CN116790560A - 一种芒果果实的几丁质酶过敏原及其应用 - Google Patents
一种芒果果实的几丁质酶过敏原及其应用 Download PDFInfo
- Publication number
- CN116790560A CN116790560A CN202210254140.6A CN202210254140A CN116790560A CN 116790560 A CN116790560 A CN 116790560A CN 202210254140 A CN202210254140 A CN 202210254140A CN 116790560 A CN116790560 A CN 116790560A
- Authority
- CN
- China
- Prior art keywords
- chitinase
- allergen
- mango
- gly
- protein
- 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.)
- Pending
Links
- 235000014826 Mangifera indica Nutrition 0.000 title claims abstract description 70
- 235000004936 Bromus mango Nutrition 0.000 title claims abstract description 67
- 235000009184 Spondias indica Nutrition 0.000 title claims abstract description 67
- 239000013566 allergen Substances 0.000 title claims abstract description 67
- 102000012286 Chitinases Human genes 0.000 title claims abstract description 63
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 63
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 30
- 240000007228 Mangifera indica Species 0.000 title description 4
- 241001093152 Mangifera Species 0.000 claims abstract description 66
- 208000026935 allergic disease Diseases 0.000 claims abstract description 21
- 206010020751 Hypersensitivity Diseases 0.000 claims abstract description 20
- 230000007815 allergy Effects 0.000 claims abstract description 19
- 239000003814 drug Substances 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108020004414 DNA Proteins 0.000 claims description 19
- 102000053602 DNA Human genes 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 2
- 230000002265 prevention Effects 0.000 claims 2
- 150000001413 amino acids Chemical class 0.000 abstract description 17
- 230000027455 binding Effects 0.000 abstract description 9
- 206010070834 Sensitisation Diseases 0.000 abstract description 5
- 230000008313 sensitization Effects 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 24
- 229960004784 allergens Drugs 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 10
- 230000000172 allergic effect Effects 0.000 description 8
- 208000010668 atopic eczema Diseases 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 8
- 238000003745 diagnosis Methods 0.000 description 7
- 238000003119 immunoblot Methods 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 238000003759 clinical diagnosis Methods 0.000 description 4
- 108010016616 cysteinylglycine Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 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 3
- 235000018185 Betula X alpestris Nutrition 0.000 description 3
- 235000018212 Betula X uliginosa Nutrition 0.000 description 3
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 108010047857 aspartylglycine Proteins 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241001247197 Cephalocarida Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010002537 Fruit Proteins Proteins 0.000 description 2
- QBEWLBKBGXVVPD-RYUDHWBXSA-N Gln-Phe-Gly Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCC(=O)N)N QBEWLBKBGXVVPD-RYUDHWBXSA-N 0.000 description 2
- APQYGMBHIVXFML-OSUNSFLBSA-N Ile-Val-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N APQYGMBHIVXFML-OSUNSFLBSA-N 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 2
- FQNUWOHNGJWNLM-QWRGUYRKSA-N Tyr-Cys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)NCC(O)=O FQNUWOHNGJWNLM-QWRGUYRKSA-N 0.000 description 2
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 2
- MDYSKHBSPXUOPV-JSGCOSHPSA-N Val-Gly-Phe Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MDYSKHBSPXUOPV-JSGCOSHPSA-N 0.000 description 2
- 108010092854 aspartyllysine Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 108010087823 glycyltyrosine Proteins 0.000 description 2
- 108091006086 inhibitor proteins Proteins 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 1
- 101710149506 28 kDa protein Proteins 0.000 description 1
- AAQGRPOPTAUUBM-ZLUOBGJFSA-N Ala-Ala-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O AAQGRPOPTAUUBM-ZLUOBGJFSA-N 0.000 description 1
- PIPTUBPKYFRLCP-NHCYSSNCSA-N Ala-Ala-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PIPTUBPKYFRLCP-NHCYSSNCSA-N 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- MKZCBYZBCINNJN-DLOVCJGASA-N Ala-Asp-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MKZCBYZBCINNJN-DLOVCJGASA-N 0.000 description 1
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- IORKCNUBHNIMKY-CIUDSAMLSA-N Ala-Pro-Glu Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O IORKCNUBHNIMKY-CIUDSAMLSA-N 0.000 description 1
- AUFACLFHBAGZEN-ZLUOBGJFSA-N Ala-Ser-Cys Chemical compound N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O AUFACLFHBAGZEN-ZLUOBGJFSA-N 0.000 description 1
- KLKARCOHVHLAJP-UWJYBYFXSA-N Ala-Tyr-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CS)C(O)=O KLKARCOHVHLAJP-UWJYBYFXSA-N 0.000 description 1
- 244000257727 Allium fistulosum Species 0.000 description 1
- 235000008553 Allium fistulosum Nutrition 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 1
- UAOSDDXCTBIPCA-QXEWZRGKSA-N Arg-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UAOSDDXCTBIPCA-QXEWZRGKSA-N 0.000 description 1
- 235000003826 Artemisia Nutrition 0.000 description 1
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 1
- FAEFJTCTNZTPHX-ACZMJKKPSA-N Asn-Gln-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FAEFJTCTNZTPHX-ACZMJKKPSA-N 0.000 description 1
- CTQIOCMSIJATNX-WHFBIAKZSA-N Asn-Gly-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O CTQIOCMSIJATNX-WHFBIAKZSA-N 0.000 description 1
- AMRANMVXQWXNAH-ZLUOBGJFSA-N Asp-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC(O)=O AMRANMVXQWXNAH-ZLUOBGJFSA-N 0.000 description 1
- KPSHWSWFPUDEGF-FXQIFTODSA-N Asp-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(O)=O KPSHWSWFPUDEGF-FXQIFTODSA-N 0.000 description 1
- UXIPUCUHQBIQOS-SRVKXCTJSA-N Asp-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UXIPUCUHQBIQOS-SRVKXCTJSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 101710137943 Complement control protein C3 Proteins 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- ATPDEYTYWVMINF-ZLUOBGJFSA-N Cys-Cys-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O ATPDEYTYWVMINF-ZLUOBGJFSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- VGTDBGYFVWOQTI-RYUDHWBXSA-N Gln-Gly-Phe Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VGTDBGYFVWOQTI-RYUDHWBXSA-N 0.000 description 1
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 1
- DOQUICBEISTQHE-CIUDSAMLSA-N Gln-Pro-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O DOQUICBEISTQHE-CIUDSAMLSA-N 0.000 description 1
- OKARHJKJTKFQBM-ACZMJKKPSA-N Gln-Ser-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OKARHJKJTKFQBM-ACZMJKKPSA-N 0.000 description 1
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 1
- GPSHCSTUYOQPAI-JHEQGTHGSA-N Glu-Thr-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O GPSHCSTUYOQPAI-JHEQGTHGSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 1
- BULIVUZUDBHKKZ-WDSKDSINSA-N Gly-Gln-Asn Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O BULIVUZUDBHKKZ-WDSKDSINSA-N 0.000 description 1
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 1
- QITBQGJOXQYMOA-ZETCQYMHSA-N Gly-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QITBQGJOXQYMOA-ZETCQYMHSA-N 0.000 description 1
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- VBOBNHSVQKKTOT-YUMQZZPRSA-N Gly-Lys-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O VBOBNHSVQKKTOT-YUMQZZPRSA-N 0.000 description 1
- GAFKBWKVXNERFA-QWRGUYRKSA-N Gly-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 GAFKBWKVXNERFA-QWRGUYRKSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- DNVDEMWIYLVIQU-RCOVLWMOSA-N Gly-Val-Asp Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O DNVDEMWIYLVIQU-RCOVLWMOSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- LJUIEESLIAZSFR-SRVKXCTJSA-N His-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N LJUIEESLIAZSFR-SRVKXCTJSA-N 0.000 description 1
- LKACSKJPTFSBHR-MNXVOIDGSA-N Ile-Gln-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N LKACSKJPTFSBHR-MNXVOIDGSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 1
- 125000003580 L-valyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(C([H])([H])[H])(C([H])([H])[H])[H] 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- WMTOVWLLDGQGCV-GUBZILKMSA-N Leu-Glu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N WMTOVWLLDGQGCV-GUBZILKMSA-N 0.000 description 1
- QJUWBDPGGYVRHY-YUMQZZPRSA-N Leu-Gly-Cys Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N QJUWBDPGGYVRHY-YUMQZZPRSA-N 0.000 description 1
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 1
- LFXSPAIBSZSTEM-PMVMPFDFSA-N Leu-Trp-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N LFXSPAIBSZSTEM-PMVMPFDFSA-N 0.000 description 1
- FZIJIFCXUCZHOL-CIUDSAMLSA-N Lys-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN FZIJIFCXUCZHOL-CIUDSAMLSA-N 0.000 description 1
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 1
- JGAMUXDWYSXYLM-SRVKXCTJSA-N Lys-Arg-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O JGAMUXDWYSXYLM-SRVKXCTJSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- GHKXHCMRAUYLBS-CIUDSAMLSA-N Lys-Ser-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O GHKXHCMRAUYLBS-CIUDSAMLSA-N 0.000 description 1
- FPQMQEOVSKMVMA-ACRUOGEOSA-N Lys-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CCCCN)N)O FPQMQEOVSKMVMA-ACRUOGEOSA-N 0.000 description 1
- MDDUIRLQCYVRDO-NHCYSSNCSA-N Lys-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN MDDUIRLQCYVRDO-NHCYSSNCSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- VOOINLQYUZOREH-SRVKXCTJSA-N Met-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N VOOINLQYUZOREH-SRVKXCTJSA-N 0.000 description 1
- 102100026933 Myelin-associated neurite-outgrowth inhibitor Human genes 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- YRKFKTQRVBJYLT-CQDKDKBSSA-N Phe-Ala-His Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 YRKFKTQRVBJYLT-CQDKDKBSSA-N 0.000 description 1
- KAJLHCWRWDSROH-BZSNNMDCSA-N Phe-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=CC=C1 KAJLHCWRWDSROH-BZSNNMDCSA-N 0.000 description 1
- AMBLXEMWFARNNQ-DCAQKATOSA-N Pro-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@@H]1CCCN1 AMBLXEMWFARNNQ-DCAQKATOSA-N 0.000 description 1
- XJROSHJRQTXWAE-XGEHTFHBSA-N Pro-Cys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XJROSHJRQTXWAE-XGEHTFHBSA-N 0.000 description 1
- OZAPWFHRPINHND-GUBZILKMSA-N Pro-Cys-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O OZAPWFHRPINHND-GUBZILKMSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 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
- DOSZISJPMCYEHT-NAKRPEOUSA-N Ser-Ile-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O DOSZISJPMCYEHT-NAKRPEOUSA-N 0.000 description 1
- RWDVVSKYZBNDCO-MELADBBJSA-N Ser-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CO)N)C(=O)O RWDVVSKYZBNDCO-MELADBBJSA-N 0.000 description 1
- ZKBKUWQVDWWSRI-BZSNNMDCSA-N Ser-Phe-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZKBKUWQVDWWSRI-BZSNNMDCSA-N 0.000 description 1
- 101710152003 Suppressor of silencing P0 Proteins 0.000 description 1
- GLQFKOVWXPPFTP-VEVYYDQMSA-N Thr-Arg-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GLQFKOVWXPPFTP-VEVYYDQMSA-N 0.000 description 1
- RCEHMXVEMNXRIW-IRIUXVKKSA-N Thr-Gln-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N)O RCEHMXVEMNXRIW-IRIUXVKKSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- BJJRNAVDQGREGC-HOUAVDHOSA-N Thr-Trp-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O BJJRNAVDQGREGC-HOUAVDHOSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- MICFJCRQBFSKPA-UMPQAUOISA-N Trp-Met-Thr Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)=CNC2=C1 MICFJCRQBFSKPA-UMPQAUOISA-N 0.000 description 1
- NSTPFWRAIDTNGH-BZSNNMDCSA-N Tyr-Asn-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NSTPFWRAIDTNGH-BZSNNMDCSA-N 0.000 description 1
- LVFZXRQQQDTBQH-IRIUXVKKSA-N Tyr-Thr-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LVFZXRQQQDTBQH-IRIUXVKKSA-N 0.000 description 1
- KSGKJSFPWSMJHK-JNPHEJMOSA-N Tyr-Tyr-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSGKJSFPWSMJHK-JNPHEJMOSA-N 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- HHSILIQTHXABKM-YDHLFZDLSA-N Val-Asp-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](Cc1ccccc1)C(O)=O HHSILIQTHXABKM-YDHLFZDLSA-N 0.000 description 1
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 1
- SVFRYKBZHUGKLP-QXEWZRGKSA-N Val-Met-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SVFRYKBZHUGKLP-QXEWZRGKSA-N 0.000 description 1
- QZKVWWIUSQGWMY-IHRRRGAJSA-N Val-Ser-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QZKVWWIUSQGWMY-IHRRRGAJSA-N 0.000 description 1
- TVGWMCTYUFBXAP-QTKMDUPCSA-N Val-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N)O TVGWMCTYUFBXAP-QTKMDUPCSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 229940074608 allergen extract Drugs 0.000 description 1
- 230000002009 allergenic effect Effects 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
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 1
- 244000030166 artemisia Species 0.000 description 1
- 235000009052 artemisia Nutrition 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 208000011318 facial edema Diseases 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229940068517 fruit extracts Drugs 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 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
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 108010025153 lysyl-alanyl-alanine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种芒果果实的几丁质酶过敏原及其应用，涉及生物技术相关技术领域。所述几丁质酶过敏原的氨基酸序列如SEQ ID No.1所示。本发明所述几丁质酶过敏原为第Ⅳ类几丁质酶蛋白，是芒果中鉴定到的第一个拥有完整分子信息和致敏性数据的过敏原，与现有芒果几丁质酶存在两个氨基酸差异，两个氨基酸差异位置有高于现有芒果几丁质酶的预测表位得分(0.503(85Val)和0.461(193Lys))，且具有高于现有芒果几丁质酶的IgE结合能力，对过敏原的潜在致敏性存在影响。该几丁质酶过敏原可被用于制备治疗或预防过敏症的药物，或者用于制备过敏原检测的试剂盒。
Description
技术领域
本发明涉及生物技术相关技术领域，特别是涉及一种芒果果实的几丁质酶过敏原及其应用。
背景技术
芒果是世界上最重要的水果作物之一，随着芒果消费量的增加，其引发的过敏问题也愈加严重。芒果引发的过敏可分为三类，分别是由IgE介导的Ⅰ型速发型过敏反应，由免疫细胞介导的Ⅳ型迟发型过敏反应，以及蛋白质接触过敏，其中由IgE介导的速发型超敏反应近年来的发生率持续增长。而目前临床上用芒果提取物对患者进行体内和体外诊断的效率低下，漏诊率较高，存在安全隐患。
过敏原组分的应用能够大大提高临床诊断的精确度和灵敏度，已经成为过敏性疾病诊断的发展趋势。而芒果果实过敏原分子鉴定的研究远远落后于其它水果。很多研究中利用患者血清免疫印迹显示，芒果中存在14kDa、17kDa、30kDa、40kDa、50kDa、60kDa和67kDa等多个过敏原，但目前为止，仅有两种过敏原分子得到鉴定，分别是14kDa的抑制蛋白以及37kDa的3-磷酸甘油醛脱氢酶，然而这两种过敏原以前并没有被纳入国际过敏原命名委员会(WHO/IUIS Allergen Nomenclature Home Page)，也没有应用于芒果过敏的诊断中。国内外的研究表明，芒果过敏常伴随着蒿花粉，桦树花粉，以及乳胶、胡萝卜、芹菜等的过敏。免疫抑制实验表明，芒果与蒿花粉、桦树花粉、乳胶之间均存在交叉反应，其中14kDa，27kDa，40kDa和67kDa的过敏原能够引起芒果与蒿花粉之间的交叉反应，而桦树花粉中的病程相关蛋白第10家族过敏原Bet v 1以及35kDa，40kDa，67kDa的过敏原能够引发与芒果之间的交叉反应。结合蛋白质组学及质谱技术，有研究鉴定到抑制蛋白，几丁质酶，β-1-3-葡聚糖酶等是芒果果实中潜在的过敏原，尤其是几丁质酶。
而已发表的研究中所报道的几丁质酶均只包含部分序列，N-端和C-端都不完整(NCBI序列号为ACD69683)，N-端缺少27个氨基酸，C-端缺少11个氨基酸(图1)，用不完整序列重组生产的过敏原对蛋白质的结构和IgE结合表位都有影响，无法应用于临床诊断。目前芒果中几丁质过敏原的完整分子信息还未得到表征，过敏原组分的缺乏使芒果过敏的临床诊断存在很大问题，国内外的研究均表明，使用芒果提取物(如ImmunoCAP系统)进行诊断时，其阳性检出率均不足30％，因此亟需对芒果果实过敏原信息进行表征并将其应用于组分诊断，以此提高诊断效率。
发明内容
为解决现有技术中的不足，本发明提供了一种芒果果实的几丁质酶过敏原及其应用。
本发明的技术方案如下：
本发明提供了一种芒果果实的几丁质酶过敏原，几丁质酶的氨基酸序列如SEQ IDNo.1所示，属于第Ⅳ类几丁质酶。
本发明又提供了编码上述几丁质酶过敏原的DNA分子，核苷酸序列如SEQ ID No.2所示。
本发明还提供了包含上述DNA分子的重组表达载体。所述重组表达载体可以为各种用于蛋白表达的质粒。
本发明还提供了包含上述DNA分子或包含上述重组表达载体的宿主生物体。所述宿主生物体可以为用于原核表达的大肠杆菌、用于真核表达的酵母、昆虫细胞、哺乳动物细胞(如CHO细胞)等各种蛋白表达系统。
本发明还提供了上述几丁质酶过敏原或上述DNA分子在制备治疗或预防芒果过敏症的药物中的应用。
本发明还提供了一种用于治疗或预防芒果过敏症的药物，包含上述几丁质酶过敏原或上述DNA分子中的至少一种。
优选的，所述药物可为脱敏免疫疫苗。
有效治疗过敏症的传统方法是特异性免疫治疗或脱敏，即以递增的剂量对过敏患者皮下注射天然过敏原提取物。
本发明还提供了上述几丁质酶过敏原或上述DNA分子在制备过敏原检测的试剂盒中的应用。
本发明还提供了一种用于过敏原检测的试剂盒，包含上述几丁质酶过敏原或上述DNA分子中的至少一种。
对于过敏症患者，一个重要的手段是检查出引起过敏的过敏原，通过有针对性的减少与过敏原的接触可有效预防过敏的发生。本发明所述几丁质酶过敏原或所述DNA分子可以用于单独或者与其他过敏原一起使用，制备成用于筛查过敏症患者的过敏原的试剂盒。
本发明一种芒果果实的几丁质酶过敏原，为第Ⅳ类几丁质酶蛋白，是芒果中鉴定到的第一个拥有完整分子信息和致敏性数据的过敏原，与现有芒果几丁质酶存在两个氨基酸差异，两个氨基酸差异位置有高于现有芒果几丁质酶的预测表位得分(0.503(85Val)和0.461(193Lys))，且具有高于现有芒果几丁质酶的IgE结合能力，对过敏原的潜在致敏性存在影响。该几丁质酶过敏原可被用于制备治疗或预防过敏症的药物，或者用于制备过敏原检测的试剂盒。
附图说明
图1为芒果两种几丁质酶序列比对图。
图2为实施例1中芒果果实提取物电泳和免疫印迹图，a图为果实提取物电泳图，ME表示果实提取物；图b为芒果果实提取物与过敏患者血清免疫印迹图，1-5代表不同的阳性血清与提取物的结合，N代表阴性对照。
图3为实施例3芒果天然几丁质酶的纯化图；图a表示分子筛纯化曲线，箭头所指的峰包含天然几丁质酶；图b-c表示天然几丁质酶电泳检测和免疫印迹；图d表示纯化后的天然几丁质酶的二级质朴结果，高亮部分为质谱匹配肽段。
图4为芒果果实天然几丁质酶分子量质谱图。
图5为重组几丁质酶电泳检测图。
图6为重组几丁质酶二级质谱图，高亮部分代表质谱匹配肽段。
具体实施方式
芒果果实“凯特”：采集自湛江。
芒果过敏阳性血清：来源于浙江大学医学院附属第二医院以及科研合作的山西省大同市第三人民医院临床病例，山西医科大学附属第一医院，伦理批号No.2020-050，2015-001，2019K-K0007。所有患者都同意并签有知情同意书。
实施例1
提取“凯特”芒果(Mangifera indica)果实总蛋白。蛋白提取纯化方法：取1g液氮研磨后的果实粉末用5mL预冷的丙酮在4℃下脱脂三次，每次1h，离心后加入5mL的PBS缓冲液在4℃下提取3h，离心取上清并用0.22μm Millipore膜过滤即为芒果果实蛋白。SDS-PAGE电泳对提取物进行检测，BCA法测定总蛋白含量。
将提取物好的芒果果实蛋白用12％浓度的SDS-PAGE电泳分离，然后将蛋白转膜到PVDF膜上，5％脱脂奶粉封闭，然后加入芒果过敏阳性血清和血清池进行孵育。结合的IgE用羊抗人IgE HRP(Thermo Fisher，货号A18793)(1∶3000)，加底物ECL化学发光检测(BioRadChemiDoc)，SDS-PAGE胶染色和免疫印迹的检测结果如图2所示，通过免疫印迹在芒果中发现了三种过敏原，分别为14kDa，17kDa以及28kDa，其中14和17kDa条带的亮度明显强于28kDa。
实施例2
分别提取“凯特”芒果果皮和果肉的mRNA，使用Illumina HiSeqTM2000进行转录组测序。通过SOAPnuke和trimmomatic对测序数据进行过滤，然后使用Trinity对序列进行组装和注释。利用Triple TOF 5600平台对芒果果实总蛋白进行测序，用Mascot将所得谱图与转录组数据库中的序列搜索匹配，最终共鉴定到5354个蛋白，19559个肽段。
利用Tbtools鉴定芒果果实中潜在的过敏原：将果实蛋白组数据比对到COMPARE过敏原数据库(http://db.comparedatabase.org/)，设定e值为10-5，序列相似度大于50％，最终鉴定到芒果果实中潜在的过敏原120种。
将实施例1中经SDS-PAGE鉴定分离到的分子量为28kD的蛋白条带割胶，进行蛋白酶解质谱测试，参照转录组和蛋白组鉴定的所有可能蛋白氨基酸序列，鉴定到该蛋白为第Ⅳ类几丁质酶，编码氨基酸278个，理论分子量为26468.49Da，等电点为5.78，为酸性蛋白，氨基酸序列如SEQ ID No.1所示，所编码的基因序列如SEQ ID No.2所示。
实施例3
将“凯特”芒果果实提取物(按实施例1所述方法提取)在冷冻干燥机上干燥至粉末状，用20mM Tris-HCl缓冲液(pH 7.0)溶解，用0.22μm滤膜过滤后，在AKTA蛋白纯化系统上用阴离子交换柱(HiPrep DEAE Sepharose Fast Flow，GE公司)进行层析，上样缓冲液为20mM Tris-HCl(pH 7.0)，洗脱缓冲液为20mM Tris-HCl、1M NaCl溶液(pH 7.0)，收集洗脱后的蛋白样品，冷冻干燥后溶解于PBS缓冲液中，SDS-PAGE检测，将含有28kDa蛋白的样品收集起来，上样至分子筛柱(Super 75 10/300GL型，GE公司)分离纯化目标蛋白，获得大小约为28kDa的蛋白(图3)。
纯化所得目标蛋白质谱分析，总体蛋白质分子量质谱为26445Da(图4)，与理论计算的相差0.8‰，胰肽酶解肽段质谱匹配的氨基酸覆盖度(下划线部分)达到整个氨基酸序列长度的39.6％(图3)，根据国际过敏原命名委员会的规则，将该过敏原命名为Man i 1。对应氨基酸序列，得到编码基因全长。纯化所得的天然几丁质酶按照实施例1中的方法，与芒果过敏患者血清免疫印迹，对其IgE结合活性进行检测，结果在28kDa处有结合(图3)，芒果果实天然几丁质酶分子量质谱如图4所示。
申请人通过多组学结合的手段，鉴定到了芒果几丁质酶过敏原的完整序列结构，该蛋白与已报道的芒果几丁质酶序列存在两个氨基酸差异(85Val/Ala和193Lys/Thr)(如图1所示)，根据空间结构预测，这两个差异位点均位于几丁质酶空间结构的表面，可能会对IgE抗体的识别和结合产生影响，赖氨酸(K)是过IgE抗体结合敏原蛋白最常见的抗原表位(具体参见文献Oezguen N,Zhou B,Negi SS,Ivanciuc O,et al.(2008).Comprehensive3D-modeling of allergenic proteins and amino acid composition of potentialconformational IgE epitopes.Molecular Immunology,45:3740-3747.)，此外，利用IEDB(https://www.iedb.org/)数据库对两种亚型的几丁质酶的表位进行预测发现，在本申请的过敏原Man i 1中，两个变异氨基酸所在的位置，其表位预测得分分别为0.503(85Val)和0.461(193Lys)，而GenBank号为ACD69683的几丁质酶序列的对应位置氨基酸预测表位得分分别为0.472(Ala)和0.436(Thr)，均低于Man i 1表位得分，如表1所示，这些均说明这两个位置上的氨基酸变异对过敏原的潜在致敏性存在影响，且Man i 1的IgE结合能力要高于GenBank号为ACD69683的几丁质酶。这也是以前的芒果几丁质酶过敏原抑制没有成功应用于临床诊断的原因。
表1几丁质酶过敏原亚型的表位预测
实施例4
提取“凯特”芒果果实mRNA，逆转录为cDNA，在转录组中找到芒果果实中编码Man i1蛋白的序列全长，并设计相应的特异性引物，上游引物为cagcaaatgggtcgcggatccATGGCTTTCAACATGAGAAAAAAT，下游引物为gtggtggtggtggtgctcgagCTAGCAGGACAAATTCTGGC，(下划线为引物接头)用2×I-5高保真酶对该序列进行克隆，用同源重组酶将回收后的基因片段连接到pET28a载体上，转化到大肠杆菌中，挑取单菌落测序。
测序正确的重组质粒转化到Rosetta菌株中，在200mL的含卡那霉素的LB培养基中扩增，37℃培养至OD600达到0.6-0.8，加入终浓度为0.2mM的IPTG，16℃诱导12h，离心后将菌体重悬至PBS缓冲液中，超声破碎机破碎20min，离心取上清，加入200μL的Ni-NTA，4℃孵育1h，用20-250mM的咪唑缓冲液梯度洗脱，SDS-PAGE检测(如图5所示)后收集含有28kDa的蛋白透析至PBS缓冲液，胶条上的样品进行LC-MS/MS质谱验证，与理论蛋白序列比对，覆盖度达到57.9％(如图6所示)。
实施例5
取实施例3中分离纯化的天然几丁质酶和实施例4中的重组几丁质酶3μg在4℃下过夜包板，5％脱脂奶粉37℃封闭2h后，加入100μL芒果过敏患者血清37℃孵育2h，然后加入100μL HRP标记的羊抗人IgE二抗(Thermo Fisher，货号A18793)(1：3000)37℃孵育2h，TMB避光显色15min后，用2M HCl终止反应，测定450nm处的吸光值，三个健康人的血清作为阴性对照，测试值大于阴性对照平均值加上标准差的三倍(阴性对照平均值+3SD，其中SD表示标准差)即视为阳性。ELISA测定后发现，在68位芒果过敏患者中，35位(52.2％)对天然几丁质酶(nMan i 1)阳性，而23位患者(35.4％)对重组的几丁质酶(rMan i 1)阳性(表2)，这个测定结果说明几丁质酶是芒果果实中的主要过敏原(超过50％的阳性率)。
表2
注：症状缩写：U——荨麻疹；C——结膜炎；OAS——口腔综合症；G——胃肠道症状；D——气紧；FA——面部水肿；S——打喷嚏。N1～N3为阴性对照血清。ImmunoCAP是测试过敏原常用方法，一般是大于0.35为阳性。CAP和ELISA所测定的sIgE值中，加粗的数值表示阳性值。ND-因为缺乏血清或者试剂没有检测。
序列表
<110> 浙江大学
<120> 一种芒果果实的几丁质酶过敏原及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 278
<212> PRT
<213> 芒果(Mangifera indica)
<400> 1
Met Ala Phe Asn Met Arg Lys Asn Leu Leu Thr Phe Ala Leu Leu Gly
1 5 10 15
Leu Phe Ser Leu Ala Ile Val Pro Lys Asn Val Met Ser Gln Asn Cys
20 25 30
Asp Cys Ala Pro Asn Leu Cys Cys Ser Gln Phe Gly Tyr Cys Gly Thr
35 40 45
Gly Glu Ala Tyr Cys Gly Leu Gly Cys Lys Gly Gly Pro Cys Thr Ser
50 55 60
Thr Pro Ser Thr Pro Ser Pro Thr Pro Thr Gly Gly Gly Ser Val Ala
65 70 75 80
Ser Ile Val Thr Val Asp Phe Phe Asp Gly Ile Lys Asn Gln Ala Ala
85 90 95
Ala Ser Cys Ala Gly Lys Ser Phe Tyr Thr Arg Asp Gly Phe Leu Asn
100 105 110
Ala Ala Asn Ser Phe Pro Gln Phe Gly Ser Gly Ser Ala Asp Glu Ser
115 120 125
Lys Arg Glu Ile Ala Ala Phe Phe Ala His Val Thr His Glu Thr Gly
130 135 140
His Leu Cys Tyr Thr Glu Glu Ile Asp Lys Ser Asn Ala Tyr Cys Asp
145 150 155 160
Gln Ser Asn Thr Gln Tyr Pro Cys Val Pro Gly Lys Lys Tyr Tyr Gly
165 170 175
Arg Gly Pro Met Gln Leu Thr Trp Asn Tyr Asn Tyr Gly Ala Cys Gly
180 185 190
Lys Ala Val Gly Phe Asp Gly Leu Asn Ala Pro Glu Thr Val Ser Asn
195 200 205
Asp Pro Ala Val Ser Phe Lys Ala Ala Leu Trp Phe Trp Met Thr Asn
210 215 220
Val His Ser Val Met Asn Gln Gly Phe Gly Ala Thr Ile Gln Lys Ile
225 230 235 240
Asn Gly Ala Leu Glu Cys Gly Gly Lys Gln Pro Asp Lys Val Asn Ala
245 250 255
Arg Ile Gly Tyr Tyr Thr Asp Tyr Cys Gln Lys Phe Gly Val Asp Pro
260 265 270
Gly Gln Asn Leu Ser Cys
275
<210> 2
<211> 834
<212> DNA
<213> 芒果(Mangifera indica)
<400> 2
atggctttca acatgagaaa aaatctactt acgtttgctc ttctgggact tttttccttg 60
gctattgttc ctaaaaatgt catgtcccaa aactgtgact gtgctcccaa cttgtgttgc 120
agtcagtttg gttactgtgg caccggcgaa gcctactgtg gattggggtg taaggggggt 180
ccttgcacct cgacgccatc gacaccgtca cctacaccaa ccggtggtgg ttcagttgcc 240
agtattgtta cggttgattt ctttgatggg ataaagaatc aagctgctgc aagctgtgct 300
ggaaagagct tctacacaag agatggattt cttaatgcag ccaattcgtt tcctcagttt 360
ggatcaggct ctgccgacga atccaagcgt gagattgctg cattttttgc ccatgttact 420
catgaaactg gacatttatg ctacaccgag gagattgaca agtcaaatgc ctactgtgac 480
caatcaaaca cacagtaccc atgtgtcccc ggaaagaagt attacgggcg tggaccgatg 540
cagctcacct ggaactacaa ctacggtgcc tgtggaaaag ccgtcgggtt cgatggactc 600
aacgctcccg aaacagtgtc taatgaccct gctgtctcct tcaaggctgc cttgtggttc 660
tggatgacca atgttcactc agtcatgaac caaggctttg gggctaccat tcagaaaatt 720
aatggcgctc ttgaatgcgg tggcaagcaa cctgataaag ttaatgctcg tattggttat 780
tacactgatt attgccagaa atttggtgtt gatcccggcc agaatttgtc ctgc 834
<210> 3
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cagcaaatgg gtcgcggatc catggctttc aacatgagaa aaaat 45
<210> 4
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtggtggtgg tggtgctcga gctagcagga caaattctgg c 41
<210> 5
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Val Ala Ser Ile Val Thr Val Asp Phe Phe Asp Gly
1 5 10
<210> 6
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Ala Cys Gly Lys Ala Val Gly Phe Asp Gly Leu
1 5 10
<210> 7
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Val Ala Ser Ile Val Thr Ala Asp Phe Phe Asp Gly
1 5 10
<210> 8
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Ala Cys Gly Thr Ala Val Gly Phe Asp Gly Leu
1 5 10
Claims (9)
1.一种芒果果实的几丁质酶过敏原，其特征在于，几丁质酶的氨基酸序列如SEQ IDNo.1所示。
2.编码如权利要求1所述几丁质酶过敏原的DNA分子，其特征在于，核苷酸序列如SEQID No.2所示。
3.包含如权利要求2所述DNA分子的重组表达载体。
4.包含如权利要求2所述DNA分子或包含如权利要求3所述重组表达载体的宿主生物体。
5.如权利要求1所述几丁质酶过敏原或如权利要求2所述DNA分子在制备治疗或预防芒果过敏症的药物中的应用。
6.一种用于治疗或预防芒果过敏症的药物，其特征在于，包含如权利要求1所述几丁质酶过敏原或如权利要求2所述DNA分子中的至少一种。
7.如权利要求6所述的药物，其特征在于，所述药物为脱敏免疫疫苗。
8.如权利要求1所述几丁质酶过敏原或如权利要求2所述DNA分子在制备过敏原检测的试剂盒中的应用。
9.一种用于过敏原检测的试剂盒，其特征在于，包含如权利要求1所述几丁质酶过敏原或如权利要求2所述DNA分子中的至少一种。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210254140.6A CN116790560A (zh) | 2022-03-15 | 2022-03-15 | 一种芒果果实的几丁质酶过敏原及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210254140.6A CN116790560A (zh) | 2022-03-15 | 2022-03-15 | 一种芒果果实的几丁质酶过敏原及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790560A true CN116790560A (zh) | 2023-09-22 |
Family
ID=88042487
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210254140.6A Pending CN116790560A (zh) | 2022-03-15 | 2022-03-15 | 一种芒果果实的几丁质酶过敏原及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790560A (zh) |
-
2022
- 2022-03-15 CN CN202210254140.6A patent/CN116790560A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Riascos et al. | The seed biotinylated protein of soybean (Glycine max): A boiling-resistant new allergen (Gly m 7) with the capacity to induce IgE-mediated allergic responses | |
| Diaz‐Perales et al. | cDNA cloning and heterologous expression of the major allergens from peach and apple belonging to the lipid‐transfer protein family | |
| Ahrazem et al. | Lipid transfer proteins and allergy to oranges | |
| Ciardiello et al. | Influence of the natural ripening stage, cold storage, and ethylene treatment on the protein and IgE-binding profiles of green and gold kiwi fruit extracts | |
| CN106967696A (zh) | 一种蒿属植物花粉的过敏原及其应用 | |
| US20230295251A1 (en) | Allergy antigen and epitope for same | |
| US20240108719A1 (en) | Allergy antigen and epitope thereof | |
| CN109942689B (zh) | 表位 | |
| CN112358536A (zh) | 拟穴青蟹致敏蛋白Scy p 1衍生物及其应用 | |
| Cui et al. | Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China | |
| Zhang et al. | Enhanced sensitivity of capture IgE‑ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens | |
| JP2009521910A (ja) | ＢｅｔｕｌａＶｅｒｒｕｃｏｓａ花粉の主要アレルゲンの低アレルギー性変種 | |
| WO2013171268A1 (en) | Allergen variants | |
| Nouri et al. | Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens | |
| Madan et al. | Use of a synthetic peptide epitope of Asp f 1, a major allergen or antigen of Aspergillus fumigatus, for improved immunodiagnosis of allergic bronchopulmonary aspergillosis | |
| CN116789776A (zh) | 一种芒果果实的抑制蛋白过敏原及其应用 | |
| CN116790560A (zh) | 一种芒果果实的几丁质酶过敏原及其应用 | |
| Nour Ibrahim et al. | Molecular cloning and immunochemical characterization of a new Japanese cedar pollen allergen homologous to plant subtilisin-like serine protease | |
| Chiu et al. | Secretome analysis of novel IgE‐binding proteins from Penicillium citrinum | |
| JPH09503125A (ja) | 組み換えクラドスポリウムヘルバルム（Ｃｌａｄｏｓｐｏｒｉｕｍｈｅｒｂａｒｕｍ）アレルゲン | |
| CN116554288A (zh) | 一种芒果果实的过敏原及其应用 | |
| Lee et al. | Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen | |
| EP3854456A1 (en) | Allergy antigen and epitope thereof | |
| CN114685636B (zh) | 蒿属植物花粉过敏原、应用及试剂盒 | |
| US7279295B2 (en) | Allergen |
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 |