CN102220311B - Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer - Google Patents
Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer Download PDFInfo
- Publication number
- CN102220311B CN102220311B CN201110109343.8A CN201110109343A CN102220311B CN 102220311 B CN102220311 B CN 102220311B CN 201110109343 A CN201110109343 A CN 201110109343A CN 102220311 B CN102220311 B CN 102220311B
- Authority
- CN
- China
- Prior art keywords
- cry1ab
- fit
- albumen
- screening
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Disclosed is a BT-transgene paddy rice Cry1Ab/Ac protein aptamer, which is an aptamer C03 having a basic group sequence of: ccgcggttcc tcggccccct atccacgccg agtcccgaat actccccaga tgragtagcc ctcagcatag. The BT-transgene paddy rice CryAb/Ac protein aptamer has the advantages that the aptamer C03 and the BT-transgene paddy rice CryAb/Ac protein have higher specific combination activity.
Description
[technical field]
The present invention relates to a kind of albumen fit, relating in particular to one, to turn BT trans-genetic hybrid rice Cry1Ab/Ac albumen fit.
[background technology]
Tribactur (Bacillus thuringiensis, BT) be to be found in 1901 by Japanese scientist S Ishiwata, and the very important insect pathogenic bacteria of one of being named in 1915 by Germany scientist Berliner, can secrete a kind of toxalbumin, lepidopteran coleopteron (such as small cabbage moth) is had to very strong lethal effect.Turn the crop of BT gene can be by the intestinal epithelial cell of cracking insect kill insects, for overcoming the threat of the plague of insects to farm crop, entered the mankind's life containing the food that turns BT toxin gene crop.Nineteen ninety-five, genetically modified crops started to enter the large-scale commercial plantation stage, and turning until now BT gene crops is the fastest insect-resistant transgenic crop of global commercialized degree, as tobacco, corn, cotton, paddy rice etc.In China, BT gene is imported into paddy rice--" extensive No. 1 of China ", " BT Shanyou 63 " transgenosis rice.BT albumen is for special population, as need protein content greatly and just the children of skeleton development and the crowd of gastric acid secretion deficiency, may cause immunity system to grow and function serious hindrance, therefore carry out the detection of genetically modified crops BT transgenosis particularly important, avoid providing the food containing BT albumen to these crowds.
Fit (aptamer) etymology is to fit in Latin aptus, means " being applicable to ", is called again aptamer.It has the advantages such as storage capacity is large, target molecule scope is wide, avidity is high, high specificity.In recent years, fit (aptamer) alternative antibody is used widely in protein detection.Fit is to be to screen ssDNA library by SELEX technology from the oligonucleotide library of synthetic, with the high-affinity single stranded oligonucleotide sequence of non-nucleic acid target material specific combination.And SELEX technology is to utilize Protocols in Molecular Biology, by random oligonucleotide library and target matter interaction, retain the oligonucleotide sequence of combination, through repeated amplification, screening, make to obtain enrichment with the oligonucleotide sequence of this target material specific combination.At present, show wide application prospect in the research field aspect the research of gene regulating research, protein function, nucleic acids research, disease treatment, be successfully applied to the screening that much albumen is fit, but rare report is applied in screening on the foreign protein of transgenic product.
For these reasons, the applicant has published respectively the article that is called " screening that Listeria monocytogenes is fit and structural analysis ", " technology of preparing that Listeria monocytogenes aptamer is fit screening " on " Food science " (the 30th volume in 2009) and " Chinese public health " (the 25th volume in 2009), and it discloses ssDNA pool is carried out to SELEX screening; Preparation ssDNA enriched library; Utilize digoxin-anti-digoxin alkaline phosphatase Color Appearance System to measure the combination rate of ssDNA enriched library and listeria spp; Screening product is cloned, is checked order after pcr amplification, and analyzes its primary structure conserved sequence and secondary structure with Macaw2.05 and DNAMAN software package; Carrying out the contents such as anti-sieve with Ying Nuoke listeria bacteria, thereby obtain the method with Listeria monocytogenes with the fit group of high-affinity specific binding, is the fit screening method solid basis of having established for further study.
[summary of the invention]
It is fit that technical problem to be solved by this invention is to provide one to turn BT trans-genetic hybrid rice Cry1Ab/Ac albumen, and turns BT trans-genetic hybrid rice Cry1Ab/Ac albumen and have higher specific combination activity.
The present invention solves the problems of the technologies described above by the following technical programs: it is fit that one turns BT trans-genetic hybrid rice Cry1Ab/Ac albumen, described fit be fit C03; The base sequence of described fit C03 is: ccgcggttcc tcggccccct atccacgccg agtcccgaat actccccaga tgtagtagcc ctcagcatag.
The invention has the advantages that: fit C03 with turn BT trans-genetic hybrid rice Cry1Ab/Ac albumen and there is higher specific combination activity, method program that the present invention sets up is simple, criterion is clear and definite, method simple and feasible, technical requirements is not high, and fit easy external preparation, circulation preparation are easy to obtain very much.
[embodiment]
The ssDNA library Shi You Shanghai Sangon Biological Engineering Technology And Service Co., Ltd that the present invention adopts synthesizes, ssDNA library is 5 '-GGG AGC TCA GAA TAA ACG CTC AA-N70-TT CGA CAT GAG GCC CGG ATC-3 ', its two ends are fixed sequence program, the i.e. stochastic sequence of 70 bases in the random district that middle N70 is library, total length is totally 113 bases, and the target product that wherein N70 also finally will obtain for the present invention is fit.
The present invention turns the fit screening method of BT trans-genetic hybrid rice Cry1Ab/Ac albumen, and concrete operation step is as follows:
Step 1:
A. will turn BT trans-genetic hybrid rice Cry1Ab/Ac proteopexy on oxyethane vinylformic acid pearl, be prepared into Cry1Ab/Ac albumen-acrylic acid epoxy ethane pearl, be prepared into 5.6mg/mL Padil-empty pearl with the coated Cry1Ab/Ac albumen-acrylic acid epoxy ethane pearl of hydration mercaptoethanol simultaneously, take turns and all use blank pearl in each of screening, its object is to remove is combined ssDNA with the pearl part beyond target protein, reduce to screen background.In addition the protein bound optimum concn of oxyethane vinylformic acid pearl and Cry1Ab/Ac is 5.6mg/mL, adopts this concentration, and the fixed effect of Cry1Ab/Ac albumen on carrier rings oxidative ethane vinylformic acid pearl is best.
B. the first run is chosen 600pmol ssDNA pool, adds binding buffer liquid dilution ssDNA to 200 μ L, then solution is placed in to 95 DEG C of sex change 5min.
C. the solution of processing through step B is transferred in the test tube that 200 μ L Padil-empty pearls are housed, again above-mentioned test tube vortex is hatched to 30min, then precipitation is removed in centrifugation, then supernatant liquor is transferred in the test tube that 100 μ L Cry1Ab/Ac albumen-acrylic acid epoxy ethane pearls are housed, under room temperature, hatch 30min, liquid phase is removed in last centrifugation.
D. wash the pearl processed through step C 8 times with the binding buffer liquid of 500 μ L, then add 400 μ L TE wash-outs, then suspension is hatched to 30min, last centrifugation discards precipitation.
E. the supernatant liquor that transfer step D obtains is in being equipped with the test tube of pre-cooled 3mol/L sodium-acetate and dehydrated alcohol, follow mixing solutions in 4 DEG C, 13400rpm centrifugation 30min, remove liquid phase, then add 200 μ L70% ethanolic soln rapid stirring 1min, again by gained solution in 4 DEG C, 13400rpm centrifugation 5min, and remove liquid phase, and finally adding 50 μ L deionized water dissolvings, gained solution is as the template of pcr amplification.Solution after step D wash-out is carried out to this step process again, can further wash away impurity, make the template purifying more obtaining.
F. the annealing temperature in PCR reaction system and template concentrations are optimized, wherein thermodynamic cycle parameter is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 66 DEG C～74 DEG C annealing 30s, 72 DEG C are extended 20s, circulate 26 times, and then, in 72 DEG C of extension 3min, wherein annealing temperature gradient is 66.0 DEG C, 66.6 DEG C, 67.7 DEG C, 69.2 DEG C, 71.0 DEG C, 72.5 DEG C, 73.5 DEG C and 74.0 DEG C; Template to E step gained is diluted.The laggard performing PCR amplification of question response system optimization.Before pcr amplification, all need annealing temperature be optimized and the template of E step gained is diluted, then select an optimum annealing temperature and optimum diluting multiple to dilute laggard performing PCR amplification.
G. prepare ssDNA enriched library, concrete operation method is referring to the step ' preparation of 1.2.5ssDNA enriched library ' in the article " technology of preparing that Listeria monocytogenes aptamer is fit screening " of " Chinese public health " (the 25th volume in 2009).
H. the above-mentioned steps that circulates B is to step G14 time, wherein often takes turns the 50 μ L ssDNA enriched libraries that described in circulation step B, 600pmol ssDNA pool makes with last round of step G and substitutes; After wherein taking turns since the 6th, every wheel hatched 30min and changed into and hatch 20min described in step C; After wherein starting from fourth round, the every Cry1Ab/Ac of TE described in step D protein solution of taking turns substitutes.
Step 2: take turns products therefrom ssDNA enriched library and the combination rate that turns BT trans-genetic hybrid rice Cry1Ab/Ac albumen with odd number wheel and the 14th in digoxin-anti-digoxin alkaline phosphatase Color Appearance System determination step 1, concrete operation method is referring to the step in the article " technology of preparing that Listeria monocytogenes aptamer is fit screening " of " Chinese public health " (the 25th volume in 2009) ' ssDNA of 1.2.6 screening and the mensuration of Listeria monocytogenes avidity '.
Step 3: take turns by the highest the combination rate recording in step 2 one the ssDNA enriched library that screening obtains and reclaim purifying after pcr amplification, products therefrom each fit fit group who mixes that serves as reasons, then fitly clone, check order each, and analyze its primary structure conserved sequence and secondary structure with Macaw2.05 and DNAMAN software package, concrete operation method is referring to the step in the article " screening that Listeria monocytogenes is fit and structural analysis " of " Food science " (the 30th volume in 2009) ' 1.2.2 clone and order-checking '.
Step 4: the fit combination rate with turning BT trans-genetic hybrid rice Cry1Ab/Ac albumen of each ssDNA by digoxin-anti-digoxin alkaline phosphatase Color Appearance System mensuration after step 2 order-checking, and it is fit therefrom to select the highest one of combination rate, and carry out anti-screening with transgenic corns EPSPS protein solution and survey its specificity, concrete operation method is referring to the step in the article " technology of preparing that Listeria monocytogenes aptamer is fit screening " of " Chinese public health " (the 25th volume in 2009) ' ssDNA of 1.2.6 screening and the mensuration of Listeria monocytogenes avidity ' and step ' 1.2.2SELEX screening process '.
Wherein, the component of described binding buffer liquid is: NaCl:250mmol/L, MgCl
2: 1mmol/L, EDTA:0.1mmol/L, Tris-HCl:20mmol/L, all the other components are water, and the value of pH value of solution is 7.5, and in binding buffer liquid, the concentration of each component is that total solution taking binding buffer liquid is as benchmark.
The component of described TE damping fluid is: Tris:10mmol/L, and EDTA:1mmol/L, all the other components are water, the pH of solution is that the concentration of each component in 8.0, TE damping fluid is that total solution taking TE damping fluid is as benchmark.
Wherein, each wheel template concentrations of PCR reaction system optimization and annealing temperature result table 1:
The every PCR that takes turns of table 1 optimizes the optimum annealing temperature and the template extension rate that react
After reaction system is optimized, carry out again pcr amplification and can make more single, the clear and assorted band of the ssDNA band that obtains after SELEX screening, make to avoid in pcr amplification reaction process the generation of non-specific amplification and primer dimer.
Take turns the combination rate of products therefrom ssDNA and Cry1Ab/Ac albumen with odd number wheel in digoxin-anti-digoxin alkaline phosphatase Color Appearance System determination step 2 and the 14th, it just reacts from absorbance of microplate reader mensuration, and concrete outcome is in table 2:
The combination rate of table 2 each wheel product ssDNA and Cry1Ab/Ac albumen
As can be seen from the table, along with the increase of screening wheel number, the protein bound absorbancy of ssDNA and Cry1Ab/Ac is totally in rising trend, by the 1st take turns 0.3769 rise to the 13rd take turns 1.7421, nearly 4.6 times are improved, illustrate along with constantly carrying out of screening, being adsorbed onto amount fit on Cry1Ab/Ac albumen is also increasing.The 14th absorbance of taking turns declines to some extent compared with the 13rd absorbance of taking turns, illustrate in the time that the specific combination site on ssDNA and Cry1Ab/Ac albumen reaches a state of saturation, being adsorbed onto ssDNA on this albumen just no longer increases, and the 13rd combination rate of taking turns the ssDNA that filters out and the Cry1Ab/Ac albumen state that reaches capacity is described.
The present invention makes screening vector with oxyethane vinylformic acid pearl, the avidity of fit C03, C05, C06, D03, D08, D09, D10, D11 and the Cry1Ab/Ac albumen filtering out is higher, the numerical value of absorbancy that the height of its avidity is measured from microplate reader reacts, and concrete outcome is in table 3:
The absorbancy of the avidity of table 3 each fit and Cry1Ab/Ac albumen
Wherein the absorbance of C03 is each fit apparently higher than other, shows that fit C03 is the highest with the avidity that turns BT trans-genetic hybrid rice Cry1Ab/Ac albumen.
In addition, fit C03 and Cry1Ab/Ac protein binding are dose-dependence, and particular case is in table 4:
Table 4: the absorbancy of the avidity of the Cry1Ab/Ac albumen of fit C03 and various dose
As can be seen from the above table, Cry1Ab/Ac albumen dosage is between 3 μ g～120 μ g, and the light absorption value of both combinations increases with the increase of target protein amount, shows that the protein bound avidity of C03 and Cry1Ab/Ac and Cry1Ab/Ac protein content are dosage correlation.
The present invention adopts oxyethane vinylformic acid pearl to make screening vector, SELEX screening method is further improved, experimental result shows to search out the fit CO3 of being of specificity that turns BT trans-genetic hybrid rice Cry1Ab/Ac albumen, and in certain Cry1Ab/Ac protein agent weight range, the protein bound avidity of C03 and Cry1Ab/Ac and Cry1Ab/Ac protein content are dosage correlation.Method program that the present invention sets up is simple, criterion is clear and definite, method simple and feasible, and technical requirements is not high, and fit easy external preparation, circulation preparation, is easy to obtain very much.
In the present invention related fit C03, C05, C06, D03, D08, D09, D10, D11 and turn the Nucleotide of BT trans-genetic hybrid rice Cry1Ab/Ac albumen or aminoacid sequence respectively referring to shown in the SEQ ID NO:1 in sequence table, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9.
Claims (1)
- One kind to turn BT trans-genetic hybrid rice Cry1Ab/Ac albumen fit, it is characterized in that: the described fit fit C03 of being, the base sequence of described fit C03 is: ccgcggttcc tcggccccct atccacgccg agtcccgaat actccccaga tgtagtagcc ctcagcatag.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110109343.8A CN102220311B (en) | 2011-04-27 | 2011-04-27 | Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110109343.8A CN102220311B (en) | 2011-04-27 | 2011-04-27 | Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102220311A CN102220311A (en) | 2011-10-19 |
| CN102220311B true CN102220311B (en) | 2014-10-01 |
Family
ID=44777015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110109343.8A Expired - Fee Related CN102220311B (en) | 2011-04-27 | 2011-04-27 | Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102220311B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665821B (en) * | 2008-09-01 | 2012-02-08 | 中国人民解放军军事医学科学院基础医学研究所 | Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof |
| CN101748210A (en) * | 2009-11-11 | 2010-06-23 | 兰州大学 | Single-strand DNA aptamer for detecting chronic myeloid leukemia and preparation method |
| CN102010866B (en) * | 2010-09-29 | 2012-10-31 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing shigella |
-
2011
- 2011-04-27 CN CN201110109343.8A patent/CN102220311B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102220311A (en) | 2011-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ferrer-González et al. | Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy | |
| Liu et al. | Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector | |
| Li et al. | Rare fungus, Mortierella capitata, promotes crop growth by stimulating primary metabolisms related genes and reshaping rhizosphere bacterial community | |
| Díaz-Muñoz et al. | Bacteria–phage interactions in natural environments | |
| Ma et al. | Selection, characterization and application of aptamers targeted to Aflatoxin B2 | |
| CN104630230B (en) | The aptamer of one group of specific recognition okadaic acid | |
| CN106834295A (en) | The wide spectrum aptamer and its directed screening method of a kind of specific recognition bacteria lipopolysaccharide | |
| Pan et al. | Root exudates and rhizosphere soil bacterial relationships of Nitraria tangutorum are linked to k-strategists bacterial community under salt stress | |
| Ling et al. | Viral metagenomics reveals significant viruses in the genital tract of apparently healthy dairy cows | |
| CN102010865B (en) | Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof | |
| CN102010866B (en) | Nucleic acid aptamer capable of specifically recognizing shigella | |
| Steven et al. | What’s under the Christmas tree? A soil sulfur amendment lowers soil pH and alters fir tree rhizosphere bacterial and eukaryotic communities, their interactions, and functional traits | |
| CN110088282A (en) | With the method for magnetic-particle separating high-purity nucleic acid | |
| Farci et al. | The role of iron and copper on the oligomerization dynamics of DR_2577, the main S-layer protein of Deinococcus radiodurans | |
| Hoh et al. | Comparative genomic and transcriptomic analyses of trans-kingdom pathogen Fusarium solani species complex reveal degrees of compartmentalization | |
| CN106916821A (en) | A kind of ssDNA aptamers and its application | |
| Chauhan et al. | RNA beyond humans | |
| CN102220311B (en) | Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer | |
| CN109134635A (en) | A kind of antibacterial peptide and the preparation method and application thereof with composite bio-active | |
| CN102323400A (en) | Method for detecting and identifying variety of snake venom by utilizing adaptor technology | |
| CN107916265A (en) | One aptamer and its screening technique that can be specifically bound with Cefquinome | |
| CN105986025A (en) | Research method for relation between microorganisms and uranium mineralization in sandstone type uranium ore deposit | |
| Liu | Handbook of nucleic acid purification | |
| CN107385117A (en) | A kind of sweet potato potato seed complex disease viral disease（SPVD）The detection method of cause of disease | |
| Wang et al. | Mucoromycotina fungi possess the ability to utilize plant sucrose as a carbon source: evidence from Gongronella sp. w5 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141001 Termination date: 20200427 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |