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O WDTLNWHPIPCMMP-AVGNSLFASA-N 0.000 description 1
- 241000525154 Micrathena beta Species 0.000 description 1
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 1
- 101100370002 Mus musculus Tnfsf14 gene Proteins 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- MHNBYYFXWDUGBW-RPTUDFQQSA-N Phe-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O MHNBYYFXWDUGBW-RPTUDFQQSA-N 0.000 description 1
- GNZCMRRSXOBHLC-JYJNAYRXSA-N Phe-Val-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N GNZCMRRSXOBHLC-JYJNAYRXSA-N 0.000 description 1
- 241000224017 Plasmodium berghei Species 0.000 description 1
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 1
- DBALDZKOTNSBFM-FXQIFTODSA-N Pro-Ala-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DBALDZKOTNSBFM-FXQIFTODSA-N 0.000 description 1
- AJLVKXCNXIJHDV-CIUDSAMLSA-N Pro-Ala-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O AJLVKXCNXIJHDV-CIUDSAMLSA-N 0.000 description 1
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- IMNVAOPEMFDAQD-NHCYSSNCSA-N Pro-Val-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IMNVAOPEMFDAQD-NHCYSSNCSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- QVOGDCQNGLBNCR-FXQIFTODSA-N Ser-Arg-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O QVOGDCQNGLBNCR-FXQIFTODSA-N 0.000 description 1
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- HEUVHBXOVZONPU-BJDJZHNGSA-N Ser-Leu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HEUVHBXOVZONPU-BJDJZHNGSA-N 0.000 description 1
- FLONGDPORFIVQW-XGEHTFHBSA-N Ser-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FLONGDPORFIVQW-XGEHTFHBSA-N 0.000 description 1
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 1
- JURQXQBJKUHGJS-UHFFFAOYSA-N Ser-Ser-Ser-Ser Chemical compound OCC(N)C(=O)NC(CO)C(=O)NC(CO)C(=O)NC(CO)C(O)=O JURQXQBJKUHGJS-UHFFFAOYSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- PQEQXWRVHQAAKS-SRVKXCTJSA-N Ser-Tyr-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=C(O)C=C1 PQEQXWRVHQAAKS-SRVKXCTJSA-N 0.000 description 1
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 1
- AQAMPXBRJJWPNI-JHEQGTHGSA-N Thr-Gly-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AQAMPXBRJJWPNI-JHEQGTHGSA-N 0.000 description 1
- XTCNBOBTROGWMW-RWRJDSDZSA-N Thr-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XTCNBOBTROGWMW-RWRJDSDZSA-N 0.000 description 1
- XYFISNXATOERFZ-OSUNSFLBSA-N Thr-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XYFISNXATOERFZ-OSUNSFLBSA-N 0.000 description 1
- MECLEFZMPPOEAC-VOAKCMCISA-N Thr-Leu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MECLEFZMPPOEAC-VOAKCMCISA-N 0.000 description 1
