CN106434619A - Biochemical reaction material and device and method for treating blood or plasma in vitro - Google Patents
Biochemical reaction material and device and method for treating blood or plasma in vitro Download PDFInfo
- Publication number
- CN106434619A CN106434619A CN201510815421.4A CN201510815421A CN106434619A CN 106434619 A CN106434619 A CN 106434619A CN 201510815421 A CN201510815421 A CN 201510815421A CN 106434619 A CN106434619 A CN 106434619A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- biochemical reaction
- blood plasma
- ldl
- neuraminidase
- 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
- 239000000463 material Substances 0.000 title claims abstract description 121
- 238000005842 biochemical reaction Methods 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims description 79
- 210000004369 blood Anatomy 0.000 title claims description 56
- 239000008280 blood Substances 0.000 title claims description 39
- 238000000338 in vitro Methods 0.000 title claims description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 184
- 108090000790 Enzymes Proteins 0.000 claims abstract description 184
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims abstract description 48
- 229940106189 ceramide Drugs 0.000 claims abstract description 48
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims abstract description 48
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims abstract description 48
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims abstract description 42
- 108010071584 oxidized low density lipoprotein Proteins 0.000 claims abstract description 6
- 108090000751 Ceramidases Proteins 0.000 claims description 85
- 108010007622 LDL Lipoproteins Proteins 0.000 claims description 85
- 102000007330 LDL Lipoproteins Human genes 0.000 claims description 85
- 102100023996 Neutral ceramidase Human genes 0.000 claims description 71
- 239000002585 base Substances 0.000 claims description 64
- 210000002381 plasma Anatomy 0.000 claims description 58
- 239000000470 constituent Substances 0.000 claims description 56
- 101001123851 Homo sapiens Sialidase-2 Proteins 0.000 claims description 55
- 102100028755 Sialidase-2 Human genes 0.000 claims description 54
- 102100024005 Acid ceramidase Human genes 0.000 claims description 46
- 101000975753 Homo sapiens Acid ceramidase Proteins 0.000 claims description 41
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 108010006232 Neuraminidase Proteins 0.000 claims description 28
- 102000005348 Neuraminidase Human genes 0.000 claims description 28
- -1 acryl Chemical group 0.000 claims description 25
- 229920002678 cellulose Polymers 0.000 claims description 24
- 239000001913 cellulose Substances 0.000 claims description 24
- 238000000746 purification Methods 0.000 claims description 24
- 101000601384 Homo sapiens Sialidase-4 Proteins 0.000 claims description 19
- 102100037729 Sialidase-4 Human genes 0.000 claims description 19
- 235000009508 confectionery Nutrition 0.000 claims description 18
- 102000004201 Ceramidases Human genes 0.000 claims description 16
- 239000012510 hollow fiber Substances 0.000 claims description 15
- 230000000844 anti-bacterial effect Effects 0.000 claims description 14
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims description 13
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims description 13
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims description 13
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 13
- 239000004695 Polyether sulfone Substances 0.000 claims description 12
- 102100028760 Sialidase-1 Human genes 0.000 claims description 12
- 241000700605 Viruses Species 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 229920002492 poly(sulfone) Polymers 0.000 claims description 12
- 229920006393 polyether sulfone Polymers 0.000 claims description 11
- JVIPLYCGEZUBIO-UHFFFAOYSA-N 2-(4-fluorophenyl)-1,3-dioxoisoindole-5-carboxylic acid Chemical compound O=C1C2=CC(C(=O)O)=CC=C2C(=O)N1C1=CC=C(F)C=C1 JVIPLYCGEZUBIO-UHFFFAOYSA-N 0.000 claims description 10
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 claims description 10
- 101001123859 Homo sapiens Sialidase-1 Proteins 0.000 claims description 10
- 101001123847 Homo sapiens Sialidase-3 Proteins 0.000 claims description 10
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 claims description 10
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 claims description 10
- 102100028756 Sialidase-3 Human genes 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 102100034050 Alkaline ceramidase 2 Human genes 0.000 claims description 9
- 101710200054 Alkaline ceramidase 2 Proteins 0.000 claims description 9
- 102100034082 Alkaline ceramidase 3 Human genes 0.000 claims description 9
- 101710200049 Alkaline ceramidase 3 Proteins 0.000 claims description 9
- 101000784192 Homo sapiens Putative inactive neutral ceramidase B Proteins 0.000 claims description 9
- 102100021030 Putative inactive neutral ceramidase B Human genes 0.000 claims description 9
- 239000011347 resin Substances 0.000 claims description 9
- 229920005989 resin Polymers 0.000 claims description 9
- 102000004366 Glucosidases Human genes 0.000 claims description 8
- 108010056771 Glucosidases Proteins 0.000 claims description 8
- 101000601385 Mus musculus Sialidase-4 Proteins 0.000 claims description 8
- 239000004793 Polystyrene Substances 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 8
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 8
- 229920002223 polystyrene Polymers 0.000 claims description 8
- 101000975757 Homo sapiens Neutral ceramidase Proteins 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 7
- 102100034083 Alkaline ceramidase 1 Human genes 0.000 claims description 6
- 101710200052 Alkaline ceramidase 1 Proteins 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 108010026867 Oligo-1,6-Glucosidase Proteins 0.000 claims description 6
- 102100027918 Sucrase-isomaltase, intestinal Human genes 0.000 claims description 6
- 150000001783 ceramides Chemical class 0.000 claims description 6
- 229940031098 ethanolamine Drugs 0.000 claims description 6
- HZAXFHJVJLSVMW-UHFFFAOYSA-N monoethanolamine hydrochloride Natural products NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 6
- 239000004743 Polypropylene Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 150000004676 glycans Chemical class 0.000 claims description 5
- 229920001155 polypropylene Polymers 0.000 claims description 5
- 229920001282 polysaccharide Polymers 0.000 claims description 5
- 239000005017 polysaccharide Substances 0.000 claims description 5
- 239000004698 Polyethylene Substances 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 4
- 229920000573 polyethylene Polymers 0.000 claims description 4
- 101001112203 Arabidopsis thaliana Neutral ceramidase 1 Proteins 0.000 claims description 3
- 229920001661 Chitosan Polymers 0.000 claims description 3
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims description 3
- 101000979695 Dictyostelium discoideum Neutral ceramidase B Proteins 0.000 claims description 3
- 101000958771 Homo sapiens N-acylethanolamine-hydrolyzing acid amidase Proteins 0.000 claims description 3
- 102100038360 N-acylethanolamine-hydrolyzing acid amidase Human genes 0.000 claims description 3
- 239000000835 fiber Substances 0.000 claims description 3
- 229910000851 Alloy steel Inorganic materials 0.000 claims description 2
- 102000004157 Hydrolases Human genes 0.000 claims description 2
- 108090000604 Hydrolases Proteins 0.000 claims description 2
- 101710128612 Sialidase-1 Proteins 0.000 claims description 2
- 229910001069 Ti alloy Inorganic materials 0.000 claims description 2
- 239000010959 steel Substances 0.000 claims description 2
- 235000013339 cereals Nutrition 0.000 claims 6
- YDNKGFDKKRUKPY-TURZORIXSA-N N-hexadecanoylsphingosine Chemical compound CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)\C=C\CCCCCCCCCCCCC YDNKGFDKKRUKPY-TURZORIXSA-N 0.000 claims 3
- 239000003513 alkali Substances 0.000 claims 3
- CERZMXAJYMMUDR-QBTAGHCHSA-N 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid Chemical compound N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO CERZMXAJYMMUDR-QBTAGHCHSA-N 0.000 claims 2
- 240000000111 Saccharum officinarum Species 0.000 claims 2
- 235000007201 Saccharum officinarum Nutrition 0.000 claims 2
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 claims 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims 1
- 239000004382 Amylase Substances 0.000 claims 1
- 108010065511 Amylases Proteins 0.000 claims 1
- 102000013142 Amylases Human genes 0.000 claims 1
- 239000004593 Epoxy Substances 0.000 claims 1
- 125000000822 N-acylsphingosine group Chemical group 0.000 claims 1
- 241000209140 Triticum Species 0.000 claims 1
- 235000021307 Triticum Nutrition 0.000 claims 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 1
- 235000019418 amylase Nutrition 0.000 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- 229930182470 glycoside Natural products 0.000 claims 1
- 150000002338 glycosides Chemical class 0.000 claims 1
- 210000005036 nerve Anatomy 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 7
- 125000003147 glycosyl group Chemical group 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 description 34
- 239000000523 sample Substances 0.000 description 29
- 239000007853 buffer solution Substances 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 26
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 239000008055 phosphate buffer solution Substances 0.000 description 22
- 230000008569 process Effects 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 238000012545 processing Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 238000003756 stirring Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 108010062497 VLDL Lipoproteins Proteins 0.000 description 12
- 241000894007 species Species 0.000 description 12
- 239000007787 solid Substances 0.000 description 11
- 230000006907 apoptotic process Effects 0.000 description 10
- 238000000703 high-speed centrifugation Methods 0.000 description 10
- 210000002706 plastid Anatomy 0.000 description 10
- 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 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 150000002632 lipids Chemical class 0.000 description 9
- 238000001890 transfection Methods 0.000 description 9
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 230000004087 circulation Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 7
- 108010010234 HDL Lipoproteins Proteins 0.000 description 7
- 102000015779 HDL Lipoproteins Human genes 0.000 description 7
- 102000004895 Lipoproteins Human genes 0.000 description 7
- 108090001030 Lipoproteins Proteins 0.000 description 7
- 239000001110 calcium chloride Substances 0.000 description 7
- 229910001628 calcium chloride Inorganic materials 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 238000011049 filling Methods 0.000 description 7
- 238000001914 filtration Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000013467 fragmentation Methods 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- 238000004811 liquid chromatography Methods 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 210000003556 vascular endothelial cell Anatomy 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 101100380298 Caenorhabditis elegans asah-2 gene Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 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 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 102000013918 Apolipoproteins E Human genes 0.000 description 4
- 108010025628 Apolipoproteins E Proteins 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000011514 reflex Effects 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- 101150037123 APOE gene Proteins 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N CHCl3 Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- 101100216294 Danio rerio apoeb gene Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000004814 polyurethane Substances 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 238000000035 BCA protein assay Methods 0.000 description 2
- 108010004103 Chylomicrons Proteins 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 108010046315 IDL Lipoproteins Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- 108010028554 LDL Cholesterol Proteins 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 101150084651 Neu2 gene Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000013098 chemical test method Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000132 electrospray ionisation Methods 0.000 description 2
- 238000011013 endotoxin removal Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000002350 fibrinopeptide Substances 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000010422 painting Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 229920002635 polyurethane Polymers 0.000 description 2
- KCTAWXVAICEBSD-UHFFFAOYSA-N prop-2-enoyloxy prop-2-eneperoxoate Chemical compound C=CC(=O)OOOC(=O)C=C KCTAWXVAICEBSD-UHFFFAOYSA-N 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000007725 thermal activation Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- XIAYFENBYCWHGY-UHFFFAOYSA-N 2-[2,7-bis[[bis(carboxymethyl)amino]methyl]-3-hydroxy-6-oxoxanthen-9-yl]benzoic acid Chemical compound C=12C=C(CN(CC(O)=O)CC(O)=O)C(=O)C=C2OC=2C=C(O)C(CN(CC(O)=O)CC(=O)O)=CC=2C=1C1=CC=CC=C1C(O)=O XIAYFENBYCWHGY-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 101150102415 Apob gene Proteins 0.000 description 1
- 101710095339 Apolipoprotein E Proteins 0.000 description 1
- 102100029470 Apolipoprotein E Human genes 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101100516397 Homo sapiens NEU4 gene Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 238000008214 LDL Cholesterol Methods 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- HCDLRJUFVJOBJP-ACEXITHZSA-N N-[(2S,3R)-1,3-Dihydroxyoctadecan-2-yl]pentacosanimidic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)CCCCCCCCCCCCCCC HCDLRJUFVJOBJP-ACEXITHZSA-N 0.000 description 1
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 238000001994 activation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- XVVLAOSRANDVDB-UHFFFAOYSA-N formic acid Chemical compound OC=O.OC=O XVVLAOSRANDVDB-UHFFFAOYSA-N 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- BCVXHSPFUWZLGQ-UHFFFAOYSA-N mecn acetonitrile Chemical compound CC#N.CC#N BCVXHSPFUWZLGQ-UHFFFAOYSA-N 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 230000000444 normolipidemic effect Effects 0.000 description 1
- 229960002378 oftasceine Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- WHPMALGCHJRYKZ-UHFFFAOYSA-N pentanedial Chemical compound O=CCCCC=O.O=CCCCC=O WHPMALGCHJRYKZ-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- YRWWOAFMPXPHEJ-OFBPEYICSA-K sodium L-ascorbic acid 2-phosphate Chemical compound [Na+].[Na+].[Na+].OC[C@H](O)[C@H]1OC(=O)C(OP([O-])([O-])=O)=C1[O-] YRWWOAFMPXPHEJ-OFBPEYICSA-K 0.000 description 1
- 229940048058 sodium ascorbyl phosphate Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a biochemical reaction material, comprising: a substrate; and an enzyme composition immobilized on the substrate, wherein the enzyme composition is selected from the group consisting of: a first enzyme for removing glycosyl of electronegative low-density lipoprotein (L5); a second enzyme to scavenge ceramide (ceramide) carried by electronegative low density lipoproteins; and the combination of the above, wherein the biochemical reaction material has the capability of removing electronegative low density lipoprotein.
