CN114107316A - 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 - Google Patents
一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 Download PDFInfo
- Publication number
- CN114107316A CN114107316A CN202111485819.8A CN202111485819A CN114107316A CN 114107316 A CN114107316 A CN 114107316A CN 202111485819 A CN202111485819 A CN 202111485819A CN 114107316 A CN114107316 A CN 114107316A
- Authority
- CN
- China
- Prior art keywords
- lambda
- recombinant
- interferon
- porcine
- poifn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010050904 Interferons Proteins 0.000 title claims abstract description 64
- 102000014150 Interferons Human genes 0.000 title claims abstract description 49
- 229940079322 interferon Drugs 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 title abstract description 76
- 230000014509 gene expression Effects 0.000 claims abstract description 25
- 241000588724 Escherichia coli Species 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000013598 vector Substances 0.000 claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 34
- 102000053602 DNA Human genes 0.000 claims description 27
- 241000282898 Sus scrofa Species 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 201000010099 disease Diseases 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 230000003612 virological effect Effects 0.000 claims description 8
- 239000012620 biological material Substances 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 230000009261 transgenic effect Effects 0.000 claims description 7
- 230000000840 anti-viral effect Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 3
- 239000000203 mixture Substances 0.000 claims 3
- 206010012735 Diarrhoea Diseases 0.000 claims 1
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 claims 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims 1
- 208000005577 Gastroenteritis Diseases 0.000 claims 1
- 241000202347 Porcine circovirus Species 0.000 claims 1
- 206010037660 Pyrexia Diseases 0.000 claims 1
- 241000702670 Rotavirus Species 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 208000032625 disorder of ear Diseases 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 208000003265 stomatitis Diseases 0.000 claims 1
- 230000004083 survival effect Effects 0.000 claims 1
- 208000005925 vesicular stomatitis Diseases 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 16
- 239000013612 plasmid Substances 0.000 abstract description 14
- 230000006698 induction Effects 0.000 abstract description 7
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 238000010367 cloning Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 33
- 102000004169 proteins and genes Human genes 0.000 description 23
- 235000018102 proteins Nutrition 0.000 description 19
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 14
- 239000004202 carbamide Substances 0.000 description 14
- 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 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 210000003000 inclusion body Anatomy 0.000 description 7
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 6
- 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 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 5
- 238000004153 renaturation Methods 0.000 description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 108010018844 interferon type III Proteins 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 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 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- 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 2
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 2
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 108010085059 glutamyl-arginyl-proline Proteins 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- 108010050343 histidyl-alanyl-glutamine Proteins 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 2
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- WXPZDDCNKXMOMC-AVGNSLFASA-N (2s)-1-[(2s)-2-[[(2s)-1-(2-aminoacetyl)pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carboxylic acid Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@H](C(O)=O)CCC1 WXPZDDCNKXMOMC-AVGNSLFASA-N 0.000 description 1
- FJVAQLJNTSUQPY-CIUDSAMLSA-N Ala-Ala-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN FJVAQLJNTSUQPY-CIUDSAMLSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- FXKNPWNXPQZLES-ZLUOBGJFSA-N Ala-Asn-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O FXKNPWNXPQZLES-ZLUOBGJFSA-N 0.000 description 1
- IKKVASZHTMKJIR-ZKWXMUAHSA-N Ala-Asp-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IKKVASZHTMKJIR-ZKWXMUAHSA-N 0.000 description 1
- BLGHHPHXVJWCNK-GUBZILKMSA-N Ala-Gln-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O BLGHHPHXVJWCNK-GUBZILKMSA-N 0.000 description 1
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 1
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 1
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- YYOVLDPHIJAOSY-DCAQKATOSA-N Arg-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N YYOVLDPHIJAOSY-DCAQKATOSA-N 0.000 description 1
- VBFJESQBIWCWRL-DCAQKATOSA-N Arg-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VBFJESQBIWCWRL-DCAQKATOSA-N 0.000 description 1
- OTCJMMRQBVDQRK-DCAQKATOSA-N Arg-Asp-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OTCJMMRQBVDQRK-DCAQKATOSA-N 0.000 description 1
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 1
- PPPXVIBMLFWNSK-BQBZGAKWSA-N Arg-Gly-Cys Chemical compound C(C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N PPPXVIBMLFWNSK-BQBZGAKWSA-N 0.000 description 1
- ZJEDSBGPBXVBMP-PYJNHQTQSA-N Arg-His-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZJEDSBGPBXVBMP-PYJNHQTQSA-N 0.000 description 1
- OFIYLHVAAJYRBC-HJWJTTGWSA-N Arg-Ile-Phe Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O OFIYLHVAAJYRBC-HJWJTTGWSA-N 0.000 description 1
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 1
- WKPXXXUSUHAXDE-SRVKXCTJSA-N Arg-Pro-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O WKPXXXUSUHAXDE-SRVKXCTJSA-N 0.000 description 1
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 1
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 1
- SNYCNNPOFYBCEK-ZLUOBGJFSA-N Asn-Ser-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O SNYCNNPOFYBCEK-ZLUOBGJFSA-N 0.000 description 1
- NECWUSYTYSIFNC-DLOVCJGASA-N Asp-Ala-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 NECWUSYTYSIFNC-DLOVCJGASA-N 0.000 description 1
- CJUKAWUWBZCTDQ-SRVKXCTJSA-N Asp-Leu-Lys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O CJUKAWUWBZCTDQ-SRVKXCTJSA-N 0.000 description 1
- NVXLFIPTHPKSKL-UBHSHLNASA-N Asp-Trp-Asn Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(O)=O)N)C(=O)N[C@@H](CC(N)=O)C(O)=O)=CNC2=C1 NVXLFIPTHPKSKL-UBHSHLNASA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- XLLSMEFANRROJE-GUBZILKMSA-N Cys-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CS)N XLLSMEFANRROJE-GUBZILKMSA-N 0.000 description 1
- YNJBLTDKTMKEET-ZLUOBGJFSA-N Cys-Ser-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O YNJBLTDKTMKEET-ZLUOBGJFSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- DTCCMDYODDPHBG-ACZMJKKPSA-N Gln-Ala-Cys Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(O)=O DTCCMDYODDPHBG-ACZMJKKPSA-N 0.000 description 1
- KVYVOGYEMPEXBT-GUBZILKMSA-N Gln-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O KVYVOGYEMPEXBT-GUBZILKMSA-N 0.000 description 1
- NUMFTVCBONFQIQ-DRZSPHRISA-N Gln-Ala-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NUMFTVCBONFQIQ-DRZSPHRISA-N 0.000 description 1
- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 description 1
- GLAPJAHOPFSLKL-SRVKXCTJSA-N Gln-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)N)N GLAPJAHOPFSLKL-SRVKXCTJSA-N 0.000 description 1
- LGIKBBLQVSWUGK-DCAQKATOSA-N Gln-Leu-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LGIKBBLQVSWUGK-DCAQKATOSA-N 0.000 description 1
- JRHPEMVLTRADLJ-AVGNSLFASA-N Gln-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JRHPEMVLTRADLJ-AVGNSLFASA-N 0.000 description 1
- JUUNNOLZGVYCJT-JYJNAYRXSA-N Gln-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JUUNNOLZGVYCJT-JYJNAYRXSA-N 0.000 description 1
- XQDGOJPVMSWZSO-SRVKXCTJSA-N Gln-Pro-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N XQDGOJPVMSWZSO-SRVKXCTJSA-N 0.000 description 1
- YJSCHRBERYWPQL-DCAQKATOSA-N Gln-Pro-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N YJSCHRBERYWPQL-DCAQKATOSA-N 0.000 description 1
- OGMQXTXGLDNBSS-FXQIFTODSA-N Glu-Ala-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O OGMQXTXGLDNBSS-FXQIFTODSA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- PBFGQTGPSKWHJA-QEJZJMRPSA-N Glu-Asp-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O PBFGQTGPSKWHJA-QEJZJMRPSA-N 0.000 description 1
- IQACOVZVOMVILH-FXQIFTODSA-N Glu-Glu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O IQACOVZVOMVILH-FXQIFTODSA-N 0.000 description 1
- AAJHGGDRKHYSDH-GUBZILKMSA-N Glu-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O AAJHGGDRKHYSDH-GUBZILKMSA-N 0.000 description 1
- HZISRJBYZAODRV-XQXXSGGOSA-N Glu-Thr-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O HZISRJBYZAODRV-XQXXSGGOSA-N 0.000 description 1
- KIEICAOUSNYOLM-NRPADANISA-N Glu-Val-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KIEICAOUSNYOLM-NRPADANISA-N 0.000 description 1
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 1
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 1
- ZWRDOVYMQAAISL-UWVGGRQHSA-N Gly-Met-Lys Chemical compound CSCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCCN ZWRDOVYMQAAISL-UWVGGRQHSA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- QIVPRLJQQVXCIY-HGNGGELXSA-N His-Ala-Gln Chemical compound C[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](CCC(N)=O)C(O)=O QIVPRLJQQVXCIY-HGNGGELXSA-N 0.000 description 1
- SYMSVYVUSPSAAO-IHRRRGAJSA-N His-Arg-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O SYMSVYVUSPSAAO-IHRRRGAJSA-N 0.000 description 1
- BZKDJRSZWLPJNI-SRVKXCTJSA-N His-His-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O BZKDJRSZWLPJNI-SRVKXCTJSA-N 0.000 description 1
- UQTKYYNHMVAOAA-HJPIBITLSA-N His-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N UQTKYYNHMVAOAA-HJPIBITLSA-N 0.000 description 1
- VYUXYMRNGALHEA-DLOVCJGASA-N His-Leu-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O VYUXYMRNGALHEA-DLOVCJGASA-N 0.000 description 1
- CCUSLCQWVMWTIS-IXOXFDKPSA-N His-Thr-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O CCUSLCQWVMWTIS-IXOXFDKPSA-N 0.000 description 1
- LNVILFYCPVOHPV-IHPCNDPISA-N His-Trp-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O LNVILFYCPVOHPV-IHPCNDPISA-N 0.000 description 1
- FHPZJWJWTWZKNA-LLLHUVSDSA-N Ile-Phe-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N FHPZJWJWTWZKNA-LLLHUVSDSA-N 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 1
- JUWJEAPUNARGCF-DCAQKATOSA-N Leu-Arg-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O JUWJEAPUNARGCF-DCAQKATOSA-N 0.000 description 1
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 1
- AOFYPTOHESIBFZ-KKUMJFAQSA-N Leu-His-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O AOFYPTOHESIBFZ-KKUMJFAQSA-N 0.000 description 1
- OYQUOLRTJHWVSQ-SRVKXCTJSA-N Leu-His-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O OYQUOLRTJHWVSQ-SRVKXCTJSA-N 0.000 description 1
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- DCGXHWINSHEPIR-SRVKXCTJSA-N Leu-Lys-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N DCGXHWINSHEPIR-SRVKXCTJSA-N 0.000 description 1
- GCXGCIYIHXSKAY-ULQDDVLXSA-N Leu-Phe-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GCXGCIYIHXSKAY-ULQDDVLXSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 1
- ZDJQVSIPFLMNOX-RHYQMDGZSA-N Leu-Thr-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZDJQVSIPFLMNOX-RHYQMDGZSA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- WQWZXKWOEVSGQM-DCAQKATOSA-N Lys-Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN WQWZXKWOEVSGQM-DCAQKATOSA-N 0.000 description 1
- WXJKFRMKJORORD-DCAQKATOSA-N Lys-Arg-Ala Chemical compound NC(=N)NCCC[C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CCCCN WXJKFRMKJORORD-DCAQKATOSA-N 0.000 description 1
- HKCCVDWHHTVVPN-CIUDSAMLSA-N Lys-Asp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O HKCCVDWHHTVVPN-CIUDSAMLSA-N 0.000 description 1
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 1
- UQRZFMQQXXJTTF-AVGNSLFASA-N Lys-Lys-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O UQRZFMQQXXJTTF-AVGNSLFASA-N 0.000 description 1
- RXWPLVRJQNWXRQ-IHRRRGAJSA-N Met-His-His Chemical compound C([C@H](NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 RXWPLVRJQNWXRQ-IHRRRGAJSA-N 0.000 description 1
- CNAGWYQWQDMUGC-IHRRRGAJSA-N Met-Phe-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CNAGWYQWQDMUGC-IHRRRGAJSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- AGYXCMYVTBYGCT-ULQDDVLXSA-N Phe-Arg-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O AGYXCMYVTBYGCT-ULQDDVLXSA-N 0.000 description 1
- MECSIDWUTYRHRJ-KKUMJFAQSA-N Phe-Asn-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O MECSIDWUTYRHRJ-KKUMJFAQSA-N 0.000 description 1
- HOYQLNNGMHXZDW-KKUMJFAQSA-N Phe-Glu-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O HOYQLNNGMHXZDW-KKUMJFAQSA-N 0.000 description 1
- KYYMILWEGJYPQZ-IHRRRGAJSA-N Phe-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KYYMILWEGJYPQZ-IHRRRGAJSA-N 0.000 description 1
- SCKXGHWQPPURGT-KKUMJFAQSA-N Phe-Lys-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O SCKXGHWQPPURGT-KKUMJFAQSA-N 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- AJLVKXCNXIJHDV-CIUDSAMLSA-N Pro-Ala-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O AJLVKXCNXIJHDV-CIUDSAMLSA-N 0.000 description 1
- 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
- JFNPBBOGGNMSRX-CIUDSAMLSA-N Pro-Gln-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O JFNPBBOGGNMSRX-CIUDSAMLSA-N 0.000 description 1
- DIFXZGPHVCIVSQ-CIUDSAMLSA-N Pro-Gln-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O DIFXZGPHVCIVSQ-CIUDSAMLSA-N 0.000 description 1
- LHALYDBUDCWMDY-CIUDSAMLSA-N Pro-Glu-Ala Chemical compound C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O LHALYDBUDCWMDY-CIUDSAMLSA-N 0.000 description 1
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 1
- FYPGHGXAOZTOBO-IHRRRGAJSA-N Pro-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 FYPGHGXAOZTOBO-IHRRRGAJSA-N 0.000 description 1
- HBBBLSVBQGZKOZ-GUBZILKMSA-N Pro-Met-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O HBBBLSVBQGZKOZ-GUBZILKMSA-N 0.000 description 1
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 1
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- FUMGHWDRRFCKEP-CIUDSAMLSA-N Ser-Leu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O FUMGHWDRRFCKEP-CIUDSAMLSA-N 0.000 description 1
- NLOAIFSWUUFQFR-CIUDSAMLSA-N Ser-Leu-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O NLOAIFSWUUFQFR-CIUDSAMLSA-N 0.000 description 1
- IUXGJEIKJBYKOO-SRVKXCTJSA-N Ser-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N IUXGJEIKJBYKOO-SRVKXCTJSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- PJIQEIFXZPCWOJ-FXQIFTODSA-N Ser-Pro-Asp Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O PJIQEIFXZPCWOJ-FXQIFTODSA-N 0.000 description 1
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- RCOUFINCYASMDN-GUBZILKMSA-N Ser-Val-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O RCOUFINCYASMDN-GUBZILKMSA-N 0.000 description 1
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- PKUJMYZNJMRHEZ-XIRDDKMYSA-N Trp-Glu-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PKUJMYZNJMRHEZ-XIRDDKMYSA-N 0.000 description 1
- IQXWAJUIAQLZNX-IHPCNDPISA-N Trp-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N IQXWAJUIAQLZNX-IHPCNDPISA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 1
- XBJKAZATRJBDCU-GUBZILKMSA-N Val-Pro-Ala Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XBJKAZATRJBDCU-GUBZILKMSA-N 0.000 description 1
- YTNGABPUXFEOGU-SRVKXCTJSA-N Val-Pro-Arg Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O YTNGABPUXFEOGU-SRVKXCTJSA-N 0.000 description 1
- QSPOLEBZTMESFY-SRVKXCTJSA-N Val-Pro-Val Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O QSPOLEBZTMESFY-SRVKXCTJSA-N 0.000 description 1
- UFCHCOKFAGOQSF-BQFCYCMXSA-N Val-Trp-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N UFCHCOKFAGOQSF-BQFCYCMXSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108010060175 trypsinogen activation peptide Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
-
- 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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/217—IFN-gamma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用。具体地公开了密码子优化的猪λ3干扰素基因PoIFNλ3‑MD及利用该基因制备重组猪λ3干扰素的方法。本发明优化的基因PoIFNλ3‑MD在宿主菌的表达能力远高于未优化的基因PoIFNλ3‑WT。将优化后的PoIFNλ3‑MD***到载体pET30a(+)的多克隆位点得到了重组质粒，并将重组质粒导入大肠杆菌得到了重组菌。利用该重组菌通过诱导培养就可以制备出大量高活性的重组猪λ3干扰素蛋白。本发明提供的基因、重组质粒和重组菌对于重组猪λ3干扰素的生产极具经济价值，对于生猪养殖业也具有重大应用价值和广泛的应用前景。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用。
背景技术
近年来，我国高密度、集约化生猪养殖方式导致了各种病害频繁发生，特别是各种病毒性疾病，已经成为制约我国养殖业可持续性发展的关键因素。长期以来人们一直使用各种化学物品来进行猪类病毒性疾病的治疗，但是此类药物极易导致药物残留超标，寻找新的安全无残留的抗病毒药已然是迫在眉睫，因此研发能够有效预防和治疗畜禽病毒性疾病新型药物，有强大的市场需求和重要的社会意义。
猪作为我国主要的肉类来源，在我国的市场中有着举足轻重的地位。在当前规模化养殖的背景下，猪病毒性疾病一直困扰和威胁着生猪产业的发展。干扰素(Interferon，IFN)作为一种高效的非特异性免疫因子，在猪的抗感染免疫中发挥重要的作用。Ⅲ型干扰素即λ-干扰素(IFN-λ)，是2003年由两个独立的研究小组在脊椎动物中同时发现的分类为Ⅲ型干扰素的细胞因子，因其基因结构与IL-10相似，又被归类为IL-10家族成员。Ⅲ型干扰素包括IFN-λ1(IL-29)、IFN-λ2(IL-28α)、IFN-λ3(IL-28β)。Ⅲ型干扰素λ具有广谱抗病毒能力，与Ⅰ型干扰素相比，其受体仅局限分布于上皮细胞和某些树突状细胞亚群，其作用具有显著的组织特异性，毒副作用小，具有应用于临床抗病毒治疗的潜力。目前国内外已有猪干扰素基因序列、氨基酸序列方面的报道，并实现了干扰素基因在原核或真核表达***中的表达，为猪基因工程干扰素的规模化生产和应用提供了依据，但仍然存在表达量低、抗病毒活性较低、产量低等问题，因此，进一步优化猪干扰素基因以提高目前猪干扰素的表达量，开发更高效的蛋白表达***，具有重要的经济意义和产业化应用前景。
发明内容
本发明所要解决的技术问题是如何通过优化编码猪λ3干扰素基因来提高重组猪λ3干扰素表达水平。所要解决的技术问题不限于如所描述的技术主题，本领域技术人员通过以下描述可以清楚地理解本文未提及的其它技术主题。
为解决上述技术问题，本发明首先提供了一种制备重组猪λ3干扰素的方法，所述方法包括将DNA分子导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组猪λ3干扰素，所述DNA分子的核苷酸序列为SEQ ID No.1或SEQ ID No.4。
进一步地，所述方法可为将含优化后DNA分子的重组质粒导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组猪λ3干扰素，所述DNA分子的核苷酸序列可为SEQ ID No.1或SEQ ID No.4。
进一步地，本发明提供了高效制备重组猪λ3干扰素的方法，所述方法包括将含优化后的DNA分子(SEQ ID No.1或SEQ ID No.4)的重组质粒导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组猪λ3干扰素。
所述DNA分子可为密码子优化的猪λ3干扰素基因(PoIFNλ3-MD)；所述PoIFNλ3-MD核苷酸序列为SEQ ID No.1；
所述DNA分子可为优化的猪λ3干扰素基因(PoIFNλ3-MD)与标签蛋白的融合基因；
进一步地，所述标签蛋白可为His标签蛋白；
所述融合基因可为融合基因His-PoIFNλ3-MD，所述融合基因His-PoIFNλ3-MD的核苷酸序列为SEQ ID No.4。
本发明还提供了所述优化的猪λ3干扰素基因编码的蛋白质，所述蛋白质的氨基酸序列为SEQ ID No.2。
本发明还提供了所述融合基因His-PoIFNλ3-MD编码的蛋白质，所述蛋白质的氨基酸序列为SEQ ID No.5。
进一步地，上述方法中，所述大肠杆菌可为大肠杆菌BL21(DE3)；
进一步地，所述诱导表达可为用0.5mmol/L的IPTG液体，16～37℃诱导4～16小时，如用0.5mmol/L的IPTG液体，37℃诱导6小时。
上述方法中，所述方法具体可包括如下步骤：
(1)将所述DNA分子PoIFNλ3-MD(SEQ ID No.1)导入到大肠杆菌中，得到重组大肠杆菌(BL21pET30a-PoIFNλ3-MD)；
(2)将所述重组大肠杆菌加入0.5mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)，16～37℃诱导4～16小时，然后超声破碎菌体，收集包涵体、洗涤包涵体、溶解包涵体并纯化后将其复性，得到含有重组猪λ3干扰素的溶液。
上述方法中，所述方法还包括纯化所述重组猪λ3干扰素的步骤。
本发明还提供了生物材料，所述生物材料可为下述任一种：
A1)含有所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD的表达盒；
A2)含有所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD的重组载体、或含有A1)所述表达盒的重组载体；
A3)含有所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD的重组微生物、或含有A1)所述表达盒的重组微生物、或含有A2)所述重组载体的重组微生物；
A4)含有所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD的转基因细胞系、或含有A1)所述表达盒的转基因细胞系、或含有A2)所述重组载体的转基因细胞系。
所述重组载体可为将SEQ ID No.1所示的DNA分子PoIFNλ3-MD***到载体pET30a(+)的多克隆位点得到的重组载体(即重组质粒pET30a-PoIFNλ3-MD)。
进一步地，所述重组载体(即重组质粒pET30a-PoIFNλ3-MD)可为用SEQ ID No.1所示的DNA分子PoIFNλ3-MD替换pET30a(+)的Xho I识别位点和EcoR V识别位点间的片段(XhoI识别位点和EcoRV识别位点间的小片段)，保持pET30a(+)的其它序列不变得到的重组表达载体。
所述重组菌可为将SEQ ID No.1所示的DNA分子PoIFNλ3-MD导入到大肠杆菌BL21(DE3)得到的重组菌；
所述重组菌可为将SEQ ID No.4所示的DNA分子His-PoIFNλ3-MD导入到大肠杆菌BL21(DE3)得到的重组菌；
进一步地，所述重组菌可为将所述重组载体(即重组质粒pET30a-PoIFNλ3-MD)导入到大肠杆菌BL21(DE3)得到的重组菌，其名称为大肠埃希氏菌(Escherichia coli)BL21(DE3)pET30a-PoIFNλ3-MD(简称BL21(DE3)pET30a-PoIFNλ3-MD)。
本发明还提供了一种制备重组猪λ3干扰素的方法，所述方法包括利用所述重组微生物或转基因细胞系制备重组猪λ3干扰素。
本文中所述的DNA分子也在本发明的保护范围内。
本文中任一所述方法制备的重组猪λ3干扰素也在本发明的保护范围内。
本发明还提供了所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD，和/或，所述生物材料在制备用于治疗和/或预防猪病毒性疾病的药物或制剂中的应用。
上述应用中，所述制剂可为抗病毒抑制剂。
本发明还提供了所述DNA分子PoIFNλ3-MD或His-PoIFNλ3-MD，和/或，所述生物材料在制备重组猪λ3干扰素中的应用。
本发明提供了一种优化的编码PoIFNλ3-MD的基因序列，所述基因在原核表达***中表达能力远高于未优化的基因。将该基因***载体pET30a(+)的多克隆位点得到了重组质粒，将重组质粒导入大肠杆菌BL21(DE3)，得到了重组菌。该重组菌通过简单的诱导培养可制备出大量重组猪λ3干扰素蛋白。本发明提供的基因、重组质粒和重组菌对于重组猪λ3干扰素的生产极具经济价值，对于生猪养殖业也具有重大价值。
附图说明
图1为PoIFNλ3-MD基因(SEQ ID No.1所示DNA)与PoIFNλ3-WT基因(SEQ IDNo.3所示DNA)的序列比对。
图2为SDS-PAGE检测猪干扰素基因(PoIFNλ3-MD和PoIFNλ3-WT)在大肠杆菌中表达产物结果；UI代表未诱导，I代表诱导，M为低分子量蛋白Marker。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所有引物与基因合成及测序工作由生工生物工程(上海)股份有限公司与苏州金唯智生物科技有限公司完成。
下述实施例中的载体pET30a(+)购自Addgene公司，货号#85761。
实施例1、优化的猪λ3干扰素基因和对照基因的人工合成
一、优化的猪λ3干扰素基因(PoIFNλ3-MD基因)的人工合成
根据氨基酸密码子的兼并性和大肠杆菌对密码子的偏好性，参考Genebank中猪λ3干扰素基因序列(Gene ID：100310828)，设计了含大肠杆菌偏好密码子的猪λ3干扰素的编码基因。优化后编码猪λ3干扰素基因(PoIFNλ3-MD基因)的核苷酸序列如SEQ ID No.1所示，共含519个核苷酸。
SEQ ID No.1所示的优化的猪λ3干扰素基因编码的蛋白质的氨基酸序列为SEQ IDNo.2。
二、对照基因(PoIFNλ3-WT基因)的克隆
对照基因即Genebank中猪λ3干扰素基因，参考Genebank中猪λ3干扰素基因序列(Gene ID：100310828)，设计引物扩增对照基因。对照基因(PoIFNλ3-WT)的核苷酸序列如SEQ ID No.3所示，共含519个核苷酸。
三、PoIFNλ3-MD基因和PoIFNλ3-WT基因的序列比对
PoIFNλ3-MD基因和PoIFNλ3-WT基因的序列比对见图1，PoIFNλ3-MD基因和PoIFNλ3-WT基因均编码SEQ ID No.2所示的重组猪λ3干扰素。
实施例2、重组质粒和重组菌的构建以及基因的表达
一、含有PoIFNλ3-MD基因的重组质粒和工程菌的构建
用核苷酸序列是SEQ ID No.1的DNA分子(PoIFNλ3-MD基因)替换pET30a(+)的XhoI识别位点和EcoR V识别位点间的片段(Xho I识别位点和EcoR V识别位点间的小片段)，保持pET30a(+)的其它序列不变得到重组载体pET30a-PoIFNλ3-MD。由生工生物工程(上海)股份有限公司测序经SnapGene软件分析被测序列与优化后的序列一致。pET30a-PoIFNλ3-MD含有核苷酸序列是SEQ ID No.4的His-PoIFNλ3-MD融合基因，所述融合基因是融合了His标签及载体上的一段基因，His-PoIFNλ3-MD基因的编码序列是SEQ ID No.4，His-PoIFNλ3-MD基因编码氨基酸序列是SEQ ID No.5的蛋白质His-PoIFNλ3-MD。SEQ ID No.5的第48-219位是蛋白质PoIFNλ3-MD的氨基酸序列(即SEQ ID No.2)。SEQ ID No.4的第142-660位是PoIFNλ3-MD基因的核苷酸序列(即SEQ ID No.1)。
将重组质粒pET30a-PoIFNλ3-MD转化进入大肠杆菌BL21(DE3)，得到重组菌BL21(DE3)pET30a-PoIFNλ3-MD。
二、PoIFNλ3-MD基因的表达：
将过夜培养的BL21(DE3)pET30a-PoIFNλ3-MD按1:100的比例转接到400ml含有卡那霉素的LB培养基中(卡那霉素浓度为0.5ug/ml)，37℃，220r/min振荡培养3小时(至菌液OD600＝1)，加入0.5mmol/L IPTG后37℃，220r/min振荡诱导6小时，得到BL21(DE3)pET30a-PoIFNλ3-MD的发酵液(诱导后的培养液)。分别取诱导前后的培养液1ml于离心管，12000r/min，4℃离心10分钟，弃上清收集沉淀。加入6×Loading Buffer 100℃煮10分钟后，进行SDS-PAGE电泳检测。电泳检测结果如图2所示，诱导后收集的菌体在26kD左右处出现一条蛋白条带，与预期大小相符。
三、重组猪λ3干扰素蛋白变性、纯化和复性
1、变性
经IPTG诱导表达的菌体超声(Φ6探头，超声4秒间隔5秒，30分钟)破碎后，收集包涵体沉淀，工程菌收集菌体重量为0.78g，对照组为0.75g。按照每克菌重加入20ml溶液比例，使用溶液A洗涤包涵体沉淀2～3遍，使用溶液B在4℃条件下搅拌过夜，充分溶解包涵体蛋白。所述溶液A的pH值为8.0，由尿素、Tris-HCl、EDTA、NaCl、β-巯基乙醇、Triton-100和水组成，尿素浓度为2mol/L，Tris-HCl浓度为50mmol/L、EDTA浓度为0.3mmol/L、NaCl浓度为50mmol/L、β-巯基乙醇浓度为2mmol/L、Triton-100浓度为1％；所述溶液B的pH值为8.0，由尿素、Tris-HCl、β-巯基乙醇和水组成，尿素浓度为8mol/L，Tris-HCl浓度为50mmol/L、β-巯基乙醇浓度为2mmol/L。
2、纯化
变性后溶液4℃条件下12000r/min离心10min收集上清，使用0.45μm滤膜过滤后，经Ni-NTA亲和层析柱纯化。将含有重组猪λ3干扰素的溶液，使用GE AKTA Pure蛋白质分离纯化***进行亲和层析柱纯化，使用结合缓冲液以0.5ml/min流速结合目的蛋白，然后用洗杂缓冲液冲洗10个柱体积，最后使用洗脱缓冲液冲洗5-10个柱体积，收集280nm光照下紫外吸收值大于200的洗脱液，得到重组猪λ3干扰素溶液；所述结合缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为10mmol/L，尿素为8M；所述洗杂缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为80mmol/L，尿素为8M；所述洗脱缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为500mmol/L，尿素为8M。
3、复性
纯化后蛋白在尿素浓度梯度递减的复性缓冲液中透析，每隔3h换一次复性缓冲液，最后在PBS缓冲液中透析12～24h。将透析袋放入大平皿中用PEG8000浓缩，将浓缩好的蛋白过滤除菌并测定浓度，于-80℃保存备用。尿素复性缓冲液pH值为8.0，由尿素、Tris-HCl、NaCl、L-精氨酸、氧化型谷胱甘肽、还原型谷胱甘肽和水组成，尿素浓度为6mol/L、4mol/L、2mol/L、0mol/L梯度递减，Tris-HCl浓度为50mmol/L，NaCl浓度为0.15mol/L，L-精氨酸浓度为0.5mol/L，氧化型谷胱甘肽浓度为0.4mmol/L，还原型谷胱甘肽浓度为2mmol/L。
经过步骤1、2、3得到含有重组猪λ3干扰素的溶液(PoIFNλ3-MD溶液和PoIFNλ3-WT溶液)。
四、优化后基因序列PoIFNλ3-MD在宿主菌中蛋白量的测定
待测溶液为步骤三得到的PoIFNλ3-MD溶液或PoIFNλ3-WT溶液。
按50:1的体积比取BCA蛋白定量试剂盒(购自上海索莱宝生物科技有限公司，货号：PC0020)中的溶液A和溶液B混合成工作液，并将BSA标准品梯度稀释至500mg/ml，400mg/ml，300mg/ml，200mg/ml，150mg/ml，100mg/ml，50mg/ml，取各个浓度的标准品或待测溶液20μl，加200μl的工作液混合，用保鲜膜封好37℃孵育30min，恢复至室温后用酶标仪读562nm处吸光值。根据标准品浓度和吸光值绘制标准曲线，再根据标准曲线计算待测蛋白样品中的蛋白浓度。
结果表明从400mL的BL21(DE3)pET30a-PoIFNλ3-MD发酵液(诱导后的培养液，OD600nm＝1.4)，按照上述步骤纯化得到3mL PoIFNλ3-MD溶液的蛋白浓度为0.6mg/ml，共收获1.8mg重组蛋白；与之相比，从400mL的BL21(DE3)pET30a-PoIFNλ3-WT的发酵液(诱导后的培养液OD600nm＝1.4)，按照上述步骤纯化得到3mL PoIFNλ3-WT溶液的蛋白浓度为0.3mg/ml，共收获0.9mg重组蛋白。结果表明，本发明提供的优化后的基因PoIFNλ3-MD在宿主菌中的表达能力远远高于未优化的基因PoIFNλ3-WT。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
SEQUENCE LISTING
<110> 山东农业大学
<120> 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 519
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 1
gttccagttc cggaagcgct gcgtgcgctg cccggcgcgc gtggttgtca tctggcgcag 60
ttcaaaagtc tgagcccgca agcgctgcaa gcgttcaaac gcgcgaaaga cgccttcgag 120
gaaagtctgc tggaagattg gaactgcagc agccgtatct tcccacgcag tcgcgatctg 180
aagcagctgc aagtttggga acgcccagtt gcgctggaag cggaagttgc gctgacgctg 240
agcgttctgg gtagtctggc caatagcagt ctgcatagca gtctggatca gccactccat 300
accctgcgcc atatccatgc ccagctgcaa gcgtgcgttc cggcccaacc aatggcgggt 360
ccgcgtccac gtggtcgtct ccaccactgg ctgcaccgcc tccaagaagc gcagaaaaaa 420
gagccgcaga gctgtctgga agcgagcgtg atgtttaatc tgtttcgtct gctgacccgc 480
gatctgaaat gcgtggccag tggtgatctg tgcgtgtaa 519
<210> 2
<211> 172
<212> PRT
<213> 人工序列（Artificial sequence）
<400> 2
Val Pro Val Pro Glu Ala Leu Arg Ala Leu Pro Gly Ala Arg Gly Cys
1 5 10 15
His Leu Ala Gln Phe Lys Ser Leu Ser Pro Gln Ala Leu Gln Ala Phe
20 25 30
Lys Arg Ala Lys Asp Ala Phe Glu Glu Ser Leu Leu Glu Asp Trp Asn
35 40 45
Cys Ser Ser Arg Ile Phe Pro Arg Ser Arg Asp Leu Lys Gln Leu Gln
50 55 60
Val Trp Glu Arg Pro Val Ala Leu Glu Ala Glu Val Ala Leu Thr Leu
65 70 75 80
Ser Val Leu Gly Ser Leu Ala Asn Ser Ser Leu His Ser Ser Leu Asp
85 90 95
Gln Pro Leu His Thr Leu Arg His Ile His Ala Gln Leu Gln Ala Cys
100 105 110
Val Pro Ala Gln Pro Met Ala Gly Pro Arg Pro Arg Gly Arg Leu His
115 120 125
His Trp Leu His Arg Leu Gln Glu Ala Gln Lys Lys Glu Pro Gln Ser
130 135 140
Cys Leu Glu Ala Ser Val Met Phe Asn Leu Phe Arg Leu Leu Thr Arg
145 150 155 160
Asp Leu Lys Cys Val Ala Ser Gly Asp Leu Cys Val
165 170
<210> 3
<211> 519
<212> DNA
<213> 猪（Sus scrofa）
<400> 3
gtgcctgtcc ctgaagccct cagggccctc ccaggagcaa ggggctgcca cttggcccag 60
ttcaagtctc tgtccccaca agcgctgcag gccttcaaga gggccaagga tgcctttgaa 120
gagtccctct tggaggactg gaactgcagc tcccgcatct tccccaggag cagggacctg 180
aagcagctgc aggtgtggga gcgccccgtg gccttggagg ccgaggtggc cctgaccctc 240
agcgtcctgg gctccttggc gaactcatcc ctgcacagca gcctggacca gccccttcac 300
acgctgcgcc acatccacgc ccagctccag gcctgtgtcc cagctcagcc catggcaggc 360
ccccggcccc ggggccgcct ccaccactgg ctgcaccggc tccaggaggc ccagaagaag 420
gagccccaga gctgcctgga agcctctgtc atgttcaacc tcttccgcct cctcacccgg 480
gacctgaaat gtgtcgccag tggagacctg tgtgtctga 519
<210> 4
<211> 660
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 4
atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60
accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120
gacgacaagg ccatggctga tgttccagtt ccggaagcgc tgcgtgcgct gccgggcgcg 180
cgtggttgtc atctggcgca gttcaaaagt ctgagcccgc aagcgctgca agccttcaaa 240
cgcgcgaaag acgccttcga ggaaagtctg ctggaagatt ggaactgcag cagccgtatc 300
ttcccacgca gtcgcgatct gaaacagctg caagtttggg aacgcccagt tgcgctggaa 360
gcggaagttg cgctgacgct gagcgttctg ggtagtctgg ccaatagcag tctgcatagc 420
agtctggatc agccactcca tacgctgcgc catatccatg cccagctgca agcgtgcgtt 480
ccggcccaac caatggcggg tccacgtcca cgtggtcgtc tccaccactg gctgcaccgc 540
ctccaagaag cgcagaaaaa agaaccgcag agctgtctgg aagcgagcgt gatgtttaat 600
ctgttccgtc tgctgacccg cgatctgaaa tgcgtggcca gtggtgatct gtgcgtgtaa 660
<210> 5
<211> 219
<212> PRT
<213> 人工序列（Artificial sequence）
<400> 5
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Val
35 40 45
Pro Val Pro Glu Ala Leu Arg Ala Leu Pro Gly Ala Arg Gly Cys His
50 55 60
Leu Ala Gln Phe Lys Ser Leu Ser Pro Gln Ala Leu Gln Ala Phe Lys
65 70 75 80
Arg Ala Lys Asp Ala Phe Glu Glu Ser Leu Leu Glu Asp Trp Asn Cys
85 90 95
Ser Ser Arg Ile Phe Pro Arg Ser Arg Asp Leu Lys Gln Leu Gln Val
100 105 110
Trp Glu Arg Pro Val Ala Leu Glu Ala Glu Val Ala Leu Thr Leu Ser
115 120 125
Val Leu Gly Ser Leu Ala Asn Ser Ser Leu His Ser Ser Leu Asp Gln
130 135 140
Pro Leu His Thr Leu Arg His Ile His Ala Gln Leu Gln Ala Cys Val
145 150 155 160
Pro Ala Gln Pro Met Ala Gly Pro Arg Pro Arg Gly Arg Leu His His
165 170 175
Trp Leu His Arg Leu Gln Glu Ala Gln Lys Lys Glu Pro Gln Ser Cys
180 185 190
Leu Glu Ala Ser Val Met Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp
195 200 205
Leu Lys Cys Val Ala Ser Gly Asp Leu Cys Val
210 215
Claims (10)
1.一种制备重组猪λ3干扰素的方法，其特征在于，所述方法包括将DNA分子导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组猪λ3干扰素，所述DNA分子的核苷酸序列为SEQ ID No.1或SEQ ID No.4。
2.根据权利要求1所述的方法，其特征在于，所述方法还包括纯化所述重组猪λ3干扰素的步骤。
3.生物材料，其特征在于，所述生物材料为下述任一种：
A1)含有权利要求1中所述DNA分子的表达盒；
A2)含有权利要求1中所述DNA分子的重组载体、或含有A1)所述表达盒的重组载体；
A3)含有权利要求1中所述DNA分子的重组微生物、或含有A1)所述表达盒的重组微生物、或含有A2)所述重组载体的重组微生物；
A4)含有权利要求1中所述DNA分子的转基因细胞系、或含有A1)所述表达盒的转基因细胞系、或含有A2)所述重组载体的转基因细胞系。
4.权利要求1中所述的DNA分子。
5.权利要求1或2所述方法制备的重组猪λ3干扰素。
6.权利要求1中所述的DNA分子，和/或，权利要求3所述的生物材料在制备用于治疗和/或预防猪病毒性疾病的药物或制剂中的应用。
7.根据权利要求6所述的应用，其特征在于，所述制剂为抗病毒抑制剂。
8.根据权利要求6所述的应用，其特征在于，所述制剂为提高仔猪存活率的制剂。
9.根据权利要求6-8中任一所述的应用，其特征在于，所述猪病毒性疾病为猪流行性腹泻、猪水疱性口炎、传染性胃肠炎、轮状病毒病、猪圆环病毒病、蓝耳病、猪***病、猪瘟或猪病毒性感冒。
10.权利要求1中所述的DNA分子，和/或，权利要求3所述的生物材料在制备重组猪λ3干扰素中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111485819.8A CN114107316A (zh) | 2021-12-07 | 2021-12-07 | 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111485819.8A CN114107316A (zh) | 2021-12-07 | 2021-12-07 | 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114107316A true CN114107316A (zh) | 2022-03-01 |
Family
ID=80367316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111485819.8A Pending CN114107316A (zh) | 2021-12-07 | 2021-12-07 | 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114107316A (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139390A (zh) * | 2007-08-14 | 2008-03-12 | 中国科学院微生物研究所 | 猪γ干扰素及其编码基因与应用 |
| CN103059124A (zh) * | 2012-12-31 | 2013-04-24 | 江苏众红生物工程创药研究院有限公司 | 一种重组猪干扰素γ及其编码基因和表达方法 |
| CN106939042A (zh) * | 2016-01-22 | 2017-07-11 | 华南农业大学 | 一种猪α干扰素及其应用 |
| CN107893087A (zh) * | 2017-11-07 | 2018-04-10 | 北京康宝利华生物科技有限公司 | 一种重组猪λ3干扰素的制备方法及其应用 |
| CN108484750A (zh) * | 2018-04-28 | 2018-09-04 | 中国农业科学院生物技术研究所 | 猪λ3干扰素突变体及其制备方法和应用 |
| CN108588111A (zh) * | 2018-04-23 | 2018-09-28 | 武汉中拓康明生物科技有限公司 | 一种毕赤酵母表达长效猪源干扰素的方法 |
-
2021
- 2021-12-07 CN CN202111485819.8A patent/CN114107316A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139390A (zh) * | 2007-08-14 | 2008-03-12 | 中国科学院微生物研究所 | 猪γ干扰素及其编码基因与应用 |
| CN103059124A (zh) * | 2012-12-31 | 2013-04-24 | 江苏众红生物工程创药研究院有限公司 | 一种重组猪干扰素γ及其编码基因和表达方法 |
| CN106939042A (zh) * | 2016-01-22 | 2017-07-11 | 华南农业大学 | 一种猪α干扰素及其应用 |
| CN107893087A (zh) * | 2017-11-07 | 2018-04-10 | 北京康宝利华生物科技有限公司 | 一种重组猪λ3干扰素的制备方法及其应用 |
| CN108588111A (zh) * | 2018-04-23 | 2018-09-28 | 武汉中拓康明生物科技有限公司 | 一种毕赤酵母表达长效猪源干扰素的方法 |
| CN108484750A (zh) * | 2018-04-28 | 2018-09-04 | 中国农业科学院生物技术研究所 | 猪λ3干扰素突变体及其制备方法和应用 |
Non-Patent Citations (5)
| Title |
|---|
| NONE: "PREDICTED: Sus scrofa interferon lambda-3-like (LOC110255217), mRNA", GENBANK * |
| NONE: "Sus scrofa interferon lambda-3-like (LOC110255217), mRNA", GENEBANK, pages 47 - 48 * |
| 周恒;沈海燕;郭鹏举;张春红;张建峰;刘志成;孙俊颖;李玉谷;: "重组猪IFN-λ3原核表达及其活性分析", 中国兽医学报, no. 01, pages 86 - 90 * |
| 张润祥: "重组牛IFN-λ3的制备及其与IFN-α/IFN-β/IFN-γ抗病毒活性比较研究", 中国博士学位论文全文数据库 农业科技辑, pages 050 - 250 * |
| 王丽;何涛;郑小莉;宋杰;谢华福;梅志强;李娟;: "猪干扰素-γ基因的合成及其在大肠杆菌中的表达", 泸州医学院学报, no. 04 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0155549B1 (en) | Dna encoding human tumor necrosis factor and human tumor necrosis factor polypeptide | |
| IE63991B1 (en) | Novel fusion proteins and their purification | |
| CN102666578A (zh) | 经过修饰的猪促生长素多肽和其用途 | |
| CN110950967A (zh) | 抗人血清白蛋白纳米抗体与il-2融合蛋白及制备方法 | |
| CN106939042B (zh) | 一种猪α干扰素及其应用 | |
| KR101949556B1 (ko) | 폴리펩티드의 제조 방법 | |
| CN102286490B (zh) | 一种鸡干扰素γ的制备复性方法 | |
| CN112553231A (zh) | 一种重组人热休克蛋白HSP90-His及其表达和纯化方法 | |
| CN114107316A (zh) | 一种密码子优化的猪λ3干扰素编码基因及其在制备猪λ3干扰素中的应用 | |
| CN107746432B (zh) | 一种Aβ42修饰蛋白及其表达与纯化方法 | |
| CN111218452B (zh) | 一种重组人tsg-6基因、重组人tsg-6蛋白标准品及其制备方法和应用 | |
| CN113980972A (zh) | 一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用 | |
| CN111748505B (zh) | 一种表达羧肽酶g2的基因工程菌及其制备方法和应用 | |
| KR20080026085A (ko) | 곤충세포에서 제조한 재조합 ｅ-셀렉틴 | |
| RU2453604C1 (ru) | Гибридный белок (варианты), штамм бактерий escherichia coli - продуцент гибридного белка (варианты) и способ получения безметионинового интерферона альфа-2 человека | |
| CN110003321A (zh) | 一种可溶性重组ngal蛋白氨基酸、基因序列及蛋白制备方法 | |
| CN104211808B (zh) | 一种肿瘤坏死因子相关凋亡诱导配体融合蛋白的制备方法 | |
| JPH02273193A (ja) | 組換えインターロイキン―２融合蛋白質類 | |
| CN117304297B (zh) | 一种重组人α-防御素5及其制备方法和应用 | |
| CN117603934B (zh) | 一种尿酸酶的纯化方法 | |
| CN113583107B (zh) | CRIg功能区蛋白变体及其应用 | |
| RU2804622C2 (ru) | Способ промышленной очистки ромиплостима | |
| CN112898407B (zh) | 重组驼源血清白蛋白的制备方法 | |
| CN109306004B (zh) | 骨保护素的s77突变体蛋白及其相关产品与应用 | |
| KR100535265B1 (ko) | 융합 단백질로부터 목적 단백질을 분리하는 방법 |
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 |