CN102109521A - Immunolatex microsphere for detecting CpTI and preparation method thereof - Google Patents
Immunolatex microsphere for detecting CpTI and preparation method thereof Download PDFInfo
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- CN102109521A CN102109521A CN2010105917288A CN201010591728A CN102109521A CN 102109521 A CN102109521 A CN 102109521A CN 2010105917288 A CN2010105917288 A CN 2010105917288A CN 201010591728 A CN201010591728 A CN 201010591728A CN 102109521 A CN102109521 A CN 102109521A
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Abstract
The invention relates to an immunolatex microsphere for detecting a cowpea trypsin inhibitor (CpTI) and a preparation method thereof. The immunolatex microsphere is coated with a CpTI-resistant protein polyclonal antibody and has the particle size of 200nm. The preparation method of the immunolatex microsphere comprises the following steps of: designing a full-length CpTI gene sequence; inserting a CpTI gene fragment into a glutathione-S-transferase (GST) gene fusion expression vector through restriction enzyme digestion to construct a bacteria fusion expression vector; purifying by using an affinity chromatographic column to obtain GST-CpTI fusion protein; fusing a GST-CpTI fusion protein antigen into a Freund adjuvant to immunize a mouse, and separating and purifying to obtain an anti-mouse CpTI polyclonal antibody; and coupling the antibody with the activated microsphere by a chemical coupling method to obtain the immunolatex microsphere for detecting the CpTI protein. The invention has the advantages that: the preparation method is high in sensitivity and repeatability and easy to operate; and the prepared immunolatex microsphere has small and uniform particle size, the lowest detectable limit of 0.2mu g/ml of the CpTI fusion protein, and the related coefficient R2 of 0.67.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of immune latex microsphere that detects Cowpea Trypsin Inhibitor and preparation method thereof.
Background technology
Protease inhibitors extensively is present in the plant, and especially the storage organ's intensive amount plant is quite high.Biological function at plant endoproteinase inhibitor mainly contains aspect two: 1. by with intrinsic protein enzyme interacting, the relevant physiological and biochemical procedure of regulation and control in the histocyte; 2. prevent that cell, in-house protein component from being degraded by exogenous protease.Its more important role is to defend the factor as a kind of chemistry of resisting extraneous plant-feed insect and microorganism attack.Studies confirm that, the protease inhibitors of most of plant origins does not have inhibiting effect to plant endogenous property proteinase, but exogenous protease is had special inhibition activity, and this just provides basis in the natural evolution for the usefulness that improves the crops defence system by genetic engineering.
(cowpea trypsin inhibitor CpTI) belongs to serpin to Cowpea Trypsin Inhibitor.Compare with the Bt toxalbumin, its insecticidal mechanism is different fully and have the desinsection wide spectrum, and to the person poultry safety, insect is difficult for producing the advantage of resistance and is subject to people's attention.By transgenic technology the transfer-gen plant that the CpTI gene transferred crop obtains has been obtained large-scale application in China some areas, but few for the detection method of Cowpea Trypsin Inhibitor in the genetically modified plants so far, and detection sensitivity, detectability and antijamming capability etc. are in urgent need to be improved.At present, about the research report of CpTI inhibitor immunoassay technology and relevant patented technology both at home and abroad report seldom, press for exploitation efficiently, novel immunoassay technology and product accurately and rapidly.
Summary of the invention
The purpose of this invention is to provide a kind of immune latex microsphere that detects Cowpea Trypsin Inhibitor and preparation method thereof, further fill up the research and development blank of CpTI inhibitor immunoassay technology; It is simple, easy to operate to draw materials; The preparation method is careful rigorous, and repeatability is high; Prepared immune latex microsphere particle is little, and particle diameter is even, can detect CpTI fusion minimum 0.2 μ g/ml, its coefficient R
2=0.67.
The objective of the invention is to be achieved through the following technical solutions:
A kind of immune latex microsphere that detects Cowpea Trypsin Inhibitor, immune microsphere are by anti-CpTI protein polyclone antibody bag quilt, and the microsphere particle size is 200nm, and lowest detection is limited to CpTI fusion 0.2 μ g/ml, coefficient R
2=0.67.