- NZRUWPIYECBYRK-HTUGSXCWSA-N Thr-Phe-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O NZRUWPIYECBYRK-HTUGSXCWSA-N 0.000 description 1
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 1
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 0.000 description 1
- VBMOVTMNHWPZJR-SUSMZKCASA-N Thr-Thr-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O VBMOVTMNHWPZJR-SUSMZKCASA-N 0.000 description 1
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 1
- PWONLXBUSVIZPH-RHYQMDGZSA-N Thr-Val-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O PWONLXBUSVIZPH-RHYQMDGZSA-N 0.000 description 1
- ACGIVBXINJFALS-HKUYNNGSSA-N Trp-Phe-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N ACGIVBXINJFALS-HKUYNNGSSA-N 0.000 description 1
- OOEUVMFKKZYSRX-LEWSCRJBSA-N Tyr-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N OOEUVMFKKZYSRX-LEWSCRJBSA-N 0.000 description 1
- QARCDOCCDOLJSF-HJPIBITLSA-N Tyr-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QARCDOCCDOLJSF-HJPIBITLSA-N 0.000 description 1
- FZSPNKUFROZBSG-ZKWXMUAHSA-N Val-Ala-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O FZSPNKUFROZBSG-ZKWXMUAHSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 1
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 1
- JXCOEPXCBVCTRD-JYJNAYRXSA-N Val-Tyr-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N JXCOEPXCBVCTRD-JYJNAYRXSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- -1 hnRNA Proteins 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 108010034507 methionyltryptophan Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 108700043516 mouse H-2Kb Proteins 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
-
- 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
- A61K39/002—Protozoa antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及一种微小隐孢子虫Gp40/15蛋白的免疫优势T细胞表位多肽,该表位多肽的氨基酸序列如SEQ ID NO.3所示。该多肽与H‑2Kb具有较高的体外亲和力且具有一定的稳定性。涉及一种重组腺病毒载体疫苗,该疫苗表达微小隐孢子虫表面糖蛋白gp40/15核苷酸,病毒载体疫苗经小鼠免疫后,能显著刺激小鼠的T细胞免疫应答。其所诱导CD8+T cell产生的IFN‑gamma,与各种阴性对照比较,P<0.01,具有显著差异。多肽免疫组特异性双阳性T细胞比例明显高于阴性对照,说明gp40/15蛋白及其相关多肽具有相应的生物免疫学功能,成为新型的微小隐孢子虫疾病表位疫苗或腺病毒载体疫苗。
Description
技术领域
本发明属于生物分子技术领域,涉及一种编码微小隐孢子虫Gp40/15蛋白的腺病毒载体疫苗及其T细胞表位多肽。
背景技术
微小隐孢子虫(Cryptosporidium parvum)隶属于顶复门(PhylumApicomplexa)的孢子虫纲(Class Sporozoa),于1912年首次由Tyzzer报道并命名,是一种重要的人畜共患性原虫。在已报道的31个人兽共患隐孢子虫种类中,能感染人和大多数哺乳动物的是微小隐孢子虫,且微小隐孢子虫宿主最为广泛,主要包括人、牛科、各种啮齿类哺乳动物、骆驼科、马科、犬科、非人灵长类和海洋哺乳动物及鱼。微小隐孢子虫子孢子表面粘附蛋白Gp60是微小隐孢子虫子孢子表面的主要抗原,2000年由Cevallos等和Strong等三个研究团队同时发现并报道,是特异性预防、治疗隐孢子虫感染的有效靶抗原。微小隐孢子虫前体糖蛋白Gp60经蛋白水解产生两种成熟的糖蛋白Gp40和Gp15,是参与隐孢子虫子孢子粘附和入侵宿主细胞的主要蛋白。Gp60羧基端保守区的Gp15通过糖基磷脂酰肌醇(glycophosphatidylinositol,GPI)锚定在子孢子表面,而胺基端多变区的Gp40具有α-N乙酰基半乳糖胺残基的序列特征,与人小肠上皮细胞受体结合。研究表明,识别Gp40的单抗具有阻止微小隐孢子虫入侵宿主细胞的能力;重组的Gp40/15蛋白可以刺激小鼠产生免疫保护性作用。
