CN103233021B - Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 - Google Patents
Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 Download PDFInfo
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Abstract
The invention discloses a preparation method of a bacillus thuringiensis crystal fusion protein Bt Cry35Ba2. The method relates to a codon-optimized bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 gene which is specifically represented by sequence 1 in the sequence list. The bacillus thuringiensis crystal fusion protein preparation method provided by the invention comprises the steps that: (1) a recombinant vector comprising a DNA fragment of the 109th-1269th site of the sequence 1 or the entire sequence 1 is introduced into Escherichia coli, such that recombinant Escherichia coli is obtained; (2) the recombinant Escherichia coli is cultured, and induction expression is carried out; and thallus is collected and cracked, such that the bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 (sequence 2) is obtained. According to the invention, protein activity of the protein obtained by expression of the codon-optimized bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) is not influenced by condon optimization.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of preparation method of bacillus thuringiensis crystal fusion rotein, particularly the preparation method of a kind of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram-positive microorganism being distributed widely in soil, in the forming process of its gemma, can form the crystallin (Cry albumen) with insecticidal activity.This albumen has higher toxicity to various pests such as lepidopteran, Diptera, Coleoptera, Hymenopteras, is the toxalbumin being most widely used.Due to this characteristic, the gene of coding Cry albumen is recognized as the alternative gene of hot topic of genetically modified crops by biologist, and existing multiple genetically modified crops kinds proceed to this gene commercialization, extensively plantation in the world.Bt fungus strain contains a large amount of different insecticidal crystal protein encoding genes, so far existing nearly 180 different Bt insecticidal crystalline genes are cloned and check order, Bt Cry35Ba2 is exactly wherein a kind of, has toxicity mainly for the coleoptera larvae of harm seeding corn and other crops root.
The plantation of genetically modified crops has met the mankind to grain yield, economic benefit demand.Meanwhile, also exist Environmental Risk, randomness and uncertainty that foreign gene imports, the change of the Physiology and biochemistry effect of genetic expression to crop, threat that ecotope is formed etc. is all difficult to prediction.There is worry to the securities of genetically modified crops in people, and the effective way of eliminating this worry is exactly that the expressed foreign protein of genetically modified crops is carried out to safety evaluation.Therefore, obtain the expressed foreign protein of various Bt insecticidal crystalline genes, significant to carrying out safety evaluation, for the follow-up method of inspection is set up, the enforcement of examination criteria provides basis, thereby can effectively prevent the generation of potential safety hazard, avoid the destruction of genetically modified crops to ecotope, give full play to the huge applications potentiality of transgenic technology in agriculture production.
Summary of the invention
An object of the present invention is to provide the gene of a kind of bacillus thuringiensis crystal protein B t Cry35Ba2 of codon optimization.
The gene of the bacillus thuringiensis crystal protein B t Cry35Ba2 that described codon is optimized, be not change under the prerequisite of bacillus thuringiensis crystal protein B t Cry35Ba2 aminoacid sequence (the 1-387 amino acids of GenBank:AY536894.1), the codon of its wild-type encoding gene (the 1-1161 position of GenBank:AY536894.1) replaced with to the gene of gained after the codon of intestinal bacteria preference (high frequency use).By optimize after unnamed gene be N-Cry35Ba2, specifically can be following a)-c) in arbitrary DNA molecular:
A) DNA molecular shown in the 109-1269 position Nucleotide of sequence 1 in sequence table;
B) DNA molecular shown in sequence 1 in sequence table;
C) DNA molecular and a) or b) limiting at least has 98% identity, and protein DNA molecule shown in the 37-423 amino acids of encoding sequence 2 or sequence 2.
