CN108823229B - Preparation method and application of recombinant Vip3Aa protein - Google Patents

Preparation method and application of recombinant Vip3Aa protein Download PDF

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CN108823229B
CN108823229B CN201810350632.9A CN201810350632A CN108823229B CN 108823229 B CN108823229 B CN 108823229B CN 201810350632 A CN201810350632 A CN 201810350632A CN 108823229 B CN108823229 B CN 108823229B
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朱家勇
刘文彬
金小宝
卢雪梅
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Abstract

The invention relates to a preparation method and application of a recombinant Vip3Aa protein, and belongs to the field of biological control. The preparation method of the recombinant Vip3Aa protein comprises the steps of carrying out PCR reaction on Vip3Aa DNA, carrying out double enzyme digestion on restriction enzymes BamH I and Xho I to obtain a target gene, connecting the target gene with pET21b to obtain a Vip 3/pET 21b expression vector, transferring the vector into E.coil BL21 competent cells to obtain a recombinant strain, adding IPTG (isopropyl-beta-thiogalactopyranoside-thiogalactoside) after the recombinant strain grows to a logarithmic growth phase, and carrying out induced expression to obtain the Vip3Aa protein.

Description

Preparation method and application of recombinant Vip3Aa protein
Technical Field
The invention relates to a preparation method and application of a recombinant Vip3Aa protein, in particular to a Vip3Aa protein prepared by inducing and expressing a Vip3Aa/pET21 b/BL21 recombinant strain and application thereof in controlling German cockroach and American cockroach, belonging to the field of biological control.
Background
Bt strains can synthesize an insecticidal protein called vegetative insecticidal proteins (Vip) at vegetative growth stages. Vips are largely divided into vip1, vip2 and vip3, with different Vips proteins having different pesticidal activities. The Vip3 protein is currently the most studied Vip protein. The Vip3Aa protein has higher insecticidal activity on crop pests such as cotton bollworm, beet armyworm, spodoptera frugiperda, tobacco budworm, diamond back moth, prodenia litura, black cutworm and the like. The action process is as follows: the Vip3 protein is hydrolyzed by midgut protease to an active protein that binds to epithelial midgut cell apical specific receptors, forming tunnels and destroying midgut cells, leading to insect death. The Vip3Aa protein has a poisoning effect on sensitive insects by triggering apoptotic-type apoptosis. The Vip3Aa protein is hydrolyzed to 4 major protein products in the insect gut, of which only one (62KD) is the toxic core structure of Vip3Aa protein. The Vip3Aa protein binds to the midgut epithelial cells of sensitive insects, initiating apoptosis, causing lysis of the midgut epithelial cells leading to insect death. It has no disease to non-sensitive insects, and will not cause apoptosis and lysis of midgut epithelial cells.
Periplaneta americana and Blattella germanica are pests widely distributed worldwide, which can carry and transmit various diseases, such as more than 40 pathogens, e.g., plague bacillus, dysentery bacillus, Escherichia coli, polio virus, etc. Currently, the main control methods for insects of the blattaceae family can be divided into physical control, chemical control and biological control. Chemical control is the most widely used method. Chemical insecticides can accumulate in human and animal organs and fats, causing chronic poisoning, while the enhanced resistance of cockroaches to insecticides affects the further use of chemical insecticides. The use of biological insecticides and their metabolites in place of chemical insecticides to prevent cockroaches is a potentially promising approach, as commercial metarhizium anisopliae insecticides (Biopath) have been used to prevent cockroaches, and the university of Wuhan developed a viral insecticide for the control of cockroaches.
Chinese patent application 2011104173078 discloses a Bacillus thuringiensis MB-15(Bacillus thuringiensis MB-15) with broad-spectrum high toxicity to vegetable lepidopteran pests, and the preservation number of the strain is CGMCC No. 5255. The strain can form diamond insecticidal crystal protein, contains cry1I, cry1Ac, cry2Ac and Vip3Aa genes, and has efficient insecticidal activity on lepidoptera pests such as diamondback moth, cabbage butterfly, beet armyworm, prodenia litura, cotton bollworm and the like.
