CN110404052A - 蓖麻毒素b的制备及其应用 - Google Patents
蓖麻毒素b的制备及其应用 Download PDFInfo
- Publication number
- CN110404052A CN110404052A CN201810957682.3A CN201810957682A CN110404052A CN 110404052 A CN110404052 A CN 110404052A CN 201810957682 A CN201810957682 A CN 201810957682A CN 110404052 A CN110404052 A CN 110404052A
- Authority
- CN
- China
- Prior art keywords
- albumen
- diabetes
- rat
- model
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 108010039491 Ricin Proteins 0.000 title abstract description 24
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 44
- 239000003814 drug Substances 0.000 claims abstract description 19
- 210000004369 blood Anatomy 0.000 claims abstract description 14
- 239000008280 blood Substances 0.000 claims abstract description 14
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 239000008103 glucose Substances 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims description 14
- 150000003626 triacylglycerols Chemical class 0.000 claims description 7
- 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 claims description 6
- 241000588722 Escherichia Species 0.000 claims description 5
- 239000003651 drinking water Substances 0.000 claims description 5
- 235000020188 drinking water Nutrition 0.000 claims description 5
- 235000012000 cholesterol Nutrition 0.000 claims description 3
- 210000001015 abdomen Anatomy 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 208000024891 symptom Diseases 0.000 claims description 2
- 102000001554 Hemoglobins Human genes 0.000 claims 1
- 108010054147 Hemoglobins Proteins 0.000 claims 1
- 230000003178 anti-diabetic effect Effects 0.000 abstract description 8
- 239000003472 antidiabetic agent Substances 0.000 abstract description 8
- 150000002632 lipids Chemical class 0.000 abstract description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 241000700159 Rattus Species 0.000 description 16
- 238000012360 testing method Methods 0.000 description 12
- 230000006798 recombination Effects 0.000 description 11
- 238000005215 recombination Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 7
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 7
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 102000016362 Catenins Human genes 0.000 description 3
- 108010067316 Catenins Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- LMMDEZPNUTZJAY-GCJQMDKQSA-N Thr-Asp-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O LMMDEZPNUTZJAY-GCJQMDKQSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 210000003000 inclusion body Anatomy 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- PQWTZSNVWSOFFK-FXQIFTODSA-N Arg-Asp-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)CN=C(N)N PQWTZSNVWSOFFK-FXQIFTODSA-N 0.000 description 2
- HYQYLOSCICEYTR-YUMQZZPRSA-N Asn-Gly-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O HYQYLOSCICEYTR-YUMQZZPRSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- TYRMVTKPOWPZBC-SXNHZJKMSA-N Gln-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCC(=O)N)N TYRMVTKPOWPZBC-SXNHZJKMSA-N 0.000 description 2
- 235000004443 Ricinus communis Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108010015792 glycyllysine Proteins 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002218 hypoglycaemic effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- ZOBPZXTWZATXDG-UHFFFAOYSA-N 1,3-thiazolidine-2,4-dione Chemical compound O=C1CSC(=O)N1 ZOBPZXTWZATXDG-UHFFFAOYSA-N 0.000 description 1
- 108010036211 5-HT-moduline Proteins 0.000 description 1
- HCEQQASHRRPQFE-UHFFFAOYSA-N 5-chloro-n-[2-[4-(cyclohexylcarbamoylsulfamoyl)phenyl]ethyl]-2-methoxybenzamide;3-(diaminomethylidene)-1,1-dimethylguanidine;hydrochloride Chemical compound Cl.CN(C)C(=N)N=C(N)N.COC1=CC=C(Cl)C=C1C(=O)NCCC1=CC=C(S(=O)(=O)NC(=O)NC2CCCCC2)C=C1 HCEQQASHRRPQFE-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-N 0.000 description 1
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 1
- RGQCNKIDEQJEBT-CQDKDKBSSA-N Ala-Leu-Tyr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 RGQCNKIDEQJEBT-CQDKDKBSSA-N 0.000 description 1
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 description 1
- UISQLSIBJKEJSS-GUBZILKMSA-N Arg-Arg-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(O)=O UISQLSIBJKEJSS-GUBZILKMSA-N 0.000 description 1
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 1
- FIQKRDXFTANIEJ-ULQDDVLXSA-N Arg-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FIQKRDXFTANIEJ-ULQDDVLXSA-N 0.000 description 1
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 1
- WPOLSNAQGVHROR-GUBZILKMSA-N Asn-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N WPOLSNAQGVHROR-GUBZILKMSA-N 0.000 description 1
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 1
- JQBCANGGAVVERB-CFMVVWHZSA-N Asn-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N JQBCANGGAVVERB-CFMVVWHZSA-N 0.000 description 1
- ZMUQQMGITUJQTI-CIUDSAMLSA-N Asn-Leu-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZMUQQMGITUJQTI-CIUDSAMLSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 1
- QUMKPKWYDVMGNT-NUMRIWBASA-N Asn-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QUMKPKWYDVMGNT-NUMRIWBASA-N 0.000 description 1
- GWTLRDMPMJCNMH-WHFBIAKZSA-N Asp-Asn-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GWTLRDMPMJCNMH-WHFBIAKZSA-N 0.000 description 1
- FOXXZZGDIAQPQI-XKNYDFJKSA-N Asp-Pro-Ser-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FOXXZZGDIAQPQI-XKNYDFJKSA-N 0.000 description 1
- 229940123208 Biguanide Drugs 0.000 description 1
- 244000248349 Citrus limon Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- XZKJEOMFLDVXJG-KATARQTJSA-N Cys-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)N)O XZKJEOMFLDVXJG-KATARQTJSA-N 0.000 description 1
- CITDWMLWXNUQKD-FXQIFTODSA-N Gln-Gln-Asn Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CITDWMLWXNUQKD-FXQIFTODSA-N 0.000 description 1
- NXPXQIZKDOXIHH-JSGCOSHPSA-N Gln-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N NXPXQIZKDOXIHH-JSGCOSHPSA-N 0.000 description 1
- RGAOLBZBLOJUTP-GRLWGSQLSA-N Gln-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N RGAOLBZBLOJUTP-GRLWGSQLSA-N 0.000 description 1
- YPMDZWPZFOZYFG-GUBZILKMSA-N Gln-Leu-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YPMDZWPZFOZYFG-GUBZILKMSA-N 0.000 description 1
- JYXKPJVDCAWMDG-ZPFDUUQYSA-N Glu-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)O)N JYXKPJVDCAWMDG-ZPFDUUQYSA-N 0.000 description 1
- DTRUBYPMMVPQPD-YUMQZZPRSA-N Gly-Gln-Arg Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DTRUBYPMMVPQPD-YUMQZZPRSA-N 0.000 description 1
- QLQDIJBYJZKQPR-BQBZGAKWSA-N Gly-Met-Cys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN QLQDIJBYJZKQPR-BQBZGAKWSA-N 0.000 description 1
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 1
- IZVICCORZOSGPT-JSGCOSHPSA-N Gly-Val-Tyr Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IZVICCORZOSGPT-JSGCOSHPSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- DMHGKBGOUAJRHU-RVMXOQNASA-N Ile-Arg-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N DMHGKBGOUAJRHU-RVMXOQNASA-N 0.000 description 1
- DMHGKBGOUAJRHU-UHFFFAOYSA-N Ile-Arg-Pro Natural products CCC(C)C(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O DMHGKBGOUAJRHU-UHFFFAOYSA-N 0.000 description 1
- PARSHQDZROHERM-NHCYSSNCSA-N Ile-Lys-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)O)N PARSHQDZROHERM-NHCYSSNCSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 description 1
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 1
- PNPYKQFJGRFYJE-GUBZILKMSA-N Lys-Ala-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNPYKQFJGRFYJE-GUBZILKMSA-N 0.000 description 1
- 108010062166 Lys-Asn-Asp Proteins 0.000 description 1
- BYPMOIFBQPEWOH-CIUDSAMLSA-N Lys-Asn-Asp Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N BYPMOIFBQPEWOH-CIUDSAMLSA-N 0.000 description 1
- SSYOBDBNBQBSQE-SRVKXCTJSA-N Lys-Cys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O SSYOBDBNBQBSQE-SRVKXCTJSA-N 0.000 description 1
- ZXFRGTAIIZHNHG-AJNGGQMLSA-N Lys-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CCCCN)N ZXFRGTAIIZHNHG-AJNGGQMLSA-N 0.000 description 1
- GHKXHCMRAUYLBS-CIUDSAMLSA-N Lys-Ser-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O GHKXHCMRAUYLBS-CIUDSAMLSA-N 0.000 description 1
- XBAJINCXDBTJRH-WDSOQIARSA-N Lys-Val-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCCCN)N XBAJINCXDBTJRH-WDSOQIARSA-N 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- DNDVVILEHVMWIS-LPEHRKFASA-N Met-Asp-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DNDVVILEHVMWIS-LPEHRKFASA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- CYQQWUPHIZVCNY-GUBZILKMSA-N Pro-Arg-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CYQQWUPHIZVCNY-GUBZILKMSA-N 0.000 description 1