Wherein, sequence 1 is made up of 1296 Nucleotide altogether, and wherein 109-1269 position Nucleotide is the gene order that the codon of the 1-1161 position of GenBank:AY536894.1 is replaced with to gained after intestinal bacteria preference codon.Protein shown in sequence 2, wherein the 37-423 amino acids of the 109-1269 position nucleotide coding sequence 2 of sequence 1 in sequence 1 code sequence list.Sequence 2 is made up of 431 amino acid altogether.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described DNA molecular (N-Cry35Ba2 gene) also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can be used existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, can add any enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer.For the ease of transgenic cell being identified and being screened, can process expression carrier used thereof, as add can luminophor gene (GFP gene, luciferase genes etc.), there is antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance etc.
In the present invention, in described recombinant expression vector, start the promotor that described DNA molecular (N-Cry35Ba2 gene) transcribes and be specially T7 promotor.
Described recombinant expression vector is specially described DNA molecular (N-Cry35Ba2 gene) is inserted into the recombinant plasmid that the multiple clone site place of plasmid pET28a obtains.In one embodiment of the invention, described multiple clone site is EcoR I and Xho I.More concrete, the nucleotide sequence of (pET28a-35Ba2) of described recombinant expression vector is as shown in sequence in sequence table 3.
Wherein, sequence 3 is made up of 6502 Nucleotide altogether, and 137-1432 position Nucleotide is the reverse complementary sequence of sequence 1.
The RNA molecule of being transcribed gained by described DNA molecular (N-Cry35Ba2 gene) also belongs to protection scope of the present invention.
A further object of the present invention is to provide a kind of protein.
Protein provided by the present invention, has bacillus thuringiensis crystal protein B t Cry35Ba2 activity, is specially the protein shown in sequence 2 in sequence table.
The encoding gene of described protein also belongs to protection scope of the present invention.
Described DNA molecular, or described recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium, or described RNA molecule, or the application that described protein has in the product of insecticidal activity in preparation also belongs to protection scope of the present invention.
In described application, described worm is corn rootworm (as three initial stage in age corn rootworms); Described product can be test kit.
The application in bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 expression amount in raising intestinal bacteria of described DNA molecular also belongs to protection scope of the present invention; Described bacillus thuringiensis crystal fusion rotein BtCry35Ba2 is specially protein shown in sequence 2 in sequence table.
Also object of the present invention is to provide the preparation method of a kind of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
The preparation method of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 provided by the present invention, specifically comprises the steps:
1) the described recombinant vectors (as recombinant expression vector pET28a-35Ba2) of expressing described DNA molecular is imported to intestinal bacteria, obtain recombination bacillus coli;
2) cultivate described recombination bacillus coli, carry out abduction delivering, collect and cracking thalline, obtain bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2;
Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is specially protein shown in sequence 2 in sequence table.
In aforesaid method, the described expression vector in step 1) can be can expressed sequence table in other recombinant prokaryotic expression vectors of DNA molecular shown in sequence 1.
In aforesaid method, step 2) in, described abduction delivering is specially: have to cultivation that in the nutrient solution of described recombination bacillus coli, to add IPTG be 1mM to final concentration, 22 DEG C jolt and cultivate 6 hours.
It is between 0.6-0.8 that described cultivation has the OD600 value of the nutrient solution of described recombination bacillus coli.
The rotating speed that described concussion is cultivated is 180rpm, and rotation radius is 1.5 centimetres.
In aforesaid method, step 2) in, afterwards, also comprise centrifugal rear collection the step of dissolving inclusion body described " collecting and cracking thalline ".
At 1mM IPTG, under 22 DEG C of inductions condition of 6 hours, described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is mainly expressed in inclusion body.
Using gene engineering method of the present invention is synthesized bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) at home first.Experiment showed, in intestinal bacteria, the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) that codon provided by the present invention is optimized is expressed gained albumen not because of codon optimized its protein-active that affects.The present invention is that detection method, physiological function and mechanism of action thereof, the degradation mechanism of further inquiring into bacillus thuringiensis crystal protein B t Cry35Ba2 lay the foundation.
