CN107011420B - 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 - Google Patents
可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 Download PDFInfo
- Publication number
- CN107011420B CN107011420B CN201710354026.XA CN201710354026A CN107011420B CN 107011420 B CN107011420 B CN 107011420B CN 201710354026 A CN201710354026 A CN 201710354026A CN 107011420 B CN107011420 B CN 107011420B
- Authority
- CN
- China
- Prior art keywords
- protein
- tumor cells
- inducing
- programmed necrosis
- preparation
- 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.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 85
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 85
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 45
- 230000017074 necrotic cell death Effects 0.000 title claims abstract description 39
- 230000001939 inductive effect Effects 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 9
- 238000011033 desalting Methods 0.000 claims abstract description 7
- 239000000284 extract Substances 0.000 claims abstract description 6
- 244000062793 Sorghum vulgare Species 0.000 claims description 18
- 235000019713 millet Nutrition 0.000 claims description 18
- 230000006907 apoptotic process Effects 0.000 claims description 15
- 239000002244 precipitate Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 11
- 235000019750 Crude protein Nutrition 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 101800000263 Acidic protein Proteins 0.000 claims description 4
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 4
- 238000012784 weak cation exchange Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 230000000259 anti-tumor effect Effects 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000002246 antineoplastic agent Substances 0.000 abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
- 206010009944 Colon cancer Diseases 0.000 abstract description 2
- 238000010298 pulverizing process Methods 0.000 abstract description 2
- 210000000582 semen Anatomy 0.000 abstract description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 abstract 1
- 238000005277 cation exchange chromatography Methods 0.000 abstract 1
- 238000005227 gel permeation chromatography Methods 0.000 abstract 1
- 201000007270 liver cancer Diseases 0.000 abstract 1
- 208000014018 liver neoplasm Diseases 0.000 abstract 1
- 235000007199 Panicum miliaceum Nutrition 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 18
- 244000022203 blackseeded proso millet Species 0.000 description 17
- 230000000694 effects Effects 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 8
