CN102094038A - Prokaryotic expression and purification method of transgenic rice Bt protein crylc - Google Patents
Prokaryotic expression and purification method of transgenic rice Bt protein crylc Download PDFInfo
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Abstract
The invention discloses a prokaryotic expression and purification method of transgenic rice Bt protein crylc. The method comprises the following steps: 1) forward and reverse primers are designed according to the genes of the crylc which has been sequenced by the Huazhong Agricultural University and the multiple cloning sites of the vector PMD18-T to be connected, polymerase chain reaction (PCR) amplification and recovery are performed, the recovered material of PCR performs DNA ligation; 2) the competent cell DH5 alpha of Escherichia coli is prepared and transformed; 3) the recombination and construction of expression plasmid and the inducible expression of the recombinant strain are performed; and 4) the affinity chromatography and purification of the expression protein are performed. The purity of the target protein prepared by the method of the invention is up to more than 90%; and the prepared high purity transgenic rice Bt protein crylc can be utilized to obtain antibody and can also be used to prepare the immunochromatographic test strip, thus the Bt gene can be detected fast and accurately in the detection of transgenic plant.
Description
Technical field
The present invention relates to prokaryotic expression and the purification process of a kind of transgenic paddy rice Bt albumen crylc.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis Berliner, be called for short Bt) be a kind of distribution extremely widely, in its life history, can form the brood cell and produce multiple protein crystalline gram positive bacterium.Bt studies microbial pesticide the most deep, that be most widely used in the present microbial pesticide, it has activity to nine purpose harmful organisms in 4 doors such as invertebrates neutral line animal door, Protozoa, Platyhelminthes and the arthropods at least, nontoxic to people, animal, insect be difficult for to produce resistance, cost low, be easy to suitability for industrialized production, be widely used in the various agricultural in the whole world and the biological control of forestry pest since half a century.
Bt can produce various insects or the deleterious toxin of invertebrates, and these toxin have plenty of protein, have plenty of small-molecule substance, what have produces in bacteriotrophy vegetative period, what have produced in the gemma formation phase, and what have is secreted into outside the bacterium, and what have is stored in the cell.Present Bt toxin is modal to be insecticidal crystal protein (Insecticidal CrystalProteins, ICPs), also claim delta-endotoxin (delta-endotoxin), form the phase in the formation of the one or both ends of thalline in the brood cell, most or the ICPs that places one's entire reliance upon (deciding according to insect) of the desinsection of Bt, its shape, structure and size all have substantial connection with its virulence.Wherein crylc is exactly a kind of of ICPs gene, and the crylc albumen of generation can be used for the pest-resistant protection of insecticidal test and transgenic plant.
Summary of the invention
Technical problem to be solved by this invention provides prokaryotic expression and the purification process of a kind of transgenic paddy rice Bt albumen crylc, choosing pest-resistant Bt--crylc is object, utilize prokaryotic expression system to express crylc albumen, and use the affinity chromatography purification expression product, to obtain the expressing protein crylc of higher degree.
In order to achieve the above object, the present invention realizes by the following technical solutions:
The prokaryotic expression method of a kind of transgenic paddy rice Bt albumen crylc may further comprise the steps:
1) the forward and reverse primer that the gene of the crylc that checks order according to Hua Zhong Agriculture University, and the multiple clone site of the carrier PMD18-T that will connect designs and synthesizes carries out pcr amplification, recovery, and this PCR regenerant is carried out the DNA ligation;
2) preparation of competent escherichia coli cell and conversion;
3) abduction delivering of the recombination to construct of expression plasmid, reorganization bacterium.
The sequence of described forward and reverse primer is shown in SEQ ID No 1 and SEQ ID No 2, and is specific as follows:
Forward primer: 5 '-CGGGATCC (BamH I) ATGGAGGAGAACAATCAGAACCAGTG-3 '
Reverse primer: 5 '-CGGAATTC (EcoRI) CTACTTTTGTGCTCTTTCAAGGTCAG-3 '.
Described competent escherichia coli cell is E.coli DH5 α and E.coil Transetta (DE3).
Further, carry out the expressing protein purifying by the Ni-NTA affinity chromatography.
The application of described transgenic paddy rice Bt albumen crylc in colloidal gold immuno-chromatography test paper strip.
The present invention reaches the purpose that detects the BT gene by the preparation proteic antibody of crylc should be used in the colloid gold immune technology.The colloid gold immune technology is emerging a kind of immunological method nineties, and now biomedical each field has obtained increasingly extensive application.It comes down to bag that polymers such as protein are adsorbed to the colloid gold particle surface by process, and the colour developing degree is directly proportional with antigenic content.