A kind of preparation method who detects the immune latex microsphere of Cowpea Trypsin Inhibitor may further comprise the steps:
1) full gene synthesis technology structure total length CpTI expressed sequence is modified and adopted to design total length CpTI gene order again according to the preferential codon of procaryotic cell expression;
2) the CpTI genetic fragment that step 1) is obtained is gone in glutathione-S-transferase (GST) the gene fusion expression carrier by the restriction enzyme digestion cleft cutting, makes up the bacterium fusion expression vector;
3) by step 2) expression vector carried out bacterial expression after, utilize the affinity chromatographic column purifying to obtain the GST-CpTI fusion;
4) will obtain mouse-anti CpTI polyclonal antibody through separation and purification through immune mouse behind the fused Freund of the GST-CpTI fusion antigen that step 3) obtains;
5) will activate microballoon by carboxyl-amino chemical coupling method coupling through the mouse-anti CpTI polyclonal antibody that step 4) obtains, obtain detecting the immune latex microsphere of CpTI albumen.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, in step 2) in glutathione-S-transferase (GST) gene fusion expression carrier be pGEX-4T-2, the fusion expression vector that makes up after finishing is pGEX-4T-2-CpTI.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, the affinity chromatographic column in step 3) is glutathione agarose gel (Glutathione Sepharose) 4B.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, adopting the monodisperse carboxyl polystyrene microsphere in step 5) is host material, carboxylic polystyrene microsphere size 200nm, it is carboxyl that group is carried on the surface, and the molecular formula of carboxylic polystyrene is as follows:
The activation of this microballoon is at first to use activator EDC(carbodiimides) the activation microballoon, add protective agent N-hydroxy thiosuccinimide (Sulfo-NHS) then and make microballoon form stable active ester intermediate, in order to connecting the usefulness of antibody molecule.
Beneficial effect of the present invention is: adopting the monodisperse carboxyl polystyrene microsphere is host material, obtains with the anti-CpTI polyclonal antibody of chemical coupling method coupling, and it is simple, easy to operate to draw materials; The preparation method is careful rigorous, and repeatability is high; Prepared immune latex microsphere particle is little, and particle diameter is even, can detect CpTI fusion minimum 0.2 μ g/ml, its coefficient R
2=0.67.
Description of drawings
With reference to the accompanying drawings the present invention is described in further detail below.
Fig. 1 is the sem photograph of the described carboxylic polystyrene microsphere of the embodiment of the invention;
Fig. 2 is the former sequence total length collection of illustrative plates of CpTI gene among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 3 is the CpTI gene order collection of illustrative plates after optimizing among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 4 is that bacterium colony PCR checking CpTI gene inserts the fragment electrophoresis pattern among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 5 is a plasmid Xba I restriction enzyme digestion and electrophoresis collection of illustrative plates among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 6 is the order-checking collection of illustrative plates of CpTI expression vector among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 7 is the detection collection of illustrative plates of GST-CpTI fusion SDS-PAGE among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 8 is the detection collection of illustrative plates of GST-CpTI fusion Western Blotting among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Fig. 9 is antiserum titre mensuration figure among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention;
Figure 10 adopts the latex enhancing immune turbidimetry to detect CpTI fusion figure as a result among the preparation method of immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention.
Embodiment
The immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention, immune microsphere is by anti-CpTI protein polyclone antibody bag quilt, the microsphere particle size is 200nm(Fig. 1), lowest detection is limited to CpTI fusion 0.2 μ g/ml, coefficient R
2=0.67.
The preparation method of the immune latex microsphere of the described detection Cowpea Trypsin Inhibitor of the embodiment of the invention may further comprise the steps:
1) full gene synthesis technology structure total length CpTI expressed sequence is modified and adopted to design total length CpTI gene order again according to the preferential codon of procaryotic cell expression;
2) the CpTI genetic fragment that step 1) is obtained is gone in glutathione-S-transferase (GST) the gene fusion expression carrier by the restriction enzyme digestion cleft cutting, makes up the bacterium fusion expression vector;
3) by step 2) expression vector carried out bacterial expression after, utilize the affinity chromatographic column purifying to obtain the GST-CpTI fusion;
4) will obtain mouse-anti CpTI polyclonal antibody through separation and purification through immune mouse behind the fused Freund of the GST-CpTI fusion antigen that step 3) obtains;
5) will activate microballoon by carboxyl-amino chemical coupling method coupling through the mouse-anti CpTI polyclonal antibody that step 4) obtains, obtain detecting the immune latex microsphere of CpTI albumen.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, in step 2) in glutathione-S-transferase (GST) gene fusion expression carrier be pGEX-4T-2, the fusion expression vector that makes up after finishing is pGEX-4T-2-CpTI.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, the affinity chromatographic column in step 3) is glutathione agarose gel (Glutathione Sepharose) 4B.
In the preparation method of the immune latex microsphere of above-mentioned described detection Cowpea Trypsin Inhibitor, the microballoon in step 5) is the monodisperse carboxyl polystyrene microsphere, big or small 200nm, and it is carboxyl that a group is carried on the surface; The activation of this microballoon is at first to use activator EDC(carbodiimides) the activation microballoon, add protective agent N-hydroxy thiosuccinimide (Sulfo-NHS) then and make microballoon form stable active ester intermediate, in order to connecting the usefulness of antibody molecule.
Example
The present invention may further comprise the steps in the specific implementation:
1) make up total length CpTI expressed sequence:
(a) design total length CpTI gene order, the result is as shown in Figure 2;
(b) modify again according to the preferential codon of procaryotic cell expression, adopt full gene synthesis technology to make up total length CpTI gene, totally 333 pairs of bases are cloned into carrier pGEM-T Easy, and the result as shown in Figure 3;
(c) correctness of inserting by bacterium colony PCR checking CpTI gene segment, the result is as shown in Figure 4;
(d) will clone CpTI gene bacterium and implement amplification cultivation, and extract and carry out that enzyme is cut and agarose electrophoretic analysis behind the plasmid, the result as shown in Figure 5;
(e) dna sequencing guarantees that the full sequence 100% of gene is accurate, and the result as shown in Figure 6.
2) expression and purifying extract the GST-CpTI fusion:
(a) the CpTI genetic fragment is gone in the pGEX-4T-2 carrier by the restriction enzyme digestion cleft cutting, make up bacterium fusion expression vector pGEX-4T-2-CpTI;
(b) with fusion expression vector pGEX-4T-2-CpTI transformed into escherichia coli BL21(DE3) cell, the cell growth conditions is 230 rev/mins, 37 ° of C of temperature;
(c) as thalli growth OD
595When value is 0.5-0.8, add the derivant IPTG of 1.0mM, induced 3 hours at 25 ° of C; When 4 ° of C, with 5,000 * g centrifugal 5 minutes, collect thalline;
(d) thalline is after PBS buffer solution for cleaning 3 times, ultrasonication; Smudge cells with 14,000 ' g centrifugal 1 hour, is collected supernatant when 4 ° of C;
(e) supernatant is cycled through glutathione agarose gel (Glutathione Sepharose) 4B affinity chromatographic column, 4 ° of C spend the night; Not in conjunction with the 1 * PBS flushing of albumen with 10 times of bed volumes;
(f) the GST-CpTI fusion of combination is also collected, preserved with the eluent wash-out; And the GST-CpTI fusion of expressing and purifying extracts done SDS-PAGE and Western Blotting analysis, the result is shown in Fig. 7 and 8.
3) the anti-CpTI polyclonal antibody of preparation:
(a) with GST-CpTI fusion antigen with after Freund mixes, fully emulsified; Adopt back multi-point injection method immunity BALB/c mouse; The about 10 μ g of antigen amount of each immunity, immunity three times; Each immune two weeks are surveyed antibody titer after arteria caudalis is got blood examination;
(b) with the antigen coated ELISA Plate of suitable concentration, 100 μ l/ holes, 4 ° of C spend the night;
(c) after the washing, add blood serum sample to be checked, 100 μ l/ holes, 37 ° of C 1 hour; If negative control hole;
(d) after the washing, add the antibody reagent of the sheep anti-mouse igg of HRP mark, 100 μ l/ holes, 37 ° of C lucifuges developed the color 15 minutes; Use 2M H
2SO
4After the cessation reaction, read the A490 value in each hole; And the anti-GST-CpTI fusion antibody to preparing, detect through the ELISA method, antibody capable and GST-CpTI, the combination of GST protein-specific, its related coefficient reaches the level of signifiance, tires〉1:8000, the result is as shown in Figure 9.
4) preparation is used to detect the immune latex microsphere of CpTI albumen:
(a) the monodisperse carboxyl polystyrene microsphere is used activator EDC(carbodiimides) activation, add protective agent N-hydroxy thiosuccinimide (Sulfo-NHS) then and make microballoon form stable active ester intermediate;
(b) add anti-CpTI protein polyclone antibody, incubation 1-2 hour;
(c) after the washing, collect immune microsphere;
(d) by CA-1680 type Biochemical Analyzer, adopt the latex enhancing immune turbidimetry, detect the CpTI fusion protein sample.
Adopt the preparation method of the immune latex microsphere of detection Cowpea Trypsin Inhibitor of the present invention, the immune latex microsphere of preparing detects through Biochemical Analyzer, the immune latex microsphere lowest detection that is used to detect CpTI albumen is limited to CpTI fusion 0.2 μ g/ml, its coefficient R
2=0.67, the result as shown in figure 10.This shows that the present invention is prepared, and to be used to detect the immune latex microsphere detection sensitivity of CpTI albumen higher, and simple to operate, repeatability is strong, has shown good prospects for application.
Claims (6)
1. immune latex microsphere that detects Cowpea Trypsin Inhibitor, it is characterized in that: immune latex microsphere is by anti-CpTI protein polyclone antibody bag quilt, and the microsphere particle size is 200nm, and lowest detection is limited to CpTI fusion 0.2 μ g/ml, coefficient R
2=0.67.
2. the immune latex microsphere of detection Cowpea Trypsin Inhibitor according to claim 1 is characterized in that, described immune latex microsphere is the monodisperse carboxyl polystyrene microsphere, and grain size is 200nm, and it is carboxyl that group is carried on the surface.
3. a preparation method who detects the immune latex microsphere of Cowpea Trypsin Inhibitor is characterized in that, may further comprise the steps:
1) full gene synthesis technology structure total length CpTI expressed sequence is modified and adopted to design total length CpTI gene order again according to the preferential codon of procaryotic cell expression;
2) the CpTI genetic fragment that step 1) is obtained is gone in glutathione-S-transferase (GST) the gene fusion expression carrier by the restriction enzyme digestion cleft cutting, makes up the bacterium fusion expression vector;
3) by step 2) expression vector carried out bacterial expression after, utilize the affinity chromatographic column purifying to obtain the GST-CpTI fusion;
4) will obtain mouse-anti CpTI polyclonal antibody through separation and purification through immune mouse behind the fused Freund of the GST-CpTI fusion antigen that step 3) obtains;
5) will activate microballoon by carboxyl-amino chemical coupling method coupling through the mouse-anti CpTI polyclonal antibody that step 4) obtains, obtain detecting the immune latex microsphere of CpTI albumen.
4. the preparation method of the immune latex microsphere of detection Cowpea Trypsin Inhibitor according to claim 3, it is characterized in that, in step 2) in: described glutathione-S-transferase (GST) gene fusion expression carrier is pGEX-4T-2, and the fusion expression vector that makes up after finishing is pGEX-4T-2-CpTI.
5. the preparation method of the immune latex microsphere of detection Cowpea Trypsin Inhibitor according to claim 3 is characterized in that, in step 3): described affinity chromatographic column is glutathione agarose gel 4B.
6. according to the preparation method of the immune latex microsphere of each described detection Cowpea Trypsin Inhibitor of claim 3-5, it is characterized in that, in step 5): described immune latex microsphere is the monodisperse carboxyl polystyrene microsphere, and grain size is 200nm, and it is carboxyl that group is carried on the surface; The activation of described immune latex microsphere is at first to activate microballoon with carbodiimides, adds the N-hydroxy thiosuccinimide then and makes microballoon form stable active ester intermediate, in order to connecting the usefulness of antibody molecule.
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Cited By (4)
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| CN102279272A (en) * | 2011-07-06 | 2011-12-14 | 中国环境科学研究院 | Preparation method of immuno-gold particle reagent strip for detecting cowpea trypsin inhibitor |
| CN102432685A (en) * | 2011-11-01 | 2012-05-02 | 中国环境科学研究院 | Immune latex microspheres for detecting recombined mouse basic fibroblast growth factor and preparation method and application of immune latex microspheres |
| CN104569434A (en) * | 2015-01-14 | 2015-04-29 | 复旦大学附属中山医院 | Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof |
| CN107449905A (en) * | 2017-08-09 | 2017-12-08 | 华中农业大学 | A kind of artificial microballoon for detecting rice Cowpea Trypsin Inhibitor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102279272A (en) * | 2011-07-06 | 2011-12-14 | 中国环境科学研究院 | Preparation method of immuno-gold particle reagent strip for detecting cowpea trypsin inhibitor |
| CN102432685A (en) * | 2011-11-01 | 2012-05-02 | 中国环境科学研究院 | Immune latex microspheres for detecting recombined mouse basic fibroblast growth factor and preparation method and application of immune latex microspheres |
| CN104569434A (en) * | 2015-01-14 | 2015-04-29 | 复旦大学附属中山医院 | Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof |
| CN104569434B (en) * | 2015-01-14 | 2016-08-17 | 复旦大学附属中山医院 | Golgi apparatus protein gp73 latex strengthens immunoturbidimetry detection kit and preparation method thereof |
| CN107449905A (en) * | 2017-08-09 | 2017-12-08 | 华中农业大学 | A kind of artificial microballoon for detecting rice Cowpea Trypsin Inhibitor |
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Application publication date: 20110629 |