Brief description of the drawings
Fig. 1 is that recombinant plasmid EcoR I and Xho I carry out DNA molecular electrophorogram after double digestion.Wherein, swimming lane Marker is that the DNA molecular amount standard 10Kbp that buys is to 1000bp band (sky root biotechnology (Beijing) company limited, catalog number MD100-1Kb Ladder).Swimming lane 1 carries out band after double digestion with EcoR I and Xho I for recombinant expression vector pET28a-35Ba2.
Fig. 2 is prokaryotic expression and the purification result (SDS-PAGE) of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
In A, swimming lane Marker is for buying molecular weight of albumen standard 10KD to 170KD band (Tuo Ying mill, Beijing development in science and technology company limited, catalog number TF413-01); Swimming lane 1 proceeds to the inclusion body that the control group thalline of pET28a empty carrier makes for cracking.Swimming lane 2 proceeds to the inclusion body that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes for cracking.Swimming lane 3 is Cry35Ba2 albumen after purifying.
In B, swimming lane Marker is for buying molecular weight of albumen standard 10KD to 170KD band (Tuo Ying mill, Beijing development in science and technology company limited, catalog number TF413-01).Swimming lane 1 proceeds to the supernatant that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes for cracking.Swimming lane 2 proceeds to the supernatant that the control group thalline of pET28a empty carrier makes for cracking; Swimming lane 3 proceeds to the inclusion body that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes for cracking.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
PGOV4 cloning vector: provided by the Gene Oracle Inc. of gene Synesis Company.Be documented in " Kalle Kantola; Mohammadreza Sadeghi; Anne Lahtinen et al.Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples:Implications for respiratory transmission and latency.Journal of Clinical Virology; 45 (2009) 292-295. " literary composition, the public can obtain from China Agricultural University.One of advantage of this carrier is to remove most conventional restriction enzyme site in its sequence, is conducive in the time of synthetic gene, add required site, and can conflict with existing site in sequence.In being connected of carrier and object fragment, also not adopting enzyme to cut the method for connection, but directly connect with concordant end.
PET28a carrier: be purchased from MERKE NOVAGEN company, catalog number is 69864-3.
Corn rootworm (D.baberi): 3 initial stages in age, be documented in " G.MALLOCH; B.FENTON.Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of Wollbachia.Molecular Ecology; (2005) 14; 627-637. " literary composition, the public can obtain from China Agricultural University.
Codon optimized and the full gene of embodiment 1, bacillus thuringiensis crystal protein B t Cry35Ba2 gene is synthetic
According to the wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 gene order (the 1-1161 position Nucleotide of GenBank:AY536894.1) in bacillus thuringiensis (Bacillus thuringiensis), through designing and repeatedly verifying and obtain being suitable for the gene order at the Optimization-type bacillus thuringiensis crystal protein B t of expression in escherichia coli Cry35Ba2, under the prerequisite that does not change aminoacid sequence (the 1-387 amino acids of GenBank:AY536894.1), be transformed into the codon optimized sequence of intestinal bacteria preference (high frequency use) by wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 gene order, thereby improve the expression level of bacillus thuringiensis crystal protein B t Cry35Ba2 in intestinal bacteria culture environment.
According to aforesaid method, final acquisition by the Optimization-type bacillus thuringiensis crystal protein B t Cry35Ba2 gene of intestinal bacteria preference design, called after N-Cry35Ba2 gene, its gene order is the 109-1269 position of sequence 1 in sequence table, is made up of altogether 1161 Nucleotide.The albumen of described N-Cry35Ba2 genes encoding is the albumen of the 37-423 amino acids composition of sequence 2 in sequence table, identical with the albumen of the wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 genes encoding shown in the 1-1161 position Nucleotide of GenBank:AY536894.1.
The prokaryotic expression of embodiment 2, bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
One, the structure of RT-PCR expression vector pET28a-35Ba2
For the needs of construction of expression vector, after embodiment 1 gained optimization, the recognition sequence of restriction enzyme site EcoR I and Xho I is added respectively at the two ends of bacillus thuringiensis crystal protein B t Cry35Ba2 gene (N-Cry35Ba2 gene), obtains " the 103-1275 position of sequence 1 ".This sequence of synthetic, and DNA fragmentation shown in this sequence is connected with pGOV4 cloning vector, recombinant plasmid obtained.To show to insert through order-checking the recombinant plasmid called after pGOV4-35Ba2 of DNA fragmentation shown in " the 103-1275 position of sequence 1 " in pGOV4 cloning vector.
The recombinant plasmid pGOV4-35Ba2 of above-mentioned structure is carried out to double digestion by restriction enzyme EcoR I and Xho I, reclaim goal gene fragment; Gained goal gene fragment is connected with the skeleton large fragment of the pET28a carrier of cutting through same enzyme, connects after product and proceed in bacillus coli DH 5 alpha competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), coated plate screening.Picking positive monoclonal, shakes bacterium, extracts plasmid, carries out double digestion with EcoR I and Xho I.Enzyme is cut result as shown in Figure 1.Obtain by cut qualification through enzyme the recombinant plasmid sample presentation order-checking that size is about 5341bp and two object bands of 1161bp, will prove between the multiple clone site EcoR of pET28a carrier I and Xho I the recombinant plasmid called after pET28a-35Ba2 of the 109-1269 position nucleotide sequence of sequence 1 in insertion sequence table through order-checking.The nucleotide sequence of recombinant expression vector pET28a-35Ba2 is as shown in sequence in sequence table 3, and wherein 137-1432 position Nucleotide is the reverse complementary sequence of sequence 1.In recombinant expression vector pET28a-35Ba2, be all connected with at the upstream and downstream of described N-Cry35Ba2 gene (the 109-1269 position of sequence 1) the His label carrying on pET28a carrier, to carry out the purifying of target protein while carrying out prokaryotic expression; Particularly, in recombinant expression vector pET28a-35Ba2, described N-Cry35Ba2 gene (the 109-1269 position of sequence 1) is present in the ORF shown in whole sequence 1, and wherein the 1-108 position of sequence 1 and the 1270-1296 position of sequence 1 are the sequence that carries on pET28a carrier.The expressed albumen of whole sequence 1 is the albumen shown in sequence 2 in sequence table (being designated as " bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 ").In recombinant expression vector pET28a-35Ba2, the promotor that starts described N-Cry35Ba2 genetic transcription is T7 promotor.
Two, prokaryotic expression, purifying and the qualification of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
1, the prokaryotic expression of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
A) recombinant expression vector pET28a-35Ba2 step 1 being obtained proceeds in e. coli bl21 (DE3) competent cell (TIANGEN Biotech (Beijing) Co., Ltd.) as experimental group, arrange simultaneously proceed to empty carrier pET28a e. coli bl21 (DE3) as a control group.By experimental group and control group, respectively at the LB substratum that contains 50 μ g/mL Kan, 37 DEG C of shaking culture 16h, obtain culture.
B) step a) gained culture is linked into containing in the LB substratum of 50 μ g/mL Kan by 1% volume, 37 DEG C of 200rpm shaking culture 2h, between 0.6-0.8, obtain nutrient solution to bacterium liquid OD600 value.
C) be 1mM to adding IPTG in step b) gained nutrient solution to its final concentration, 22 DEG C jolt and cultivate 6 hours, and its rotating speed is 180rpm, and shaking table rotation radius is 1.5 centimetres.
D), by 4 DEG C of step c) products therefroms, the centrifugal 20min of 12000g, collects thalline.
E) in thalline, add BugBuster Master Mix(MERCK company, catalog number is 71456-3), consumption is 1mg thalline: 5ml BugBuster Master Mix.Under room temperature, beat or gentle vortex mixes BugBuster Master Mix and thalline with inhaling, 50-60rpm shaking culture 20min, obtains lysate.
F) by step e) gained lysate with 4 DEG C, the centrifugal 20min of 16000g.On the one hand, collect supernatant; On the other hand, collecting precipitation (inclusion body), to adding after BugBuster Master Mix fully vortex (1mg precipitates corresponding 5ml lysate) in precipitation, 4 DEG C, 5000g are centrifugal, and supernatant is removed in 10min hypsokinesis, in precipitation, add the BugBuster Master Mix diluent with 10 times of deionized water dilutions that adds before 6 times of BugBuster Master Mix volumes again, fully after vortex 4 DEG C, 5000g centrifugal supernatant is removed in 10min hypsokinesis, 4 DEG C, the centrifugal 15min of 12000g after repeating 3 times, the supernatant that inclines obtains high purity inclusion body; In inclusion body, add solubilization of inclusion bodies liquid (0.05M CAPS damping fluid, containing 0.3%(0.3g/100mL) N-dodecyl musculamine acid sodium; The corresponding 1ml solubilization of inclusion bodies of 10mg inclusion body liquid), light and slow resuspended inclusion body; After 4 DEG C, the centrifugal 20min of 12000g, draw supernatant, obtain solubilization of inclusion bodies liquid.
Step f) gained supernatant and solubilization of inclusion bodies liquid are carried out respectively to SDS-PAGE electrophoresis detection; SDS-PAGE method is with reference to " biochemical test method and technology " second editions of showing such as Zhang Longxiang, Higher Education Publishing House (2006), 100-106 page.Adopt 5% concentrated glue, voltage 50V.Separation gel adopts 12% gum concentration, voltage 120V.Applied sample amount is 15 μ l.
Result is as shown in B in Fig. 2.As can be seen from the figure, compared with proceeding to the control group of pET28a empty carrier, proceeding to the position that the experimental group of the recombinant expression vector pET28a-35Ba2 that step 1 obtains is about 48KD in size has object band to occur, consistent with the target protein molecular size range calculating according to aminoacid sequence in theory.In addition, from result, it can also be seen that, at 1mM IPTG, under 22 DEG C of inductions condition of 6 hours, bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 mainly expresses in inclusion body.
2, the purifying of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
In above-mentioned steps 1, f) collected solubilization of inclusion bodies liquid is the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 slightly carrying, use BugBuster Ni-NTA His Bind purification kit (purchased from Novagen company, catalog number is 70751-3) and BugBuster His*Bind purification kit (purchased from Novagen company, catalog number is 70899-3) it is carried out to purifying, obtain the bacillus thuringiensis crystal fusion rotein BtCry35Ba2 after purifying.
In purge process, the resin that adopts BugBuster Ni-NTA His Bind test kit to provide, the purifying damping fluid that adopts BugBuster His*Bind purification kit to provide.The method operation providing according to BugBuster His*Bind purification kit.
3, the qualification of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
(1) sequence verification
Bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 after step 2 gained purifying is carried out to the order-checking of N end, and result confirms that its sequence conforms to sequence 2, has confirmed the exactness of sequence.
(2) SDS-PAGE qualification
The purification result of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 after adopting SDS-PAGE to step 2 gained purifying is verified.Taking the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that slightly carries before purifying as contrast.
SDS-PAGE method is with reference to " biochemical test method and technology " second editions of showing such as Zhang Longxiang, Higher Education Publishing House (2006), 100-106 page.Adopt 5% concentrated glue, voltage 50V.Separation gel adopts 12% gum concentration, voltage 120V.Applied sample amount is 10 μ l.
SDS-PAGE result is as shown in A in Fig. 2.As can be seen from the figure,, compared with (swimming lane 2 of A in Fig. 2) before purifying, albumen (swimming lane 3 of A in Fig. 2) the object band after purifying is single, has effectively removed foreign protein.
4, the output of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 detects
Adopt BCA protein quantification test kit (BCA Protein Assay Kit, purchased from Novagen company, catalog number is 71285-3) the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 after step 2 gained purifying is carried out to concentration determination, and then obtain the ultimate production of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.Concentration determination concrete operations are carried out referring to test kit specification sheets.3 repetitions are established in the determination of yield experiment of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 altogether, and result is got the mean value repeating three times.
Result shows that the output of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is 0.0236mg albumen/mg thalline, and wherein " thalline " is collected bacterial sediment after IPTG abduction delivering is cultivated.
The determination of activity of the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 3, embodiment 2 obtain
The Cry35Ba2 albumen that uses embodiment 2 purifying to obtain, use 0.1%(volume ratio) dilution of the Triton-100 aqueous solution, (protein concentration is followed successively by 10mg/L to be made into 5 concentration, 5.0mg/L, 2.5mg/L, the diluent of 1.25mg/L and 0.625mg/L, simultaneously with 0.1%(volume ratio) the Triton aqueous solution compares, after being immersed to 10 seconds of diluent, naturally dries the root of water planting corn seedling, being paved with bottom is covered with in the culture dish of water-soaked filter paper, 10 three initial stage in age corn rootworms (D.baberi) of every ware access, be placed in 25 DEG C, relative humidity 65%, in dark incubator, cultivate.After 120 hours, add up the death condition of corn rootworm larva and (touch larva abdomen end with pen, if its head can not swing, can not creep forward and be considered as death), and calculate accordingly bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 2 the obtains LC50 value (median lethal concentration represents the protein concentration that kills 50% corn rootworm larva) to corn rootworm larva.Each concentration arranges 4 replicate treatment groups, results averaged.
Result shows, the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 2 obtains is 2.53mg/L to the LC50 value of corn rootworm larva.
Claims (12)
1.DNA molecule, for following a) or b):
A) DNA molecular shown in the 109-1269 position Nucleotide of sequence 1 in sequence table;
B) DNA molecular shown in sequence 1 in sequence table.
2. contain the recombinant vectors of DNA molecular described in claim 1.
3. contain the expression cassette of DNA molecular described in claim 1.
4. contain the transgenic cell line of DNA molecular described in claim 1.
5. contain the recombinant bacterium of DNA molecular described in claim 1.
6. recombinant vectors according to claim 2, is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
7. recombinant vectors according to claim 6, is characterized in that: in described recombinant expression vector, starting the promotor that described DNA molecular transcribes is T7 promotor.
8. recombinant vectors according to claim 7, is characterized in that: described recombinant expression vector is that described DNA molecular is inserted into the recombinant plasmid that the multiple clone site place of plasmid pET28a obtains.
9. transcribed the RNA molecule of gained by DNA molecular described in claim 1.
10. DNA molecular application in bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 expression amount in raising intestinal bacteria described in claim 1; Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is protein shown in sequence 2 in sequence table.
The preparation method of 11. 1 kinds of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2, comprises the steps:
1) arbitrary described recombinant vectors in claim 2 and 6-8 is imported to intestinal bacteria, obtain recombination bacillus coli;
2) cultivate described recombination bacillus coli, carry out abduction delivering, collect and cracking thalline, obtain bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2;
Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is protein shown in sequence 2 in sequence table.
12. methods according to claim 11, is characterized in that step 2) in, described abduction delivering is: have to cultivation that in the nutrient solution of described recombination bacillus coli, to add IPTG be 1mM to final concentration, 22 DEG C jolt and cultivate 6 hours.
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| CN101240285A (en) * | 2008-03-19 | 2008-08-13 | 清华大学 | Cephalosporin C acrylase and its vector and application |
| CN102140444A (en) * | 2010-01-28 | 2011-08-03 | 复旦大学 | Low temperature alkaline phosphatase and preparation method thereof |
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