微小隐孢子虫为胞内寄生性原虫,其CTL表位由MHCⅠ分子呈递给CD8+T细胞,从而激活机体的细胞免疫应答。细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)介导的特异性细胞免疫应答在机体抗胞内微生物感染的过程中起着关键作用。微小隐孢子虫卵囊感染细胞后,其表面Gp40/15、Cp15、Cp23等蛋白在被感染细胞内经过一系列酶解过程,降解为8-11个氨基酸的多肽,再与相应的主要组织相容性复合体(Major histocompatibilitycomplex,MHC)I类分子抗原多肽结合槽结合,形成MHCI-抗原肽复合体,并呈递于感染细胞表面。MHCI-抗原肽复合体(pMHCI)被表达于T细胞表面的受体(T cell receptor,TCR)识别并与之结合。细胞毒性T淋巴细胞(CTL)可通过释放穿孔素或颗粒酶溶解胃肠道内感染细胞,释放未成熟的胞囊,切断隐孢子虫在细胞内的繁殖,从而有效控制了隐孢子虫的感染。研究表明,与体液免疫应答相比,细胞免疫应答在抗隐孢子虫感染中具有更重要的作用,且CD4+T细胞和CD8+T细胞在抗微小隐孢子虫(C.parvum)免疫中均具有重要作用。如Pantenburg等在对人CD8+T细胞清除肠上皮细胞内微小隐孢子虫感染的功能研究中发现,人HLAⅠ分子可通过呈递隐孢子Gp15蛋白的免疫优势表位,激活CD8+T细胞免疫应答。
发明内容
有鉴于此,本发明的目的在于提供一种编码微小隐孢子虫Gp40/15蛋白的腺病毒载体疫苗,及其相关表位多肽。
为达到上述目的,本发明提供如下技术方案:
1.一种微小隐孢子虫Gp40/15蛋白表位多肽,该多肽的氨基酸序列如SEQ ID NO.3所示。
2.编码氨基酸序列如SEQ ID NO.3所示微小隐孢子虫Gp40/15蛋白表位多肽的核酸。
进一步,所述核酸的核苷酸序列如SEQ ID NO.4所示。
3.含有编码氨基酸序列如SEQ ID NO.3所示微小隐孢子虫Gp40/15蛋白表位多肽的核酸的重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体。
进一步,所述核酸的核苷酸序列如SEQ ID NO.4所示。
本发明的核酸可以是RNA形式(例如mRNA、hnRNA、tRNA或任意其它形式),也可以是DNA形式(包括但不限于通过克隆或合成产生,或其任意组合而产生的cDNA和基因组DNA)。DNA可以是三链、双链或单链,或其任意组合。DNA或RNA的至少一条链的任意部分可以是编码链,也称作有义链,或可以是非编码链,也称作反义链。
4.氨基酸序列如SEQ ID NO.4所示的微小隐孢子虫Gp40/15蛋白表位多肽在制备预防或治疗微小隐孢子虫病的疫苗中的应用。
进一步,编码氨基酸序列如SEQ ID NO.3所示微小隐孢子虫Gp40/15蛋白表位多肽的核酸在制备预防或治疗微小隐孢子虫病的疫苗中的应用。
5.技术方案3所述重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体在制备预防或治疗微小隐孢子虫病的疫苗中的应用。
6.一种微小隐孢子虫疫苗,所述微小隐孢子虫疫苗的活性成分为如下物质中的至少一种:
a.微小隐孢子虫Gp40/15蛋白表位的多肽,氨基酸序列如SEQ ID NO.3所示;
b.编码氨基酸序列如SEQ ID NO.3所示表位多肽的核酸;
c.核苷酸序列如SEQ ID NO.4所示的核酸;
d.技术方案3所述的重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体。
进一步,所述疫苗为腺病毒疫苗。
7.技术方案1所述微小隐孢子虫Gp40/15蛋白表位的多肽在制备CD8+T淋巴细胞增殖剂中的应用。
8.技术方案2或3所述核酸、权利要求4所述重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体在制备CD8+T淋巴细胞增殖剂中的应用。
本发明的有益效果在于:本发明提供一种微小隐孢子Gp40/15蛋白表位P2,该多肽在小鼠体内能够引起特异性T细胞免疫应答,且与H-2Kb分子在体外具有一定的体外亲和力和稳定性,说明其具有相应的生物免疫学功能,为制备微小隐孢子虫疫苗提供一种新的方向。制备Gp40/15腺病毒载体疫苗,免疫C57BL/6N小鼠,实验结果表明,多肽P2能刺激C57BL/6N小鼠脾细胞特异性CTL免疫应答,其所诱导CD8+T cell产生的IFN-gamma与其它多肽和阴性对照比较,P<0.01,具有显著差异,说明多肽P2作为疫苗活性物质能有效刺激机体反应,并刺激特异性T细胞特异性分泌出IFN-gamma。通过流式细胞检测FITC标记的CD8+T与PE标记的IFN-γ双阳性细胞比例,其中阴性对照组为0.087%;P2多肽刺激组为1.23%,具有极显著差异。体外制备P2多肽的四聚体蛋白,检测结果表明,疫苗免疫组多肽特异性双阳性T细胞比例为0.84%,明显高于阴性对照(PBS免疫组)0.23%。说明本发明含有微小隐孢子Gp40/15蛋白表位多肽P2核酸制备的腺病毒载体疫苗能诱导产生对靶细胞具有杀伤活性的CTL,在微小隐孢子虫特异性免疫治疗领域有着巨大的开发应用潜力。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为H-2Kb、mβ2m与多肽P2复性后分子筛层析和离子交换层析结果。其中,A为为H-2Kb、mβ2m与多肽P2复性后分子筛层析结果;B为H-2Kb、mβ2m与多肽P2复性后离子交换层析结果;插入图片为分子筛层析后SDS-PAGE鉴定结果。
图2为P2多肽生物素化和四聚体蛋白的SDS-PAGE电泳鉴定图;其中A为生物素化结果鉴定图;B为多肽P2的四聚体蛋白SDS-PAGE电泳鉴定图。
图3为ELISPOT鉴定隐孢子虫表位的结果。其中,横坐标为4条多肽和阳性对照多肽(伯氏疟原虫,PbTRAP130-138:SALLNVDNL),阴性对照(未加入多肽刺激的细胞),纵坐标为斑点形成细胞数量,以斑点形成细胞(spot forming cells)SFC/106表示。其中多肽P2(Gp40/15-AIF9)产生的斑点数高于阳性对照多肽,与其他三条多肽相比亦具有显著差异(p<0.01)。
图4为P2多肽细胞内细胞因子染色结果。其中A为不加多肽时产生的斑点;B为加多肽刺激产生的斑点,C为P2多肽的细胞细胞表面FITC标记的CD8抗体和PE标记的IFN-γ双染结果。
