CN100348616C - Bt CrylA antibody, and its preparing method and special antigen and use - Google Patents
Bt CrylA antibody, and its preparing method and special antigen and use Download PDFInfo
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- CN100348616C CN100348616C CNB2005101300589A CN200510130058A CN100348616C CN 100348616 C CN100348616 C CN 100348616C CN B2005101300589 A CNB2005101300589 A CN B2005101300589A CN 200510130058 A CN200510130058 A CN 200510130058A CN 100348616 C CN100348616 C CN 100348616C
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Abstract
The present invention discloses a Bt Cry 1A antibody, a preparation method thereof, an antigen special for the Bt Cry 1A antibody and the application, which has the purpose to provide a Bt Cry 1A antibody and a preparation method thereof, an antigen special for the Bt Cry 1A antibody and the application for preparing a Bt immunity detection 3.1 kit. The antigen special for the Bt Cry 1A antibody is prepared by the linking polypeptide of an amino acid residue sequence containing SEQ ID No. 1 in the sequence table and carrier proteins. The Bt Cry 1A antibody is prepared by separating and purifying blood serum in immune animals by immune animals special for the antigen. The antibody has the following advantages: (1) the specificity is high, the detection sensitivity can reach 52.7 ng/ml<-1>, and the antibody can be used for the qualitative analysis and the quantitative analysis of poison protein of transgenic Bt plants (such as cotton, maize, rice, tobacco, etc.) (2) the preparation method is simple and has the feasibility of industrial production. The present invention plays an important role in the biological detection field of transgenic Bt plants and has broad market prospects.
Description
Technical field
The present invention relates to Bt Cry1A antibody and preparation method thereof and special antigen and application, particularly relate to a kind of BtCry1A antibody and preparation method thereof and special antigen and the application in preparation Bt immunity detection reagent thereof.
Background technology
The insecticidal mechanism that changes Bt plant (cotton, corn, paddy rice, tobacco) be since plant interior expression toxalbumin.The Bt immunity detection reagent is mainly used in the extensive height that accurately detects the content that has that it's too late of Bt toxalbumin, and the detection method of changeing the Bt plant mainly contains three kinds: (1) biological examination method; (2) DNA detection; (3) proteic immunodetection.In these three kinds of methods, biological examination method is more loaded down with trivial details, and the measurement result variation is big, and is difficult to carry out horizontal and vertical comparison.DNA detection just detects gene, does not represent protein level, there is no directive significance for the height of resistance.Immunologic detection method is convenient, practical, quick, economical.
The Bt immunity detection reagent has been widely used in transgenic pest-resistant cotton, corn seed quality examination, authenticity, transgene component analysis in the food, the research of biological safety.The quality of Bt test kit depends on the quality of Bt insecticidal crystal protein Cry1A antibody fully.Cry1A antibody in the domestic and foreign literature report all is that the albumen by bacillus thuringiensis fermentation, extraction, purifying obtains as antigen-immunized animal.Because proteic separation and purifying be difficulty very, in the process of purifying, unavoidably can mix some foreign proteins, even the unusual albumen of purifying, because the existence of a plurality of antigenic determinants, prepared antibody often also has cross reaction, so the specificity of antibody is affected.Be to solve the specificity problem of antibody, the Cry1A antibody in the at present external Bt test kit is that the method by synthetic polypeptide prepares, but what utilized is the not open mostly or patent applied for of which kind of polypeptide.
Summary of the invention
First purpose of the present invention provides a kind of special antigen that is used to prepare Bt Cry1A antibody.
The special antigen of preparation Bt Cry1A antibody provided by the present invention is to have SEQ ID № in the sequence table: the polypeptide of 1 amino acid residue sequence and carrier proteins connection obtain.
SEQ ID № in the sequence table: 1 is made up of 16 amino-acid residues.The synthetic of peptide used solid-phase synthesis usually, adopts uncle's fourth oxygen (acyl) carbonyl (t-BOC) chemical method and 9-fluorine methoxycarbonyl (FMOC) chemical method more.
Described carrier proteins is linked in the aminoterminal (N end) of described polypeptide.
Carrier proteins can be any one carrier proteins commonly used, as bovine serum albumin (BSA), human serum albumin (HSA), albumen keyhole limpet hemocyanin (KLH) or oralbumin (OVA) etc.
The method that polypeptide and carrier proteins are connect also is conventional glutaraldehyde method.
Second purpose of the present invention provides a kind of Bt Cry1A antibody.
Bt Cry1A antibody provided by the present invention is with above-mentioned special antigen immune animal, again from separate the immune animal, antibody that purified blood serum obtains.
The described immune animal that is used to prepare Bt Cry1A antibody can be immune animal commonly used such as chicken, rabbit, mouse, sheep or horse.
Another object of the present invention provides a kind of preparation method of Bt Cry1A antibody.
The preparation method of Bt Cry1A antibody provided by the present invention may further comprise the steps:
1) with above-mentioned special antigen immune animal;
2) from step 1) through the animal of immunity, separate, purified blood serum, obtain Bt Cry1A antibody.
In above-mentioned preparation method, the immune animal that is used to prepare Bt Cry1A antibody in the step 3) can be immune animal commonly used such as chicken, rabbit, mouse, sheep or horse.
The 4th purpose of the present invention provides a kind of Bt immunity detection reagent.
Bt immunity detection reagent provided by the present invention, its activeconstituents are above-mentioned Bt Cry1A antibody.
In actual applications, detect, detection reagent such as negative serum contrast, serum dilution, washing composition, terminator, developer, Bt standard specimen and HRP-goat anti-rabbit igg antibody also can be packaged into the mentioned reagent box for convenient.
The invention provides a kind of Bt Cry1A antibody.This antibody has following advantage: 1) have higher specificity, detection sensitivity can reach 52.7ngmL
-1, can be used for changeing proteic qualitative, the quantitative analysis of Bt plant (as cotton, corn, paddy rice, tobacco etc.) poisoning; 2) preparation method is simple, has the feasibility of suitability for industrialized production.The present invention will play a significant role in the field of biological detection of changeing the Bt plant, have vast market prospect.
The invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is a Bt Cry1A protein concentration typical curve
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The preparation of embodiment 1, Bt Cry1A antibody
One, the selection of Bt insecticidal crystal protein Cry1A polypeptide fragments and synthetic
Bt insecticidal crystal protein Cry1A (comprising Cry1Aa, Cry1Ab, Cry1Ac) gene in the GenBank database (is respectively for GenBank number: D17518, X54939, U87397) coded aminoacid sequence carries out physico-chemical analysis, therefrom select one section specific for 16 amino-acid residues (SEQ ID № in the sequence table: the polypeptide fragment of 1) forming, synthesize with solid-phase synthesis, molecular weight is 1943.14.
Two, the preparation of immunizing antigen
Adopt the glutaraldehyde method to connect step 1 synthetic polypeptide fragment and bovine serum albumin (BSA), concrete grammar may further comprise the steps:
(1) gets 10mg BSA, be dissolved in fully among the 5mL 0.01mol/L PBS, add the synthetic polypeptide of 4mg step 1 again, it is dissolved fully.
(2) slowly add the glutaraldehyde solution of 5mL 0.2%, the solution that obtains with step (1) mixes, and at room temperature, stirring reaction 2 hours.
(3) add 0.2mL 1M glycine, at room temperature stirred 1 hour, with termination reaction.
(4) during the solution that step (3) is obtained is packed dialysis tubing into, carried out purifying in 4 days, change dialyzate every day three times with the PBS dialysis.The polypeptide-BSA that obtains after the dialysis is connect thing carries out packing, through-40 ℃ freezing after, the vacuum concentration drying is stored in-20 ℃ of refrigerators.
Three, preparation Bt Cry1A antibody
Immune animal is adopted New Zealand's large ear rabbit, and rabbit is about three months ages, more than the body weight 2kg.With the connection thing of the polypeptide-BSA of step 2 preparation as antigen, immune animal, preparation Bt Cry1A antibody, immunization method and dosage are: immunization is six times altogether, and antigen is diluted to 2mgmL with PBS
-1(calculating with carrier proteins) mixes back emulsification with isopyknic Fu Shi Freund's complete adjuvant (immunity for the first time) or freund 's incomplete adjuvant (for the second time to the 5th immunity) then, uses the immunizing antigen direct immunization for the last time, and immunizing dose is the 1mL/ rabbit.Immunity back auricular vein is got blood for the third time, detects antiserum titre, and detected result shows that antiserum titre reaches 1/50000.Carotid artery bloodletting in the 7th day after the last immunity obtains antiserum(antisera) behind separation, the purifying, detects antiserum titre, and detected result shows that antiserum titre reaches 1/50000, can be used for the detection of Bt Cry1A content.
The foundation of the evaluation of embodiment 2, Bt Cry1A antibody and Bt protein concentration typical curve
With the ELISA method Bt Cry1A antibody is identified, and drawn out Bt protein concentration typical curve, detailed process may further comprise the steps:
(1) bag is by the Bt standard protein
Bt Cry1A standard protein is cushioned liquid (1.5g Na with bag
2CO
3, 2.93g NaHCO
3, 0.2g NaN
3, add 1000mL distilled water, pH is 9.6) be diluted to five concentration, be followed successively by 2640,880,293,97,32ngmL
-1, being cushioned liquid with bag is that blank (is 0ngmL
-1), add in the 96 hole enzyme plates, every hole 100 μ l, each concentration repeats incubation 3h in 37 ℃ of incubators three times.Discard liquid in the hole, with washings (8.0g NaCl, 0.2gKH
2PO
4, 2.96g Na
2HPO
412H
2O, 1mL Tween-20 adds 1000mL distilled water) dry after washing plate four times.