Description
Technical field
The present disclosure generally relates to the method for biochemical reaction material and device and extracorporeal treatment blood or blood plasma.
Background technology
Low density lipoprotein, LDL (low-density lipoprotein, LDL) is a kind of lipoprotein particles, is to hydrolyze the product after enzyme effect via lipoprotein.The role that delivery fatty acid molecule uses to whole body for cell in blood played the part of on normal physiological by low density lipoprotein, LDL.There is close positive correlation in the cholesterol levels that known low density lipoprotein, LDL carries and cardiovascular disease.
At present medical circle still with plasma low density lipoprotein cholesterol (LDL cholesterol, LDL-C) as assessment cardiovascular disease index, but the patient of acute myocardial infarction, the low density lipoprotein, LDL in its blood plasma does not have increased trend.
Because of extraneous factors such as excessive oxidative pressures, low density lipoprotein, LDL can be modified after being translated, and assumes higher elecrtonegativity, becomes elecrtonegativity low density lipoprotein, LDL (electronegative LDL or L5).
LDL-5 (L5) elecrtonegativity low density lipoprotein, LDL is the principal element causing cardiovascular disease, L5 is not almost detected in normal human, again, cell experiment and zoopery are all proved to injure vascular endothelial cell and activated blood platelet L5 in vitro, cause arteriosclerosis and myocardial infarction.
Therefore, a kind of material in order to remove elecrtonegativity low density lipoprotein, LDL of novelty, device and/or method are needed at present badly.
Content of the invention
The present invention provides a kind of biochemical reaction material, including：Base material；And enzyme constituent is fixed on this base material, wherein, this enzyme constituent is selected from：In order to remove the first enzyme of the candy base of elecrtonegativity low density lipoprotein, LDL (electronegative low-density lipoprotein, electronegative LDL)；In order to remove the second enzyme of the ceramide (ceramide) entrained by elecrtonegativity low density lipoprotein, LDL；And combinations of the above, wherein this biochemical reaction material have remove elecrtonegativity low density lipoprotein, LDL ability.
The present invention also provides for a kind of biochemical reaction device, including：Biochemical reaction material as described in front；And container, in order to accommodate this biochemical reaction material, wherein this container has at least one entrance and at least one outlet；Wherein, liquid sample enters this biochemical reaction device from this at least one opening, and flows through this biological respinse material to react with this biochemical reaction material, and flows out from this at least one outlet afterwards.
The present invention also provides a kind of extracorporeal treatment blood or the method for blood plasma, including：A () makes blood or blood plasma contact with enzyme constituent in vitro, so that this enzyme constituent reacts to this blood or blood plasma, wherein, this enzyme constituent has the ability removing elecrtonegativity low density lipoprotein, LDL, and this enzyme constituent is selected from：In order to remove the first enzyme of the candy base of elecrtonegativity low density lipoprotein, LDL；In order to remove the second enzyme of the ceramide entrained by elecrtonegativity low density lipoprotein, LDL；And combinations of the above；And (b) is terminated this blood or blood plasma and contacted with this enzyme constituent, with terminating this enzyme constituent, this blood or blood plasma are reacted.
In order to above and other purpose, feature and the advantage of the present invention can be become apparent, preferred embodiment cited below particularly, and coordinate appended diagram, it is described in detail below：
Brief description
Figure 1A is the generalized section of an embodiment of biochemical reaction device of the present invention.
Figure 1B is the generalized section of another embodiment of biochemical reaction device of the present invention.
Fig. 1 C is the generalized section of the still another embodiment of biochemical reaction device of the present invention.
Fig. 2, display NEU2 turns shape result.
Fig. 3 shows, ASAH2 turns shape result.
Fig. 4 shows, confirms the gene transfection results of NEU4/ASAH2 by Western blot.
Fig. 5 shows the purification result of NEU2.
Fig. 6 shows the purification result of ASAH2.
Fig. 7 shows L5 (25 μ g/mL；50 μ g/mL) and co-culture with vascular endothelial cell respectively through the L5 (1.25 μ g) of immobilization NEU2 filling device process, and carry out apoptosis mensure.
Fig. 8 is shown to be processed by not treated, with 37 DEG C or process the LDL sample being obtained by cardiac patient of 2 hours through immobilization NEU2 filling device and carries out elecrtonegativity low density lipoprotein, LDL quantitative analyses respectively.
Fig. 9 A, Fig. 9 B and Fig. 9 C show and for L5 to carry out mass spectral analyses, can detect apoE lipoprotein and have L5 distinctive candy modification.
Figure 10 A1-2 and Figure 10 B1-2 shows, the L5 processing through NEU2 is carried out mass spectral analyses, and result display apoE distinctive candy modification has been removed.
Figure 11 A, Figure 11 B show the candy modification that all can effectively remove on lipoprotein via NEU2, the NEU4 being immobilized on unlike material:The most common sequence modified by candy base of LDL is (K) AKPALEDLRQGLLPVLESFK (V) of (K) SGSSTASWIQNVDTKYQIR (I) 5.apoB100 of (K) GVISIPR (L) 4.apoB100 of (K) VLVDHFGYTK (D) 3.apoB100 of (R) IGQDGISTSATTNLK (C) 2.apoB100 of 1. apoB100, ITRI-A-01 (NEU2), ITRI-CD-01 (NEU2), ITRI-Si-Nu-01 (NEU4) all can effectively remove the candy base on apoB.
Figure 12 shows, ceramide (ceramide) content of the L5 and L5 processing 24 hours through ASAH2.
Figure 13 shows, the ceramide content of the L5 and L5 processing 24 hours through ASAH2.
Figure 14 shows, L5 and through ASAH2 in buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) presence or absence of lower process 2 or 24 hours L5 ceramide content.
Figure 15 shows, L5 and through ASAH2 in buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) presence or absence of lower process 24 hours L5 ceramide content.
Figure 16 A shows, L5 processes the L5 of 24 hours with through ASAH2, using the lipid components contained by mass spectrograph quantitative analyses, and compares the difference of ceramide (ceramide) content, schema is the result quantitative using mass spectrograph.
Figure 16 B shows, L5 and through ASAH2 in buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) in the presence of process 2 hours L5 ceramide content.
ASAH2 after Figure 17 A, Figure 17 B show via immobilization can effectively remove ceramide acid, and increases product sphingol (Sphingosine):One of L5 modal ceramide acid is Cer (d18:0/25:0) after, acting on via ASAH2, product is sphingol.ASAH2 (ITRI-EC-AS-01) after experimental result shows via immobilization can reduce the Cer (d18 contained by a LDL corpse or other object for laboratory examination and chemical testing:0/25:, and increase product sphingol 0).
Symbol description
100～biochemical reaction device
101～biochemical reaction material
103～container
105～at least one entrances
1051～first entrance
1052～second entrance
107～at least one outlets
1071～first outlet
1072～second outlet
109～filter material
Embodiment
In an embodiment of the present invention, the present invention provides a kind of biochemical reaction material, and it has the ability removing elecrtonegativity low density lipoprotein, LDL (electronegative low-density lipoprotein, electronegative LDL).Example in described elecrtonegativity low density lipoprotein, LDL, it may include, but it is not limited to L1, L2, L3, L4 and/or L5 etc..In one embodiment, described elecrtonegativity low density lipoprotein, LDL is elecrtonegativity low density lipoprotein, LDL L5.
The biochemical reaction material of the present invention, it may include, but it is not limited to, base material and enzyme constituent, wherein enzyme constituent is fixed on base material.
The example of suitable base material, may include silica gel (silica gel), cellulose (cellulose), diethylaminoethyl cellulose (diethylaminoethyl cellulose, DEAE cellulose), shitosan (chitosan), polystyrene (polystyrene), polysulfones (polysulfone), polyether sulfone (polyethersulfone), resin and polysaccharide etc., but not limited to this.Base material can have grain structure or hollow tubular structure etc..In one embodiment, base material can be cellulose pearl (bead).In another embodiment, base material is shitosan pearl.Also, in another embodiment, base material can be cellulose hollow fiber (cellulose hollow fiber).
And above-mentioned enzyme constituent may include, in order to remove the first enzyme, the second enzyme in order to remove the ceramide (ceramide) entrained by elecrtonegativity low density lipoprotein, LDL of the glycosyl of elecrtonegativity low density lipoprotein, LDL, or combinations of the above, but not limited to this.There is no particular restriction for the source of species of above-mentioned first enzyme and second enzyme.In one embodiment, the first enzyme and the source of species of second enzyme are the mankind.
Above-mentioned first enzyme can be sialidase (sialidase) or glycosidase (glycosidase).
Sialidase may include the neuraminidase 1 (neuraminidase 1 from human body, NEU1), neuraminidase 2 (neuraminidase 2, NEU2), neuraminidase 3 (neuraminidase 3, NEU3), neuraminidase 4 (neuraminidase 4, NEU4), O- sialidase (O-sialidase) etc., or turned with gene and grow, performance purification and one of above-mentioned enzyme of coming, or be from virus, sialidase (the another name of antibacterial or other species, n acetylneuraminic acid n base hydrolase (acetylneuraminyl hydrolase)) etc., but not limited to this.
