CN110331152B - 粉棒束孢Cyanovirin-N基因、重组蛋白及应用 - Google Patents
粉棒束孢Cyanovirin-N基因、重组蛋白及应用 Download PDFInfo
- Publication number
- CN110331152B CN110331152B CN201910624269.XA CN201910624269A CN110331152B CN 110331152 B CN110331152 B CN 110331152B CN 201910624269 A CN201910624269 A CN 201910624269A CN 110331152 B CN110331152 B CN 110331152B
- Authority
- CN
- China
- Prior art keywords
- gene
- ifcvn
- recombinant
- cvn
- protein
- 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.)
- Active
Links
- 108060002021 cyanovirin N Proteins 0.000 title claims abstract description 31
- 241000030456 Isaria farinosa Species 0.000 title abstract description 53
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title abstract description 19
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 230000000443 biocontrol Effects 0.000 claims abstract description 6
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 14
- 241000255896 Galleria mellonella Species 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 241000588696 Pantoea ananatis Species 0.000 claims description 6
- 241000607479 Yersinia pestis Species 0.000 claims description 6
- 241000556430 Erwinia persicina Species 0.000 claims description 5
- 241001144907 Pseudomonas trivialis Species 0.000 claims description 4
- 241001478271 Rahnella aquatilis Species 0.000 claims description 4
- 241001622809 Serratia plymuthica Species 0.000 claims description 4
- 241000588914 Enterobacter Species 0.000 claims description 3
- 241001083841 Aquatica Species 0.000 claims 1
- 241000588698 Erwinia Species 0.000 claims 1
- 241001435397 Florella Species 0.000 claims 1
- 241000422392 Laurencia Species 0.000 claims 1
- 241000589516 Pseudomonas Species 0.000 claims 1
- 241000607720 Serratia Species 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 110
- 102000004169 proteins and genes Human genes 0.000 abstract description 38
- 241001248590 Isaria Species 0.000 abstract description 10
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
- 230000000749 insecticidal effect Effects 0.000 abstract description 4
- 230000000144 pharmacologic effect Effects 0.000 abstract description 3
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 229960005486 vaccine Drugs 0.000 abstract description 3
- 235000018102 proteins Nutrition 0.000 description 32
- 239000013612 plasmid Substances 0.000 description 31
- 230000014509 gene expression Effects 0.000 description 23
- 238000012795 verification Methods 0.000 description 20
- 238000001514 detection method Methods 0.000 description 19
- 239000007788 liquid Substances 0.000 description 18
- 239000012634 fragment Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 238000000034 method Methods 0.000 description 13
- 238000011160 research Methods 0.000 description 12
- 239000000725 suspension Substances 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 10
- 238000010276 construction Methods 0.000 description 10
- 238000001976 enzyme digestion Methods 0.000 description 10
- 238000003209 gene knockout Methods 0.000 description 10
- 239000013600 plasmid vector Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 238000012258 culturing Methods 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 210000003000 inclusion body Anatomy 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 241000233866 Fungi Species 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 230000028070 sporulation Effects 0.000 description 7
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 5
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 101150103518 bar gene Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000009465 prokaryotic expression Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000232969 Sterigmatomyces pulcherrimus Species 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound 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 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 210000002468 fat body Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 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 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- CBCKQZAAMUWICA-UHFFFAOYSA-N 1,4-phenylenediamine Chemical compound NC1=CC=C(N)C=C1 CBCKQZAAMUWICA-UHFFFAOYSA-N 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- 239000005561 Glufosinate Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
- 229960001668 cefuroxime Drugs 0.000 description 3
- JFPVXVDWJQMJEE-IZRZKJBUSA-N cefuroxime Chemical compound N([C@@H]1C(N2C(=C(COC(N)=O)CS[C@@H]21)C(O)=O)=O)C(=O)\C(=N/OC)C1=CC=CO1 JFPVXVDWJQMJEE-IZRZKJBUSA-N 0.000 description 3
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 210000002615 epidermis Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 3
- 229960001225 rifampicin Drugs 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- QENJLXATANVWMR-UHFFFAOYSA-N 2-[(3-amino-3-imino-2-methylpropanethioyl)amino]acetic acid Chemical compound NC(=N)C(C)C(=S)NCC(O)=O QENJLXATANVWMR-UHFFFAOYSA-N 0.000 description 2
- 241000960320 Agriophyllum squarrosum Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 2
- 241000192700 Cyanobacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000012681 biocontrol agent Substances 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000036046 immunoreaction Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000013081 phylogenetic analysis Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- AVRPFRMDMNDIDH-UHFFFAOYSA-N 1h-quinazolin-2-one Chemical compound C1=CC=CC2=NC(O)=NC=C21 AVRPFRMDMNDIDH-UHFFFAOYSA-N 0.000 description 1
- IDQJPXPEIIEMQI-UHFFFAOYSA-N 2-[4-[bis(carboxymethyl)amino]-n-(carboxymethyl)anilino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C1=CC=C(N(CC(O)=O)CC(O)=O)C=C1 IDQJPXPEIIEMQI-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229940124718 AIDS vaccine Drugs 0.000 description 1
- BFMIRJBURUXDRG-DLOVCJGASA-N Ala-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 BFMIRJBURUXDRG-DLOVCJGASA-N 0.000 description 1
- 235000006667 Aleurites moluccana Nutrition 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- NVGWESORMHFISY-SRVKXCTJSA-N Asn-Asn-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NVGWESORMHFISY-SRVKXCTJSA-N 0.000 description 1
- JRVABKHPWDRUJF-UBHSHLNASA-N Asn-Asn-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N JRVABKHPWDRUJF-UBHSHLNASA-N 0.000 description 1
- ZVUMKOMKQCANOM-AVGNSLFASA-N Asn-Phe-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVUMKOMKQCANOM-AVGNSLFASA-N 0.000 description 1
- UTLCRGFJFSZWAW-OLHMAJIHSA-N Asp-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O UTLCRGFJFSZWAW-OLHMAJIHSA-N 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241001491790 Bupalus piniaria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 240000004957 Castanea mollissima Species 0.000 description 1
- 235000018244 Castanea mollissima Nutrition 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241000190633 Cordyceps Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000254171 Curculionidae Species 0.000 description 1
- 241001635274 Cydia pomonella Species 0.000 description 1
- BSFFNUBDVYTDMV-WHFBIAKZSA-N Cys-Gly-Asn Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O BSFFNUBDVYTDMV-WHFBIAKZSA-N 0.000 description 1
- UOEYKPDDHSFMLI-DCAQKATOSA-N Cys-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CS)N UOEYKPDDHSFMLI-DCAQKATOSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 235000005903 Dioscorea Nutrition 0.000 description 1
- 244000281702 Dioscorea villosa Species 0.000 description 1
- 235000000504 Dioscorea villosa Nutrition 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- MFHVAWMMKZBSRQ-ACZMJKKPSA-N Gln-Ser-Cys Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N MFHVAWMMKZBSRQ-ACZMJKKPSA-N 0.000 description 1
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 1
- HQRHFUYMGCHHJS-LURJTMIESA-N Gly-Gly-Arg Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N HQRHFUYMGCHHJS-LURJTMIESA-N 0.000 description 1
- OLPPXYMMIARYAL-QMMMGPOBSA-N Gly-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)CN OLPPXYMMIARYAL-QMMMGPOBSA-N 0.000 description 1
- IVSWQHKONQIOHA-YUMQZZPRSA-N Gly-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN IVSWQHKONQIOHA-YUMQZZPRSA-N 0.000 description 1
- MHXKHKWHPNETGG-QWRGUYRKSA-N Gly-Lys-Leu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O MHXKHKWHPNETGG-QWRGUYRKSA-N 0.000 description 1
- BAYQNCWLXIDLHX-ONGXEEELSA-N Gly-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN BAYQNCWLXIDLHX-ONGXEEELSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241000256257 Heliothis Species 0.000 description 1
- 241000330899 Hepialus Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-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
- HXWALXSAVBLTPK-NUTKFTJISA-N Leu-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(C)C)N HXWALXSAVBLTPK-NUTKFTJISA-N 0.000 description 1
- DBVWMYGBVFCRBE-CIUDSAMLSA-N Leu-Asn-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O DBVWMYGBVFCRBE-CIUDSAMLSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- QZONCCHVHCOBSK-YUMQZZPRSA-N Lys-Gly-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O QZONCCHVHCOBSK-YUMQZZPRSA-N 0.000 description 1
- VKCPHIOZDWUFSW-ONGXEEELSA-N Lys-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN VKCPHIOZDWUFSW-ONGXEEELSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001442207 Monochamus alternatus Species 0.000 description 1
- 240000005303 Nostoc ellipsosporum Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000743698 Pinicola Species 0.000 description 1
- 241001250596 Pleione Species 0.000 description 1
- VEUACYMXJKXALX-IHRRRGAJSA-N Pro-Tyr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VEUACYMXJKXALX-IHRRRGAJSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000321184 Raoultella Species 0.000 description 1
- 241000588756 Raoultella terrigena Species 0.000 description 1
- 241000586497 Runella Species 0.000 description 1
- WTUJZHKANPDPIN-CIUDSAMLSA-N Ser-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N WTUJZHKANPDPIN-CIUDSAMLSA-N 0.000 description 1
- KNCJWSPMTFFJII-ZLUOBGJFSA-N Ser-Cys-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KNCJWSPMTFFJII-ZLUOBGJFSA-N 0.000 description 1
- BRGQQXQKPUCUJQ-KBIXCLLPSA-N Ser-Glu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BRGQQXQKPUCUJQ-KBIXCLLPSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- AXKJPUBALUNJEO-UBHSHLNASA-N Ser-Trp-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(O)=O AXKJPUBALUNJEO-UBHSHLNASA-N 0.000 description 1
- UKKROEYWYIHWBD-ZKWXMUAHSA-N Ser-Val-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UKKROEYWYIHWBD-ZKWXMUAHSA-N 0.000 description 1
- 241000563489 Sesamia inferens Species 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 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 description 1
- QARCDOCCDOLJSF-HJPIBITLSA-N Tyr-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QARCDOCCDOLJSF-HJPIBITLSA-N 0.000 description 1
- UUBKSZNKJUJQEJ-JRQIVUDYSA-N Tyr-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O UUBKSZNKJUJQEJ-JRQIVUDYSA-N 0.000 description 1
- DNOOLPROHJWCSQ-RCWTZXSCSA-N Val-Arg-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DNOOLPROHJWCSQ-RCWTZXSCSA-N 0.000 description 1
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 description 1
- MIAZWUMFUURQNP-YDHLFZDLSA-N Val-Tyr-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N MIAZWUMFUURQNP-YDHLFZDLSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- -1 anthraquinone compounds Chemical class 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- XIURVHNZVLADCM-IUODEOHRSA-N cefalotin Chemical compound N([C@H]1[C@@H]2N(C1=O)C(=C(CS2)COC(=O)C)C(O)=O)C(=O)CC1=CC=CS1 XIURVHNZVLADCM-IUODEOHRSA-N 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- UCKZMPLVLCKKMO-LHLIQPBNSA-N cephamycin Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](C)[C@]21OC UCKZMPLVLCKKMO-LHLIQPBNSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 235000004879 dioscorea Nutrition 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000000967 entomopathogenic effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 108010076756 leucyl-alanyl-phenylalanine Proteins 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- LVWZTYCIRDMTEY-UHFFFAOYSA-N metamizole Chemical compound O=C1C(N(CS(O)(=O)=O)C)=C(C)N(C)N1C1=CC=CC=C1 LVWZTYCIRDMTEY-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000424 optical density measurement Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- 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
- A61P31/04—Antibacterial agents
-
- 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
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Polymers & Plastics (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Crystallography & Structural Chemistry (AREA)
Abstract
本发明属于分子生物学领域,具体涉及粉棒束孢Cyanovirin‑N基因、基因和重组蛋白的制备方法、重组蛋白及应用。本发明从一株具有较强杀虫活力的粉棒束孢FTRSY‑2菌株出发,首次从粉棒束孢基因组中克隆出粉棒束孢CVN基因,命名为Ifcvn,以该基因为出发点,通过分子生物学的手段构建重组载体、工程菌、重组CVN蛋白等,并通过构建Ifcvn基因敲除与回复株验证该基因的功能。本发明得到的重组CVN蛋白性质稳定,产量和纯度较高,并具有广谱杀菌效力和药理活性,有潜力开发成为生防制剂、抗菌药品、多肽疫苗、保健产品等。
Description
技术领域
本发明属于分子生物学领域,具体涉及粉棒束孢Cyanovirin-N基因、基因和重组蛋白的制备方法、重组蛋白及应用。
背景技术
粉棒束孢(Isaria farinosa),是丝状真菌棒束孢属(Isaria Fries)模式种,是一种主要的昆虫病原真菌,同时也是一种极具开发价值和商业利用价值的真菌,具体表现在:1)现有研究表明,粉棒束孢对多种类型的害虫具有良好的杀虫效果;2)粉棒束孢作为蝙蝠蛾幼虫的定殖真菌,其药用活性也越来越多地被研究和发现;3)已从粉棒束孢培养菌丝体中分离得到包括多糖、虫草酸、生物碱、吡啶酮、天然苯腙、喹唑啉酮、环状五肽、蒽醌类化合物等多种新化合物;4)研究证明粉棒束孢菌的代谢产物中具有类似生长素和细胞分裂素的物质等。然而目前针对粉棒束孢的研究主流还停留在宏观层面上,主要以粉棒束孢的代谢产物或孢子悬液或菌丝体作为研究对象,其微观分子层面的研究才刚刚起步,且现有的利用分子生物学手段对粉棒束孢进行的深入和系统研究,主要都集中在真菌分类及作为生防菌的角度进行。
申请号为201610476131.6的发明专利公开了一株粉棒束孢的发酵培养物在制备预防林业害虫的生防制剂中的应用,该发明主要是利用了菌株的发酵培养液。杨俊媛等学者从粉棒束孢培养菌丝体中分离得到多种有价值的化合物,但其研究依然停留在宏观层面。因此,有必要在全面、系统研究活性代谢产物的基础上深入开展粉棒束孢药理活性与基因功能之间的关系,以便构建出能够产生特定活性成分的超级生产菌株。
鉴于此,本发明从一株具有较强杀虫活力的粉棒束孢菌株FTRSY-2出发,在筛选分析基因信息时,发现了一个与蓝藻抗病毒蛋白-N(Cyanovirin-N)同源的基因,并首次从粉棒束孢基因组中克隆出粉棒束孢CVN基因,命名为Ifcvn。本发明以该基因为出发点,通过分子生物学的手段构建重组载体、工程菌、重组CVN蛋白等,并通过构建Ifcvn基因敲除与回复株验证该基因的功能,为进一步研究粉棒束孢功能基因及其潜在的药用价值奠定了基础,具有重要的研究意义和潜在的商业价值。
发明内容
有鉴于此,本发明的目的之一,在于提供粉棒束孢的Cyanovirin-N基因。
粉棒束孢的Cyanovirin-N基因,所述的Cyanovirin-N基因的核苷酸序列包括SEQID NO.1所示和/或其中的片段。该基因由本发明的课题组首次从粉棒束孢基因组中克隆得到,命名为Ifcvn。
本发明的目的之二,在于提供粉棒束孢的Cyanovirin-N基因的制备方法。
上述Cyanovirin-N基因的制备方法,根据粉棒束孢全基因组序列预测得到SEQ IDNO.1所示序列,根据所述SEQ ID NO.1序列设计若干引物对;提取粉棒束孢总RNA,反转cDNA,进行PCR扩增,获得如SEQ ID NO.1所示的Cyanovirin-N基因。Cyanovirin-N基因的cDNA ORF全长为369bp,编码122个氨基酸,分子量约为12.9kDa,与现有报道的天然成熟的CVN氨基酸残基数目及相对分子质量有一定差异。
进一步,所述引物对包括如序列SEQ ID NO.2和SEQ ID NO.3所示的一组引物对,所述序列SEQ ID NO.2的引物命名为Ifcvn F;所述序列SEQ ID NO.3的引物命名为IfcvnR。
本发明的目的之三,在于提供一种重组质粒载体。
重组质粒载体,所述重组质粒载体包含权利要求1所述的SEQ ID NO.1序列和原核表达质粒;所述SEQ ID NO.1序列与原核表达质粒连接。
作为一种优选,所述原核表达质粒采用SUMO-pET28a质粒。
本发明的目的之四,在于提供一种基因工程菌。
重组质粒载体转化得到的产重组Cyanovirin-N蛋白的基因工程菌。
进一步,所述重组Cyanovirin-N蛋白在所述基因工程菌的包涵体和培养上清液中表达。本发明的基因工程菌用大肠杆菌BL21(DE3)制备。
本发明的目的之五,在于提供一种重组CVN蛋白。
粉棒束孢的重组Cyanovirin-N蛋白,所述重组Cyanovirin-N蛋白的氨基酸序列包括SEQ ID NO.4所示和/或其中的片段。
粉棒束孢的重组Cyanovirin-N蛋白,所述重组Cyanovirin-N蛋白由上述的SEQ IDNO.1核苷酸序列翻译得到。
天然的CVN蛋白最初是从蓝藻椭孢念株藻(Nostoc elliposporum)中分离得到的一种水溶性糖蛋白。CVN具有较广谱的抗病毒活性,同时还具有稳定的理化特性,能抵抗变性剂、去污剂和有机溶剂的处理。CVN的基因工程研究在CVN被发现后就开始了,但椭孢念株藻养殖困难,从中分离纯化CVN不仅成本高、工艺复杂,而且原料浪费严重。因此本发明首次从粉棒束孢基因组中克隆出粉棒束孢CVN基因,并借助分子生物学手段得到稳定高产的重组CVN蛋白,具有突破性的意义。
本发明的目的之六,在于提供一种多克隆抗体。
一种多克隆抗体,所述多克隆抗体包括上述的重组Cyanovirin-N蛋白。
本发明的目的之七,在于提供上述重组CVN蛋白的一系列应用。
上述重组Cyanovirin-N蛋白在制备用于防治林业害虫的生防制剂中的应用。所述应用还可以由上述SEQ ID NO.1的核苷酸序列和上述重组质粒载体得到。
进一步,所述林业害虫包括油松毛虫、松梢螟、苹果蠹蛾、美国白蛾、落叶松卷蛾、黄刺蛾幼虫、板栗象甲、松墨天牛、柑桔粉蚧中的一种或多种。传统的生防制剂主要是以菌株或菌株代谢产物为基础制备,而以本发明的重组CVN蛋白为基础来制备,效力更强,可控性更高。
上述重组Cyanovirin-N蛋白在制备广谱抗菌药物中的应用。CVN蛋白具有广谱的抗病毒活性和稳定的生化特性,是一种新型的抗菌活性物。所述应用还可以由上述SEQ IDNO.1的核苷酸序列和上述重组质粒载体得到。
进一步,所述细菌包括水生拉恩氏菌(Rahnella aquatilis)、E.persicina、普城沙雷菌(Serratia plymuthica)、平凡假单胞菌(Pseudomonas trivialis)、菠萝泛菌(Pantoea ananatis)、土生柔特勒菌(Raoultella terrigena)、河生肠杆菌(Lelliottiaamnigena)中的一种或多种。
进一步,所述重组Cyanovirin-N蛋白可以为任意药学可接受剂型,所述广谱抗菌药物还包括药学上可接受的载体和/或助剂。
上述重组Cyanovirin-N蛋白在制备多克隆抗体中的应用。所述应用还可以由上述SEQ ID NO.1的核苷酸序列和上述重组质粒载体得到。
上述重组Cyanovirin-N蛋白在制备抗艾滋病疫苗中的应用。所述应用还可以由上述SEQ ID NO.1的核苷酸序列和上述重组质粒载体得到。天然的CVN蛋白从椭孢念株藻(Nostocelliposporum)的培养基提取物中分离得到,前期临床研究证实其能够不可逆地阻止HIV进入宿主细胞,因此有望成为第1种多肽类的天然抗HIV药物,但目前对其他藻类及真菌是否能分离出CVN少见报道。本发明成功构建出一种稳定性较强、产量和纯度较高的重组CVN蛋白,为后续进一步制备抗艾滋病疫苗提供了高纯度、高效、廉价的核心原材料。
上述重组Cyanovirin-N蛋白在制备提高人体免疫功能的保健品中的应用。所述应用还可以由上述SEQ ID NO.1的核苷酸序列和上述重组质粒载体得到。通过对CVN蛋白的系统进化分析发现粉棒束孢与冬虫夏草菌进化关系最近,进化树聚为一枝,表明在虫生真菌尤其是虫草真菌中具有同一起源,而冬虫夏草被公认为具有调节免疫、抗疲劳等多种功效。因此本发明的重组Cyanovirin-N蛋白及相应菌株具备了与冬虫夏草相当的开发成保健品的潜力。
上述重组Cyanovirin-N蛋白在增强虫草抗逆性能中的应用。所述应用还可以由上述SEQ ID NO.1的核苷酸序列和上述重组质粒载体得到。
本发明的有益效果在于:
1)本发明从一株具有较强杀虫活力的粉棒束孢FTRSY-2菌株出发,首次从粉棒束孢基因组中克隆出粉棒束孢CVN基因,命名为Ifcvn。以该基因为出发点,通过分子生物学的手段构建重组载体、工程菌、重组CVN蛋白等,并通过构建Ifcvn基因敲除与回复株验证该基因的功能。从而为粉棒束孢功能基因的研究及其对寄主、环境互作分子机制的阐明、药用价值的深入研究奠定理论基础,同时为构建产生特定功能的活性成分的超级菌株奠定了基础。
2)本发明得到的重组CVN蛋白性质稳定,产量和纯度较高,并具有广谱杀菌效力和药理活性,有潜力开发成为生防制剂、抗菌药品、多肽疫苗、保健产品。
附图说明
图1:Ifcvn基因的DNA序列及所对应的氨基酸序列(框中为信号肽)。
图2:重组CVN蛋白二级结构预测。
图3:重组CVN蛋白三维结构图。
图4:粉棒束孢重组CVN与其他真菌CVN的氨基酸序列相似性分析(*标注为活性位点)。
图5:粉棒束孢重组CVN蛋白与其他真菌CVN蛋白的系统进化树。
图6:粉棒束孢Ifcvn基因扩增。
图7:Ifcvn基因同源敲除及回复载体构建过程图。
图8:Ifcvn基因同源敲除载体图谱(左)及PCR验证(右)。
图9:Ifcvn基因回复载体图谱(左)及PCR验证(右)。
图10:Ifcvn基因同源敲除转化子bar基因PCR验证(M:DL2000marker;1-12:Ifcvn基因敲除转化子;P:pCambiaMX9-IfcvnUBD;W:FTRSY-2)。
图11:Ifcvn基因同源敲除、回复转化子PCR验证(左)及回复转化子荧光观察(右)(M:DL2000marker;1-3:Ifcvn基因敲除转化子;4-6:Ifcvn基因回复转化子)。
图12:Ifcvn基因同源敲除、回复转化子菌落形态及逆境胁迫下的生长情况(WT:野生菌株;△Ifcvn:Ifcvn基因敲除突变株;RC:回复突变株;1-7:PDA正常培养基;刚果红;NaCl;山梨醇;pH11;UV;35℃)。
图13:Ifcvn基因在感病大蜡螟不同时间及不同组织中的表达量。
图14:Ifcvn基因扩增、表达载体酶切验证及重组蛋白表达(A、B:M:DL2000marker;1:Ifcvn扩增产物;2:BamH I/Sal I酶切验证;C:1:0.4mg/mL BSA;2:上清;3marker;4:SUMO-pET28a-SUMO空载诱导表达;5:上清2(2M尿素溶解包涵体);6:包涵体2倍稀释(2M尿素溶解包涵体);7:包涵体2倍稀释(2M尿素溶解包涵体))。
图15:CVN重组蛋白特异性检测及感病大蜡螟血液中Ifcvn基因表达量测定(A:多克隆抗体特异性及灵敏度检测;B:感病大蜡螟血液中CVN蛋白表达量检测;(M:marker;1-2:FTRSY-2野生株总蛋白;3:CK;4-8:0h、12h、24h、48h、72h感病大蜡螟血液总蛋白;9:阴性对照)。
图16:不同浓度重组CVN的WST测试及(左)对sf9细胞影响的增殖的影响(右)(A:cell+CCK8;B:cell+SUMO-CVN+CCK8;C:SUMO-CVN+CCK8;a-h:SUMO-CVN使用浓度梯度)。
具体实施方式
以下将参照附图,对本发明的优选实施例进行详细描述。按照常规条件,所举实施例是为了更好地对本发明的内容进行说明,但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
本发明的实验材料
1.实验材料
1.1供试菌株及昆虫
粉棒束孢FTRSY-2菌株;大肠杆菌BL21(DE3);大蜡螟幼虫
1.2质粒载体
pCambiaMX9质粒(GenBank accession number:KX755248,具kana抗性);
pGapneoR12质粒(GenBank accessionnumber:KY363244,具G418抗性)。
实施例1
1.基因组DNA提取
基因组DNA提取采用天根植物基因组提取试剂盒,按说明书步骤提取。
2.基因组RNA提取
基因组RNA提取采用天根植物基因组提取试剂盒,按说明书步骤提取。
3.微量快速PCR验证
采取微量菌丝PCR扩增:无菌牙签挑取少量粉棒束孢菌丝至EP管中,液氮速冻研磨后,加入50μL Lysis Buffer,混合均匀,95℃,10min;取菌丝裂解液2μL进行常规PCR扩增。
4.生物信息学分析
运用常规的生物信息分析软件进行。
5.生物信息学分析结果
Ifcvn cDNA ORF全长为369bp,编码122个氨基酸(图1),分子量约为12.9kDa,等电点为5.99,平均疏水性为-0.236,且稳定性比较好(instability index(II))为27.64。信号肽预测结果表明该序列具有信号肽,其切割位点在N端第26和27个氨基酸之间。二硫键预测表明该序列2个二硫键分别位于第8位和第22位及第53位和第79位的半胱氨酸残基上;二级结构预测如图2所示,表明该蛋白序列由α螺旋、β折叠、β转角及无规则卷曲构成;三级结构预测结果如图3所示,其主要由β折叠片形成的链状蛋白,折叠以后的构象呈现假二重对称;多序列比对结果显示粉棒束孢CVN氨基酸序列和稻曲霉、巴西青霉、层生镰刀菌、底栖蓝藻、亚麻色单歧藻、霍氏伪枝蓝细菌的CVN氨基酸序列相似度不高,序列较特异(图4),保守性最突出的区域为Cyanovirin-N的活性中心(亮氨酸Leu L、异亮氨酸Ile I、天冬氨酸Asp D、甘氨酸Gly G),这些保守结构域和氨基酸残基对CVN功能起着非常重要的作用;对CVN蛋白系统进化分析发现,粉棒束孢与冬虫夏草菌(Cordyceps confragosa)聚为一枝(图5)。
实施例2
基因cDNA ORF全长克隆:
查找粉棒束孢全基因组序列得到预测的Ifcvn基因全长序列,根据该序列设计引物Ifcvn F/R。提取粉棒束孢总RNA,反转cDNA,进行PCR扩增,获得Ifcvn基因cDNA ORF全长。取5μLPCR扩增产物进行电泳验证。
实验结果:
扩增得到全长为369bp大小的片段,PCR扩增结果如图6所示。
实施例3
1.Ifcvn基因敲除盒的构建
(1)Ifcvn基因上、下游及草丁膦抗性基因片段克隆
以野生型菌株FTRSY-2基因组为模板,使用引物Ifcvnqcup F和Ifcvnqcup R扩增Ifcvn基因上游序列,产物命名为IfcvnU;使用引物Ifcvnqcdown F和Ifcvnqcdown R扩增Ifcvn基因下游序列,产物命名为IfcvnD;以pCambaregfp质粒为模板,使用引物Bar5/6扩增草丁膦抗性基因,产物命名为B。电泳检测后切胶回收备用。
(2)质粒pCambiaMX9-IfcvnU构建
将提取的质粒pCambiaMX9和回收的Ifcvn基因上游回收片段IfcvnU分别进行SacI和Hind III双酶切,电泳检测后切胶回收,将回收的线性化质粒pCambiaMX9和Ifcvn基因上游片段IfcvnU进行连接,并将连接产物转化大肠杆菌DH5α,用引物MX9F/R对进行菌液PCR验证;提取PCR检测结果为阳性菌落的质粒,用Sac I和BamH I对质粒进行双酶切验证。
(3)质粒pCambiaMX9-IfcvnUD构建
将提取的质粒pCambiaMX9-IfcvnU和回收的Ifcvn基因下游回收片段IfcvnD分别进行HindIII和BamH I双酶切,电泳检测后切胶回收,将回收的线性化质粒pCambiaMX9-IfcvnU和Ifcvn基因下游片段IfcvnD进行连接,并转化大肠杆菌DH5α,用引物MX9F/R对进行菌液PCR验证;提取PCR检测结果为阳性菌落的质粒,用Sac I和BamH I对质粒进行双酶切验证。
(4)质粒pCambiaMX9-IfcvnUBD构建
将提取的质粒pCambiaMX9-IfcvnUD和回收的草丁膦抗性基因片段B分别进行HindIII酶切。电泳检测后切胶回收,将回收的线性化质粒pCambiaMX9-IfcvnUD和草丁膦抗性基因片段B连接,将连接产物转化大肠杆菌DH5α,用引物MX9F/R对进行菌液PCR验证;提取PCR检测结果为阳性的质粒,用Sac I和BamH I对质粒进行双酶切验证。
实验结果:
敲除载体构建及验证过程如图7所示,成功扩增获得Ifcvn基因左右臂侧翼序列和Bar基因元件,测序正确后按顺序依次连接后形成Ifcvn基因敲除盒,成功连接到敲除骨架载体pCambiaMX9上,图谱及验证结果见图8。
2.Ifcvn基因回补盒的构建
采用同源重组方法构建基因回补盒。
(1)Ifcvn基因回补片段扩增回收
以野生型菌株FTRSY-2基因组为模板,用引物HB 1/2扩增Ifcvn及其假定的启动子序列,产物命名为IfcvnH。PCR反应电泳检测后,切胶回收。
(2)回补质粒pGapneocvn构建
将提取的质粒pGapneoR12进行BamH I酶切线性化。电泳检测后切胶回收,将回收的线性化质粒pGapneoR12和Ifcvn基因回补片段IfcvnH进行同源重组连接,并产物转化大肠杆菌DH5α,用引物R12F/R对转化子进行菌液PCR验证;PCR检测结果为阳性的菌落,提取质粒,用BamH I对质粒进行酶切验证。
实验结果:
回复载体构建及验证过程如图9所示。
实施例4
1.粉棒束孢Ifcvn基因敲除及回复突变株筛选
(1)转化子筛选用G418浓度的确定
确定突变株筛选时的抗生素浓度,分别测试野生菌株FTRSY-2菌株对草丁膦的敏感度、对头孢霉素的敏感度及头孢霉素对农杆菌LBA4404的毒性,从而确定筛选转化子的草丁膦和头孢霉素的最适使用浓度。由于回复采用的pGapneoR12质粒带有遗传霉素(G418)抗性标签,因此需要对野生株进行G418敏感度测定,确定筛选转化子所需G418的最适浓度。
(2)农杆菌介导转化FTRSY-2菌株
(a)将已转入载体的农杆菌在加有利福平(100μg/mL)、链霉素(100μg/mL)、卡那霉素(100μg/mL)的YEB固体平板上活化,28℃,黑暗培养2-3d;
(b)挑取单菌落到加有利福平(100μg/mL)、链霉素(100μg/mL)、卡那霉素(100μg/mL)的1mL YEB液体的1.5mL离心管中,28℃,200rpm培养过夜;
(c)第二天待菌液浑浊后,吸取100uL菌液至10mL加有利福平(100μg/mL)、链霉素(100μg/mL)、卡那霉素(100μg/mL)的YEB液体培养基中,28℃,200rpm避光培养至OD600约为0.15。
(d)取5mL的菌液于10mL的离心管中,6000rpm离心5min,弃上清。再往离心管中加入IM液体,调整菌液OD600为0.15;
(e)用Tween-20制备粉棒束孢孢子悬液,并稀释至1×106分生孢子/mL;
(f)取400μL农杆菌菌液、400μL孢子悬液混匀,加入200μL AIM液体培养基,200rpm28℃黑暗诱导培养2d;
(g)吸取100μL农杆菌与孢子悬浮液混合液,涂布于铺有灭菌玻璃纸的AIM固体平板上,28℃倒置黑暗培养3-4d;
(h)将玻璃纸正向转移到含有草丁膦与头孢霉素的CZM平板培养基上,28℃倒置黑暗培养。
(3)粉棒束孢突变株的筛选及验证
当CZM平板的玻璃纸上长出粉棒束孢菌落时,用牙签挑取,并接种到PPDA平板培养基上,28℃培养;待菌落长大后,挑取少量菌丝,压片法在荧光显微镜下观察;选取带有绿色荧光的转化子,单孢分离并多次传代培养,确保EGFP是稳定遗传的荧光标记;随机挑选10株突变株接种到无筛选药物的PDA平板上,重复转接10代后,再接种PPDA平板上,观察是否能够正常生长,以确定抗性基因能否稳定遗传。随机选取15个突变株,提取基因组DNA,采用基于草丁膦抗性基因设计的特异性引物Bar F/R对突变株进行检测;随机挑取转化子在荧光显微镜下观察是否有绿色荧光。
(4)表型突变的转化子筛选及抗逆性测定
以野生FTRSY-2菌株为对照,根据菌落形态及颜色、孢子产量等特征,从突变体库中筛选表型差异较大的突变株。将表型差异较大的突变株,接种于含有筛选药物的PPDA培养基上,28℃条件下培养7d,测定其菌落直径和产孢量,计算日平均生长速率及单位面积产孢量,每个处理3次重复,以野生菌株为对照,确定由于T-DNA的插入对突变株生长和产孢的影响。
将FTRSY-2的转化子培养7d后,刮取菌丝孢子,用终浓度为0.05%吐温-20的ddH20制作孢子悬浮液,使其浓度为1×105分生孢子/mL,分别取1μL点接于pH11及分别含有刚果红(500μg/mg)、NaCl(1mol/L)、山梨醇(1mol/L)的PDA制成的十二孔板上,设立空白对照,28℃培养7d,观察其生长状态。设置紫外线照射1h处理及亚致死温度35℃培养7d,以观察转化子对紫外线及亚致死温度的敏感性。
实验结果:
利用bar基因引物bar F/R微量快速PCR验证初筛敲除转化子,以pCambiaMX9为阳性对照,野生菌株为阴性对照,结果表明野生菌株无扩增片段,而阳性质粒及随机挑选的阳性同源敲除突变子能够扩增出500bp左右的bar基因片段,证明bar基因已经成功插入,为进一步验证敲除转化子的准确性,同时采用原目的基因引物IfcvnF/R进行PCR扩增,结果表明野生菌株能扩到约360bp左右的目的基因片段,而突变株由于目的基因破坏的缘故,没有扩增出条带,因此表明Ifcvn基因已经成功被敲除(图10)。采用原目的基因引物Ifcvn F/R进行PCR扩增,结果表明野生菌株及回复菌株均能扩到约360bp左右的目的基因片段,表明Ifcvn基因已经重新整合到基因组上,由于回复载体带有EGFP基因盒,可使用倒置荧光显微镜于400-500nm波长激发光下进行观察,突变株菌丝及孢子形态如图11所示。
Ifcvn基因对粉棒束孢生长和产孢的影响:结果如表1所示,粉棒束孢野生型、Ifcvn基因同源敲除及回复转化子的生长速率差异显著,其中敲除株生长较慢,但三者之间产孢量无差异;逆境胁迫实验结果如图12所示,与野生对照相比,敲除株对刚果红及碱性环境表现敏感,菌丝生长速率减慢,对NaCl、山梨醇等高渗条件胁迫不敏感,能够正常生长并产孢,回复株与野生株表现一致,而紫外线照射1h和35℃处理时野生株和敲除株及回复株的生长速度均受到抑制。
表1转化子的生长速率和产孢量
2.Ifcvn基因敲除株毒力测定
(1)体表侵染
以0.02%吐温-80为溶剂,使用新鲜活化的粉棒束孢孢子配制浓度为1×107分生孢子/mL的WT、敲除菌株分生孢子悬液。实验对象为4龄大蜡螟幼虫,接种方式为体表浸渍,每个处理3次重复,每个重复20头,浸于事先准备好的分生孢子悬浮液25S后取出,滤纸吸干体表水分并放入150mm培养皿中,置于28℃恒温培养箱保湿培养,逐日观察统计幼虫死亡数,及时清理病死幼虫,避免二次感染,将死亡个体挑出保湿培养,连续观察7d。
(2)体内注射
以PBS为溶剂,使用新鲜活化的粉棒束孢孢子配制浓度为1×107分生孢子/mL的WT、敲除菌株分生孢子悬液。实验对象为4龄大蜡螟幼虫,接种方式为血腔注射,使用微量注射器从幼虫第三腹节气孔处注射孢悬液至幼虫血腔,接种量为10μL孢悬液/头虫(对照组为10μL PBS),每处理30头虫,每12小时观察取样。直至幼虫全部死亡(或停止死亡)时停止取样。
实验结果:
结果如表2所示。在采用浸渍法处理时,由于Ifcvn基因的敲除,导致粉棒束孢对大蜡螟侵染能力增强,其校正死亡率由24.44%增加到33.33%,LT50值由26.53d降低到12.64d;采用注射法处理时与野生株无差异。由此表明Ifcvn基因影响粉棒束孢的致病力,主要通过影响粉棒束孢对昆虫体壁吸附、穿透的过程来调控其毒力。
表2野生株与ΔIfcvn菌株处理大蜡螟幼虫的校正死亡率及致死中时间(LT50)
3.Ifcvn基因在感病大蜡螟组织中表达分析
配制浓度为1×107分生孢子/mL的WT菌株孢悬液,注射接种,28℃恒温培养箱保湿培养,接种0h、12h、24h、48h、72h之后取血淋巴、表皮、脂肪体及中肠组织,提RNA后,反转cDNA,进行RT-qPCR。
实验结果:
结果如图13所示:随侵染时间延长,Ifcvn基因在大蜡螟幼虫各个组织中的表达量不同,在侵染后12h各组织中的表达量均达到最高,其中在血液中的表达量最高,在中肠中的表达量次之,在脂肪体中表达量较少,在表皮的表达量最低;随侵染时间延长,所有组织中Ifcvn基因表达量均开始降低,其中在血液及脂肪体中的表达量迅速降低,而中肠中表达量的下降幅度较小,侵染后72h在表皮检测到少量Ifcvn基因表达,而血液、中肠及脂肪体中Ifcvn基因表达量均降到最低。
4.Ifcvn基因原核表达及多克隆抗体制备
(1)原核表达载体SUMO-pET28a-cvn构建
按步骤提取SUMO-pET28a质粒,并以引物Ifcvn F/R扩增Ifcvn基因cDNA ORF全长,命名为cvn。电泳验证后回收目的条带。
将提取的质粒SUMO-pET28a和回收的Ifcvn基因片段cvn进行BamH I/Sal I双酶切。电泳验证后回收目的条带。将回收的线性化质粒SUMO-pET28a和Ifcvn基因cDNA回收片段cvn进行连接,转化大肠杆菌DH5α,用引物T7F/R对转化子进行菌液PCR验证;阳性菌落扩繁后提取质粒,用BamH I/Sal I对质粒进行双酶切验证后,转化大肠杆菌BL21(DE3)备用。
(2)CVN蛋白诱导表达及表达形式鉴定
将含Ifcvn基因的重组大肠杆菌BL21(DE3)菌液在液体LB培养基中按1%接种量接种,37℃摇床振荡过夜培养至对数生长期,OD600值为0.5-0.6范围时,加入终浓度为1mM/L的ITPG诱导剂,再培养5-8h后离心收集菌体,ddH2O重悬后破碎,8000rpm离心10min,沉淀用5mL ddH2O重悬。取10μL上清液及沉淀重悬液进行SDS-PAGE分析,考马斯亮蓝R-250染色4h后进行脱色,鉴定表达产物的存在形式。纯化蛋白并制备相应多克隆抗体。
(3)抗原及内源CVN蛋白Western Blot检测
以制备的多克隆抗体(1:1000)为一抗、羊抗兔IgG-HRP(1:5000)为二抗,采用Western-blot方法分析多克隆抗体的特异性;分别提取粉棒束孢侵染大蜡螟不同时期各组织的总蛋白,检测感病大蜡螟组织中粉棒束孢CVN蛋白的表达量。
实验结果:
扩增获得Ifcvn基因cDNA ORF全长共369bp,PCR产物电泳鉴定大小正确(图14A),测序成功后克隆到SUMO-pET28a载体上,酶切鉴定(图14B)后诱导表达。重组大肠杆菌BL21(DE3)菌液在浓度达到OD600值为0.5-0.6范围时,加入终浓度为1mM/L的ITPG诱导剂,以空载体为对照,37℃诱导培养8h后,经SDS-PAGE分析,结果如图14C所示。由可以看出,目标蛋白大小大约为32kDa左右,在上清液中检测到少量目标蛋白的表达,但大多数均以聚集的形式存在,即包涵体,说明在大肠杆菌BL21(DE3)中CVN蛋白表达在包涵体和上清中。
Western-blot方法分析抗Ifcvn基因重组蛋白多克隆抗体的特异性,结果显示,重组蛋白能与多克隆抗体发生特异性血清学反应,在32kDa处可见一明显的免疫反应条带,其大小与预测Ifcvn基因重组蛋白分子量相近,同时抗体灵敏度高,1:1000稀释后可检测到500pg抗原(图15A)。以多克隆抗体检测粉棒束孢侵染后大蜡螟幼虫血液中的总蛋白,来表征Ifcvn基因在感病大蜡螟组织中表达量(图15B),结果显示侵染后12h和24h可检测到CVN蛋白。
实施例5
CVN重组蛋白活性检测
(1)抑菌活性检测
采用生长抑制法进行CVN重组蛋白的体外抑菌活性检测(马驰,2014)。用双蒸水作空白对照,以氨苄青霉素(Ampicillin)、链霉素(Streptomycin)、头孢霉素(Cefalothin)、硫酸卡那霉素(Kanamycin)为阳性对照,对照药剂和重组蛋白均采用1×PBS稀释,使其终浓度为100μg/mL。在37℃的条件下,将水生拉恩氏菌(Rahnella aquatilis)、E.persicina、普城沙雷菌(Serratia plymuthica)、平凡假单胞菌(Pseudomonas trivialis)、菠萝泛菌(Pantoea ananatis)、土生柔特勒菌(Raoultella terrigena)、河生肠杆菌(Lelliottiaamnigena)等从粉棒束孢生境及宿主虫尸上分离的供试菌株培养12h,再用LB培养基将其稀释至OD600值为0.001;取稀释菌液加入96孔板中,加入供试药物,每个测试菌株三次重复;37℃培养12h后测量OD600值,观察重组蛋白的抑菌活性。
实验结果:
抑菌活性实验结果见表3,与阳性对照相比,重组SUMO-CVN在100μg/mL的使用浓度下,对P.ananatis的抑制作用最为明显,抑制率为11.05%;对E.persicina的抑制率为10.41%,稍强于氨苄青霉素阳性对照的抑制作用,表明其对E.persicina的效价与氨苄青霉素相当。
表3 CVN重组蛋白对不同细菌的抑制率
(2)细胞毒性检测
采用WST-8试剂盒法测试重组CVN蛋白的细胞增值毒性检测,试验采用草地贪夜蛾细胞sf9细胞系,向96孔板各孔中加入100μL细胞悬液,28℃下预孵育培养板24h,待细胞贴壁后,加入10μL不同浓度的重组蛋白稀释液,使其终浓度依次为100μg/mL、10μg/mL、1μg/mL、100ng/mL、10ng/mL、1ng/mL、100pg/mL;28℃下孵育不同时间后,向各孔中加入10μL的CCK-8溶液,继续28℃下孵育1-4h,用酶标仪测定450nm处的OD值,分析重组蛋白的细胞毒性。
实验结果:
WST-8实验表明,在所测试的使用浓度及作用时间下,除在最高使用浓度100μg/mL下作用72h时,重组CVN蛋白影响草地贪夜蛾细胞sf9细胞正常增值外,其余各试验条件下,重组CVN蛋白对sf9细胞生长无影响,同时短时间内一定浓度的重组CVN蛋白能够促进sf9的增值,因此表明重组CVN蛋白对正常sf9细胞具有一定的保护效应(图16)。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
<110>太极集团有限公司;西南大学
<120>粉棒束孢Cyanovirin-N基因、重组蛋白及应用
<160> 4
<170> PatentIn Version 3.5
<210> 1
<211> 369
<212> DNA
<213>人工序列(Artificial sequence)
<400> 1
ATGCGCTGGA CAACAAACAC CGTCGCGGCC GCTCTGGCTT TTGCTACTCT CGGTAGCTGC 60
GTCCACTACA CAGACAGCTG CGACGACACC AACCTGTCTG GCACCACGCT GTCTGGCCAC 120
TGCGGTGACA ACAAGGGCAA CAGTCCCTAC AGCAGCGTTG ACCTGGCCCA AAAGGTTGGC 180
AACAACTGGG GTGTTTTGGC TTGGGGCGGT GTCAACTTCC AGCAGAGTTG CTCCGAGATT 240
GTATACAACT CTTGGAACGG TGTTTTGTCC GCCAAGTGCG GCAACGGCGG TGGCCGTGAT 300
GTCCGCACAG TTCTGAATCT GAACAACTAC ATTTCCAACA ACTTTGGCAA ACTAGCATTT 360
GACTCGTAG 369
<210> 2
<211>30
<212> DNA
<213>人工序列(Artificial sequence)
<400> 2
ATATGGATCC ATGCGCTGGA CAACAAACAC 30
<210> 3
<211>30
<212> DNA
<213>人工序列(Artificial sequence)
<400> 3
ATATGTCGAC CTACGAGTCA AATGCTAGTT 30
<210> 4
<211>122
<212> PRT
<213>人工序列(Artificial sequence)
<400> 4
Met Arg Trp Thr Thr Asn Thr Val Ala Ala Ala Leu Ala Phe Ala Thr
1 5 10 15
Leu Gly Ser Cys Val His Tyr Thr Asp Ser Cys Asp Asp Thr Asn Leu
20 25 30
Ser Gly Thr Thr Leu Ser Gly His Cys Gly Asp Asn Lys Gly Asn Ser
35 40 45
Pro Tyr Ser Ser Val Asp Leu Ala Gln Lys Val Gly Asn Asn Trp Gly
50 55 60
Val Leu Ala Trp Gly Gly Val Asn Phe Gln Gln Ser Cys Ser Glu Ile
65 70 75 80
Val Tyr Asn Ser Trp Asn Gly Val Leu Ser Ala Lys Cys Gly Asn Gly
85 90 95
Gly Gly Arg Asp Val Arg Thr Val Leu Asn Leu Asn Asn Tyr Ile Ser
100 105 110
Asn Asn Phe Gly Lys Leu Ala Phe Asp Ser
115 120
Claims (2)
1.Cyanovirin-N基因在制备用于防治林业害虫的生防制剂中的应用,其特征在于,所述Cyanovirin-N基因的核苷酸序列如SEQ ID NO.1所示,所述林业害虫为大蜡螟。
2.Cyanovirin-N基因在制备广谱抗细菌药物中的应用,其特征在于,所述Cyanovirin-N基因的核苷酸序列SEQ ID NO.1所示,所述细菌为水生拉恩氏菌(Rahnella aquatilis)、桃色欧文氏菌(E.persicina)、普城沙雷菌(Serratia plymuthica)、平凡假单胞菌(Pseudomonas trivialis)、菠萝泛菌(Pantoea ananatis)、土生柔特勒菌(Raoultellaterrigena)、河生肠杆菌(Lelliottia amnigena)中的一种或多种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910624269.XA CN110331152B (zh) | 2019-07-11 | 2019-07-11 | 粉棒束孢Cyanovirin-N基因、重组蛋白及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910624269.XA CN110331152B (zh) | 2019-07-11 | 2019-07-11 | 粉棒束孢Cyanovirin-N基因、重组蛋白及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110331152A CN110331152A (zh) | 2019-10-15 |
