CN110423770A - A kind of recombinant vector and expression of ustilago zeae effect protein CMU1 gene - Google Patents
A kind of recombinant vector and expression of ustilago zeae effect protein CMU1 gene Download PDFInfo
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- CN110423770A CN110423770A CN201910358656.3A CN201910358656A CN110423770A CN 110423770 A CN110423770 A CN 110423770A CN 201910358656 A CN201910358656 A CN 201910358656A CN 110423770 A CN110423770 A CN 110423770A
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- 230000014509 gene expression Effects 0.000 title claims abstract description 49
- 244000301083 Ustilago maydis Species 0.000 title claims abstract description 28
- 235000015919 Ustilago maydis Nutrition 0.000 title claims abstract description 28
- 101150031336 CMU1 gene Proteins 0.000 title claims abstract description 25
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 239000013598 vector Substances 0.000 title claims abstract description 17
- 101100060492 Ustilago maydis (strain 521 / FGSC 9021) CMU1 gene Proteins 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 31
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- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
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- 239000002773 nucleotide Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- VBUNOIXRZNJNAD-UHFFFAOYSA-N ponazuril Chemical compound CC1=CC(N2C(N(C)C(=O)NC2=O)=O)=CC=C1OC1=CC=C(S(=O)(=O)C(F)(F)F)C=C1 VBUNOIXRZNJNAD-UHFFFAOYSA-N 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- 231100000397 ulcer Toxicity 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of recombinant vectors for expressing ustilago zeae effect protein CMU1 gene, and invention additionally discloses a kind of methods for expressing ustilago zeae effect protein CMU1 gene.The present invention constructs the prokaryotic expression carrier of ustilago zeae effect protein CMU1 gene, and a series of optimization is carried out for protein expression condition, finally obtain CMU1 soluble protein, as a result further research albumin crystal structure and biological characteristics lay the foundation, thinking is provided for the pesticide manufacturing of target for CMU1 for design, and then provides foundation for prevention and treatment ustilago zeae.
Description
Technical field
The present invention relates to the recombinant vectors and expression of a kind of ustilago zeae effect protein CMU1 gene, belong to biology
Technical field.
Background technique
Ustilago zeae is one of main disease of corn, can all be occurred during the entire fertility of corn.But corn
Smut can also be eaten raw when children is tender with prepared food, pleasantly sweet, stir-fry and eat and have a distinctive flavor.It is often edible to prevent and treat liver
System and gastrointestinal ulceration, and can aid digestion and defaecation.Contain glutamic acid, lysine, alanine, smart ammonia in ustilago zeae
16 kinds of amino acid such as acid, methionine, threonine, histidine.This ustilago zeae can be processed medicinal, and fresh sorus is taken
Or will be aging after collection refined honey ball, cold in nature, sweet in flavor, the effect of advantageous liver benefit liver stomach, and neurasthenia, children's infantile malnutrition due to digestive disturbances or intestinalparasites can be controlled
Product.
During plant and pathogen are confronted with each other, pathogen can secrete some albumen into host cell.These
Albumen is referred to as " effect protein (Avr) ", plant immune can be inhibited to react in several ways, cause of disease is promoted successfully to infect plant
Object.In recent years, the report that continuous pathogen effect protein gene is cloned, such as rice blast fungus, flax rust, oomycetes, tomato
Effect protein gene in leaf mycete, Fusarium oxysporum, real-time fluorescent RCR ulcer bacteria, barley leaf blotch bacterium and ustilago zeae etc.
Also it is successively cloned by separation.
The prokaryotic expression of gene is one of the common technology for studying gene function.Large intestine is usually present when albumen pronucleus expression
Since rare codon causes expression to limit in bacillus, inclusion body is formed；It can inhibit albumen when the stronger protein expression of toxicity
Expression causes expression yield too low；Another easy the problem of ignoring, which is that reproducibility is excessively high in Escherichia coli, will lead to two sulphur
Key cannot be properly formed, and cause expression product insoluble.For above-mentioned problem, it is common practice to after dissolving inclusion body with urea
It is purified in the case of a degenerated, then renaturation.But the method complex steps, success rate are low.Ustilago zeae effect at present
PROTEIN C MU1 gene has been found, but how it to be expressed with soluble form be still this field problem.The present invention is logical
It crosses and selects different bacterium bacterial strains, to screen CMU1 albumen using the concentration of different inducing temperatures and inducer IPTG solvable
Property expression condition, lay the foundation for the researchs such as gene function, protein crystal.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of ustilago zeae effect protein CMU1 genes
Recombinant vector and expression.
The technical scheme is that a kind of recombinant vector of ustilago zeae effect protein CMU1 gene, the recombination
Carrier the preparation method is as follows: extracting corn RNA, and reverse transcription goes out cDNA；Using cDNA as template amplification CMU1 gene；It will
CMU1 amplified production is inserted into the pET-28a table through same double digestion after EcoRI and XhoI double digestion, through DNA ligase
Up in carrier, recombinant plasmid pET-28a-CMU1 is obtained.
Preferably, using cDNA as template, using following primer amplification CMU1 gene, primer sequence is as follows:
Upstream primer: CMU1-F:GCGAATTCATGGCGGCTGTATCTGGC underscore is EcoRI restriction enzyme site；
Downstream primer: CMU1-R:CGCTCGAGTTAGGTGCACTTGTTGGCG underscore is XhoI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
The present invention also provides a kind of methods for expressing ustilago zeae effect protein CMU1 gene, comprising the following steps:
(1) Bacillus coli cells are converted with recombinant vector of any of claims 1 or 2, the recombination for obtaining expression CMU1 is former
Nuclear expression bacterial strain；
(2) recombined pronucleus expression strain inoculated to kanamycins fluid nutrient medium or kanamycins+chloramphenicol liquid are trained
It supports in base, is incubated overnight activated strains；
(3) the recombined pronucleus expression bacterial strain of activation is transferred to kanamycins fluid nutrient medium again or kanamycins+chlorine is mould
The expression of IPTG induction recombinant C MU1 is added in plain fluid nutrient medium, after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed CMU1 recombinant protein.
Preferably, when earthquake culture is to OD600=0.5 in step (3), the IPTG of final concentration of 1.0mM, In is added
25 DEG C of Fiber differentiations.
Preferably, kanamycins is 50ug/mL in the kanamycins fluid nutrient medium.
Preferably, kanamycins is 50ug/mL, chloramphenicol 50ug/ in the kanamycins+chloramphenicol fluid nutrient medium
mL。
The beneficial effects of the present invention are: the present invention is to obtain CMU1 soluble protein, ustilago zeae effect egg is constructed
The prokaryotic expression carrier of white CMU1 gene, and a series of optimization is carried out for protein expression condition, inducing temperature is worked as in discovery
When being 25 DEG C, expression quantity is best；With the increase of IPTG concentration, expressing quantity is gradually increased, and best induced concentration is
1.0mM；At 25 DEG C, when IPTG concentration is 1.0, expression quantity is best.Through SDS-PAGE electrophoresis detection, obtaining molecular weight is about
The purpose band of 33kDa finally obtains CMU1 soluble protein for the expected size of CMU1 albumen.Meanwhile present invention albumen
Purification system purifies it, as a result further research albumin crystal structure and biological characteristics lay the foundation, to set
Meter provides thinking for CMU1 for the pesticide manufacturing of target, and then provides foundation for prevention and treatment ustilago zeae.
Detailed description of the invention
Fig. 1 is pET-28a Vector map；
Fig. 2 is digestion result schematic diagram, and 1 is the Plasmid DNA of digestion, and 2 be the digestion of EcoRI and XhoI inscribe as a result, M is
DNA Marker；
Fig. 3 is the albumen peak figure that CMU1 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 4 is the electrophoresis result figure after CMU1 protein purification；
Fig. 5 is the result figure that SDS-PAGE electrophoresis tests optimum expression bacterial strain；
Fig. 6 is the result figure that SDS-PAGE electrophoresis tests best IPTG induced concentration；
Fig. 7 is the result figure that SDS-PAGE electrophoresis tests best inducing temperature；
Fig. 8 is western blot detection figure.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
Embodiment 1: the building and identification of ustilago zeae effect protein CMU1 gene recombined vector
1, corn RNA is extracted, and reverse transcription goes out cDNA
Ustilago zeae material is taken, extracts seedling using RNA extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.)
Phase total serum IgE obtains cDNA with reverse transcription reagent box (Promega) reverse transcription.
2, using cDNA as template amplification CMU1 gene；
Design primer expands CMU1 gene using cDNA as template, the nucleotide sequence of CMU1 gene such as SEQ ID
Shown in NO.1, the amino acid sequence of the protein encoded as CMU1 gene is as shown in SEQ ID NO.2.
Primer is as follows:
Upstream primer: CMU1-F:GCGAATTCATGGCGGCTGTATCTGGC underscore is EcoRI restriction enzyme site；
Downstream primer: CMU1-R:CGCTCGAGTTAGGTGCACTTGTTGGCG underscore is XhoI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
PCR amplification system used in gene magnification for vector construction is as follows: 5 × buffer 10 μ L, FastPfu
4 μ L, cDNA template of Fly DNA Polymerase 1 μ L (Quan Shijin), 2.5mmol dNTPs, 0.5 μ L, 10 μm of ol upstream and downstream
Primer each 1 μ L, ddH2O 32.5μL。
The following are PCR amplification conditions: 95 DEG C of 2min, 35 circulation (95 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 30s-1min, 72
℃5min。
3, CMU1 gene recombined vector is constructed
After PCR product is purified, it is connected to pET-28a carrier.PCR product is detected using 1% agarose.PCR
Product is recycled with reference to Nanjing Vazyme Biotechnology Co., Ltd.'s Ago-Gel QIAquick Gel Extraction Kit specification.It is after the recovery
Product is inserted into the pET-28a expression vector through same double digestion after EcoRI and XhoI double digestion by DNA ligase
(expression vector map is as shown in Figure 1), 16 DEG C connect 16 hours, construct pET-28a-CMU1 recombinant plasmid.
By recombinant plasmid transformed to coli strain DH5a, trained on 50ug/mL kanamycins solid plate in 37 DEG C
It supports, picking individual colonies hold up the limited public affairs of section's biotechnology after digestion identification is correct (digestion result is as shown in Figure 2), then through Beijing
Take charge of its correct sequence of sequence verification.Sequencing result shows that CMU1 coding sequence obtained fulfills the expectation, and says
Bright construction of recombinant plasmid success is simultaneously named as pET-28a-CMU1, i.e. recombinant vector.
The inducing expression of embodiment 2:CMU1 albumen
1, the recombined pronucleus expression bacterial strain of CMU1 is obtained
It selects and successful monoclonal is sequenced in embodiment 1 is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight, according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit by pET-28a-CMU1
Recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), Rosetta (DE3), BL21
(DE3) pLysS bacterial strain detects the expression of CMU1 albumen.
2, activated strains are incubated overnight
It is incubated overnight and activates above-mentioned recombined pronucleus expression bacterial strain.For example, BL21 (DE3) bacterial strain is transferred to 50ug/mL
In kanamycins fluid nutrient medium, by Rosetta (DE3), BL21 (DE3) pLysS bacterial strain be transferred to 50ug/mL kanamycins+
In 50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.
3, inducing expression
The recombined pronucleus expression bacterial strain of activation is transferred in the fluid nutrient medium containing corresponding antibiotic, for example, will activation
BL21 (DE3) bacterial strain be transferred in 50ug/mL kanamycins fluid nutrient medium, by Rosetta (DE3), BL21 (DE3)
PLysS bacterial strain is transferred in 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, 37 DEG C of shake cultures to OD600
When=0.5, the IPTG of final concentration of 1.0mM is added, 25 DEG C Fiber differentiation 10 hours, obtain the recombinant protein of CMU1.
4, the purifying of CMU1 recombinant protein
After the completion of induction, expressed CMU1 recombinant protein is recycled and purified, is carried out using AKTA protein purification system pure
Change and desalination, it is preferable that protein purification is carried out using AKTA purifier10 fast protein purification system, steps are as follows:
Since pET28a has 6 His coded sequences before terminator, so pET28a-CMU1 recombinant expression carrier obtains
The albumen taken can use Ni-NTA and carry out affinitive layer purification.Affinity chromatography is with protein or large biological molecule and to be incorporated in
Based on special affinity on medium between aglucon, because being connected to a NTA in the matrix of chromatography gel used in His-tags
[(nitrilotriacetic acid) nitrilotriacetic acid], can with Ni ions binding, and the 6 of Ni ion and fusion protein ×
Attraction is generated between His amino acid, so that the fusion protein with His-tags histidine tag be distinguished with other albumen
It comes, five yuan of imidazole rings of histidine residues are the key that albumen and Ni ionization, therefore when we use the imidazoles of high concentration
(Imidazole) solution elution when marquis (such as 400mM), imidazoles be convenient for protein His-tags imidazole ring competitive binding, most
Fusion protein is eluted from gel at last.
Concrete operations: taking out the thallus of -80 DEG C of preservations, and Lysis buffer is added and is resuspended, the bacterium solution after resuspension is in ice bath
Middle carry out ultrasonic disruption, power 350W, duty ratio 50%, each circulation 30s, total time 35min.It is broken even
Slurry, 4 DEG C, 1,2000rpm centrifugation 30min.Supernatant is adsorbed on Ni-NTA adsorption column with peristaltic pump, 5mL/min, repeats one
It is secondary, retain 20 μ L by the sample of adsorption column and carries out protein adhesive electrophoresis；Ni-NTA adsorption column is connected to fast protein purifying system
On system, eluted；Albumen is collected according to UV absorption curve, and samples and carries out protein adhesive electrophoresis detection.
Fig. 3 is AKTA protein purification system purifying protein peak figure.CMU1 albumen is purified by AKTA protein purification system
The albumen peak figure arrived.
Fig. 4 is the electrophoretogram after CMU1 protein purification.It is the expression and purification CMU1 egg at 25 DEG C, IPTG concentration 1.0mM
White result.1 is the albumen after Ni column is affine after purification；2 be efflux；M is albumen marker；Arrow meaning is shown as
CMU1 albumen.Fig. 4 shows that CMU1 albumen often has in supernatant under this condition, is solubility expression, passes through AKTA albumen
The CMU1 purity of protein that purification system obtains after purification reaches requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 3:CMU1
1, the recombinant strains of CMU1 are obtained
It selects and successful monoclonal is sequenced in embodiment 1 is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight, according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit by pET-28a-CMU1
Recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), Rosetta (DE3), BL21
(DE3) pLysS bacterial strain is coated on kanamycins containing 50ug/mL (Rosetta (DE3) need to add 50ug/mL chloramphenicol) plate
It is incubated overnight in 37 DEG C, picking single colonie is inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm mistake
50% glycerol of sterilizing is added in bacterium solution, the prokaryotic expression for obtaining expression CMU1 in -80 DEG C of refrigerators is frozen after mixing for night culture
Bacterial strain.
2, the determination of CMU1 Primary structure optimum strain
It is inoculated with above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3), Rosetta (DE3), BL21 (DE3) pLysS respectively
Bacterial strain, into 50ug/mL kanamycins (Rosetta (DE3) need to add 50ug/mL chloramphenicol) fluid nutrient medium, 37 DEG C,
200rpm is incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to 50ug/mL kanamycins again
In (Rosetta (DE3) need to add 50ug/mL chloramphenicol) fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is added
The IPTG of final concentration of 1mM, 25 DEG C Fiber differentiation 10 hours (bacterium not induced is taken to make negative control).4 after the completion of induction
DEG C, 1,2000rpm centrifugation 2min collects thallus, bacterial precipitation and negative control 2mL and PBS buffer is pre-chilled (with preceding addition
1mM PMSF) it suspends, clasmatosis is carried out with Ultrasonic Cell Disruptor, each 10sec, totally 10 times, every minor tick 30sec；1,
2000rpm is centrifuged 10min, takes supernatant, and SDS-PAGE electrophoresis detection tests optimum expression bacterial strain, as a result as shown in figure 5, CMU1
The optimum strain of Primary structure is Rosetta (DE3).
3, the determination of the best IPTG concentration of CMU1 Primary structure
It is mould to 50ug/mL kanamycins+50ug/mL chlorine to be inoculated with above-mentioned recombined pronucleus expression bacterial strain Rosetta (DE3)
In plain fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to
In 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is separately added into
The IPTG of final concentration of 0,0.4,0.8,1.0,1.2mM (take the bacterium not induced to make negative right in Fiber differentiation 4-16 hours at 25 DEG C
According to).4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min collects thallus, bacterial precipitation and negative control with 2mL and PBS is pre-chilled
Buffer (with preceding addition 1mM PMSF) suspends, and carries out clasmatosis with Ultrasonic Cell Disruptor, each 10sec, and totally 10 times, between each
Every 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-PAGE electrophoresis detection tests best IPTG induced concentration, as a result
As shown in fig. 6, the best IPTG of CMU1 Primary structure is 1.0mM.
4, the determination of CMU1 Primary structure optimum temperature
It is mould to 50ug/mL kanamycins+50ug/mL chlorine to be inoculated with above-mentioned recombined pronucleus expression bacterial strain Rosetta (DE3)
In plain fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to
In 50ug/mL kanamycins+50ug/mL chloramphenicol fluid nutrient medium, when 37 DEG C of shake cultures are to OD600=0.5, it is added dense eventually
Degree is the IPTG of 1.0mM, (takes pET-28a bacterium to make negative right within Fiber differentiation 8-16 hours at 16,20,25,30 and 37 DEG C respectively
According to).4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min collects thallus, bacterial precipitation and negative control with 2mL and PBS is pre-chilled
Buffer (with preceding addition 1mM PMSF) suspends, and carries out clasmatosis with Ultrasonic Cell Disruptor, each 10sec, and totally 10 times, every time
It is spaced 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-PAGE electrophoresis detection is tested best inducing temperature, as a result seen
The best inducing temperature of Fig. 7, CMU1 Primary structure is 25 DEG C.
The western blot of embodiment 4:CMU1 recombinant protein is detected
(1) it extracts by CMU1 albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-CMU1 plasmid, IPTG inducing expression；
2. protein lysate (50mM Tris pH 8.0,120mM NaCl, 1mM DTT, 0.5% NP-40, before is added
1mM PMSF, 1mM DTT is added), after ultrasonic disruption cell, supernatant is collected by centrifugation, places 20min on ice；
3. 15,000g, 4 DEG C of centrifugation 5min abandon precipitating；
4. taking 200 μ L supernatants that 5 × Loading Buffer is added, concussion is mixed, and -80 DEG C of remaining sample freeze；
5. boiling water boiling 5min, ice bath, 12,000rpm centrifugation 5min draw 15 μ L of supernatant and carry out protein electrophoresis, remaining sample
- 20 DEG C of product freeze；
6. prepare SDS-PAGE glue, first carry out electrophoresis 20min with 90V, when sample enters separation gel, with 150V voltage into
Row electrophoresis, until bromophenol blue stops electrophoresis when running out of separation gel.
(2) transferring film
Cut with a paper cutter out the pvdf membrane more somewhat larger than glue (for pvdf membrane, first with methanol impregnate 30 seconds, then
ddH23-5min is impregnated in O, is then placed in transferring film buffer) rubber plate clamp is put into electrophoretic blotting groove.Transferring film buffer is poured into,
4 DEG C, 100V 90min.
(3) it closes
After the completion of transferring film, pvdf membrane is taken out, is put in the PBST solution with 5% skimmed milk power, 2h is closed on shaking table.
(4) primary antibody is incubated for
Confining liquid is discarded, is added Anti His antibody (1:5000), shaking table is incubated for 12h.
(5) secondary antibody is incubated for
Primary antibody is outwelled, washs pvdf membrane 3 times with 1 × PBST, each 5min；Corresponding secondary antibody (sheep anti-Mouse) is added, shakes
Bed is incubated for 1h.
(6) ECL exposes
Secondary antibody is abandoned, washs pvdf membrane 3 times with 1 × PBST, each 5min；HRP chemiluminescent substrate is added on pvdf membrane
Liquid can detect protein expression signal (as shown in Figure 8) on 5200 chemiluminescence detectors in day, and succeed table as can be seen from the results
Up to CMU1 albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of recombinant vector and expression of ustilago zeae effect protein CMU1 gene
<160> 4
<210>1
<211>873
<212>DNA
<213>ustilago zeae effect protein CMU1
<400>1
ATGAAGTTGA GCGTGTCCAT CTTTGTGCTG CTGGCGGTGT CGGCGTTCGG TGGTGGCAGC 60
GCGGCGGCTG TATCTGGCAA GTCGGAGGCG GCAGAAATCG AAGCGGGCGA TCGACTGGAT 120
GCACTTCGTG ATCAACTGCA GAGGTACGAG ACGCCTATTA TTCAGACGAT TCTTGCACGC 180
TCTGCGTTGG GTGGGCGCGC GCCATCCGAA CAAGATGAGG TGCGTGCGGC ACTGTCTCGC 240
AATGCATTCG AGCCGTCAGA GGTAATCAGC GAGTGGCTGC AGACCGAGTC GGGAGCACGC 300
TTCCGGTCGA CGCGACCGTT GCCGCCTGCT GTCGAGTTCA TCACGCCCGT CGTCCTCTCG 360
CGAGACACAG TGCTGGACAA ACCAGTTGTC GGCAAAGGCA TCTTCCCGAT CGGTCGACGT 420
CCGCAGGATC CGACCAACAT GGACGAGTTC TTGGACACAT CTCTGCTGTC TCTCAACCAA 480
TCGTCCACGG TGGATCTCGC ATCCGCCGTC TCGCTCGACG TGAGCCTGCT CCATCTCGTC 540
TCGGCGCGTG TGCTGCTCGG CTATCCGATC GCGCTCGCTA AATTCGACTG GCTCCACGAC 600
AACTTCTGCC ACATCCTCAC CAACACTACT CTATCGAAAT CCCAAAAACT CGCCAACATC 660
ATCCAACAAC TTACCGACCA CAAACAAGAA GTCAACGTAC TCAGTCGTGT CGAGCAAAAG 720
TCCAAGTCGC TCTCACACCT CTTCCGCAAC GACATCCCTT ACCCTCCGCA CACCCAAGAC 780
CGCATCCTCA GACTCTTCCA AGCTTACCTC ATCCCCATCA CCACCCAAAT CGAAGCGGCC 840
GCTATCCTCG ACCACGCCAA CAAGTGCACC TAA 873
<210>2
<211>290
<212>PRT
<213>ustilago zeae effect protein CMU1
<400>2
MKLSVSIFVL LAVSAFGGGS AAAVSGKSEA AEIEAGDRLD ALRDQLQRYE TPIIQTILAR 60
SALGGRAPSE QDEVRAALSR NAFEPSEVIS EWLQTESGAR FRSTRPLPPA VEFITPVVLS 120
RDTVLDKPVV GKGIFPIGRR PQDPTNMDEF LDTSLLSLNQ SSTVDLASAV SLDVSLLHLV 180
SARVLLGYPI ALAKFDWLHD NFCHILTNTT LSKSQKLANI IQQLTDHKQE VNVLSRVEQK 240
SKSLSHLFRN DIPYPPHTQD RILRLFQAYL IPITTQIEAA AILDHANKCT 290
<210>3
<211>26
<212>DNA
<213>artificial sequence
<400>3
GCGAATTCAT GGCGGCTGTA TCTGGC 26
<210>4
<211>27
<212>DNA
<213>artificial sequence
<400>4
CGCTCGAGTT AGGTGCACTT GTTGGCG 27
Claims (6)
1. a kind of recombinant vector of ustilago zeae effect protein CMU1 gene, which is characterized in that the preparation of the recombinant vector
Method is as follows: extracting corn RNA, and reverse transcription goes out cDNA；Using cDNA as template amplification CMU1 gene；CMU1 amplified production is passed through
It after EcoRI and XhoI double digestion, is inserted into the pET-28a expression vector through same double digestion by DNA ligase, obtains weight
Group plasmid pET-28a-CMU1.
2. recombinant vector according to claim 2, which is characterized in that using cDNA as template, utilize following primer amplification
CMU1 gene, primer sequence are as follows:
Upstream primer: CMU1-F:GCGAATTCATGGCGGCTGTATCTGGC underscore is EcoRI restriction enzyme site；
Downstream primer: CMU1-R:CGCTCGAGTTAGGTGCACTTGTTGGCG underscore is XhoI restriction enzyme site；
Upstream and downstream primer is respectively as shown in SEQ ID NO.3,4.
3. a kind of method for expressing ustilago zeae effect protein CMU1 gene, which comprises the following steps:
(1) Bacillus coli cells are converted with recombinant vector of any of claims 1 or 2, obtains the recombination protokaryon table of expression CMU1
Up to bacterial strain；
(2) by recombined pronucleus expression strain inoculated to kanamycins fluid nutrient medium or kanamycins+chloramphenicol fluid nutrient medium
In, it is incubated overnight activated strains；
(3) the recombined pronucleus expression bacterial strain of activation is transferred to kanamycins fluid nutrient medium or kanamycins+chloromycetin solution again
The expression of IPTG induction recombinant C MU1 is added in body culture medium, after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed CMU1 recombinant protein.
4. the method for expression ustilago zeae effect protein CMU1 gene according to claim 3, which is characterized in that step
(3) when earthquake culture is to OD600=0.5 in, the IPTG of final concentration of 1.0mM is added, in 25 DEG C of Fiber differentiations.
5. the method for expression ustilago zeae effect protein CMU1 gene according to claim 3, which is characterized in that described
Kanamycins is 50ug/mL in kanamycins fluid nutrient medium.
6. the method for expression ustilago zeae effect protein CMU1 gene according to claim 3, which is characterized in that described
Kanamycins is 50ug/mL, chloramphenicol 50ug/mL in kanamycins+chloramphenicol fluid nutrient medium.
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| CN110117607A (en) * | 2019-04-29 | 2019-08-13 | 贵州大学 | A kind of recombinant vector and expression of ustilago zeae effect protein Pit2 gene |
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