CN110042115A - A kind of recombinant vector and expression of potato aphid effect protein Me23 gene - Google Patents
A kind of recombinant vector and expression of potato aphid effect protein Me23 gene Download PDFInfo
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- CN110042115A CN110042115A CN201910358654.4A CN201910358654A CN110042115A CN 110042115 A CN110042115 A CN 110042115A CN 201910358654 A CN201910358654 A CN 201910358654A CN 110042115 A CN110042115 A CN 110042115A
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Abstract
The invention discloses a kind of recombinant vectors of Potato Aphid effect protein Me23 gene, and invention additionally discloses a kind of expressions of Potato Aphid effect protein Me23 gene.The present invention constructs the prokaryotic expression carrier of Potato Aphid effect protein Me23 gene, and a series of optimization is carried out for protein expression condition, finally obtain Me23 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 Me23 for design, and then provides foundation for prevention and treatment Potato Aphid.
Description
Technical field
The present invention relates to the recombinant vectors and expression of a kind of potato aphid effect protein Me23 gene, belong to life
Object technical field.
Background technique
Potato is that a kind of important industrial crops prevent potato disease with the rapid growth that potato produces
Controlling just more seems important.Potato aphid (Macrosiphum euphorbiae) is the important pests on potato.They
Not only feeding plant phloem juice but also many destructive viroses of plant can be propagated, vegetables and grain-production was caused great
Damage.
Severity whether generation with prevalence of entomophila disease, depends on virus-Insect-Plant three's interaction.
In plant and plant-feed insect defence with the anti-game defendd, when plant experiences the signal of plant-feed insect, energy is rapidly
The relevant immune signal transduction of plant-feed insect molecular pattern is activated, the transcriptional expression of numerous resistance related genes is then regulated and controled,
The synthesis of defensive substance is mediated, to improve the resilience of plants against insects infringement.In order to which the resistance for coping with plant is immune, plant
Feeding habits insect can secrete the saliva effector of specificity to inhibit the immune activation of plant, to inhibit to plant during feeding
The expression of object defensin gene and the synthesis of resisting substance, so guarantee insect on host plant normal feeding, grow with it is numerous
Spread out.In recent years, the report for constantly thering is insect effect protein gene to be cloned, such as Bemisia tabaci, root-knot nematode, cereal crop spore
Effect protein gene in capsule nematode etc. has also successively been 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.Potato aphid is imitated at present
Answer albumen Me23 gene to have been found, but how it to be expressed with soluble form be still this field problem.The present invention
By select different bacterium bacterial strain, Me23 albumen is screened using the concentration of different inducing temperatures and inducer IPTG can
The condition of dissolubility expression, lays the foundation for researchs such as gene function, protein crystals.
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 potato aphid effect protein Me23 genes
Recombinant vector and expression.
The technical scheme is that a kind of recombinant vector of potato aphid effect protein Me23 gene, described heavy
Group carrier the preparation method is as follows: extracting horse bell RNA, and reverse transcription goes out cDNA；Using cDNA as template amplification Me23 gene；It will
Me23 amplified production is inserted into the pET-28a table through same double digestion after BamHI and XhoI double digestion, through DNA ligase
Up in carrier, recombinant plasmid pET-28a-Me23 is obtained.
Preferably, using cDNA as template, using following primer amplification Me23 gene, primer sequence is as follows:
Upstream primer: Me23-F:GCGAATTCATGAGGGTTTCCCAAGGATTT underscore is BamHI restriction enzyme site；
Downstream primer: Me23-R:CGCTCGAGTTAGCAACATTGGTCCTTTAACAGT underscore is XhoI digestion position
Point；
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 potato aphid effect protein Me23 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 Me23 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 recombination Me23 is added in plain fluid nutrient medium, after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed Me23 recombinant protein.
Preferably, when earthquake culture is to OD600=0.5 in step (3), the IPTG of final concentration of 0.8mM is added, 37
DEG C Fiber differentiation.
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 Me23 soluble protein, potato aphid effect is constructed
The prokaryotic expression carrier of albumen Me23 gene, and a series of optimization is carried out for protein expression condition, discovery is when induction temperature
When degree is 37 DEG C, expression quantity is best；With the increase of IPTG concentration, expressing quantity is gradually increased, and best induced concentration is
0.8mM；At 37 DEG C, when IPTG concentration is 0.8, expression quantity is best.Through SDS-PAGE electrophoresis detection, obtaining molecular weight is about
The purpose band of 16kDa finally obtains Me23 soluble protein for the expected size of Me23 albumen.Meanwhile present invention albumen is pure
Change system purifies it, as a result further research albumin crystal structure and biological characteristics lay the foundation, for design
Thinking is provided for the pesticide manufacturing of target for Me23, and then provides foundation for prevention and treatment Potato Aphid.
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 BamHI and XhoI inscribe as a result, M is
DNA Marker；
Fig. 3 is the albumen peak figure that Me23 albumen is obtained by the purifying of AKTA protein purification system and desalination；
Fig. 4 is the electrophoresis result figure after Me23 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 potato aphid effect protein Me23 gene recombined vector
1, horse bell RNA is extracted, and reverse transcription goes out cDNA
Potato aphid tissue is taken, is extracted using RNA extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.)
Seedling stage total serum IgE obtains cDNA with reverse transcription reagent box (Promega) reverse transcription.
2, using cDNA as template amplification Me23 gene；
Design primer expands Me23 gene using cDNA as template, the nucleotide sequence of Me23 gene such as SEQ ID
Shown in NO.1, the amino acid sequence of the protein encoded as Me23 gene is as shown in SEQ ID NO.2.
Primer is as follows:
Upstream primer: Me23-F:GCGAATTCATGAGGGTTTCCCAAGGATTT underscore is BamHI restriction enzyme site；
Downstream primer: Me23-R:CGCTCGAGTTAGCAACATTGGTCCTTTAACAGT underscore is XhoI digestion position
Point；
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 are drawn
Object 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 DEG C
5min。
3, Me23 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 BamHI 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-Me23 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 Me23 coding sequence obtained fulfills the expectation, explanation
Construction of recombinant plasmid success is simultaneously named as pET-28a-Me23, i.e. recombinant vector.
The inducing expression of embodiment 2:Me23 albumen
1, the recombined pronucleus expression bacterial strain of Me23 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-Me23
Recombinant expression carrier extracts, and recombinant expression carrier is taken to convert e. coli bl21 (DE3), Rosetta (DE3), BL21
(DE3) pLysS, JM109 (DE3) bacterial strain detects the expression of Me23 albumen.
2, activated strains are incubated overnight
It is incubated overnight and activates above-mentioned recombined pronucleus expression bacterial strain.For example, BL21 (DE3), JM109 (DE3) bacterial strain are turned
It is connected in 50ug/mL kanamycins fluid nutrient medium, Rosetta (DE3), BL21 (DE3) pLysS bacterial strain is transferred to 50ug/
In mL kanamycins+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), JM109 (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 concussion trainings
When supporting to OD600=0.5, the IPTG of final concentration of 0.8mM is added, 25 DEG C Fiber differentiation 10 hours, obtain the recombination of ME10
Albumen.
4, the purifying of Me23 recombinant protein
After the completion of induction, expressed Me23 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-Me23 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.Me23 albumen is purified by AKTA protein purification system
The albumen peak figure arrived.
Fig. 4 is the electrophoretogram after Me23 protein purification.It is the expression and purification Me23 egg at 37 DEG C, IPTG concentration 0.8mM
White result.1 is the efflux after Ni column is affine after purification；2 albumen after Ni column is affine after purification；M is albumen
marker；Arrow meaning is shown as Me23 albumen.Fig. 4 shows that Me23 albumen often has in supernatant under this condition, is solvable
Property expression, the Me23 purity of protein obtained after purification by AKTA protein purification system reaches requirement, can be used for subsequent experimental.
The verifying of the protein induced expression condition of embodiment 3:Me23
1, the recombinant strains of Me23 are obtained
It chooses the successful monoclonal of sequencing to be inoculated into 50ug/mL kanamycins fluid nutrient medium, 37 DEG C, 200rpm trains overnight
It supports, according to Nanjing Vazyme Biotechnology Co., Ltd.'s small amount plasmid extraction kit by pET-28a-Me23 recombinant expression carrier
Extract, take recombinant expression carrier convert respectively e. coli bl21 (DE3), Rosetta (DE3), BL21 (DE3) pLysS,
JM109 (DE3) bacterial strain, being coated on the plate of kanamycins containing 50ug/mL, (Rosetta (DE3), BL21 (DE3) pLysS bacterial strain need
Add 50ug/mL chloramphenicol) on be incubated overnight in 37 DEG C, picking single colonie is inoculated into the Liquid Culture containing corresponding antibiotic
In base, 37 DEG C, 200rpm be incubated overnight, in bacterium solution be added sterilizing 50% glycerol, frozen after mixing in -80 DEG C of refrigerators, obtained
Express the prokaryotic expression bacterial strain of Me23.
2, the determination of Me23 Primary structure bacterial strain
Be inoculated with respectively above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3), Rosetta (DE3), BL21 (DE3) pLysS,
To 50ug/mL kanamycins, (Rosetta (DE3), that BL21 (DE3) pLysS bacterial strain need to add 50ug/mL chlorine to JM109 (DE3) is mould
Element) in fluid nutrient medium, 37 DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to
50ug/mL kanamycins (Rosetta (DE3), BL21 (DE3) pLysS bacterial strain need to add 50ug/mL chloramphenicol) fluid nutrient medium
In, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 1mM is added, (is taken within Fiber differentiation 13 hours at 16 DEG C
PET-28a bacterium makees negative control).4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min collects thallus, bacterial precipitation and feminine gender
Control is suspended with 2mL pre-cooling PBS buffer (with preceding addition 1mM PMSF), carries out clasmatosis with Ultrasonic Cell Disruptor, every time
10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes supernatant, and SDS-PAGE electrophoresis detection tests table
Up to bacterial strain, as a result as shown in figure 5, BL21 (DE3), Rosetta (DE3), BL21 (DE3) pLysS, JM109 (DE3) can be preferable
Express Me23 albumen.
3, the determination of the best IPTG concentration of Me23 Primary structure
Above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3) is inoculated with into 50ug/mL kanamycins fluid nutrient medium, 37
DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to the training of 50ug/mL kanamycins liquid
It supports in base, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0,0.6,0.8,1.0mM is separately added into, 25
DEG C Fiber differentiation 8 hours (bacterium not induced is taken to make negative control).4 DEG C after the completion of induction, 1,2000rpm centrifugation 2min is collected
Thallus, bacterial precipitation and negative control 2mL are pre-chilled PBS buffer (with preceding addition 1mM PMSF) and suspend, and use Ultrasonic Cell Disruptor
Progress clasmatosis, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes supernatant, SDS-
PAGE electrophoresis detection tests best IPTG induced concentration, as a result sees Fig. 6, and the best IPTG concentration of Me23 inducing expression is 0.8mM.
4, the determination of Me23 Primary structure optimum temperature
Above-mentioned recombined pronucleus expression bacterial strain BL21 (DE3) is inoculated with into 50ug/mL kanamycins fluid nutrient medium, 37
DEG C, 200rpm be incubated overnight activated strains.The recombined pronucleus expression bacterial strain of activation is transferred to the training of 50ug/mL kanamycins liquid
Support in base, when 37 DEG C of shake cultures are to OD600=0.5, the IPTG of final concentration of 0.8mM be added, respectively 16,20,25,30,
With 37 DEG C Fiber differentiation 8-16 hours (pET-28a bacterium is taken to make negative control).4 DEG C after the completion of induction, 1,2000rpm rpm centrifugation
2min collects thallus, bacterial precipitation and negative control 2mL and PBS buffer (with preceding addition 1mM PMSF) suspension is pre-chilled, uses
Ultrasonic Cell Disruptor progress clasmatosis, each 10sec, totally 10 times, every minor tick 30sec；1,2000rpm centrifugation 10min, takes
Clearly, SDS-PAGE electrophoresis detection tests best inducing temperature, as a result as shown in fig. 7, best inducing temperature is 25 DEG C.
The western blot of embodiment 4:Me23 recombinant protein is detected
(1) it extracts by Me23 albumen after purification:
1. cultivating the E. coli expression strains containing pET-28a-Me23 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 Me23 albumen out.
Nucleotide series table:
SEQUENCE LISTING
Sequence table
<110>Guizhou University
<120>a kind of recombinant vector and expression of potato aphid effect protein Me23 gene
<160> 4
<210>1
<211>636
<212>DNA
<213>potato aphid effect protein Me23
<400>1
ATGAGGGTTT CCCAAGGATT TCCCTACGAG TCTCTCGACT GGCCGACAAT GCCCGACATT 60
TCTGGATCGT CGCCCACAGG ATACGACAAA TCGTCGTCCT CCGGATACGA CAAATCGTCG 120
GATGAGGATA ACTGCAATGA GGACTCGTGC GGCAACATTT ACAAATTCAC CGCAAGAAAA 180
CCCAACGGAC AAAACGTTTG TTTGCAACAA TACGTTGGAA AAGTGTTGAT CATTGTGAAT 240
TACGCTTCAG CATGTGGTTT CACATACGAC AACGTTTGCA CTCTATCGGA CTTTGCTCAA 300
AGGTACAGGA AATGTGGCTT GGAGATACTG GTATTTCCCA GCAACGACTT CTTACAGAAC 360
TTCGGTGGGG ATATCGCAGC CGAAGAGTTC GCAAATAATC ATCCTGAATT TGAAGTATTT 420
TCGGAAATTT GTGTAAATGG CAGATCACAA CATCCTTTGT ACAGATTTCT CAAGAATAAG 480
TTACCGGGTG CTTGTAACGC AAAACCTATT AAATGGAATT TTACCAAATT CGTAGTAGAC 540
AGGAACGGAT GTCCCGTTCA ACGATATGCG GCCACAGACA GTTTTAAGGA CATAGAAGAT 600
TTAGTACAAG AACTGTTAAA GGACCAATGT TGCTAA 636
<210>2
<211>210
<212> PRT
<213>potato aphid effect protein Me23
<400>2
RVSQGFPYES LDWPTMPDIS GSSPTGYDKS SSSGYDKSSD EDNCNEDSCG NIYKFTARKP 60
NGQNVCLQQY VGKVLIIVNY ASACGFTYDN VCTLSDFAQR YRKCGLEILV FPSNDFLQNF 120
GGDIAAEEFA NNHPEFEVFS EICVNGRSQH PLYRFLKNKL PGACNAKPIK WNFTKFVVDR 180
NGCPVQRYAA TDSFKDIEDL VQELLKDQCC 210
<210>3
<211>29
<212>DNA
<213>artificial sequence
<400>3
GCGAATTCAT GAGGGTTTCC CAAGGATTT 29
<210>4
<211>33
<212>DNA
<213>artificial sequence
<400>4
CGCTCGAGTT AGCAACATTG GTCCTTTAAC AGT 33
Claims (6)
1. a kind of recombinant vector of potato aphid effect protein Me23 gene, which is characterized in that the system of the recombinant vector
Preparation Method is as follows: extracting horse bell RNA, and reverse transcription goes out cDNA；Using cDNA as template amplification Me23 gene；By Me23 amplified production
After BamHI and XhoI double digestion, it is inserted into the pET-28a expression vector through same double digestion, is obtained by DNA ligase
Recombinant plasmid pET-28a-Me23.
2. recombinant vector according to claim 2, which is characterized in that using cDNA as template, utilize following primer amplification
Me23 gene, primer sequence are as follows:
Upstream primer: Me23-F:GCGAATTCATGAGGGTTTCCCAAGGATTT underscore is BamHI restriction enzyme site；
Downstream primer: Me23-R:CGCTCGAGTTAGCAACATTGGTCCTTTAACAGT 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 potato aphid effect protein Me23 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 Me23
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 recombination Me23 is added in body culture medium, after shake culture；
(4) it after the completion of inducing, recycles and purifies expressed Me23 recombinant protein.
4. the method for expression potato aphid effect protein Me23 gene according to claim 3, which is characterized in that step
Suddenly when earthquake culture is to OD600=0.5 in (3), the IPTG of final concentration of 0.8mM is added, in 37 DEG C of Fiber differentiations.
5. the method for expression potato aphid effect protein Me23 gene according to claim 3, which is characterized in that institute
Stating kanamycins in kanamycins fluid nutrient medium is 50ug/mL.
6. the method for expression potato aphid effect protein Me23 gene according to claim 3, which is characterized in that institute
Stating kanamycins in kanamycins+chloramphenicol fluid nutrient medium is 50ug/mL, chloramphenicol 50ug/mL.
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| CN110117606A (en) * | 2019-04-29 | 2019-08-13 | 贵州大学 | A kind of recombinant vector and expression of Potato Aphid effect protein Me10 gene |
Citations (6)
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