The present invention is by preparation transgenic paddy rice Bt albumen crylc and then obtain antibody, and is made into colloidal gold immuno-chromatography test paper strip, so that accurate detection goes out the Bt gene in transgenic plant detection.
The colloidal gold immuno-chromatography test paper strip that the present invention developed is gold to be marked bacillus thuringiensis crylc monoclonal antibody be sprayed on the glass fibre membrane.Because capillary action, sample will move along nitrocellulose filter, and the antibody that is fixed then when bacillus thuringiensis crylc exists is painted, observe district (S district) at sample and can form a colour band, detect existing of Bt gene with this.Built-in Quality Control colour band (C district) is in order to confirm whether this experiment sets up simultaneously.Characteristics such as that the colloid gold immune technology has is convenient, quick, sensitive, safety, low cost, owing to do not need the substrate of enzymatic reaction, single part of mensuration need not any special plant and instrument except that reagent, and stable reagent, development in recent years is rapid.This method detection speed is fast, can go out the result in general one or two minute, and is highly sensitive, can reach 50IU/L.
Prokaryotic expression and the purification process of a kind of transgenic paddy rice Bt albumen crylc of the present invention, simple to operate, cost is low, and prepare purity at the expressing protein crylc more than 90%.Further, the high purity transgenic paddy rice Bt albumen crylc that utilizes the present invention to prepare can obtain antibody, and can be made into colloidal gold immuno-chromatography test paper strip, provides possibility for accurate detection in transgenic plant detection goes out the Bt gene.
Description of drawings
Fig. 1 is prokaryotic expression and the purifying route map of crylc.
Fig. 2 is a crylc gene PCR amplification.
Fig. 3 is a crylc-T recombinant clone plasmid PCR qualification result, and wherein swimming lane 1-8 is the reorganization cloned plasmids; Swimming lane 9 is a blank; Swimming lane 10 positive contrasts.
Fig. 4 is a crylc-T recombinant clone plasmid enzyme restriction qualification result, after wherein swimming lane 2 and 3 is the crylc-T double digestion; Swimming lane 6 is the crylc-T recombinant plasmid.
Fig. 5 is the protein purification result, and wherein swimming lane 1 is host bacterium (E.coli Transetta (DE3)); Swimming lane 2 is PET-30a (+); Swimming lane 3 is crylc-pET30a (+); After swimming lane 6 and 7 is purifying.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is described in further detail.
The used restriction enzyme of the present invention, solution 1 (Solution I) are all available from precious biotechnology (Dalian) limited liability company, primer is synthetic by Shanghai Shenergy Biocolor BioScience ﹠ Technology Company, plasmid DNA is extracted test kit, DNA recovery test kit and affinity column all available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company, competent escherichia coli cell Transetta (DE3) is available from the Beijing Quanshijin Biotechnology Co., Ltd, and other used chemical reagent are available from Chemical Reagent Co., Ltd., Sinopharm Group.
The proteic prokaryotic expression of crylc and the purification process of embodiment 1 bacillus thuringiensis crylc genes encoding
The prokaryotic expression of transgenic paddy rice Bt albumen crylc and purifying route map are referring to Fig. 1.
1. primer design is with synthetic
According to the gene of crylc, and the multiple clone site of the carrier PMD18-T that will connect, PrimerPremier 5.0 design a pair of Auele Specific Primer-forward and reverse primers utilized.Wherein, add BamH I site at forward primer (crylc-F), (crylc-R) adds the EcoRI site at reverse primer.
Forward and reverse primer is synthetic by Shanghai Shenergy Biocolor BioScience ﹠ Technology Company, and wherein, the sequence of forward and reverse primer is shown in SEQ ID No 1 and SEQ ID No 2, and is specific as follows:
Forward primer: 5 '-CGGGATCC (BamHI) ATGGAGGAGAACAATCAGAACCAGTG-3 ';
Reverse primer: 5 '-CGGAATTC (EcoRI) CTACTTTTGTGCTCTTTCAAGGTCAG-3 '.
2.PCR amplification
The plasmid DNA of pCAMBIA 1300 carriers that will contain the crylc gene is carried out pcr amplification as template, and the pcr amplification result is referring to Fig. 2.
The pcr amplification system is as follows:
10×PCR?Buffer 5μL
dNTP(10mM) 4μL
Template DNA (100ng/ μ L) 1 μ L
Forward primer (10 μ M) 1 μ L
Forward primer (10 μ M) 1 μ L
Ex-Taq polysaccharase (5U/ μ L) 0.5 μ L
ddH
2O 37.5μL
Cumulative volume 50 μ L
The PCR response procedures is as follows:
94℃ 5min
72℃ 10min
3.PCR the recovery of product and ligation
Separate the PCR product with 1% agarose gel electrophoresis, electrophoresis finishes, and gel is placed in the uv analyzer, cuts the blob of viscose that contains the PCR product, reclaims test kit with dna gel and reclaims.
Add in aseptic Eppendorf pipe: 4 μ L reclaim the PCR product, 1 μ L pMD18-T carrier, and 5 μ L SolutionI, cumulative volume are 10 μ L.With each component mixing, 16 ℃ of connections are spent the night, and obtain DNA and connect product.
4.E.coli the preparation of DH5 α competent cell
The single bacterium colony of picking bacillus coli DH 5 alpha (E.coli DH5 α) spends the night in 37 ℃ of shaking culture in the triangular flask that contains 50mL LB substratum; The bacterium liquid 10 μ L that get overnight incubation are in the triangular flask that contains 50mL LB substratum, and 37 ℃ of shaking culture are 0.4-0.6 to the OD600 value; Cultured bacterium liquid is transferred in the centrifuge tube of 50mL ice bath 10min; At 4 ℃, centrifugal 5min removes supernatant under the 5000rpm condition, collecting cell; 0.1M CaCl with the precooling of 20mL ice
2Suspend gently and precipitate, place 10min on ice; At 4 ℃, centrifugal 10min under the 5000rpm condition is with 2mL 0.1M CaCl
2Suspend and precipitate, in aseptic Eppenforf pipe, use the liquid nitrogen quick freezing, be stored in-70 ℃ by every pipe 200 μ L packing cell suspending liquids.
5. connect product Transformed E .coli DH5 α competent cell and evaluation
In 100 μ L competent escherichia coli cell E.coli DH5 α, add 10 μ L DNA and connect product, light finger bomb tube wall, the mixing mixture places 30min on ice; 90sec is handled in 42 ℃ of water-bath heat shocks, puts back on ice ice bath 5min rapidly; Add 800 μ L LB nutrient solutions, mixing, 37 ℃, 150rmp shaking culture 1h; Discard unnecessary LB nutrient solution behind the centrifugal 2min of 4000rpm, remaining 200 μ L LB suspends competent cell gently and adds the IPTG (24mg/mL) of 20 μ L, the X-gal of 40 μ L (20mg/mL), be coated on the solid nutrient agar that contains 100 μ g/mL Amp, smear evenly with glass stick, treat that fully absorbing back 37 ℃ of inversions hatched 12-16 hour.
Carry out the screening of recon with the α-Hu Bu phenomenon, the white colony of growing on the picking flat board carries out PCR and identifies that qualification result is referring to Fig. 3.Wherein, PCR reaction system and response procedures are checked amplified production with step 2PCR amplification with 1% agarose gel electrophoresis.Inoculation is accredited as the male bacterium colony in the LB substratum that contains penbritin through PCR, and 37 ℃, the 240rpm shaking culture is spent the night.Extract plasmid PMD18-T-crylc with the DNA extraction test kit.
6. the recombination to construct of expression plasmid
After the result that waits to check order was correct, with BamH I/EcoR I enzymes double zyme cutting plasmid PMD18-T-crylc, enzyme was cut the result referring to Fig. 4.Endonuclease reaction carries out with reference to the concrete operations in the restriction enzyme specification sheets (precious biotechnology (Dalian) limited liability company).Reaction finishes, and separates enzyme by agarose gel electrophoresis and cuts product, and reclaimer operation is identical with PCR product recovery in the step 3.The expression vector pET-30a (+) of (BamH I/EcoR I) is connected behind the crylc gene fragment that reclaims and the double digestion, linked system is carried out with reference to the concrete operations in the ligase enzyme test kit specification sheets (the DNA Ligation Kit Ver.2.0 of precious biotechnology (Dalian) limited liability company), obtain to be transformed in E.coli Transetta (DE3) competent cell behind recombinant expression plasmid pET-30a (+)-crylc, after double digestion and PCR evaluation correctly, carrying out sequential analysis identifies, obtain the bacterium of recombinating after frame is correct, begin then to express.
The reorganization bacterium abduction delivering
To recombinate colony inoculation in 5mL LB substratum (containing card that penicillin 50 μ g/mL and paraxin 34 μ g/mL), and 37 ℃ of shaking tables are cultured to logarithmic growth early stage (the OD value is about 0.6), add 0.6mmol/L IPTG and induce 5h.Carry out SDS-PAGE analysis expression after getting the centrifugal collection of an amount of bacterium liquid bacterium.Get cultured bacterium liquid 1mL, with centrifugal 3 minutes collection of 10000rpm room temperature bacterium, abandon supernatant, 200 μ L pure water have hanged precipitation, get 20 μ L suspension and add 20 μ L, 2 * sds gel sample loading buffer, boil ten minutes, get 20 μ L and carry out the SDS-PAGE analysis.Voltage: 80V, 20min; 120V, 80min dyes, decolours.After gel-colored 30 minutes, analyze behind the decolouring 1h.
8. the Ni-NTA affinitive layer purification of expressing protein
To recombinate colony inoculation in 5mL LB substratum (containing card that penicillin 50 μ g/mL and paraxin 34 μ g/mL), after 37 ℃ of shaking table overnight incubation by the reorganization bacterium: LB substratum=1: 100 was inoculated in the big triangular flask to logarithmic growth early stage (OD value is about 0.6), and adding 0.6mmol/L IPTG induces 5h.Add ultrasonic Buffer after collecting thalline, the 100mL substratum thalline add the ultrasonic Buffer of 20mL.After bacterium hanged, ultrasonication (ultrasonication 30 times, ultrasonic 10s, 20s at interval).4 ℃, 12000rpm, 20min, fully centrifugal, supernatant is analyzed with SDS-PAGE with precipitation and is determined the albumen position, and major part is arranged in precipitation as a result, with the inclusion body formal representation.Precipitation with the 5mL lysis buffer in 4 ℃ of stirring and dissolving slowly.4 ℃, 12000rpm, centrifugal 20min pours out supernatant and is used for purifying in 4 ℃, and purification result is referring to Fig. 5.
Concrete purge process is as follows:
1. in 1mL 50%Ni-NTA His Bind Slurry, add 4mL Bind Buffer mixing gently,, move and abandon suspension by the action of gravity natural subsidence.
2. with the supernatant collected after the thalline ultrasonication and Ni-NTA at 4 ℃ of jolting 2h, collect behind the dress post and penetrate liquid, be SDS-PAGE and analyze.
3. it is assorted to use 4 * 4mL Wash Buffer to wash, pack into wash behind the assorted liquid earlier block the exit and leave standstill half an hour with rubber cap after, remove rubber cap, flow rate control is collected effluent liquid respectively at 30mL/h, is SDS-PAGE and analyzes.
4. use 4 * 0.5mL Elute Buffer wash-out, collect elutriant with 4 eppendorf pipes respectively, flow rate control is SDS-PAGE and is analyzed at 15mL/h, and the result shows that target protein is eluted with this understanding.
The target protein purity that purifying comes out reaches more than 90%.
9. the renaturation of recombinant protein crylc is with concentrated
Reorganization inclusion body protein purifying is intact, and after SDS-PAGE analyzes content and purity, selects content height and purity to meet the requirements of recombinant protein with the renaturation solution renaturation of dialysing.Adopt the mode that urea content successively decreases in the renaturation dialysis mother liquor to remove denaturing agent gradually, each stage all places 4 ℃ of dialysis 2-4h.Concentrate with the Millipore evaporating pipe, 4000rpm, 4 ℃ of centrifugal 30min-60min get a certain amount of albumen after concentrating and measure protein content with the Bradford method, and all the other are loaded on-70 ℃ of preservations in the Eppendorf pipe.
Attached explanation:
1. the used Buffer of protein purification is as follows:
Ultrasonic Buffer:20mM Tris-HCl, 0.3M NaCl, 10%Glycerol, pH8.0.
Lysis buffer (Buffer): 50mM Tris-HCl, 0.1M NaCl, 5%Glycerol, 8M urea, pH8.0.
Bind?Buffer：20mM?Tris-HCl、0.3M?NaCl、10％Glycerol、10mM?Imidazonle、pH8.0。
Wash?Buffer：20mM?Tris-HCl、0.3M?NaCl、10％Glycerol、8M?urea、20mMImidazonle、pH8.0。
Elute?Buffer：20mM?Tris-HCl、0.3M?NaCl、10％Glycerol、8M?urea、500mMImidazonle、pH8.0。
Renaturation dialysis mother liquor: 20mM Tris-HCl, 0.3M NaCl, 6M, 4M, 2M and 0M urea, pH8.0.
2. the preparation of affinity column
1. put upside down mixing gently and solidify Ni
2+Resin is got the 5mL chromatography column of packing into, resin natural subsidence.
2. use the deionized water rinsing resin (being a column volume after the resin settled) of 3 times of volumes.
3. use the Bind Buffer balance resin of 3 times of column volumes, leave standstill.
3. the thorough regeneration of affinity column:
The all liquid that drains off from the chromatography column lower end is with the regeneration damping fluid (acetate of 6M Guanidinium hydrochloride or 0.2M) of 2 times of NTA resin volumes
The deionized water of 2 times of column volumes
The 2%SDS of 3 times of column volumes
25% ethanol of 1 times of column volume
50% ethanol of 1 times of column volume
75% ethanol of 1 times of column volume
100% ethanol of 5 times of column volumes
75% ethanol of 1 times of column volume
50% ethanol of 1 times of column volume
25% ethanol of 1 times of column volume
The deionized water of 1 times of column volume
The 0.1M EDTA of 1 times of column volume (PH 8.0)
The deionized water of 1 times of column volume
The 0.1M NiSO of 1 times of column volume
4
The deionized water of 3 times of column volumes
20% ethanol is washed after the volume of 1 times of column volume, adds 4 ℃ of preservations of ethanol of 20% of 1 times of column volume.
Use solution 4.SDS-PAGE analyze
1. 2 * sds gel sample loading buffer (stock solution)
4 * Tris-HCI (pH6.8) 25mL, glycerine 20mL, SDS 4g, tetrabromophenol sulfonphthalein (0.001%W/v) 2-3mL
Deionized water is settled to 100mL, gets 95 μ L during application and adds 5 μ L beta-mercaptoethanols.
2. 5 * Tris-glycine electrophoretic buffer
12.1g Tris, 94g Gly, 5g SDS, adding distil water is settled to 1L, uses 5 times of distilled water dilutings during application.
3. the gel-colored liquid of SDS-PAGE (100mL)
Methyl alcohol: water (1: 1) 90mL, acetate 10mL; Coomassie brilliant blue R250 0.25g.
4. the quick destainer of SDS-PAGE gel (100mL)
Methyl alcohol 30mL, acetate 10mL; Distilled water 60mL; With the Whatmanl filter paper filtering to remove particulate material, room temperature preservation.
5.SDS polyacrylamide gel preparation
Two 15% polyacrylamide gels of table 1 (15mL)
Attention: the last TEMED that adds when recording polypropylene phthalein amine gel.
6. gel casting of polypropylene phthalein amine and electrophoresis:
As above the polyacrylamide gel reagent for preparing of method is put stand-by on one side, cleaned electrophoresis sheet glass is dried up (being divided into two kinds of thin and thicks) earlier, thin sheet glass will be placed on the outstanding side on the thick sheet glass, can leave the space in the middle of the one thin one thick and heavy poststack, be contained in the green shelf after the alignment below the glass and slowly step up, the upper limb of thin glass is lower than heavy sheet glass, with hang down some thin glass this face and be fixed on to the outside on the big rack of joining glue.
With pasting the wall of sheet glass behind the separation gel adding TEMED for preparing with suction pipe, slowly be added in two spaces between glass, liquid level slowly adds pure water then and flattens liquid level apart from end margin 2cm on glass place.(note in the operation: the polypropylene phthalein amine gel that adds behind the TEMED can very fast polymerization).
After waiting 30min, can be clearly seen that separation gel polymerization, and a tangible line of delimitation is arranged between the pure water, at this moment can pour out water, with the filter paper edge glass intermediary water is fully blotted then, firmly too not big in order to avoid destroy the plane of glue.
To join above the polymeric separation gel with suction pipe behind the spacer gel adding TEMED that prepare, liquid level will be filled it up with, and then the electrophoresis comb is inserted between the glass, bubble do not occur.(note in the operation: the polypropylene phthalein amine gel that adds behind the TEMED can very fast polymerization)
The polymerization of 30min backset bed glue.
After the spacer gel polymerization, carefully extract comb, on electrophoresis apparatus, interior water jacket respectively adds Tris one glycine buffer with gel sets.
After sample added isopyknic 2 * SDS sample loading buffer, boil 5min in 100 ℃, get sample on the 10 μ L.
Electrophoresis under 80V voltage after the bromjophenol blue forward position enters separation gel, improves voltage to 120V, arrives separation gel bottom, powered-down until bromjophenol blue.
An amount of Coomassie brilliant blue staining fluid is put in the separation gel taking-up, dyeed half an hour on the room temperature condition shaking table.
Gel is immersed in the destainer, on shaking table, decolour under the room temperature condition and spend the night, in water, soak again, take out the back and under UVP Bioimagingsystem, take pictures and saving result.
Should be noted that at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
Claims (5)
1. the prokaryotic expression method of a transgenic paddy rice Bt albumen crylc may further comprise the steps:
1) gene of the crylc that checks order according to Hua Zhong Agriculture University, and the multiple clone site of the carrier PMD18-T that will connect designs forward and reverse primer, carries out pcr amplification and recovery, and the PCR regenerant is carried out the DNA ligation;
2) preparation of competent escherichia coli cell and conversion;
3) abduction delivering of the recombination to construct of expression plasmid, reorganization bacterium.
2. method according to claim 1 is characterized in that, described forward and reverse primer sequence is shown in SEQ ID No1 and SEQ ID No 2, and is specific as follows:
Forward primer: 5 '-CGGGATCC (BamHI) ATGGAGGAGAACAATCAGAACCAGTG-3 ';
Reverse primer: 5 '-CGGAATTC (EcoRI) CTACTTTTGTGCTCTTTCAAGGTCAG-3 '.
3. method according to claim 1 is characterized in that, described competent escherichia coli cell is E.coliDH5 α and E.coil Transetta (DE3).
4. method according to claim 1 is characterized in that, carries out being further purified of expressing protein by the Ni-NTA affinity chromatography.
5. the application of transgenic paddy rice Bt albumen crylc in colloidal gold immuno-chromatography test paper strip of the method for claim 1 preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343163A (en) * | 2013-06-06 | 2013-10-09 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC detection primer and detection method for transgenic rice line CRY1C |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6809078B2 (en) * | 1996-11-27 | 2004-10-26 | Monsanto Technology Llc | Compositions encoding lepidopteran-toxic polypeptides and methods of use |
| CN101250519A (en) * | 2007-11-27 | 2008-08-27 | 江苏省原子医学研究所 | Prokaryotic expression of soluble interleukins 15 acceptor alpha sub-unit and purifying method |
| CN101691582A (en) * | 2009-10-20 | 2010-04-07 | 上海市农业科学院 | Prokaryotic expression and purification method for listeria monocytogenes hemolysin O |
-
2010
- 2010-12-08 CN CN 201010578268 patent/CN102094038A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6809078B2 (en) * | 1996-11-27 | 2004-10-26 | Monsanto Technology Llc | Compositions encoding lepidopteran-toxic polypeptides and methods of use |
| CN101250519A (en) * | 2007-11-27 | 2008-08-27 | 江苏省原子医学研究所 | Prokaryotic expression of soluble interleukins 15 acceptor alpha sub-unit and purifying method |
| CN101691582A (en) * | 2009-10-20 | 2010-04-07 | 上海市农业科学院 | Prokaryotic expression and purification method for listeria monocytogenes hemolysin O |
Non-Patent Citations (4)
| Title |
|---|
| 《Nature biotechnology》 20031231 Jian-Zhou Zhao Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution 1493-1497 1-5 第21卷, 第12期 * |
| 《Nature biotechnology》 20031231 Jian-Zhou Zhao Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution 1493-1497 1-5 第21卷, 第12期 2 * |
| 《植物保护学报》 20030630 宋福平等 对鳞翅目害虫有活性的cry1C基因的克隆和表达 161-165 1-4 第30卷, 第2期 * |
| 《植物保护学报》 20030630 宋福平等 对鳞翅目害虫有活性的cry1C基因的克隆和表达 161-165 1-4 第30卷, 第2期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343163A (en) * | 2013-06-06 | 2013-10-09 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC detection primer and detection method for transgenic rice line CRY1C |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| CN109781992B (en) * | 2018-12-27 | 2021-04-06 | 中国农业科学院生物技术研究所 | Colloidal gold immunochromatographic assay rapid test card for insect-resistant protein Cry1C |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
| CN109917032B (en) * | 2019-03-13 | 2021-08-24 | 杭州老爸评测科技有限公司 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
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Application publication date: 20110615 |