And the example of glycosidase, then may include and come from the α and β glucosidase (alpha-and beta-glucosidase) of human body or animal body, maltase-glucoamylase (maltase-glucoamylase), Sucrase-isomaltase (sucrase-isomaltase) etc., or turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, or come from N- glycosidase F (the N-glycosidase F of virus, antibacterial or other species, PNGase F), glucosidase (glucosidase) etc., but not limited to this.
Also, above-mentioned second enzyme can be ceramidase (ceramidase).
Ceramidase then may include ASAH1 (N-acylsphingosine amidohydrolase)，Such as ASAH1 1 (N-acylsphingosine amidohydrolase 1,ASAH1)、ASAH1 2 (N-acylsphingosine amidohydrolase 2,ASAH2)、ASAH1 2B (N-acylsphingosine amidohydrolase 2B,ASAH2B)、ASAH1 2C (N-acylsphingosine amidohydrolase 2C,ASAH2C) etc.、N- acyl ethanol amine acylating acid enzyme (N-acylethanolamine acid amidase)、Alkaline ceramidase 1 (alkaline ceramidase 1)、Alkaline ceramidase 2 (alkaline ceramidase 2)、Alkaline ceramidase 3 (alkaline ceramidase 3)，But not limited to this.
In one embodiment, the enzyme constituent in biochemical reaction material of the present invention is above-mentioned first enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this.
In another embodiment, the enzyme constituent in biochemical reaction material of the present invention is above-mentioned second enzyme.In this embodiment, described second enzyme can be ASAH1 2, but not limited to this.
In still another embodiment, the enzyme constituent in biochemical reaction material of the present invention is above-mentioned first enzyme and the combining of second enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this, and described second enzyme can be ASAH1 2, but it is also not necessarily limited to this.
In an alternative embodiment of the invention, the present invention provides a kind of biochemical reaction device, and this device can be used for removing the elecrtonegativity low density lipoprotein, LDL in liquid sample.
The example of aforesaid liquid sample, it may include aqueous solution, buffer solution, blood, blood plasma etc., but not limited to this.
The example of the described elecrtonegativity low density lipoprotein, LDL in front again, it may include, but be not limited to, L1, L2, L3, L4 and/or L5 etc..In one embodiment, described elecrtonegativity low density lipoprotein, LDL is elecrtonegativity low density lipoprotein, LDL L5.
The section of structure of the biochemical reaction device of the present invention is as shown in Figure 1.
Referring to Figure 1A.The biochemical reaction device 100 of the invention described above, it may include, biochemical reaction material 101, container 103, in order to accommodate this biochemical reaction material 101.Container 103 has at least one entrance 105 and at least one outlet 107.Aforementioned liquids sample enters biochemical reaction device 100 from least one opening 105, and flows through biological respinse material 101 to react with biochemical reaction material 101, and afterwards from least one outlet 107 outflow.
Above-mentioned biochemical reaction material 101, it may include, but it is not limited to, base material and enzyme constituent, wherein enzyme constituent is fixed on base material.
Aforementioned substrates, it may include, but be not limited to, silica gel, cellulose, diethylaminoethyl cellulose, shitosan, polystyrene, polysulfones, polyether sulfone, resin and polysaccharide etc..Base material can have grain structure or hollow tubular structure etc., but not limited to this.
Enzyme constituent then may include, but is not limited to, in order to remove the first enzyme, the second enzyme in order to remove the ceramide entrained by elecrtonegativity low density lipoprotein, LDL of the candy base of elecrtonegativity low density lipoprotein, LDL, or combinations of the above.There is no particular restriction for the source of species of above-mentioned first enzyme and second enzyme.In one embodiment, the first enzyme and the source of species of second enzyme are the mankind.
Above-mentioned first enzyme can be sialidase or glycosidase.
Sialidase may include, but it is not limited to, from the neuraminidase 1 of human body, neuraminidase 2, neuraminidase 3, neuraminidase 4, O- sialidase etc., or turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, or it is the sialidase etc. from virus, antibacterial or other species.
And glycosidase, then may include, but it is not limited to, come from the α and β glucosidase of human body or animal body, maltase-glucoamylase, Sucrase-isomaltase etc., or turn with gene and grow, show and purification and one of above-mentioned enzyme of coming, or come from the N- glycosidase F of virus, antibacterial or other species, glucosidase etc..
Also, above-mentioned second enzyme can be ceramidase.Ceramidase may include; but it is not limited to; ASAH1; such as ASAH1 1, ASAH1 2, ASAH1 2B, ASAH1 2C etc., N- acyl ethanol amine acylating acid enzyme, alkaline ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3, but not limited to this.
In one embodiment, the enzyme constituent in aforementioned biochemical reaction material 101 is above-mentioned first enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this.
In another embodiment, the enzyme constituent in aforementioned biochemical reaction material 101 is above-mentioned second enzyme.In this embodiment, described second enzyme can be ASAH1 2, but not limited to this.
In still another embodiment, the enzyme constituent in aforementioned biochemical reaction material 101 is above-mentioned first enzyme and the combining of second enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this, and described second enzyme can be ASAH1 2, but it is also not necessarily limited to this.
Additionally, the material of the container 103 in biochemical reaction device 100 of the present invention, it may include glass, acryl, polypropylene, polyethylene, rustless steel or titanium alloy etc., but not limited to this.In one embodiment, the material of the container 103 in biochemical reaction device 100 of the present invention is polypropylene.Also, the shape of container is not particularly limited, and in one embodiment, container is hollow column.
In one embodiment, as shown in Figure 1B, the biochemical reaction device 100 of the present invention, also can further include filter material 109, and it is arranged at above-mentioned at least one entrance 105 rear and above-mentioned at least one outlet 107 rears.Also, the aperture of above-mentioned filter material is less than above-mentioned biochemical reaction material 101, to avoid biological respinse material 101 to spill from above-mentioned at least one entrance 105 and/or at least one outlet 107, so it can make liquid sample pass through.The material of above-mentioned filter material includes filter paper, glass, acryl, polypropylene, polyethylene etc., but not limited to this.In this embodiment, the base material of biological respinse material 101 can have grain structure or hollow tubular structure.In a specific embodiment, the base material of biological respinse material 101 has grain structure, and in this specific embodiment, the base material of biological respinse material 101 can be cellulose pearl or shitosan pearl, but not limited to this.
When the base material of biological respinse material 101 is hollow tubular structure, also using the encapsulation of polyurethanes (polyurethane, PUR) glue, and can not use filter material.
In one embodiment, container 103 can be hollow column, and the two ends of its hollow column are respectively provided with the first entrance 105 of above-mentioned at least one entrance 1051First outlet 107 with above-mentioned at least one outlet 1071.In this embodiment, the base material of biological respinse material 101 can have grain structure or hollow tubular structure.
In another embodiment, as shown in Figure 1 C, container 103 can be hollow column, and the two ends of its hollow column are respectively provided with the first entrance 105 of above-mentioned at least one entrance1First outlet 107 with above-mentioned at least one outlet1, and the second entrance 105 of above-mentioned at least one entrance2Second outlet 107 with above-mentioned at least one outlet2On the side wall of cylindrical body.In this embodiment, first liquid sample can be from the first entrance 105 of container 1031Flow into biochemical reaction device 100, flow through biological respinse material 101, and from first outlet 1071Flow out.Separately there is a second liquid, can be water, dialysis solution, contain salts solution by second entrance 1052Flow into biochemical reaction device 100, flow through biological respinse material 101, and from second outlet 1072Flow out.Second liquid can take the by-product after reaction or dialysis out of.
In this embodiment, the base material of biological respinse material 101 can have grain structure or hollow tubular structure.In a specific embodiment, the base material of biological respinse material 101 has hollow tubular structure, and in this specific embodiment, the base material of biological respinse material 101 can be cellulose hollow fiber, but not limited to this.
In still another embodiment of the present invention, the present invention provides a kind of extracorporeal treatment blood or the method for blood plasma.By the method for extracorporeal treatment blood of the present invention or blood plasma, the elecrtonegativity low density lipoprotein, LDL in blood can be removed.Aforementioned elecrtonegativity low density lipoprotein, LDL, it may include, but be not limited to, L1, L2, L3, L4 and/or L5 etc..In one embodiment, described elecrtonegativity low density lipoprotein, LDL is elecrtonegativity low density lipoprotein, LDL L5.
The method of the extracorporeal treatment blood of the present invention or blood plasma may include following step, but not limited to this.
First, blood or blood plasma is made to contact with enzyme constituent in vitro, so that enzyme constituent reacts to blood or blood plasma, wherein, described enzyme constituent has the ability removing elecrtonegativity low density lipoprotein, LDL.
Above-mentioned enzyme constituent may include, in order to remove the first enzyme, the second enzyme in order to remove the ceramide entrained by elecrtonegativity low density lipoprotein, LDL of the glycosyl of elecrtonegativity low density lipoprotein, LDL, or combinations of the above, but not limited to this.There is no particular restriction for the source of species of above-mentioned first enzyme and second enzyme.In one embodiment, the first enzyme and the source of species of second enzyme are the mankind.
Above-mentioned first enzyme can be sialidase or glycosidase.
Sialidase may include neuraminidase 1 from human body, neuraminidase 2, neuraminidase 3, neuraminidase 4, O- sialidase etc., or turned with gene grow, show and purification and come above-mentioned enzyme one, or be from virus, sialidase of antibacterial or other species etc., but not limited to this.
And the example of glycosidase, then may include and come from the α and β glucosidase of human body or animal body, maltase-glucoamylase, Sucrase-isomaltase etc., or turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, or come from the N- glycosidase F of virus, antibacterial or other species, glucosidase etc., but not limited to this.
Also, above-mentioned second enzyme can be ceramidase.
Ceramidase then may include ASAH1 (N-acylsphingosine amidohydrolase)，Such as ASAH1 1 (N-acylsphingosine amidohydrolase 1,ASAH1)、ASAH1 2 (N-acylsphingosine amidohydrolase 2,ASAH2)、ASAH1 2B (N-acylsphingosine amidohydrolase 2B,ASAH2B)、ASAH1 2C (N-acylsphingosine amidohydrolase 2C,ASAH2C) etc.、N- acyl ethanol amine acylating acid enzyme (N-acylethanolamine acid amidase)、Alkaline ceramidase 1 (alkaline ceramidase 1)、Alkaline ceramidase 2 (alkaline ceramidase 2)、Alkaline ceramidase 3 (alkaline ceramidase 3)，But not limited to this.
In one embodiment, the enzyme constituent used in the method for extracorporeal treatment blood of the present invention or blood plasma is above-mentioned first enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this.
In another embodiment, the enzyme constituent used in the method for extracorporeal treatment blood of the present invention or blood plasma is above-mentioned second enzyme.In this embodiment, described second enzyme can be ASAH1 2, but not limited to this.
Additionally, in one embodiment, the enzyme constituent used in the method for extracorporeal treatment blood of the present invention or blood plasma is securable on base material.The example of above-mentioned base material, it may include silica gel, cellulose, diethylaminoethyl cellulose, shitosan, polystyrene, polysulfones, polyether sulfone, resin and polysaccharide, but not limited to this.Also, above-mentioned base material can have chondritic or hollow tubular structure.
In still another embodiment, the enzyme constituent used in the method for extracorporeal treatment blood of the present invention or blood plasma is above-mentioned first enzyme and the combining of second enzyme.In this embodiment, described first enzyme can be neuraminidase 2, but not limited to this, and described second enzyme can be ASAH1 2, but it is also not necessarily limited to this.