图5为四聚体实验检测P2特异性T细胞。其中A为空白为染色对照;B为FITC标记的CD8+抗体单染对照;C为PE标记的四聚体的单染对照;D为多肽P2刺激组的双染色结果;F为阴性对照的双染色结果。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1微小隐孢子虫蛋白表位多肽的选择与合成。
对微小隐孢子虫的主要致病蛋白和表面蛋白Gp40/15、CSL、Gp900、Cp15和Cp23、进行扫描,通过以下网站预测(https://services.healthtech.dtu.dk/service.php?NetMHC-4.0)。共获得203个表位,其中H-2Kb限制性多肽表位107个,HLA-A*0201限制性多肽表位123个,预测多肽亲和力以IC50(nM)表示,多肽亲和力范围在2.9-1179.35nM之间,其中包括17条HLA-A*0201和H-2Kb交叉表位。(强结合多肽SB,亲和力阈值%Rank<0.500),弱结合多肽WB,0.500<亲和力阈值%Rank<2.000)。采用固相多肽合成法(Fmoc/tBu策略)合成多肽,合成后用HPLC纯化,其纯度为98%。合成后进一步采取实验验证其免疫原性。实验数据过于繁多,在此只描述与本发明相关的表位多肽P2(Gp40/15-AIF9),多肽的氨基酸序列如SEQ ID NO.3所示,其核苷酸序列如SEQ ID NO.4所示。多肽P2来源于Gp40/15蛋白。表1为部分预测的表位。
表1
多肽P2氨基酸序列如序列表中SEQ ID NO.3所示,核苷酸序列如序列表中SEQ IDNO.4所示。序列SEQ ID NO.1和SEQ ID NO.2分别是微小隐孢子虫Gp40/15蛋白的氨基酸序列和核苷酸序列。
实施例2蛋白体外复性实验检测P2表位多肽与小鼠轻链β2m(mouseβ2m,mβ2m)分子的亲和力
一、H-2Kb、mβ2m和多肽P2的折叠复性
将小鼠MHC I类分子重链(H-2Kb)和轻链(mβ2m)的包涵体蛋白以及多肽P2按照摩尔比1:1:3的比例加入包涵体复性试剂(Refolding Buffer)中。具体过程如下所述:
(1)将包涵体复性试剂(一般500ml体系,可按常规视复性效果而定)置于4℃冰箱(或冷库中)预冷。复性过程均在4℃条件下进行(4℃冰箱或冷库)。
(2)将盛有包涵体复性试剂的烧杯置于磁力搅拌器上,加入磁力转子,设置合适的搅拌速度(100-200rpm为宜)。
(3)取1mL盐酸胍溶解后的mβ2m包涵体(30mg/ml)加入注射器内,使之缓慢滴入包涵体复性试剂内,慢慢搅拌6-8h。
(4)将5mg多肽P2分别溶解在200μL DMSO中,然后用移液器直接加入到包涵体复性试剂中。
(5)慢慢搅拌30min后,再分别于注射器内加入3mL H-2Kb包涵体(30mg/ml),慢慢搅拌8-12h,可适当延长作用时间。
二、复合物蛋白的浓缩
复性结束后,在4℃冰箱或冷库中小心将复性液移至压力搅拌式浓缩杯中,采用氮气提供压力,在10kDa超滤膜上的浓缩溶液至体积约为30mL时加入120mL预冷的分子筛缓冲液。最后浓缩至30mL左右时将溶液转移至50mL离心管中,4℃,12000rpm离心15min除去沉淀,将上清转移至10KD蛋白浓缩管中,4℃,2400rpm进一步浓缩至2-5ml。如果复性后复性液变得浑浊,可低温离心去除沉淀后,再用压力搅拌式浓缩杯浓缩。
三、复合物蛋白的分子筛层析和离子交换层析鉴定
用分子筛缓冲液平衡Superdex 200 16/60HiLoad凝胶层析柱,平衡至基线(一般1ml/min,需2h左右),将浓缩并换液完毕之后的复合物蛋白样品离心除去沉淀(4℃,12000rpm,10min),用分子筛缓冲液洗涤快速蛋白液相色谱仪(FPLC)的上样泵后,取上清注入。在层析柱上以适当的流速运行(一般1ml/min),根据分子筛层析的结果检测复性效果,收集相应分子量目标峰蛋白,通过SDS-PAGE鉴定。将鉴定后为目的蛋白(复合物)的蛋白峰收集物用阴离子交换柱Resource Q进一步分析。先用离子交换液B(10mM Tris-HCl,1MNaCl,pH 8.0)平衡柱子,再用离子交换液A(10mM Tris-HCl,10mM NaCl,pH 8.0)平衡柱子后并上样,样品挂上柱子后,用由离子交换液A和离子交换液B组成的洗脱液进行线性梯度洗脱60分钟。在该60分钟内,洗脱液中离子交换液A的体积比由100%线性下降到50%,洗脱液中离子交换液B的体积比由50%线性上升到100%。收集洗脱峰,并进行SDS-PAGE鉴定。H-2Kb蛋白、小鼠mβ2m蛋白和多肽P2蛋白的复合物,记为H-2Kb/Gp40/15-AIF9。
结果如图1所示,其中A为H-2Kb、mβ2m与多肽P2(H-Kb分子量约32KD,mβ2m 10KD左右,P2只是一个短肽,可忽略不计)复性后分子筛层析结果。蛋白层析在280nm的紫外吸收条件下共出现3个主峰,其中峰1为H-2Kb重链的多聚体峰,峰2(目标蛋白峰)为H-2Kb、mβ2m和多肽形成的三分子复合体峰,收集其峰值处的蛋白用12%SDS-PAGE电泳分析,插入图片为多肽P2复性后分子筛层析图及收集的蛋白峰SDS-PAGE鉴定结果。结果显示在82mL洗脱体积处的峰2为H-2Kb/P2/mβ2m复合体单体,蛋白分子量约为42kDa,说明多肽与H-2Kb具有一定的体外亲和力;最后一个峰(峰3)在105mL洗脱体积左右,为mβ2m单体蛋白。
收集目标峰,进一步通过离子交换层析鉴定,发现多肽复合物在电导率12-16mS/cm下解离(结果见图1B),说明多肽在体外与H-Kb分子结合的稳定性较低,但后续实验表明,体外结合的稳定性并不影响多肽在体内的免疫原性。
实施例3、阳性多肽四聚体蛋白的构建
一、目的基因的扩增
根据H-2Kb的核苷酸序列,设计了一条上游引物SU1和两条下游引物SL-BSP1和SL-BSP2,其中上游引物引入BamHⅠ酶切位点,下游引物XhoI酶切位点。利用两条下游引物在H-2Kb下游引物序列区引入编码长度为15个氨基酸(GSLHHILDAQKMVWNH)的BSP(BirA酶的底物肽)的DNA序列以及编码Gly-Ser接头的碱基序列,引物信息详见表2。构建pET21a/H-2Kb-BSP质粒,提取H-2Kb-BSP包涵体,纯化H-2Kb-BSP-mβ2m/阳性多肽复合物;应用BirA酶将受体蛋白进行生物素化,通过分子筛层析除去生物素化的蛋白中多余的亲和素,生物素化蛋白测浓度后,按照一定比例加入PE-标记的链霉亲和素,制备阳性多肽的四聚体,4℃保存备用。
表2扩增小鼠H-2Kb-BSP基因所用引物