Chinese patent application 2013102898486 relates to an insecticidal gene and its use, the nucleotide sequence of the insecticidal gene includes: (a) has the sequence shown in SEQ ID NO: 4; or (b) encodes the same nucleotide sequence as (a) and is not SEQ ID NO: 22; or (c) a nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) or (b) under stringent conditions and encodes a protein having pesticidal activity. The insecticidal gene is particularly suitable for expression in monocotyledons, and the expression quantity, stability and toxicity of Vip3 Aa-02 insecticidal protein are obviously improved; meanwhile, the invention controls the pests of the sesamia inferens by generating Vip3Aa protein capable of killing the sesamia inferens in the plant body, and protects the plant in the whole growth period and the whole plant to prevent and control the damage of the pests of the sesamia inferens. Through retrieval, no application report of Vip3Aa protein in cockroach control is found at present.
Disclosure of Invention
One of the objects of the present invention is to provide a method for producing a recombinant Vip3Aa protein.
A preparation method of a recombinant Vip3Aa protein specifically comprises the following steps:
1) cloning of Vip3Aa gene and construction of double-restriction enzyme recombinant plasmid
Using Vip3Aa DNA as a template, and a reference sequence GenBank: l48811.1, Bacillus thuringiensis AB88 immunogenic protein designed primer for PCR reaction, introducing BamH I and Xho I enzyme cutting site. The Vip3 AaDNA sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.
Wherein the forward primer of the PCR reaction is shown as SEQ ID NO.3, and the reverse primer is shown as SEQ ID NO. 4. The reaction system of the PCR reaction is shown in Table 1.
TABLE 1 reaction System for PCR reaction
Figure BDA0001633317510000021
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, amplification at 72 ℃ for 3min (1000bp per minute), 30 cycles, and extension at 72 ℃ for 10 min.
Bt genome DNA is used for PCR amplification to obtain a gene about 2370bp, which accords with a theoretical result. The F end of the gene is introduced into a BamH I restriction site, and the R end is introduced into an Xho I restriction site. Subsequently, a recombinant plasmid Vip3 Aa/pMD20-T is constructed, and then positive transformant PCR identification is carried out. Construction and identification of PET (2) Vip3Aa/pET21b expression vector
Performing double enzyme digestion on the PCR product and the prokaryotic expression plasmid pET21b by respectively adopting restriction enzymes BamH I and XhoI, recovering enzyme digestion fragments by adopting a DNA purification recovery kit, detecting the concentration of the recovered product by using a full-wavelength spectrophotometer and calculating the mole number of the enzyme digestion fragments, and performing the following steps of: the expression vector molar ratio is 3: 1, carrying out ligation reaction by using T4 ligase to construct a Vip3Aa/pET21b expression vector. The prepared 50. mu.L enzyme digestion system is shown in Table 2:
TABLE 2 restriction systems for the double cleavage of the restriction enzymes BamH I and Xho I
Figure BDA0001633317510000031
The components are fully mixed in the enzyme digestion reaction, the reaction is carried out for 1h at 37 ℃, 1.5% agarose gel electrophoresis separation is carried out for 15min, a target band is carefully cut, a DNA purification recovery kit is adopted to recover enzyme digestion fragments, a full-wavelength spectrophotometer is used to detect the concentration of the recovered product, the molar number of the enzyme digestion fragments is calculated according to the formula 1pmol 1000bp DNA which is equal to 0.66 mu g, and the target gene is used: the expression vector molar ratio is 3: 1, ligation reaction was carried out by T4 ligase to prepare reaction systems as shown in Table 3.
TABLE 3 ligation reaction System composition of target genes and expression vectors
Figure BDA0001633317510000032
Figure BDA0001633317510000041
After the sample is added, the mixture is flicked and mixed, and then the mixture is instantaneously centrifuged and connected at 4 ℃ overnight. mu.L of DH 5. alpha. was competed with 10. mu.L of the ligation product, gently mixed, placed on ice for 30min, and heat-shocked in a metal bath at 42 ℃ for 90 s. Placing on ice for 1min, adding 900 μ l LB culture medium, and performing low speed shaking culture (or water bath) at 37 deg.C for 1-1.5 h. Centrifuging at 5000rpm, removing supernatant, reserving 200 μ L of culture medium, blowing to precipitate uniformly, and coating on ampicillin screening plate uniformly. After 16-18h, picking single colony for colony PCR and enzyme digestion identification. Positive clones were picked for sequencing by the Weijie funding company. FIG. 1 identification of Vip3Aa/PET21 b-positive transformants revealed that colony 7 had a positive band, and a 2370bp band, indicating that the Vip3Aa gene had been correctly introduced. Therefore, the colony 7 is further shaken to extract plasmid double enzyme digestion. FIG. 2 shows the restriction enzyme identification of positive transformant, the restriction enzyme identification of Vip3Aa/PET21b positive transformant, the double restriction enzyme of plasmid extracted from sample 7, and the agarose gel electrophoresis after the double restriction enzyme is observed to respectively obtain 5443bp vector and target gene band of about 2400. The vector has a 2370bp band, and the PET21b vector is correctly inserted into a Vip3Aa gene.
(3) Obtaining of Vip3Aa/pET21 b/BL21 recombinant Strain
Extracting expression vector Vip3Aa/pET21b identified as positive, adding 10 μ L Vip3Aa/pET21b into 100 μ L E.coil BL21(DE3) competence, flicking, mixing, standing on ice for 30min, and heating in metal bath at 42 deg.C for 90 s. Standing on ice for 1min, adding 900 μ l LB, and low speed shaking culturing (or water bath) at 37 deg.C for 1 h-1.5. Centrifuging at 5000rpm, removing supernatant, reserving 200 μ L of culture medium, blowing to precipitate uniformly, and coating on ampicillin screening plate uniformly. After 16-18h, picking a single colony, and carrying out colony PCR and enzyme digestion identification to obtain a Vip3Aa/pET21 b/BL21 recombinant strain.
(4) Inducible expression of recombinant strains
A single colony of the Vip3Aa/pET21 b/BL21 recombinant strain is picked up and inoculated into 5ml of LB liquid culture medium with 100 mu g/ml of ampicillin, the mixture is shake-cultured for 12 to 16 hours at 37 ℃ and 180rpm, and the bacterial liquid is prepared according to the weight ratio of 1: 100 percent of the total weight of the culture medium is inoculated into a fresh LB liquid culture medium, the temperature is 37 ℃, the rpm is 180, and the culture is performed with shaking until the OD is reached600The value was 0.6, and induction was carried out by adding IPTG at a final concentration of 0.1mM and shaking-culturing at 20 ℃ and 180rpm for 24 hours. FIG. 3 is Western-Blot identification of Vip3Aa, which shows Vip3Aa/PET21 b/BL21 shows Vip3Aa protein expression around 88Kda after induction.
Taking 1ml of bacterial liquid before and after induction, centrifuging at 4 ℃ and 10000rpm for 10min, collecting thalli, suspending in 150 mu L of deionized water, adding 50 mu L of 4 xSDS loading buffer solution, boiling in boiling water for 10min, and performing 10% SDS-PAGE electrophoretic identification. FIG. 4 shows SDS-page analysis of purified protein, showing a band around 88 Kda.
(5) Separation and purification and freeze drying of Vip3Aa
The overnight cultured Vip3Aa/pET21b strain was cultured in the following manner of 1: 100 were inoculated into 1L of medium, cultured for 2.5h to logarithmic growth phase, and induced to express using the optimum conditions determined in the above experiment. Centrifugation was carried out at 12000rpm for 10min to collect the cells. Washed with PBS and centrifuged. 40mL of lysis buffer (25 mmol. L-1Tris-Cl, 300 mmol. L-1NaCl, 5 mmol. L-1. beta. -mercaptoethanol, pH8.0) was added per liter of the culture medium to suspend the cells, PMSF and 1% Triton X-100 were added, the cells were disrupted with an ultrasonic cell disrupter for 12min (power 250w, work 6s, off6s), and after cell disruption, the cells were centrifuged at 12000rpm for 30min, and the supernatant was subjected to SDS-PAGE.