In the method for extracorporeal treatment blood of the present invention or blood plasma, the time that blood or blood plasma are contacted with enzyme constituent is made to can be about 0.25-8 hour in vitro.In one embodiment, can be about 2 hours.
Also, in the method for extracorporeal treatment blood of the present invention or blood plasma, making the temperature that blood or blood plasma are contacted with enzyme constituent can be about 4-40 DEG C in vitro.In one embodiment, the temperature that blood or blood plasma are contacted with enzyme constituent is made to can be about 37 DEG C in vitro.
In addition, in the method for extracorporeal treatment blood of the present invention or blood plasma, in external, blood or blood plasma can be made to be contacted under about pH5-10 with enzyme constituent.In one embodiment, in external, blood or blood plasma can be made to be contacted under about pH7.4 with enzyme constituent.
Then, terminate blood or blood plasma is contacted with enzyme constituent, with terminating enzyme constituent, blood or blood plasma are reacted.
Terminate the mode that blood or blood plasma contacted with enzyme constituent to be not particularly limited, for example, blood or blood plasma are separated with enzyme constituent, or so that enzyme constituent is lost activity.
Embodiment
Embodiment 1
A. method
1. the acquirement of low density lipoprotein, LDL (low-density lipoprotein, electronegative LDL)
(1) purification of low density lipoprotein, LDL (low-density lipoprotein, electronegative LDL)
It is individual acquisition for the detached blood sample of low density lipoprotein, LDL.After preliminary screening, avoiding condensing blood under the preventive measure aoxidizing with vitro (ex vivo) from individual removal.Complete Protease
Inhibitor Cocktail(Roche；Cat.No.05056489001；1tablet/100mL) processing blood plasma to avoid protein degradation.Prepare (Lipoprotein preparation from human) from the lipoprotein of the mankind.Blood plasma is covered and in 20,000rpm centrifugation 2 hours with 2mL Milli-Q water.Remove upper strata white fraction, Chylomicron (chylomicrons), and retain and contain very low density lipoprotein (VLDL) (very-low-density lipoprotein, VLDL), middle low density lipoprotein, LDL (intermediate-density lipoproteins, IDL), low density lipoprotein, LDL and high density lipoprotein (high-density lipoprotein, HDL remaining lower floor), for a series of separating step.
In order to further will be separated from one another with high density lipoprotein (1.063-1.210g/dL) to very low density lipoprotein (VLDL) (d=0.93-1.006), middle low density lipoprotein, LDL (d=1.006-1.019), low density lipoprotein, LDL (1.019-1.063g/dL), by interpolation potassium bromide, respectively remaining sample is one after the other adjusted to d=1.006, d=1.019, d=1.063, d=1.210, and be then centrifuged 20 hours with 45,000rpm in 4 DEG C.After the centrifugation of each separating step, middle low density lipoprotein, LDL can be dropped, and very low density lipoprotein (VLDL), low density lipoprotein, LDL and high density lipoprotein then can be collected.Separated very low density lipoprotein (VLDL), low density lipoprotein, LDL and high density lipoprotein sample are processed with 5mM EDTA and nitrogen to avoid in vitro (ex vivo) oxidation.By very low density lipoprotein (VLDL), low density lipoprotein, LDL and high density lipoprotein sample with buffer solution A (0.02M Tris-HCl, pH 8.0 and 0.5mM EDTA) carry out dialysing 24 hours, continuously repeat above dialysis step to amount to 3 times, to remove excessive potassium bromide, and the filter membrane (Sartorius with 0.22- μm of aperture；) filtered so that sample is aseptic.
(2) LDL subfraction (subfractions):
By useFast protein liquid chromatographic analyses (fast-protein liquid chromatography, FPLC) pump (GE Healthcare Life Sciences, Pittsburgh, PA), the low density lipoprotein, LDL material of about 30mg is injected into UnoQ12anion-exchange column (BioRad).By the buffer solution B (1mol/L NaCl is in buffer solution A) using multi-step gradient (multistep gradient), under the flow velocity of 2mL/ minute, LDL can be eluted out according to electronegativity (electronegativity).In brief, sample can be balanced 10 minutes with buffer solution A, then, be linearly increasing to 15% buffer solution B (fraction (fraction) 1) in 10 minutes, be linearly increasing to 20% buffer solution B (fraction 2,3) in 30 minutes, be maintained at 20% buffer solution B and reach, 10 minutes (fraction 4) be linearly increasing to 100% buffer solution B (fraction 5) in 20 minutes.Finally, monitoring eluant (effluents) under 280nm.
(3) purification of the low density lipoprotein, LDL being classified:
According to gradient curve (gradient profile), each low density lipoprotein, LDL fraction is collected.The volume of each subfraction (subfraction) is fixing.During chromatographic analyses, the dilution of low density lipoprotein, LDL is according to injected slurry volume.Can by other fraction withfilters(YM-30；EMD Millipore Corp., Billerica, MA) concentrate, carry out dialysing 24 hours with buffer solution A (0.02M Tris-HCl, pH 8.0 and 0.5mM EDTA), continuously repeat above dialysis step to amount to 3 times, and pass through 0.22- μm of filter membrane (Sartorius；) so that it is aseptic.By Lowry method, separated fraction is quantified its protein concentration and be stored in 4 DEG C afterwards.
2.NEU2 or the screening of NEU4
(1) turn shape (Transformation) (the gene choosing having NEU2 with the pCMV6 carrier of NEU4 gene is grown)：
NEU2 (neuraminidase 2) and NEU4 (neuraminidase 4) is purchased from Origene, RC219858 and RC203948.It is competent at cell (Yeastern, FYE608) by ECOSTM 101DH5 α, indicate to amplify gene according to manufacturer.
In brief, the competent cell of 1 vial (vial) and 5 μ L plastids are shaken one second, and be incubated at 5 minutes on ice afterwards.After 42 DEG C of 45 seconds heat shock (heat-shoc), by mixture painting disk on the LB agar with kanamycin (kanamycin).
Bacterium colony (colonies) is confirmed by VP1.5 and XL39 introduction with Polymerase Chain Reaction, 95 DEG C of Polymerase Chain Reaction, 1 minute, for pre-polymerization enzyme chain reflex degeneration (pre-PCR denaturation)；95 DEG C of 2 circulations, 10 seconds, 62 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 2 circulations, 10 minutes, 60 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 2 circulations, 10 seconds, 58 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 15 circulations, 10 seconds, 56 DEG C, 20 seconds, 72 DEG C, 4 minutes；In 72 DEG C, cultivate in order to post-polymerization enzyme chain reflex and be maintained at 4 DEG C within 10 minutes.
(2) plastid extraction
After confirming insertion through the bacterium colony turning shape, will be placed in the 5mL LB culture fluid with 25mg/mL kanamycin through turning shape cell, 37 DEG C of culture overnight afterwards.
Plastid DNA is extracted according to the step of Plasmid Miniprep Plus Purification Kit (GeneMark, DP01P).In brief, antibacterial is centrifuged 1 minute under 14,000x g, removes culture medium.Precipitate is resuspended in 200 μ L SolutionI, adds 200 μ L SolutionII afterwards, and to mix by being inverted test tube.Add 200 μ L SolutionIII and to mix by being inverted test tube 5 thing.By cell solute (lysate) with high speed centrifugation 5 minutes, and a close white precipitate can be formed along tube wall.Tubing string (spin column) insertion one collection tubing string will be centrifuged, and limpid cell solute will be moved to centrifugation tubing string, and with high speed centrifugation 1 minute.Effluent is abandoned, 500 μ L Endotoxin Removal Wash Solution is loaded in centrifugation tubing strings and waits 2 minutes with balance film, then with high speed centrifugation 1 minute.Filter liquor is abandoned and adds 700 μ L Washing Solution, in high speed centrifugation 1 minute, and repeat this step.Abandon filter liquor and in 5 minutes to remove the micro ethanol of residual.Centrifugation tubing string is moved in a new test tube and adds 35 μ L water, waits 1-2 minute, and in high speed centrifugation 2 minutes to elute out DNA.By micropore disk spectrogrph (microplate spectrophotometer) (Epoch, BioTek), DNA is quantitative.
(3) transfection for HEK cell (transfection) and protein purification
In day before transfection, by 1.25*105HEK293T cell is placed in 500 μ L DMEM culture medium in 24- porose disc.For cell in each hole that will be transfected, the DNA of 1 μ g is diluted in 100 μ L serum-free mediums, and adds Lipofectamine 2000Transfection Reagent (Invitrogen) of 1.5 μ L, gently mixes and be incubated at room temperature 30 minutes.After incubation, add above-mentioned complex to mix to each hole containing cell and gently.By cell culture in 37 DEG C of CO220 hours in incubator.By the RIPA containing protease inhibitor by the cell dissolving through transfection, and prepare standardization protein.
In brief, 80 μ L ANTI-FLAG M2Magnetic Beads (Sigma-Aldrich) are balanced for single hole (one-well) cell lysate purification, in protein-resin-bonded after 4 DEG C overnight, by winning the competition elution of peptide with 150 μ g/mL 3X FLAG twice to elute out combined FLAG fusion protein, collect eluant and to confirm protein by Western blot (western blot).
3.NEU2 or the efficacy test of NEU4
(1) quantification of protein:
Using Pierce BCA Protein Assay Kit (Thermo), indicated according to manufacturer, protein is carried out quantitation.
Briefly, the BSA standard substance of 25 μ L serial dilutions and 5 μ L samples in 20uL sample diluting liquid are drawn to 96- hole culture plate.In order to prepare BCA working reagent, the BCA reagent A of 50 parts of mixing with 1 part of BCA reagent B and is placed on ice until using.The BCA working reagent adding 200 μ L to every hole and is thoroughly mixed, and covers culture plate and cultivates 30 minutes in 37 DEG C.Measure light absorption value by spectrogrph (Epoch, BioTek) in 562nm.
(2) apoptosis (Apoptosis) measure
Vascular endothelial cell, nephrocyte are used after 3 or 4 generations and are maintained at the DMEM containing 10% hyclone (Invitrogen).Hyclone is reduced to 5% in DMEM during processing.By 1 × 104Individual cell kind enter 24 hours sparse cultures (subconfluent cultures) of 96 porose disc cultured cells be exposed to PBS (no lipoprotein, negative control group) or classification (25,50 and 100 μ g/mL) LDL subfraction (subfractions), (unfractionated normolipidemic) LDL and LDL/L1/L5 not being classified normal lipid and sialidase (sialidase) culture 24 hours.Analyze apoptosis by with the visualization of Zeiss Axiovert 200 fluorescence microscope and the optical filter of the digital image of the dyeing caught according to nucleus, the Hoechst 33342 of apoptosis DNA film integrality (integrity), propidium iodide (propidium iodide) (red) and calcein (calcein) AM (green).By using Cell Death Detection ELISA Assay (Roche), the program according to manufacturer is checking Cytoplasm histone associated clip (cytoplasmic histone-associated DNA fragmentation).
(4) it is used for the LC/MS of protein compositionEAnalysis
By using using the parallel fragment mass spectrograph of continuous coupled liquid chromatography (LC) Dynamic data exchange (serially coupled liquid chromatography data-independent parallel-fragmentation mass spectrometry, LC/MSE) Quantitative Western plastid technology come quantitative low density lipoprotein, LDL subfraction protein content.This kind of analysis is shown, with regard to, in terms of relative and/or absolute (when being incorporated to spiked internal peptide standard substance in the data collection/analysis) protein abundances in complex protein mixtures, being highly quantitative.Except Waters Xevo G2 and SynaptTMOutside HDMS mass spectrograph (Waters Corporation, MA, USA), substantially can execute quantitative analyses as discussed previously.
