CN109134626A - Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing - Google Patents
Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing Download PDFInfo
- Publication number
- CN109134626A CN109134626A CN201811031684.6A CN201811031684A CN109134626A CN 109134626 A CN109134626 A CN 109134626A CN 201811031684 A CN201811031684 A CN 201811031684A CN 109134626 A CN109134626 A CN 109134626A
- Authority
- CN
- China
- Prior art keywords
- albumen
- super quick
- concentrated
- quick albumen
- plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 33
- 241000193764 Brevibacillus brevis Species 0.000 title claims abstract description 23
- 230000003248 secreting effect Effects 0.000 title abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 239000013612 plasmid Substances 0.000 claims abstract description 23
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 23
- 241000196324 Embryophyta Species 0.000 claims description 16
- 238000000108 ultra-filtration Methods 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 13
- 230000004151 fermentation Effects 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 239000012510 hollow fiber Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 241000588694 Erwinia amylovora Species 0.000 claims description 3
- 238000004064 recycling Methods 0.000 claims description 3
- 241000534630 Brevibacillus choshinensis Species 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000011426 transformation method Methods 0.000 claims description 2
- 241000208292 Solanaceae Species 0.000 claims 1
- 235000002566 Capsicum Nutrition 0.000 description 12
- 235000013399 edible fruits Nutrition 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 240000008574 Capsicum frutescens Species 0.000 description 8
- 239000001390 capsicum minimum Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 230000009182 swimming Effects 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 241000208293 Capsicum Species 0.000 description 4
- 240000005319 Sedum acre Species 0.000 description 4
- 235000014327 Sedum acre Nutrition 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000006002 Pepper Substances 0.000 description 3
- 241000722363 Piper Species 0.000 description 3
- 235000016761 Piper aduncum Nutrition 0.000 description 3
- 235000017804 Piper guineense Nutrition 0.000 description 3
- 235000008184 Piper nigrum Nutrition 0.000 description 3
- 230000009418 agronomic effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 235000014103 egg white Nutrition 0.000 description 3
- 210000000969 egg white Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 description 2
- 229960002504 capsaicin Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 241000555281 Brevibacillus Species 0.000 description 1
- JGEVJQBAHWCNEN-UHFFFAOYSA-N C1(=CC=CC=C1)O.S(=O)(=O)(O)O.C1=CC=CC=C1 Chemical compound C1(=CC=CC=C1)O.S(=O)(=O)(O)O.C1=CC=CC=C1 JGEVJQBAHWCNEN-UHFFFAOYSA-N 0.000 description 1
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 1
- 240000008384 Capsicum annuum var. annuum Species 0.000 description 1
- 235000002568 Capsicum frutescens Nutrition 0.000 description 1
- 101710151559 Crystal protein Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101100184605 Escherichia coli mobA gene Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101100391699 Pseudomonas viridiflava gacA gene Proteins 0.000 description 1
- 101100507443 Ralstonia solanacearum (strain GMI1000) hrcU gene Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 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 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 238000009295 crossflow filtration Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 101150039191 hrpN gene Proteins 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000000077 insect repellent Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 101150055347 repA2 gene Proteins 0.000 description 1
- 101150107738 repB gene Proteins 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 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
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of methods using the super quick albumen of Brevibacillus brevis secreting, expressing, the method includes the following steps, (1) encoding gene of super quick albumen is inserted into Brevibacillus brevis as in the plasmid of host, (2) plasmid for inserting the super quick albumen is transferred in Brevibacillus brevis host, (3) Brevibacillus brevis described in fermented and cultured, super quick albumen described in (4) separation and Extraction.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of super quick using Brevibacillus brevis secreting, expressing
The method of albumen.
Background technique
Chemical fertilizer and pesticide are to improve crop yield and prevent and treat the main of pest and disease damage to select product, but produce environment, the energy simultaneously
And the problem of cost etc., become the great of the economy and society development of puzzlement countries in the world now, seriously restricts agriculture
The sustainable development of industry.And utilize prepare from the protein of microorganism or fungi biological products (including bio-feritlizer with
Biological pesticide) be contemporary crop it is high-quality, efficiently and No-harmful apple orchard major measure.In recent years, it has opened in this regard both at home and abroad
A large amount of scientific research is opened up.The microprotein of early stage is mainly the insecticidal crystal protein from bacillus thuringiensis
(ICP), novel microbial albumen is mainly albumen exciton substance, at present representative novel micro- life of research report
Object albumen mainly has allergen protein (Harpin), hidden ground albumen (Cryptogein) and activator protein (Activator), these eggs
White matter does not kill pest and pathogen directly, does not also provide nutriment directly, but in promotion plant growth and development and to battalion
The absorption for supporting substance, enhances plant at the broad spectrum resistance (including the resistance to various bacteria, fungi and virus) for enhancing plant
Insect-repellent, extends crop product at the resistance (tolerance of the plant to the poor environments such as arid, severe cold, saline and alkaline) for enhancing plant
Storage time, have wide application prospects to the moulding of potted flower and nursery stock effect aspect.Therefore, they can be configured to biological system
Product are applied to the above.
These novel microbial albumen or polypeptide are mostly realized by bioengineered strains such as fermentation recombination bacillus colis big
Batch production, next step process after fermentation are the bacterial cell in dissolution of bacteria suspension with releasing microbe albumen or more
Peptide.Cytolytic method has non-chemical method, such as high pressure or ultrasonication, using this method the high requirements on the equipment,
Energy consumption is high and is difficult to large-scale application.Alternatively, most of manufacturer uses and contacts cell suspending liquid with lysozyme
Carry out, also need to carry out 40-42 DEG C of digestion later, centrifugation go cell fragment and albuminate and protein purification technique with
Reach the purity of needs.These complex operations, it is expensive, and since step is more, process tedious can reduce microorganism egg
White and polypeptide yield, and reduce their bioactivity and stability.
It is therefore desirable to have step is easy, the production of low-cost method for this kind of novel microbial albumen or polypeptide.
Summary of the invention
To solve the above-mentioned problems, a kind of easy, quickly super quick albumen preparation method is provided, the present invention provides as follows
Technical solution.
Use Brevibacillus brevis (Brevibacillus choshinensis) as place present invention firstly relates to a kind of
Main bacterium, the method that fermented and cultured produces destination protein, the method includes the following steps,
(1) encoding gene of the destination protein is inserted into Brevibacillus brevis as in the plasmid of host,
(2) plasmid for inserting the destination protein is transferred in Brevibacillus brevis host,
(3) Brevibacillus brevis described in fermented and cultured,
(4) destination protein described in separation and Extraction.
Destination protein described in step (1) is super quick albumen, preferably from bacterial strain Erwinia amylovora's
The harpinEa albumen that GeneBank number is M92994.3；
Plasmid described in step (1) is pNC-hisT plasmid；
It is electric shock transformation method that plasmid, which is transferred to the method in Brevibacillus brevis host, described in step (2)；
The condition of culture of fermented and cultured described in step (3) are as follows: T2 culture medium, 30~33 DEG C of temperature, revolving speed 120~
200rpm, 24~60h of fermented incubation time, it is preferred that fermentation culture conditions are: T2 culture medium, 33 DEG C of temperature, revolving speed
120rpm, fermented incubation time 48h；
The step of destination protein described in separation and Extraction described in step (4) is: the bacterium solution after collecting fermentation is collected by centrifugation
Clearly, cross-flow ultrafiltration and concentration are carried out to supernatant, obtains destination protein and liquid is concentrated by ultrafiltration.
The step of described cross-flow ultrafiltration, is:
(1) ultrafiltration is carried out to supernatant using the hollow fiber column in the aperture 30KD, collects filtered solution；
(2) recycling filtrate is concentrated using the hollow fiber column in the aperture 10KD, when being concentrated into 1/10 volume, be added etc.
The albumen buffer of volume, the formula of the albumen buffer are 20mM Tri-HCl, 400mM NaCl, 1mM EDTA, are repeated
Three times；
(3) step (2) protein concentrated solution obtained is concentrated in the hollow fiber column for continuing to use the aperture 10KD, dense
It is reduced to 1/8 volume and liquid is concentrated by ultrafiltration to get destination protein.
Detailed description of the invention
Fig. 1, the pNC-harpinEa plasmid for being transferred to secreting, expressing in Brevibacillus brevis super quick albumen (harpinEa)
Map illustrates cyclic plasmid DNA molecular with annulus, and gene expression direction is shown in fragment ends with arrow；The structure of the plasmid
Region includes: harpinEa albumen, secreting signal peptide (secretion signal peptide), gene promoter
(T2promoter), ribosome binding site (RBS), replicon (repB)；The ampicillin resistance replicated in Escherichia coli
(AmpR) and copy starting area (oriC)；The neomycin resistance gene (NmR) and replication initiation replicated in Brevibacillus brevis
Area (ori).
The pH and OD of Fig. 2, different fermentations condition595With the change curve of incubation time, pH and temperature known to left figure table
It is inversely proportional, the OD known to right figure table595It is directly proportional to temperature, the pH and OD according to two charts595With incubation time on first
Downward trend after rising.
Fig. 3, Brevibacillus brevis secreting, expressing after purification harpinEa albumen electrophoretogram, swimming lane 1 is egg in figure
White molecular weight standard, unit kilodalton (KD)；Swimming lane 2 is fermented supernatant fluid；3 fermentation supernatant of swimming lane is after step ultrafiltration concentration
Sample；Swimming lane 4,5 is respectively the sample of first time and second of filter wash；HarpinEa after swimming lane 6-10 indicates filter wash three times is super
2 times of gradient dilution samples that quick protein solution (44KD) carries out from stoste to 16 times of dilutions；Swimming lane 11-15 indicates calf serum egg
White (BSA) carries out 2 times of gradient dilution samples from 2mg/ml to 0.125mg/ml.
Capsicum plant character under Fig. 4, different disposal, left figure are that the Pepper crops Agronomic trait of test group treatment region is real
Object figure；Right figure is the Pepper crops Agronomic trait pictorial diagram of control group treatment region.Spray the capsicum of the super quick albumen of harpinEa
Crop plant is more healthy and stronger, and disease incidence is lower, and volume increase is up to 40.35%.
Specific embodiment
Embodiment 1, super quick albumen (harpinEa) retrieval
Gene hrpN sequence from bacterial strain Erwinia amylovora coding harpinEa albumen has disclosed
(GeneBank number: M92994.3).The acquisition of super quick albumen (harpinEa) gene can using gene chemical synthesis, PCR amplification,
Extract chromosomal DNA and the row technology known in the industry such as establish library.The present invention uses method for synthesizing gene, by super quick albumen
(hrapinEa) amino acid sequence is sent synthesis (Chinese Suzhou Jin Weizhi biotechnology company), in the upstream of aim sequence when synthesis
I restriction enzyme site sequence of Pst is introduced, downstream introduces III restriction enzyme site sequence of Hind.
Embodiment 2, building pNC-harpinEa plasmid
The raw skeleton of plasmid is pBU110, is pNC-hisT (being purchased from TaKaRa company) through business corporate makeover, carries
There are Pst I and III restriction enzyme site of Hind each one in body multiple cloning sites area, and entire carrier has and only one I He of Pst
III restriction enzyme site of Hind.We are by above-mentioned plasmid backbone DNA and harpinEa protein gene synthesis segment through Pst I and Hind III
After restriction enzyme enzymatic treatment, cyclic DNA is connected into using T4 ligase and enters Escherichia coli using chemical transformation conversion
In JM109 competent cell.Plasmid is extracted through cell culture and alkaline lysis, obtaining can be transferred in Brevibacillus brevis
The pNC-harpinEa plasmid of the super quick albumen of secreting, expressing, the map of the plasmid are shown in Fig. 1.The correctness of plasmid sequence have passed through survey
Sequence confirms (Chinese Suzhou Jin Weizhi biotechnology company).
Correct plasmid will be sequenced and be transferred to Brevibacillus brevis (strain title through electroporated method
Brevibacillus.choshinensis SP3 is purchased from Takara Biomedical Technology (Beijing) Co.,
Ltd.) electricity turns in competent cell, through bacterium colony PCR to confirm that positive transformant, monoclonal positive transformant are containing neomycin
2SY culture medium be incubated overnight and preservation of bacteria strain.
The secreting, expressing of embodiment 3, harpinEa albumen
The positive transformant of above-mentioned acquisition is carried out to the screening optimum condition of the expression that ferments in a small amount in T2 culture medium.Setting is such as
Lower condition is screened,
(1) two temperature condition, is 30 DEG C and 33 DEG C respectively,
(2) three conditions of shaking speed are that 120rpm, 160rpm and 200rpm carry out orthogonal experiment respectively,
(3) for 24 hours, tetra- incubation time points of 48h, 72h and 90h take 1ml culture solution, wherein 200 μ L culture solutions carry out
OD595Measurement and pH value test, 800 μ L of residue are centrifugated culture supernatant and bacillus cell carries out SDS-PAGE glue
The super quick expressing quantity (run glue result figure and do not show that partial data is shown with Fig. 2) of electrophoresis detection, comprehensive analysis data result,
Have selected wherein optimal expression condition are as follows: revolving speed 120rpm, 33 DEG C of fermentation temperature, fermentation time 48h.
With above-mentioned optimal culture conditions cultivation and fermentation positive transformant, with 10000xg, 4 DEG C of pelleted by centrifugation after fermentation
10min separates cell and supernatant.
The separation of embodiment 4, harpinEa albumen
The culture supernatant of acquisition is separated in culture above-mentioned steps through cross-flow ultrafiltration technology (TFF) by super quick Protein Separation
It shifts to and is saved in buffer simultaneously.Main band is super quick albumen and the cell wall protein that falls off in supernatant, bright almost without other
Aobvious foreign protein, cell wall protein size about 120KD.This step includes that a step is concentrated by ultrafiltration and filter wash three times.Ultrafiltration system is " new
Generation KR2i tangential flow filtration system " (golden (Shanghai) Biotechnology Co., Ltd of auspicious Puli) model SYR2-U20-A.
1, hollow fiber column (D02-E030--05-N, mPES/30kd, the 115cm in the aperture 30KD are selected2) 400ml is trained
It supports supernatant and carries out ultrafiltration, cell wall protein is trapped, super quick albumen filtration；
2, recycling filtered solution selects hollow fiber column (D02-E010--05-N, mPES/10kd, the 115cm in the aperture 10KD2)
Carry out the concentration of super quick albumen, when 10 times of concentration i.e. about 40ml be added isometric 40ml buffer (20mM Tri-HCl,
400mM NaCl, 1mM EDTA), the same buffer of isometric 40ml is added when being again concentrated to 40ml, is concentrated into for the third time
The same buffer that isometric 40ml is added when 40ml finally continues to be concentrated into 10ml or so, final to obtain harpinEa egg
White liquor 14ml.This step shifts to albumen in buffer (20mM Tri-HCl, 400mM NaCl, 1mM EDTA), while cell
Wall-held protein is retained by ultrafiltration membrane and is separated, and is obtained purer harpinEa protein liquid, is as a result seen Fig. 3.
The glycerol that 50% is added into harpinEa protein liquid, in -20 DEG C of long-term preservations.
The application and effect of embodiment 5, harpinEa albumen on Pepper crops
The kind for testing selected Changyue County of Weifang City of Shandong Province town Shi Yan plantation is the Pepper crops of " green cage three ", this is peppery
Green pepper crop was sowed on October 22nd, 2017, on March 26th, 2018 is transplanted to crop field, 3600 plants/667m of planting density2。
It is designed according to randomized block experiment, plot area 15m2。
HarpinEa protein liquid will be purified according to the method described above to be applied by spraying by 50 μ g/ml of final concentration as experimental group, clear water
As a control group, parallel in 2 groups of groups, 3 repetitions are done respectively.Test is sprayed respectively at seedling stage, florescence, young fruit period, fruit expanding period
Apply processing；Every cell pinpoints 20 plants of capsicums in June 23 diseased plant number to capsicum morbidity, Agronomic trait, physiology respectively
Shape carries out data acquisition.Capsicum incidence is as shown in table 1, and statistical result discovery is anti-through harpinEa albumen treated capsicum
The ability of virus significantly improves, and virosis disease incidence is 5%, and compareing is 20%, has dropped 15 percentage points compared to control.
A situation arises for pepper virus disease under 1 different disposal of table
With ruler, vernier caliper measurement fruit is long, claims fruit mass with day chessboard, picks 1 time by the every 7d of fruit maturity, dynamic
Note produces, and each cell yield of hot pepper is counted respectively, as shown in table 2 and Fig. 4.It can be seen that capsicum Post flowering, every septum secundum 14~20 days
The super quick albumen of foliage-spray, obvious to capsicum effect of increasing production, volume increase is up to 40.35%.
Capsicum plant character under 2 different disposal of table
Pepper fruit 20 for randomly selecting exterior quality qualification smash the measurement for wearing into homogenate progress fruit quality to pieces.Use benzene
Phenol sulfuric acid method measures soluble sugar, and ultraviolet absorption method measures soluble protein, and molybdenum blue colorimetric method measures vitamin C, HPLC method
The content for measuring the capsaicine in capsicum, is repeated 3 times, takes its mean value.The wherein capsicum of the super quick albumen of foliar spray harpinEa
It is 20.12mg/g containing soluble sugar, it is higher by 64.5% than compareing；It is 52.68mg/g containing soluble protein, it is higher than compareing
33.13%；It is 20.10mg/kg containing capsaicine, it is higher by 62.09% than compareing；It is 350.13mg/kg containing vitamin C, than control
It is higher by 29.42%, see Table 3 for details.
Pepper fruit physiology character under 3 different disposal of table
Above-mentioned test result shows to express the super quick albumen obtained using method Brevibacillus brevis of the present invention
It can promote plant yield-increasing by the factors such as quantity and single fruit weight of bearing fruit of influence plant.It is opened according to recommended density in plant
The super quick protein solution that expression obtains is sprayed between florescence, and plant fruit yield and disease resistance can be improved.Because super quick albumen is enzyme
Preparation sprays between should be avoided at high temperature in practical vegetables production application, is preferably being close to vulnerable to the influence of outside environmental elements
It is used when late.In addition, super quick albumen can rapid induction plant generate resist and pre- bacteriological protection, fungi, virus caused by it is a variety of
The effect of disease, and be used for a long time and will not develop drug resistance, Nantural non-toxic is extremely easy in decomposition noresidue, to person poultry harmless, will not kill
The natural enemy and other beneficial bacteriums of worm are injured, it is green safe nuisanceless.
Finally, it should be noted that above embodiments are only used for helping skilled in the art to understand essence of the invention,
Limiting the scope of the present invention that it goes without doing.
Claims (9)
1. a kind of use Brevibacillus brevis (Brevibacillus choshinensis) as host strain, fermented and cultured production
The method of super quick albumen, the method include the following steps,
(1) encoding gene of the super quick albumen is inserted into Brevibacillus brevis as in the plasmid of host,
(2) plasmid for inserting the super quick albumen is transferred in Brevibacillus brevis host,
(3) Brevibacillus brevis described in fermented and cultured,
(4) the super quick albumen is concentrated in separation from fermentation liquid.
2. the method according to claim 1, wherein
Super quick albumen described in step (1) is to number to be from the GeneBank of bacterial strain Erwinia amylovora
The harpinEa albumen of M92994.3；
Plasmid described in step (1) is pNC-hisT plasmid.
3. the method according to claim 1, wherein
It is electric shock transformation method that plasmid, which is transferred to the method in Brevibacillus brevis host, described in step (2).
4. the method according to claim 1, wherein
The condition of culture of fermented and cultured described in step (3) are as follows: T2 culture medium, 30~33 DEG C of temperature, 120~200rpm of revolving speed,
24~60h of fermented incubation time, it is preferred that the fermentation culture conditions are: T2 culture medium, 33 DEG C of temperature, revolving speed 120rpm, hair
Ferment incubation time 48h.
5. the method according to claim 1, wherein
The step of super quick albumen described in separation and Extraction described in step (4) is: supernatant is collected by centrifugation in the bacterium solution after collecting fermentation,
Cross-flow ultrafiltration and concentration are carried out to supernatant, super quick albumen is obtained and liquid is concentrated by ultrafiltration.
6. according to the method described in claim 5, it is characterized in that, the step of described cross-flow ultrafiltration be:
(1) ultrafiltration is carried out to supernatant using the hollow fiber column in the aperture 30KD, collects filtered solution；
(2) recycling filtrate is concentrated using the hollow fiber column in the aperture 10KD, when being concentrated into 1/10 volume, is added isometric
Albumen buffer, the formula of the albumen buffer is 20mM Tri-HCl, 400mM NaCl, 1mM EDTA, in triplicate；
(3) step (2) protein concentrated solution obtained is concentrated in the hollow fiber column for continuing to use the aperture 10KD, is concentrated into
Liquid is concentrated by ultrafiltration to get super quick albumen in 1/8 volume.
7. the super quick albumen that any method of claim 1-6 prepares.
8. any method of claim 1-6 is preparing the application in Plant growth-promoting effect preparation and/or plant anti-insect preparation.
9. application according to claim 8, which is characterized in that the plant is plant of Solanaceae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811031684.6A CN109134626A (en) | 2018-09-05 | 2018-09-05 | Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811031684.6A CN109134626A (en) | 2018-09-05 | 2018-09-05 | Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109134626A true CN109134626A (en) | 2019-01-04 |
Family
ID=64827065
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811031684.6A Pending CN109134626A (en) | 2018-09-05 | 2018-09-05 | Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109134626A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1878859A (en) * | 2003-11-11 | 2006-12-13 | 日下田酱油股份有限公司 | Novel brevibacillus choshinensis and process for producing protein with use of the microbe as host |
| CN101338319A (en) * | 2008-08-15 | 2009-01-07 | 南京农业大学 | Recombinant vector pM43HF for expressing harpin protein and engineering strain thereof |
| TW201522628A (en) * | 2013-12-09 | 2015-06-16 | Univ Nat Chunghsing | Method for production of glycosidic recombinant trefoil factor 1 by brevibacillus choshinensis expression system, glycosidic recombinant trefoil factor 1 and uses thereof |
| CN107142235A (en) * | 2017-06-20 | 2017-09-08 | 江西嘉博生物工程有限公司 | A kind of restructuring Brevibacillus brevis for expressing pig growth hormone gene and construction method and application |
| CN107354191A (en) * | 2017-08-09 | 2017-11-17 | 湖北胜苗生物科技有限公司 | It is a kind of to be used to promote plant growth and the biological agent of the prevention and control of plant diseases, pest control |
-
2018
- 2018-09-05 CN CN201811031684.6A patent/CN109134626A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1878859A (en) * | 2003-11-11 | 2006-12-13 | 日下田酱油股份有限公司 | Novel brevibacillus choshinensis and process for producing protein with use of the microbe as host |
| CN101338319A (en) * | 2008-08-15 | 2009-01-07 | 南京农业大学 | Recombinant vector pM43HF for expressing harpin protein and engineering strain thereof |
| TW201522628A (en) * | 2013-12-09 | 2015-06-16 | Univ Nat Chunghsing | Method for production of glycosidic recombinant trefoil factor 1 by brevibacillus choshinensis expression system, glycosidic recombinant trefoil factor 1 and uses thereof |
| CN107142235A (en) * | 2017-06-20 | 2017-09-08 | 江西嘉博生物工程有限公司 | A kind of restructuring Brevibacillus brevis for expressing pig growth hormone gene and construction method and application |
| CN107354191A (en) * | 2017-08-09 | 2017-11-17 | 湖北胜苗生物科技有限公司 | It is a kind of to be used to promote plant growth and the biological agent of the prevention and control of plant diseases, pest control |
Non-Patent Citations (3)
| Title |
|---|
| MAKOTO MIZUKAMI ET AL.: ""Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system"", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| WEI,Z.M. ET AL.: ""accession number：M92994.3 Erwinia amylovora harpin (hrpN) gene, complete cds"", 《GENBANK》 * |
| 邓兵兵: ""外源蛋白在芽孢杆菌中分泌表达的研究进展"", 《生物工程进展》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104365461B (en) | A kind of tea grounds earthworm excrement matrix being applicable to leaf vegetables nursery and preparation method thereof | |
| CN103756975B (en) | A kind of preparation method and application thereof infecting the soybean mosaic virus of tobacco | |
| CN106480063A (en) | A kind of tea tree myb transcription factor CsAN1 and its application in regulation and control anthocyanidin metabolism | |
| CN101831481A (en) | New method for preparing Iturin A and homolugues thereof | |
| CN109081865A (en) | Moso bamboo PeVQ28 albumen and its encoding gene and application | |
| CN103843592A (en) | Breeding method for disease-resistant and heat-resistant capsicum annuum L. rootstocks of different physiological races | |
| CN108840915A (en) | Moso bamboo PeDi19-4 albumen and its encoding gene and application | |
| CN107236751A (en) | A kind of utilization bacillus subtilis expression system is quick, the method for high frequency zone antifungal genes | |
| CN107400671A (en) | Pear fruit saccharide transporter gene PbTMT4 and its application | |
| CN101864372A (en) | Preparation method of antibacterial peptide gene engineering strain | |
| CN104887615B (en) | A kind of method for preparing class mycetocyte element coarse amino acid in hair weeds cells | |
| CN109134626A (en) | Use the method for the super quick albumen of Brevibacillus brevis secreting, expressing | |
| CN107242250A (en) | A kind of wheat cold-resistant derivant and its application process | |
| CN101875940B (en) | Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof | |
| CN103044533B (en) | Protein associated with hexose transport and coding gene and application thereof | |
| CN103849605B (en) | A kind of method cultivating resistance plant and special protein thereof and gene | |
| CN107586748B (en) | A kind of China's sporangium and its application | |
| CN110106117A (en) | Regional efficient nitrogen microbial composite bacteria agent and its application in a kind of suitable Guizhou Province | |
| CN109234289A (en) | A method of initiative resistant transgenic clover | |
| CN109679982A (en) | A method of high-purity hidden ground albumen is prepared using Brevibacillus brevis | |
| CN104894084B (en) | A kind of method that Trichoderma atroviride prepares heat-resisting acidproof feruloyl esterase | |
| CN102898512A (en) | Recombinant plectasin as well as preparation method and application of recombinant plectasin | |
| CN107033228A (en) | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application | |
| CN106676131A (en) | Method for generating alfalfa genome edited homozygous plant | |
| CN107012100A (en) | A kind of snowy peak cordyceps sinensis stroma and its artificial culturing method |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191220 Address after: 436060 Ezhou province Hubei City Liangzihu District No. 9 Phoenix Road, East Lake high tech creative city B-06 Building 1 floor and 2 floor apartment layout D D apartment layout Applicant after: Hubei Wei Sheng Yuan Biotechnology Co., Ltd. Address before: 436060 Ezhou province Hubei City Liangzihu District No. 9 Phoenix Road, East Lake high tech creative city B-06 Building 1 floor and 2 floor apartment layout D D apartment layout Applicant before: Liu Sheng |
|
| TA01 | Transfer of patent application right |