- 238000011534 incubation Methods 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- TXUWMXQFNYDOEZ-UHFFFAOYSA-N 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone Chemical compound O=C1N(C)C(=S)NC1CC1=CNC2=CC=CC=C12 TXUWMXQFNYDOEZ-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 101150071716 PCSK1 gene Proteins 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 108090000317 Chymotrypsin Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 241000209117 Panicum Species 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229960002376 chymotrypsin Drugs 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 210000002784 stomach Anatomy 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 230000004565 tumor cell growth Effects 0.000 description 3
- 229940088872 Apoptosis inhibitor Drugs 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 235000006443 Panicum miliaceum subsp. miliaceum Nutrition 0.000 description 2
- 235000009037 Panicum miliaceum subsp. ruderale Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000000158 apoptosis inhibitor Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- DVWVZSJAYIJZFI-FXQIFTODSA-N Ala-Arg-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O DVWVZSJAYIJZFI-FXQIFTODSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- ZIBWKCRKNFYTPT-ZKWXMUAHSA-N Ala-Asn-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZIBWKCRKNFYTPT-ZKWXMUAHSA-N 0.000 description 1
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 1
- HQJKCXHQNUCKMY-GHCJXIJMSA-N Ala-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C)N HQJKCXHQNUCKMY-GHCJXIJMSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 1
- HKRXJBBCQBAGIM-FXQIFTODSA-N Arg-Asp-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N HKRXJBBCQBAGIM-FXQIFTODSA-N 0.000 description 1
- VXXHDZKEQNGXNU-QXEWZRGKSA-N Arg-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N VXXHDZKEQNGXNU-QXEWZRGKSA-N 0.000 description 1
- PBSOQGZLPFVXPU-YUMQZZPRSA-N Arg-Glu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PBSOQGZLPFVXPU-YUMQZZPRSA-N 0.000 description 1
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 1
- ZWASIOHRQWRWAS-UGYAYLCHSA-N Asn-Asp-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZWASIOHRQWRWAS-UGYAYLCHSA-N 0.000 description 1
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 1
- QPTAGIPWARILES-AVGNSLFASA-N Asn-Gln-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QPTAGIPWARILES-AVGNSLFASA-N 0.000 description 1
- BXUHCIXDSWRSBS-CIUDSAMLSA-N Asn-Leu-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BXUHCIXDSWRSBS-CIUDSAMLSA-N 0.000 description 1
- NCFJQJRLQJEECD-NHCYSSNCSA-N Asn-Leu-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O NCFJQJRLQJEECD-NHCYSSNCSA-N 0.000 description 1
- IXIWEFWRKIUMQX-DCAQKATOSA-N Asp-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O IXIWEFWRKIUMQX-DCAQKATOSA-N 0.000 description 1
- JDHOJQJMWBKHDB-CIUDSAMLSA-N Asp-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N JDHOJQJMWBKHDB-CIUDSAMLSA-N 0.000 description 1
- OEUQMKNNOWJREN-AVGNSLFASA-N Asp-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N OEUQMKNNOWJREN-AVGNSLFASA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- HKEZZWQWXWGASX-KKUMJFAQSA-N Asp-Leu-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HKEZZWQWXWGASX-KKUMJFAQSA-N 0.000 description 1
- NONWUQAWAANERO-BZSNNMDCSA-N Asp-Phe-Tyr Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 NONWUQAWAANERO-BZSNNMDCSA-N 0.000 description 1
- RVMXMLSYBTXCAV-VEVYYDQMSA-N Asp-Pro-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMXMLSYBTXCAV-VEVYYDQMSA-N 0.000 description 1
- HRVQDZOWMLFAOD-BIIVOSGPSA-N Asp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N)C(=O)O HRVQDZOWMLFAOD-BIIVOSGPSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229940123169 Caspase inhibitor Drugs 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 1
- CLPQUWHBWXFJOX-BQBZGAKWSA-N Gln-Gly-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O CLPQUWHBWXFJOX-BQBZGAKWSA-N 0.000 description 1
- XIYWAJQIWLXXAF-XKBZYTNZSA-N Gln-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O XIYWAJQIWLXXAF-XKBZYTNZSA-N 0.000 description 1
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 1
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 1
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 1
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 1
- UUYBFNKHOCJCHT-VHSXEESVSA-N Gly-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN UUYBFNKHOCJCHT-VHSXEESVSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- 206010019345 Heat stroke Diseases 0.000 description 1
- 208000031361 Hiccup Diseases 0.000 description 1
- LYSMQLXUCAKELQ-DCAQKATOSA-N His-Asp-Arg Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N LYSMQLXUCAKELQ-DCAQKATOSA-N 0.000 description 1
- UJWYPUUXIAKEES-CUJWVEQBSA-N His-Cys-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UJWYPUUXIAKEES-CUJWVEQBSA-N 0.000 description 1
- VUUFXXGKMPLKNH-BZSNNMDCSA-N His-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC3=CN=CN3)N VUUFXXGKMPLKNH-BZSNNMDCSA-N 0.000 description 1
- XVZJRZQIHJMUBG-TUBUOCAGSA-N His-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC1=CN=CN1)N XVZJRZQIHJMUBG-TUBUOCAGSA-N 0.000 description 1
- NULSANWBUWLTKN-NAKRPEOUSA-N Ile-Arg-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N NULSANWBUWLTKN-NAKRPEOUSA-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
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 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
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- QUAAUWNLWMLERT-IHRRRGAJSA-N Leu-Arg-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(O)=O QUAAUWNLWMLERT-IHRRRGAJSA-N 0.000 description 1
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 1
- YWKNKRAKOCLOLH-OEAJRASXSA-N Leu-Phe-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YWKNKRAKOCLOLH-OEAJRASXSA-N 0.000 description 1
- JLYUZRKPDKHUTC-WDSOQIARSA-N Leu-Pro-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JLYUZRKPDKHUTC-WDSOQIARSA-N 0.000 description 1
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 1