CN110331152B true CN110331152B (zh) | 2021-06-18 |
Family
ID=68146278
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910624269.XA Active CN110331152B (zh) | 2019-07-11 | 2019-07-11 | 粉棒束孢Cyanovirin-N基因、重组蛋白及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110331152B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114134046B (zh) * | 2021-11-12 | 2023-07-18 | 广东省科学院动物研究所 | 一株粉棒束孢giz_ph5-1及其应用 |
CN114032184B (zh) * | 2021-12-02 | 2023-03-24 | 济南栖圣农林科技有限公司 | 一株防治褐梗天牛的层生镰刀菌及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0836647B1 (en) * | 1995-04-27 | 2004-04-21 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by the SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES | Antiviral proteins, dna coding sequences therefor, and uses thereof |
CN101104851A (zh) * | 2006-07-14 | 2008-01-16 | 金宁一 | 抗病毒蛋白cv-n的制备 |
CN101705241A (zh) * | 2008-09-28 | 2010-05-12 | 暨南大学 | 重组蓝藻抗病毒蛋白的制备方法及应用 |
CN106010985A (zh) * | 2016-06-24 | 2016-10-12 | 南京林业大学 | 一株粉棒束孢hs05及其应用 |
-
2019
- 2019-07-11 CN CN201910624269.XA patent/CN110331152B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0836647B1 (en) * | 1995-04-27 | 2004-04-21 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by the SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES | Antiviral proteins, dna coding sequences therefor, and uses thereof |
CN101104851A (zh) * | 2006-07-14 | 2008-01-16 | 金宁一 | 抗病毒蛋白cv-n的制备 |
CN101705241A (zh) * | 2008-09-28 | 2010-05-12 | 暨南大学 | 重组蓝藻抗病毒蛋白的制备方法及应用 |
CN106010985A (zh) * | 2016-06-24 | 2016-10-12 | 南京林业大学 | 一株粉棒束孢hs05及其应用 |
Non-Patent Citations (3)
Title |
---|
Isaria fumosorosea ARSEF 2679 Cyanovirin-N (ISF_08283), partial mRNA;Shang,Y. 等;《Genbank Database》;20170828;CDS部分和ORIGIN部分 * |
米曲霉中蓝藻抗病毒蛋白-N基因的克隆与表达;谢露 等;《生物技术》;20160430;第26卷(第2期);摘要,第128页 第2.4节,第129页 图4A,右栏第1段 * |
蓝藻抗病毒蛋白-N 基因的克隆、表达、纯化及活性鉴定;陈伟 等;《生物工程学报》;20100425;第26卷(第4期);第538-544页 * |
Also Published As
Publication number | Publication date |
---|---|
CN110331152A (zh) | 2019-10-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Gawehns et al. | The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death | |
CN111748562B (zh) | 一种水稻纹枯病菌Atg 22蛋白编码基因及其靶标片段Rsatg22和应用 | |
CN110331152B (zh) | 粉棒束孢Cyanovirin-N基因、重组蛋白及应用 | |
CN105274130B (zh) | 一种利用基因操作提高球孢白僵菌分生孢子产量和毒力的方法 | |
Neshani et al. | Preparation and evaluation of a new biopesticide solution candidate for plant disease control using pexiganan gene and Pichia pastoris expression system | |
CN113862295A (zh) | 一个柑橘黄龙病菌效应子作为筛选抗柑橘黄龙病菌药物靶点的应用 | |
CN110938118B (zh) | 致病疫霉菌分泌的植物免疫激活蛋白pc2及其应用 | |
CN101755050A (zh) | 蝎毒素基因转化的杀虫真菌工程菌株及应用 | |
CN114480476B (zh) | 一个可以用于改良木薯抗病性的蛋白及编码基因的应用 | |
CN101153057B (zh) | 一种提高植物抗性促进植物生长的蛋白质及其编码基因 | |
CN108611352B (zh) | 一种拟禾本科根结线虫翻译延长因子Mg-eEF1A及其防治植物病害的应用 | |
WO2024037548A1 (zh) | 一种植物免疫激活蛋白PmSCR1及其应用 | |
CN100591767C (zh) | 源于稻瘟病菌的真菌致病性基因mnh6及其用途 | |
Urayama et al. | Suppressive effects of mycoviral proteins encoded by Magnaporthe oryzae chrysovirus 1 strain A on conidial germination of the rice blast fungus | |
CN110256570A (zh) | 一种重组融合抗菌肽及应用 | |
CN110759983A (zh) | 一种靶向沉默害虫模式识别蛋白gnbp3基因表达的重组真菌及在害虫防治中的应用 | |
CN110643613A (zh) | 一种靶向沉默小菜蛾gnbp2基因的重组细菌及在害虫防治中的应用 | |
CN110272908B (zh) | 粉棒束孢Kill Protion4基因、重组蛋白及应用 | |
CN109305996A (zh) | 禾谷镰刀菌分泌型蛋白激发子FgHrip1及其应用 | |
CN114751878A (zh) | 一种BcTol1基因靶向剂及其应用 | |
CN110272909B (zh) | 粉棒束孢Defensin基因、重组蛋白及应用 | |
CN109134629B (zh) | 灰霉菌分泌型蛋白激发子BcXyl1及其应用 | |
Jin et al. | Carbon catabolite repressor gene BbCre1 influences carbon source uptake but does not have a big impact on virulence in Beauveria bassiana | |
CN112641929B (zh) | 美洲大蠊宿主防御肽的抗病毒应用 | |
CN111138518B (zh) | 细菌转位子组分蛋白及其截短体的表达和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |