CN103194460B - Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb - Google Patents
Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb Download PDFInfo
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Abstract
The invention discloses a preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb. The method relates to a gene of bacillus thuringiensis crystal protein Bt Cry3Bb, and the gene is subjected to codon optimization and is specifically shown in the sequence 1 in the sequence table. The preparation method of fusion protein Bt Cry3Bb of the bacillus thuringiensis crystal comprises the following steps of: 1) introducing a recombinant vector which contains DNA (Deoxyribonucleic Acid) fragment of the 109-2064th in the sequence 1 or in the whole sequence 1, so as to obtain recombinant escherichia coli; and 2) culturing the recombinant escherichia coli, carrying out inducible expression, collecting and splitting the thallus so as to obtain the bacillus thuringiensis crystal fusion protein Bt Cry3Bb (sequence 2). In the escherichia coli, the bacillus thuringiensis crystal protein Bt Cry3Bb gene (N-Cry3Bb gene) which is subjected to codon optimization shows that the protein activity of the obtained protein is not affected by the codon 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 Cry3Bb.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) a kind of parasporal crystal can be produced in the process forming gemma, the main component of this crystal is the protein with insecticidal activity, be called as insecticidal crystal protein (ICP, Insecticidal Crystal Protein) or Bt toxalbumin.Bt toxalbumin can kill the insects such as lepidopteran, Diptera, Coleoptera, is the biotic pesticide be most widely used in the world at present.So far existing nearly 180 Bt toxoprotein genes are cloned and check order, and Cry3Bb is exactly wherein a kind of, and its major target insect is Coleoptera.
Genetically modified crops meet the demand of the mankind to grain yield and economic benefit, but due to foreign gene, on the impact of plant physiology metabolism and genetically modified crops, the threat to environment is difficult to prediction, it utilizes also exists risk simultaneously, and the effective way reducing this risk is exactly carry out safety evaluation to the foreign protein expressed by genetically modified crops.Therefore, obtain the foreign protein expressed by various Bt insecticidal crystalline gene, to carrying out, safety evaluation is significant, for the enforcement of the foundation of the follow-up method of inspection, examination criteria provides basis, thus effectively can prevent the generation of potential safety hazard, avoid genetically modified crops to the destruction of 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 the bacillus thuringiensis crystal protein B tCry3Bb that a kind of codon is optimized.
The gene of the bacillus thuringiensis crystal protein B t Cry3Bb that described codon is optimized, be under the prerequisite not changing bacillus thuringiensis crystal protein B t Cry3Bb aminoacid sequence (the 1-652 amino acids of GenBank:M89794.1), the codon of its wild-type encoding gene (the 202-2157 position of GenBank:M89794.1) replaced with the gene of gained after the codon of intestinal bacteria preference (high frequency use).By optimize after unnamed gene be N-Cry3Bb, specifically can be following a)-c) in arbitrary DNA molecular:
A) DNA molecular shown in the Nucleotide of 109-2064 position of sequence 1 in sequence table;
B) DNA molecular shown in sequence 1 in sequence table;
C) DNA molecular and a) or b) limited at least has the identity of 98%, and protein DNA molecule shown in the 37-688 amino acids of encoding sequence 2 or sequence 2.
Wherein, sequence 1 is made up of 2091 Nucleotide altogether, and wherein 109-2064 position Nucleotide is the gene order codon of the 202-2157 position of GenBank:M89794.1 being replaced with gained after intestinal bacteria preferred codons.Protein shown in sequence 2 in sequence 1 polynucleotide, wherein the 37-688 amino acids of the 109-2064 position nucleotide coding sequence 2 of sequence 1.Sequence 2 is made up of 696 amino acid altogether.
Recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium containing described DNA molecular (N-Cry3Bb 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 use existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one 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, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer.For the ease of identifying transgenic cell and screening, can process expression carrier used thereof, as add can luminophor gene (GFP gene, luciferase genes etc.), there is the antibiotic marker thing (gentamicin marker, kantlex marker etc.) etc. of resistance.
In the present invention, start the promotor that described DNA molecular (N-Cry3Bb gene) transcribes in described recombinant expression vector and be specially T7 promotor.