- ZDJQVSIPFLMNOX-RHYQMDGZSA-N Leu-Thr-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZDJQVSIPFLMNOX-RHYQMDGZSA-N 0.000 description 1
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 1
- NQCJGQHHYZNUDK-DCAQKATOSA-N Lys-Arg-Ser Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CCCN=C(N)N NQCJGQHHYZNUDK-DCAQKATOSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GVIVXNFKJQFTCE-YUMQZZPRSA-N Met-Gly-Gln Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O GVIVXNFKJQFTCE-YUMQZZPRSA-N 0.000 description 1
- ZIIMORLEZLVRIP-SRVKXCTJSA-N Met-Leu-Gln Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZIIMORLEZLVRIP-SRVKXCTJSA-N 0.000 description 1
- 208000007027 Oral Candidiasis Diseases 0.000 description 1
- 240000008114 Panicum miliaceum Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- FYQSMXKJYTZYRP-DCAQKATOSA-N Pro-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FYQSMXKJYTZYRP-DCAQKATOSA-N 0.000 description 1
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-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
- CLJLVCYFABNTHP-DCAQKATOSA-N Pro-Leu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O CLJLVCYFABNTHP-DCAQKATOSA-N 0.000 description 1
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 1
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 description 1
- 108010079005 RDV peptide Proteins 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- IDQFQFVEWMWRQQ-DLOVCJGASA-N Ser-Ala-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IDQFQFVEWMWRQQ-DLOVCJGASA-N 0.000 description 1
- VAIZFHMTBFYJIA-ACZMJKKPSA-N Ser-Asp-Gln Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O VAIZFHMTBFYJIA-ACZMJKKPSA-N 0.000 description 1
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 1
- LRWBCWGEUCKDTN-BJDJZHNGSA-N Ser-Lys-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LRWBCWGEUCKDTN-BJDJZHNGSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- BUYHXYIUQUBEQP-AVGNSLFASA-N Ser-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N BUYHXYIUQUBEQP-AVGNSLFASA-N 0.000 description 1
- FGBLCMLXHRPVOF-IHRRRGAJSA-N Ser-Tyr-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FGBLCMLXHRPVOF-IHRRRGAJSA-N 0.000 description 1
- 208000007180 Sunstroke Diseases 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- SWIKDOUVROTZCW-GCJQMDKQSA-N Thr-Asn-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C)C(=O)O)N)O SWIKDOUVROTZCW-GCJQMDKQSA-N 0.000 description 1
- ZQUKYJOKQBRBCS-GLLZPBPUSA-N Thr-Gln-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O ZQUKYJOKQBRBCS-GLLZPBPUSA-N 0.000 description 1
- MSIYNSBKKVMGFO-BHNWBGBOSA-N Thr-Gly-Pro Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N)O MSIYNSBKKVMGFO-BHNWBGBOSA-N 0.000 description 1
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- WVVOFCVMHAXGLE-LFSVMHDDSA-N Thr-Phe-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(O)=O WVVOFCVMHAXGLE-LFSVMHDDSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- 241000287411 Turdidae Species 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 1
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 108010047495 alanylglycine 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
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 101150042537 dld1 gene Proteins 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 1
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 1
- 108010020688 glycylhistidine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 230000009422 growth inhibiting effect Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000000955 peptide mass fingerprinting Methods 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc 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
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明提供了一种可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用。蛋白的制备方法：将黍子籽粒或黍糠粉碎，加入蛋白提取液低温提取，随后加入硫酸铵沉淀蛋白，再经过脱盐，阳离子交换层析和凝胶层析等步骤获得纯的蛋白。经实验这种蛋白可以诱导结直肠癌、肝癌等多种肿瘤细胞发生程序性坏死，其可以在制备抗肿瘤药物和保健食品中应用。
Description
技术领域
本发明涉及植物功能蛋白制备和应用，具体属于一种来源于黍子籽粒或黍糠的可以诱导肿瘤细胞程序性坏死的蛋白及其制备方法，以及该蛋白在制备抗肿瘤药物中的应用。
背景技术
黍子(Panicum miliaceum L.)属禾本科黍属，一年生草本植物，主要生长在北方。籽实也叫黍子，糜子，淡黄色，去皮后俗称黄米，常用于制作年糕，酿酒的原料，是重要的粮食作物之一。
黍子种子中含有17种氨基酸，含量较高，特别是必需氨基酸含量为4.76mg/100g，含量明显高于小麦，且与标准食谱相近，易于人体吸收，可以作为人体营养的补充食物。黍子籽粒中的钙、镁、磷及微量元素铁、铜、锌含量是水稻的3.4倍、小麦的2.3倍、玉米的1.05倍，并且含有一定量的硒。这些微量元素对于活化人体免疫性和正常的生理功能，抗衰老、预防心血管病等方面有着重要的作用。此外，黍子具有一定的药用价值，是我国传统的中草药之一。黍子性味甘、平、微寒、无毒。据《名医别录》记载：稷米“入脾、胃经”，功能“和中益气、凉血解暑”。煮熟和研末食，主治气虚乏力、中暑、头晕、口渴等症。黍米“入脾、胃、大肠、肺经”，功能“补中益气、健脾益肺、除热愈疮”。主治脾胃虚弱、肺虚咳嗽、呃逆烦渴、泄泻、胃痛、小儿鹅口疮、烫伤等症。
目前国内外关于黍子中活性物质的研究还较少，仅仅集中在其化学成分(植物多酚)和营养价值方面。黍糠是黍子加工过程中脱壳而得到的皮壳，也含有多种生物活性成分，是一种廉价而潜在的资源。本发明发现的来源于黍子，可诱导肿瘤细胞程序性坏死的蛋白具有较高的抗肿瘤活性，可在制备抗肿瘤药物中应用。另外，这一发现将为进一步研究开发黍子高附加值产品以及黍子资源的综合利用提供思路和依据，从而有利于推动黍子产业的规模化和持续化发展，带动我国半干旱、高海拔地区的经济发展。
发明内容
本发明的目的在于提供一种可诱导肿瘤细胞程序性坏死的蛋白及其制备方法，以及将该蛋白在制备抗肿瘤药物中得到应用。
本发明提供的一种可诱导肿瘤细胞程序性坏死的蛋白，来源于黍子(PanicummiliaceumL.)籽粒或黍糠，其分子量为37kD，氨基酸序列为SEQ ID No.1。
本发明提供的一种可诱导肿瘤细胞程序性坏死的蛋白，可以通过如下步骤的方法制得：
第一步：取干燥的黍子籽粒或黍糠，进一步粉碎后，然后按每克粉末加入10-15mL预冷的酸性蛋白提取液，4℃下搅拌4-8小时，8000-12000rpm离心10-20分钟除去沉淀，收集上清，得到蛋白粗提液；所述的酸性蛋白提取液为20mmol/L醋酸铵缓冲液，pH4.5；
第二步：在蛋白粗提液中加入硫酸铵，至60％-80％的饱和度，在4℃搅拌3-5小时；8000-12000rpm离心20-30分钟，收集沉淀；
第三步：用少量pH7.0的磷酸盐缓冲液溶解沉淀，上样于脱盐柱，除去硫酸铵，收集蛋白峰得到蛋白粗品；
第四步：将蛋白粗品上样于HiTrapTM SP弱阳离子交换进行分离纯化，收集洗脱峰，将收集的洗脱峰蛋白上样于凝胶层析柱，收集活性蛋白峰，真空冷冻干燥，得到可以诱导肿瘤细胞程序性坏死的蛋白。采用SDS-PAGE分析，该蛋白的分子量约为37kD。经胰蛋白酶和胰凝乳蛋白酶水解及肽质量指纹图谱鉴定，并结合Edman降解法测定其N端序列，确定氨基酸序列为SEQ ID No.1。
本发明提供的肿瘤细胞程序性坏死蛋白是一种热稳定性较好，经紫外-可见分光光度计检测，该蛋白在280nm和400nm处有吸收峰。
经体外抗肿瘤活性检测显示，该蛋白通过诱导多种肿瘤细胞发生程序性坏死，可以显著地抑制肿瘤细胞增殖。当用终浓度为100ug/mL的该蛋白处理结肠癌细胞HT29细胞24小时，对该细胞的增殖抑制率达到60％以上，而当作用时间为48小时时，增殖抑制率达到90％以上。
本发明获得的蛋白可以通过诱导肿瘤细胞坏死发挥抗增殖作用，因为程序性细胞坏死与经典的细胞凋亡在信号通路上存在较大的不同，当细胞凋亡通路被抑制时，在胞外信号刺激下可以诱导细胞发生程序性坏死。因此该蛋白对一些对常规药物表现出抗性的细胞已具有显著的增殖抑制作用。该蛋白可以在制备抗肿瘤药物中应用，也可以在保健食品的制备中应用。
本发明首次从黍子中分离纯化出一种可以诱导肿瘤细胞发生程序性坏死的蛋白，对多种肿瘤细胞具有显著的增殖抑制作用。这一蛋白的发现为糜子高附加值产品以及糜子资源的综合利用提供思路和依据，具有巨大的经济价值，同时也为抗肿瘤药物及保健食品的开发提供技术支持。
附图说明
图1是本发明可诱导肿瘤细胞程序性坏死的蛋白SDS-PAGE分析图
图2是本发明可诱导肿瘤细胞程序性坏死的蛋白紫外可见吸收光谱
图3是本发明可诱导肿瘤细胞程序性坏死的蛋白对肿瘤细胞生长抑制图
图4是凋亡抑制剂z-VAD和坏死抑制剂Nec-1对本发明蛋白诱导细胞死亡的影响
具体实施方式
实施例1：黍子籽粒型可诱导肿瘤细胞程序性坏死的蛋白的制备
将烘干的黍子籽粒，进行粉碎后，收集粉末；称取100g粉末，加入1000mL pH 4.5的20mM的醋酸盐缓冲液，4℃提取6小时，8000rpm离心20分钟除去沉淀，然后加入硫酸铵，至80％的饱和度；在4℃搅拌4小时，使蛋白充分沉淀，8000rpm离心30min，收集沉淀，用30mLpH7.0的PBS缓冲液将沉淀重新溶解；将溶解的沉淀上样于HiPrepTM26/10脱盐柱，除去硫酸铵，收集蛋白峰得到蛋白粗提物；将粗提物上样于HiTrapTM SP弱阳离子交换进行分离纯化，收集氯化钠洗脱峰，将收集的洗脱峰蛋白样品再次上样于HiPrepTM 26/10脱盐柱，收集蛋白峰，真空冷冻干燥，得到黍子籽粒中可诱导肿瘤细胞程序性坏死蛋白。采用Folin-酚法测定蛋白浓度，经计算采用该方法制得9.1mg可诱导肿瘤细胞程序性坏死的蛋白，经SDS-PAGE分析,分子量大小为37kD。
实施例2：黍糠型可诱导肿瘤细胞程序性坏死的蛋白的制备
将黍子加工过程中的副产物黍糠，进行进一步粉碎后，收集粉末；称取100g粉末，加入1000mL pH 4.0的20mM的醋酸盐缓冲液，4℃提取4小时，8000rpm离心20min除去沉淀，上清用纱布过滤，然后加入硫酸铵，至80％的饱和度；在4℃搅拌4小时，使蛋白充分沉淀，8000rpm离心30min，收集沉淀，用30mL pH7.0的PBS缓冲液将沉淀重新溶解；将溶解的沉淀上样于HiPrepTM 26/10脱盐柱，除去硫酸铵，收集蛋白峰得到蛋白粗提物；将粗提物上样于HiTrapTM SP弱阳离子交换柱进行分离纯化，收集洗脱峰，将收集的洗脱峰蛋白样品再次上样于HiPrepTM26/10脱盐柱，收集蛋白峰，真空冷冻干燥，得到黍糠中可诱导肿瘤细胞程序性坏死的蛋白。采用Folin-酚法测定蛋白浓度，经计算采用该方法制得16.5mg可诱导肿瘤细胞程序性坏死的蛋白。经SDS-PAGE分析,分子量大小为37kD(见图1)。经紫外-可见分光光度计检测，该蛋白在280nm和400nm两处有特征吸收峰(见图2)。
实施例3可诱导肿瘤细胞程序性坏死的蛋白氨基酸序列的测定
经SDS-PAGE分离后，切下含有实施例2制备的蛋白的条带，经胰蛋白酶和胰凝乳蛋白酶水解后，进行肽质量指纹图谱鉴定。经胰蛋白酶水解后的样品然后进行MALDI-TOF/TOF-MS检测，测得肽质量指纹谱，在数据库中查询识别的方式鉴定实施例2制备的蛋白。经过BLAST分析，找出与鉴定的蛋白同源性较高的序列。经胰凝乳蛋白酶水解后的样品测得肽质量指纹图谱，在数据库中进行搜索比对，找出与数据库中相符的肽段。将两种酶水解后得到的肽段进行拼接，得到实施例2制备的蛋白的主体序列。将实施例2制备的蛋白应用Edman降解法测定N端20个氨基酸序列。该N端序列和肽质量指纹图谱得到的序列进行拼接，得到实施例2制备的蛋白完整的氨基酸序列，该序列如SEQ ID No.1所示。
实施例4：可诱导肿瘤细胞程序性坏死的蛋白对肿瘤细胞生长抑制实验(MTT法)
应用MTT法，检测实施例2制备的蛋白对肿瘤细胞株Hep G2，HT29，SW480，HCT116等的生长抑制作用。取对数生长期的细胞，以1-5×103个/孔传入96孔培养板，37℃含5％的CO2细胞培养箱中孵育24小时后，分别加入可诱导肿瘤细胞程序性坏死的蛋白，使其终浓度分别为25μg/mL,50μg/mL,75μg/mL,100μg/mL,每组浓度设5个复孔，并用等体积的pH 7.0的20mM/L PBS缓冲液作为对照，孵育48h后，每孔加入20μL MTT试剂(5mg/mL)，继续孵育4小时后，终止培养，弃去孔内培养上清，每孔加入150μL DMSO,振荡10分钟，使结晶物充分溶解，在酶标仪上490nm波长处测定其光吸收值。本发明所得黍糠肿瘤细胞程序性坏死蛋白对HepG2,SW480，HCT116和HT29具有显著的增殖抑制作用(见图3)。
实施例5：可诱导肿瘤细胞程序性坏死的蛋白对肿瘤细胞生长抑制实验(ATP法)
细胞内ATP快速降低是细胞发生坏死的一个显著特征，通过测定细胞内ATP含量，检测实施例2制备的蛋白对肿瘤细胞株Hep G2，HT29，SW480，HCT116等的生长抑制作用。取对数生长期的细胞，以1-5×103个/孔转入不透光的96孔板，用25μg/mL,50μg/mL,75μg/mL,100μg/mL可诱导肿瘤细胞程序性坏死蛋白，分别处理细胞48h，弃去培养液，加入100μLDMEM培养基室温平衡30min,添加100μL Cell Titer-Glo试剂，摇床混合2min,诱导细胞裂解,室温孵育10min，稳定发光信号,酶标仪检测发光信号。结果显示，本发明所得可诱导肿瘤细胞程序性坏死蛋白对Hep G2，SW480，HCT116和DLD1细胞中ATP的降低有非常显著的效果，具有显著的增殖抑制作用(见图3)。
实施例6：凋亡抑制剂z-VAD和坏死抑制剂Nec-1对本发明蛋白诱导HCT116细胞死亡的影响
当细胞内Caspase酶活性被抑制，细胞的活性进一步减弱，而细胞程序性坏死抑制剂necrostatin-1(Nec-1)可以特异性抑制细胞坏死，使细胞活力恢复。取对数生长期的细胞，以1-5×103个/孔转入96孔培养板，37℃含5％的CO2细胞培养箱中孵育24小时后，分别加入终浓度为50μg/mL可诱导肿瘤细胞程序性坏死蛋白，同时加入不同浓度的抑制剂z-VAD和Nec-1，每组浓度设5个复孔，并用等体积的pH 7.0的20mM/L PBS缓冲液作为对照，孵育48h后，每孔加入20μL MTT试剂(5mg/mL)，继续孵育4小时后，终止培养，弃去孔内培养上清，每孔加入150μL DMSO,振荡10分钟，使结晶物充分溶解，在酶标仪上490nm波长处测定其光吸收值。结果显示，当Caspase抑制剂z-VAD与可诱导肿瘤细胞程序性坏死蛋白共同作用时，显著加强了可诱导肿瘤细胞程序性坏死蛋白诱导的细胞死亡，而坏死抑制剂Nec-1显著减弱了可诱导肿瘤细胞程序性坏死蛋白诱导的细胞死亡(见图4)。结果表明,可诱导肿瘤细胞程序性坏死蛋白诱导的细胞死亡是程序性坏死，而不是细胞凋亡。
SEQUENCE LISTING
<110> 山西大学
<120> 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用
<130> .
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 359
<212> PRT
<213> Panicum miliaceum L.
<400> 1
Met Ala Arg Ala Ala Ala Thr Val Ala Ala Ala Leu Ala Leu Ala Ala
1 5 10 15
Ala Leu Leu Cys Ala Ala Ser Ala Ala Pro Asn Asp Asp Val Ser Gln
20 25 30
Pro Pro Val Ala Arg Gly Leu Ser Tyr Asp Phe Tyr Lys Arg Ser Cys
35 40 45
Pro Arg Ala Glu Ala Ile Val Arg Ser Phe Val Gln Asp Ala Val Arg
50 55 60
Arg Asp Val Gly Leu Ala Ala Gly Leu Leu Arg Leu His Phe His Asp
65 70 75 80
Cys Phe Val Gln Gly Cys Asp Ala Ser Val Leu Leu Asp Gly Ser Ala
85 90 95
Thr Gly Pro Gly Glu Lys Gln Ala Pro Pro Asn Leu Thr Leu Arg Pro
100 105 110
Ser Ala Phe Lys Ala Ile Asn Asp Ile His Asp Arg Leu Thr Arg Glu
115 120 125
Cys Gly Gly Pro Val Val Ser Cys Ser Asp Val Leu Ala Leu Ala Ala
130 135 140
Arg Asp Ser Val Val Val Ser Gly Gly Pro Ser Tyr Arg Val Pro Leu
145 150 155 160
Gly Arg Arg Asp Ser Pro Ser Phe Ala Thr Gln Gln Asp Val Leu Thr
165 170 175
Gly Leu Pro Pro Pro Thr Ala Asn Val Pro Ala Leu Leu Ala Val Leu
180 185 190
Ser Lys Ile Asn Leu Asp Ala Ile Asp Leu Val Ala Leu Ser Gly Gly
195 200 205
His Thr Ile Gly Leu Gly His Cys Thr Ser Phe Glu Asp Arg Leu Phe
210 215 220
Pro Arg Pro Asp Pro Thr Leu Asn Ala Thr Phe Ala Gly Arg Leu Arg
225 230 235 240
Gln Thr Cys Pro Ala Lys Gly Thr Asp Arg Arg Thr Pro Leu Asp Val
245 250 255
Arg Thr Pro Asn Gln Phe Asp Asn Lys Tyr Tyr Val Asn Leu Val Asn
260 265 270
Arg Glu Gly Leu Phe Thr Ser Asp Gln Asp Leu Phe Thr Asn Ala Arg
275 280 285
Thr Arg Ala Leu Val Asp Lys Phe Ala Arg Ser Gln Lys Asp Phe Phe
290 295 300
Asp Gln Phe Thr Ser Ser Met Leu Gln Met Gly Gln Ile Lys Val Leu
305 310 315 320
Thr Gly Ser Gln Gly Gln Ile Arg Ser Asn Cys Ser Ala Arg Asn Asn
325 330 335
Pro Ala Gly Thr Ala Gly Leu Pro Trp Ser Ile Leu Asp Leu Glu Glu
340 345 350
Ala Asp Ser Leu Leu Val Phe
355
Claims (4)
1.一种可诱导肿瘤细胞程序性坏死的蛋白，其特征在于，氨基酸序列为SEQ ID No.1。
2.如权利要求1所述的可诱导肿瘤细胞程序性坏死的蛋白的制备方法，其特征在于，包括如下步骤：
第一步：取干燥的黍子籽粒或黍糠，进一步粉碎后，然后按每克粉末加入10-15mL预冷的酸性蛋白提取液，4℃下搅拌4-8小时，8000-12000rpm离心10-20分钟除去沉淀，收集上清，得到蛋白粗提液；
第二步：在蛋白粗提液中加入硫酸铵，至60％-80％的饱和度，在4℃搅拌3-5小时；8000-12000rpm离心20-30分钟，收集沉淀；
第三步：用少量pH7.0的磷酸盐缓冲液溶解沉淀，上样于脱盐柱，除去硫酸铵，收集蛋白峰得到蛋白粗品；
第四步：将蛋白粗品上样于HiTrapTM SP弱阳离子交换进行分离纯化，收集洗脱峰，将收集的洗脱峰蛋白上样于凝胶层析柱，收集活性蛋白峰，真空冷冻干燥，得到可以诱导肿瘤细胞程序性坏死的蛋白。
3.如权利要求2所述的可诱导肿瘤细胞程序性坏死的蛋白的制备方法，其特征在于，所述的酸性蛋白提取液为20mmol/L醋酸铵缓冲液，pH4.5。
4.如权利要求1所述的可诱导肿瘤细胞程序性坏死的蛋白在制备抗肿瘤药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710354026.XA CN107011420B (zh) | 2017-05-18 | 2017-05-18 | 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710354026.XA CN107011420B (zh) | 2017-05-18 | 2017-05-18 | 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107011420A CN107011420A (zh) | 2017-08-04 |
| CN107011420B true CN107011420B (zh) | 2020-06-12 |
Family
ID=59450599
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710354026.XA Expired - Fee Related CN107011420B (zh) | 2017-05-18 | 2017-05-18 | 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107011420B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211808A (zh) * | 2013-06-05 | 2014-12-17 | 江苏先声药物研究有限公司 | 一种肿瘤坏死因子相关凋亡诱导配体融合蛋白的制备方法 |
| CN106366169A (zh) * | 2016-10-11 | 2017-02-01 | 南开大学 | 一种葡萄状枝瑚菌中抗肿瘤蛋白提取物及其制备方法及应用 |
-
2017
- 2017-05-18 CN CN201710354026.XA patent/CN107011420B/zh not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211808A (zh) * | 2013-06-05 | 2014-12-17 | 江苏先声药物研究有限公司 | 一种肿瘤坏死因子相关凋亡诱导配体融合蛋白的制备方法 |
| CN106366169A (zh) * | 2016-10-11 | 2017-02-01 | 南开大学 | 一种葡萄状枝瑚菌中抗肿瘤蛋白提取物及其制备方法及应用 |
Non-Patent Citations (2)
| Title |
|---|
| Cationic peroxidase from proso millet induces human colon cancer cell necroptosis by regulating autocrine TNF-α and RIPK3 demethylation;Xiaodong Cui等;《Food & Function》;20180228(第3期);第1878-1888页 * |
| DNA damage and necroptosis induced by peroxidase from proso millet in human colorectal cancer cells;Xiaodong Cui等;《Tropical Journal of Pharmaceutical Research》;20190531;第18卷(第5期);第975-983页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107011420A (zh) | 2017-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107141336B (zh) | 具有dpp-iv抑制活性的牦牛骨蛋白肽及制备方法 | |
| CN108486202B (zh) | 一种辣木酶解物及其制备方法和应用 | |
| Tounkara et al. | Antioxidant activities of roselle (Hibiscus sabdariffa L.) seed protein hydrolysate and its derived peptide fractions | |
| JP4711272B2 (ja) | アンジオテンシン変換酵素阻害物質 | |
| Zheng et al. | Purification and identification of angiotensin I-converting enzyme inhibitory peptides from fermented walnut residues | |
| Tak et al. | Pulse derived bioactive peptides as novel nutraceuticals: A review | |
| Lin et al. | Ganoderma immunomodulatory proteins: mushrooming functional FIPs | |
| CN108651994B (zh) | 一种新的燕窝提取液及其制备方法和应用 | |
| CN107011420B (zh) | 可诱导肿瘤细胞程序性坏死的蛋白及其制备方法和应用 | |
| CN107828842B (zh) | 一种具有抗氧化和dpp-iv抑制功能的核桃蛋白肽 | |
| CN107224445B (zh) | 蜂胶有效部位及其复方制品和应用 | |
| CN111670995B (zh) | 一种小麦蛋白肽的制备方法 | |
| KR101256730B1 (ko) | 대두박, 함초 및 쌀을 이용한 메주의 제조방법 및 그 메주를 이용한 재래식 간장의 제조방법 | |
| CN101974394B (zh) | 纳米海参酒及其制备方法 | |
| CN114213512B (zh) | 增强藻胆蛋白光热稳定性的组合物及其制备方法与应用 | |
| CN114656522B (zh) | 一种抗氧化肽及其制备方法和应用 | |
| CN106868082A (zh) | 一种新的参苏补肾胶囊中脑蛋白水解物的制备方法 | |
| CN108048518B (zh) | 鸡血球抗氧化肽及其酶解制备方法 | |
| WO2020024594A1 (en) | Preparation method and application of recombinant mutant collagenase | |
| KR102501955B1 (ko) | 동과 발효 추출물을 유효성분으로 함유하는 면역 증진용 조성물 | |
| KR102472951B1 (ko) | 좁은잎천선과 혼합 추출물을 유효성분으로 함유하는 면역 증진용 조성물 | |
| KR102305964B1 (ko) | 좁은잎천선과 추출물을 유효성분으로 함유하는 면역 증진용 조성물 | |
| CN115124591A (zh) | 钝顶螺旋藻藻蓝蛋白血管紧张素转化酶抑制肽及其制备方法和应用 | |
| CN114752585B (zh) | 一种核桃青皮抗肿瘤活性蛋白及其制备方法和应用 | |
| CN105566446B (zh) | 一种蒲公英籽抗氧化肽及其制备方法与应用 |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200612 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |