CN1557960A - Method for separating antibiotic peptide and separated antibiotic peptide - Google Patents
Method for separating antibiotic peptide and separated antibiotic peptide Download PDFInfo
- Publication number
- CN1557960A CN1557960A CNA2004100051993A CN200410005199A CN1557960A CN 1557960 A CN1557960 A CN 1557960A CN A2004100051993 A CNA2004100051993 A CN A2004100051993A CN 200410005199 A CN200410005199 A CN 200410005199A CN 1557960 A CN1557960 A CN 1557960A
- Authority
- CN
- China
- Prior art keywords
- dna
- ser
- thr
- peptide
- pro
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000003115 biocidal effect Effects 0.000 title description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 60
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 51
- 108020004414 DNA Proteins 0.000 claims abstract description 25
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 17
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 16
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 101710163270 Nuclease Proteins 0.000 claims abstract description 14
- SBKVPJHMSUXZTA-MEJXFZFPSA-N (2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-5-amino-2-[[2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-indol-3-yl)propanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylsulfanylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 SBKVPJHMSUXZTA-MEJXFZFPSA-N 0.000 claims abstract description 12
- 108010038049 Mating Factor Proteins 0.000 claims abstract description 12
- 108010052305 exodeoxyribonuclease III Proteins 0.000 claims abstract description 10
- 230000005764 inhibitory process Effects 0.000 claims abstract description 10
- 230000004927 fusion Effects 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 230000001939 inductive effect Effects 0.000 claims abstract description 5
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 36
- 230000029087 digestion Effects 0.000 claims description 24
- 230000012010 growth Effects 0.000 claims description 21
- 230000005611 electricity Effects 0.000 claims description 18
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 16
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 16
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 15
- 150000001413 amino acids Chemical group 0.000 claims description 14
- 241000894006 Bacteria Species 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 10
- 230000000968 intestinal effect Effects 0.000 claims description 9
- 230000003248 secreting effect Effects 0.000 claims description 9
- 210000000582 semen Anatomy 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims description 5
- 102000057593 human F8 Human genes 0.000 claims description 5
- 229940047431 recombinate Drugs 0.000 claims description 5
- IEVXCWPVBYCJRZ-IXOXFDKPSA-N Lys-Thr-His Chemical group NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IEVXCWPVBYCJRZ-IXOXFDKPSA-N 0.000 claims description 4
- MMLHRUJLOUSRJX-CIUDSAMLSA-N Ala-Ser-Lys Chemical group C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN MMLHRUJLOUSRJX-CIUDSAMLSA-N 0.000 claims description 3
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical group C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 claims description 3
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 claims description 2
- ZIYGTCDTJJCDDP-JYJNAYRXSA-N Glu-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)O)N ZIYGTCDTJJCDDP-JYJNAYRXSA-N 0.000 claims description 2
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 claims description 2
- AXNGDPAKKCEKGY-QPHKQPEJSA-N Ile-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N AXNGDPAKKCEKGY-QPHKQPEJSA-N 0.000 claims description 2
- PKGGWLOLRLOPGK-XUXIUFHCSA-N Ile-Leu-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PKGGWLOLRLOPGK-XUXIUFHCSA-N 0.000 claims description 2
- HJDZMPFEXINXLO-QPHKQPEJSA-N Ile-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N HJDZMPFEXINXLO-QPHKQPEJSA-N 0.000 claims description 2
- 108010065920 Insulin Lispro Proteins 0.000 claims description 2
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 claims description 2
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims description 2
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 claims description 2
- VIBXMCZWVUOZLA-OLHMAJIHSA-N Thr-Asn-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VIBXMCZWVUOZLA-OLHMAJIHSA-N 0.000 claims description 2
- YOOAQCZYZHGUAZ-KATARQTJSA-N Thr-Leu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YOOAQCZYZHGUAZ-KATARQTJSA-N 0.000 claims description 2
- 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 claims description 2
- 108010091092 arginyl-glycyl-proline Proteins 0.000 claims description 2
- 238000005267 amalgamation Methods 0.000 claims 4
- YSMPVONNIWLJML-FXQIFTODSA-N Ala-Asp-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(O)=O YSMPVONNIWLJML-FXQIFTODSA-N 0.000 claims 1
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 claims 1
- ORZGPQXISSXQGW-IHRRRGAJSA-N His-His-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O ORZGPQXISSXQGW-IHRRRGAJSA-N 0.000 claims 1
- JJHWJUYYTWYXPL-PYJNHQTQSA-N His-Ile-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CN=CN1 JJHWJUYYTWYXPL-PYJNHQTQSA-N 0.000 claims 1
- WLRJHVNFGAOYPS-HJPIBITLSA-N Ile-Ser-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N WLRJHVNFGAOYPS-HJPIBITLSA-N 0.000 claims 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 claims 1
- SNGZLPOXVRTNMB-LPEHRKFASA-N Pro-Ser-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N2CCC[C@@H]2C(=O)O SNGZLPOXVRTNMB-LPEHRKFASA-N 0.000 claims 1
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 claims 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 claims 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 claims 1
- 108010062796 arginyllysine Proteins 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 9
- 238000000746 purification Methods 0.000 abstract description 9
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 8
- 229920001184 polypeptide Polymers 0.000 abstract description 6
- 108010038807 Oligopeptides Proteins 0.000 abstract description 4
- 102000015636 Oligopeptides Human genes 0.000 abstract description 4
- 229940088710 antibiotic agent Drugs 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 240000004922 Vigna radiata Species 0.000 abstract 1
- 235000010721 Vigna radiata var radiata Nutrition 0.000 abstract 1
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 45
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 21
- 230000001186 cumulative effect Effects 0.000 description 20
- 239000000047 product Substances 0.000 description 20
- 230000003321 amplification Effects 0.000 description 19
- 238000003199 nucleic acid amplification method Methods 0.000 description 19
- 239000012530 fluid Substances 0.000 description 17
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 14
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 14
- 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 description 14
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 14
- 239000008103 glucose Substances 0.000 description 14
- 238000013016 damping Methods 0.000 description 12
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 12
- 230000002779 inactivation Effects 0.000 description 12
- 238000002156 mixing Methods 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 230000004087 circulation Effects 0.000 description 9
- 230000008859 change Effects 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 239000011573 trace mineral Substances 0.000 description 8
- 235000013619 trace mineral Nutrition 0.000 description 8
- 239000011782 vitamin Substances 0.000 description 8
- 235000013343 vitamin Nutrition 0.000 description 8
- 229930003231 vitamin Natural products 0.000 description 8
- 229940088594 vitamin Drugs 0.000 description 8
- 150000003722 vitamin derivatives Chemical class 0.000 description 8
- 230000008901 benefit Effects 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C([O-])=O)(C)C)=CC=CC=C1 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 230000000845 anti-microbial effect Effects 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 239000006035 Tryptophane Substances 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 102220369445 c.668T>C Human genes 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229960004799 tryptophan Drugs 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 206010027336 Menstruation delayed Diseases 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002611 lead compounds Chemical class 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 102220023257 rs387907546 Human genes 0.000 description 2
- 102220023256 rs387907547 Human genes 0.000 description 2
- 102220023258 rs387907548 Human genes 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- NGTYEHIRESTSRX-UWVGGRQHSA-N Arg-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NGTYEHIRESTSRX-UWVGGRQHSA-N 0.000 description 1
- AMIQZQAAYGYKOP-FXQIFTODSA-N Arg-Ser-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O AMIQZQAAYGYKOP-FXQIFTODSA-N 0.000 description 1
- 108090000328 Arrestin Proteins 0.000 description 1
- 102000003916 Arrestin Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 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 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 240000004859 Gamochaeta purpurea Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe 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)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- APKRGYLBSCWJJP-FXQIFTODSA-N Pro-Ala-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O APKRGYLBSCWJJP-FXQIFTODSA-N 0.000 description 1
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 1
- JUJGNDZIKKQMDJ-IHRRRGAJSA-N Pro-His-His Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O JUJGNDZIKKQMDJ-IHRRRGAJSA-N 0.000 description 1
- BFXZQMWKTYWGCF-PYJNHQTQSA-N Pro-His-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BFXZQMWKTYWGCF-PYJNHQTQSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- WXVIGTAUZBUDPZ-DTLFHODZSA-N Thr-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 WXVIGTAUZBUDPZ-DTLFHODZSA-N 0.000 description 1
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- ABSXSJZNRAQDDI-KJEVXHAQSA-N Tyr-Val-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ABSXSJZNRAQDDI-KJEVXHAQSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000010359 gene isolation Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000002070 germicidal effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012745 toughening agent Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method of isolating antibacterial peptides and the isolated peptides thereof. The method comprises: constructing a fusion DNA sequence of yeast alpha mating factor leader sequence and a random peptide, inserting the sequence into a saccharomyces cerevisiae vector which contains an inducible promotor, digesting the unlinked linear DNA with exonuclease III and mungbean sprout nuclease, electrotransforming the yeast vector after purification, culturing the transformant 24h and covering the transformant with the upper medium which contains E. coli K12D31, and detecting the bacterial inhibition ring and the bacterial inhibition rate after inducing the expression by liquid medium, selecting the positive clones and amplifying by PCR, and determining the sequence. The method of the present invention can be used easily and effectively to isolate antibacterial oligopeptides and polypeptides and can be used as an alternative way to find antibiotics.
Description
Technical field
The present invention relates to a kind of method and isolating antibacterial peptide that separates antibacterial peptide.
Background technology
Microbiotic is under lower concentration, can optionally suppress or kill and wound him and plant the microbial secondary metabolites of microorganism or tumour cell and similar compound and the structural modification thing that adopts chemistry or biological method to make.Various antibiotic antibacterial effects, majority is bacteriostatic action, germicidal action of minority tool or bacteriolysis.Antibiotic anti-microbial effect mainly is the physiology aspect that acts on mushroom, suppresses metabolic some link of microorganism cells or some enzyme system.The antibiotic mechanism of action can reduce following several types: (1) suppresses the synthetic of nucleic acid.(2) arrestin matter is synthetic.(3) permeability of change cytolemma.(4) formation of interference cell wall.(5) act on energy metabolism system or as metabolic antagonist.
Microbiotic is playing an important role aspect the guarantee human health as the important clinical drug application.Yet, along with antibiotic widespread use, cause two problems clinically: the one, bacterial drug resistance increases year by year, causes some microbiotic curative effects to reduce, even invalid, and multi-drug resistant constantly worsens.The 2nd, some non-pathogenic bacteriums become conditioned pathogen.Increasing microbiotic lost efficacy to many patients, needed the new antibiosis of exploitation usually to be prevented and treated.Traditional microbiotic finds that the resulting new antibiotic of approach is fewer and feweri, need set up some new methods and approach and develop.New development approach comprises structural modification, the new role mechanism and the screening of target site, material such as the antimicrobial enhancing agent that strengthens and protect the antibacterials performance, antibiotic enzyme inhibitor, membrane permeability toughener, efflux pump inhibitor etc. of microbiotic and synthetic antibacterial drug now.The present new target site of finding is kind surplus in the of 10 only.
Polypeptide antibiotics is one of direction of antibiotics medicine development.Traditional microbiotic is all produced by mould.Studies show that recently also has the antibiotic gene of coding in the genome of animal, plant and human body.These genes encodings mostly be some small peptides, be called antibacterial peptide.It is strong that antibacterial peptide has an anti-microbial activity, characteristics such as broad-spectrum antibacterial property.Antibacterial peptide is widespread in nature, and represents the ancient mechanism of host defense system.From genomes such as insect, batrachians, Mammals, plant, hundreds of antibacterial peptides have been separated so far.Antibacterial peptide can be used for fields such as medicine, fodder additives, food preservatives, plant genetic engineering.Because antibacterial peptide is to the particularly procaryotic toxicity of microorganism, general gene isolation method is unsuitable for separating antibacterial peptide gene.The method of traditional separation antibacterial peptide adopts repeatedly crosses post from amino acid sequencing or the mass spectrum order-checking then of various bioseparation purifying, very loaded down with trivial details.The another one method is according to known antibacterial peptide design homology fragment, carries out activity then and detects, and has significant limitation.Can also seek antibacterial peptide according to the method for combinatorial chemistry, the expense costliness, modification reaches maximum activity if antibacterial peptide needs glycosylation etc., and this method can't satisfy requirement of experiment.Be badly in need of setting up advantages of simplicity and high efficiency antibacterial peptide separation method at present.The general length of antibacterial peptide is 10 to 40 amino acid, has stronger immunogenicity, and patient is used effectively for the first time, and later result of use reduces gradually, must improve constantly dosage.The small molecules antibacterial peptide is these shortcomings of tool not.
Summary of the invention
The object of the present invention is to provide a kind of method of high efficiency separation antibacterial peptide, so that isolate oligopeptides and polypeptide with strong anti-microbial activity.The present invention also provides isolating antibiotic tripeptides and polypeptid coding sequence, to be used for the microbiotic exploitation.
The objective of the invention is to realize: make up the yeast α mating factor leading peptide and the fusion dna fragment of peptide at random, be used for the secreting, expressing of peptide at random by following technical measures; Insert the yeast saccharomyces cerevisiae carrier that contains inducible promoter with EcoR I and Xba I double digestion; Recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, it is carried out digestion process with exonuclease III and Semen Phaseoli radiati Germinatus nuclease; Electricity swashs transformed saccharomyces cerevisiae behind the purifying; Transformant is forwarded to raffinose/semi-lactosi abduction delivering flat board, covers the observation inhibition zone after the overnight growth with the upper strata substratum that contains intestinal bacteria bacterial classification K12D31; Or with surveying bacteriostasis rate behind the liquid nutrient medium abduction delivering; Positive colony is carried out pcr amplification, order-checking; Chemosynthesis part antibacterial peptide is used the agar diffusion method detection of active.
The present invention includes following concrete steps:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.With having the active Pfu archaeal dna polymerase of high-fidelity, PyrobestDNA polysaccharase or other has the active thermostable DNA polymerases amplification of high-fidelity, amplification template is that (plasmid pPICZ α A contains yeast α mating factor leading peptide dna sequence dna to plasmid pPICZ α A, the Invitrogen product), amplified production ethanol sedimentation; Hair dryer dries up; Be suspended in the sterile distilled water; With EcoR I and Xba I double digestion 6 hours to spending the night; 70 ℃ of heat inactivations.
(2) pYES2/CT (Invitrogen product) with EcoR I and Xba I double digestion 6 hours to spending the night; 70 ℃ of heat inactivations; Get the pYES2/CT of double digestion, add the PCR product and the T4 dna ligase of double digestion, 6 ℃ or 16 ℃ of connections are spent the night.
(3) cause the segmental transformant of nothing insertion too much for the linear DNA that does not connect of avoiding quantity to occupy the majority with pastoris genomic dna reorganization takes place after conversion, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company; Use H
2The O wash-out; Adding the exonuclease III of 10-20 unit handled 1 hour in 37 ℃; 70 ℃ of inactivations 15 minutes; Add 10-20 unit's Semen Phaseoli radiati Germinatus nuclease again, handled 40 minutes to 1 hour 20 minutes for 37 ℃; With the PCR purification column purifying of Qiagen company, use H
2The O wash-out; Getting 10 μ l does electric sharp;
(4) at first prepare the yeast competent cell.It is centrifugal to cultivate the S. cervisiae of OD value 1.3-1.5 at 30 ℃ of shaking tables, removes supernatant; Be resuspended in Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 20-30 ℃ is incubated 1 hour; Ice-cold sterilized water washed twice; Washing once in the ice-cold again 1mol/L sorbyl alcohol; Be resuspended in the 1mol/L sorbyl alcohol of 0.2-0.5ml ice precooling ice bath; Add 80 μ l yeast suspensions in each sterile tube and be no more than the DNA (cumulative volume is less than 10 μ l) of 100ng, mixing gently, ice bath 5 minutes; The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom; Doing electricity in 1.5 kilovolts, 25 microfarads and 200 ohm swashs; Add 450 μ l to 1ml 1mol/L sorbyl alcohols at once and swash in the cup to electricity, mixing is transferred to culture tube gently;
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days; Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped;
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day; Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth;
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed; Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant; Leave standstill and solidified in 10 minutes; Make a call to an aperture, add 0.5 μ l 50mg/ml penbritin, put super clean bench 5 minutes as over against photograph; Be inverted overnight growth at 37 ℃ of incubators; Observe inhibition zone, record is taken pictures;
(8) glucose that in addition yeast transformant is never contained sorbyl alcohol selects plating to 5-10ml raffinose/semi-lactosi abduction delivering liquid nutrient medium; Abduction delivering 7 days; Get the culture of inducing 7 days, centrifugal 1 minute of 13000rpm gets supernatant; With e. coli k12 D31 and LB substratum mixing; Put into 37 ℃ of shaking tables, 200rpm shook 3 hours, took out ice bath; The OD. value of 600nm place measure sample and contrast on spectrophotometer is a blank with the LB and the yeast raffinose/semi-lactosi abduction delivering liquid nutrient medium of equivalent; Calculate bacteriostasis rate according to formula (bacteriostasis rate=1-sample OD. value/contrast OD. value);
(9) insert fragment from the yeast transformant amplification, utilize boiling method to prepare pcr template; According to carrier pYES2/CT sequences Design upstream and downstream primer, amplification, purifying and full-automatic order-checking.Chemosynthesis part antibacterial peptide is used the agar diffusion method detection of active.
The inventive method is applicable to separates various antibacterial peptides, behind the fusion dna fragment secreting, expressing of yeast α mating factor leading peptide and peptide at random a Kex2 proteolytic enzyme restriction enzyme site is arranged, and will downcut peptide at random, secretes to born of the same parents, thereby can suppress colibacillary growth.3 antibiotic tripeptide sequences that record are:first clone is tripeptides, and dna sequence dna is AAGACCCAC, and aminoacid sequence is Lys Thr His; Second clone is tripeptides, and dna sequence dna is CCAACGCCT, and aminoacid sequence is Pro Thr Pro; The 3rd clone is tripeptides, and dna sequence dna is GCGTCTAAA, and aminoacid sequence is Ala Ser Lys; The 4th clone is 68 peptides; Dna sequence dna is ACCAACAATGATCTAGAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTC GGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCG CTGATCCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCTTACATTCACGCCC TCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGT, and amino acid sequence is Thr Asn Asn Asp Leu Glu Gly Pro Ser Lys Val Ser LeuSer Leu Thr Leu Ser Ser Val Ser Ile Leu Arg Val Pro Val Ile Ile Thr IleThr Ile Glu Phe Lys Pro Ala Asp Pro Arg Gly Pro His His Val Ile Ser TyrVal Thr Leu Thr Phe Thr Pro Ser Pro His Ile Arg Ser Asn Arg Lys Gly ArgSer.
Three separating obtained antibiotic 3 peptides of the inventive method can be used as the lead compound of microbiotic exploitation.
Following table 1 is the bacteriostasis rate that records with antibacterial peptide clone juice.
| First clone | Second clone | The 3rd clone | Sequence table No. 1 and No. 2 | Blank: the pYES2/CT transformant | |
| OD600nm | ?0.024 | ?0.029 | ?0.056 | ?0.037 | ?0.27 |
| Bacteriostasis rate | ?91.1％ | ?89.2％ | ?79.2％ | ?86.3％ |
Proved absolutely that from table 1 and each experiment diagram the inventive method can separate antibacterial peptide effectively.
The method that the inventive method is set up can antibiotic oligopeptides of high efficiency separation and polypeptide.The frequency of finding strong antibiotic clone is about 20%, and about 10% records sequence.A general people 1 year separable antibiotic oligopeptides and polypeptide more than 40, and the general people of traditional method just can be separated to a kind of antibacterial peptide in several years.The tripeptides that is separated to is not had an immunogenicity.Because molecular weight is little, can see through bacteria cell wall, biological accessibility is good.Owing to carry out secreting, expressing in the yeast saccharomyces cerevisiae system, be equivalent to utilize eukaryotic system to carry out a preliminary Biosafety property testing.And, can utilize Yeast gene engineering production.Because encoding sequence can produce a large amount of combinations, nearly all target position that these little peptides can directed toward bacteria.Albumen and polypeptide at yeast saccharomyces cerevisiae system secreting, expressing are subjected to glycosylation modified through the Serine of being everlasting, Threonine and N site.And all there is glycosylation site in the antibacterial peptide that the present invention is separated to, and has shown strong anti-microbial activity, and antibiotic 3 peptides can be used as the in addition chemically modified of lead compound of microbiotic exploitation, develop potent antibiotics.
Description of drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d are that separating obtained 4 antibacterial peptides clone covers formed inhibition zone behind the upper strata substratum that contains e. coli k12 D31;
Fig. 1 e is the negative contrast after empty carrier pYES2/CT yeast conversion subcovering contains the upper strata substratum of e. coli k12 D31;
Fig. 1 f for add in the upper strata substratum punching that contains e. coli k12 D31 0.5 μ l 50mg/ml penbritin over against photograph;
Fig. 2 upper left for full-automatic synthetic, purity be that 95.8% antibiotic 3 peptide Lys Thr His point sample 10ng are containing the formed inhibition zone of upper strata substratum of e. coli k12 D31;
Fig. 2 bottom right be 0.5 μ l 50mg/ml penbritin point sample the upper strata substratum that contains e. coli k12 D31 over against photograph.
Embodiment
Embodiment 1:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PfuDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 50ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 20 seconds, 15 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and 1 μ l (20 unit) exonuclease III, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 1 μ l (20 unit) Semen Phaseoli radiati Germinatus nuclease again, handles 1 hour 20 minutes for 37 ℃.With the PCR purification column purifying of Qiagen company, with 13 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 200ml YPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 100ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 25 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 200ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 100ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.2ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 doing electricity, kilovolt, 25 microfarads, 200 ohm swash.Adding 450 μ l 1mol/L sorbyl alcohols at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day.Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth.
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed.Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant.Leave standstill and solidified in 10 minutes.Make a call to an aperture in the place that does not have transformant with rifle head point, add 0.5 μ l50mg/ml penbritin, put super clean bench 5 minutes as over against photograph.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
(8) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the Pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAATACGACTCACTATAGGG;＜6〉TAGGATCAGCGGGTTTAAACTC.2 antibiotic tripeptide sequences that record are: first clone is tripeptides, and dna sequence dna is AAGACCCAC, and aminoacid sequence is Lys ThrHis.Second clone is tripeptides, and dna sequence dna is CCAACGCCT, and aminoacid sequence is Pro Thr Pro.
(9) synthesize kyrine L ys Thr His in Shanghai Bo Ya company with Peptide synthesizer.The intestinal bacteria bacterial classification K12D31 of 5 μ l overnight growth is added to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed, dull and stereotyped, leave standstill and solidified in 10 minutes.Make a call to two apertures with rifle head point, an aperture adds 10ng kyrine L ysThr His, and another aperture adds 0.5 μ l 50mg/ml penbritin as over against photograph, puts super clean bench 5 minutes.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
Embodiment 2:
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PyrobestDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 100ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 49 ℃ 30 seconds, 72 ℃ 30 seconds, 18 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 40 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 40 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and the 10 exonuclease III of unit, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 20 unit Semen Phaseoli radiati Germinatus nucleases again, handles 1 hour 20 minutes for 37 ℃.With the PCR purification column purifying of Qiagen company, with 20 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 50mlYPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 50ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 25 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 50ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 30ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.2ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 kilovolt, 25 microfarads and 200 ohm do electricity and swash.Adding 1ml 1mol/L sorbyl alcohol at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) glucose that yeast transformant is never contained sorbyl alcohol selects plating to 10ml raffinose/semi-lactosi abduction delivering liquid nutrient medium.Abduction delivering 7 days.Get each 1.5ml of culture that has induced 7 days, centrifugal 1 minute of 13000rpm gets supernatant, adds 0.75ml in every sterile eppendorf tubes.Get e. coli k12 D31 overnight culture and LB substratum mixing, final concentration is 50 μ l e. coli k12 D31 overnight culture/5mlLB substratum, wherein gets 0.75ml and is added to the eppendorf pipe that sample is housed, and making cumulative volume is 1.5ml.Put into 37 ℃ of shaking tables, 200rpm shook 3 hours, took out ice bath.The OD. value of 600nm place measure sample and contrast on 752 spectrophotometers is a blank with the LB and the yeast raffinose/semi-lactosi abduction delivering liquid nutrient medium of equivalent.Calculate bacteriostasis rate according to formula (bacteriostasis rate=1-sample OD. value/contrast OD. value).
(7) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAGGATCAGCGGGTTTAAACTC;＜6〉TAGGATCAGCGGGTTTAAACTC.1 the antibiotic tripeptides dna sequence dna that records is GCGTCTAAA, and aminoacid sequence is Ala Ser Lys.
Embodiment 3
(1) the structure yeast α mating factor leading peptide and the fusion dna fragment of peptide at random are used for the secreting, expressing of peptide at random.Two primers are respectively＜and 1〉GCGAATTCAAAAATGAGATTTCCTTCAA.<2>CGTCTAGATCA(N)
9TCTTTTCTCGAGAGAT。With having the active PfuDNA polymeric enzymatic amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 50ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 51 ℃ 30 seconds, 72 ℃ 20 seconds, 20 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 2.5M NaAc, pH5.2 and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and the 20 exonuclease III of unit, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 10 unit Semen Phaseoli radiati Germinatus nucleases again, handles 1 hour for 37 ℃.With the PCR purification column purifying of Qiagen company, with 11 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 100ml YPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 100ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 30 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 100ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 50ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.3ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) that adds 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently, ice bath 5 minutes.The aseptic electricity of 0.2-cm that is transferred to the ice precooling swashs cup, is added to the bottom.1.5 kilovolt, 25 microfarads and 200 ohm do electricity and swash.Adding 650 μ l 1mol/L sorbyl alcohols at once swashs in the cup to electricity.Mixing is transferred to culture tube gently.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) be forwarded to raffinose/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day.Propose inoculation the day before yesterday and be used to detect the active intestinal bacteria bacterial classification of antibacterial peptide K12D31, in 37 ℃ of overnight growth.
(7) the intestinal bacteria bacterial classification K12D31 with 5 μ l overnight growth adds to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed.Being poured on length gently has on the raffinose/semi-lactosi abduction delivering flat board of yeast transformant.Leave standstill and solidified in 10 minutes.Make a call to an aperture in the place that does not have transformant with rifle head point, add 0.5 μ l50mg/ml penbritin, put super clean bench 7 minutes as over against photograph.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.
(8) insert fragment from the yeast transformant amplification.The yeast 1.5ml that takes the logarithm and grow late period, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ l TE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the Pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp, by the Shen, Shanghai technology company limited purifying and full-automatic order-checking is arranged.Sequencing primer is＜5〉TAATACGACTCACTATAGGG;＜6〉TAGGATCAGCGGGTTTAAACTC.1 antibiotic 68 peptide that records, its dna sequence dna and aminoacid sequence are seen sequence table.This clone's stochastic sequence is lost a base and is caused reading frame to change when primer is synthetic.
Lithium Acetate/dithiothreitol (DTT) pretreatment fluid prescription used in the foregoing description is (every 100ml): 0.1M Lithium Acetate (final concentration), 10mM Tris (final concentration), pH7.5,1mM EDTA (final concentration) adds the 10ml 0.1M dithiothreitol (DTT) of filtration sterilization to the above-mentioned solution of 90ml (final concentration is 10mM) behind the autoclaving.
Glucose/sorbyl alcohol selection plate culture medium prescription used in the foregoing description is (every a liter): 100ml10X yeast salt (containing trace element), 1M sorbyl alcohol, 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Add VITAMIN and in the time of 50-60 ℃, add penicillin, fall dull and stereotyped with bacteria growing inhibiting.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
The used glucose that does not contain sorbyl alcohol selects dull and stereotyped prescription to be (every liter) in the foregoing description: 100ml10X yeast salt (containing trace element), 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Add VITAMIN and in the time of 50-60 ℃, add penicillin, fall dull and stereotyped with bacteria growing inhibiting.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
The dull and stereotyped prescription of raffinose/semi-lactosi abduction delivering used in the foregoing description is (every liter): 100ml10X yeast salt (containing trace element), 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.The final concentration that adds filtration sterilization is 1% raffinose and 1% semi-lactosi.Add VITAMIN, fall dull and stereotyped.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
Raffinose/semi-lactosi abduction delivering liquid culture based formulas used in the foregoing description is (every a liter):: 100ml 10X yeast salt (containing trace element), 30mg leucine, 20mg Histidine, 30mg tryptophane.Autoclaving.Add 1% raffinose of filtration sterilization and 1% semi-lactosi.Add VITAMIN.10X yeast salt (containing trace element), vitamin formula are seen " yeast genetics and molecular biology guide ", Enzymology method, 1991,194 volumes.
LB prescription used in the foregoing description is (500ml): 5g peptone, 2.5g yeast extract, 5g sodium-chlor, distilled water, pH7.0.
Antibacterial peptide sequence table .seq
＜110〉Liu Qiuyun, impressively
＜120〉a kind of method and isolating antibacterial peptide that separates antibacterial peptide
<160>2
<210>1
<211>207
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(204)
<400>1
ACC?AAC?AAT?GAT?CTA?GAG?GGC?CCT?TCG?AAG?GTA?AGC?CTA?TCC?CTA?ACC???48
Thr?Asn?Ash?Asp?Leu?Glu?Gly?Pro?Ser?Lys?Val?Ser?Leu?Ser?Leu?Thr
1???????????????5???????????????????10??????????????????15
CTC?TCC?TCG?GTC?TCG?ATT?CTA?CGC?GTA?CCG?GTC?ATC?ATC?ACC?ATC?ACC???96
Leu?Ser?Ser?Val?Ser?Ile?Leu?Arg?Val?Pro?Val?Ile?Ile?Thr?Ile?Thr
20??????????????????25??????????????????30
ATT?GAG?TTT?AAA?CCC?GCT?GAT?CCT?AGA?GGG?CCG?CAT?CAT?GTA?ATT?AGT???144
Ile?Glu?Phe?Lys?Pro?Ala?Asp?Pro?Arg?Gly?Pro?His?His?Val?Ile?Ser
35??????????????????40??????????????????45
TAT?GTC?ACG?CTT?ACA?TTC?ACG?CCC?TCC?CCC?CAC?ATC?CGC?TCT?AAC?CGA???192
Tyr?Val?Thr?Leu?Thr?Phe?Thr?Pro?Ser?Pro?His?Ile?Arg?Ser?Asn?Arg
50??????????????????55??????????????????60
AAA?GGA?AGG?AGT?TAG?207
Lys?Gly?Arg?Ser?***
65
<210>2
<211>68
<212>PRT
＜213〉artificial sequence
<400>2
Thr?Asn?Asn?Asp?Leu?Glu?Gly?Pro?Ser?Lys?Val?Ser?Leu?Ser?Leu?Thr
1???????????????5???????????????????10??????????????????15
Leu?Ser?Ser?Val?Ser?Ile?Leu?Arg?Val?Pro?Val?Ile?Ile?Thr?Ile?Thr
20??????????????????25??????????????????30
Ile?Glu?Phe?Lys?Pro?Ala?Asp?Pro?Arg?Gly?Pro?His?His?Val?Ile?Ser
35??????????????????40??????????????????45
Tyr?Val?Thr?Leu?Thr?Phe?Thr?Pro?Ser?Pro?His?Ile?Arg?Ser?Asn?Arg
50??????????????????55??????????????????60
Lys?Gly?Arg?Ser
65
Claims (5)
1, a kind of method of separating antibacterial peptide is characterized in that utilizing high-fidelity PCR to make up the yeast α mating factor leading peptide and the fusion dna fragment of peptide at random, is used for the secreting, expressing of peptide at random; Insert the yeast saccharomyces cerevisiae carrier that contains inducible promoter with EcoR I and Xba I double digestion; Recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, it is carried out digestion process with exonuclease III and Semen Phaseoli radiati Germinatus nuclease; Electricity swashs transformed saccharomyces cerevisiae behind the purifying; Transformant is forwarded to raffinose/semi-lactosi abduction delivering flat board, covers the observation inhibition zone after the overnight growth with the upper strata substratum that contains intestinal bacteria bacterial classification K12D31; Or with surveying bacteriostasis rate behind the liquid nutrient medium abduction delivering; Positive colony is carried out pcr amplification, order-checking.
2, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is AAGACCCAC, aminoacid sequence is Lys Thr His.
3, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is CCAACGCCT, aminoacid sequence is Pro Thr Pro.
4, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is a tripeptides, its dna sequence dna is GCGTCTAAA, aminoacid sequence is Ala Ser Lys.
5, the isolating antibacterial peptide of the described method of claim 1, but it is characterized in that amalgamation and expression is 68 peptides, and its dna sequence dna is
ACCAACAATGATCTAGAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTC
TCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGT
TTAAACCCGCTGATCCTAGAGGGCCGCATCATGTAATTAGTTATGTCACGCT
TACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGT, aminoacid sequence are Thr Asn Asn Asp Leu Glu Gly Pro Ser Lys Val Ser Leu Ser Leu Thr LeuSer Ser Val Ser Ile Leu Arg Val Pro Val Ile Ile Thr Ile Thr Ile Glu Phe Lys Pro Ala AspPro Arg Gly Pro His His Val Ile Ser Tyr Val Thr Leu Thr Phe Thr Pro Ser Pro His IleArg Ser Asn Arg Lys Gly Arg Ser.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100051993A CN1294258C (en) | 2004-01-16 | 2004-02-10 | Method for separating antibiotic peptide and separated antibiotic peptide |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200410015163.3 | 2004-01-16 | ||
| CN200410015163 | 2004-01-16 | ||
| CNB2004100051993A CN1294258C (en) | 2004-01-16 | 2004-02-10 | Method for separating antibiotic peptide and separated antibiotic peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1557960A true CN1557960A (en) | 2004-12-29 |
| CN1294258C CN1294258C (en) | 2007-01-10 |
Family
ID=34378870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100051993A Expired - Fee Related CN1294258C (en) | 2004-01-16 | 2004-02-10 | Method for separating antibiotic peptide and separated antibiotic peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1294258C (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345865C (en) * | 2005-09-16 | 2007-10-31 | 中山大学 | Antibiotic dipeptide and its chemical modifier, its preparation method and uses |
| CN102382173A (en) * | 2011-11-12 | 2012-03-21 | 山东大学 | Oligopeptide C and antibacterial application thereof |
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for expressing antibacterial peptide G13 by using saccharomyces cerevisiae |
| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093998B (en) * | 2010-12-09 | 2012-11-07 | 山东大学 | Preparation method of antibacterial peptide cecropin feed additive |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07203975A (en) * | 1994-01-23 | 1995-08-08 | Showa Denko Kk | Gene encoding antimicrobial peptide and production thereof |
| JP3459314B2 (en) * | 1994-08-31 | 2003-10-20 | 社団法人農林水産技術情報協会 | New peptide, antimicrobial agent, new peptide gene, new recombinant DNA and novel peptide production method |
| CN1269837C (en) * | 2002-09-02 | 2006-08-16 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
-
2004
- 2004-02-10 CN CNB2004100051993A patent/CN1294258C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100345865C (en) * | 2005-09-16 | 2007-10-31 | 中山大学 | Antibiotic dipeptide and its chemical modifier, its preparation method and uses |
| CN102382173A (en) * | 2011-11-12 | 2012-03-21 | 山东大学 | Oligopeptide C and antibacterial application thereof |
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for expressing antibacterial peptide G13 by using saccharomyces cerevisiae |
| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
| CN103360467B (en) * | 2013-07-16 | 2015-06-03 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1294258C (en) | 2007-01-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1037844C (en) | Tnf-muteins | |
| CN104497119A (en) | Natural antibacterial peptide and application thereof | |
| RU2003126290A (en) | FAMILY BACTERIA ENTEROBACTERIACEAE-PRODUCER OF L-HISTIDINE AND METHOD FOR PRODUCING L-HISTIDINE | |
| CN114015676B (en) | Construction method of cellulase adapting to traditional Chinese medicine feed additive | |
| CN101985605A (en) | Saprolegnia antagonist and application thereof | |
| CN1294258C (en) | Method for separating antibiotic peptide and separated antibiotic peptide | |
| CN100351270C (en) | Antibacterial peptide of brown-spotted torrential frog, its gene and application in drug preparation | |
| CN1177055C (en) | Musca domestica antimicrobial peptide gene and its cloning method | |
| CN1657627A (en) | Fusarium graminearum resistance carbendazim detection gene and its detection method | |
| CN1763175A (en) | Deletion of glycerol channel protein gene reduces glycerol production and improves ethanol production Saccharomyces cerevisiae strain and construction method | |
| CN104381010B (en) | Cultivation method of fruiting body of Cordyceps sinensis and its composition and application | |
| CN1306031C (en) | Novel natural antibacterial peptide Hippocampusin and its preparation and application | |
| CN1262559C (en) | Housefly phylaxin gene, and its cloning method and recombinant application | |
| CN113832117A (en) | An enzyme for degrading oxytetracycline and its encoding gene and application | |
| CN1763081A (en) | A kind of antibacterial dipeptide and its chemical modification, preparation method and application | |
| CN101818191B (en) | Vibrio harveyi specific molecular genetic marker and application thereof | |
| CN1696279A (en) | Method for preparing epsilon-polylysine and salts thereof by using northern sporotrichum PL 6-3 | |
| CN101164431A (en) | Feed additive with antibiotic function and application | |
| CN1238377C (en) | Acidic antibacterial peptides and gene as well as its application in pharmacy | |
| CN1157477C (en) | Escherichia coli containing chinook salmon growth factor | |
| CN100351271C (en) | Active polypeptide of brown-spotted torrential frog, its gene and application in drug preparation | |
| CN100350045C (en) | Corn tyrosin protein phosphatase gene and its coding protein and use | |
| CN1632120A (en) | Hepcidin Antimicrobial Peptide Gene of Mariculture Sea Bass | |
| CN110003318B (en) | Sebastes pomiferus antibacterial peptide moronecidin and application thereof | |
| CN1760203A (en) | Antibacterial tripeptides and modified chemicals, and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070110 Termination date: 20120210 |