Described recombinant expression vector is specially the recombinant plasmid obtained at the multiple clone site place that described DNA molecular (N-Cry3Bb gene) is inserted into plasmid pET28a.In one embodiment of the invention, described multiple clone site is EcoR I and XhoI.
The RNA molecule of being transcribed gained by described DNA molecular (N-Cry3Bb gene) also belongs to protection scope of the present invention.
Another 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 Cry3Bb active, 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 described protein also belongs to protection scope of the present invention in the preparation application had in the product of insecticidal activity.
In the application, described worm can be coleopteran pest; In one embodiment of the invention, described worm be specially tenebrio molitor (as 2 age tenebrio molitor).Described product can be test kit.
The application of described DNA molecular in raising intestinal bacteria in bacillus thuringiensis crystal fusion rotein Bt Cry3Bb expression amount also belongs to protection scope of the present invention; Described bacillus thuringiensis crystal fusion rotein Bt Cry3Bb 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 Cry3Bb.
The preparation method of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb provided by the present invention, specifically comprises the steps:
1) the described recombinant vectors (as recombinant expression vector pET28a-3Bb) of expressing described DNA molecular is imported 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 Cry3Bb;
Described bacillus thuringiensis crystal fusion rotein Bt Cry3Bb is specially protein shown in sequence 2 in sequence table.
In the above-mentioned methods, the described expression vector in step 1) can be can other recombinant prokaryotic expression vectors of DNA molecular shown in sequence 1 in expressed sequence table.
In the above-mentioned methods, step 2) in, described abduction delivering is specially: have to cultivation in the nutrient solution of described recombination bacillus coli that to add IPTG to final concentration be 0.1mM, and 18 DEG C jolt cultivation 12 hours.
Described cultivation has the OD600 value of the nutrient solution of described recombination bacillus coli between 0.6-0.8.
The rotating speed that described concussion is cultivated is 120rpm, and rotation radius is 1.5cm.
In the above-mentioned methods, step 2) in, after described " collecting and cracking thalline ", also comprise collected after centrifugation and dissolve the step of inclusion body.At 0.1mM IPTG, under 18 DEG C of inductions condition of 12 hours, described bacillus thuringiensis crystal fusion rotein Bt Cry3Bb is mainly expressed in inclusion body.
Using gene engineering method of the present invention synthesizes bacillus thuringiensis crystal fusion rotein BtCry3Bb gene (N-Cry3Bb gene) at home first.Experiment proves, in intestinal bacteria, bacillus thuringiensis crystal fusion rotein Bt Cry3Bb gene (N-Cry3Bb gene) that codon provided by the present invention is optimized expresses gained albumen does not affect its protein-active because of codon optimized.The present invention be inquire into the detection method of bacillus thuringiensis crystal protein B t Cry3Bb, physiological function and mechanism of action thereof further, degradation mechanism lays the foundation.
Accompanying drawing explanation
Fig. 1 is that recombinant plasmid EcoR I and Xho I carry out DNA molecular electrophorogram after double digestion.Wherein, swimming lane M is DNA molecular amount standard 10Kbp to 1000bp band (sky root biotechnology (Beijing) company limited, catalog number MD100-1Kb Ladder).Swimming lane 1 carries out band after double digestion for recombinant expression vector pET28a-3Bb EcoR I and Xho I.
Fig. 2 is the prokaryotic expression result (SDS-PAGE) of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb.Wherein, swimming lane M is Protein Marker 10KD to 170KD band (Tuo Ying mill, Beijing development in science and technology company limited, catalog number TF413-01).The supernatant liquor that the control group thalline that swimming lane 1 proceeds to pET28a empty carrier for cracking obtains; The supernatant liquor that the experimental group thalline that swimming lane 2 proceeds to recombinant expression vector pET28a-3Bb for cracking obtains; The inclusion body that the experimental group thalline that swimming lane 3 proceeds to recombinant expression vector pET28a-3Bb for cracking obtains.Albumen for the purpose of arrow indication.
Fig. 3 is the purification result (SDS-PAGE) of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb.Wherein, swimming lane M is Protein Marker 10KD to 170KD band (Tuo Ying mill, Beijing development in science and technology company limited, catalog number TF413-01).Swimming lane 1 is inclusion body protein coarse body fluid before purifying (solubilization of inclusion bodies liquid).Swimming lane 2 is inclusion body protein after purifying.Albumen for the purpose of arrow indication.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
PGOV4 cloning vector: provided by gene chemical synthesis company Gene Oracle Inc..Be documented in " Kalle Kantola; Mohammadreza Sadeghi; Anne Lahtinen et al.Merkel cell polyomavirus DNA in tumor-freetonsillar tissues and upper respiratory tract samples:Implications for respiratorytransmission and latency.Journal of Clinical Virology; 45 (2009) 292-295. " civilian, 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 to adding required site when synthetic gene, and can not conflict with site existing in sequence.In the connection of carrier and object fragment, also not adopt enzyme to cut the method for connection, but directly connect with concordant end.
PET28a carrier: Novagen company, article No. is 69864-3.
Tenebrio molitor: 2 instar larvaes, Plant Protection institute, Chinese Academy of Agricultral Sciences.
Codon optimized and the full genome synthesis of embodiment 1, bacillus thuringiensis crystal protein B t Cry3Bb gene
According to the Wild-type B. thuringiensis crystallin Bt Cry3Bb gene order (the 202-2157 position Nucleotide of GenBank:M89794.1) in bacillus thuringiensis (Bacillus thuringiensis), obtain being suitable for the Optimization-type bacillus thuringiensis crystal protein B t Cry3Bb gene order at expression in escherichia coli with repeatedly verifying through design, under the prerequisite not changing aminoacid sequence (the 1-652 amino acids of GenBank:M89794.1), the codon optimised sequence of intestinal bacteria preference (high frequency use) is transformed into by Wild-type B. thuringiensis crystallin Bt Cry3Bb gene order, thus improve the expression level of bacillus thuringiensis crystal protein B t Cry3Bb in intestinal bacteria culture environment.
According to aforesaid method, final acquisition is by the Optimization-type bacillus thuringiensis crystal protein B t Cry3Bb gene of intestinal bacteria preference design, called after N-Cry3Bb gene, its gene order is the 109-2064 position of sequence 1 in sequence table, is made up of altogether 1956 Nucleotide.The albumen of described N-Cry3Bb genes encoding is the albumen of the 37-688 amino acids composition of sequence 2 in sequence table, identical with the albumen of the Wild-type B. thuringiensis crystallin Bt Cry3Bb genes encoding shown in the 202-2157 position Nucleotide of GenBank:M89794.1.
The prokaryotic expression of embodiment 2, bacillus thuringiensis crystal fusion rotein Bt Cry3Bb
One, the structure of RT-PCR expression vector pET28a-3Bb
In order to the needs of construction of expression vector, after the optimization of embodiment 1 gained, the recognition sequence of restriction enzyme site EcoR I and Xho I is added at the two ends of bacillus thuringiensis crystal protein B tCry3Bb gene (N-Cry3Bb gene) respectively, obtains " the 103-2070 position of sequence 1 ".This sequence of synthetic, and DNA fragmentation shown in this sequence is connected with pGOV4 cloning vector, obtain recombinant plasmid.The recombinant plasmid called after pGOV4-3Bb inserting DNA fragmentation shown in " the 103-2070 position of sequence 1 " in pGOV4 cloning vector will be shown through order-checking.
The recombinant plasmid pGOV4-3Bb restriction enzyme EcoR I of above-mentioned structure and Xho I is carried out double digestion, reclaims goal gene fragment; The skeleton large fragment of gained goal gene fragment with the pET28a carrier cut through same enzyme be connected, connect after product and proceed in bacillus coli DH 5 alpha competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), coated plate screens.Picking positive monoclonal, shakes bacterium, extracts plasmid, carries out double digestion with EcoR I and XhoI.Restriction enzyme mapping as shown in Figure 1.The recombinant plasmid sample presentation order-checking identified and obtain size and be about 5335bp and 1962bp two object bands will be cut through enzyme.The recombinant plasmid called after pET28a-3Bb of the 109-2064 position nucleotide sequence of sequence 1 in insertion sequence table between the multiple clone site EcoR I and Xho I of pET28a carrier will be shown through order-checking.In recombinant expression vector pET28a-3Bb, be all connected with at the upstream and downstream of described N-Cry3Bb gene (the 109-2064 position of sequence 1) the His label that pET28a carrier carries, to carry out the purifying of target protein when carrying out prokaryotic expression; Specifically, in recombinant expression vector pET28a-3Bb, described N-Cry3Bb gene (the 109-2064 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 2065-2091 position of sequence 1 are that pET28a carrier carries sequence.Albumen expressed by whole sequence 1 is the albumen (being designated as " bacillus thuringiensis crystal fusion rotein Bt Cry3Bb ") shown in sequence in sequence table 2.In recombinant expression vector pET28a-3Bb, the promotor starting described N-Cry3Bb genetic transcription is T7 promotor.
Two, prokaryotic expression, the Isolation and characterization of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb
1, the prokaryotic expression of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb
A) recombinant expression vector pET28a-3Bb step one obtained proceeds to as experimental group in e. coli bl21 (DE3) competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), and setting simultaneously proceeds to the e. coli bl21 (DE3) of empty carrier pET28a as a control group.By experimental group and control group respectively at the LB substratum containing 50 μ g/mL Kan, 37 DEG C of shaking culture 16h, obtain culture.
B) by step a) gained culture by 1% volume be linked in the LB substratum containing 50 μ g/mL Kan, 37 DEG C of 200rpm shaking culture between 0.6-0.8, obtain nutrient solution to bacterium liquid OD600 value.
C) in step b) gained nutrient solution, adding IPTG to its final concentration is 0.1mM, and 18 DEG C jolt cultivation 12 hours, and its rotating speed is 120rpm, and shaking table rotation radius is 1.5cm.
D) by step c) products therefrom 4 DEG C, the centrifugal 10min of 10000g, 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, 60rpm shaking culture 20min, obtains lysate.
F) by step e) gained lysate with 4 DEG C, the centrifugal 20min of 12000g.On the one hand, supernatant is collected; On the other hand, collecting precipitation (inclusion body), abundant vortex (1mg precipitates corresponding 5ml lysate) add BugBuster Master Mix in precipitation after, 4 DEG C, centrifugal supernatant is removed in 10min hypsokinesis to 5000g, the BugBuster Master Mix diluent diluting 10 times with deionized water of added BugBuster Master Mix volume 6 times before adding in precipitation again, after abundant vortex 4 DEG C, centrifugal supernatant is removed in 10min hypsokinesis to 5000g, 4 DEG C, the centrifugal 15min of 12000g after repeating 3 times, the supernatant that inclines obtains high purity inclusion body; Solubilization of inclusion bodies liquid (0.05M CAPS damping fluid, containing 0.3%(0.3g/100mL) N-dodecyl musculamine acid sodium is added in inclusion body; 10mg inclusion body corresponding 1ml solubilization of inclusion bodies liquid), light and slow resuspended inclusion body; 4 DEG C, draw supernatant after the centrifugal 20min of 12000g, obtain solubilization of inclusion bodies liquid.
Step f) gained supernatant and solubilization of inclusion bodies liquid are carried out SDS-PAGE electrophoresis detection respectively; SDS-PAGE method with reference to Zhang Longxiang etc. show " biochemical test Method and Technology " second edition, Higher Education Publishing House (2006), 100-106 page.Adopt 5% concentrated glue, voltage 50V.Separation gel adopts 10% gum concentration, voltage 120V.Applied sample amount is 10 μ l.
Result as shown in Figure 2.As can be seen from the figure, compared with proceeding to the control group of pET28a empty carrier, the position that the experimental group proceeding to the recombinant expression vector pET28a-3Bb that step one obtains is about 76KD in size has object band to occur, consistent with the target protein molecular size range calculated according to aminoacid sequence in theory.In addition, it can also be seen that from result, at 0.1mM IPTG, under 18 DEG C of inductions condition of 12 hours, bacillus thuringiensis crystal fusion rotein Bt Cry3Bb mainly expresses in inclusion body.
2, the purifying of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb
In above-mentioned steps 1 f) collected by solubilization of inclusion bodies liquid be the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb slightly carried, 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) purifying is carried out to it, obtain the bacillus thuringiensis crystal fusion rotein BtCry3Bb after purifying.
In purge process, the resin adopting BugBuster Ni-NTA His Bind test kit to provide, adopts the purification buffer that BugBusterHis*Bind purification kit provides.The method operation provided according to BugBuster His*Bind purification kit.
3, the qualification of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb
(1) sequence verification
Carry out the order-checking of N end to the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb after step 2 gained purifying, result confirms that its sequence conforms to sequence 2, confirms the exactness of sequence.
(2) SDS-PAGE qualification
The purification result of SDS-PAGE to the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb after step 2 gained purifying is adopted to verify.Bacillus thuringiensis crystal fusion rotein Bt Cry3Bb(solubilization of inclusion bodies liquid slightly to carry before purifying) be contrast.
SDS-PAGE method with reference to Zhang Longxiang etc. show " biochemical test Method and Technology " second edition, Higher Education Publishing House (2006), 100-106 page.Adopt 5% concentrated glue, voltage 50V.Separation gel adopts 10% gum concentration, voltage 120V.Applied sample amount is 30 μ l.
SDS-PAGE result as shown in Figure 3.As can be seen from the figure, compared with before purifying, the albumen object band after purifying is single, effectively eliminates foreign protein.
4, the output of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb detects
Adopt BCA protein quantification test kit (BCA Protein Assay Kit, purchased from Novagen company, catalog number is 71285-3) concentration determination is carried out to the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb after step 2 gained purifying, and then obtain the ultimate production of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb.Concentration determination concrete operations are carried out see test kit specification sheets.3 repetitions are established in the determination of yield experiment of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb altogether, and result gets three mean values repeated.
Result shows that the output of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb is 0.0276mg/mg thalline, and wherein " thalline " is bacterial sediment collected after IPTG abduction delivering is cultivated.
The determination of activity of the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb that embodiment 3, embodiment 2 obtain
The bacillus thuringiensis crystal fusion rotein Bt Cry3Bb obtained in Example 2, use 0.1%(volume ratio) dilution of the Triton-100 aqueous solution, be made into 5 dilution diluents, simultaneously with 0.1%(volume ratio) the Triton aqueous solution compares, naturally dry after the clean cabbage leaves be slightly smaller than bottom culture dish being immersed diluent 10 second, put into bottom and be covered with culture dish through water-soaked filter paper, every ware access 10 2 age Yellow meal worm larva, cultivate under room temperature condition.The death condition of adding up Yellow meal worm larva after 72 hours (touches larva abdomen end with pen, if its head can not swing, can not creep forward, be considered as death), and the bacillus thuringiensis crystal fusion rotein Bt Cry3Bb utilizing POLO computed in software embodiment 2 to obtain accordingly is to the LC50 value (median lethal concentration represents the protein concentration killing 50% Yellow meal worm larva) of Yellow meal worm larva.Each concentration arranges 4 replicate treatment groups, results averaged.
Result shows, the LC50 value of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb to Yellow meal worm larva that embodiment 2 obtains is 587.3mg/L.
Claims (10)
1.DNA molecule, for following a) or b):
A) DNA molecular shown in the Nucleotide of 109-2064 position of sequence 1 in sequence table;
B) DNA molecular shown in sequence 1 in sequence table.
2. the recombinant vectors containing DNA molecular described in claim 1.
3. the expression cassette containing DNA molecular described in claim 1.
4. the transgenic cell line containing DNA molecular described in claim 1.
5. the recombinant bacterium containing DNA molecular described in claim 1.
6. recombinant vectors according to claim 2, is characterized in that: described recombinant vectors is the recombinant plasmid that the multiple clone site place that described DNA molecular is inserted into plasmid pET28a obtains.
7. the RNA molecule of gained is transcribed by DNA molecular described in claim 1.
8. DNA molecular described in claim 1 is improving the application in intestinal bacteria in bacillus thuringiensis crystal fusion rotein Bt Cry3Bb expression amount; Described bacillus thuringiensis crystal fusion rotein Bt Cry3Bb is protein shown in sequence in sequence table 2.
9. a preparation method of bacillus thuringiensis crystal fusion rotein Bt Cry3Bb, comprises the steps:
1) recombinant vectors described in claim 2 or 6 is imported 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 Cry3Bb;
Described bacillus thuringiensis crystal fusion rotein Bt Cry3Bb is protein shown in sequence in sequence table 2.
10. method according to claim 9, is characterized in that, step 2) in, described abduction delivering is: have to cultivation in the nutrient solution of described recombination bacillus coli that to add IPTG to final concentration be 0.1mM, and 18 DEG C jolt cultivation 12 hours.
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