二、H-2Kb-BSP/AIF9四聚体的制备
1.肽复合物单体的制备
按照实施例2中的方法制备四聚体蛋白所需要的单体(H-2Kb蛋白、小鼠mβ2m蛋白和多肽蛋白的复合物,简称H-2Kb复合体蛋白),提取包涵体并进行体外复性。通过分子筛层析和离子交换层析纯化蛋白复合物。按照以下体系进行肽复合物的生物素化。
室温孵育过夜进行生物素化。
2.生物素化后H-2Kb-BSP/AIF9的纯化
分别将H-2Kb-BSP/AIF9复合物于4℃,12000rpm离心10min,去除沉淀,使用SuperdexTM200 16/60GL凝胶柱进行纯化,去除未结合D-生物素(D-biotin)。纯化后的生物素化复合物蛋白用10kDa的超滤管浓缩至500-1000μl,用BCA试剂盒测定其浓度,蛋白浓度为2-3mg/ml。
3.体外生物素化结果鉴定
取30μl链酶亲和素平均分成两份,其中15μl与上一步纯化浓缩后的生物素化复合物混合,4℃过夜结合。另一份作为链酶亲和素对照,再取相同质量的生物素化后的复合物蛋白稀释后作为样品对照,用12%SDS-PAGE对上述三份样品进行电泳分析,结果见图2中A。泳道1和3为作为对照的复合物和链霉亲和素,泳道2为生物素化和链霉亲和素的相结合的复合物,与泳道1和3相比,泳道2出现大于170KD的条带,说明生物素化是成功的,另一条100KD左右的条带,表示链霉亲和素仅结合一个复合物分子,也证实其生物素化是成功的。
4.四聚体的制备与鉴定
将生成四聚体所需的PE标记链霉亲和素分成10等份,取一份加入生物素化复合物纯化浓缩样品中,4℃避光孵育30min,期间不断混匀,重复上述步骤直至反应结束。然后将多聚化后的反应产物加至孔径为100kDa大小的超滤离心管中,4℃,2400g离心至体积小于50μl。再用PBS(pH8.0)将样品稀释至4ml,然后再次离心至体积小于50μl;用PBS(pH8.0)重复稀释四次后,再次加PBS(pH8.0)至4ml,再向体系中加8μl Na-EDTA(储存液浓度:0.5mol/L),2μl胃蛋白酶抑制剂(储存液浓度:1mg/mL),2μl抑亮蛋白酶肽(储存液浓度:2mg/mL)。继续浓缩四聚体浓度到2-2.5mg/mL,4℃避光保存。以生物素化复合物和PE标记的链酶亲和素为对照,取2μl四聚体于采用12%SDS-PAGE进行结果检测(结果见图2中B)。泳道1和3分别为等量的H-2Kb复合物和PE标记的链霉亲和素,泳道2为PE标记的四聚体分子;泳道2的复合物明显减少而且有比PE标记链霉亲和素要大的蛋白条带出现,说明生物素化后的H-2Kb-BSP/AIF9复合物与PE标记的链霉亲合素结合形成了四聚体。但是,图中可以发现四聚体中仍有少部分游离的生物素化复合物分子,采用100kDa的超滤离心管离心去除多余的蛋白,然后用PBS充分换液浓缩4次后,测定浓度约2-2.5mg/mL,避光保存于4℃。
实施例4、实验动物特异性CTL免疫应答功能检测。
一、实验动物免疫
病毒包装转染过程如下:
1.细胞铺板:
传代293T/(293FT),铺细胞前,将6厘米Dish用明胶包被,加0.5mL明胶,置于37℃培养箱包被20min,完全吸净,即可将细胞铺上(若第二天进行转染,铺1*106细胞,此时细胞密度大约为50-60%,第二天会达到80-90%)。第二天,观察细胞密度,80-90%满即可进行转染,转染前换培养基为10%血清无抗生素DMEM培养基。
2.转染过程
(1)在转染前2小时,移除细胞上原有的培养基,换为新鲜的完全培养基。
(2)将2μg质粒DNA用100μL无血清稀释液稀释,充分混匀后制成DNA稀释液。
注意:无血清稀释液建议采用Opti-MEM,无血清DMEM或150mM NaCl溶液。
(3)向DNA稀释液中直接加入2μL NeofectTM转染试剂,轻柔混匀,室温静置15-30分钟,转染复合物制备完成。
(4)将转染复合物加入细胞培养基中,轻柔混匀。
(5)继续培养24-48小时,收取细胞进行鉴定或加入相应抗生素筛选稳定克隆。
(6)分别收集转染后24h,48h(以转染后时间为开始时间)的病毒上清,用0.45um的滤器过滤病毒上清或3000rpm离心5分钟,收集后置于4度冰箱保存,4℃可保存14天。
应用构建的腺病毒载体疫苗免疫6-8周龄C57BL/6N小鼠,免疫剂量为109个病毒粒子/只,免疫方法为小鼠腿部肌肉注射。免疫后14天分离小鼠脾脏细胞免疫,并通过ELISPOT、细胞内细胞因子染色和四聚体技术检测小鼠体内T细胞免疫应答水平。
二、小鼠脾脏细胞的分离
颈椎脱臼法处死小鼠,放入75%酒精中,备用;分离脾脏,用镊子慢慢剥离脾脏;将分离的脾脏加入1640培养基中,用注射器头,采用40um的细胞筛进行研磨;将研磨液慢慢吸入离心管中,1500rpm(350-400g),4℃离心5min,离心后弃上清,用食指轻弹剩余液体4-5次,将细胞选起;加入细胞裂解液10ml,室温裂解10min,1500rpm离心5min;用1640培养基洗涤细胞1-2次,去除红细胞和结缔组织;离心后将上清小心移至新的15ml离心管中,取10ul细胞计数(显微镜下观察成亮的圆形白点)。
三、酶联免疫斑点技术(Enzyme-linked Immunospot Assay,ELISPOT)技术检测小鼠IFN-γ
取制备好的淋巴细胞(约106cells),铺板于处理好的ELISPOT板中,加入刺激物多肽(浓度为10ug/ml),并分别设阳性对照(ConA)、阴性对照(不加多肽)和空白对照(空白培养基);置于37℃CO2培养基中培养12-48h,在培养期间不能移动ELISPOR平板;培养结束后,弃掉细胞,用PBS洗涤5遍,每孔加入200ul;加入生物素化抗体(1ug/ml),37℃孵育2h;PBS洗涤5次,加入HRP-Streptavidin后于37℃孵育1h,PBS洗涤5次后加入底物显色;室温晾干后于ELISpot读数机上读数,结果见附图3,其中第4个为多肽P2,1-3是其余的多肽结果,1氨基酸序列为MIYDYNSGL,2为MIWHKSVNL,3为KMLDKYTRM,第5为阳性对照,第6为阴性对照。
四、细胞内细胞因子染色检测小鼠体内IFN-γ
取分离的小鼠脾脏细胞,阳性对照加入PMA(5ng/ml)和Ionomycin(500ng/ml)用37℃6h来培养和孵育细胞,多肽组加入多肽10ug/ml,37度5%CO2孵育12h,阴性对照加入同体积的DMSO。培养结束前5-6h,每1ml培养细胞加入3μl BrefeldinA(5mg/ml),充分混匀,抑制细胞因子的转运,培养5h。1500rpm离心3minutes来收集细胞,用含2%FBS的PBS溶液将细胞稀释到1x107细胞/mL分装入流式管;用PBS重悬两次洗涤,加入PE标记的CD8抗体,避光4度孵育30min,PBS洗涤细胞洗一遍;加入固定剂,室温固定细胞10min,FACS重悬;加破膜剂permeabilization wash buffer,室温避光反应30min,1000rpm离心5min,4℃离心,加入洗液1ml/管,洗细胞2次;用100ul FITC标记的抗小鼠IFN-gamma抗体重悬(抗体推荐稀释倍数:1:100in 1X Perm/Wash),孵育30min,500g,3minutes离心,加入洗液1ml/管洗涤细胞2次,1000rpm离心,5min;最后用300-400μl FACS buffer重悬细胞用于流式检测,结果见附图4,为P2多肽细胞内细胞因子染色结果。其中A为不加多肽时产生的斑点;B为加多肽刺激产生的斑点,C为P2多肽的细胞细胞表面FITC标记的CD8抗体和PE标记的IFN-γ双染结果。
五、四聚体技术检测多肽特异性T淋巴细胞水平
取2*106个细胞(约100ul),加入FITC标记的抗小鼠CD8抗体(浓度0.5mg/ml)1ul,4度避光作用30min后补加900ul PBS,1500rpm离心5min,加入FACS液体重悬细胞,加入PE标记的四聚体(1mg/ml)2ul,4度避光作用30min,4度用PBS洗涤三次后染色,流式细胞仪分析FITC和PE双阳性T细胞比例。同时设置空白调剂组,FITC标记的CD8单染组,PE标记的四聚体单染组,结果见图5。
分离多肽免疫组脾脏细胞和佐剂免疫组脾脏细胞,用上述制备的PE标记的H-2Kb-BSP/AIF9四聚体与FITC标记的抗小鼠CD8单抗进行避光染色,用流式细胞仪检测双阳性T细胞的比例,同时分别设立空白组细胞、FITC和PE标记的单染对照(图5A,5B,5C)。结果表明,多肽免疫组特异性双阳性T细胞比例为0.84%(图5D,5E),明显高于阴性对照(PBS免疫组)(0.23%)(图5F)。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 周口师范学院
<120> 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 324
<212> PRT
<213> 微小隐孢子虫(Cryptosporidium parvum)
<400> 1
Met Arg Leu Ser Leu Ile Ile Val Leu Leu Ser Val Ile Val Ser Ala
1 5 10 15
Val Phe Ser Ala Pro Ala Val Pro Leu Arg Gly Thr Leu Lys Asp Val
20 25 30
Pro Val Glu Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
35 40 45
Ser Ser Ser Ser Ser Thr Ser Thr Val Ala Pro Ala Asn Lys Ala Arg
50 55 60
Thr Gly Glu Asp Ala Glu Gly Ser Gln Asp Ser Ser Gly Thr Glu Ala
65 70 75 80
Ser Gly Ser Gln Gly Ser Glu Glu Glu Gly Ser Glu Asp Asp Gly Gln
85 90 95
Thr Ser Ala Ala Ser Gln Pro Thr Thr Pro Ala Gln Ser Glu Gly Ala
100 105 110
Thr Thr Glu Thr Ile Glu Ala Thr Pro Lys Glu Glu Cys Gly Thr Ser
115 120 125
Phe Val Met Trp Phe Gly Glu Gly Thr Pro Ala Ala Thr Leu Lys Cys
130 135 140
Gly Ala Tyr Thr Ile Val Tyr Ala Pro Ile Lys Asp Gln Thr Asp Pro
145 150 155 160
Ala Pro Arg Tyr Ile Ser Gly Glu Val Thr Ser Val Thr Phe Glu Lys
165 170 175
Ser Tyr Asn Thr Val Lys Ile Lys Val Asn Gly Gln Asp Phe Ser Thr
180 185 190
Leu Ser Ala Asn Ser Ser Ser Pro Thr Glu Asn Gly Gly Ser Ala Gly
195 200 205
Gln Ala Ser Ser Arg Ser Arg Arg Ser Leu Ser Glu Glu Thr Ser Glu
210 215 220
Ala Ala Ala Thr Val Asp Leu Phe Ala Phe Thr Leu Asp Gly Gly Lys
225 230 235 240
Arg Ile Glu Val Ala Val Pro Asn Val Glu Asp Ala Ser Lys Arg Asp
245 250 255
Lys Tyr Ser Leu Val Ala Asp Asp Lys Pro Phe Tyr Thr Gly Ala Asn
260 265 270
Ser Gly Thr Thr Asn Gly Val Tyr Arg Leu Asn Glu Asn Gly Asp Leu
275 280 285
Val Asp Lys Asp Asn Thr Val Leu Leu Lys Asp Ala Gly Ser Ser Ala
290 295 300
Phe Gly Leu Arg Tyr Ile Val Pro Ser Val Phe Ala Ile Phe Ala Ala
305 310 315 320
Leu Phe Val Leu
<210> 2
<211> 975
<212> DNA
<213> 微小隐孢子虫(Cryptosporidium parvum)
<400> 2
atgagattgt cgctcattat cgtattactc tccgttatag tctccgctgt attctcagcc 60
ccagccgttc cactcagagg aactttaaag gatgttcctg ttgagggctc gtcatcgtca 120
tcatcatcat catcatcatc atcatcatca tcatcatcaa catcaaccgt cgcaccagca 180
aataaggcaa gaactggaga agacgcagaa ggcagtcaag attctagtgg tactgaagct 240
tctggtagcc agggttctga agaggaaggt agtgaagacg atggccaaac tagtgctgct 300
tcccaaccca ctactccagc tcaaagtgaa ggcgcaacta ccgaaaccat agaagctact 360
ccaaaagaag aatgcggcac ttcatttgta atgtggttcg gagaaggtac cccagctgcg 420
acattgaagt gtggtgccta cactatcgtc tatgcaccta taaaagacca aacagatccc 480
gcaccaagat atatctctgg tgaagttaca tctgtaacct ttgaaaagag ttataataca 540
gttaaaatca aggttaacgg tcaggatttc agcactctct ctgctaattc aagtagtcca 600
actgaaaatg gcggatctgc gggtcaggct tcatcaagat caagaagatc actctcagag 660
gaaaccagtg aagctgctgc aaccgtcgat ttgtttgcct ttacccttga tggtggtaaa 720
agaattgaag tggctgtacc aaacgtcgaa gatgcatcta aaagagacaa gtacagtttg 780
gttgcagacg ataaaccttt ctataccggc gcaaacagcg gcactaccaa tggtgtctac 840
aggttgaatg agaacggaga cttggttgat aaggacaaca cagttctttt gaaggatgct 900
ggttcctctg cttttggact cagatacatc gttccttccg tttttgcaat ctttgcagcc 960
ttattcgtgt tgtaa 975
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Ile Phe Ala Ala Leu Phe Val Leu
1 5
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctatttttg cggcgctgtt tgtgctg 27
Claims (10)
1.一种微小隐孢子虫Gp40/15蛋白表位多肽,其特征在于,该多肽的氨基酸序列如SEQIDNO.3所示。
2.编码权利要求1所述微小隐孢子虫Gp40/15蛋白表位多肽的核酸。
3.根据权利要求2所述的核酸,其特征在于,所述核酸的核苷酸序列如SEQ ID NO.4所示。
4.含有权利要求2或3所述核酸的重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体。
5.权利要求1所述微小隐孢子虫Gp40/15蛋白表位多肽、权利要求2或3所述核酸在制备预防或治疗微小隐孢子虫病的疫苗中的应用。
6.权利要求4所述重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体在制备预防或治疗微小隐孢子虫病的疫苗中的应用。
7.一种微小隐孢子虫疫苗,其特征在于,所述微小隐孢子虫疫苗的活性成分为如下物质中的至少一种:
a.微小隐孢子虫Gp40/15蛋白表位多肽,氨基酸序列如SEQ ID NO.3所示;
b.编码氨基酸序列如SEQ ID NO.3所示表位多肽的核酸;
c.核苷酸序列如SEQ ID NO.4所示的核酸;
d.权利要求4所述的重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体。
8.权利要求7所述的微小隐孢子虫疫苗,其特征在于,所述疫苗为腺病毒疫苗。
9.权利要求1所述微小隐孢子虫Gp40/15蛋白表位多肽在制备CD8+T淋巴细胞增殖剂中的应用。
10.权利要求2或3所述核酸、权利要求4所述重组表达载体、表达盒、转基因细胞系、重组菌或重组病毒载体在制备CD8+T淋巴细胞增殖剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111179827.XA CN113754749B (zh) | 2021-10-09 | 2021-10-09 | 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111179827.XA CN113754749B (zh) | 2021-10-09 | 2021-10-09 | 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113754749A true CN113754749A (zh) | 2021-12-07 |
CN113754749B CN113754749B (zh) | 2024-02-09 |
Family
ID=78799046
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111179827.XA Active CN113754749B (zh) | 2021-10-09 | 2021-10-09 | 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113754749B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6323020B1 (en) * | 1996-08-23 | 2001-11-27 | North Carolina State University | Neutralization-sensitive epitopes of Cryptosporidium parvum |
CN101880328A (zh) * | 2010-06-02 | 2010-11-10 | 中国农业科学院上海兽医研究所 | 微小隐孢子虫Th多表位基因和融合蛋白及其应用 |
CN102276725A (zh) * | 2010-06-13 | 2011-12-14 | 中国农业科学院上海兽医研究所 | 隐孢子虫CTL和Th混合多表位基因和融合蛋白及应用 |
CN109942693A (zh) * | 2019-04-03 | 2019-06-28 | 周口师范学院 | 一种微小隐孢子虫的ctl表位多肽及其应用和疫苗 |
-
2021
- 2021-10-09 CN CN202111179827.XA patent/CN113754749B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6323020B1 (en) * | 1996-08-23 | 2001-11-27 | North Carolina State University | Neutralization-sensitive epitopes of Cryptosporidium parvum |
CN101880328A (zh) * | 2010-06-02 | 2010-11-10 | 中国农业科学院上海兽医研究所 | 微小隐孢子虫Th多表位基因和融合蛋白及其应用 |
CN102276725A (zh) * | 2010-06-13 | 2011-12-14 | 中国农业科学院上海兽医研究所 | 隐孢子虫CTL和Th混合多表位基因和融合蛋白及应用 |
CN109942693A (zh) * | 2019-04-03 | 2019-06-28 | 周口师范学院 | 一种微小隐孢子虫的ctl表位多肽及其应用和疫苗 |
Non-Patent Citations (1)
Title |
---|
GENBANK数据库: "gp40/15 [Cryptosporidium parvum], GenBank: AAM64092.1", GENBANK数据库 * |
Also Published As
Publication number | Publication date |
---|---|
CN113754749B (zh) | 2024-02-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2721574C2 (ru) | Вакцинная композиция против злокачественной опухоли | |
EP1923463B1 (en) | Cancer-rejection antigen peptide derived from glypican-3 (gpc3) for use in hla-a2-positive patient and pharmaceutical comprising the antigen | |
TW202003032A (zh) | 包含s t i n g 促效劑之胞外囊泡 | |
KR20130119952A (ko) | 감염성 질환, 자가면역 질환, 동종인자에 대한 면역 반응, 알레르기 질환, 종양, 이식편 거부 및 유전자 요법 또는 유전자 백신접종을 위해 사용되는 바이러스 벡터에 대한 면역 반응의 예방 및/또는 치료에 사용하기 위한 면역원성 펩티드 | |
CN108884152A (zh) | 鉴定结合细胞表面表位的抗体的筛选方法 | |
CN112426526B (zh) | 一种nk细胞的制备方法及其在治疗癌症中的应用 | |
TW201825122A (zh) | 用精胺酸耗盡劑進行之組合癌症免疫療法 | |
CA2462951A1 (en) | Isolation and purification of plasmodium falciparum merozoite protein-142 | |
CN109942693B (zh) | 一种微小隐孢子虫的ctl表位多肽及其应用和疫苗 | |
KR20220004115A (ko) | 항체로 장식된 자가-조립 단백질 나노케이지(sapna) 및 이의 일부 | |
CA2583361C (en) | Gene immunization using an antigen-chaperonin fusion | |
CN113754749A (zh) | 一种微小隐孢子虫Gp40/15蛋白表位多肽及其腺病毒载体疫苗 | |
US8378071B2 (en) | Peptide epitopes of VEGFR-2/KDR that inhibit angiogenesis | |
JP2021527419A (ja) | 免疫細胞操作用の安定化されたmhc分子を有するスカフォールド | |
CN109758575B (zh) | 充分多样的双亲性mhc ii结合多肽、免疫载体微球及其制备方法和应用 | |
CN112755051B (zh) | 一种nk细胞的制备及治疗癌症中的应用 | |
CN112300248B (zh) | 一类促进猪机体产生广谱性免疫应答的多肽及其应用 | |
TWI776048B (zh) | 預防豬瘟病毒感染之重組蛋白質及含其之組合物及細胞 | |
WO2020215266A1 (zh) | 预防猪瘟病毒感染的重组蛋白质及含其的组合物及细胞 | |
US20060233789A1 (en) | Process for producing antigenic substance | |
CN117659140A (zh) | 新冠病毒hla-a2限制性表位肽及应用 | |
JP2023527067A (ja) | 免疫調節複合体および治療のためのその使用 | |
JPH02264731A (ja) | アイメリア膜結合タンパク質免疫原 | |
KR101535704B1 (ko) | 항원성 단백질 고정화를 통한 돈단독 백신 제조방법 | |
CN117778363A (zh) | 一种α-烯醇化酶肿瘤抗原多肽及其肿瘤多肽疫苗及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |