CN109055418A - A kind of construction method recombinating Brevibacillus brevis - Google Patents
A kind of construction method recombinating Brevibacillus brevis Download PDFInfo
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- CN109055418A CN109055418A CN201710467689.2A CN201710467689A CN109055418A CN 109055418 A CN109055418 A CN 109055418A CN 201710467689 A CN201710467689 A CN 201710467689A CN 109055418 A CN109055418 A CN 109055418A
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- Prior art keywords
- brevibacillus brevis
- porcine
- pny326
- plasmid
- csf
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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Abstract
A kind of construction method recombinating Brevibacillus brevis, comprising the following steps: synthesis pig granulocyte macrophage colony stimulating factor (Porcine GM-CSF) gene and Brevibacillus brevis (pNY326) plasmid first；Double digestion is carried out to Porcine GM-CSF gene and pNY326 plasmid respectively with BamHI and EcoRI again, reacts 3h in 37 DEG C of incubators；4 DEG C of connection reactions overnight are carried out using the digestion products of 1.2% agarose gel electrophoresis recycling Porcine GM-CSF gene and pNY326 plasmid, and by the double enzyme digestion product of recycling；The direct electrotransformation Brevibacillus brevis SP3 of reaction product will be connected, electricity carries out the slow recovery 1h of 30 DEG C of shaking table 80rpm after the completion of turning；The converted product recovered neomycin resistance is screened into transformant, obtains recombination Brevibacillus brevis.The present invention can quick, simplicity acquisition pig granulocyte macrophage colony stimulating factor.
Description
Technical field
The present invention relates to a kind of construction methods for recombinating Brevibacillus brevis.
Background technique
Brevibacillus brevis is a kind of probiotics of safe and nontoxic property, while having protein secretion ability by force and extracellular egg
The low feature of white enzymatic activity, therefore be particularly suitable for as exocytosis protein expression system.
Colony stimulating factor (CSF) is one group can stimulate marrow hemopoietic stem cells to be divided into cell colony in vivo and in vitro
Low molecule cell factor.Granulocyte macrophage colony stimulating factor can stimulate the differentiation of Progranulocyte monocyte to generate granulocyte
And monocyte.Colony stimulating factor is one of most important cell factor of body, and weight is played during immune cell differentiation
It acts on, can be used as immunity enhancement adjuvant to enhance the immunogenicity of vaccine, the present invention has synthesized pig granular leukocyte macrophage collection
G-CSF gene, and recombinant plasmid is constructed with plasmid pNY326, success electrotransformation Brevibacillus brevis SP3 is obtained
Express the recombination Brevibacillus brevis of pig granulocyte macrophage colony stimulating factor, Recombinant Swine granular leukocyte macrophage colony thorn
The high efficient expression for swashing the factor is that it lays a good foundation as being widely used for pig vaccine immunopotentiator.
Summary of the invention
Its purpose of the invention, which is that, provides a kind of construction method for recombinating Brevibacillus brevis, has quick, simplicity
The characteristics of obtaining pig granulocyte macrophage colony stimulating factor.
It adopts the technical scheme that achieve the above object, a kind of construction method recombinating Brevibacillus brevis, including
Following steps:
(1) pig granulocyte macrophage colony stimulating factor (Porcine GM-CSF) gene and Brevibacillus brevis are synthesized
(pNY326) plasmid；
(2) double digestion is carried out to Porcine GM-CSF gene and pNY326 plasmid respectively with BamHI and EcoRI, in 37 DEG C of temperature
Case reacts 3h；
(3) using the digestion products of 1.2 % agarose gel electrophoresis recycling Porcine GM-CSF gene and pNY326 plasmid, and
The double enzyme digestion product of recycling is subjected to 4 DEG C of connection reactions overnight；
(4) the direct electrotransformation Brevibacillus brevis SP3 of reaction product will be connected, it is slow that electricity carries out 30 DEG C of shaking table 80rpm after the completion of turning
Slow recovery 1h；
(5) converted product recovered neomycin resistance is screened into transformant, obtains recombination Brevibacillus brevis.
Beneficial effect
The present invention has the following advantages that compared with prior art.
The present invention can quick, simplicity acquisition pig granulocyte macrophage colony stimulating factor.
Detailed description of the invention
Below in conjunction with attached drawing, the invention will be further described.
Fig. 1 is the PCR qualification figure of Porcine GM-CSF-pNY326 recombinant plasmid in the present invention；
Fig. 2 is the western blot figure that Brevibacillus brevis is SP3 expression Porcine GM-CSF purifying protein in the present invention；
Fig. 3 is pNY326 plasmid map in the present invention；
Fig. 4 is the multiple cloning sites map of pNY326 plasmid in the present invention.
