CN107417774A - 一种伊维菌素偶联的Bt杀虫毒素及其应用 - Google Patents
一种伊维菌素偶联的Bt杀虫毒素及其应用 Download PDFInfo
- Publication number
- CN107417774A CN107417774A CN201710859935.9A CN201710859935A CN107417774A CN 107417774 A CN107417774 A CN 107417774A CN 201710859935 A CN201710859935 A CN 201710859935A CN 107417774 A CN107417774 A CN 107417774A
- Authority
- CN
- China
- Prior art keywords
- ivermectin
- cry2ab
- insecticidal proteins
- succinoyl
- ivermectins
- 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
- 108700012359 toxins Proteins 0.000 title claims abstract description 36
- 239000003053 toxin Substances 0.000 title claims abstract description 34
- 231100000765 toxin Toxicity 0.000 title claims abstract description 34
- AZSNMRSAGSSBNP-UHFFFAOYSA-N 22,23-dihydroavermectin B1a Natural products C1CC(C)C(C(C)CC)OC21OC(CC=C(C)C(OC1OC(C)C(OC3OC(C)C(O)C(OC)C3)C(OC)C1)C(C)C=CC=C1C3(C(C(=O)O4)C=C(C)C(O)C3OC1)O)CC4C2 AZSNMRSAGSSBNP-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 229960002418 ivermectin Drugs 0.000 title claims abstract description 33
- SPBDXSGPUHCETR-JFUDTMANSA-N 8883yp2r6d Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O[C@@H]([C@@H](C)CC4)C(C)C)O3)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1C[C@H](C)[C@@H]([C@@H](C)CC)O[C@@]21O[C@H](C\C=C(C)\[C@@H](O[C@@H]1O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C1)[C@@H](C)\C=C\C=C/1[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\1)O)C[C@H]4C2 SPBDXSGPUHCETR-JFUDTMANSA-N 0.000 title claims abstract description 32
- 230000000361 pesticidal effect Effects 0.000 title claims abstract description 28
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 14
- 230000008878 coupling Effects 0.000 title claims abstract description 11
- 238000010168 coupling process Methods 0.000 title claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 35
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 34
- 239000007853 buffer solution Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000004913 activation Effects 0.000 claims abstract description 6
- 239000000417 fungicide Substances 0.000 claims abstract description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 5
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 239000002904 solvent Substances 0.000 claims abstract description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 101100275683 Bacillus thuringiensis subsp. kurstaki cry2Ab gene Proteins 0.000 claims description 7
- 150000002460 imidazoles Chemical class 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 229940014800 succinic anhydride Drugs 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 5
- 229940086542 triethylamine Drugs 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 231100000614 poison Toxicity 0.000 claims description 3
- 239000002574 poison Substances 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 150000003851 azoles Chemical class 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 claims 1
- 229940092714 benzenesulfonic acid Drugs 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 238000000855 fermentation Methods 0.000 claims 1
- 230000004151 fermentation Effects 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- 241000500437 Plutella xylostella Species 0.000 abstract description 11
- 241000607479 Yersinia pestis Species 0.000 abstract description 5
- 230000001018 virulence Effects 0.000 abstract description 3
- -1 succinoyl Chemical group 0.000 abstract 3
- 241000238631 Hexapoda Species 0.000 description 9
- 239000003905 agrochemical Substances 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 4
- 241000500441 Plutellidae Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 241000305071 Enterobacterales Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 239000005875 Acetamiprid Substances 0.000 description 1
- 241000724266 Broad bean mottle virus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Dentistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供一种伊维菌素偶联的Bt杀虫毒素，其制备方法如下：称取4”‑O‑succinoyl伊维菌素、EDC和NHS，且溶于DMSO溶剂内，得活化后的4”‑O‑succinoyl伊维菌素，待用；取Bt Cry2Ab杀虫蛋白配置含5mmol/L BtCry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即Bt Cry2Ab杀虫蛋白溶液，待用；分别量取配制好的活化后的4”‑O‑succinoyl伊维菌素与Bt Cry2Ab杀虫蛋白溶液进行偶联反应，获得Bt杀虫毒素。本发明Bt杀虫毒素具有较强的毒力，其对小菜蛾具有更好的杀虫效果，能够作为农业杀虫剂，即对防治农业害虫具有更好的应用前景。
Description
【技术领域】
本发明属于农业害虫防治领域，具体涉及一种伊维菌素偶联的Bt杀虫毒素及其应用。
【背景技术】
近些年，农业科学的快速发展，农药防治起一定程度的促进作用。农药可以有效的防治农业病、虫、草、鼠等有害生物，保证了农业增产丰收这一重要环节。但病害抗药性的产生，是无论化学农药还是生物农药都不能避免的难题；害虫对农药抗药性就是克服农药位点作用，使之失效、钝化。在实际应用中，农药轮换施用、农药混配施用、增加农药对害虫的接触面积、培育害虫的敏感品系、增加生态多样性以增加害虫的多样性等等，实质上是使农药作用位点保持足够的敏感性，寻求克服抗性的手段。生物偶联是利用生物偶联技术将两种毒素偶联形成新的毒素，对保持农药位点的敏感性等具有重要的意义。
苏云金杆菌(Bacilus thuringiensis，简称Bt)是目前世界上生产量最大、应用最为广泛的一类微生物杀虫剂，其晶体蛋白对570余种鳞翅目害虫及双翅目、膜翅目和鞘翅目等数十种害虫有杀虫毒力。一般认为，昆虫在取食晶体蛋白后，通过自身的中肠蛋白酶将其酶解为活性的毒素蛋白，毒素蛋白再和中肠上皮细胞刷状缘膜(BBMVs)上的受体结合，并进一步插入膜内，形成孔洞或离子通道，引起离子渗漏，破坏肠壁上皮细胞，使肠道内溶物渗入血腔，引起败血症，致使昆虫死亡；但昆虫的抗药性使得现有的Bt毒素的防效急剧下降，因此，对Bt毒素进行改造，提供一种具有较强毒力的Bt毒素是从业人员所迫切希望地。
【发明内容】
本发明所要解决的技术问题在于提供一种伊维菌素偶联的Bt杀虫毒素及其应用。
本发明是通过以下技术方案解决上述技术问题的：一种伊维菌素偶联的Bt杀虫毒素，其制备方法的具体操作步骤如下：按1:1.5:1.5的摩尔比分别称取4”-O-succinoyl伊维菌素、EDC和NHS，且溶于DMSO溶剂内，以对4”-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”-O-succinoyl伊维菌素，待用；取Bt Cry2Ab杀虫蛋白配置含5mmol/L Bt Cry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即Bt Cry2Ab杀虫蛋白溶液，待用；分别量取配制好的活化后的4”-O-succinoyl伊维菌素与所述Bt Cry2Ab杀虫蛋白溶液，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
进一步地，所述Bt Cry2Ab杀虫蛋白的制备步骤如下：
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将其接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，并先采用Ni50缓冲液除去杂蛋白，后采用Ni500缓冲液洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到BtCry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
进一步地，所述Ni50缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑50mM；Ni500缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑500mM。
进一步地，所述4”-O-succinoyl伊维菌素制备过程为：按摩尔比1:3:6称取伊维菌素、叔丁基二甲基氯硅烷和咪唑，并溶解于四氢呋喃，于室温下搅拌反应2小时，从而对伊维菌素的5-OH进行保护；取保护后的伊维菌素溶解于二氯甲烷中，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且保护后的伊维菌素、4-二甲氨基吡啶、三乙胺、琥珀酸酐的摩尔比为1:4:8:16，之后避光水浴回流3小时，以对伊维菌素4”-OH进行羧基化改造，接着用乙醚萃取产物，柱层析分离产物；然后取层析分离后的伊维菌素溶解于甲醇中，并加入对甲苯磺酸，且层析分离后的伊维菌素与对甲苯磺酸的摩尔比为1:6，室温搅拌反应30分钟进行脱保护，再采用乙酸乙酯萃取产物，最后薄层色谱分离即得4”-O-succinoyl伊维菌素。
本发明还揭露了所述Bt杀虫毒素作为农业杀虫剂的应用。
本发明的有益效果在于：提供一种伊维菌素偶联的Bt杀虫毒素，该Bt杀虫毒素具有较强的毒力，其对小菜蛾具有更好的杀虫效果，能够作为农业杀虫剂，即对防治农业害虫具有更好的应用前景；且同时揭露了该Bt杀虫毒素的制备方法，该制备方法过程简单便捷、可操作性强。
【具体实施方式】
为了更好地理解本发明，下面结合实施例和应用例进一步地阐述说明本发明的内容，但这些实施例与应用例仅是示例性的，并不用于限制本发明的范围。且需要说明的是，本发明中所涉及的培养基在无特殊说明的情况下均为现有常规性培养基。
实施例1
4”-O-succinoyl伊维菌素的制备
按摩尔比1:3:6称取伊维菌素、叔丁基二甲基氯硅烷和咪唑，并溶解于四氢呋喃，于室温下搅拌反应2小时，从而对伊维菌素的5-OH进行保护；取保护后的伊维菌素溶解于二氯甲烷中，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且保护后的伊维菌素、4-二甲氨基吡啶、三乙胺、琥珀酸酐的摩尔比为1:4:8:16，之后避光水浴回流3小时，以对伊维菌素4”-OH进行羧基化改造，接着用乙醚萃取产物，柱层析分离产物；然后取层析分离后的伊维菌素溶解于甲醇中，并加入对甲苯磺酸，且层析分离后的伊维菌素与对甲苯磺酸的摩尔比为1:6，室温搅拌反应30分钟进行脱保护(即脱掉羟基保护)，再采用乙酸乙酯萃取产物，最后薄层色谱分离即得4”-O-succinoyl伊维菌素。
通过MS和NMR技术鉴定4”-O-succinoyl伊维菌素的结构：
1H NMR(400MHz,CDCl3)：δ5.88(d，J＝8.7Hz,1H)，5.80-5.69(m，2H)，5.39(dd，J＝18.3，9.9Hz，3H)，5.31(s，1H)，5.15(d，J＝13.2Hz，1H)，5.00(d，J＝9.9Hz，1H)，4.79(s，1H)，4.71(d，J＝10.2Hz，3H)，4.30(s，1H)，4.15(s，1H)，3.98(d，J＝6.7Hz，3H)，3.92-3.82(m，3H)，3.68(d，J＝12.7Hz，5H)，3.66-3.61(m，2H)，3.49(s，1H)，3.41(d，J＝26.5Hz，7H)，3.26(dd，J＝19.9，11.1Hz，4H)，2.74-2.62(m，5H)，2.54(s，2H)，2.39-2.20(m，7H)，2.09-1.96(m，6H)，1.89(s，5H)，1.78(d，J＝11.6Hz，3H)，1.67(d，J＝12.7Hz，5H)，1.63-1.47(m，15H)，1.27(d，J＝4.7Hz，9H)，1.17(dd，J＝10.7，6.7Hz，8H)，1.04-0.71(m，14H)；
ESI-MS(m/z):997.39[M+Na]+。
从而确定了该4”-O-succinoyl伊维菌素的化学结构，其化学结构如下：
实施例2
苏云金芽胞杆菌的杀虫蛋白即Bt Cry2Ab杀虫蛋白的制备
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将该含有cry2Ab基因的大肠杆菌工程菌接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，先采用Ni50缓冲液(NaCl 300mM、NaH2PO4 50mM、咪唑50mM)除去杂蛋白；后采用Ni500缓冲液(NaCl 300mM、NaH2PO4 50mM、咪唑500mM)洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到Bt Cry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
实施例3
Bt杀虫毒素的制备
取实施例1制备所得的4”-O-succinoyl伊维菌素(0.5mmol)，用1mL DMSO溶解，并加入EDC(0.75mmol)和NHS(0.75mmol)，室温搅拌2h，对4”-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”-O-succinoyl伊维菌素，待用；取实施例2制备所得的Bt Cry2Ab杀虫蛋白配置含5mmol/L Bt Cry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即Bt Cry2Ab杀虫蛋白溶液，待用；之后在4℃条件下，将100μL活化后的4”-O-succinoyl伊维菌素加入所述BtCry2Ab杀虫蛋白溶液1mL中，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
应用例1
Bt杀虫毒素的杀虫活性分析
(1)小菜蛾的准备
采集室内种群的小菜蛾蛹，羽化，收集成虫产卵，每24小时收集一次小菜蛾卵，收集的同批的小菜蛾卵在人工气候箱内以同样条件饲养以备LC50测定，该人工气候箱的条件为：温度25℃、相对湿度70％、光周期16：8(L：D)。
(2)验证方法与结果
采用24孔平板饲喂法测定Bt Cry2Ab杀虫蛋白、啶虫脒与实施例3制备所得的Bt杀虫毒素对小菜蛾二龄幼虫的死亡率，同时设置实验组和对照组。具体实验方法为，实验组：将Bt杀虫毒素进行稀释，得到不同浓度的Bt杀虫毒素溶液；接着在无菌环境下，吸取1mL的饲料分装于24孔板中并自然晾干，之后分别吸取100μL配置好的不同浓度的Bt杀虫毒素溶液并均匀的涂布于饲料表面，以100μL Na2CO3/NaHCO3缓冲液作为空白对照，各组做好标记、自然状态下晾干；二龄小菜蛾接种到24孔板(每孔5-7只)，每个浓度梯度4孔重复，48小时测定后小菜蛾的死亡率(虫的死亡以用毛笔轻触小菜蛾不动为准)，得出LC50。在对照组，与实验组的唯一区别在于以Bt Cry2Ab杀虫蛋白作为效应物。试验结果见下表1。
表1 Bt杀虫毒素的杀虫效果
经由表1并利用SPSS软件的回归模型进行分析，得出：对照组即Bt Cry2Ab杀虫蛋白对二龄小菜蛾的LC50为0.922μg/cm2，实验组即本发明Bt杀虫毒素对二龄小菜蛾的LC50为0.652μg/cm2，杀虫毒力大概提高了1.41倍这个结果表明本发明Bt杀虫毒素确实能够提高对小菜蛾的杀虫毒力。
综上所述，本发明通过偶联而形成的Bt杀虫毒素具有较强的毒力，本发明Bt杀虫毒素对小菜蛾具有更好的杀虫效果，换而言之，其能够作为农业杀虫剂用于杀害小菜蛾，对防治农业害虫具有更好的应用前景。
Claims (5)
1.一种伊维菌素偶联的Bt杀虫毒素，其特征在于：其制备方法的具体操作步骤如下：按1:1.5:1.5的摩尔比分别称取4”-O-succinoyl伊维菌素、EDC和NHS，且溶于DMSO溶剂内，以对4”-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”-O-succinoyl伊维菌素，待用；取Bt Cry2Ab杀虫蛋白配置含5mmol/L Bt Cry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即BtCry2Ab杀虫蛋白溶液，待用；分别量取配制好的活化后的4”-O-succinoyl伊维菌素与所述Bt Cry2Ab杀虫蛋白溶液，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
2.根据权利要求1所述的一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述BtCry2Ab杀虫蛋白的制备步骤如下：
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将其接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，并先采用Ni50缓冲液除去杂蛋白，后采用Ni500缓冲液洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到Bt Cry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
3.根据权利要求2所述的一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述Ni50缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑50mM；Ni500缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑500mM。
4.根据权利要求1所述的一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述4”-O-succinoyl伊维菌素制备过程为：按摩尔比1:3:6称取伊维菌素、叔丁基二甲基氯硅烷和咪唑，并溶解于四氢呋喃，于室温下搅拌反应2小时，从而对伊维菌素的5-OH进行保护；取保护后的伊维菌素溶解于二氯甲烷中，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且保护后的伊维菌素、4-二甲氨基吡啶、三乙胺、琥珀酸酐的摩尔比为1:4:8:16，之后避光水浴回流3小时，以对伊维菌素4”-OH进行羧基化改造，接着用乙醚萃取产物，柱层析分离产物；然后取层析分离后的伊维菌素溶解于甲醇中，并加入对甲苯磺酸，且层析分离后的伊维菌素与对甲苯磺酸的摩尔比为1:6，室温搅拌反应30分钟进行脱保护，再采用乙酸乙酯萃取产物，最后薄层色谱分离即得4”-O-succinoyl伊维菌素。
5.一种权利要求1所述伊维菌素偶联的Bt杀虫毒素的应用，其特征在于：所述Bt杀虫毒素作为农业杀虫剂的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710859935.9A CN107417774B (zh) | 2017-09-21 | 2017-09-21 | 一种伊维菌素偶联的Bt杀虫毒素及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710859935.9A CN107417774B (zh) | 2017-09-21 | 2017-09-21 | 一种伊维菌素偶联的Bt杀虫毒素及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107417774A true CN107417774A (zh) | 2017-12-01 |
| CN107417774B CN107417774B (zh) | 2021-04-09 |
Family
ID=60433360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710859935.9A Active CN107417774B (zh) | 2017-09-21 | 2017-09-21 | 一种伊维菌素偶联的Bt杀虫毒素及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107417774B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817008A (zh) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | 新型琥珀酰基十六元大环内酯的制备方法及用途 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007595A2 (en) * | 2004-07-01 | 2006-01-19 | E.I. Dupont De Nemours And Company | Synergistic mixtures of anthranilamide invertebrate pest control agents |
| CN101029067A (zh) * | 2006-03-01 | 2007-09-05 | 中国农业科学院兰州畜牧与兽药研究所 | 伊维菌素人工抗原的合成新方法 |
| CN102675411A (zh) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | 一种生物毒素的制备方法 |
| CN103228670A (zh) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | 用于昆虫控制的cry1i蛋白和基因 |
-
2017
- 2017-09-21 CN CN201710859935.9A patent/CN107417774B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007595A2 (en) * | 2004-07-01 | 2006-01-19 | E.I. Dupont De Nemours And Company | Synergistic mixtures of anthranilamide invertebrate pest control agents |
| CN101029067A (zh) * | 2006-03-01 | 2007-09-05 | 中国农业科学院兰州畜牧与兽药研究所 | 伊维菌素人工抗原的合成新方法 |
| CN103228670A (zh) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | 用于昆虫控制的cry1i蛋白和基因 |
| CN102675411A (zh) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | 一种生物毒素的制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| PAN Z等: "Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.", 《 WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817008A (zh) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | 新型琥珀酰基十六元大环内酯的制备方法及用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107417774B (zh) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singer | Insecticidal activity of recent bacterial isolates and their toxins against mosquito larvae | |
| US10028507B2 (en) | Sixteen-membered macrolide compound and applications thereof | |
| BR112019019694A2 (pt) | usos de uma yersinia entomophaga, de um filtrado de cultura de yersinia entomophaga isento de células, de uma toxina de yersinia entomophaga isolada, método, e, composição | |
| CN108330071B (zh) | 具昆虫肠道感染作用的球孢白僵菌菌株及其害虫防治应用 | |
| CN113142244B (zh) | 球孢白僵菌sb063和乙基多杀菌素对普通大蓟马的协同防治 | |
| Varo et al. | Development and validation of an inoculation method to assess the efficacy of biological treatments against Verticillium wilt in olive trees | |
| MARQUES et al. | Antimicrobial and insecticidal activities of cashew tree gum exudate | |
| Ratnaweera et al. | Antifeedant, contact toxicity and oviposition deterrent effects of phyllostine acetate and phyllostine isolated from the endophytic fungus Diaporthe miriciae against Plutella xylostella larvae | |
| CN113142245A (zh) | 球孢白僵菌sb038和乙基多杀菌素对普通大蓟马的协同防治 | |
| KR20180033774A (ko) | 곤충병원성 곰팡이 보베리아 바시아나 anu1 및 이를 이용한 파밤나방, 배추좀나방, 담배나방 또는 좁은가슴잎벌레 방제용 미생물제제 | |
| Ramyabharathi et al. | Potential of a rhizobacterium Bacillus subtilis (Bbv 57) on Fusarium oxysporum f. sp. gerberae and Meloidogyne incognita infecting Gerbera grown in protected cultivation | |
| Sharma et al. | Effect of Alternaria alternata on the coccinellid pest Henosepilachna vigintioctopunctata and its implications for biological pest management | |
| CN107058120A (zh) | 对黄瓜根结线虫具强致病力的淡紫紫孢菌及其应用 | |
| CN107417774A (zh) | 一种伊维菌素偶联的Bt杀虫毒素及其应用 | |
| Otsu et al. | Stable phylloplane colonization by entomopathogenic bacterium Pseudomonas fluorescens KPM-018P and biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) | |
| KR101773339B1 (ko) | 이사리아 푸모소로세아 Pf212 균주 또는 이를 이용한 진딧물, 잘록병균 및 고추 탄저병균의 동시방제용 조성물 | |
| CN107602678A (zh) | 一种啶虫脒偶联的Bt杀虫毒素及其制备方法与应用 | |
| CN107446030A (zh) | 一种伊维菌素偶联的Bt杀虫毒素及其应用 | |
| Sagar et al. | Evaluation of prodigiosin pigment for antimicrobial and insecticidal activities on selected bacterial pathogens & household pests | |
| CN107417775A (zh) | 一种阿维菌素偶联的Bt杀虫毒素及其应用 | |
| CN108967445A (zh) | 一种含多杀霉素和苏云金杆菌的可分散油悬浮剂及其制备方法 | |
| CN107474121A (zh) | 一种阿维菌素偶联的Bt杀虫毒素及其应用 | |
| CN108179123B (zh) | 一株白眉野草螟罹病虫体来源的粘质沙雷氏菌及其应用 | |
| Arain et al. | Toxicity of butene-fipronil, in comparison with seven other insecticides, in Leptinotarsa decemlineata and Drosophila melanogaster | |
| CN109699683A (zh) | 一种滑石基质爪哇虫草孢子制剂 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231214 Address after: Comprehensive Experimental Building (Crop Institute), No. 104 Pudang Road, Xindian Town, Jin'an District, Fuzhou City, Fujian Province, 350013 Patentee after: Crop Research Institute of Fujian Academy of Agricultural Sciences (Fujian Provincial Germplasm Resources Center) Address before: 350000 No. 54, 247, Fuzhou, Fujian Patentee before: AGRICULTURAL BIORESOURCES INSTITUTE OF FUJIAN ACADEMY OF AGRICULTURAL SCIENCES |