After disruption of Vip3Aa/pET21b strain, the supernatant was filtered through a 0.22. mu.M filter. 10ml of Vip3Aa/pET21b strain is sucked by an A pump, supernatant after the strain is crushed is adopted, the chromatographic column is balanced by a 2 percent imidazole blanking buffer with five times of column volume, and linear gradient elution is carried out.
Desalting: the desalting column was connected to a chromatography system and 5 column volumes were washed with 4ml/min deionized water. The column was equilibrated with 5 column volumes of 0.15M NaCl buffer. Loading with 2ml loading ring, desalting with desalting procedure:
the desalted sample solution was collected and frozen at-80 ℃ for 2 h. Pre-cooling to-40 deg.c in a freeze drier, setting the frozen sample inside a vacuum cavity, covering the vacuum cavity with cover and turning on the vacuum pump. And freeze-drying for 48 hours to obtain the sample protein powder.
Another object of the present invention is to claim the Vip3Aa protein prepared according to the above preparation method.
The invention also aims to protect the application of the Vip3Aa protein in preventing and treating American cockroach or German cockroach.
The Vip3Aa prepared by the preparation method of the Vip3Aa protein has high purity and greatly improved production efficiency, and the prepared Vip3Aa protein has good effect of preventing and treating Periplaneta americana or Blattella germanica and good agricultural application prospect.
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FIG. 1 shows PCR identification of positive transformants.
FIG. 2 restriction identification of positive transformants.
FIG. 3 Western-Blot identification of Vip3Aa, where M, protein marker; 1. before induction, Vip3Aa/pET21 b/BL 21; 2. vip3Aa/pET21 b/BL21 is induced into whole bacteria.
FIG. 4 SDS-page analysis of purified protein.
FIG. 5 the insecticidal activity of Vip3Aa protein on Periplaneta americana.
FIG. 6 shows the insecticidal activity of Vip3Aa protein on German cockroach.
Figure 7 effect of Vip3Aa protein on periplaneta americana periplaneta digestive tract, where a: a, a control group; b, administration group; b: an administration group; c: and (4) a control group.
Figure 8 effect of Vip3Aa protein on crop of periplaneta americana, where a: control crop (× 200); b: control crop (× 400); c: dosing with crop (× 200); d: crop (x 400) dosed.
Figure 9 effect of Vip3Aa protein on the midgut of periplaneta americana, where a: intestine in control group (× 200); b: intestine in control group (× 400); c: intestine (× 200) in the administration group; d: intestine (. times.400) in the group administered.
Figure 10 effect of Vip3Aa protein on periplaneta americana columnar colon, where a: control columnar colon (× 200); b: control columnar colon (× 400); c: columnar colon (x 200) of the group administered; d: the group administered had a columnar colon (. times.400).
Figure 11 effect of Vip3Aa protein on crop of periplaneta americana (sem), where a: control crop (× 500); b: control crop (× 1000); c: dosing with crop (× 500); d: dosing crop combination (× 1000)
Figure 12 effect of Vip3Aa protein on the midgut of periplaneta americana (scanning electron microscopy), where a: intestine in control group (× 500); b: intestine in control group (× 1000); c: intestine in the group administered (× 500); d: intestine (. times.1000) in the group administered.
Figure 13 effect of Vip3Aa protein on periplaneta americana columnar colon (sem), where a: intestine in control group (× 500); b: intestine in control group (× 1000); c: intestine in the group administered (× 500); d: intestine (. times.1000) in the group administered.
Detailed Description
The invention is further described below by means of specific examples, which do not limit the scope of the patent protection of the invention in any way.
Experimental example 1 insecticidal activity of Vip3Aa protein of the invention against Periplaneta americana and Blattella germanica
Preparing 1ml solution from 2mg/ml, 0.5mg/ml, 0.125mg/ml and 0.03125mg/ml Vip3Aa protein, dividing into 5 groups, placing the German cockroach into a bacteria culture dish, adding 1ml drug solution, freely drinking water for 12h, 24h, 36h and 48h respectively, and observing the death rate of the German cockroach.
Preparing 1ml solution from 2mg/ml, 0.5mg/ml, 0.125mg/ml and 0.03125mg/m Vip3Aa protein, dividing into 5 groups, placing Periplaneta americana into conical bottles, adding 1ml of the drug solution, and freely drinking water for 12h, 24h and 36h respectively to observe the death rate of Periplaneta americana.
3.3.1Vip 3Aa protein Activity assay for killing Blatta Seu Periplaneta
12h, 24h and 36h LD50 of Vip3Aa to Periplaneta americana are 0.70302mg/ml, 0.15906mg/ml and 0.15906mg/ml respectively; 12h, 24h and 36h LD of Vip3Aa against German cockroach500.54077mg/ml, 0.27321mg/ml and 0.17914mg/ml, respectively. See tables 3-1 and 3-2 or FIGS. 5 and 6.
TABLE 3-1 insecticidal Activity of Vip3Aa protein against Periplaneta americana (mg/ml)
Figure BDA0001633317510000071
TABLE 3-2 insecticidal Activity of Vip3Aa protein against German cockroach
Figure BDA0001633317510000072
Experimental example 2 Effect of Vip3Aa protein on the digestive system of Periplaneta americana
(A) HE staining
(1) Anesthesia and dissection: placing Blatta Seu Periplaneta on ice for anesthesia, fixing neck of Blatta Seu Periplaneta with insect needle, removing wing and foot of Blatta Seu Periplaneta, cutting body wall from abdomen edge with ophthalmologic scissors, removing excessive abdominal tissue with small forceps, straightening digestive tract, and cutting along pharynx top and rectum end to obtain complete digestive system of Blatta Seu Periplaneta.
(2) Material taking: the crop, the midgut and the columnar colon are respectively cut by an operating blade, and the sample tissue is less than 0.5cm multiplied by 0.1 cm.
(3) Fixing and embedding: fixation with 4% PFA (0.01MPBS, pH7.4) was performed in 70% ethanol for 60 min, 85% ethanol for 60 min, 95% ethanol for 60 min, 100% ethanol for 50 min twice, xylene for 30min twice, paraffin for 30min at 55 ℃ for three times, and the tissue blocks were embedded in stainless steel molds.
(4) Paraffin section: slicing paraffin, attaching tissue slices with the thickness of 4 mu m on a glass slide coated with polylysine, and baking for 2h at 65 ℃;
(5) HE staining: dewaxing and entering water: immersing the slices in xylene for 10 minutes twice, 100% ethanol for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and tap water for 5 minutes twice; immersing into alum hematoxylin staining solution for 15 minutes, and flushing the section for 30 seconds by running water. The slices were differentiated with 1% hydrochloric acid alcohol for 3 seconds and rinsed with running water for 30 seconds. The light ammonia water turns blue for 1 minute. Staining with 1% eosin alcohol for 3 min. Dehydrating with 95% ethanol for 30 s. 100% alcohol was dehydrated twice for 30 seconds. Xylene was three times in 60 seconds. And (5) sealing the neutral gum.
(B) Observation by scanning electron microscope
(1) Material taking: after separating the midgut and crop of cockroach, the cockroach is rinsed clean with normal saline of insects, 15min each time. Fixing with 2.5% glutaraldehyde at room temperature for 30min, carefully cutting the tissue to facilitate unfolding, and fixing in a refrigerator at 4 deg.C for 24 h.
(2) Rinsing: the fixed samples were rinsed 3 times with 0.1M PBS for 15min each.
(3) Post-fixing: the rinsed midgut of cockroach and crop were fixed in 1% osmic acid for 2 h.
(4) And (3) dehydrating: dehydrating samples of Blatta Seu Periplaneta midgut and crop with 50% ethanol-70% ethanol-80% ethanol-85% ethanol-90% ethanol-95% ethanol-anhydrous ethanol for 1 hr, respectively, replacing the samples with pure acetone for 20min, and adding the samples into isoamyl acetate for 30 min.
(5)CO2Critical point drying: the cockroach midgut and crop samples were taken out of isoamyl acetate, placed in the sample chamber of a critical point desiccator, and covered with a lid. After reaching the critical state (31 ℃, 72.8 atm), the temperature was raised by another 10 ℃ to vaporize liquid CO2, and then the gas valve was opened to gradually vent the gas, and the sample was completely dried.
(6) Ion sputtering coating: and (3) placing the dried skin sample under an ion sputtering instrument, and sputtering metal particles to the surfaces of the midgut sample and the crop sample to form a layer of metal film. And observing under an electron microscope and taking a picture.
The experimental results are as follows:
(1) effect of Vip3Aa protein on Periplaneta americana digestive tract
FIG. 7 shows the effect of Vip3Aa protein on the periplaneta americana digestive tract, and the administration group periplaneta americana digestive tract is obviously shortened compared with the normal periplaneta americana digestive tract by dissection; most of the post-dose, american cockroach crop dilates.
(2) Pathological observation of Vip3Aa protein
Fig. 8 is the effect of Vip3Aa protein on the crop of periplaneta americana, showing that no significant change was seen in the cells on the upper layer of the crop of periplaneta americana after dosing. FIG. 9 shows the effect of Vip3Aa protein on the periplaneta americana midgut, showing that the periplaneta americana midgut cell epithelial cells ruptured and BBMV shriveled off the epithelial cells after administration. FIG. 10 shows the effect of Vip3Aa protein on the columnar colon of Periplaneta americana, showing that the columnar colon cells of Periplaneta americana have ruptured epithelial cells and BBMV has collapsed, leaving the epithelial cells. Figure 11 influence of Vip3Aa protein on american cockroach crop (scanning electron microscopy), results show that cells on top of american cockroach crop do not change significantly after administration, and the surface covers dentate structure. Figure 12 effect of Vip3Aa protein on periplaneta americana midgut (scanning electron microscopy), results show disappearance of dentate structure on the surface of periplaneta americana midgut, exposing the underlying tissue structure. FIG. 13 shows the effect of Vip3Aa protein on the columnar colon of Periplaneta americana (scanning electron microscopy), showing the disappearance of the dentate structure on the surface of the columnar colon of Periplaneta americana, exposing the underlying tissue structure.
Sequence listing
<110> university of Guangdong department of pharmacy
<120> preparation method and application of recombinant Vip3Aa protein
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 2430
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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 (3)

1. The application of the Vip3Aa protein in controlling American cockroaches or German cockroaches is characterized in that the preparation method of the Vip3Aa protein comprises the following steps:
1) cloning of Vip3Aa Gene:
using Vip3Aa DNA as a template, and a reference sequence GenBank: l48811.1, Bacillus thuringiensis strain nAB88 induced chemical protein design primer for PCR reaction, introducing BamH I and Xho I enzyme cutting sites;
2) construction and identification of Vip3Aa/pET21b expression vector:
performing double enzyme digestion on the PCR product and the prokaryotic expression plasmid pET21b by respectively adopting restriction enzymes BamH I and Xho I, recovering enzyme digestion fragments by adopting a DNA purification recovery kit, detecting the concentration of the recovered product by using a full-wavelength spectrophotometer, and calculating the mole number of the enzyme digestion fragments by using a target gene: the expression vector molar ratio is 3: 1, carrying out ligation reaction by using T4 ligase to construct a Vip3Aa/pET21b expression vector;
3) obtaining of Vip3Aa/pET21 b/BL21 recombinant strain:
adding 10 μ L of Vip3Aa/pET21b expression vector identified as positive after extraction into 100 μ L of E.coil BL21 competence, placing on ice for 30min after flicking and mixing uniformly, performing heat shock for 90s at 42 ℃ in a metal bath, adding 900 μ L of LB culture medium after placing on ice for 1min, and performing low-speed shaking culture for 1h-1.5h at 37 ℃; centrifuging at 5000rpm to remove supernatant, uniformly beating the precipitate with 200 μ L LB culture medium, and uniformly coating on ampicillin screening plate; after continuous culture for 16-18h, selecting a single colony for colony PCR and enzyme digestion identification to obtain a Vip3Aa/pET21 b/BL21 recombinant strain;
4) induced expression of recombinant strains:
a single colony of the Vip3Aa/pET21 b/BL21 recombinant strain is picked up and inoculated into 5mL LB liquid culture medium containing 100 mug/mL ampicillin, shake culture is carried out for 12-16h under the condition of 37 ℃ and 180rpm, and the bacterial liquid is mixed according to the ratio of 1: inoculating 100% of the total amount of the extract into a fresh LB liquid culture medium, performing shake culture at 37 ℃ and 180rpm until the OD600 value is 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM for induction, and continuing shake culture at 20 ℃ and 180rpm for 24 hours;
5) separation and purification of Vip3Aa protein and freeze drying:
centrifuging the Vip3Aa/pET21 b/BL21 recombinant strain subjected to induced expression at 12000rpm for 10min, collecting thalli, washing with PBS and centrifuging; adding 40mL of lysis buffer solution into each liter of culture solution to suspend thallus, adding PMSF and 1% Triton X-100, crushing for 12min by using an ultrasonic cell crusher, centrifuging at 12000rpm for 30min after cell crushing, and taking supernatant to perform SDS-PAGE;
filtering the supernatant obtained after the Vip3Aa/pET21b strain is crushed by adopting a 0.22 mu M filter, sucking 10ml of the supernatant obtained after the Vip3Aa/pET21b strain is crushed to a chromatographic column, balancing the chromatographic column by adopting a blanking buffer of 2 percent imidazole with five times of the column volume, and then carrying out linear gradient elution;
desalting and freeze-drying of Vip3Aa protein: connecting a desalting column with a chromatography system, washing 5 column volumes with 4mL/min deionized water, balancing the chromatography column with 0.15M NaCl buffer solution with 5 column volumes, loading the sample with 2mL loading rings, desalting by adopting a desalting program, collecting a desalted sample solution, freezing at-80 ℃ for 2h, pre-cooling to-40 ℃ by using a freeze-drying instrument, and freeze-drying for 48h to obtain Vip3Aa protein;
the identification method of the positive product of the ligation reaction in the step 2) comprises the following steps: adding 10 μ L of the ligation product into 100 μ L of DH5 α, flicking, mixing, standing on ice for 30min, performing heat shock at 42 deg.C for 90s, standing on ice for 1min, adding 900 μ L of LB culture medium, and performing low-speed shaking culture at 37 deg.C for 1-1.5 h; centrifuging at 5000rpm, removing supernatant, keeping 200 μ L of culture medium, blowing to precipitate uniformly, coating on ampicillin screening plate uniformly, 16-18h, and selecting single colony for colony PCR and enzyme digestion identification;
the forward primer of the PCR reaction in the step 1) is shown by SEQ ID NO.3, and the reverse primer of the PCR reaction is shown by SEQ ID NO. 4; the composition of the lysis buffer in the step 5) is 25 mmol.L-1 Tris-HCl，300mmol·L-1NaCl，5mmol·L-1Beta-mercaptoethanol, pH 8.0.
2. The use of the Vip3Aa protein according to claim 1 for preventing and treating Periplaneta americana or Periplaneta germanica, wherein the composition of the PCR reaction system in step 1) of the preparation method of the Vip3Aa protein is as follows: template 1.0. mu.L, 10 XPCR buffer 5.0. mu.L, ddH2O35.5. mu.L, 10mM dNTP Mix 4.0. mu.L, F primer 2.0. mu.L, R primer 2.0. mu.L, La Taq 0.5. mu.L; and (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, amplification at 72 ℃ for 3min, 30 cycles, and extension at 72 ℃ for 10 min.
3. The use of the Vip3Aa protein in the prevention and treatment of Periplaneta americana or Periplaneta germanica according to claim 1, wherein the reaction system of the ligation reaction in step 2) of the preparation method of the Vip3Aa protein is Vip3Aa enzyme-cleaved fragment 0.3pmol, pET21b enzyme-cleaved fragment 0.1pmol, ddH2O Up to 10μL，10×buffer 1μL，T4 DNA Ligase 0.5μL。
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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