(2) with Bt Cry1A antibody response
Bt Cry1A antibody sample diluting liquid (8.0g NaCl, 0.2g KH with embodiment 1 preparation
2PO
4, 2.96gNa
2HPO
412H
2O, 1mL Tween-20, the 1g gelatin adds 1000mL distilled water, pH is 7.5) and by 1: 500 dilution proportion, add 100 μ l in the every hole of enzyme plate, put into 37 ℃ of incubator incubation 30min.Discard liquid in the hole, dry after washing plate four times with washings.
(3) with the ELIAS secondary antibody reaction
(sigma company) dilutes in 1: 1000 ratio with sample diluting liquid with the HRP-goat anti-rabbit igg antibody, and every hole adds 100 μ l, puts into 37 ℃ of incubator incubation 30min.Discard liquid in the hole, wash plate four times, dry with washings.
(4) colour developing
(ortho-phenylene diamine, colour developing under the OPD) solution, normal temperature, colour developing be back 2N sulfuric acid termination reaction fully, measures the OD value at each 492nm place, hole with microplate reader to add 100 μ l O-Phenylene Diamines in the every hole of enzyme plate.
Detected result show embodiment 1 preparation Bt Cry1A antibody can with Bt Cry1A standard protein specific reaction, concentration gradient and corresponding OD value drawing standard curve thereof according to Bt Cry1A standard protein, (the y axle is the natural logarithm value of each concentration of Bt Cry1A standard protein as shown in Figure 1, the x axle is its corresponding OD value), the regression equation of typical curve is: y=3.9016x+3.185, coefficient R
2=0.982, IC
50Be 169.9ngmL
-1, sensing range is 52.7ngmL
-1To 547.8ngmL
-1
The detection of embodiment 3, commentaries on classics Bt cotton insecticidal crystal protein Cry1A
With the ELISA method insecticidal crystal protein Cry1A that changes in the pest-resistant cotton of Bt is detected, used Insect Resistant Cotton flower variety comprises: in 41, in 45, in 29, GK36, GK12,33B, with in the conventional cotton 12, in 43 be that contrast (Cotton Inst., Chinese Agricultural Academy's purchase) detailed process may further comprise the steps:
(1) take by weighing Bt transgene cotton sample 0.3g, fully grind behind the adding 3mL PBS extracting solution, the centrifugal 10min of 3000rpm behind the extraction 4h gets supernatant liquor and is used for analyzing.
(2) in the every hole of enzyme plate, add the supernatant liquor 100 μ l that step (1) obtains, incubation 3h in 37 ℃ of incubators.Discard liquid in the hole, wash plate four times, dry with washings.
(3) the Bt Cry1A antibody with embodiment 1 preparation dilutes in 1: 500 ratio with sample diluting liquid, adds 100 μ l in the every hole of enzyme plate, puts into 37 ℃ of incubator incubation 30min.Discard liquid in the hole, dry after washing plate four times with washings.
(4) the HRP-goat anti-rabbit igg antibody is diluted in 1: 1000 ratio with sample diluting liquid, in the every hole of enzyme plate, add 100 μ l, place 37 ℃ of incubator incubation 30min.Discard liquid in the hole, wash plate four times, dry with washings.
(5) add 100 μ l OPD solution in the every hole of enzyme plate, normal temperature colour developing down with 2N sulfuric acid termination reaction, is measured the OD value at each 492nm place, hole on microplate reader.
Experimental result shows, in the transgenic cotton kind 41, in 45, in 29, GK36, GK12,33B obviously than in the conventional cotton variety 12 and in 43 colour developings dark.The Bt Cry1A protein concentration typical curve of drawing according to embodiment 2 calculates the proteic concentration of Bt Cry1A in the above-mentioned cotton sample, the result is as shown in table 1, show that BtCry1A insecticidal crystal protein content is apparently higher than above-mentioned two kinds of conventional cotton varieties in the transgenic cotton, the result is consistent with colour developing.Proof can be distinguished conventional cotton and Insect Resistant Cotton effectively with Bt Cry1A antibody of the present invention and ELISA method, and can measure comparatively accurately Bt Cry1A insecticidal crystal protein content wherein.
Bt toxalbumin content in conventional cotton of table 1 and the Insect Resistant Cotton
| Kind | Conventional cotton | Transgenic cotton against pests | ||||||
| Toxalbumin content (ngmL -1) | In 12 | In 43 | In 41 | In 45 | In 29 | GK36 | GK12 | 33B |
| 0 | 0 | 44.6 | 42.9 | 29.0 | 50.3 | 19.3 | 52.7 | |
Sequence table
<160>1
<210>1
<211>16
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met
1 5 10 15
Claims (8)
1, the special antigen of preparation Bt Cry1A antibody is to adopt glutaraldehyde method to connect the polypeptide that obtains amino acid residue sequence shown in the SEQ ID NO:1 and carrier proteins.
2, special antigen according to claim 1 is characterized in that: described carrier proteins is linked in the aminoterminal of described SEQID NO:1.
3, special antigen according to claim 1 and 2 is characterized in that: described carrier proteins is bovine serum albumin, human serum albumin, albumen keyhole limpet hemocyanin or oralbumin.
4, Bt Cry1A antibody is with claim 1-3 any described special antigen immune animal, again from separate the immune animal, antibody that purified blood serum obtains.
5, Bt Cry1A antibody according to claim 4 is characterized in that: the described immune animal that is used to prepare Bt Cry1A antibody is chicken, rabbit, mouse, sheep or horse.
6, a kind of method for preparing the described Bt Cry1A of claim 4 antibody, its step is as follows:
1) with the described special antigen immune animal of claim 1;
2) from step 1) through the animal of immunity, separate, purified blood serum, obtain Bt Cry1A antibody.
7, preparation method according to claim 6 is characterized in that: the immune animal that is used to prepare Bt Cry1A antibody in the described step 1) is chicken, rabbit, mouse, sheep or horse.
8, Bt immunity detection reagent, its activeconstituents are the described Bt Cry1A of claim 4 antibody.
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| CNB2005101300589A CN100348616C (en) | 2005-12-12 | 2005-12-12 | Bt CrylA antibody, and its preparing method and special antigen and use |
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| CN100348616C true CN100348616C (en) | 2007-11-14 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914156A (en) * | 2010-08-20 | 2010-12-15 | 中华人民共和国北京出入境检验检疫局 | Protein chip kit and method for detecting transgenic crops |
| CN102156193B (en) * | 2011-03-31 | 2013-12-11 | 中国科学院植物研究所 | Method for detecting target protein in plants and special SPR (selective posterior rhizotomy) biosensor for method |
| CN102816235B (en) * | 2011-05-18 | 2013-12-18 | 北京华大蛋白质研发中心有限公司 | Preparation method of hybrid tumor of monoclonal antibody inhibiting common bacillus thuringiensis CryI, and application of monoclonal antibody thereof |
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN117147882B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260397A (en) * | 1999-08-26 | 2000-07-19 | 中国科学院遗传研究所 | Insecticidal protein gene and its use |
| CN1465596A (en) * | 2002-06-06 | 2004-01-07 | 北京市创伤骨科研究所 | Establishment of recombination human bone morphogenetic protein-2 microimmunication and antibody preparation thereof |
| CN1501082A (en) * | 2002-11-18 | 2004-06-02 | 中国农业科学院原子能利用研究所 | Bt crystallin CrylAc radio-immunity test reagent box, preparing and detecting method thereof |
| CN1525173A (en) * | 2003-02-27 | 2004-09-01 | 重庆金标生物技术有限公司 | Insecticidal crystallin BT-Cry1Ab/1Ac rapid detection test bar |
| CN1672049A (en) * | 2002-05-31 | 2005-09-21 | 印度农业研究委员会 | Rapid detection of bt-cry toxins |
-
2005
- 2005-12-12 CN CNB2005101300589A patent/CN100348616C/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260397A (en) * | 1999-08-26 | 2000-07-19 | 中国科学院遗传研究所 | Insecticidal protein gene and its use |
| CN1672049A (en) * | 2002-05-31 | 2005-09-21 | 印度农业研究委员会 | Rapid detection of bt-cry toxins |
| CN1465596A (en) * | 2002-06-06 | 2004-01-07 | 北京市创伤骨科研究所 | Establishment of recombination human bone morphogenetic protein-2 microimmunication and antibody preparation thereof |
| CN1501082A (en) * | 2002-11-18 | 2004-06-02 | 中国农业科学院原子能利用研究所 | Bt crystallin CrylAc radio-immunity test reagent box, preparing and detecting method thereof |
| CN1525173A (en) * | 2003-02-27 | 2004-09-01 | 重庆金标生物技术有限公司 | Insecticidal crystallin BT-Cry1Ab/1Ac rapid detection test bar |
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