CN108823229A - A kind of preparation method and applications recombinating Vip3 Aa albumen - Google Patents

A kind of preparation method and applications recombinating Vip3 Aa albumen Download PDF

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CN108823229A
CN108823229A CN201810350632.9A CN201810350632A CN108823229A CN 108823229 A CN108823229 A CN 108823229A CN 201810350632 A CN201810350632 A CN 201810350632A CN 108823229 A CN108823229 A CN 108823229A
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朱家勇
刘文彬
金小宝
卢雪梅
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Guangdong Pharmaceutical University
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Abstract

The present invention relates to a kind of preparation method and applications for recombinating Vip3 Aa albumen, belong to field of biological control.After the preparation method of recombination Vip3 Aa albumen of the present invention is by carrying out PCR reaction for Vip3 Aa DNA, then target gene is obtained using restriction enzyme BamH I and Xho I progress double digestion and connect to obtain Vip3 Aa/pET21b expression vector with pET21b, carrier is transferred to E.coil BL21 competent cell and obtains recombinant bacterial strain, Vip3 Aa albumen is prepared in addition IPTG inducing expression after recombinant bacterial strain grows to logarithmic growth phase, the albumen has good biological control effect for Groton bug and American cockroach, is suitble to agriculturally promoting and applying.

Description

A kind of preparation method and applications recombinating Vip3 Aa albumen
Technical field
The present invention relates to a kind of preparation method and applications for recombinating Vip3 Aa albumen, and in particular to a kind of inducing expression Vip3 Aa/pET21b/BL21 recombinant bacterial strain prepares Vip3 Aa albumen and its applies in Groton bug and American cockroach prevention and treatment, Belong to field of biological control.
Background technique
Bt bacterial strain can synthesize a kind of insecticidal proteins in vegetative growth phase, and insecticidal proteins are referred to as trophism insecticidal proteins (vegetative insecticidal proteins, Vip).Vips is broadly divided into vip1, vip2 and vip3, different vips Albumen has different insecticidal activities.Vip3 albumen is the Vip albumen of current most study.Vip3 Aa albumen is to bollworm, sweet tea The crop pests such as dish noctuid, Spodopterafrugiperda, tobacco budworm, diamondback moth, prodenia litura and black cutworm desinsection with higher Activity.Mechanism is：Vip3 albumen is hydrolyzed to activated protein, activated protein and epithelium midgut epithelial cells top by midgut proteinase Specific receptor combines, and forms duct and destroys midgut epithelial cells, leads to insect death.Vip3 Aa albumen passes through excitation apoptosis class The apoptosis of type has poisoning effect to sensibility insect.Vip3 Aa albumen is hydrolyzed to 4 kinds in insect gut Major protein product, wherein only a kind of protein hydrolysate (62KD) is the toxicity nuclear structure of Vip3 Aa albumen.Vip3 The midgut epithelial cell of Aa protein binding sensitive insect, active cell programmed death cause the dissolution of midgut epithelial cell to lead Cause insect death.Any illness is not generated to non-sensitive insect, not will lead to apoptosis and the dissolution of midgut epithelial cell.
American cockroach and the Groton bug pest widely distributed in the whole world, can carry and propagate a variety of diseases, such as mouse 40 multiple pathogens such as epidemic disease bacillus, shigella dysenteriae, Escherichia coli and polio virus.Currently, Blattidae insect is main Control method can be divided into physical control, chemical prevention and biological control.Chemical prevention is most popular method.Pest control with insecticide Agent can accumulate in human body and animal organ and fat, cause slow poisoning, while blattaria enhances shadow to the resistance of insecticide Further using for chemical insecticide is rung.It is to prevent blattaria instead of chemical insecticide using biological insecticides and its metabolin Potential promising method, such as commercial green muscardine fungus insecticide (Biopath) can be used for it is pre- control blattaria, Wuhan University opens The prevention and treatment that a kind of viral insecticide is used for cockroach is sent out.
Chinese patent application 2011104173078 discloses one plant of Soviet Union to vegetables lepidoptera pest with the high virulence of wide spectrum Cloud gold bacillus MB-15 (BacillusthuringiensisMB-15), bacterial strain deposit number are CGMCC № .5255.The bacterial strain Diamond shape insecticidal crystal protein can be formed, containing tetra- kinds of genes of cry1I, cry1Ac, cry2Ac and Vip3Aa, to diamondback moth, vegetable powder The lepidoptera pests such as butterfly, beet armyworm, prodenia litura, bollworm have efficient insecticidal activity.
Chinese patent application 2013102898486 is related to a kind of killing gene and application thereof, the nucleosides of the killing gene Acid sequence includes：(a) there is SEQ ID NO：Nucleotide sequence shown in 4；Or (b) with the nucleotide sequence isocoding of (a), It and is not SEQ ID NO：22；Or the nucleotide sequence hybridization and coding (c) limited under strict conditions with (a) or (b) has The nucleotide sequence of the protein of insecticidal activity.Killing gene of the present invention is particularly suitable for expressing in monocotyledon, significantly mentions High expression quantity, stability and the virulence of Vip3 Aa-02 insecticidal proteins；The present invention can be killed by generating in plant simultaneously The Vip3 Aa albumen of dead pink rice borer controls pink rice borer pest, to plant carries out the protection in the time of infertility, whole plant to prevent and treat pink rice borer evil The infringement of worm.By retrieval, the application report in Vip3 Aa albumen cockroach prevention and cure is had not yet to see.
Summary of the invention
One of the objects of the present invention is to provide the preparation methods of recombination Vip3 Aa albumen.
A kind of preparation method recombinating Vip3 Aa albumen, specifically includes following step：
1) building of the clone of Vip3 Aa gene and double digestion recombinant plasmid
Using Vip3 Aa DNA as template, reference sequences GenBank：L48811.1, Bacillus Thuringienisstrain AB88insecticidal protein design primer carry out PCR reaction, introduce BamH I and Xho I digestion position.Vip3 AaDNA sequence is as shown in SEQ ID NO.1, the amino acid sequence such as SEQ of the protein of coding Shown in ID NO.2.
Wherein the forward primer of PCR reaction is as shown in SEQ ID NO.3, and reverse primer is as shown in SEQ ID NO.4.PCR The reaction system of reaction is as shown in table 1.
The reaction system of 1 PCR of table reaction
PCR reaction condition：94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 60 annealing reaction 30s, 72 DEG C of amplification 3min (1000bp is per minute), 30 circulations, 72 DEG C of extension 10min.
With Bt genomic DNA, PCR amplification obtains a 2370bp or so gene, meets with notional result.The end gene F quilt A BamH I restriction enzyme site is introduced, the end R is introduced into an Xho I restriction enzyme site.Subsequent construction recombination plasmid Vip3 Aa/ Then pMD20-T carries out positive transformant PCR identification.The building and identification of PET (2) Vip3Aa/pET21b expression vector
Restriction enzyme BamH I and XhoI is respectively adopted in PCR product and prokaryotic expression plasmid pET21b and carries out double enzymes It cuts, endonuclease bamhi is recycled using DNA purification and recovery kit, all-wave length spectrophotometer detection recovery product concentration simultaneously calculates enzyme It is sliced section molal quantity, with target gene：Expression vector molar ratio is 3：1, reaction building is attached by T4 ligase and is obtained Vip3 Aa/pET21b expression vector.The 50 μ L digestion systems wherein prepared are as shown in table 2：
The digestion system of 2 restriction enzyme BamH I and Xho I of table progress double digestion
It carries out after mixing well component when endonuclease reaction, 37 DEG C of reaction 1h, the separation of 1.5% agarose gel electrophoresis 15min carefully cuts purpose band, recycles endonuclease bamhi, the detection of all-wave length spectrophotometer using DNA purification and recovery kit Recovery product concentration calculates endonuclease bamhi molal quantity according to formula 1pmol 1000bp DNA=0.66 μ g, with target gene：Table It is 3 up to carrier molar ratio：1, reaction is attached by T4 ligase, it is as shown in table 3 to prepare reaction system.
3 target gene of table and expression vector coupled reaction system form
After sample adds well, mixing is flicked, brief centrifugation, 4 DEG C of connections are overnight.10 μ L connections are added in 100 μ L DH5 α competence Product flicks mixing, places 30min, 42 DEG C of thermal shock 90s of metal bath on ice.1min is placed on ice, and 900 μ l LB culture is added Base, 37 DEG C of low-speed oscillation culture (or water-bath) 1h-1.5h.5000rpm centrifugation, removes supernatant, and it is uniform to retain the piping and druming of 200 μ L culture mediums Precipitating, is spread evenly across ammonia benzyl screening flat board.After 16-18h, picking single colonie carries out bacterium colony PCR and digestion identification.Picking is positive Clone send Invitrogen Corp. to be sequenced.The identification display of Fig. 1 Vip3 Aa/PET21b positive transformant, bacterium colony 7 have positive band, have There is 2370bp band, prompts to have imported properly Vip3 Aa gene.Therefore it subsequent bacterium is further shaken to bacterium colony 7 extract the double enzymes of plasmid It cuts.Fig. 2 is positive transformant digestion identification, and Vip3 Aa/PET21b positive transformant identifies that sample 7 extracts plasmid double digestion, It can be seen that agarose gel electrophoresis obtains 5443bp carrier and 2400 or so target gene band respectively after double digestion.With 2370bp Band, PET21b carrier are correctly inserted into Vip3 Aa gene.
(3) acquisition of Vip3 Aa/pET21b/BL21 recombinant bacterial strain
It extracts and is accredited as positive expression vector Vip3 Aa/pET21b, 100 μ L E.coil BL21 (DE3) competence add Enter 10 μ L Vip3 Aa/pET21b, flick mixing, places 30min, 42 DEG C of thermal shock 90s of metal bath on ice.1min is placed on ice, 900 μ l LB, 37 DEG C of low-speed oscillation culture (or water-bath) 1h-1.5 are added.5000rpm centrifugation, removes supernatant, retains 200 μ L culture Base blows and beats homogeneous precipitation, is spread evenly across ammonia benzyl screening flat board.After 16-18h, picking single colonie carries out bacterium colony PCR and digestion mirror Surely Vip3 Aa/pET21b/BL21 recombinant bacterial strain is obtained.
(4) inducing expression of recombinant bacterial strain
Picking Vip3 Aa/pET21b/BL21 recombinant bacterial strain single colonie is inoculated in the LB liquid training of 100 μ g/ml ammonia benzyl of 5ml It supports in base, 37 DEG C, 180rpm, shake culture 12-16h, this bacterium solution is pressed 1：100 ratio is inoculated in fresh LB Liquid Culture In base, 37 DEG C, 180rpm, shake culture to OD600Value is 0.6, and the IPTG that final concentration of 0.1mM is added is induced, 20 DEG C, Shake culture is for 24 hours under the conditions of 180rpm.The Western-Blot of Fig. 3 Vip3 Aa identifies that Western-bolt identification display induces Visible 88Kda of Vip3 Aa/PET21b/BL21 or so Vip3 Aa protein expression afterwards.
Each 1ml of bacterium solution of induction front and back is taken, 4 DEG C of 10000rpm centrifugation 10min collection thallus are resuspended in 150 μ L deionizations In water, 4 × SDS sample-loading buffer of 50 μ L is added, boiling water boiling 10min carries out 10% SDS-PAGE electroresis appraisal.Fig. 4 is Purifying protein SDS-page analysis, it is seen that 88Kda or so band.
(5) Vip3 Aa isolating and purifying and is freeze-dried
By the Vip3 Aa/pET21b bacterial strain being incubated overnight by 1：100 are inoculated into 1L culture medium, culture 2.5h to logarithm Growth period carries out inducing expression using the measured optimal conditions of above-mentioned test.12000rpm is centrifuged 10min, collects thallus.PBS Washing, centrifugation.Lysis buffer (25mmolL-1Tris-Cl, the 300mmolL- of 40mL are added by every liter of culture solution 1NaCl, 5mmolL-1 beta -mercaptoethanol, pH8.0) suspension thalline, PMSF and 1% triton x-100 is added, using ultrasonic thin Born of the same parents are crushed instrument and are crushed 12min (power 250w, work 6s, off6s), and after clasmatosis, 12000rpm is centrifuged 30min, take supernatant Carry out SDS-PAGE.
Supernatant is using 0.22 μM of filter filtering after Vip3 Aa/pET21b bacterial strain is broken.It is pumped using A and draws 10mlVip3 Supernatant after Aa/pET21b bacterial strain is broken carries out chromatographic column using the Blinding buffer of 2% imidazoles of five times of column volumes Balance carries out linear gradient elution.
Desalination：Desalting column is connected with tomographic system, 5 column volumes are rinsed with 4ml/min deionized water.5 times of column volumes 0.15M NaCl buffer chromatographic column is balanced.Using 2ml loading ring loading, using desalination program desalination：
The sample solution for collecting desalination, is placed in -80 DEG C of freezing 2h.Freeze drier is cooled to -40 DEG C in advance, and frozen samples are put Enter in vacuum chamber, cover vacuum chamber lid, opens vacuum pump.Freeze-drying 48h can reach sample albumen powder.
The present invention is another object is that be claimed the Vip3 Aa albumen being prepared according to above-mentioned preparation method.
Still a further object of the present invention is the application that Vip3 Aa albumen is claimed in prevention and treatment American cockroach or Groton bug.
The Vip3 Aa purity that the preparation method of Vip3 Aa albumen of the present invention is prepared is higher, and production efficiency is big Big to be promoted, the Vip3 Aa albumen being prepared has the effect of preferably preventing and treating American cockroach or Groton bug, has preferable Application prospect in agriculture.
Detailed description of the invention
Fig. 1 is positive transformant PCR identification.
Fig. 2 positive transformant digestion identification.
The Western-Blot of Fig. 3 Vip3 Aa identifies, wherein M, protein marker；1,Vip3 Aa/pET21b/ Before BL21 induction；2, full bacterium after Vip3 Aa/pET21b/BL21 induction.
Fig. 4 purifying protein SDS-page analysis.
Fig. 5 Vip3 Aa albumen is to American cockroach insecticidal activity.
Fig. 6 Vip3 Aa albumen is to Groton bug insecticidal activity.
Fig. 7 Vip3 Aa albumen is on the gastral influence of American cockroach American cockroach, wherein A：A, control group；B, administration group； B：Administration group；C：Control group.
Influence of Fig. 8 Vip3 Aa albumen to American cockroach crop, wherein A：Control group crop (× 200)；B：Control group crop Capsule (× 400)；C：Administration group crop (× 200)；D：Administration group crop (× 400).
Influence of Fig. 9 Vip3 Aa albumen to intestines in American cockroach, wherein A：Intestines (× 200) in control group；B：In control group Intestines (× 400)；C：Intestines (× 200) in administration group；D：Intestines (× 400) in administration group.
Influence of Figure 10 Vip3 Aa albumen to American cockroach column colon, wherein A：Control group column colon (× 200)； B：Control group column colon (× 400)；C：Administration group column colon (× 200)；D：Administration group column colon (× 400).
Influence (scanning electron microscope (SEM) photograph) of Figure 11 Vip3 Aa albumen to American cockroach crop, wherein A：Control group crop (× 500)；B：Control group crop (× 1000)；C：Administration group crop (× 500)；D：Administration group crop (× 1000)
Influence (scanning electron microscope (SEM) photograph) of Figure 12 Vip3 Aa albumen to intestines in American cockroach, wherein A：Intestines in control group (× 500)；B：Intestines (× 1000) in control group；C：Intestines (× 500) in administration group；D：Intestines (× 1000) in administration group.
Influence (scanning electron microscope (SEM) photograph) of Figure 13 Vip3 Aa albumen to American cockroach column colon, wherein A：Intestines in control group (×500)；B：Intestines (× 1000) in control group；C：Intestines (× 500) in administration group；D：Intestines (× 1000) in administration group.
Specific embodiment
The present invention is further described below by way of specific embodiment, but the embodiment does not limit this hair in any way The range of bright patent protection.
Insecticidal activity of the Vip3 Aa albumen of the present invention of experimental example 1 to American cockroach and Groton bug
2mg/ml, 0.5mg/ml, 0.125mg/ml and 0.03125mg/ml concentration Vip3 Aa albumen is taken to be configured to 1ml molten Liquid is divided into 5 groups, and Groton bug is put into Micro-Organism Culture Dish by every group of 15 Groton bugs, and 1ml drug solution is added, freely drinks Water observes the Groton bug death rate respectively at 12h, for 24 hours with 36h and 48h.
2mg/ml, 0.5mg/ml, 0.125mg/ml and 0.03125mg/m concentration Vip3 Aa albumen is taken to be configured to 1ml molten Liquid is divided into 5 groups, and American cockroach is put into conical flask by every group of 10 American cockroaches, addition 1ml drug solution, free water, Respectively at 12h, for 24 hours and 36h, observe the American cockroach death rate.
3.3.1Vip3 Aa albumen kills blattaria activity analysis
Vip3 Aa to the 12h of American cockroach, for 24 hours with 36h LD50 be respectively 0.70302mg/ml, 0.15906mg/ml and 0.15906mg/ml；Vip3 Aa to the 12h of Groton bug, for 24 hours with 36h LD50Respectively 0.54077mg/ml, 0.27321mg/ Ml and 0.17914mg/ml.It is shown in Table 3-1 and table 3-2 or Fig. 5 and Fig. 6.
Table 3-1 Vip3 Aa albumen is to American cockroach insecticidal activity (mg/ml)
Table 3-2 Vip3 Aa albumen is to Groton bug insecticidal activity
Influence of the experimental example 2Vip3 Aa albumen to American cockroach digestive system
(A) HE is dyed
(1) it anaesthetizes and dissects：Blattaria is placed in and is anaesthetized on ice, is fixed using insect needle insertion blattaria neck, removes blattaria Wing and foot cut off body wall from the non-edge of abdomen with eye scissors, and pincet rejects abdominal cavity excess tissue, alimentary canal is straightened, along pharynx Top and rectum end, which are cut, obtains the complete digestive system of blattaria.
(2) it draws materials：Knife blade cuts crop and middle intestines, column colon respectively, and sample tissue is less than 0.5cm × 0.5cm ×0.1cm。
(3) fixed and embedding：It is fixed with 4%PFA (0.01MPBS, PH7.4), through 70% ethyl alcohol 60 minutes, 85% Ethyl alcohol 60 minutes, 95% ethyl alcohol 60 minutes, 100% ethyl alcohol 50 minutes twice, transparent 30 minutes of dimethylbenzene twice, in 55 DEG C of paraffin 30 minutes three times, with stainless steel mould investing tissue block.
(4) paraffin section：4 μm of histotomy of thickness is invested the glass slide through poly-D-lysine membrane by paraffin section On, 65 DEG C of baking 2h；
(5) HE is dyed：It dewaxes, enter water：By slice be dipped in dimethylbenzene 10 minutes twice, 100% ethyl alcohol 5 minutes, 95% Ethyl alcohol 5 minutes, 85% ethyl alcohol 5 minutes, 75% ethyl alcohol 5 minutes, originally washes 5 minutes twice at 90% ethyl alcohol 5 minutes；It immerses bright Alum haematoxylin dye liquor is disseminated 15 minutes, and flowing water rinses slice 30 seconds.The differentiation of 1% hydrochloride alcohol slice 3 seconds, flowing water flushing was cut Piece 30 seconds.Light ammonium hydroxide returns 1 minute blue.1% Yihong alcohol liquid dyes 3 minutes.95% dehydration of alcohol 30 seconds.100% alcohol It is dehydrated 30 seconds twice.Dimethylbenzene 60 seconds is three times.Neutral gum mounting.
(B) scanning electron microscopic observation
(1) it draws materials：It separates in blattaria after intestines and crop, using insect physiological saline rinsed clean, each 15min.Using 2.5% glutaraldehyde fixes 30min at room temperature, and carefully tissue shear evolution is just unfolded, and it is fixed for 24 hours to be put into 4 DEG C of refrigerators.
(2) it rinses：The sample fixed is rinsed 3 times using 0.1M PBS, each 15min.
(3) it fixes afterwards：Intestines and crop are put into 1% osmic acid and fix 2h in blattaria after rinsing.
(4) it is dehydrated：Intestines and crop sample are through -85% alcohol -90% of -80% alcohol of -70% alcohol of 50% alcohol in blattaria - 95% alcohol of alcohol-absolute alcohol is dehydrated each 1h, and sample, which is put into pure acetone, replaces 20min, and it is different that sample is finally put into acetic acid 30min in pentyl ester.
(5)CO2Critical point drying：It is dry to be put into critical point after taking out in isoamyl acetate for intestines and crop sample in blattaria In the sample room of dry instrument, close the lid.After reaching critical state (31 DEG C, 72.8 atmospheric pressure), temperature is increased to 10 DEG C again, is made Liquid CO 2 gasification, then opens deflation valve, and gas is gradually discharged, and sample is completely dried.
(6) ion sputtering film coating：Skin samples after drying are placed under ion sputtering instrument, metallic is splashed to Intestines and crop sample surfaces form layer of metal film.It observes, takes pictures under Electronic Speculum.
Experimental result：
(1) Vip3 Aa albumen is on the gastral influence of American cockroach
Fig. 7 is Vip3 Aa albumen on the gastral influence of American cockroach, is found by dissection, big relative to normal America Lian alimentary canal, administration group American cockroach alimentary canal are obviously shortened；After most of administration, the expansion of American cockroach crop.
(2) Vip3 Aa albumen is to pathological observation
Fig. 8 is influence of the Vip3 Aa albumen to American cockroach crop, and American cockroach crop upper layer is thin after being administered as the result is shown Born of the same parents, which have no, to be substantially change.Fig. 9 is influence of the Vip3 Aa albumen to intestines in American cockroach, after being administered as the result is shown in American cockroach The rupture of enterocyte epithelial cell, BBMV shrinkage are detached from epithelial cell.Figure 10 is Vip3 Aa albumen to American cockroach column colon Influence, as the result is shown American cockroach column colon cell epithelial cell rupture, BBMV shrinkage, be detached from epithelial cell.Figure Influence (scanning electron microscope (SEM) photograph) of the 11Vip3 Aa albumen to American cockroach crop, American cockroach crop upper layer after being administered as the result is shown Cell, which has no, to be substantially change, and surface covers dentalation.Influence (scanning electricity of Figure 12 Vip3 Aa albumen to intestines in American cockroach Mirror figure), intestines surface dentalation disappears in American cockroach as the result is shown, exposure lower-hierarchy structure.Figure 13 is Vip3 Aa albumen Influence (scanning electron microscope (SEM) photograph) to American cockroach column colon, American cockroach column colon surface dentalation disappears as the result is shown It loses, exposure lower-hierarchy structure.
Sequence table
<110>Guangdong pharmaceutical university
<120>A kind of preparation method and applications recombinating Vip3 Aa albumen
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<170> SIPOSequenceListing 1.0
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<213> Bacillus thuringienis
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atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgaacaa gaataatact 60
aaattaagca caagagcctt accaagtttt attgattatt ttaatggcat ttatggattt 120
gccactggta tcaaagacat tatgaacatg atttttaaaa cggatacagg tggtgatcta 180
accctagacg aaattttaaa gaatcagcag ttactaaatg atatttctgg taaattggat 240
ggggtgaatg gaagcttaaa tgatcttatc gcacagggaa acttaaatac agaattatct 300
aaggaaatat taaaaattgc aaatgaacaa aatcaagttt taaatgatgt taataacaaa 360
ctcgatgcga taaatacgat gcttcgggta tatctaccta aaattacctc tatgttgagt 420
gatgtaatga aacaaaatta tgcgctaagt ctgcaaatag aatacttaag taaacaattg 480
caagagattt ctgataagtt ggatattatt aatgtaaatg tacttattaa ctctacactt 540
actgaaatta cacctgcgta tcaaaggatt aaatatgtga acgaaaaatt tgaggaatta 600
acttttgcta cagaaactag ttcaaaagta aaaaaggatg gctctcctgc agatattctt 660
gatgagttaa ctgagttaac tgaactagcg aaaagtgtaa caaaaaatga tgtggatggt 720
tttgaatttt accttaatac attccacgat gtaatggtag gaaataattt attcgggcgt 780
tcagctttaa aaactgcatc ggaattaatt actaaagaaa atgtgaaaac aagtggcagt 840
gaggtcggaa atgtttataa cttcttaatt gtattaacag ctctgcaagc ccaagctttt 900
cttactttaa caacatgccg aaaattatta ggcttagcag atattgatta tacttctatt 960
atgaatgaac atttaaataa ggaaaaagag gaatttagag taaacatcct ccctacactt 1020
tctaatactt tttctaatcc taattatgca aaagttaaag gaagtgatga agatgcaaag 1080
atgattgtgg aagctaaacc aggacatgca ttgattgggt ttgaaattag taatgattca 1140
attacagtat taaaagtata tgaggctaag ctaaaacaaa attatcaagt cgataaggat 1200
tccttatcgg aagttattta tggtgatatg gataaattat tgtgcccaga tcaatctgaa 1260
caaatctatt atacaaataa catagtattt ccaaatgaat atgtaattac taaaattgat 1320
ttcactaaaa aaatgaaaac tttaagatat gaggtaacag cgaattttta tgattcttct 1380
acaggagaaa ttgacttaaa taagaaaaaa gtagaatcaa gtgaagcgga gtatagaacg 1440
ttaagtgcta atgatgatgg ggtgtatatg ccgttaggtg tcatcagtga aacatttttg 1500
actccgatta atgggtttgg cctccaagct gatgaaaatt caagattaat tactttaaca 1560
tgtaaatcat atttaagaga actactgcta gcaacagact taagcaataa agaaactaaa 1620
ttgatcgtcc cgccaagtgg ttttattagc aatattgtag agaacgggtc catagaagag 1680
gacaatttag agccgtggaa agcaaataat aagaatgcgt atgtagatca tacaggcgga 1740
gtgaatggaa ctaaagcttt atatgttcat aaggacggag gaatttcaca atttattgga 1800
gataagttaa aaccgaaaac tgagtatgta atccaatata ctgttaaagg aaaaccttct 1860
attcatttaa aagatgaaaa tactggatat attcattatg aagatacaaa taataattta 1920
gaagattatc aaactattaa taaacgtttt actacaggaa ctgatttaaa gggagtgtat 1980
ttaattttaa aaagtcaaaa tggagatgaa gcttggggag ataactttat tattttggaa 2040
attagtcctt ctgaaaagtt attaagtcca gaattaatta atacaaataa ttggacgagt 2100
acgggatcaa ctaatattag cggtaataca ctcactcttt atcagggagg acgagggatt 2160
ctaaaacaaa accttcaatt agatagtttt tcaacttata gagtgtattt ttctgtgtcc 2220
ggagatgcta atgtaaggat tagaaattct agggaagtgt tatttgaaaa aagatatatg 2280
agcggtgcta aagatgtttc tgaaatgttc actacaaaat ttgagaaaga taacttttat 2340
atagagcttt ctcaagggaa taatttatat ggtggtccta ttgtacattt ttacgatgtc 2400
tctattaagc atcatcatca tcatcattaa 2430
<210> 2
<211> 809
<212> PRT
<213> Bacillus thuringienis
<400> 2
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Asn
1 5 10 15
Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile Asp
20 25 30
Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met
35 40 45
Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu
50 55 60
Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys Leu Asp
65 70 75 80
Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn Leu Asn
85 90 95
Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln Asn Gln
100 105 110
Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met Leu
115 120 125
Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met Lys
130 135 140
Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys Gln Leu
145 150 155 160
Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu Ile
165 170 175
Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr
180 185 190
Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser Ser
195 200 205
Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr
210 215 220
Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val Asp Gly
225 230 235 240
Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly Asn Asn
245 250 255
Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Thr Lys
260 265 270
Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn Phe
275 280 285
Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr Leu Thr
290 295 300
Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile
305 310 315 320
Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn Ile
325 330 335
Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys Val
340 345 350
Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro Gly
355 360 365
His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr Val Leu
370 375 380
Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp Lys Asp
385 390 395 400
Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu Cys Pro
405 410 415
Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe Pro Asn
420 425 430
Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys Thr Leu
435 440 445
Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile
450 455 460
Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr Arg Thr
465 470 475 480
Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile Ser
485 490 495
Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp Glu
500 505 510
Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Leu
515 520 525
Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile Val Pro
530 535 540
Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile Glu Glu
545 550 555 560
Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val Asp
565 570 575
His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys Asp
580 585 590
Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys Thr Glu
595 600 605
Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His Leu Lys
610 615 620
Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu
625 630 635 640
Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr Asp Leu
645 650 655
Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu Ala Trp
660 665 670
Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys Leu Leu
675 680 685
Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly Ser Thr
690 695 700
Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg Gly Ile
705 710 715 720
Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg Val Tyr
725 730 735
Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser Arg Glu
740 745 750
Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val Ser Glu
755 760 765
Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu Leu Ser
770 775 780
Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr Asp Val
785 790 795 800
Ser Ile Lys His His His His His His
805
<210> 3
<211> 29
<212> DNA
<213> Bacillus thuringienis
<400> 3
cgcggatcca tgaacaagaa taatactaa 29
<210> 4
<211> 47
<212> DNA
<213> Bacillus thuringienis
<400> 4
ccgctcgagt taatggtgat ggtgatgatg cttaatagag acatcgt 47

Claims (8)

1. a kind of preparation method for recombinating Vip3 Aa albumen, specifically includes following step：
1) clone of Vip3 Aa gene：
Using Vip3 Aa DNA as template, reference sequences GenBank：L48811.1, Bacillus thuringienis strain AB88 insecticidal protein design primer carries out PCR reaction, introduces BamH I and Xho I restriction enzyme site；
2) building and identification of Vip3 Aa/pET21b expression vector：
Restriction enzyme BamH I and Xho I is respectively adopted in PCR product and prokaryotic expression plasmid pET21b and carries out double digestion, Endonuclease bamhi is recycled using DNA purification and recovery kit, all-wave length spectrophotometer detection recovery product concentration simultaneously calculates digestion Segment molal quantity, with target gene：Expression vector molar ratio is 3：1, reaction building is attached by T4 ligase and is obtained Vip3 Aa/pET21b expression vector；
3) acquisition of Vip3 Aa/pET21b/BL21 recombinant bacterial strain
10 μ L are added into 100 μ L E.coil BL21 competence and extract the Vip3 Aa/pET21b expression load for being accredited as the positive Body places 30min, thermal shock 90s at 42 DEG C of metal bath after flicking mixing on ice, and 900 μ are added thereto after placing 1min on ice LLB culture medium, low-speed oscillation culture 1h-1.5h at 37 DEG C；Supernatant is removed in 5000rpm centrifugation, and precipitating is cultivated using 200 μ L LB Base piping and druming uniformly, is spread evenly across ammonia benzyl screening flat board；After continuous culture 16-18h, picking single colonie carries out bacterium colony PCR and enzyme It cuts identification and obtains Vip3 Aa/pET21b/BL21 recombinant bacterial strain；
4) inducing expression of recombinant bacterial strain
Picking Vip3 Aa/pET21b/BL21 recombinant bacterial strain single colonie is inoculated in the LB liquid training that 5ml contains 100 μ g/ml ammonia benzyls It supports in base, shake culture 12-16h under the conditions of 37 DEG C of 180rpm, this bacterium solution is pressed 1：100 ratio is inoculated in fresh LB liquid In culture medium, shake culture is to OD under the conditions of 37 DEG C of 180rpm600Value is 0.6, and the IPTG that final concentration of 0.1mM is added is lured It leads, 20 DEG C, continues shake culture for 24 hours under the conditions of 180rpm；
5) Vip3 Aa albumen isolating and purifying and is freeze-dried
The Vip3 Aa/pET21b/BL21 recombinant bacterial strain 12000rpm of inducing expression is centrifuged 10min, collects thallus, PBS washing Centrifugation；40mL ratio is added in every liter of culture solution, lysis buffer (25mmolL-1Tris-Cl, 300mmolL- is added 1NaCl, 5mmolL-1 beta -mercaptoethanol, pH8.0) suspension thalline, PMSF and 1% triton x-100 is added, using ultrasonic thin Born of the same parents are crushed instrument and are crushed 12min, and after clasmatosis, 12000rpm is centrifuged 30min, and supernatant is taken to carry out SDS-PAGE；
Supernatant draws 10ml Vip3 Aa/pET21b using 0.22 μM of filter filtering after Vip3 Aa/pET21b bacterial strain is broken Chromatographic column on supernatant after bacterial strain is broken, carries out chromatographic column using the Blinding buffer of 2% imidazoles of five times of column volumes Balance, then carries out linear gradient elution；
Vip3 Aa albumen desalination and freeze-drying：Desalting column is connected with tomographic system, rinses 5 with 4ml/min deionized water The 0.15M NaCl buffer of column volume, 5 times of column volumes is balanced chromatographic column, using 2ml loading ring loading, using desalination The sample solution of desalination is collected in program desalination, is placed in -80 DEG C of freezing 2h, and freeze drier is cooled to -40 DEG C in advance, is freeze-dried 48h Up to Vip3 Aa albumen.
2. the preparation method of recombination Vip3 Aa albumen according to claim 1, which is characterized in that PCR in the step 1) The forward primer of reaction is shown in SEQ ID NO.3, and the reverse primer of PCR reaction is shown in SEQ ID NO.4.
3. the preparation method of recombination Vip3 Aa albumen according to claim 1, which is characterized in that PCR in the step 1) The group of reaction system becomes：Template 1.0 μ l, 10 × PCRbuffer 5.0 μ l, ddH235.5 μ l, 10mM dNTP Mix 4.0 of O 0.5 μ l of μ l, F primer 2 .0 μ l, R primer 2 .0 μ l, La Taq；PCR reaction condition：94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 60 annealing reaction 30s, 72 DEG C of amplification 3min, 30 recycle, 72 DEG C of extension 10min.
4. the preparation method of recombination Vip3 Aa albumen according to claim 1, which is characterized in that connect in the step 2) The reversed reaction system answered is Vip3 Aa endonuclease bamhi 0.3pmol, pET21b endonuclease bamhi 0.1pmol, ddH2O Up to 10 1 μ L, T4 DNA Ligase of μ L, 10 × buffer, 0.5 μ L.
5. the preparation method of recombination Vip3 Aa albumen according to claim 1, which is characterized in that connect in the step 2) The reversed identification method for answering positive products is：10 μ L connection products are added in 100 μ L DH5 α competence, flick mixing, place on ice 30min, 42 DEG C of thermal shock 90s of metal bath place 1min on ice, and 900 μ l LB culture mediums, 37 DEG C of low-speed oscillation culture 1h- are added 1.5h；5000rpm centrifugation, removes supernatant, retains 200 μ L culture mediums and blows and beats homogeneous precipitation, is spread evenly across ammonia benzyl screening flat board, After 16-18h, picking single colonie carries out bacterium colony PCR and digestion identification.
6. the preparation method of recombination Vip3 Aa albumen according to claim 1, which is characterized in that split in the step 5) Solve buffer group become 25mmolL-1Tris-HCl, 300mmolL-1NaCl, 5mmolL-1 beta -mercaptoethanol, pH8.0。
7. a kind of Vip3 Aa albumen being prepared according to any preparation method of claim 1-6.
Application of the 8.Vip3 Aa albumen in prevention and treatment American cockroach or Groton bug.
CN201810350632.9A 2018-04-18 2018-04-18 Preparation method and application of recombinant Vip3Aa protein Expired - Fee Related CN108823229B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114907458A (en) * 2022-05-10 2022-08-16 山东大学 Vip3A mutant protein with improved activity and application thereof
CN115785235A (en) * 2022-09-19 2023-03-14 隆平生物技术(海南)有限公司 Vip3Aa truncated protein variant, and vector and application thereof
CN116693700A (en) * 2023-08-01 2023-09-05 百葵锐(深圳)生物科技有限公司 Protein RNA complex for hair directional binding and delivery

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103204912A (en) * 2013-04-27 2013-07-17 中国农业科学院植物保护研究所 Insecticidal gene cry2Ah-like with high toxicity on lepidoptera pest and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103204912A (en) * 2013-04-27 2013-07-17 中国农业科学院植物保护研究所 Insecticidal gene cry2Ah-like with high toxicity on lepidoptera pest and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ESTRUCH,J.J. ET AL: "Bacillus thuringienis strain AB88 insecticidal protein (vip3A(a)) gene, complete cds", 《GENBANK: L48811.1》 *
JUAN J. ESTRUCH ET AL: "Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects", 《PROC. NATL. ACAD. SCI. USA》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114907458A (en) * 2022-05-10 2022-08-16 山东大学 Vip3A mutant protein with improved activity and application thereof
CN114907458B (en) * 2022-05-10 2023-12-22 山东大学 Vip3A mutant protein with improved activity and application thereof
CN115785235A (en) * 2022-09-19 2023-03-14 隆平生物技术(海南)有限公司 Vip3Aa truncated protein variant, and vector and application thereof
CN115785235B (en) * 2022-09-19 2023-11-17 隆平生物技术(海南)有限公司 Vip3Aa truncated protein variant and vector and application thereof
CN116693700A (en) * 2023-08-01 2023-09-05 百葵锐(深圳)生物科技有限公司 Protein RNA complex for hair directional binding and delivery
CN116693700B (en) * 2023-08-01 2023-09-29 百葵锐(深圳)生物科技有限公司 Protein RNA complex for hair directional binding and delivery

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