Specific embodiment
A kind of construction method recombinating Brevibacillus brevis, as shown in Figs 1-4, comprising the following steps:
(1) pig granulocyte macrophage colony stimulating factor (Porcine GM-CSF) gene and Brevibacillus brevis are synthesized
(pNY326) plasmid；
(2) double digestion is carried out to Porcine GM-CSF gene and pNY326 plasmid respectively with BamHI and EcoRI, in 37 DEG C of temperature
Case reacts 3h；
(3) using the digestion products of 1.2 % agarose gel electrophoresis recycling Porcine GM-CSF gene and pNY326 plasmid, and
The double enzyme digestion product of recycling is subjected to 4 DEG C of connection reactions overnight；
(4) the direct electrotransformation Brevibacillus brevis SP3 of reaction product will be connected, it is slow that electricity carries out 30 DEG C of shaking table 80rpm after the completion of turning
Slow recovery 1h；
(5) converted product recovered neomycin resistance is screened into transformant, obtains recombination Brevibacillus brevis.
Embodiment
1, in pig granulocyte macrophage colony stimulating factor gene (NP_999283), after removing stream signal peptide sequence
Mature peptide see sequence 1 in sequence table, totally 127 amino acid, optimizes according to Brevibacillus brevis codon preference, on
The nucleotide sequence of trip 6 histidines of addition, terminator codon is added in downstream, and adds BamHI and EcoRI two respectively at both ends
Restriction enzyme site and protectiveness base, the artificial synthesized sequence, are shown in sequence 2 in sequence table:
Sequence 1
APTRPPSPVTRPWQHVDAIKEALSLLNNSNDTAAVMNETVDVVCEMFDPQEPTCVQTRLNLYKQGLRGSLTRL
KSPLTLLAKHYEQHCPLTEETSCETQSITFKSFKDSLNKFLFTIPFDCWGPVKK
Sequence 2
CGGGATCCCATCATCATCATCATCATGCACCAACGCGCCCACCAAGCCCGGTGACGCGTCCATGGCAGCACGT
CGATGCGATTAAAGAAGCGCTGAGCCTGCTGAACAACAGCAACGATACGGCGGCGGTCATGAACGAAACGGTGGATG
TGGTGTGCGAGATGTTCGACCCGCAAGAACCGACGTGCGTGCAAACGCGCCTGAACCTGTATAAACAGGGCCTGCGC
GGCAGCCTGACGCGCCTGAAAAGCCCGCTGACGCTGCTGGCGAAACATTATGAACAGCACTGCCCGCTGACGGAGGA
GACGAGCTGCGAGACGCAGAGCATCACGTTTAAAAGCTTTAAAGATAGCCTGAACAAGTTTCTGTTCACGATTCCGT
TCGACTGCTGGGGCCCGGTGAAAAAATAAGAATTCGC
2, with BamHI and EcoRI respectively to Porcine GM-CSF gene and pNY326 plasmid double digestion, double enzyme digestion reaction system
Are as follows: BamHI 2 uL, EcoRI 2 uL, Porcine GM-CSF gene/pNY326 plasmid 20 uL, ddH2O 12 uL, 10 ×
4 uL of M buffer, totally 40 uL, after 37 DEG C of incubators react 3h；
It 3, will using the digestion products of 1.2 % agarose gel electrophoresis recycling Porcine GM-CSF gene and pNY326 plasmid
The digestion products of recycling are attached reaction, linked system are as follows: 8 uL of gene, 8 uL of plasmid, T4DNA ligase 2 uL, 10 ×
2 uL of buffer, total volume are 20 uL, 4 DEG C of connections overnight；
4, connection Porcine GM-CSF gene and the direct electricity of pNY326 plasmid connection product are turned into Brevibacillus brevis SP3,
Step are as follows: 10uL connection product is added in 90uL Brevibacillus brevis SP3 competent cell, mixes gently；By connection product and
Competence mixture is slowly added to 2mm electricity revolving cup bottom, ice bath 10min；Parameter voltages 2000V, capacitor 50uF, resistance are set
200 Ω, electricity turn after the completion of after be transferred in 1.5mlEP pipe, the slow recovery 1h of 30 DEG C of shaking table 80rpm；
5, the converted product recovered passes through coating neomycin (30ug/mL) plate screening, 37 DEG C of culture 15-18h.Single colonie
Expand culture in neomycin (30ug/mL) resistance fluid nutrient medium, bacterium adds appropriate lysozyme short to destroy before applying upgrading grain
Bacillus cell wall extracts plasmid as PCR amplification template, passes through amplification Porcine GM-CSF sequence and be sequenced to identify
Recombinate Brevibacillus brevis.PCR amplification primer are as follows:
Porcine GM-CSF-F:GGATCCCATCATCATCATCAT
Porcine GM-CSF-R:GAATTCTTATTTTTTCACCGGG
PCR system are as follows: Porcine GM-CSF-F 5uL, Porcine GM-CSF-R 5uL, dNTP 5uL, 10 × buffer
1 uL of 5uL, transT-Taq enzyme, transformant bacterium solution 1 uL, ddH2O 28 uL, 50 uL of total volume
PCR amplification program are as follows: initial denaturation, 95 DEG C, 5min；Denaturation, 95 DEG C, 30sec；Annealing, 55 DEG C, 30sec；Extend, 72 DEG C,
1min；Overall elongation, 72 DEG C, 5min.
Wherein, it is denaturalized, anneals, extend, 30 cycle.
After the completion of PCR amplification, PCR stoste carries out agarose gel electrophoresis, has clear band at 414bp, PCR stoste is same
When be sequenced, sequencing result with synthesis gene it is consistent.
Recombinate the expression of Brevibacillus brevis SP3
Picking recombination Brevibacillus brevis single colonie is inoculated in the 3mL T2 fluid nutrient medium of the 30ug/mL containing neomycin on plate
(glucose 1.0%, peptone 1.0%, beef extract 0.5%, yeast extract 0.2%), 30 DEG C of 120rpm shaking table culture 48-64h,
The Porcine GM-CSF of expression is secreted into culture medium supernatant.
Recombinate the purifying and verifying of Porcine GM-CSF albumen
1, the albumen in saturated ammonium sulphate culture medium supernatant
The bacterium solution maximum (top) speed that culture is completed is centrifuged 30min, bacterial sediment is abandoned, by isometric saturated ammonium sulfate solution
(4.1mol/L, 25 DEG C) is slowly added into while stirring, is added to put and is stirred overnight for 4 DEG C on magnetic stirring apparatus, precipitates albumen sufficiently.
4 DEG C of centrifugation 30min of 10000rpm abandon supernatant and retain precipitating, and precipitating is dissolved in a small amount of (10-20mL) PBS-0.2g/L Sodium azide
In, precipitating is dissolved, 4 DEG C of dialysis 48h of bag filter are reused, every 6 hours replacement elution buffers, to completely remove sulfuric acid
Ammonium.
2, Porcine GM-CSF albumen ni-sepharose purification
Related reagent is as follows: mother liquor 1:29.22g sodium chloride, and 2.42gTris-Base, ddH2O are dissolved to 1L, adjusts pH=8.0
Mother liquor 2:6.06gTris-Base, ddH2O are dissolved to 1L, adjust pH=8.0
Binding buffer:0.1702g imidazoles is dissolved in 500 mL mother liquors 1.
Elution buffer:17.02g imidazoles is dissolved in 500 mL mother liquors 1.
Washing buffer:2.3828g imidazoles is dissolved in 500 mL mother liquors 2.
Nickel ion affinity chromatograph column first is balanced with Binding buffer, sample to be purified is then splined on nickel column,
In conjunction with 30min or so, collection reserves liquid Flow Through, washes 3 column volumes with Washing buffer, collects Wash
Throngh finally elutes albumen with Elution buffer, collects Eluent. for Binding buffer, Wash
Throngh, Eluent.Device is concentrated by ultrafiltration with Milipore pressure in the albumen that nickel column is collected to retain by 30kDa ultrafiltration membrane
It is concentrated into 10mL or so, sample is divided by the equilibrated Superdex200 molecular sieve of Gel filtration buffer
From.
3, the western blot verifying of Porcine GM-CSF albumen
SDS-PAGE loading buffer, boiling water bath 15min will be added in the albumen of purifying, carry out western blot, step
It is rapid as follows:
3.1 cleaning Bio-Rad glass plates, are installed after dry, prepare separation gel by upper formula, 2ml separation gel is added in 1mm offset plate
Liquid is exhausted remaining until 1.5cm under comb teeth, covers a small amount of ddH2O, the ddH2O of covering is outwelled after to be solidified with filter paper
ddH2O；
3.2 same methods prepare concentration glue by upper formula, record on separation gel, are immediately inserted into 1mm glue comb, coagulate at room temperature
Gu；
3.3 glue preparations to be concentrated are completed, and pull up glue comb, offset plate is fixed on electrophoretic apparatus immediately, electrophoresis is added in electrophoresis tank
Buffer drains the bubble between gel bottom glass plate；
3.4 every holes are loaded 20 μ L, power on, and set voltage 80V/cm, after dyestuff enters concentration glue, adjust voltage to
120V/cm, until bromjophenol blue dyestuff is away from stopping electrophoresis at the 2cm of glue bottom；
3.5, which remove gel, is put in transferring film buffer for use, while by filter paper, NC film, and fiber mat is put into transferring film buffer.
3.6 install film transfer device by anode-fiber mat-filter paper-NC film-gel-filter paper-fiber mat sequence, drive away
Bubble notices that the area of two layers of filter paper is less than gel and NC film, 100V electrophoresis 4h；
Membrane-transferring device is unloaded after the completion of 3.7 transferring films, cleans film 3 times with TBST cleaning solution, each 5min；
3.8 are placed on film in confining liquid, and 4 DEG C of closings overnight, discard confining liquid, cleaned 3 times with cleaning solution, each 5min；
3.9 dilute His-tag monoclonal antibody using TBST 1:5000, and room temperature shaker is incubated for 2h, and cleaning solution washs 3 times, each 5min；
It is incubated for sheep anti mouse lgG-HRP secondary antibody 30min, TBST is washed 5 times, each 5min；It is carried out using HRP-DAB substrate colour reagent box
Colour developing, is as a result shown in Fig. 2.
Recombinate the measurement of Porcine GM-CSF protein active
With MTT(nitrogen blue tetrazole salt) activity of colorimetric method for determining recombinant protein, specific steps are as follows: connect in 96 also cell culture analysis
The NFS-60 cell that 50uL cell concentration is 3 × 104, by GM-CSF standard items (being purchased from Boechringer company) and recombination weight
Group Porcine GM-CSF dilutes respectively, and each 50uL is added in 96 orifice plates, and negative control is without GM-CSF, and blank control is only
Containing culture solution, 37 DEG C, 5% CO2 cultivates 48h, adds the hole MTT lysate 100Ul/, and next day measures each hole A570/630 value.MTT knot
Brilliant lysate is 10% SDS-0.01M HCl.
The recombination Porcine GM-CSF protein active of MTT colorimetric method for determining purifying is 1.5 × 107 U/mg.
Claims (1)
1. a kind of construction method for recombinating Brevibacillus brevis, which comprises the following steps:
Synthesize pig granulocyte macrophage colony stimulating factor (Porcine GM-CSF) gene and Brevibacillus brevis
(pNY326) plasmid；
Double digestion is carried out to Porcine GM-CSF gene and pNY326 plasmid respectively with BamHI and EcoRI, it is anti-in 37 DEG C of incubators
Answer 3h；
Using the digestion products of 1.2 % agarose gel electrophoresis recycling Porcine GM-CSF gene and pNY326 plasmid, and will
The double enzyme digestion product of recycling carries out 4 DEG C of connection reactions overnight；
The direct electrotransformation Brevibacillus brevis SP3 of reaction product will be connected, it is slowly multiple that electricity carries out 30 DEG C of shaking table 80rpm after the completion of turning
Revive 1h；
The converted product recovered neomycin resistance is screened into transformant, obtains recombination Brevibacillus brevis.
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| CN201710467689.2A CN109055418A (en) | 2017-06-20 | 2017-06-20 | A kind of construction method recombinating Brevibacillus brevis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921483A (en) * | 2022-06-17 | 2022-08-19 | 山东柏佳薇生物科技有限公司 | Gene for coding archizurin, engineering bacteria and biosynthesis method thereof |
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| CN106086048A (en) * | 2016-08-16 | 2016-11-09 | 吉林大学 | A kind of acid resistance high temperature alpha amylase and gene, engineering bacteria and preparation method |
| CN106459953A (en) * | 2014-06-09 | 2017-02-22 | 株式会社钟化 | Method for producing recombinant proteins using recombinant brevibacillus |
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2017
- 2017-06-20 CN CN201710467689.2A patent/CN109055418A/en active Pending
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| 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 |
| CN101050448A (en) * | 2006-12-26 | 2007-10-10 | 华中农业大学 | Oral recombined DNA vaccine for accelerating growth of animal, and application |
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| CN106459953A (en) * | 2014-06-09 | 2017-02-22 | 株式会社钟化 | Method for producing recombinant proteins using recombinant brevibacillus |
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| AKIRA YAMAMOTO ET AL.: "Expression and purification of canine granulocyte colony-stimulating factor (cG-CSF)", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921483A (en) * | 2022-06-17 | 2022-08-19 | 山东柏佳薇生物科技有限公司 | Gene for coding archizurin, engineering bacteria and biosynthesis method thereof |
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