- XYAFCOJKICBRDU-JYJNAYRXSA-N Pro-Phe-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O XYAFCOJKICBRDU-JYJNAYRXSA-N 0.000 description 1
- WVXQQUWOKUZIEG-VEVYYDQMSA-N Pro-Thr-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O WVXQQUWOKUZIEG-VEVYYDQMSA-N 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108090000829 Ribosome Inactivating Proteins Proteins 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- RNFKSBPHLTZHLU-WHFBIAKZSA-N Ser-Cys-Gly Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N)O RNFKSBPHLTZHLU-WHFBIAKZSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- OQPNSDWGAMFJNU-QWRGUYRKSA-N Ser-Gly-Tyr Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OQPNSDWGAMFJNU-QWRGUYRKSA-N 0.000 description 1
- IXZHZUGGKLRHJD-DCAQKATOSA-N Ser-Leu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IXZHZUGGKLRHJD-DCAQKATOSA-N 0.000 description 1
- 229940100389 Sulfonylurea Drugs 0.000 description 1
- 229940123464 Thiazolidinedione Drugs 0.000 description 1
- VOGXLRKCWFLJBY-HSHDSVGOSA-N Thr-Arg-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O VOGXLRKCWFLJBY-HSHDSVGOSA-N 0.000 description 1
- WPAKPLPGQNUXGN-OSUNSFLBSA-N Thr-Ile-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WPAKPLPGQNUXGN-OSUNSFLBSA-N 0.000 description 1
- GMXIJHCBTZDAPD-QPHKQPEJSA-N Thr-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N GMXIJHCBTZDAPD-QPHKQPEJSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 1
- NHQVWACSJZJCGJ-FLBSBUHZSA-N Thr-Thr-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NHQVWACSJZJCGJ-FLBSBUHZSA-N 0.000 description 1
- GRIUMVXCJDKVPI-IZPVPAKOSA-N Thr-Thr-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GRIUMVXCJDKVPI-IZPVPAKOSA-N 0.000 description 1
- VYVBSMCZNHOZGD-RCWTZXSCSA-N Thr-Val-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O VYVBSMCZNHOZGD-RCWTZXSCSA-N 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- DDJHCLVUUBEIIA-BVSLBCMMSA-N Trp-Met-Phe Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)CCSC)C(O)=O)C1=CC=CC=C1 DDJHCLVUUBEIIA-BVSLBCMMSA-N 0.000 description 1
- KXIQQAWIPDDVOE-BPUTZDHNSA-N Trp-Pro-Cys Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)N1CCC[C@H]1C(=O)N[C@@H](CS)C(O)=O KXIQQAWIPDDVOE-BPUTZDHNSA-N 0.000 description 1
- HTGJDTPQYFMKNC-VFAJRCTISA-N Trp-Thr-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)[C@@H](C)O)=CNC2=C1 HTGJDTPQYFMKNC-VFAJRCTISA-N 0.000 description 1
- HGEHWFGAKHSIDY-SRVKXCTJSA-N Tyr-Asp-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)O HGEHWFGAKHSIDY-SRVKXCTJSA-N 0.000 description 1
- SYFHQHYTNCQCCN-MELADBBJSA-N Tyr-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O SYFHQHYTNCQCCN-MELADBBJSA-N 0.000 description 1
- PFNZJEPSCBAVGX-CYDGBPFRSA-N Val-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C(C)C)N PFNZJEPSCBAVGX-CYDGBPFRSA-N 0.000 description 1
- KXUKIBHIVRYOIP-ZKWXMUAHSA-N Val-Asp-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N KXUKIBHIVRYOIP-ZKWXMUAHSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- NYTKXWLZSNRILS-IFFSRLJSSA-N Val-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N)O NYTKXWLZSNRILS-IFFSRLJSSA-N 0.000 description 1
- JTWIMNMUYLQNPI-WPRPVWTQSA-N Val-Gly-Arg Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N JTWIMNMUYLQNPI-WPRPVWTQSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- YTUABZMPYKCWCQ-XQQFMLRXSA-N Val-His-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N YTUABZMPYKCWCQ-XQQFMLRXSA-N 0.000 description 1
- MGVYZTPLGXPVQB-CYDGBPFRSA-N Val-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N MGVYZTPLGXPVQB-CYDGBPFRSA-N 0.000 description 1
- BZDGLJPROOOUOZ-XGEHTFHBSA-N Val-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N)O BZDGLJPROOOUOZ-XGEHTFHBSA-N 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000004283 biguanides Chemical class 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940112611 glucovance Drugs 0.000 description 1
- 108091005995 glycated hemoglobin Proteins 0.000 description 1
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229960004329 metformin hydrochloride Drugs 0.000 description 1
- OETHQSJEHLVLGH-UHFFFAOYSA-N metformin hydrochloride Chemical compound Cl.CN(C)C(=N)N=C(N)N OETHQSJEHLVLGH-UHFFFAOYSA-N 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin hydrochloride Natural products CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000000837 restrainer Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- YROXIXLRRCOBKF-UHFFFAOYSA-N sulfonylurea Chemical class OC(=N)N=S(=O)=O YROXIXLRRCOBKF-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Diabetes (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种蓖麻毒素B的制备及其应用,涉及抗糖尿病药物的技术领域,具体为一种用于治疗2型糖尿病的重组蛋白药物,其能降低2型糖尿病大鼠的血糖和血脂,可以开发成治疗2型糖尿病的药物。
Description
技术领域
本发明涉及抗糖尿病蛋白药物技术领域,具体为一种用于对2型糖尿病的重组蓖麻毒素B链蛋白突变体。
背景技术
糖尿病是一种因胰岛素分泌不足和或胰岛素抵抗而引起的糖、脂肪和蛋白质代谢紊乱的免疫系统疾病,涉及全身各个系统,甚至可诱发许多致命性并发症,严重危害人类健康,糖尿病已经成为当前危害人类健康的第三大疾病。90%以上的糖尿病患者为2型糖尿病(type 2diabetes mellitus,T2DM)。目前,全世界约有2.46亿人患有T2DM,预计在20年内T2DM患者数目将增至3.8亿。中国是全球糖尿病患者人数最多的国家,但中国T2DM患者的血糖达标率严重不足。现阶段治疗T2DM的药物主要为传统抗糖尿病药物,包括:磺酰脲类、格列奈类、双胍类、噻唑烷二酮、α-葡萄糖苷酶抑制剂及胰岛素等,但这些药物均存在不同程度的不良反应,如引发低血糖、胃肠道不适、肥胖等。因而开发更优良的糖尿病治疗新药显得尤为必要,随着对糖尿病基础理论研究的不断深入,抗糖尿病新药的开发成为国内外研究的热点。许多制药公司正在开发新的抗糖尿病药物,以满足目前的糖尿病治疗需求。
由于糖尿病复杂的发病机制,使得医学工作者在药物研发的过程中,逐渐发现了许多潜在的抗糖尿病疗法的新型目标。与此同时,据统计,2013至今进入Ⅲ期临床试验或递交NDA/BLA或获得批准用于治疗糖尿病的药物有65种,分为非胰岛素类抗糖尿病药(41种,包括用于治疗糖尿病相关疾病的药物10种,占63.1%)、胰岛素类药(8种,12.3%)和复方制剂(16种,24.6%)。这些数据表明,国际制药公司将开发糖尿病新药的重点集中在非胰岛素类抗糖尿病药物上,本发明重组蓖麻毒素B链蛋白在一定程度上契合国际药物开发的需求,应用前景广阔。
蓖麻毒素(Ricin)是一种由A链(RTA)和B链(RTB)两条链通过二硫键连接的异源二聚体糖蛋白。其中,RTA是一种在哺乳动物细胞内可抑制蛋白质合成的核糖体灭活蛋白。RTB是一个两叶形结构的分子,由两个相同折叠的拓扑学球状结构域构成,每一个区域又包括三个亚区(ɑ,β,γ),只有1ɑ和2β有明显的半乳糖结合活性,无毒,具有凝集素活性,可协助A链进入细胞内发挥生物学功能。RTB可以结合不同的糖结构,调节各种生物过程,包括细胞与宿主、病原体相互作用和先天免疫反应。
目前,通过国内外公开出版物的检索并未发现重组蓖麻毒素B链蛋白突变体治疗2型糖尿病以及并发症的相关报道。
发明内容
为了解决目前治疗2型糖尿病药物效果不理想,副作用较多的问题,本发明提供一种重组蓖麻毒素B链蛋白突变体及其表达方法,并验证其对2型糖尿病大鼠的降糖效果。
本发明的技术方案为:
包含SEQ ID:1序列的蛋白在制备治疗2型糖尿病药物中的应用。
包含SEQ ID:1序列的蛋白的插入、删除、替换等突变体在制备治疗2型糖尿病药物中的应用。
所述SEQ ID:1蛋白序列的编码核苷酸为SEQ ID:2。
SEQ ID:1序列的蛋白可降低2型糖尿病模型的大鼠空腹血糖水平。
SEQ ID:1序列的蛋白可降低2型糖尿病模型的大鼠糖化血红蛋白水平。
SEQ ID:1序列的蛋白可降低2型糖尿病模型的大鼠的饮水量。
SEQ ID:1序列的蛋白可增加2型糖尿病模型的大鼠的体重,改善消瘦症状。
SEQ ID:1序列的蛋白可改善2型糖尿病模型的大鼠的甘油三酯(TG)和总胆固醇(TCH)水平。
SEQ ID:1序列的蛋白可改善2型糖尿病模型的大鼠的耐糖能力。
SEQ ID:1序列的蛋白的制备方法,通过大肠杆菌表达系统制备。
所述大肠杆菌表达系统所用载体为PET系列载体,所用表达菌种为BL21。
本发明提供了一种重组蓖麻毒素B链蛋白突变体的表达方法,通过大肠杆菌表达系统表达重组蓖麻毒素B链蛋白突变体。
本发明还提供了重组蓖麻毒素B链蛋白突变体抗2型糖尿病大鼠的应用。
本发明的有益效果是:本发明的重组蓖麻毒素B链蛋白突变体具有改善2型糖尿病大鼠的体重和饮水量,同时还具有显著的降低血糖和血脂的效果,糖耐量实验证明该蛋白对2型糖尿病大鼠有治疗作用。
具体实施方式
本发明提供了重组蓖麻毒素B链蛋白突变体抗2型糖尿病的应用,通过重组蓖麻毒素B链蛋白突变体对2型糖尿病大鼠的实验证明:本发明的重组蓖麻毒素B链蛋白突变体可以改善2型糖尿病。
实施例1重组蓖麻毒素B链蛋白突变体制备
1大肠杆菌表达工程菌BL21(DE3)/PET28a-tRTB的获得
在氨基酸序列不变的条件下,根据已有的蓖麻毒素B链(rRTB)突变体进行密码子优化,根据上述的蓖麻毒素B链密码子优化后的全序列作为模板,用tRTB sense primer和tRTB anti-sense primer进行PCR扩增,获得的PCR产物连接至pMD19-T simple克隆载体(宝生物工程(大连)有限公司),获得pMD19T-tRTB。与经过同样NdeI与HindIII限制性酶切的PET-28a表达载体链接,得到的链接产物转入大肠杆菌感受态细胞BL21(DE3),筛选获得阳性菌,对阳性菌提取质粒后进行测序,结果该质粒为序列表中序列1插入PET28a表达载体的NdeI与HindIII的酶切位点间得到的质粒,将该质粒命名为PET28a-tRTB,其对应的阳性菌命名为BL21(DE3)/PET28a-tRTB。
2重组蓖麻毒素B链蛋白突变体的诱导表达与纯化
将上述获得的阳性菌BL21(DE3)/PET28a-tRTB按1:100v/v的比例接种于5mL含Kan+(浓度为50μg/mL)的TB培养基中,在37℃下,180r/min恒温振荡培养至OD600=0.6时,诱导组添加IPTG至终浓度为1mmol/L,同上述培养条件即在37℃下,180r/min恒温振荡诱导10h;经诱导10h后,在4℃下,8000r/min离心获得菌体沉淀。
获得的菌体沉淀重悬用超声破碎方法将其裂解,离心获得包涵体沉淀;经12%SDS-PAGE电泳分析,重组蓖麻毒素B链蛋白在包涵体有特异性的表达,且其蛋白分子量为38Kda,与预期的蛋白大小相符合。将上述获得的包涵体溶解液进行亲和层析,当咪唑浓度为300mmol/L时,收集洗脱峰即为纯化后的重组蓖麻毒素B链蛋白。纯化后的重组蓖麻毒素B链蛋白进行透析梯度复性。复性结束后用PEG20000浓缩蛋白,BCA法测定蛋白浓度,用0.22μm滤器过滤除菌后保存于-80℃。
实例2重组蓖麻毒素B链蛋白突变体抗2型糖尿病大鼠效果实验
1实验动物和分组
1.1模型的制作
试验用的雄性wistar大鼠,随机留取10只作为正常对照组,其他大鼠高脂饲料喂养4W后,其余单次小剂量(30mg/kg)腹腔注射STZ诱导2型糖尿病模型(STZ溶解在柠檬酸-柠檬酸钠缓冲液中,在半小时内注射完),1W后,禁食但不禁水12h,尾静脉取血,应用血糖仪测定空腹血糖值(FBG),其中FBG≥7.8mmol/l者为2型糖尿病模型成功。试验动物在动物中心12小时明暗交替的标准化饲养间喂养,室温在20-25℃,室内相对的湿度45%-60%,饲养间内通风良好,试验期间给予标准颗粒的正常饲料和高脂高糖饲料,试验动物自由饮水。
1.2试验动物分组及给药
成功造模的大鼠选取50只,随机分为5组,分别是:2型糖尿病模型组、阳性药物对照组、蛋白高浓度组、蛋白中浓度组及蛋白低浓度组。阳性对照组药物为盐酸二甲双胍,浓度200mg/kg,蛋白给药组分别给予腹腔注射蛋白400ug/kg,300ug/kg,200ug/kg。正常对照组,模型组等体积腹腔注射纯净水,蛋白给药每周一次,二甲双胍每天一次,连续给药6W。试验期间给予标准颗粒的正常饲料,高脂高糖饲料,试验动物自由饮水。
2实验步骤
试验给药前,禁食但不禁水12h,血糖仪测空腹血糖值。试验结束时,禁食但不禁水12h,测空腹血糖值。腹腔注射葡萄糖在0、30、60、90、120min测血糖值,2天后禁食8h杀鼠,腹主动脉取血1.5ml,3000r/min x 5min x2,离心后取血清及下沉的红细胞。大鼠采取的血清主要用于生化指标的检测。
3结果
结果表1所示,RTB突变体干预后2型糖尿病大鼠的饮水量和体重较模型组明显改善;结果表2所示,给予RTB蛋白突变体干预后,空腹血糖(FBG)和糖化血红蛋白(GHb)水平明显改善;结果表3所示,RTB蛋白有较好的改善甘油三酯(TG)和总胆固醇(TCH)的效果;结果表4所示,糖耐量实验证明RTB有较高的改善2型糖尿病耐糖水平的效果。
表1各试验组试验前后饮水量和体重的比较(均数±标准差)
注:与正常组比较,△P<0.05,△△P<0.01;与模型组比较,#P<0.05,##P<0.01。
表2各试验组试验前后FBG水平与试验后GHb水平的比较(均数±标准差)
注:与正常组比较,△P<0.05,△△P<0.01;与模型组比较,#P<0.05,##P<0.01。
表3各组TG和TCH比较
注:与模型组比较,#P<0.05,##P<0.01。
表4各试验组试验前后糖耐量水平的比较(均数±标准差)
注:与正常组比较,△P<0.05,△△P<0.01;与模型组比较,#P<0.05,##P<0.01。
以上对本发明的一个实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
SEQUENCE LISTING
<110> 侯峰
<120> 蓖麻毒素B的制备及其应用
<130> 123
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 259
<212> PRT
<213> Ricinus communis
<400> 1
Met Asp Pro Glu Pro Ile Val Arg Ile Val Gly Arg Asn Gly Leu Cys
1 5 10 15
Val Asp Val Arg Asp Gly Arg Phe His Asn Gly Asn Ala Ile Gln Leu
20 25 30
Trp Pro Cys Lys Ser Asn Thr Asp Ala Asn Gln Leu Trp Thr Leu Lys
35 40 45
Arg Asp Asn Thr Ile Arg Ser Asn Gly Lys Cys Leu Thr Thr Tyr Gly
50 55 60
Tyr Ser Pro Gly Val Tyr Val Met Ile Tyr Asp Cys Asn Thr Ala Ala
65 70 75 80
Thr Asp Ala Thr Arg Trp Gln Ile Trp Asp Asn Gly Thr Ile Ile Asn
85 90 95
Pro Arg Ser Ser Leu Val Leu Ala Ala Thr Ser Gly Tyr Ser Gly Thr
100 105 110
Thr Leu Asp Val Gln Thr Asn Ile Tyr Ala Val Arg Gln Gly Trp Leu
115 120 125
Pro Thr Asn Asn Thr Gln Pro Phe Val Thr Thr Ile Val Gly Leu Tyr
130 135 140
Gly Met Cys Leu Gln Ala Asn Ser Gly Lys Val Trp Leu Val Asp Cys
145 150 155 160
Thr Ser Glu Lys Ala Glu Gln Leu Ser Ala Leu Tyr Ala Asp Ala Ser
165 170 175
Ile Arg Pro Gln Gln Asn Arg Asp Asn Cys Leu Thr Thr Asp Ala Asn
180 185 190
Ile Lys Gly Thr Val Val Lys Ile Leu Ser Cys Gly Pro Ala Ser Ser
195 200 205
Gly Gln Arg Trp Met Phe Lys Asn Asp Gly Thr Ile Leu Asn Leu Tyr
210 215 220
Asn Gly Leu Val Thr Cys Val Arg Arg Ser Asp Pro Ser Ser Leu Lys
225 230 235 240
Gln Ile Ile Val His Pro Val His Gly Asn Leu Asn Gln Ile Trp Leu
245 250 255
Pro Leu Phe
<210> 2
<211> 777
<212> DNA
<213> 人工序列
<400> 2
atggaccctg aaccgattgt tcgcattgtt ggtcgcaatg gcctgtgcgt ggatgtgcgc 60
gatggtcgtt ttcataatgg taatgccatt cagctgtggc cgtgtaaaag caataccgat 120
gcaaatcagc tgtggaccct gaaacgtgat aataccattc gtagcaatgg caaatgcctg 180
accacctatg gctatagtcc gggtgtttat gtgatgatct atgattgtaa taccgcagcc 240
accgatgcca cccgctggca gatttgggat aatggtacaa ttattaaccc gcgcagtagt 300
ctggtgctgg cagcaaccag cggatatagt ggtacaaccc tggacgttca gaccaatatc 360
tatgccgtta ggcagggttg gctgccgacc aataataccc agccgtttgt taccaccatt 420
gttggtctgt atggcatgtg tctgcaagcc aatagtggta aagtgtggct ggttgattgt 480
accagcgaaa aagccgaaca gctatccgcc ctgtatgccg atgctagcat tcgcccgcag 540
cagaatcgcg ataattgcct gaccacagat gccaatatta agggcaccgt tgttaaaatt 600
ctgagctgcg gcccggccag tagcggccaa cgttggatgt ttaaaaatga tggcaccatt 660
ctgaatctgt ataatggcct ggttacctgt gtgcgtcgca gcgatccgag cagtctgaaa 720
cagattattg ttcatccggt tcatggcaat ctgaatcaga tttggttacc gctgttt 777
Claims (10)
1.包含SEQ ID:1序列的蛋白在制备治疗2型糖尿病药物中的应用。
2.根据权利要求1所述的的蛋白,其特征在于:该蛋白可降低2型糖尿病模型的大鼠空腹血糖水平。
3.根据权利要求1所述的的蛋白,其特征在于:该蛋白可降低2型糖尿病模型的大鼠糖化血红蛋白水平。
4.根据权利要求1所述的的蛋白,其特征在于:该蛋白可降低2型糖尿病模型的大鼠的饮水量。
5.根据权利要求1所述的的蛋白,其特征在于:该蛋白可增加2型糖尿病模型的大鼠的体重,改善消瘦症状。
6.根据权利要求1所述的的蛋白,其特征在于:该蛋白可改善2型糖尿病模型的大鼠的甘油三酯(TG)和总胆固醇(TCH)水平。
7.根据权利要求1所述的的蛋白,其特征在于:该蛋白可改善2型糖尿病模型的大鼠的耐糖能力。
8.根据权利要求1所述的的蛋白,其特征在于:该蛋白通过大肠杆菌表达系统制备。
9.根据权利要求8所述的蛋白的制备方法,所述大肠杆菌表达系统所用载体为PET系列载体。
10.根据权利要求8所述的蛋白的制备方法,所述大肠杆菌表达系统所用表达菌种为BL21。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810957682.3A CN110404052B (zh) | 2018-08-22 | 2018-08-22 | 蓖麻毒素b的制备及其应用 |
PCT/CN2018/111237 WO2020037809A1 (zh) | 2018-08-22 | 2018-10-22 | 蓖麻毒素b的制备及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810957682.3A CN110404052B (zh) | 2018-08-22 | 2018-08-22 | 蓖麻毒素b的制备及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110404052A true CN110404052A (zh) | 2019-11-05 |
CN110404052B CN110404052B (zh) | 2020-10-09 |
Family
ID=68358046
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810957682.3A Active CN110404052B (zh) | 2018-08-22 | 2018-08-22 | 蓖麻毒素b的制备及其应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN110404052B (zh) |
WO (1) | WO2020037809A1 (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102552876A (zh) * | 2012-02-17 | 2012-07-11 | 中国人民解放军军事医学科学院军事兽医研究所 | 蓖麻毒素b链蛋白的免疫调节新用途 |
CN105481956A (zh) * | 2015-12-31 | 2016-04-13 | 中国人民解放军军事医学科学院军事兽医研究所 | 重组蓖麻毒素b链截短蛋白及其表达方法和应用 |
-
2018
- 2018-08-22 CN CN201810957682.3A patent/CN110404052B/zh active Active
- 2018-10-22 WO PCT/CN2018/111237 patent/WO2020037809A1/zh active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102552876A (zh) * | 2012-02-17 | 2012-07-11 | 中国人民解放军军事医学科学院军事兽医研究所 | 蓖麻毒素b链蛋白的免疫调节新用途 |
CN105481956A (zh) * | 2015-12-31 | 2016-04-13 | 中国人民解放军军事医学科学院军事兽医研究所 | 重组蓖麻毒素b链截短蛋白及其表达方法和应用 |
Non-Patent Citations (2)
Title |
---|
GENBANK: "AAA63506.1", 《GENBANK》 * |
JAMES E. CARTER III等: "Expression of a Ricin Toxin B Subunit: Insulin Fusion Protein in Edible Plant Tissues", 《MOL BIOTECHNOL.》 * |
Also Published As
Publication number | Publication date |
---|---|
CN110404052B (zh) | 2020-10-09 |
WO2020037809A1 (zh) | 2020-02-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113265007B (zh) | 一种治疗代谢疾病的融合蛋白及其制备方法和应用 | |
CN107980043A (zh) | 多肽用于作用于免疫信号转导和/或影响肠屏障功能和/或调节代谢状态的用途 | |
EP2246064A1 (en) | Recombinant ganoderma lucidium immunomodulatory protein (rlz-8) and uses thereof | |
CN102010473A (zh) | 重组胃泌酸调节素融合蛋白及其制备和应用 | |
JP3960393B2 (ja) | 健康組成物 | |
CN105925598A (zh) | 分泌表达glp-1的减毒鼠伤寒沙门氏菌的制备方法及应用 | |
CN107033234B (zh) | 酰化的glp-1衍生物 | |
CN105367664B (zh) | 激活GLP-1受体和Amylin受体双功能作用的融合蛋白制备及其用途 | |
CN110386974A (zh) | Glp-1衍生物及其治疗用途 | |
CN106608915A (zh) | Glp-1(7-37)多肽类似物 | |
CN113583142A (zh) | 双靶点融合蛋白、编码基因、载体或宿主细胞及其应用与表达和纯化方法 | |
CN102127160B (zh) | 蝎活性多肽及其制备方法与应用 | |
CN110404052A (zh) | 蓖麻毒素b的制备及其应用 | |
CN101580846A (zh) | 一种用于防治肝硬化的人源细胞珠蛋白及其制备方法 | |
CN1978466B (zh) | 转导肽-人脑源性神经营养因子融合蛋白及其应用 | |
CN101665799A (zh) | 一种Exendin-4衍生物的重组制备方法和应用 | |
CN1985854A (zh) | 一种含有植物乳杆菌的组合物及其在增强肠道免疫屏障中的应用 | |
US7947313B2 (en) | Compositions for diabetes treatment and prophylaxis | |
CN108218978A (zh) | 一种重组白细胞介素18及其制备方法与应用 | |
Taylor et al. | Poisoning with cadmium fumes after smelting lead. | |
CN101906158B (zh) | 一种聚乙二醇化降糖多肽及其制法和用途 | |
CN101906156B (zh) | 一种双功能蛋白及其衍生物的结构及用途 | |
CN1318587C (zh) | 酰胺化Exendin-4多肽的重组制备方法 | |
CN103613657B (zh) | 缩短肽链的Exendin4及其基因工程应用 | |
EP1567174B1 (en) | Compositions for diabetes treatment and prophylaxis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20210915 Address after: 101200 no.6-2004, Xinggu a district, Pinggu Park, Zhongguancun Science and Technology Park, Pinggu District, Beijing (cluster registration) Patentee after: Beijing Hai Mu Group Co.,Ltd. Address before: No.19, Hongda street, Binhai New Area, Tianjin, 300457 Patentee before: Hou Feng |