Briefly, the gross protein isolated from each low density lipoprotein, LDL fraction is decomposed by trypsin, and produced trypsin decompose victory peptide by by chromatographic separation by Nano-Acquity separations module (Waters Corporation, MA, USA), the 50fmol-on-column tryptic digest merging yeast alcoholdehydrogenase (yeast alcohol dehydrogenase) is inherent spiked protein plasmid standards for quantitation.Via 75 ì m × 25cm BEH C-18 tubing string under a gradient condition, in 30 minutes flow velocitys with 300nL/ minute, in 35 DEG C of execution victory peptide elutions.Mobile phase is to be made up of with the formic acid (formic acid) (0.1%v/v) for Molecular Mass (molecule protonation) the acetonitrile (acetonitrile) by Organic modification agent (organic modifier).Mass spectrograph is performed in and is equipped with the Synapt HDMS instrument that nanometer EFI spills ionizing interface (nano-electrospray ionization interface), and operate in Dynamic data exchange collection mode (data-independent collection mode, MSE).Formulate parallel ion fragmentation (Parallel ion fragmentation) to switch between low (4eV) and high (15-45eV) energy in collision cell, and calibrate (separate data channel lock mass calibrant) using glu- fibrinopeptide (glu-fibrinopeptide) B for separately data channel fixed mass, collect data from 50 to 2000m/z.With ProteinLynx GlobalServer v2.4 (Waters) processing data.Search from Uniprot (www.uniprot.org) human protein database and remove isotope result (Deisotoped results
Will albumen qualitative correlation).
The screening of 4.ASAH2
(1) turn shape (the gene choosing with the pCMV6 carrier of ASAH2 gene is grown)：
ASAH2 (N-acylsphingosine amidohydrolase 2) is purchased from Origene, RC203706.It is competent at cell (Yeastern, FYE608) by ECOSTM 101DH5 α, indicate to amplify gene according to manufacturer.
In brief, the competent cell of 1 vial (vial) and 5 μ L plastids are shaken one second, and be incubated at 5 minutes on ice afterwards.After 42 DEG C of 45 seconds heat shock (heat-shoc), by mixture painting disk on the LB agar with kanamycin.
Bacterium colony (colonies) is confirmed by VP1.5 and XL39 introduction with Polymerase Chain Reaction, 95 DEG C of Polymerase Chain Reaction, 1 minute, for pre-polymerization enzyme chain reflex degeneration (pre-PCR denaturation)；95 DEG C of 2 circulations, 10 seconds, 62 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 2 circulations, 10 minutes, 60 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 2 circulations, 10 seconds, 58 DEG C, 20 seconds, 72 DEG C, 4 minutes；95 DEG C of 15 circulations, 10 seconds, 56 DEG C, 20 seconds, 72 DEG C, 4 minutes；In 72 DEG C, cultivate in order to post-polymerization enzyme chain reflex and be maintained at 4 DEG C within 10 minutes.
(2) plastid extraction
After confirming insertion through the bacterium colony turning shape, will be placed in the 5mL LB culture fluid with 25mg/mL kanamycin through turning shape cell, 37 DEG C of culture overnight afterwards.
Plastid DNA is extracted according to the step of Plasmid Miniprep Plus Purification Kit (GeneMark, DP01P).In brief, antibacterial is centrifuged 1 minute under 14,000x g, removes culture medium.Precipitate is resuspended in 200 μ L Solution I, adds 200 μ L Solution II afterwards, and to mix by being inverted test tube.Add 200 μ L Solution III and to mix by being inverted test tube 5 thing.By cell solute (lysate) with high speed centrifugation 5 minutes, and a close white precipitate can be formed along tube wall.Tubing string (spin column) insertion one collection tubing string will be centrifuged, and limpid cell solute will be moved to centrifugation tubing string, and with high speed centrifugation 1 minute.Effluent is abandoned, 500 μ L Endotoxin Removal Wash Solution is loaded in centrifugation tubing strings and waits 2 minutes with balance film, then with high speed centrifugation 1 minute.Filter liquor is abandoned and adds 700 μ L Washing Solution, in high speed centrifugation 1 minute, and repeat this step.Abandon filter liquor and in 5 minutes to remove the micro ethanol of residual.Centrifugation tubing string is moved in a new test tube and add 35 μ L water, waits 1-2 minute, and in high speed centrifugation 2 minutes to elute out DNA.By micropore disk spectrogrph (microplate spectrophotometer) (Epoch, BioTek), DNA is quantitative.
(3) transfection for HEK cell (transfection) and protein purification
In day before transfection, by 1.25*105Individual HEK293T cell is placed in 500 μ L DMEM culture medium in 24- porose disc.For cell in each hole that will be transfected, the DNA of 1 μ g is diluted in 100 μ L serum-free mediums, and adds the Lipofectamine 2000 Transfection Reagent (Invitrogen) of 1.5 μ L, gently mixes and be incubated at room temperature 30 minutes.After incubation, add above-mentioned complex to mix to each hole containing cell and gently.By cell culture in 37 DEG C of CO220 hours in incubator.By the RIPA containing protease inhibitor by the cell dissolving through transfection, and prepare standardization protein.
In brief, 80 μ L ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) are balanced for single hole (one-well) cell lysate purification.
In protein-resin-bonded after 4 DEG C overnight, by winning the competition elution of peptide with 150 μ g/mL 3X FLAG twice to elute out combined FLAG fusion protein, collect eluant and to confirm protein by Western blot (western blot).
5.ASAH2 efficacy test
(1) quantification of protein:
Using Pierce BCA Protein Assay Kit (Thermo), indicated according to manufacturer, protein is carried out quantitation.
Briefly, the BSA standard substance of 25 μ L serial dilutions and 5 μ L samples in 20 μ L sample diluents are drawn to 96- hole culture plate.In order to prepare BCA working reagent, the BCA reagent A of 50 parts of mixing with 1 part of BCA reagent B and is placed on ice until using.The BCA working reagent adding 200 μ L to every hole and is thoroughly mixed, and covers culture plate and cultivates 30 minutes in 37 DEG C.Measure light absorption value by spectrogrph (Epoch, BioTek) in 562nm.
(2) lipid extraction
The ASAH2 of 30 μ g LDL/L1/L5 and 5 μ g is rushed solution in ASAH2, and (200mM Tris-HCl is in pH 8.4,1.5M NaCl, 25mM CaCl2) in be incubated at 37 DEG C.After culture 2 or 24 hours, transfer samples in teat glass.By 1mL water, 2.5mL methanol and 1.25mL CHCl3Add in sample, shake 15 seconds.Afterwards, extra 0.9mL water and 1.25mL CHCl are provided3To sample, shake 15 seconds and be centrifuged 10 minutes in 3000rpm.Using glass syringe, bottom organic solvent is moved to 2.0mL teat glass.Precipitation is rushed each sample until drying with nitrogen, and with 0.25mL sample solution (isopropanol/acetonitrile/ water=2:1:1) dissolve.
(3) it is used for the LC/MS of lipid compositionEAnalysis
By using liquid chromatography (LC) Dynamic data exchange parallel fragment mass spectrograph (LC/MSE) to be quantitatively derived from the lipid content of the TL of each subfraction of low density lipoprotein, LDL, phospholipid (phospholipids), neutral lipid (neutral lipids) and free fatty (free fatty acid).Quantitative analyses substantially can be executed as discussed previously.
Briefly, lipid is via merging CSHTM1.7 μm, the Acquity separations module (Waters Corporation, MA, USA) of 2.1mm × 10cm C-18 tubing string, under a gradient condition, with the flow velocity of 400 μ L/ minutes in 18 minutes, in 55 DEG C of chromatography.Mobile phase A is by ACN/H210mM NH in O (60/40)4HCO2Constituted with 0.1% formic acid (0.1%v/v), mobile phase B is by the 10mM NH in IPA/ACN (90/10)4HCO2Constituted (0.1%v/v) with 0.1% formic acid for Molecular Mass to be constituted.Mass spectrograph is performed in and is equipped with the Synapt HDMS instrument that nanometer EFI spills ionizing interface (nano-electrospray ionization interface), and operate in Dynamic data exchange collection mode (data-independent collection mode, MSE).Formulate parallel ion fragmentation (Parallel ion fragmentation) to switch between low (4eV) and high (35-55eV) energy in collision cell, and calibrate (separate data channel lock mass calibrant) using leucine (leucin) for separately data channel fixed mass, collect data from 50 to 1600m/z.With MarkerLynx (Waters) processing data.
B. result
1. turn shape
(1) NEU2 and NEU4
NEU2 turn shape result such as Fig. 2 with shown.
From Fig. 2A, the NEU2 of bacterium colony 3,5 and 6 (respectively referring to electrophoresis track (lane) 3,5 and 6) turns formation work(.Therefore select the plastid that bacterium colony 3,5 and 6 amplifies and preserves NEU2.
(1)ASAH2
ASAH2 to turn shape result as shown in Figure 3.
Select the plastid that bacterium colony 7 amplifies and preserves ASAH2.
2. transfect
Confirm the gene transfection of NEU4/ASAH2 by Western blot, result is as shown in Figure 4.
Transfection conditions are as follows：
HEK293T 1.25x105Individual cell is in 24 holes
Plastid：NEU4 and ASAH2
Amount of DNA：1μg
Transfected by Lipofectamine.
Western condition is as follows：
SDS-PAGE：Using 5 μ L samples
Antibody：Anti- DDK (1:2000)
3. protein purification
(1) purification of NEU2
The purification result of NEU2 is as shown in Figure 5.Fig. 5 shows and is really purified into NEU2.The aminoacid sequence of NEU2 such as sequence identification number：Shown in 1.
(2) ASAH2 purification
The purification result of ASAH2 is as shown in Figure 6.Fig. 6 shows and is really purified into ASAH2 (extraction 1 and the protein that obtained by different batches of extraction 2).The aminoacid sequence of ASAH2 such as sequence identification number：Shown in 2.
Embodiment 2 enzyme is fixed
Method 1
0.4454g silica gel after thermal activation is placed in 7mL CHCl3, and add 1/5 part of weight APTS stirring at normal temperature after 24 hours by filtration, gained solid is drained for lower 50 DEG C in vacuum, solidss after draining add 5% glutaraldehyde (glutaraldehyde) (the phosphate buffer solution 1X TBS of pH=8) to stir 21 hours under room temperature, filtration is washed and is taken out wherein solidss, NEU21/100-10000 (wt%) is added to be diluted to volume as 2mL with phosphate buffer solution 1X TBS pH=8, finally solid is filtered within 24 hours in normal-temperature reaction, enzyme immobilization product (ITRI-Siw-Nu-01) be can get with phosphate buffer solution pH=8 washing.
Method 2
Thermal activation silica gel add in the toluene (toluene) 1/5 part of weight 3- glycidyl ether oxygen propyl trimethoxy silicane (3-glycidoxypropyltrimethoxysilane) to flow back 20 hours after by filtration, and gained solid to be drained under vacuum after washing with acetone.After NEU2 adds above-mentioned process, resin 1/100～1/10000 (wt%) stirs 2 hours in phosphate buffer solution and filters solid after 15 points, and washs with the buffer solution of deionized water and pH=8 respectively to obtain enzyme and fix product.
Method 3
1g cellulose pearl is taken to adjust pH to 11 about with NaOH solution in 15mL water, 1g Bromine cyanide. (cyanogen bromide) is added under the temperature remains within the normal range, after about 30 minutes, pearl is cleaned with the phosphate buffer solution of deionized water, pH=8 in regular turn, this pearl is added 1/600 weight than NEU2 in phosphate buffer solution, after stirred overnight, again enzyme immobilization product is cleaned to obtain with the phosphate buffer solution of pH=8.
Method 4
0.5g cellulose pearl is taken to flow back 4 hours in 1.5mL APTS 10mL toluene solvant, cellulose pearl is filtered and is cleaned with the phosphate buffer solution of acetone and pH=8 respectively, the phosphate buffer solution that the pearl obtaining is added 5% (w/v) glutaraldehyde/pH=8 stirs 21 hours under room temperature, filter and take out solidss, and glutaraldehyde activated silica gel is obtained with phosphate buffer solution (pH=8) washing, this pearl is added 2/1000 weight again with the phosphate buffer solution of pH=8 to clean to obtain enzyme immobilization product after stirred overnight than NEU2 in the phosphate buffer solution.
Method 5
Take5% glutaraldehyde (phosphate buffer solution of pH=8) is added to stir 21 hours under room temperature, filter and take out solidss, and glutaraldehyde activated silica gel is obtained with phosphate buffer solution (pH=8) washing, NEU21/10000 (wt%) is taken to be diluted to volume as 15mL with phosphate buffer solution pH=8, glutaraldehyde activated silica gel stirs 20 hours under room temperature with above-mentioned 1.13g again, finally solid is filtered, enzyme immobilization product be can get with phosphate buffer solution pH=8 washing.
Method 6
1g diethylaminoethyl cellulose (diethylaminoethyl cellulose, DEAE cellulose) it is suspended in NaOH (1M 10mL aqueous solution) with after the washing of 10mL water, stirring 10 minutes after by filtrations wash, the solid of acquisition is suspended in 10mL dioxane (dioxane), in addition 2g Cyanuric Chloride (cyanuric chloride) and 10mL toluene are added stirring in above-mentioned solid aaerosol solution will wherein filter solid after 30 minutes, respectively again with drying under reduced pressure after dioxane, water, acetone cleaning.Then, by this activating solid (activated solid support) add NEU21/10000 (wt%) stir 18 hours by filtration, washing obtain enzyme immobilization product.
Method 7
0.5g shitosan (chitosan) pearl adds 10mL 0.5% glutaraldehyde, under room temperature, stirring thoroughly washs acquisition enzyme immobilization product by reactant filtration and with deionized water afterwards continuously with water thoroughly cleaning after this activating solid is added NEU21/3500 (wt%) normal-temperature reaction 2 hours in 1 hour.
Method 8
1g cellulose hollow fiber (cellulose hollow fiber) with example 4 method respectively through APTS, glutaraldehyde activated after add NEU23/10000 (wt%) pH=8 phosphate buffer solution stirred overnight after again enzyme immobilization product is cleaned to obtain with the phosphate buffer solution of pH=8.
Method 9
1g cellulose hollow fiber, cleans to obtain enzyme immobilization product with the phosphate buffer solution of pH=8 after the phosphate buffer solution stirred overnight not adding NEU2pH=8 with the method for example 3 after CNBr activation again.
Method 10
Take ECR-8204F epoxy-acrylate (epoxy-acrylate) resin with deionized water wash after, add ASAH2 1/10000 (wt%), and with 0.2M sodium ascorbyl phosphate buffer solution adjustment cumulative volume to 2ml, and rotate 24hr by filtration, and respectively with deionized water, 2M pH=8 phosphate buffer solution clean.Obtain wet product about 52mg.(ITRI-EC-AS-01)
Method 11
Again respectively with 3 after taking Iontosorb MT200 fiber pearl first to clean with deionized water:7 water/dioxane, 7:3 water/dioxane, dioxane is added after 100% dioxane cleaning, and add 1/3 part of CDI rotation about 0.5-1hr, extract dioxane under decompression, add Neu2 Stirring about 2hr 15min immediately, after reaction, solution is filtered and rinse wet product about 0.2g (ITRI-CD-01) with pH of buffer=6.5.
Method 12
Take 0.5ug Neu2 to add in 2%w/v alginate (aliginate) aqueous solution and the 2%CaCl in stirring is instilled with syringe needle2(wt/v) aqueous solution, continues stirring 30min after dripping off, particles in solution is filtered and washes to obtain wet product (ITRI-A-01) with deionized water.
Embodiment 3
Effect of immobilization NEU2 filling device
With the method 2 shown in embodiment 2, NEU2 is fixed, and fixing enzyme is filled in a tubing string to be formed as the biochemical reaction device (immobilization NEU2 filling device) in Figure 1B.
(1) apoptosis measure
By L5 (25 μ g/mL；50 μ g/mL) and through immobilization NEU2 filling device process L5 (1.25 μ g) (37 DEG C of the treatment temperature of 2 hours, pH 7.4) co-culture 24 hours with vascular endothelial cell respectively, and carrying out apoptosis mensure, result is as shown in Figure 7.
Can learn that 25 μ g/mL L5 cause about 15% apoptosis of vascular endothelial cell reaction according to Fig. 7,50 μ g/mL L5 cause about 30% apoptosis, and after 1.25 μ g NEU2 effects, L5 lowers for the apoptosis impact of vascular endothelial cell.
(2) elecrtonegativity low density lipoprotein, LDL quantitative analyses
Will be not treated, be not added with 37 DEG C ferment treatment 2 hours or through NEU2 process 2 hours (37 DEG C for the treatment of temperature, pH 7.4) the LDL sample being obtained by cardiac patient carry out L5 quantitative analyses respectively, to determine the content of L5, result is as shown in Figure 8.
According to Fig. 8, by a LDL corpse or other object for laboratory examination and chemical testing through NEU2 enzyme effect 2 hours later, the content of L5 reduces, and 12.4% is reduced to 8.48% from originally.
(3) mass spectral analyses
L5 is carried out mass spectral analyses with the L5 through processing 2 hours (37 DEG C for the treatment of temperature, pH 7.4) through NEU2, result is respectively shown in Fig. 9 A, Fig. 9 B and Fig. 9 C.
The feature of known L5 is that the serine (serine) of apo E (apolipoprotein E, apoE) is usually modified by candy base with threonine (threonine).
Referring to Fig. 9 A and Fig. 9 B, molecular weight 1497 is avirulence LDL, and LDL adds that a glycosyl molecule is that 1700, LDL adds that two candy base molecules are 1884, and LDL adds that three glycosyl molecules are 2154.Fig. 9 C shows the aminoacid sequence of apo E by candy modification, and making script win peptide chain charge-mass ratio 1497.8009 increases as 1700.8868,1884.9021 and 2154.0300.
Figure 10 A1-2 shows, processes the L5 of 2 hours through immobilization NEU2 filling device, do not detected charge-mass ratio be 1700,1884 or 2154 molecule, represent and do not had candy base on the serine of apo E and threonine and modify, represent that the candy base of LDL is removed.
Similarly, Figure 10 B1-2 shows, processes the L5 of 2 hours through immobilization NEU2 filling device, candy base modification also in other sites of apo E, the candy base representing LDL is removed, the candy base representing LDL is removed.
Embodiment 4
Effect of ASAH2
(1) LC/MS of the L5 of 24 hours is processed with ASAH2EAnalysis
L5 is processed 24 hours with ASAH2, and undressed L5 is carried out LC/MS with the L5 through aforementioned processingEAnalysis is to measure its ceramide (ceramide) content (Detailed Experimental mode is referring to " the 5.ASAH2 efficacy test " in " the A. method " of embodiment 1).
LC/MSEAnalysis result is as shown in table 1 (four shown in every group numerical value be derived from by identical sample measures 4 times).Sample signal each in table 1 is converted and is obtained various kinds this ceramide content percentage ratio (with the highest signal the most 100% of undressed L5), result is as shown in figure 12.The ASAH2 that ASAH2#1 in Figure 12 and ASAH2#2 is obtained by different batches.
Table 1, untreated L5 and the LC/MS with the ASAH2 process L5 of 24 hoursEAnalysis result
| 24 little base lines | With ASAH2#1 process | With ASAH2#2 process |
| Signal | 459.6464 | 295.3353 | 202.1154 |
| 443.4776 | 236.9632 | 177.1598 | |
| 449.8201 | 230.7273 | 173.031 | |
| 451.5772 | 249.0337 | 175.7823 | |
| Meansigma methodss | 451.1303 | 253.0149 | 182.0221 |
| Standard deviation | 6.658431 | 29.21906 | 13.50503 |
| Reduce | 43.9 | 59.7 |
24 little base lines represent the ceramide content contained by undressed L5
According to table 1 and Figure 12, after L5 is processed 24 hours through ASAH2, its ceramide content is decreased obviously.
(2) LC/MS of the L5 of 24 hours is processed with ASAH2EAnalysis (Detailed Experimental mode is referring to " the 5.ASAH2 efficacy test " in " the A. method " of embodiment 1).
L5 is processed 24 hours with ASAH2, and the L5 that undressed L5 is processed with premenstrual number carries out LC/MSEAnalysis is to measure its ceramide content.
LC/MSEAnalysis result is as shown in table 2 (four shown in every group numerical value be derived from by identical sample measures 4 times).Sample signal each in table 2 is converted and is obtained various kinds this ceramide content percentage ratio (with the highest signal the most 100% of undressed L5), result is as shown in figure 13.
Table 2, untreated L5 and the LC/MS with the ASAH2 process L5 of 24 hoursEAnalysis result
| 24 little base lines | With ASAH2#1 process | With ASAH2#2 process | |
| Signal | 2008.465 | 1827.823 | 1638.186 |
| 2007.321 | 1747.422 | 1627.067 | |
| 1946.985 | 1725.032 | 1622.848 | |
| 1989.728 | 1688.382 | 1616.651 | |
| Meansigma methodss | 1988.125 | 1747.165 | 1626.188 |
| Standard deviation | 28.73594 | 59.02271 | 9.070668 |
| Reduce | 12.11997 | 18.20495 |
24 little base lines represent the ceramide content contained by undressed L5
According to table 2 and Figure 13, after L5 is processed 24 hours through ASAH2, its ceramide content is decreased obviously.
(3) LC/MS of the L5 of 2 or 24 hours is processed under buffer solution is presence or absence of with ASAH2EAnalysis
In buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) presence or absence of lower, L5 is processed 2 or 24 hours with ASAH2, and undressed L5 is carried out LC/MS with the L5 through aforementioned processingEAnalysis is to measure its ceramide content (in addition to having or not buffer solution mixing, Detailed Experimental mode is referring to " the 5.ASAH2 efficacy test " in " the A. method " of embodiment 1).
LC/MSEEach sample signal of analysis is converted and is obtained various kinds this ceramide content percentage ratio (the highest signal the most 100% after being placed 2 hours with undressed L5), and result is as shown in figure 14.In Figure 14, LDL baseline refers to the contained ceramide content after placing 0 hour of undressed L5；LDL refers to the contained ceramide content after placing 2 hours of undressed L5 for 2 hours；LDL refers to the contained ceramide content after placing 24 hours of undressed L5 for 24 hours.
According to Figure 14, in the presence of buffer solution, after L5 is processed 2 hours through ASAH2, its ceramide content can be decreased obviously.And under buffer solution is presence or absence of, after L5 is processed 24 hours through ASAH2, its ceramide content all can be decreased obviously.
(4) LC/MS of the L5 of 24 hours is processed under buffer solution is presence or absence of with ASAH2EAnalysis
In buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) presence or absence of lower, L5 is processed 24 hours with ASAH2, and undressed L5 is carried out LC/MS with the L5 through aforementioned processingEAnalysis is to measure its ceramide content (in addition to having or not buffer solution mixing, Detailed Experimental mode is referring to " the 5.ASAH2 efficacy test " in " the A. method " of embodiment 1).
LC/MSEAnalysis result is as shown in table 4 (four shown in every group numerical value be derived from by identical sample measures 4 times).Sample signal each in table 4 is converted and is obtained various kinds this ceramide content percentage ratio (with the highest signal the most 100% of undressed L5), result is as shown in figure 15.
Table 4, untreated L5 with buffer solution presence or absence of under with ASAH2 process 24 hours L5 LC/MSEAnalysis result
| 24 little base lines | With ASAH2#1 process | With ASAH2#2 process | |
| Signal | 217.36 | 122.40 | 121.87 |
| 220.16 | 122.08 | 121.65 | |
| 214.80 | 117.11 | 123.68 | |
| 215.96 | 121.81 | 136.51 | |
| Meansigma methodss | 217.07 | 120.85 | 125.93 |
| Standard deviation | 2.31 | 2.51 | 7.11 |
| Reduce | 44.33 | 41.99 |
24 little base lines represent the ceramide content contained by undressed L5
According to table 4 and Figure 15, under buffer solution is presence or absence of, after L5 is processed 24 hours through ASAH2, its ceramide content all can be decreased obviously.
(5) LC/MS of the L5 of 24 hours is processed with ASAH2EAnalysis
The L5 and L5 processing 24 hours through ASAH2, using the lipid components contained by mass spectrograph quantitative analyses, and compares the difference of ceramide (ceramide) content.Result is as shown in Figure 16 A.
(6) LC/MS of the L5 of 2 hours is processed in the presence of buffer solution with ASAH2EAnalysis
In buffer solution (200mM Tris-HCl pH 8.4,1.5M NaCl, 25mM CaCl2) in the presence of L5 is processed 2 hours with ASAH2, and undressed L5 and L5 through aforementioned processing is carried out LC/MSEAnalysis is to measure its ceramide content (Detailed Experimental mode is referring to " the 5.ASAH2 efficacy test " in " the A. method " of embodiment 1) LC/MSEAnalysis result is as shown in table 5 (four shown in every group numerical value be derived from by identical sample measures 4 times).Sample signal each in table 5 is converted and is obtained various kinds this ceramide content percentage ratio (with the highest signal the most 100% of undressed L5), result is as shown in fig 16b.
Table 5, untreated L5 with buffer solution presence or absence of under with ASAH2 process 2 hours L5 LC/MSEAnalysis result
| Sample names | Signal |
| L5 0 hour | 529.0532 |
| L5 0 hour | 498.5066 |
| L5 0 hour | 478.2745 |
| L5 0 hour | 432.8346 |
| L5+ASAH 2 hours | 266.8874 |
| L5+ASAH 2 hours | 276.2790 |
| L5+ASAH 2 hours | 282.9767 |
| L5+ASAH 2 hours | 283.6284 |
According to table 5 and Figure 16 B, in the presence of buffer solution, after L5 is processed 2 hours through ASAH2, its ceramide contains and can be decreased obviously.
Although the present invention is disclosed above with preferred embodiment; so it is not limited to the present invention, any is familiar with this those skilled in the art, without departing from the spirit and scope of the present invention; when making a little change and retouching, therefore protection scope of the present invention is worked as and is defined depending on the defined person of appended claims.
Claims (63)
1. a kind of biochemical reaction material, including：
Base material；And
Enzyme constituent is fixed on this base material, and wherein, this enzyme constituent is selected from：
In order to remove elecrtonegativity low density lipoprotein, LDL (electronegative low-density
Lipoprotein, electronegative LDL) candy base the first enzyme；
In order to remove ceramide (ceramide) entrained by elecrtonegativity low density lipoprotein, LDL
Second enzyme；And
Combinations of the above,
Wherein this biochemical reaction material has the ability removing elecrtonegativity low density lipoprotein, LDL.
2. biochemical reaction material as claimed in claim 1, wherein this base material include silica gel (silica gel),
Cellulose (cellulose), diethylaminoethyl cellulose (diethylaminoethyl cellulose, DEAE
Cellulose), shitosan (chitosan), polystyrene (polystyrene), polysulfones (polysulfone), poly-
Ether sulfone (polyethersulfone), acryl resin or polysaccharide.
3. biochemical reaction material as claimed in claim 1, wherein this base material has grain structure or hollow
Tubular structure.
4. biochemical reaction material as claimed in claim 1, wherein this base material are cellulose pearl (bead).
5. biochemical reaction material as claimed in claim 1, wherein this base material are shitosan pearl.
6. biochemical reaction material as claimed in claim 1, wherein this base material are cellulose hollow fiber
(cellulose hollow fiber), polysulfone hollow fibre (polysulfone hollow fiber) or polyether sulfone hollow
Fiber (polyethersulfone hollow fiber).
7. biochemical reaction material as claimed in claim 1, wherein this first enzyme are sialidase
Or glycosidase (glycosidase) (sialidase).
8. biochemical reaction material as claimed in claim 7, wherein this sialidase is selected from：
Neuraminidase 1 (neuraminidase 1, NEU1), neuraminidase 2 from human body
(neuraminidase 2, NEU2), neuraminidase 3 (neuraminidase 3, NEU3), neuraminic acid
Enzyme 4 (neuraminidase 4, NEU4) and O- sialidase (O-sialidase), turned with gene and grow, show simultaneously
Purification and one of above-mentioned enzyme of coming, and sialidase (another name, the acetyl nerve from virus or antibacterial
Propylhomoserin base hydrolase (acetylneuraminyl hydrolase)).
9. biochemical reaction material as claimed in claim 7, wherein this glycosidase is selected from：
Come from α and β glucosidase (alpha-and beta-glucosidase), the wheat of human body or animal body
Bud enzyme-glucose amylase (maltase-glucoamylase) and Sucrase-isomaltase
(sucrase-isomaltase), turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, and be derived from
In virus, the N- glycosidase F (N-glycosidase F, PNGase F) of antibacterial and glucosidase
(glucosidase).
10. biochemical reaction material as claimed in claim 1, wherein this second enzyme are ceramidase
(ceramidase).
11. biochemical reaction materials as claimed in claim 10, wherein this ceramidase is selected from：
ASAH1 1 (N-acylsphingosine amidohydrolase 1,
ASAH1), ASAH1 2 (N-acylsphingosine amidohydrolase 2,
ASAH2), ASAH1 2B (N-acylsphingosine amidohydrolase 2B,
ASAH2B), ASAH1 2C (N-acylsphingosine amidohydrolase
2C, ASAH2C), N- acyl ethanol amine acylating acid enzyme (N-acylethanolamine acid amidase), alkali
Property ceramidase 1 (alkaline ceramidase 1), alkaline ceramidase 2 (alkaline ceramidase
2) and alkaline ceramidase 3 (alkaline ceramidase 3).
12. biochemical reaction materials as claimed in claim 1, wherein this enzyme constituent are this first enzyme.
The 13. biochemical reaction materials as described in claim the 12, wherein this first enzyme are neuraminic acid
Enzyme 2.
14. biochemical reaction materials as claimed in claim 1, wherein this enzyme constituent are this second enzyme.
15. biochemical reaction materials as claimed in claim 14, wherein this second enzyme are N- acyl sphingosine
Hydroamidase 2.
16. biochemical reaction materials as claimed in claim 1, wherein this enzyme constituent are this first enzyme and are somebody's turn to do
The combination of second enzyme.
17. biochemical reaction materials as claimed in claim 16, wherein this first enzyme are neuraminidase 2,
And this second enzyme is ASAH1 2.
18. biochemical reaction materials as claimed in claim 1, wherein this elecrtonegativity low density lipoprotein, LDL include
L1, L2, L3, L4 and L5.
19. biochemical reaction materials as claimed in claim 18, wherein this elecrtonegativity low density lipoprotein, LDL are
L5.
A kind of 20. biochemical reaction device, including：
Biochemical reaction material as claimed in claim 1；And
Container, in order to accommodate this biochemical reaction material, wherein this container has at least one entrance and at least
Outlet；
Wherein, liquid sample enters this biochemical reaction device from this at least one opening, and it is anti-to flow through this biology
Answer material to react with this biochemical reaction material, and flow out from this at least one outlet afterwards.
21. biochemical reaction device as claimed in claim 20, the material of wherein this container include glass,
Acryl, polypropylene, polyethylene, rustless steel or titanium alloy.
22. biochemical reaction device as claimed in claim 20, further include：
Filter material, is arranged in this container, in this at least one entrance rear and this at least one outlet rear,
The aperture of wherein this filter material is less than this biological respinse material, to avoid this biological respinse material from this extremely
A few entrance and/or this at least one outlet spill.
23. biochemical reaction device as claimed in claim 22, the material of wherein this filter material include filter paper,
Glass, acryl, polypropylene or polyethylene.
24. biochemical reaction device as claimed in claim 20, wherein this container are hollow column, and
The two ends of its hollow column are respectively provided with first entrance and this at least one outlet of this at least one entrance
First outlet.
25. biochemical reaction device as claimed in claim 24, wherein, the one second of this at least one entrance
Entrance is located on the side wall of this hollow column with a second outlet of this at least one outlet.
26. biochemical reaction device as claimed in claim 20, wherein this base material have grain structure or in
Hollow tube-like structure.
27. biochemical reaction device as claimed in claim 22, wherein this base material have grain structure or in
Hollow tube-like structure.
28. biochemical reaction device as claimed in claim 22, wherein this base material have grain structure.
29. biochemical reaction device as claimed in claim 24, wherein this base material have grain structure or in
Hollow tube-like structure.
30. biochemical reaction device as claimed in claim 25, wherein this base material have hollow tubular structure.
31. biochemical reaction device as claimed in claim 20, wherein this base material include silica gel, cellulose,
Diethylaminoethyl cellulose, shitosan, polystyrene, polysulfones, polyether sulfone, acryl resin or many
Sugar.
32. biochemical reaction device as claimed in claim 28, wherein this base material are cellulose pearl.
33. biochemical reaction device as claimed in claim 28, wherein this base material are shitosan pearl.
34. biochemical reaction device as claimed in claim 30, wherein this base material be cellulose hollow fiber,
Polysulfone hollow fibre (polysulfone hollow fiber), epoxy acryl resin or Polyethersulfone Hollow Fiber Plasma
(polyethersulfone hollow fiber).
35. biochemical reaction device as claimed in claim 20, wherein this first enzyme are sialidase or sugar
Glycosides enzyme.
36. biochemical reaction device as claimed in claim 35, wherein this sialidase are selected from：
From the neuraminidase 1 of human body, neuraminidase 2, neuraminidase 3, neuraminidase 4 with
O- sialidase, turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, and from virus or
The sialidase of antibacterial.
37. biochemical reaction device as claimed in claim 35, wherein this glycosidase are selected from：
Come from α and β glucosidase, maltase-glucoamylase and the sugarcane of human body or animal body
Carbohydrase-isomaltase, turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, and be derived from
In virus, the N- glycosidase F of antibacterial and glucosidase.
38. biochemical reaction device as claimed in claim 20, wherein this second enzyme are ceramidase.
39. biochemical reaction device as claimed in claim 38, wherein this ceramidase are selected from：
ASAH1 1, ASAH1 2, N- acyl sphingosine
Hydroamidase 2B, ASAH1 2C, N- acyl ethanol amine acylating acid enzyme, alkali
Property ceramidase 1, alkaline ceramidase 2 and alkaline ceramidase 3.
40. biochemical reaction device as claimed in claim 20, wherein this enzyme constituent are this first enzyme,
And this first enzyme is neuraminidase 2.
41. biochemical reaction device as claimed in claim 20, wherein this enzyme constituent are this second enzyme,
And this second enzyme is ASAH1 2.
42. biochemical reaction device as claimed in claim 20, wherein this enzyme constituent be this first enzyme with
The combination of this second enzyme, and this first enzyme is neuraminidase 2, and this second enzyme is N- acyl sphingosine
Hydroamidase 2.
43. biochemical reaction device as claimed in claim 20, wherein this elecrtonegativity low density lipoprotein, LDL bag
Include elecrtonegativity low density lipoprotein, LDL L1, L2, L3, L4 and L5.
44. biochemical reaction device as claimed in claim 20, wherein this elecrtonegativity low density lipoprotein, LDL are
Elecrtonegativity low density lipoprotein, LDL L5.
45. biochemical reaction device as claimed in claim 20, wherein this liquid sample include aqueous solution,
Blood, blood plasma.
A kind of 46. extracorporeal treatment bloods or the method for blood plasma, including：
A () makes blood or blood plasma contact with enzyme constituent in vitro so that this enzyme constituent to this blood or
Blood plasma is reacted, and wherein, this enzyme constituent has the ability removing elecrtonegativity low density lipoprotein, LDL, and
This enzyme constituent is selected from：
In order to remove the first enzyme of the candy base of elecrtonegativity low density lipoprotein, LDL；
In order to remove the second enzyme of the ceramide entrained by elecrtonegativity low density lipoprotein, LDL；With
And
Combinations of the above；And
B () is terminated this blood or blood plasma and is contacted with this enzyme constituent, to terminate this enzyme constituent to this blood
Or blood plasma is reacted.
47. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein execute this step
(a) about 0.25-8 hour.
48. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein execute this step
A () is in for about 4-40 DEG C.
49. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein execute this step
A () is in about pH5-10.
50. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this first enzyme are
Sialidase or glycosidase.
51. extracorporeal treatment bloods as claimed in claim 50 or the method for blood plasma, wherein this sialidase
It is selected from：
From the neuraminidase 1 of human body, neuraminidase 2, neuraminidase 3, neuraminidase 4 with
O- sialidase, turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, and from virus or
The sialidase of antibacterial.
52. extracorporeal treatment bloods as claimed in claim 51 or the method for blood plasma, wherein this glycosidase are selected
From：
Come from α and β glucosidase, maltase-glucoamylase and the sugarcane of human body or animal body
Carbohydrase-isomaltase, turned with gene and grow, show and purification and one of above-mentioned enzyme of coming, and be derived from
In virus, the N- glycosidase F of antibacterial and glucosidase.
53. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this second enzyme is
Ceramidase.
54. extracorporeal treatment bloods as claimed in claim 53 or the method for blood plasma, wherein this ceramide
Enzyme is selected from：
ASAH1 1, ASAH1 2, N- acyl sphingosine
Hydroamidase 2B, ASAH1 2C, N- acyl ethanol amine acylating acid enzyme, alkali
Property ceramidase 1, alkaline ceramidase 2 and alkaline ceramidase 3.
55. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this enzyme constituent
For this first enzyme.
56. extracorporeal treatment bloods as claimed in claim 55 or the method for blood plasma, wherein this first enzyme are
Neuraminidase 2.
57. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this enzyme constituent
For this second enzyme.
58. extracorporeal treatment bloods as claimed in claim 57 or the method for blood plasma, wherein this second enzyme is
ASAH1 2.
59. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this enzyme constituent
For combining of this first enzyme and this second enzyme.
60. extracorporeal treatment bloods as claimed in claim 59 or the method for blood plasma, wherein this first enzyme are
Neuraminidase 2, and this second enzyme is ASAH1 2.
61. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this enzyme constituent
It is fixed on base material
62. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this base material includes
Silica gel, cellulose, diethylaminoethyl cellulose, shitosan, polystyrene, polysulfones, polyether sulfone,
Resin or polysaccharide.
63. extracorporeal treatment bloods as claimed in claim 46 or the method for blood plasma, wherein this base material has
Grain structure or hollow tubular structure.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14/984,938 US20170044516A1 (en) | 2015-08-11 | 2015-12-30 | Biochemistry reactive material and device for eliminating electronegative low-density lipoprotein (ldl) and method for treating blood or plasma ex vivo to eliminate electronegative low-density lipoprotein therein |
| US16/137,925 US11242516B2 (en) | 2015-08-11 | 2018-09-21 | Method for ex vivo treating blood or plasma |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW104126050A TWI580782B (en) | 2015-08-11 | 2015-08-11 | Biochemistry reactive materialand device for eliminating low-density lipoprotein (ldl) and method for treating blood or plasma ex vivo to eliminate low-density lipoprotein therein |
| TW104126050 | 2015-08-11 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106434619A true CN106434619A (en) | 2017-02-22 |
| CN106434619B CN106434619B (en) | 2020-03-24 |
Family
ID=58094004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510815421.4A Active CN106434619B (en) | 2015-08-11 | 2015-11-23 | Biochemical reaction material and device and method for treating blood or plasma in vitro |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN106434619B (en) |
| TW (1) | TWI580782B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108642037A (en) * | 2018-05-18 | 2018-10-12 | 暨南大学 | A kind of neuraminidase microreactor and its preparation method and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5998183A (en) * | 1997-07-07 | 1999-12-07 | Le Fevre; Gerard N. | Enzyme immobilization on a siliceous support with a polyaldehyde cross-linking agent |
| CN101370536A (en) * | 2005-12-13 | 2009-02-18 | 埃克塞拉有限责任公司 | Method for extracorporeal removal of a pathogenic microbe, an inflammatory cell or an inflammatory protein from blood |
| US20100190146A1 (en) * | 2009-01-29 | 2010-07-29 | Bynum Magdalena A | Microfluidic Glycan Analysis |
| CN102091595A (en) * | 2011-01-12 | 2011-06-15 | 华东理工大学 | Method for preparing spherical carbon aerogel with specific absorbability for low density lipoprotein |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6029B2 (en) * | 1978-09-06 | 1985-01-05 | 藤沢薬品工業株式会社 | Immobilized enzyme column |
| US5232696A (en) * | 1987-09-25 | 1993-08-03 | Massachusetts Institute Of Technology | Reduction of low density lipoproteins in biological fluids |
| IL182956A0 (en) * | 2007-05-03 | 2008-01-20 | Yeda Res & Dev | Glycan modified soluble receptors and binding proteins and their use |
-
2015
- 2015-08-11 TW TW104126050A patent/TWI580782B/en active
- 2015-11-23 CN CN201510815421.4A patent/CN106434619B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5998183A (en) * | 1997-07-07 | 1999-12-07 | Le Fevre; Gerard N. | Enzyme immobilization on a siliceous support with a polyaldehyde cross-linking agent |
| CN101370536A (en) * | 2005-12-13 | 2009-02-18 | 埃克塞拉有限责任公司 | Method for extracorporeal removal of a pathogenic microbe, an inflammatory cell or an inflammatory protein from blood |
| US20100190146A1 (en) * | 2009-01-29 | 2010-07-29 | Bynum Magdalena A | Microfluidic Glycan Analysis |
| CN102091595A (en) * | 2011-01-12 | 2011-06-15 | 华东理工大学 | Method for preparing spherical carbon aerogel with specific absorbability for low density lipoprotein |
Non-Patent Citations (4)
| Title |
|---|
| 任晻 等: "神经酰胺与代谢综合征", 《国际内分泌代谢杂志》 * |
| 侯丽娟 等: "糖基化低密度脂蛋白与动脉粥样硬化", 《华北煤炭医学院学报》 * |
| 张迎庆 等: "低密度脂蛋白血液净化研究进展", 《药物生物技术》 * |
| 范刚 等: "血清唾液酸与动脉粥样硬化的相关性研究", 《医学检验与临床》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108642037A (en) * | 2018-05-18 | 2018-10-12 | 暨南大学 | A kind of neuraminidase microreactor and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI580782B (en) | 2017-05-01 |
| CN106434619B (en) | 2020-03-24 |
| TW201706408A (en) | 2017-02-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5688043B2 (en) | Method for producing sugar chain derivative, structure analysis method, and sugar chain derivative | |
| EP4092417A1 (en) | Lectin-macromolecule carrier coupling complex for separating glycosylated exosome in clinical sample | |
| CN109642229A (en) | Extracellular vesica is separated from biofluid and isolates the automatically and manually method of Cell-free DNA | |
| KR20100001826A (en) | Nucleic acid extraction apparatus | |
| CN107254430A (en) | A kind of method based on positive charge adsorbing separation excretion body | |
| EP2875348B1 (en) | Method for the isolation of microvesicles | |
| CN104152436B (en) | DNA isolation and purification methods and its kit | |
| CN106434619A (en) | Biochemical reaction material and device and method for treating blood or plasma in vitro | |
| CN101780276B (en) | Method for removing residual DNA in hydrophobia vaccine product by utilizing ultrasound combined with EDTA solution | |
| JP2011095069A (en) | Clinical examination system, clinical examination method, and equipment for pretreatment | |
| US11242516B2 (en) | Method for ex vivo treating blood or plasma | |
| JP6238087B2 (en) | Method for preparing labeled sugar chain sample | |
| CN102115739B (en) | Method, reagent and kit for separating nucleic acids | |
| CN112941067A (en) | Lysis binding solution for whole blood nucleic acid extraction and kit and application thereof | |
| Mycroft-West et al. | A glycosaminoglycan extract from portunus pelagicus inhibits BACE1, the β secretase implicated in Alzheimer’s disease | |
| Jurado-Sánchez et al. | Gas chromatographic determination of N-nitrosamines in beverages following automatic solid-phase extraction | |
| Lee et al. | Low concentration of polyethylene glycol facilitates separation of extracellular vesicles from bronchoalveolar lavage fluid | |
| DE202007018852U1 (en) | Product of a serum | |
| JP6048036B2 (en) | Purification method of sugar chain | |
| Esteve et al. | Analytical approaches for the characterization and identification of olive (Olea europaea) oil proteins | |
| CN105586330B (en) | A kind of Halase and preparation method thereof | |
| JP2007139759A (en) | Protein fraction device, and protein fraction and concentration method | |
| CN110484450A (en) | A kind of preparation method of Eimeria unsporulated oocysts wall | |
| KR102370468B1 (en) | A composition and a method for removal of residual anionic surfactant, protein-detergent complexes, and micelles, in biological tissues | |
| CN109355286A (en) | A method of exempting from pre-treatment automation and extracts large volume whole blood DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |